TANG-8-93-002 C3

CICtlUIfHgggpy SeaQt'act pepository

Design,Operation and TrainingManual for an Intensive Culture

Granvil D. Treece Joe M. Fox

Texas ARM University Sea Grant College Program Design, Qperation and Training Manual for an Intensive Culture Shrimp Hatchery with emphasison Penaeusmonodon and Penaeusvannamei!

Granvil D. Treece SeaGrant College Program Texas A&M University

and

Joe lVl. Fox Pesca S.A. Guatemala, C.A.

TAMU-SG-93-505 July 1993

Publicationof this documentsupported in part by Institutional Grant NA16RG0457-01to TexasA&M University SeaGrant College Program by thc National ScaG rant Office, National Oceanic and Atmospheric Administration, US. Department of Commcrce, 0~1992. Copyright@1993 Texas A&M UniversitySea Grant College Program All rights reserved. Printed in the United States of America.

TexasA&M UniversitySea Grant College Program Publication TAMU-SG-93-505 ISBN 1-883550-02-5

For more information on Texas A&M SeaGrant publications,contact: SeaGrant Program Texas A &M University TAM U-SG-93-503 2nd Floor, Building 306 1 M July1993 4700 Avenue U NA16RG0457-01 Galveston, Texas 77551 A/F-1 Purpose of this Manual

In 1986there was a perceived need for more written information on intensive shrimp hatchery management, especially with P. monodomand P. vannmnei,and especially for training purposes, In compiling the information in this manual, the intent hasbeen to survey existing information abou t intensive penaeid shrimp hatcheries to develop the production procedures needed to maximize the efficiency of the hatchery and minimize the length of time that it takes to train personnel in the procedures. The underlying question is: what is presently known and what needs to be known to operate a commercial hatchery efficiently? This manual has been organized into three main parts Design, Operation and Training! within nine chapters, The designpart focuseson two hatcheries one for eachspecies! and givesdetailed plans of their constructionas well asother options. The operation portion of the manual details the procedures for most efficient operation of a specific hatchery. The training portion of the manual consists of compiled, presently known background or historical! information thought to be important for training new personnel.Hopefully this compiledmaterial will alsoserve as reference material if future problems are encountered, Having historical or background material as well as current or "state-of-the-art" technology availablemakes it easierto solveproblems. Each hatchery is unique with its own setof requirements,The nextbest thing to experience is having a broad-based reference collection or library at hand. It is the intent of this manual to provide a single publication that will cover most of the important steps in intensive larval shrimp culture, will act as a single reference to our present knowledge on the subject and/or will be a source of information. It is hoped that there is enough detail in this manual to provide the newcomer with an adequate amount of knowledge presentedin laymen's terms to run a hatcheryand, at the sametime, provide details and new technologyto assistthe experienced hatchery manager. Acknowledgements

In addition to those acknowledged at the end of each chapter and in the literature citations, the authors would like to thankthose "pioneers" in the shrimpaquaculture field who havepaved the road to penaeidshrimp larval culture those peoplewho havespent their livesdiscovering, testing, publishing and sharing results. These are the people who have discoveredand published the information through the years that has been compiled in this manual. It is also their discoveriesthat wereused as a basisforthcspccific procedures that worked to makethe Dian Desahatchery more efficient. Someof these"pioneers" are Fujinaga,Cook, Murphy, Mock, Motoh, Platon, Kittaka, Liao, Salser, Yang, Shigueno, Shokita,Tabb, and Villaluz, just to namea few. Someof themore recent workers in thisfield whoalso contributed significantly through their work and publications areJohns, McVey, Tseng, Howell, Simon, Wilkenfeld, Lightner, Johnson, Redman, Primavera, Quinitio, Laramore, Tang, and many morc. Specialthanks go out to the U S.Agency for InternationalDevelopment, Development Alternatives Inc., Central JAVA EnterpriseDevelopment Project, and the Indonesian Government. Members of theseagencies tha t deservean extra special thankyou areJim Boomgard, Anton Sudjarwo, Mumuk Soebagiyo and Pa Dibo for theirassistance and friendship. We alsowould like to thank the SEAFDECpeople for their cooperationin releasingcopyrights to someof the information presentedherein. Thanksgo to Sea Grant for providinga workingatmosphere that allowed this manual to becompleted, to Cindy Liles, Lynn P ropesand DebbieHermann for processingthe manuscript, Amy Broussard,Melissa Hightower and David O'Ncal for editingand to Drs.James McVcy, Robert Stickncy and Bart Baca for reviewingthe manuscript and offering valuable suggestions. Table of Contents

Hatchery Site Selection, Design Criteria, Engineering and Construction. .1 Introduction I B ackground. 1 Small Hatcheries 2 Big Hatcheries 3 Maturation Facilities. ....,., 3 The Hatchery Cycle 3 The Future .... 4 FreshwaterAvailability . Electrical Support ...,... 5 Market Considerations. Availability of SkiBed Labor ...... 5 Availability of Building Materials. ...5 Availability of Mated Females...... 6 Feed Distributors ...... 6 Extension Assistance .6 Competition with Other Operations. ,....6 Expansion Potential ...... 6 Evaluating the Site. ,6 Hatchery Site Evaluation/Selection...... 6 DesignCriteria, Engineeringand Construction. 11 Preliminary Considerations .. ,....11 Approach to Culture ll General Layout of the Hatchery . ,, 11 Modular Concept for Future Expansion, 11 Culture Tanks. 12 Sizing the Hatchery 12 Specific Hatchery Background. ...,.20 Central American Facility Hatchery Example! . 20 Maturation and Hatching . ...51 SpeciesMatured in Captivity. ....51 Maturation ...,.51 ResearchHistory. 51 Maturation-Hatchery ResearchHighlights in the United States 52 Important Parameters for Maturation ,..,53 Other Parameters tc Consider . 54 Additional Considerationsfor Optimum Maturation . ....54 Sources of Broodstock ...,.....,...... 55 Sourcing P. monodonin Indonesia . ....55 ExternalCharacteristics of Adult PenaeidShrimp . 55 General Reproducti ve Characteristics 55 Male Reproductive System ...... 55 Female Reproductive System. .,58 Size at First Maturity and Mating ...... 59 Ovarian Maturation/Staging Mature Shrimp. The Shrimp Egg Mating of Open-TheIycum Shrimp ...... 61 Ma ting of Closed-Thelycum Shrimp . 62 Spawningand Rematuration ..., ...,...... ,...... ,..... 62 Fecundity ...... 63 Hormonal Control ...... 63 Eyestalk Ablation Background and Research . Maturation Feeds B roodstock Operations . ..,65 Broodstock Receiving and Shipping .....65 Broodstock HoIding Ponds 65 Broodstock Distribution and Feeding,. Ablation af Female Broodstock at Dian Desa Hatchery Tank Volume Determination, .70 Record Keeping. 77 Spawningand Hatching . .78 Transferring Nauplii . .78 Disinfection Procedures. .78 Selected Maturation References. .78 Larval Rearing .83 Introduction and Background. Daily Larval and PostlarvalRearing Schedule .83 Larval-RearingTank PreparationBefore Use .84 Other EnvironmentalConditions of Importance. .87 Larval Staging, Feeding and Observations.. .89 Artemia Enrichment 92 Formula 92 For Use with Hatching Artemia 92 For Usewith HatchedSeparated Artemia in seawater!. 93 Algae Culture in Support of Larval Rearing! 93 Growth Medium, 93 CommercialApplication and Community Method of AlgaeProduction used by Continental Obtaining Cultures. 93 Fisheries, Ltd., Panama City, Florida. 95 Growth Characteristics 95 Culture Techniques .....,.... 95 Filtration, Sterilization, Disinfection, 96 Comparisonof Disinfection Methods.. 99 Other Disinfection Methods . 99 CentrifugationWashing, Antibiotics or other Chemicals. ,, 99 Counting Alagal Cells and DeterminingAmount to FeedLarvae 99 AlternateExplanation of AlgaeCell Counting and Determining Amount of Feed. . 100 The Use of the Hemacytometer . 100 Preparationof Sampleand Hemacytometer 100 How to CalculateAlgae Culture RequirementsBased on 60,000Liter Larviculture Capacity 101 Alternate Larval Diets 101 Egg Diet Preparationfor PenaeidLarvae Mysis I or older! 101 Cooking Directions 102 Feeding Directions and Application Rates 102 Microencapsulated Diets . 102 OmegaYeast 102 Signs of LarvalStress, Diseases and Treatmentsat Dian Desa 102 More Detail of Treatments . 103 Harvesting Larval-Rearing Tanks at Dian Desa 103 Selected References ...... ,..... 103 Postlarval Holding and Rearing in Circular Raceways .107 Raceway Description 107 RacewayTank Preparatioonand Operation 107 Raceway Stocking. 108 Postlarval Feeding . 108 Daily Inspection . 109 Diseases and Treatments at Dian Desa. 110 Records 110 Identificationof Postlarvaeand RecognizingHealthy, Desirableor UndesirableLarvae 110 Harvesting Postlarvae 113 Alternate Production Strategiesfor the YayasanDian DesaHatchery 115 Maturation 115 Larva] Rearing 115 Postlarval Rearing . 115 Alternate Production Strategies .. t ~two oct 115 Shrimp Toxicology and Shrimp Diseases, 119 Shrimp Toxicology,. ~ 92tt++M II 0+i ~ +tttI~ It 119 Organic ChemicalsOther than Petroleum 119 Industrial Organic Chemicals 119 Polychlorinated Biphenyls . 119 Pesticidal Chemicals. 121 Organochlorines 121 Organophosphates and Carbamates.. 122 Petroleum . 122 Heavy Metals. 123 Cadmium Cd! . l23 Iron Fe! . 124 Copper Cu! 124 Mercury Hg! . 124 Zinc Zn! . 125 Lead Pb! 125 Nickel Ni!. 125 Maganese Mn!, 125 Magnesium Mg! 125 Aluminum Al!. 125 Treatment of Heavy Metals. 125 Biological Agents 126 Biochemical Agents . 126 Infectious Pathological Agents 126 Interactions of Pollutants, Pollutant Mixes and Other Factors in Penaeid Shrimp 127 Pollutant-SalinityStress Interactions 127 Pollutant-Temperature StressInteractions 127 Pollutant-Pollutant Interactions .... 127 Pollutant-Natural Disease Interaction 127 Larval Diseases Overview. 128 Larval and Juvenile Shrimp DiseasesDetail 131 Infectious Diseases...... , 1 31 Bacterial and Fungal Diseases 134 Fluorescent Bacteria .,.136 Luminous Bacteria . ... 136 Fungi...... I 37 Protozoan Parasi tic Diseases. 1 38 Noninfectious Diseases . 138 Bacterial epicommensals,. 1 38 Protozoan epicommensals...... I 39 Algae ...... 1 39 Nutritional, Toxic and Environmental Diseases Detail...... 139 Nutrihonal Diseases ...... 139 Toxic Diseases, ...... 1 39 Methods for Routine Examinationof Larval Shrimp ...... 1 41 Particular Examinations .. ...142 Methods for Routine Examination of Juvenile and Adult Size Shrimp 142 Particular Examinations .. 143 Media and test proceduresaf specialimportance inpresumptiv e edentification of bacteria . ..143 Virological...... ,...... ,...... ,...... 146 Rickettsia/Chlamidia detecation ..... 147 Fungal ...... 147 Protozoological ....147 Helminthological Flukes, Acanthocephalans, and Tapeworms! 147 Specific Pathogen Free SPF! Shrimp...... 148 Acknowledgments .....148 Selected Recent Shrimp Toxicology and Disease References...... 148 Drugs Used in Shrimp Hatcheries as Chemotherapeutartts 155 Necessityof Antibiotics Chamberlain,1988!. ,.,155 Problems with Antibiotics . ,155 General Use of Antibiotics. ... 155 CommonDrugs Usedin World Hatcheriesas Chemotherapeutants and Applicable Dosages 155 Chernotherapeutants in United States Shrimp Hatcheries ...... ...156 Oxytetracycline 159 TrifluraIin Treflan! . 159 Compoundsto be Replaced. 159 Needed Chemotherapeutants. 160 Results of Larval Rearing and Postlarval Rearing Trial Runs at Yayasan Dian Desa Hatchery .161 Other Information about the Raceways 168 Recommendations . 168 The Hatchery As A 8usiness . ...169 Introduction 169 The Manager's Role. 169 Planning 169 Sample Annual BusinessPlan . 169 Accounting/Bookkeeping 170 Hatchery Capital Flow. 170 The Purpose of Accounting , 170 The Monthly Profit and Loss Statement. 170 Notes on Operating Expenses. , 170 The Yearly Profit and LossStatement 171 The Annual Balance Sheet. 171 Marketing, 171 Packaging . 171 Delivery. 173 Customer Relations . 173 When To Do Marketing 173 How Much Is Necessary 173 Types of Marketing, 173 Summary. 173 Hatchery Cash Flow Example .174 Monthly Cash Flow ...... 176 Large U.S. Hatchery Description 179 Large Hatchery Postlar val Disposition 179 Large Hatchery Production Summary ...... .....,...... ,.....,...... ,...... ,...... 1 79 Large Hatchery Postlarvae Costs ...... I 80 Large Hatchery Postlarvae Production and Disposition by Month s! . ., 1 80 Actual and Projected Large Hatchery Operating Costs 1989-1995 ...... 1 81 1990-1991Equipment Price List for CentralAmerican Shrimp Hatchery in U.S.$! with a ProductionCapacity of 10 Million Postlarvae/Month. ,...,...... ,...... ,...... 182 Aquaculture Equipment Suppliers ...... 186 New ResearchDevelopments for 1993 ...... 186 Adding Enteromorpha to Broodstock Diet ,.........., .... ........... '187 List of Figures

1 Location of Java. 21 Location of Hatchery on Java 22 Dian Desa Hatchery 23 Front and Side View with Elevations. 24 Front and Back View with Elevation 24 246 35ja 25 HatcheryOverview with Detail Original SeawaterSystems 26 7b Final Seawater System 27 Artemia Production Area. 29 810 9Laboratory and Algae Production in FiberglassPolytanks 29 LaboratoryWork Bench 30 11 Carboy Standfor Algae Production ...... 30 12 Various Maturation Systems ...... ,. 31 13 Maturation Tank Detail ...... 31 14 TypicalRaceway Design Postlarvae!. 32 15 SpawningTank Detail, 32 16 Larval Culture Area ... 33 17 Larval RearingApproach ¹2 ContinuousFeeding! ...... 33 'l8 Various Larval RearingTanks 34 19 Larval Rearing Tank Detail. 34 20 RacewayTank Detail 35 20a Central American HatcheryFacility. 35 20b ConceptualDrawing of SeawaterIntake Central American Facility 36 20c Central American Hatchery Foundation/Levels 36 20d Central American HatcheryRooms and Dimensioning. 37 20e Central American Hatchery Tanks/Location 37 20f ConceptualDrawing of Central AmericanHatchery SeawaterTreatment . 38 20g Central AmericanHatchery Seawater Elevated Header Distribution System...... 38 20h Central AmericanHatchery Maturation Facility SeawaterDistribution ... 39 20i Central AmericanHatchery Blower Aeration System 39 20j Central AmericanMaturation Facility Aeration Distribution System .40 20k Central American Hatchery Drainage System. .40 201 Central American Facility Maturation Tank Design .41 20m Central American Hatchery Larval RearingTank ....41 21 Lateral View of Adult Female Penaeussetiferus 56 22 Dorsal View of Adult Shrimp. 56 23 Details of Male and Female Reproductive System. 57 24 Details of Male Shrimp ReproductiveSystem . 57 25 Male ReproductiveSystem 58 26 Details of Male and Female Reproductive System ...... 58 27 Quick Identification of SelectedSpecies . , 59 28 ExternalAppearance of theOvaries of P. monodonat DifferentStages of Maturity ...... 60 as SeenThrough the Dorsal Exoskeleton 29 Developmentof DifferentEgg Types of Penaeusmonodon , 61 30 Courtship and Mating of P. monodon 62 31 Peneausvannamei and monodonLength to Weight Conversion .66 32 Length and Weight Data Sheet .68 33 Feed Rate Determination, .69 34 Weekly Schedule of Maturation Activities 72 35 Maturation Tank Log. 73 36 Actual Egg-Nauplii Data Log. 74 37 Maturation/Hatchery Egg-Nauplii Data Log, 75 38 Larval-State of Health, 76 39 Larval Rearing Tank Data Log . 84 40 Shrimp Larval Stages...... -----"-.--.-"-""- -" -"-"-- ...... 86 41 Naupliar Substages ...... 88 42 Zoeal Substages...... "---"-.--"-"-"- "-" - " -"" - -" -" ""-" .,88 43a SomeCommonly Cultured Microalgae...... 88 43b Typical Phytoplankton Culture Growth ,88 44 Myses and Postlarval Substages ...... 89 45a Stagesof ArfemiaDevelopment ...... 90 45b ArtemiaHatching Cones...... 90 46 Counting Artemia When Concentrated and Hensen-Stemple Pipet 91 47 Typical Cycle Used in Algae Culture 92 48 Glass Carboy 96 49 Typical Algae Culture Rack 96 50 Recommended Ozone Levels and Formula for Checking Ozone Residual .....98 51 Hemacytometer . .100 52 Chemicaland Antibiotic TreatmentsUsed in Philippine Hatcheries ,156 53 FormsUsed for the Calculationof Drug and ChemicalRequirements for EachTank, Morning and Evening. ....158 List of Tables

1 Suggested Larval-CultureSequences and Postlarval Feeding for Yayasan. 85 Dian DesaHatchery, Jepara, Indonesia 2 Classes and Speciesof MicroalgaePresently Under Culture 89 3a Guilllard's F/2 Algal Culture Medium . 94 3b The Philippines Method of Algae Culture 94 3c IndonesianMethod of Algae Culture . 94 4 Alternate PostlarvalFeeding Guideline/Feed Requirement/Feed Costs per Month,...... ...111 5 Postlarval Holding and RearingData Log . 112 6a PolychlorinatedBiphenyls and PenaeidShrimp. 120 6b OrganochlorinePesticides and PenaeidShrimp . 120 6c Petroleum and Penaeid Shrimp 122 7 Diseases Foundin theDevelopmental Stages of PenaeidLarvae and Their Control Meth ods ...... 1 29 8 Penaeid Virusesand Their Known Natural and ExperimentallyInfected Hosts 132 9 Routine ProphylacticTreatments for Shrimp Larvae 156 10 Alternate Method of Larval Culture Sequences,Postlarval Feeding Regime and Disease PreventiveSchedule Used by JoshVilkenfeld, Aquatic Farms,Ltd., 1989. 157 1 l Actual Larval RearingTank Log for Tank Number 1 162 12 Actual Larval RearingTank Data for Tank Number 2 164 13 Actual PostlarvalRearing Tank Log for RacewayNumber 7 .. 166 14 Actual PostlarvalHolding and RearingData for RacewayNumber 8 167

36. Broodshrimpsettling in and eating squid; someare more stressedthan othersupon arrival and appeared red in color and swim at the surface until they too settle in 37. Healthy Broodshrimpabout one week after stocking;ready for ablation and to be placedin production 38. Demonstratingproper ablation technique holding 450gram femaleBroodstock just under water surface with one hand and removing contents of one eye with the other hand. Contents have been removed in the right eye! 39. A soft nylon net is used to capturebrood from ablation or just for inspection 40. Constant water level maintained in maturation tank by this dual standpipe water being drawn from the bottom of the tank at this point, but by removing the outer standpipe water can be "skimmed" from the upper surface in case there is excessfood floating! 41. Effluent flow rate is checked daily at the maturation tank in order to maintain a constant, stable environment 42. Fresh molt on the bottom of maturation tank 43.. Salinity readings are taken routinely once a week unless during the rainy season then refractometer readings may be required more often! Molts are removed after they are no longer a good food source usually within 24 hours and they have turned yellow-gold! 45. Using inexpensive underwater light attached to PVC pole! to locate late stage female shrimp 46. Flashlight can be placed such that light beams shows through exoskeleton and stage of developing female is determined 47. Records are kept for the day on a formica board hung near each tank 48. The daily records from the formica boards are kept on a clipboard for easy reference in the event there is a problem 49. Late stage females are placed individually in a series of 800 liter spawning tanks, each equipped with water inflow and screen standpipe 50. Counting egg and nauplii out of a 100 ml beaker subsample taken from the spawning tank note inflow and effluent screen with air collar! 51. Larval rearing tank approximately one-half full micron filter cartridge on inflow; central, screen standpipe with air collar and 100,000cells/ml algae! Larval rearing area Seriesof larval rearing tanks;some covered with plastic during evening hours to keep temperature up 53. 4,000 liter Larval rearing tank filled to capacity 54. Siphoningoff freshly hatchedArtemia nauplii after settling note the stratified layers;the bright orange layer is nauplii! 55. Laboratory work bench with balance and chemical storage 56. Algae lab behind closed doors in air-conditioned room 57. Algae production in small flasks 58, Algae production in glassjars wide mouth allows easyaccess for cleaningjar! 59. Figure 50, typical algae room arrangement 60. Figure 50, typical algae room arrangement 61. Postlarval rearing and holding area 62, Postlarvalholding and rearing tank 5ft.-dia., ferro cement,circular, holding approximately 12 tons, airlifts and screen habitats! 63. Feedingpostlarvae a finely ground pellet 64. Siphonused to cleanPL holding tank when necessary 65. Postlarvalrearing and holding data board formica board locatednear the tank! 66. 1,000liter plastic containers used to transport postlarvae 67. Example of a fiberglass postlarval transport tank Glossary of Selected Terms ablation Extripation, removalor otherwisedamaging the eye in somemanner to promote maturation. Anemia Brine shrimp. aseptic Sterile. axenic Free from other living organisms. bacteria One-celledorganisms that canonly be seenwith a microscope.Compared to protozoans they are of less complex organization and are much smaller in size. broodstock Larger animalsthat are sourcedand expectedto produceoffspring in the maturation facility. carboy Glass bottle. cysts Eggs of Arternia that are in a dormant stage. de capsulation Removal of the thick outer layer on Artemia cysts. disinfection Reduction of bacterial numbers to a "safe" or "acceptable" level. exoskeleton Shell of the shrimp. grooves There are numerousgrooves on a shrimp.Some are usedas an aid in identifying the species. Examples: The "thumbnail" grooves on the last abdominal segment of tropical Atlantic brown shrimp distinguish them from similar white shrimp; Thelength of the rostral grooveis shorterin P. monodonthan in P. semisulcatusand canbe used asone way to distinguishthe two similar species.Other distinguishing characteristics can be seen in Figure 27. hemacytometer Device used for counting algae cells Figure 51!. hemoiymph Blood. Hensen-Stemple pipet Pipet used to take an objectivewater sample Figure 46!. hep atop ancreas Digestive gland. larvae Plural of larva and the stage in the development of a shrimp's life cycle between the egg and the juvenile. maturation The act of maturing In the caseof white shrimp, egg development, mating and then spawning; with brown shrimp, mating, egg development and spawning!. media Plural of medium and in this case refers to saltwater and nutrients or food added to promote proper growth either of algae,larvae, etc. For shrimp, sheddingof the exoskeleton. nauplii Plural of naupliusand the first of threemajorlarval stages.Itisa non-feeding stageand isthebest stage to transport the animals until they reach the postlarval stage. penaeid The family, superfamily and an infraorder of shrimp distinguished from the caridean shrimp by the shape of the second segment of the abdomen. The sides of the penaeid shrimp shell known as the pleura! overlap each segment that is behind it. In the caridean shrimp the pleurum of the secondsegment overlaps both the first segmentand the third, making the secondsegment look very large. There are 109 species of penaeid shrimp listed by the Food and Agriculture Organizationof the United Nations F.A.O.!. petasma Male shrimp reproductive structure. prawn According to Dore and Frimod t 987!, different people use this name to mean and to apply to quite different species. Examples: U.K.: larger than shrimp, U.S.: restaurants use it to mean large shrimp and other places mean small shrimp or freshwater shrimp Norway: producerspromote the northern shrimp Pandalusas a prawn, and the "Dublin Bay prawn" is evenused to describeNephrops, which is a langoustineor Norway lobster.

Xiv The OxfordDictionary defines prawn as '1argerthan shrimp" whereas Webster's Dictionary describesit as "a small, edible crustaceanof the shrimp family." SouthAfrica. larger animals are prawns; smaller animals are shrimp. F A O. attemptedto introduce a clearcutdefini tion as early as 1967, At theWorld Conference on thebiology and culture of shrimpsand prawns held in MexicoCity, it wasagreed that the term "prawn"was to be reserved for freshwater creatures only, while their marine/brackish water relativeswere to be called"shrimps."Unfortunately, despite all of theirefforts, the confusion continues.The only point on whicheveryone can surely agree is thatthe use of "prawn"in the Englishlanguage is confusingand unclear, and shouldbe avoided. protozoea New world term of zoea or the secondmajor larval stageof penaeid shrimp. raceway A smallpond or tank that is usually rectangular with a centerdivider or circulartank with a water flow that "races" around the tank. rostrum Thepointed prow that extends from the head of mostshrimp. setae Hair-likestructures that appear to branchoff of appendagesor legs. sourcing Obtaininganimals to be usedfor broodstock. spore A small cell that can develop into a new individual. spp. or sp. Plura]and singularabbreviations for species, respectively. sterilization Total inactivation of all microbial life. thelycuxn Femaleshrimp reproductive structure, Chapter 1 Hatchery Site Selection, Design Criteria, Engineering and Construction

Introduction There is a perceivedneed for more written information on intensive shrimp hatchery management, especially with P. manadanand P. vannamei,and especially for train- ing purposes. In compiling this manual, existing information on intensive penaeid shrimp hatcheries hasbeen surveyed to develop efficient production procedures and minimize personnel training time. The underlying question is what is presently known and what needs to be known to oper- ate a commercial hatchery efficiently? The manual is organized into three main parts design,operation and training in nine chapters.Design focuseson two hatcherics onefor eachspecies! and gives detailed plans for construction as well as selected equip- Photo1. Seasonal haruest of zoild postlarvae arith scissor nets from the ment options. The operation portion details the most Pacificbeach of Bahia,Ecuador. efficient procedures for a specific hatchery, and the train- ing portion consists of compilations of background or In the1990sthere willbe thousands of shrimp hatcher- historical information thought tobe important in training new personnel. ies of all sizesoperating worldwide, helping to supply "seed" or postlarvae to one of the most valuable industries Hopefully, this material will also provide a referenceif future problemsare encountered,since having historical in the world. In 1992, over 700,000 MTs of farm-raised orbackground inaterial as well as current or "state-of-the- shrimp wereharvested 50,000 MTs from Thailand alone!. art" technologyavailable makes it easierto solve prob- Shrimp farmersnow produceat least28 percent of the2 6 lerns.Each hatchery is unique with its own setof require- million metric tons of shrimp placedon world markets ments.The nextbest thing to experienceis having a broad- today,and this percentagehas risen steadily since1981. based referencecollection or library at hand. It is the intent These shrimp hatcheries help provide a more reliable of this manualto provide a singlepublication that covers supply of postlarvaefor pond culture, especiallysince most of the important steps in intensive larval shrimp wild populations fluctuate unpredictably and are pres- culture, acts as a singlereference to our presentknowl- ently being harvested at or beyond their maximum sus- edge on the subject and /or is a sourceof informa tion. It is tainable yields. As a result of this exploitation of wild hopedthat thereis enoughdetail in thismanual toprovide populations,many countrieshave placed restrictions on the newcomer with adequateknowledge in laymen's the collectionof wild shrimp populations seeharvesting termsto operatea hatcheryand, at the sametime, provide wild postlarvae,Photo 1!. Shrimp hatcheriesare helping details and new technology to assist the experienced to fill thedemand for postlarvaeand to relieve someof the hatchery manager. pressuresplaced on the wild stocks.Undoubtedly, the lackof a reliablesupply ofpostlarvaewas once among the biggestsources ofuncertainty, inefficiencyand economic Background lossesfacing shrimp farmersworldwide. As time passed Shrimp of the family Penaeidaeform thebasis for one and we havebecome more familiar with the technology of the most valuable fisheriesin the world. The highly involved in shrimp reproductionand growout in captiv- esteemedquality and edibility of theseshrimp, their rela- ity, these uncertainties do not seem so monumental, We tively large size and their worldwide distribution have still havea longway to go beforewe arc totally indepen- eliciteda largeinvestment of manpowerand funds in the dentof naturalstocks, but thegap is closingquickly with shrimp industry. Worldwide, thc pcnaeidshrimp fishery the rapid technologicaladvances that have occured in is an annual multibillion dollar enterprise.According to shrimp hatcheries since 1985. Rosenberry 992! the fishery is a 2 million metric ton To solvethe majorlimiting factorof insufficientavail- MT'! marketof which 28 percent or 700,000MT'! were ability of postlarvae,mass production in hatcherieshas farm-raised in 1992. receivedmuch a ttcntionfrom researchers.The techniques 2 ... Treeceand Fox are now well known, although the results are not always satisfactory.The following threemam znethodsare used: ~ TheJapanese method ischaracterized bylow densig of aroundten larvaeper liter, largetanks up to 200m, a need for nuznerous wild-caught gravid females, direct water fertilization by inorganic nutrients to promotealgal bloom,and production of 20to 30day- oId postlarvae.This method is well adaptedto tem- perateconditions when it is necessaryto producethe seedin a shortperiod of time.Hut theselarge-volume tanks are difficult to con tz'olin tropical conditions and resultsare dependenton variationsof water quality and light. If diseaseoccurs, a curative treatmentis almost impossiblewith suchlarge voluznes. ~ Incontrast, theintensive method ischaracterized b~ high density00/I!, small tanksbetween 1 to 10m, Photo2, Exampleof a largehatchery in Ecuador. few gravid females,phytoplankton or Artemiapro- ductionin separateculture systems and productionof alreadyprofitable in someparts of the world under differ- one to five day-old postlarvae can be modified to ent socio-economic conditions. We can also look forward produceolder larvaeif necessary!.It allows accurate to majorimproveznents that will leadto increased produc- controlof rearingfor water quality, foodquantity and tivity with a paralleldecrease in costs,which, in turn, will quality, anddiseasesby preventive andcurative trea t- broaden the market, ment. It is important to analyze the reasonsnot only for ~ The intermediate method is a coznbination of the first successbut also for failure. Technology can succeedonly two methodswith zneandensity 0/I!, medium-size if applied under the right conditions.Many failureshave tanks 0 to 50 ms!, and use of fertilization in the tanks been due to introduction of technology inadequate for to bloom an algal inoculum cultured separately, specificsite constraints and logistic availability. Thesethree methods differ mainly in degreeof control The management of production units is also essential. and,when practiced by experiencedpersonnel, give good Too often investors have focused their attention on bio- results. However, it must be znentioncd that many exist- logicaland technologicalproblezns, forgetting theimpor- ing hatcheries su ffer from a lackof reliability in resultsdue tance of routine decisionsand proceduresnecessary for to siteconditions, inadequate control of algal quality and any business.Success depends on good managementand lack of knowledge of thc necessary sanitary procedures technological advancements. suchas regular dry-out to eliminate the resistantpatho- Shrimp hatchery activity is just emerging from its genic bacterial strain problem that is always the most infancy.The successof the broiler industry was achieved limiting factor in a hatchery operating year round in only after more than 20 yearsof intensivescientific and tropical conditions. In recently establishedhatcheries, commercial effort, and the shrimp industry will undoubt- these results have prompted physical separation of the edly follow the samepath. Therate of developznentwill .different steps of the system in roozns that have indepen- dependon how closely researcherswill work with pro- dent water pipes and nets,that can be closedand dried ducers to identify the constraints and to integrate avail- regularly.At the commerciallevel, the resulthas been the able techniques according to the socio-economic condi- developmentof large-capacityproduction hatcheries 0 tions of each country. to 20znillion PL/nonth! in Japan,Ecuador and Panamaas The accompanying schematic represents the different well as in small-scale hatcheries in Taiwan, Thailand and feeding regimes used worldwide for the developmental the Philippines.The presentchallenge is not to be ableto stagesof penaeid shrimp and related culture parameters: producepostlarvae, but to optimize the techniques. Hatcheriessell two products:nauplii tiny,newly hatched To reach the necessary reliability level and decrease larvae! and postlazvae juveniles that have passedthrough production costs,research priorities have been focused on three larval stages!.Postlarvae are stockedin nuzsezyponds the following: or directly into growout ponds, Nau plii aresold to specialists ~ Characteriza tion o f algal quality according to culture who grow them to the postlarvae stage or to farmers who techniques stock them in nursery ponds at high densities and later ~ Replacement of algae and Artemia by inert feeds transfer them to growout ponds at lower densities.Hatcher- microparticles, microcapsules! ies come in two sizes big and small. ~ Optimization and automationof the procedures re- circulation systems! Small Hatcheries ~ Development of strict sanitary procedures like those Sznall hatcheries are usually operated by a family routinely used for pig husbandry group on a small plot of land. Called "nom-and-pop" or ~ Cryopreservationof sperm, eggsand nauplii to be "backyard" hatcheries, they adopt a green thumb, non- able to run the hatcheries by sequential period, technical approach. Their chief advantages are low con- Shrimp-hatchery techniques are far from their opti- struction and opera ting costsand their ability to open and mum efficiencya ta timewhen commercial opera tions are close, depending on the season and the supply of wild Design, Operationand Training ... 3

Larval Rearing Techniques of Penaeids

I Decreased light intensity Zoea 1! Zoea stage larvae light-sensitive / 2! Permits uniform environmental conditions t

Defused light intensity Cultured zooplankton 1! Mysis stage no longer light-sensitive Mysis x/ 2! Conditions larvae to natural environment ! /x I V Artemia naupl i i

Thinning I/ Artificial feed f! increases living space Postlarvae 2! Enhances vigor, rapid growth 3! Avoids stunting, high mortality Chopped meat of clam or shrimp indicates most prominent feeding regime indicates occasional feeding regime

* indicates community culture method only seed.They usually concentrateon just one phaseof pro- marginally successful.Some species are very difficult to duction, suchas nauplii or postlarvacproduction. They mature in captivity. Other species, such as the western use low densities and untrea tcd water. Diseases and water white shrimp, will mature in captivity, but it is a tricky, quality problemsoften knock them out of production,but expensive proposition. The Chinese white shrimp, on thc they can quickly disinfect and restart operations.Small other hand, seemsto be especially well suited to matura- hatcheries have achieved great successin SoutheastAsia, tion facilities. Viral and bacterial diseases limit the devel- particularly in Thailand,Taiwan and the Philippines. opment of successful ma turation facilities, Big Hatcheries The Hatchery Cycle Bighatcheries are multi million dollar, high-techfacili- Whether pregnant shrimp are captured in the wild or ties that produce large quantitiesof seedstockin a con- matured in a hafchery, they invariably spawn at night. trolled environment. Requiring high-paid techniciansand Depending on a number of variables temperature, spe- scientists, they work with high densities and clean water, cies, size, wild/captive and number of times previously attemptingto takeadvantage of thc cconorniesof scaleby spawned!, they produce between 100,000and 1,000,000 producing seedstockthroughout the year. When wild eggs. The next day, thc eggs hatch into nauplii, the first females and juveniles are readily available, big hatcheries larval stage. Nauplii, looking nrorc like tiny aquatic spi- have a difficult time competing with small hatcheries and ders than shrimp, feed on their yoke sac for a couple of fishermenwho supply freshly spawnednauplii or wild days. Then they metamorphose into zoeae, which have postlarvaeto farms. In Ecuador,farmers considerwild featheryappendages and elongatedbodies but few adult postlarvae the "Cadillac" of seedstock. Big hatcheries shrimp characteristics.Zocac feed on algae for about five haveproblernswithdiseascandwaterquality,andit takes days and then metamorphose into rnyses.fvtyses, the last them a long time to recover from production failures, In larval stage, have many of the characteristics of adult thewesternhemisphcrc,particularlyin Ecuador,bighatch- shrimp, such as segmented bodies, bulging eyes and a eries are the trend. Big hatcheriesalso supply most of shrimp-like tail. They feed on algaeand zooplankton. This China's seedstock, and big hatcheries are established in stage lasts another three days and then the myses meta- most of the major shrimp-farming countries secexample morphose into postlarvac. Postlarvac, looking almost like of large hatchery in Ecuador, Photo 2!. adult shrimp, feed on zooplankton, detritus and commer- cial feeds. After one to four weeks and almost daily molts, Maturation Facilities the postlarvae arc stocked in nursery or growout ponds. Maturation facilities are big hatcheries that maintain The larval period lasts abou t two weeksand the posllarvae captive broodstock to produce seedstock. They represent period four weeks, so from spawning to stocking in the future of the industry but, thus far, have been only growout ponds takes abou t six weeks. 4 .T'reeee and Fox

The Future quality seawater, Most successful hatcheries obtain their scawatcr directly from thisbody of water with theunder- Hatcheries remain the weak link in the production standing that the ocean is a fairly stable water source.The cycle. Feeding the various life stagesof developing shrimp further a hatchery is located from thc ocean, the more takes a majoreffort. Hatcheries are plagued v ith manage- likelv the facility is to experience difficulties in terms of ment, disease and water-quality problems, yet they are pumping, water quality and accessto broodstock. constantly improving and constantly increasing produc- tion. Hundreds of rcscarchcrs in a dozen countries work Distance from Ponds/Access on unraveling thc mysteries of hatchery production, and There is no optimum distance between the hatchery thousands of hatchcrymenin all thc shrimp farming coun- and the pond market it supports; however, an optimum tries work with new techniques, designs and ideas to situation could be described as a hatchery centrally lo- improve hatchery production. When hatcheries become cated among thc ponds it supplies with postlarvae PLs!. more reliable, the production of farm-raised shrimp will This helps to reduce PL transport costs and transport take another leap forward Bob Rosenberry, Aquaculture morta1ity, and generally helps public relations with buy- Digest!, ers. In Indonesia, hatcheries have been known to sell PLs The svstcrn described in this manual is called the to ponds more than l,000 km from the hatchery, which intensive culture system and can be conducted in either requires air transport. This is also complicated by inter- small or large hatcheries. The advantages nf this system island licensing procedures. A morc practical situation include the following: would involve the majority of ponds being less than two ~ Low initial investment to threchours,by car, from the hatchery.If road conditions ~ Only a small number of' spawncrsrequired for onc are favorable paved, well-maintained, low traffic, etc.!, operation the rnaxirnum distance is primarily dependent upon qual- ~ Larvae can be reared at high densities ity of packaging. Some hatcheries in Ecuador transport ~ Ea sicr to control d isea ses PLs from the hatchery to the ponds by boat,but many are This manual presents information on the intensive sent by air freight using direct runways near the pond method first by introducing hatchery site sclcction and sites. design criteria, engineering and construction of two very similar hatcheries onc for P, rnnnodon and onc for P, Elevation and Topography r anrurnrei!to show the similarities of procedures and de- Thc chosen site should bc flat and elevated above the sign criteria as well as the subtle differences, Maturation, maximum high tide mark but within reasonablepumping hatching, larval rearing, postlarval hoIding and rearing, distance. A good rule of thumb is hatchery floor elevation d iscascs, chemo thcrapeu tan ts, and thc hatchery as a busi- of less than 3 to 4 meters above the maximum high tide ness is also discussed. The technologies discussed in this mark and an overall pumping head height! of less than 6 manual also can be modified slightly to include intensive meters. Pumping cost verses elevation appears to be a culture of all penacid species. fairly linear relationship up to a certainpoint. At times, the The hatchery site seIection, design criteria, engineer- pumping requirement for even a small hatchery is in ing and construction plans that follow are from two inten- excessof 600to 7001itersper minute, if operated properly. sive hatcheries one P. nronodonhatchery in Indonesia and If theland is not flat, the expenditureon sitedevelopment onc P, vannanreihatchery in Central America!. will increase due to increased excavation or filling. The site should be on fairly solid ground. A coral or stone base Site Selection may be difFicult to level, but will eventually mean a reduction in overall concrete required for the foundation. Because the shrimp hatchery is concerned with the In some cases, concrete may not even be required. A production of living animals that would otherwise be foundation that uses a sand base will have a tendency to found in a marine or brackish-v,ater situation, special settleand will require additional concretesupport. consideration must be given tow ard selection of its loca- tion. Theprimary concernin situatinga hatcheryis water Water Quality quality. Sinceshrimp spawn in the open oceanand larvae The primary water quality considerations are pollu- remain there until they become postlarvae, hatcheries tion and fluctuations in seawater components within an should be able to accessoceanic-quality seawaterdirectly optimum range.Very few bodiesof water are pollution and have a year-round, constant salinity. Proper site selec- free and caution should be exercisedwhen reading re- tion also rncans proximity to thc local market farmers, ports that make this claim. In choosinga hatcherysite, middlemen,buyers, etc,!, accessto support industries and locations adjacent to or within 30 km of a major river utilities, simplified engineeringand construction,and systemshould be avoided unlesscareful analysisof the integra tion with gro wout ponds. Another majorconsider- watershedhas been performed. This involves surveying ation for hatchery site location is accessto a disease-free all potentialsources of industrial, municipaland agricul- broodstrock supply. The folIowing are importantsite tural pollution that impact that particular river. Nearby sclcction criteria. factoryor plant personnel,farmers, etc., shouldbe inter- viewedto identi fy possiblepollutants. Most hydrocarbon, Distance from Ocean pesticide or heavy metal pollution is quite serious and As mentioned previously, hatcheries should be lo- should bc avoided at all costs.The extent to which organic cated on the coast adjacent to a large body of oceanic- poilu tion e.g.sewage ou tfalls! hasa ffecteda waterwayis Design, Operationand Training ....'>

somewhat morc difficult to assess due to the inherent ability of the waterway to absorb nutrient offloading. A useful index of organic pollution is biochemial oxygen demand BOD!,which isan indicatorof thebiomassbeing supported by a body of water and how much oxygen it requires over a given period of time. High nutrient loads will support a high biamass primary productivity, macrospecies,etc.! as Iong as oxygenis availabIe.If this nu trient load is releasedinto thc oceannear the hatchery, it could result in blooms of disease-causing microbial species and place an excessive load on filtration equip- rnent, Therefore, it is recommended that water chemistry sampling take place not only in wa ter ways impacting the hatchery's near-shore environment, but also in the area aroundthepotentialhatcheryintake. Thissarnplingshould occur during the rainy season, since runoff is greatest Photo 3, Generators during this time of the year. Water chemistry sampling should bc continued on a monthly basis once the hatchery generator sct large enough to run the entire hatchery is is in operation. also recommended. If no rural electricity is available, two Although watersalinity is fairly easyto determine,it is generatorsets should be purchased.Rural electricpower oneof the majorproblems encountered as a resultof poor can be distributed more easily and can accommodate a hatchery site selection. As a rule-of-thumb, all hatcheries larger demand than gcncrator power, but it often can be should use water taken directly from the ocean, and morc costly to run lines from the main power pole to the prospective sites should not be located within 30 km of a hatcherythan to purchasetwo generatars Photo3!. major river-mouth. Hatcheries in Indonesia are often situ- ateddnear rivcrmouths and, as a result,experience tremen- Market Considerations dousseasonal fluctuations in salinity.The optimum range Hatcheries often are built without consideration of in salinity for hatcheryculture of penaeidshrimp is about demandor markets.If the hatcheryis owned by a com- 26 to 32 parts per thousand ppt!. Mature adult and pany that alsoowns growout ponds,the market is fairly postlarval shrimp are naturally found in the ocean,where secure most of the fry will be stockedin company-owned the salinity is approximately35 ppt 5 gramsof salt per pond s!, If not, the best possible approach is to estimate the liter of water!. numberof pondsin the localarea that would be willing to Water pH is seldom a problem in hatcheries thatobtain buy PLs from a hatchery.This can be accomplishedby their water directly from the ocean, Thc pH of seawater visiting the local fisheriesrepresentatives or by talking should fluctuate around 8.0 or slightly higher unless the with local pond farmers. Many times the market demand body of water usedas a sourceis polluted or is receiving is basedon a seasonalstocking strategy. There also could a largeamount of freshwaterrunoff. Occasionally,algal be somereluctance on the part of thepond farmersto buy culture will be modified by enrichment with carbon diox- PLsthat havebeen produced in a hatchery.These factors ide resulting in lower pH, but this is not a characteristic must be determinedprior to siteselechon. required of the water source. A very thorough water analysisfor a potentialhatch- Availability of Skilled Labor ery site in SouthTexas can be scenin Appendix A, B and Sinceit is usually quiteexpensive to impo~tlabor from C of the Hatchery Site Evaluation/Selection section of this outside the pond site area,and sometimessocially unac- chapter. ceptable,most hatchery developers turn to thelocal labor forcefor hatcherypersonnel. It is highly unlikely thata FreshwaterAvailability hatchery manager can be found in most rural areas,but a Freshwatermust be availableat thehatchery site both substantialproportion of the remainingpersonnel can be for cleaningand for the workers. Also, many nutrient- hired locally. It helpsif this local group hasat leastsome enrichmentmedias require preparationin fresh or dis- familiarity with hatcheryconcepts. Otherwise, extensive tilled water. Often hatcheries are located in areas with training is necessary,which increaseshatchery start-up very little or no ground water, or with water that is not time and overalI s tar -up costs. potable and may fluctuate in its concentration af dis- solved solids. The extent of the water table mus t bc known Availabilityof BuildingMaterials beforea wellcan be considered. It is too expensiveto truck Availability of suitablebuilding materials concrete, water to a site for this to be considered a viable alternative. sand,gravel, wood, roofing material,paint, piping,etc,! at reasonablccostsisanotherimportantcansidera tion.Many Electrical Support times hatcheriesare built in areasthat possessexcellent Continuity is themost important criterion for electrical water quality yet are so far from material outlets that support. Electricity is used to power aeration devices, constructioncos ts double. Sometimes con tractors request seawater pumps, algal culture lights and heaters should specificmaterials in order to achievea certaindesign but theyberequired!,Ruralelectricityis certainly a bonus,but neglectto determine whether these materials canbebought should not be relied upon as the only sourceof power.A locally.The further one locates a hatcheryFrom a large 6 ... Treeeearid Fox metropolitan area, the higher the cost of the material if to receive some assistancein circumventing disease i.e. locally bought! or the higher the cost of transport. In remote identification, prevention, treatment! it would certainly areas,the quality of material can often suffer. Poor storage bc advantageous to have some research institution in the conditions can leave the contractor with lit tie to choose from. vicinity. Identifying a localfisheries department represen- An exainpleis the use of volcanicsand in foundationand tative in the areacould help the hatcheryowner/operator tank construction. This sand, if purchased in Indonesia keep abreast of industry or fisheries development. Yogyakarta area! would cost about Rp, 4,000 per cubic tneter U.S. $4.68!.In building a hatchery near Semarang, Competition with Other Operations Indonesia,the contractor rcquircdroughly 1~ cubic meters If the local market for postlarvae is quite large, then a of this sand.Instead of paying the Rp. 4 million U.S.$4,680! little competition could be quite healthy in terms of the heexpected to pay,he eventually spent Rp. 14 million U.S. developmentof a shrimp hatchery. Often one hatchery $16~!. The more remote areas of the world where hatch- mayencounterunfamiliarproblems tha tanadjacenthatch- ery site locations and water quality may be more desirable ery has already solved. If these two hatcheries have a may also cost the owner double to build. This must be history of information exchange,both wiII benefit. Inter- consideredduring the planning stage. action is something tha t could be beneficial for the entire industry.A clandestineapproach to assistancecould prove Availability of Mated Females dctr imcntal later, Some potential hatchery sites are located adjacent to Should the local PL market be so small that it can large spawning grounds and can easily obtain a ycar- support only one hatchcry, it may be best to go elsewhere round supply of mated females for production of eggs/ if such a hatchery already exists. larvae!. This enablessuch ha tchcrics to reduce their opcra- honal overhead since they do not need to support a large Expansion Potential maturation facility. This is also advantageousfor site One aspectof site selectionthat is seldom considered is investigators since it reduces capital outlay on construc- expansion potential should the market demand increase. tion smaller maturation facility containing only spawn- Sometimesthc site itself will not allow for expansion due to ing tanks!. Information on availability can best be ob- physical constraints or the area available for development. tained by visiting thelocal fisheries department represen- Evaluating The Site weighted ranking system! tativeeand/or a localfishingvillage. Since offshore ma ting ~ Most evaluations use this method is seasonal in nature one should also ascertain the extent of the spawning/fishing seasonsand to what extentac- ~ Assigns relative weight of iinportance to criteria cessto these grounds is limited by thc monsoon or rainy ~ Assigns a scoreto each measurement or observation season!,which usually affects the mating seasonin some ~ Resultis weighted scoreversus ideal man~er. A source ofbroodstock should be found within 100km of the proposed hatchery site, if possible, and the Hatchery Site Evaluation/Selection stock should be disease free. Site Evaluation Feed Distributors Scoring The availability of feed refers primarily to inert/for- Range of Scores %! Evaluation mulated feed and Arternia, Thc cost of these feed items will 100-80 Excellent site increasein relation to transport distance, Somehatcheries 79-60 Good to Fair bypass higher prices by buying Artemia direct from the 59-40 Marginal United States while pcllctcd fccds are purchasedfrom Less than 40 Avoid site large importer s in thc local area. However, should a local feed distributor stock the particular brand of Arterrtia or Site sclcc ionis perhapsthe most critical step in the feed to be used, that is only to the advantage of the development of a successfulhatchery business. Selection hatchcry owner, However, because of poor stockings trat- of a suitable site can have a significant impact on the cgics and supply, local distributors are sometimes found profitability of the business.Many unsuccessfulhatcher- to be without quali ty feeds. Thus, it is recommended tha t ies were troubled by site-relatedproblems that were too beforechoosingahatchery sitesomeidca of feedavailabil- d ifficult or expensive to correct. The ideal site for a hatch- iP from local distributors should be obtained, The more ery probably doesnot exist anywherein the world if all conscientious hatcheries choose to buy direct from im- site selection criteria arc considered. The decision to locate porters or manufacturers, This is not so much an attempt a shrimp hatcheryat a specificsite is usually basedon to savemoney but an cnsuranceof availability. whetherthe biological requirements of the shrimp canbe fulfillcd at a reasonablecost. Political stability and safety Extension Assistance of the investment,as well as potential difficulties in re- Hatcheries are intensive culture systems. Being such, couping profits, must also be considered before the site they are often plagued by various diseasesof a nutritional selection decision is made. Five to ten acres two to four and/or endemicnature. Furthermore, hatchery operators hcctarcs!is generallysufficient land to build a hatchery locatedin remote facilities sometimesfeel that they arc and accommodatefuture expansion. losing touch with current developmentsin the industry A list ofboth general and detailed information needed new innovations, enhanced availability of previously for proper evaluation of a site follows. limited supplies, government regulations, ctc.!. In order Design, Operatianand Troirung ... 7

General Information on Site

1. Location:

2. Map:

3. Access:

4. Wave Action:

5. Water Salinity:

6. Water pH:

7. Elevation Above Seawa ter Source:

8. Land Topography: 9, Benthic Topography/Description:

10. Seawater Inlet Distance From Site: ll. Type of Inlet Prescribed:

12. Water Depth at Inlet Site:

13. Pollution Potential:

14. Organics in Water?: 15, Turbidity:

16. Nearest River:

17. Source of Broodstock/Price/Conditions:

18. Electrical Power:

19. Freshwater Source:

20. Aquaculture Distributor:

21. Source of'Trained Workers:

22. Potential for Well: 8 ... Tnmeard Foz

Detailed Information on Site

Land Details

Total Land Available Expansion Potential? Current or Previous Use Owner Cost of Land Lease Terms Available? Topography Soil Details for constructionpurposes! 1. Soil Samples Taken by location! Map of Sample Area

Location1: Depths Location2: Depths Location 3: Depths Site Stability determined by soil type! SeawaterQuality Details at Site 1. Potential Source Distance from Site Color of Water Wave Action Storm Potential 6. BeachAppearance 7. Erosion

Accretion Freshwater Plume 10 Proximity of River 11 Salinity 12 Tidal Flux 13 Type of Tide: Diurna 1/Semi-Diurnal 14 Navigation Traffic 15 Industrial Activity On or Near Source? 16 Describe Nearest Port

17. Potential for Pollution

18. Bottom Topography Near Inlet '19. Water Depth Near Potential Inlet Design,Operation and Training ... 9

Freshwater Details at Site

1. Nearest River 2. Size of River 3. Other Sources of Freshwater

4. RainySeason River Depth/Width 5. Dry SeasonRiver Depth/Width 6. EstimatedHydraulic Flow During Seasons

7. Potentialfor Flooding 8. LastMajor Flood/Damage

9. Distance of Freshwater Source from Site 10. PotentialFor Well, Pipeline or Canal 11. Tidal Influence/Flux

12. Salinity at PotentialIntakes Low Tide! 13. Salinityat PotenhalIntakes High Tide! 14. Potential Sources of Effluent

15. Pollution Potential

16. Agricultural/FarmingPoilu tion

Meteorlogical Data at Site 1. Dry Season 2. Rainy Season 3. TemperatureVariance: Wet Season Dry Season

Flora and Fauna at Site 1. Describethe Marine Fisheries/ObtainAny RelevantData

2. Distance to BroadstockFishing Grounds 3. Typical Fishing Vessels

4. NearestFishing Port

Utilities/Resources at Site 1. ElectricalSupply 10 ... Tram and Fox

Nearest Transformer Type of Current Capacity Cost of Electricity Potentialfor PowerShortages Petroleum,Oil, Lubricant Availability

Building Materials

Equipment Laboratory Heavy Construction Computers

Socio/Economic Data at Site 1. NearestCity 2. Industries Labor Force Construction Contractors 5. Engineers 6. Aquaculturists 7. University/ResearchBackup 8. Business Education 9. Nearest Village/Town 10 Local Industries Local Government 12. Political Peaceand Tranquility/Unrest in non-U,S.locales! 13. Other AquacultureProjects

14. Extent of Government Assistance Design, Operationand Training ... 11

Design Criteria, Engineering and Construction the typeof cultureand cultuze techniques that willbe used to producenauplii, larval and postlarval shrimp, algae, If one were to travel around the world examining and Ar temia,etc. For example, some hatchery operators prefer analyzing various hatchery operations, one of the first to add water to their larval-rearing tanks on a continuous observations would be that all hatcheriesarebasically the basis.This implies the use of largeoutdoor or indooralgae same, in that there are certain areas within the overall culture tanks,an extensivedistribufion network of pipes, complex that servespecific functions. The functions, when precision valves, a mixing manifold, etc. Figure 17!. The coordinated, constitute a production system that operates engineering is somewhat simpler for batch larval culture similarly to any factozy. However, when hatcheries are Figure 16!.In someareas of the world wherethe diurnal compared at a more detailed level, they become very temperature variation can be quite extreme, the matura- dissimiliar, Design can be influencedby local tradition, tion facility must resort to the use of a heat exchanger or the availability of certainbuilding materials,desired pro- must recirculate its water througha largebiological fil tra- duction level, differences in culture of the speciestargeted tion systern. for production, a particular approach to strategy and /or These are just some of the many examples of how a the physical site. In this section, some general statements certainapproach to culture caninfluence the engineering are made about design that can be incorporated into of a hatchery. Other examples are summarized below. developingany shrimp hatchery. ~ Propagation of algal cultures as food within the larval Preliminary Considerations rearingtank can determine roof designand length-to- width orientation of the hatchery itself. Selectinga suitablehatchery design should be one of the ~ Use of microencapsulated diets can mean less space first steps in hatchery development and, for this reason,is made available for algal and Artenria culture areas. closely tied to site selection. ~ The duration that shrimp stay in the hatchery can The first question that usually coznesup after site selec- determine whether or not raceway tanks are used tion is how much capital outlay will be required for the some hatcheries keep postlarvae only until PL3-5 project; however, this question cannot be answered unless and, once harvested, they are transferred either di- somelevel of production hasbeen identified. In somecases rectly to the growout pond or to a nursery pond!, the order is reversed, but let us assume that investment will ~ If algal specieswith a wide temperature tolerance are be applied as needed.! For example, most hatcheries in not usedto feedlarval shrimp,it may notbe practical Indonesiawant to produce abou tone to two million post larvae to include a large outdoor algae culture area. PLI5-20! per month. This is a relatively small hatchery These are only a few examples and may only be rel- compared to other hatcheriesaround the world, but none- thelesswiII serveas our subsequentmodel. evant to specific situations. Thereare basicaIIy two hatchery designscurrently used General Layout of the Hatchery in Indonesia a high stocking density model intensive- Most hatcheries are contained within one building or type! andalowstockingdensitymadel extensive-type!. The perhapstwo the nurserysection sornetirnes is segregated high stockingdensity model is characterizedby thefollow- to a separatebuilding!. Hatcherydesigns usually a temptt ing: to integrate all systems under one roof with the basic ~ Optimization of space understanding that efficient use of space will lower the ~ Environmental control overall costof the hatchery.Most designscentralize one ~ Dynamic water exchange important aspectof larval culture and orient other sys- ~ System integration tems around this point. The most common strategy is to ~ A continuous and coordinated production strategy ~ High level filtra tion locate the larval culture area centrally and have support- ~ A higher level of automation ing systems algae culture, Artemiaculture, feed prepara- The basic intensive ha tchery design modeled a fterthe tion, maturationand postlarvalculture, etc.! surrounding "ladder system," Liao, 1984! was used for the Yayasan it. Noisy areas or supporting areas that need substantial Dian Desa hatchery located in Jepara, Central Java. A interaction with the rest of the operation are situa ted as far quick glanceat the designwill show that all production as possiblefrom the larviculture area i.e., the generator facilitiesare containedwithin the samebuilding. room would not be located adjacent to the maturation Thelow stockingdensity design, however, is most com- area, the algae culture area would not be located between mon in Indonesiaand zeflectsthe followingcharacteristics: the larval rearing area and the postlarval rearing area, ~ Largerrelative surfacearea based on lower stocking etc.!.This approach attempts to eliminatethe potential for cross-contamination,reduceunnecessary work,and rnain- densities tain quiet in areasdemanding quiet. The hatcheryover- ~ No accom mod a tions made for en vironmen ta1 con trol ~ Low or intermittent water exchange in culture tanks view Figure6! is that of theYayasan Dian Desa Hatchery ~ Batchproduction that is not necessarilycoordinated YDD! Unit 1! in Jepara,Indonesia; Figure 20E shows the with other systems layout of a CentralAmerican hatchery. Both figuresillus- ~ Simple, low-level filtration trate these points. ~ Low-level of automation Modular Concept for Future Expansion Many timeshatcheries are designedto accoznmodate Approach to Culture eventual expansion. Modular designs are helpful should Another factor impacting hatcherydesign relatesto a larger market for fry develop. They also lend theznselves 12 ... Tnmz and Fox

to seasonal/traditional fluctuation in the market. The tankwith a surfacearea of 120square meters has a diameter YDD hatchery typically experiencedfluctuation in fry of about 12.4meters, A stockingrate of 4,NN per square demandat theonsetofthemonsoon stocking season when meter requires a tank diameter of 6.2 meters. production demand doubled. Hatcheries in the United Thenumber of tanksused directly depends on thenum- Stateswould experiencesimilardemands duringthe spring berof postlarvaetobe produced each month. A projectgoal and early summer months and demand would decrease of 2 znillion postlarvaePL20 per month meansonly 4.5 during winter. million naupliiare required each month 5 percentsurvival Culture Tanks from PL3to PL20,60 percent survival &om N6 to PL2!.This numberof nauplii .5 mNion! meansstocking about 11 The size of the tanks used in hatcheries is determined larval-rearingtanks each month 00@00per tank!.Larval- by severalfactors. Many hatcheriessize their tanks ac- rearingtanks should be startedevery two to threedays. cordingto the single-spawn principal that is, the larval- However,this doesnot mean11 tanks are actually needed. zearingtank canhold one averagespawn of nauplii. The Withan average mme of13 days to PL3 and with a staggered postlarval-rearingtank, in turn, can hold the average approachto stocking,only six to sevenlarval-rearing tanks number of postlarvaesurviving fmm that spawn. For are required.More postlarvaltanks aze needed since they example,if the averagespawn from the maturationfacil- willbeholdingpostlarvaefor17days rather than 13 days P. ity contains400,000 viable nauplii typical for P.monodon!. rnonodonsell at PL18or 1Mayold PLs!. If stockingis at a rateof 100nauplii per liter in the larval Thevariety of shapesand sizes of culturetanks, as well as rearing tank, this tank should contain 4,000liters of wa ter. materialsused in construction,isextensive. It is necessary to If you usuallyexperience 60 percent survival to thepost- classifythem accordingto their function.The list below lazvaltransfer stage, this meansthat 240,000PL3 to PL5 summarizesthe variety of tank typesand shapescurzently will be transferredto the postlarvalholding and rearing used in hatcheries. tank.Some postlarval-rearing tanks have been stocked to densitiesin excessof 5,000PL3 to PL5 per squaremeter Sizing the Hatchery using habita ts or screenedbarriers. A safer level would be Once a production level has been determined, the next somewherearound 2,000 per squazemeter, depending pri- stepin designcalls for proper sizing of tanks,estunating marily on how long theywill be kept beforetransfer to the the numberof tanksto bebuilt, and theoverall layout/ pondand the ability to maintainproper water quality. If this dimensionsof the building itself. According to Boucher stackingdensity is used,the postlarval-rearing tank needs a unpublished data, 1987!, these determinations can and surfaceareaofabout120squaremeters.Again, this will vary shouldbe madewith consideration givento a certainlevel accordingto expectations of survival, ability to stock at high of detail.A checklistof thesecriteria, as well as design densities,water exchange rate and quality of feed,A round calculations,are shown on worksheets1 through11.

Hatchery Culture of Shrimp

Shape Volume, MT or sq. m!

Round 12 Round 12 Round 6 Round 12 Round 9-12 Square 40 Round 0.2 Round Cylindro-conicao 0.7 Round 0.5 Square 0.5 Rectangular 1.0 Cylindro-conical 2-20+ Cylindro-conical 2-5 Cylindro-conical 2-5 Half-barrel 5-15 Half-barrel 5-15 Rectangular 2-20 Rectangular 2-5 Oval raceway 5-100! Round 5-30! Rectangular 0 -100! Rectangular 00! Square -5! Design, Operationand Training . 'I3

Worksheet I - Postlarval PL! SectionDesign

PostLarvalSection Design Assumptions Number of PLsrequired per month PL/month Proposed PL delivery schedule days/tank Initial stage of PLs when stocked days Final stage of PLs when delivered days Maximum density in PL tanks PL/liter Estimated PL survival rate Average water depth in PL tanks meters Minimum freeboard in PL tanks meters Distance between individual PL tanks meters Distance between PL tank and exterior wall meters PostLarvalSection Design Calculations Duration in PL tanks days Minimum total PL tank volume Minimum number of PLs tanks used PL tanks Actual number of PLs tanks used PL tanks Minimum diameter of individual PL tank meters Actual diameter of PL tank used meters Actual volume of individual PL tank ms tank

Worksheet 2 - Larviculture LC! SectionDesign

Larviculture Section.Design Assumptions: Duration of nauplii stocking period days Duration from nauplii to PL1 stage days Number of LC tanks to stock one PL tank tanks/tank Maximum density in LC tanks PL/liter Estimated LC survival rate % PL/naup Average water depth in LC tanks meters Minimum freeboard in LC tanks meters Distance between adjacent LC tanks meters Distance between LC tank and exterior wall meters Width of central walkway in LC section meters Larviculture SelectionDesign Calculations: Duration in LC tanks days Number of nauplii required per day naup/day Minimum volume of individual LC tank m Minirnurn diameter of individual LC tank meters Actual diameter of LC tank used meters Actual volume of individual LC tank used m Minimum number of LC tanks LC tanks Actual number of LC tanks used LC tanks 14 , Treeeearrd Fox

Worksheet 3 - Maturation MT! SectionDesign

Maturation SectionDesign Assumptions: Broodstock stocking density adult/m2 Femalesas a percentageof broodstock % fern/adult Expected nauplii production rate naup/fern/rno Diameter of one maturation tank meter Averagewater depth of maturationtank meter Expected duration of broodstock viability weeks Diameter of one spawning tank meters Average water depth of one SP tank meters Number of femalesper spawning SP!tank fern/tank Distance between adjacent MT tanks meters Distance between MT tank and exterior wall meters Width of central walkway in MT section meters Maturation SectionDesign Calculations: Total number of broodstock required adults Number of female broodstock females Number of male broodstock males Minimum number of MT tanks required MT tanks Actual number of MT tanks used MT tanks Volume of one MT tank rn Broodstock replacement rate adults/week Volume of one spawning tank rrr Minimum numberof SPtanks required SP tanks Actual number of SP tanks used SP tanks

Worksheet 4 - Building Design

Building Design Assumptions; Height of exterior walls meters Spacing of columns/trusscs meters Thickness of floor slab centimeters Cross-sectional area of columns rn Building Design Calculations: Minirnrrmlength of larviculturc section meters next higher multiple of column spacing! meters Minirnurn length of maturation section meters next higher multiple of column spacing! meters Length of center area meters Total length of main hatchery building meters Minimum width of larviculture section meters Width of main hatchery building next higher multiple of column spacing! meters Width of postlarval shed meters Nurnbcr of columns columns Total exterior wall area m Total interior wall area In Design, Operationand Training ... 15

Worksheet 5 - Pipe Sizing

Pipe Sizing Design Assumptions: Water exchangerate in PL tanks %/day Water exchangerate in LC tanks %/day Waterexchange rate in MT tanks %/day Desired filling time for PL tanks hours Desired filling time for LC tanks hours Number of secondary PL feeder pipes pIpes Number of secondary LC feeder pipes pipes Number of secondary MT feeder pipes pipes Maximum velocity in pipes test/sec Estimated fraction factor Equivalent length of PL supply pipe, fittings and gate valve feet Equivalent length of LC supply pipe, fittings and gate valve feet Equivalent length of MT supply pipe, fittings and gate valve feet Pipe Sizing Design Calculations: Maxirnurn flow rate to one PL tank liters/sec f3/sec Average flow rate to one PL tank liters/sec f3/sec Maximum flow rate to one LC tank liters/sec f3/sec Average flow rate to one LC tank liters/sec f3/sec Averageflow rateto oneMT tank liters/sec f3/sec Minimum diameter of each PL supply pipe inches Next larger pipe size inches Minimum diameter of each LC supply pipe inches Next larger pipe size inches Minimum diameterof eachMT supply pipe inches Next larger pipe size inches Maximum flow rate of PL feeder pipes liters/sec f3/sec Minimum flow rate of PL feeder pipes inches Next larger pipe size inches Maximum flow rate of LC feederpipes liters/sec f3/sec Total length of LC supply pipe meters Total length of LC feeder pipe meters Total length of LC mainline meters Total length of MT supply pipe meters Total length of MT feederpipe meters Total length of MT mainline meters 'I6 ... Treeceand Fox

Worksheet 6- Header Tank Sizing

Header Tank Design Assumptions: Minimum detention time in header tank minutes Mirumum pressure of pipe at discharge psl Header Tank Design Calculations: Flow rate for PL tank water exchange ms/min Flow rate for PL tank filling ms/min Flow rate for LC tank water exchange ms/min Flow rate for MT tank water exchange ms/min Maximum total hatcheryflowrate ms/min Minimum volume of header tank m Actual volume of header tank used m Minimum elevationof header tank standpipe abovehatchery floor feet meters NOTE: If it is desiredto reduce the elevationof the headertank, try selectivelyusing largerpipe sizesin one or more of the above applications.

Worksheet7- SeawaterReservoir SR! Tank Sizing

SeawaterReservoir Design Assumptions: Miscellaneous seawater usage ms/day Average water depth on SR tank dieters Minimum freeboard in SR tank meters SeawaterReservoir Design Calculations: Water exchangerequirements for PL tanks ms/day Averagenumber of PL tanksfilled per day tanks/day rounded to next highest number! tanks/day Filling requirements for PL tanks ms/day Water exchangerequirements for LC tanks m3/day Averagenumber of LC tanksfilled per day tanks/day rounded to next highest number! tanks/day Filling requirements for LC tanks ms/day Waterexchange requirements for MT tanks ms/day Total volume of seawater reservoir ms/day Design, Operationand Training ... 17

Worksheet 8 - Algae Culture

Algae Culture Design Assumptions: Water exchange rate in LC tanks %%uo/day Algae feeding density in LC tanks cells/rnl Estimated ratio or larval algae consumption to natural algaeproduction in LC tanks cells/cell Volume of algae mass-culture AMC! tank liters Harvest cell density in AMC tanks cells/cell Minimum detention time in AMC tanks days No. of AMC tanks stocked/harvested per day tanks/day Volume of algae intermediate-culture AIC! tank liters Harvest cell density in AIC tanks cells/ml Minimum detention time in AIC tanks days No. of AIC tanks stocked/harvested per day tanks/day Volume of algae carboy-culture ACC! tank liters Harvest cell density in ACC tanks cells/rnl Minimum detention time in ACC tanks days No. of ACC tanks stocked/harvested per day tanks/day Volume of algae flask-culture AFC! tanks liters Harvestcell density in AFC tanks cells/rnl Minimum detention time in AFC tanks days No, of AFC tanks stocked/harvested per day tanks/day Maximum water depth in algal culture area meters Algae Culture Design Calculations: Total volume of AMC required liters Number of AMC tanksrequired AMC tanks Stocking cell density in AMC tanks cells/ml Total volume of AIC required liters Number of A!C tanks required tanks Stocking cell density in AIC tanks cells/ml Total volume of AIC required liters Number of AIC tanks required AIC tanks Stocking cell density in AIC tanks cells/rnl Total volume of ACC required liters Number of ACC tanksrequired ACC tanks Stocked cell density in ACC tanks cells/ml Total volume of AFC required liters Number of AFC tanks required AFC tanks 18 ... Treeeeand Fox

Worksheet 9 - Arterrtia Culture Artemia Culture Design Assumptions: Duration from Ml to M2 days Feting density FromMl to M2 Ar temia/1nl Duration from M3 to PL2 days Feeding density FromM3 to PL2 Ar temia/ml Duration from PL3 to harvest days Feeding density From PL3 to harvest Ar temia/ml Expected hatching rate of Artemia cysts Artemia/gm l lydration density of Artemia cysts gm/liter Duration of hydration period hours Volume of Artemi hatching tank AHT! liters Anemia Culture Design Calculations: Expected number of LC tanks tobe at Ml-M2 stage on any given day LC tanks Number of Artemia needed from Ml-M2 Artemia/day Expected number of LC tanks tobe at M3-PL2 stageson any given day LC tanks Number of Artemia needed from M3-PL2 A rtemia/d ay Expected number of LC tanks tobe at PL3-harvest stage on any given day LC tanks Number Of Artemia needed frOrn PL-3-harveSt A rtemia/d a y Maximum nurnbcr of Artemia needed pcr day Ar temia/day Minimum amount of Artemia cysts to be hydrated pcr day gra ms/day Total volume of AHT required liters Minimum number of AHT tanks required AHT tanks

Worksheet 10 - Aeration System

Aeration System Design Assumptions: Acra tion requiremen ts for PL tanks f'/min/ms Aeration rcquircrnents for LC tanks p/min/ms Aeration requirements for MT tanks f /min/res Aeration requirements for SP tanks f'/min/m-' Aeration requirements for algal culture p/min/ms Aeration requireme~ts for SR tank f'/min/ma Number of diffusers pcr PL tank dif/tank Number of diffusers per LC tank dif/tank Number of diffuscrs per MT tank dif/tank Number of diffusers per SP tank dif/tank Number of diffusersfor algal culturc diffusers Prcssure loss through diffuser PS1 Minimum air presssure at discharge psl Number of backup blowers per application blowers Aeration System Design Calculations: Total airflow to PL section f'/m Total airflow to LC section f'/rn Total airflow to MT section F'/m Total airflow to spawning tanks f'/m Total airflow to algal culture area f'/m Total pressure head in PL section PS1 Total pressure head in LC section PS1 Total pressure head in MT section psl Total pressure head to SP tanks PS1 Total prcssure head to algal culture area PS1 Capacity of main hatchery blower f3!rn Maximum prcssure head of main hatchery blower PS1 Capacity of seawater reservoir blower P/m Maximum prcssure head of SR blower PS1 Total number of diffuscrs diffusers Length of air supply pipe/tubing meters Length of'3" pvc Fccdcr pipe meters Length of 6" pvc air mainline meters Design, Operatiortartd Training ... 19

Worksheet 11 - Shrimp Hatchery Design - ConstructionCost Estimate

Item Units Quantity Cost per Unit Total Clear and grade m~ Excavation Rough grading mm2 m Landscaping Footings rn' Columns Concrete block wall m m~ Bond beam ms Top plate LM Prefabricated trusses EA Purlins LM Corruga ted roofing m2 Exterior doors EA Interior doors EA Overhead doors EA Postlarval tanks EA Larviculture tanks EA Ma tura tion tanks EA Spawning tanks EA AMC tanks EA AIC tanks EA ACC tanks EA AHT ta~ks EA Header tanks EA Seawater reservoir tanks EA 1" PVC pipe LM 1.5" PVC pipe LM 2" PVC pipe LM 3" PVC pipe LM 4" PVC pipe LM 6" PVC pipe LM 1" gate valves EA 1,5" gate valves EA 2" gate valves EA 3" gate valves EA 4" gate valves EA 5" gate valves EA Concrete drainage channel LM G.I. grating SM Main seawater intake pump EA Secondary lift pumps EA Main hatchery blowers EA Seawater reservoir blowers EA Water heating system LS Electrical system LS Lab suppliesand equipment LS Misc, supplies and equipment LS SUBTOTAL 3.5%Contigency TOTAL

Note: LM = Lengthin meters,EA = each,LS = Lump sum 20 ... Treeeearrd Foz

Specific Hatchery Background Indonesian Hatchery Development Program In Bandengan Jepara, there are two hatch- The following abstract appeared in the Journal of the ery units referred to as Unit I and Unit II. Unit World AquacultureSociety, Aquaculture Communiques I is a model hatchery whose construction was Volume 18, Number 1, page 20A, Cornrnuniquc number 72, 1987!. partially funded by USAID and which has 72. The Establishment of a Successful Penaerrs received most of the CJEDP program support monodorrHatchery in Jepara,Java. for hatchery development. Unit II is also oper- ated by YayasanDian Desa YDD! and was TREECE, GRANVlL D., Texas Marine Ad- built without USAID support, based on the visory Service, Texas A&M University Sea design of Unit I. YDD thereby doubled its Grant College Program, CollegeStation, Texas operational capacity and also doubled its fa- 77843;FOX, JOE M., Central Java Enterprise cilities dedicated to the researchand develop- Development Project, Gedung BAPPEDA, Jl. ment of appropria te hatchery technology Pemuda 127,Scrnarang, Indonesia, In general, both Unit I and Unit II have been A short-term technical assistance and in for- performing as expected during the past quar- mation transfer subcontractbetween Develop- ter. Unit I wasres ta rted a fterbeing ternpor a rily rncnt Alternatives, Inc., Washington, D.C., the shut down for cleaning, If the water quality United StatesAgency for International Devel- holds into next quarter, it is hoped that the opment, the Central Java Enterprise Develop- hatchery will achieve a combined output of 4 ment Project and the TexasAgricultural Exten- million postlarvae per month. sion Service-SeaGrant Advisory Program re- sulted in the establishment of a successful Unit I was constructed in 1986by Yayasan Dian Desa Perraeusmoitodori hatchery in Jepara,Java. The for approximately$90,000 U.S.!, and had aii estimated ha tchcry is now opera ting as a "model" train- production capacity of 3 million postlarvae per morith. The hatchery is located on the island of Java in the Repub- ing facility for the country and is also produc- lic of Indonesia scc Figure 1!, A recent NASA satellite ing an average of one million nauplii per day survey has shown that Indonesia, also referred to as The with only two maturation tanks in use, East Indies, Malay Archipelago or The Spice Islands, is Shortly after construction of the state-of- madeup of nearly 20,000tropical isles instead of the 13,677 the-art hatchery was completed, an intensive isles which it was once thought to have. In the words of technical information transfer was initiated. one writer, these isles "gird the Equator like a string of Broodstock was purchased from local fisher- emeralds," and they comprise the most extraordinary rnen, female shrimp were ablated and produc- collection of places, peoples, sights, tastes and natural tion began in less than onc week. Twenty rnil- wonders on earth, The model hatchery project of Yayasan lion nauplii were produced in the first 15days Dian Desa is more specifically located on the north shore and someof thebroodstock had spawned three of Java near Bandengan Jepara see Figure 2!. times in two weeks. After a production strat- Thebasic layout anddesign of the hatcherywas rnod- egy was initiated, which took into consider- eled after the "ladder system"described by Liao 984!, ation the hatchery's larval production needs, taking advantage of water levels and gravity. Even with and a continuous scheme of production was "state-of-the-art"design, and a good location, success established, the hatchery staff members were dependson good managementpractices. Management is trained using techniques to sustain produc- the key to a successfulhatchery opera tion, and it should be tion. Three training sessionswere conducted stressedthat eachhatchery will haveits own procedures with neighboring hatcheries invited. Hatchery for optimum production. procedures were incorporated into a training Figures3 through 20 give details of the hatchery,its program for future traincesby documenting seawater system, tanks and production areas, as well as tanks and systems used in other hatcheries. activities specifically needed at that hatchery. Theprocedures were compiled into a training- CentralAmerican Facility HatcheryExample! operating manual, which can be used as a Figures 20A through 20M depict a Central American guide or tool in future training programs. hatchery that was designed to produce 10

Figure 1. Location of Java 22 ... Treece and Fox

Figure 2. Location of Hatchery on Java

Photo4. Bamboofishing platformsoffshore ' nets are lowered at night with light aboveto attract catch!.

Photo 5, Palm leaf barrier " pound net" ar- rangement!with fisherman'splatform at end where net is raised.

Photo6. Fishing vi! lagelocated next to hatch- Photo7. Proudfishermen who bring in broodstockshrimp J- whenyellow flag is flying an hatcheryis i n needof newstock. Desig~, OPerationand Training ... 23

Figure 3 I3ian Desa Hatchery

Photo8. Dian DesaHatchery front entrance.. Photo 9. Dian DesaHatchery side view.

Photo10. Maturation room Unit ll!, lower left, generationor build- ing, center;seawater settling, lower right, 24...TreeczandFaxFigure4.FrOntandSideViewwithElevatianS

Figure5 Frontand SackView with Elevation Design, Operationand Training ... 25

Seawater Pumps e stion Gan-set Algae Pumpnous* Lab, Food Prep

Overllow Algae Settling Pond 00m't Storage SlawSand OutdoorA geeArea F terII 00mt MalurstionTank Primarp undinet Reservoir D rei riag I uotten ElevatedReservoir StandpipeRe<>roc sting 0 ml pump ,uVSteniikeliOn

Dialo macecal sarislitters Figure 6. Hatchery Overview with Detail

Functional Areas within the Hatchery

System/Area Description Algae Culture Provides feed algae! to the larval rearing Figures 9 and 11! tanks LRTs!. Shrimp larvae consume algae in their early stages. Algae Laboratory Servesas a multi-purpose room Forculture Figures 9 and 10! of small volumes of algae; counting; micro- scopic work; storage and weighing of chemicals, Artemia, artificial diets; washing of glassware. Artemia Culture Provides Arternia nauplii for feeding to lar- Figure 8! vae in the LRTs. Shrimp larvae consume Arterrtia in their latter stages,! Larval Rearing Contains elevated conical tanks used for Figures 16-19! the culture of larvae. Postlarvae from here Photo 11 Air blowerfor Llnit l Hatchery high are transferredto postlarvalrearing tanks volumelow pressure!,later movedinside. or "raceways." Postlarval Rearing Containsshallow cylindrical tanks used for Figures 14 and 20! production of postlarvae.From here PL 18 stageshrimp are transferred to growout ponds. Maturation Contains tanks used for maturing male Figures 12, 13 and 15! and feinale shrimp so that they will mate in captivity. Mated femalesare transferred into spawningtanks where they release fertilized eggs spawn!. Theseeggs are allowed to hatch and the nauplii trans- ferred to the LRTs. Harvesting Designed for harvesting postlarvac being transferred to pond for grow-out. Aeration Used to provide air to the various produc- Photos11 and 12! tion areas. Seawater Provides filtered seawater to all production Figures 7a and 7b! areas. Electrical Provides electricity for operatio~ of pumps, Photo 3! air blowers, light banks, room lighting and Photo 12. Close up of air blower and filtered air conditioning. in take. 26 .Treece arrd Fox

Photo 14. Concreteholds intake Pipesin place,visible at tow tide.

Photo 13. Two Schedule 80 3-inch PVC intake Pipelines oneintake in shallowwater aruf one in deeperwater!,

Seawater Pump

I + ConcreteAnchor Sub Sand Filter

M alurat i 0 n

~Algae Production and Larval Rearing Pressurized Sand Filter Diatom Pump Filter

Figure 7a. Original Seawater System Cart ridge Filter

Photo 15. Seawaterpumps work on a demarulsystem and use is Photo16."Home-made" sand filter usedin1986,which were rotated only one runs at a time!, replacedby slow sandfilWs in 1987 Photo 29!. Design, Operationand Training ... 27

Photo77. Cementsettling tank and cementslow sarrdfili'ers, Photo18, PrimaryReservoir: Note organicmatter floating and primary reservoirsand overflows, finished product iclear wafer!.

~ SeawaterLine / ..:=:=-=-T':. -- - 3 - +I Settling S owSand Tank Filter ~-- ConcreteAnchor SubSand Filter

To Algae ~ Predudtien Pu and Larval Rearing

1 gymCartridge Filter 1ym Cartridge Filter

DepthCartridges Figure 7b. Fina Seawater System 28 ... Treece and Fox

Photo 19. Close up of primary, settling, slow Photo 20. Elevated reservoir 0 MT! acts as a sandand cleanwater in overflow. constant head devicefor Unit I and Unit II U.V. treafmentfakes place in headdevice res- ervoirand pumps,and D,E.filters arehoused in structure below elevated reservoir!.

Photo27. Unit I, outsidepostlarval holding and rearingtanks, Photo22.Diatomaceous earth filfers housedinside elevated reservoir elevated reservoir. strucfure. Design, Operationand Traint'ng ... 29

1J 1. I Table Side Lr Flourescent Lights

, --, ri

Arrerrria cysts placed in Table Tcp bottles containing seawater with aeration

Figure 8 Arremi* PrOduCtian Area

300 Liter Poly Tank Al geeCulture! 'i jjIjj~,'!

Figure 9. Laboratory and Algae Production in Fiberglass Polytanks

Photo23. Algae production in 300-literfiberglass tanks, 30 ... Treeceand Fox

Figure 10 Laboratory Work Bench

FloureaoentLights located behind . bottles

Aeraticn via PVC & plastic tubing

5 gallon clear plastic bottles for algae culture

Ii

Photo 24. Algae production in 29-liter, or 5- gallon,cfear plastic bottles theseare safer to Figure t t Carboy Stand tor Algae Production handleandtransport than theglassbut aremore difft'cult to clrMn!. Design,Operation and Training. 31

MainSeawater System Filtration lFiltered Water! System

~ Drain ..Supplementary SeaWater

SeaWater Pump Alternative1: Open. Warm/Coo Climate

~ Drain

Drain Drain Drain Alternative2 Recirculating System ead

W>FloatVat~ve SeaWater Puinp Figure 12. VaricusMaturation Systems ,a -Valve Alternative3: Open, Warm Climate not drawn to scale!

Figure 13 Maturation Tank Detai 400 /90 ~erroCement 5 20 40 ini t !P6,

02

40 55+

! tst Slope tO Center Drain '

Bi = Drain 32 ... Treeceand Fox

Air Manifold

Wate Harvester

E CO

Top View

Figure 14. TypiCa Aaceway Design postlareae!

Figure 15. Spawning Tank Detail

40 45 5 120 5 50 5 120 5 45 40 fi ii 1

". FerroCement

Photo 25. Rectangularlarval rearing and postlarval rearing 400 and holding tanksin useat nearbyhatchery.

pc. CcP

! = Drain Design, Operationand Training ... 33

Diatom Fitter

ldwSaltd liter

settllntf Tank

daSand liter Ocean!

LarvalRearing Approach 1 batefeeding! i

L'pp ~ pupmumrpl~ P mp

Lmp~ puemprprprp purp

Photo26. Hatcheryeffluent flow returningto oceanudownstrearnu of intake. 34 ... Treecearid Foz

ipe Drain AerationLine

Flex bi

Cylindro-Conical Half-Barrel Shape

tand Pipe Drain

At atone GentleSlope

Square or Rectangular Larval Raceway

Figure 1tt. Various Larval Rearing Tanks

Figure 19, Larval Rearing Tank Detail

,40 45 5 200 5 5 40

FerroCement 30

02"

55'

QA!= Drain 400 Design, Operationand Training , 35

Figure 20. Raceway Tank Detail

$ ilS'

QD= Drain

Figure 20A CENTRAL AMERICAN HATCHERY FACILITY 36 . Treeceand Fox

Figure 20B CONCEPTUAL DRAWING OF SEAWATER INTAKE CENTRAL AMERICAN FACILITY

Figure 20C CENTRAL AMERICAN HATCHERY FOUNDATION /LEVELS Design, Operationand Training ... 37

Figure 20D CENTRAL AMERICAN HATCHERY ROOMS AND DIMENSIONING 0 Million PL/Mo. Production!

Figure 20E CENTRAL AMERICAN HATCHERY TANKS/LOCATION 38 . Treeceand Fox

FOR GROWOUT PONOS FOR HATCHERY

SEAWATER SOURCE SCREENE D INTAKE FILTER

~6000 GPIV!AXIAL PUMP

2,500 MT RESEVOIR UV STERILIZER E LEVA T ED! PALL FILTERS~ 12.0 IvIT MATVPATIO NI CONTACTPUMPS QVARINTINE,SPAWNING, MAT LAB LARYICVLTVFIE.ALGAE LAB HEADER WIER +SANDFILTERS MATVRATIGNII CYCLONE HYDRAULICFLOWMETER $ SCREENEDINTAKEFILTERS CYCLONICSEPARATORS

PONOS 6,000 GPM AXIAL PUMP 200 GPM CENTRIFUGALPUMPS

Figure 20F CONCEPTUAL DRAWING OF CENTRAL AMERICAN HATCHERY SEAWATER TREATMENT

Figure 2QG CENTRAL AMERICAN HATCHERY SEAWATER ELEVATED HEADER DISTRIBUTION SYSTEM Design, Operationand Training ... 39

Figure 20H CENTRAL AMERICAN HATCHERY MATURATION FACILITY SEAWATER DISTRIBUTION

Figure 201 CENTRAL AMERICAN HATCHERY BLOWER AERATION SYSTEM 40 ... Treeceand Fox

Figure 2OJ CENTRAL AMERICAN MATURATION FACILITY AERATION DISTRIBUTION SYSTEM

Figure 20K CENTRAL AMERICAN HATCHERY DRAINAGE SYSTEM Design, Operationand Training ... 41

Figure 20L CENTRAL AMERICAN FACILITY MATURATION TANK DESIGN

Figure 20M C ENTRAL AMERICAN HATC HE RY LARVA L R EA R IN G TANK 42 , Treeceand Fox

AppendicesA throughC givewater analysis results for a potentialhatchery site in SouthTexas in July1990. Appendix D, excerpted from the SafeDrinking Water Act of 1986,gives drinking water maximum-contaminant levels for metalsand should be usedfor cornpa ra tive purposes only. Appendix E gives seawater parameters for comparative purposes. Appendix F givesprimary components of seawater Riley and Chester, 1971!, Appendix G givesvalues for concentrationsof trace elements in seawater Quimby-Hunt and Turektan,1983!, Appendix H presentselements in seawater Spotte, 1970!, and Appendix I givesexamples of pesticidetevets toxic to penacid shrimp.

ysisResults for an Existing App n South Texas Jul 1990!. Sam

Concentration Found Param ! PPB parts per billion! Silver Benzene 0,01 U Bariu Toluene 0.01 U Cadm Ethyl Benzene 0.01U Chrom Xylenes 0.02 U Arsen U-Undetected,indicating not foundabove detection limit Selen given. Lead Sample ID; Low Tide Intake Site! Mercu Concentration Found Coppe Colnpound pg/L! PPB Tin Zinc Benzene 0.01 U Toluene 0.01 U Boron Ethyl Benzene 0.01 U U-Uud Xylenes 0.02U given U-Undetected,indicahng not f oundabove detection limit Samp given. Sample ID: High Tide Intake Site! Parameter Mg/L! PPM parts per million! I Concentration Found Silver 0.05 U Compound !tg/L! PPB Barium 1.0 U Benzene 0.01 U Cadmium 0.01 U Toluene 0.01 U Chromium 0.05 U Ethyl Benzene 0,01 U Arsenic 0.3 U Xylenes 0.02 U Selenium 0.16 U U-Undetected, indicating not found above detectionlimit Lead 0.566 given, Mercury 0.0002U Copper 0.009 U Appendix D. DrinkingwaterMaxilnulnContaminan Tln 2.25 U Zinc 0.075 U Levels for Metals to be used fo Boron 3.75 corn aritive osesonl ! U-Undetected, indicating not found above detection limit Maxilnum Contaminant given. Nonchlorinated Water Level" Sample ID: High Tide Potential Intake Site! Primary Standards p.g/Lor PPM Concentration Arsenic AS! 0.05 Paralneter Mg/L! PPM parts per million! Barium Ba! 1.0 Silver 0.270 Standard Cadrniurn Cd! 0.010 0.0003Mg/L Chrorniurn Cr! 0.05 Barium 1.0 U Lead Pb! 0.05 Cadmium 0.01 U Mercury Hg! 0.002 Chromium 0.050 U Nitrate N! 10.0 Arsenic 0.12 U Nitrate NO3! 45.0 Selenium 0.16 U Selenium se! 0.01 Lead 0.452 Standard Silver Ag! 0.05 0.004 Mg/L Copper Cu! 1.0 Mercury 0.0002U Iron Fe! 0.3 Copper 0,09 U Manganese Mn! 0.05 Tln 2.25 U Zn! 5.0 Zinc 0.075 U rpt from SafeDrinking WaterAct of 1986 Boron 3.70 levels adopted at 0,005 ppm 990! U-Undetected, indicating not found above detection limit Detection limits for Arsenic in two samples are differ- given. e to sample matrix effect.

44 ... Treeceand Fox

CV LI Q O A%A NI ~5 + + 9 Q Q 0 o Q LrIQ Q Q PC Q Q Q Q Q O Q < O O 4 Q Q w O Lrl O Q O o o o Q o o Q Lrl w N o o LlI o

08 c 4J l EI 0 0 20 E LI 0 ~ 0 0~ 0 X 00 a- C0 0 0'0 Pu IIL t5 ILI 0 IILW U Xzo XZZZ 0 !gal L

QWgPN Pl

LrI lA ORRwlqwlh98 ILh CAWA&NA&lhlhA QQOHQO+O

ILI

LI 04 0 U 0C4 0 0 0 .a 0 ILI 0 0 f4 0 n. 2 LLICf! CCI g CC 6L ~a U0 Z LLI 0 0 0 0L~ 8, 0 0 0 P 0 ~~ LI++ 0 rA Z V V 'LI LI V LI ~ +0P 'g p ggg5- Q ILI cv ~ A A t5 0 0 0 A w w ra K PP Z X w w 2 EzhM W LL.POZ

U I RQ lLIPQOMRGChKZRALLt R QChW N * cV AAALqPP Aab I05 0 ~ w 8 Q +g + W O Q Q m m m w Q O LPIP I LPIQ Q l,g Q O H O O O O O 0 0 O LCI CI Q O O O O Q O O O O O N N O O N O O O O

06 0 0 LL

0 U 0- 'w 0 x 0 x > IXI 0 0 U 5C ILIM '0 C UC0 ~ v0 '40 Q Q m LLI LI 0 R 4 0 g,S x 0 a aalu LLI~ ILI oooo 0. U EI 0 ~~0 0 uuuA N W CVW P! n k' a t5 0 6 6

R~58LANMOAEIJ!K~RQOCO t4

co A 0 cr! W W ~ W W Q cAlA W CqA A O A ccIA 0 O A OOO OOOOwOO c o o w a col o o o a Q o a Q o Q LA w cv o o cA o

cLc

0 D. c JOB 0 06 6c td~ 0 E 9 cc «0 p P. cn 40 0 Q. 0 g h4 2 CII 600~0 0 c- c 0 0 4 N ~ 4 - N 0 p2 c 4 4 0 0 c c 0 c c w 0 S.-xg pcEpg pc 0 L 0 0 cll 4 ~+ c 0 4 4 24c= 2 cYI w>a 0 K 0, Ea 0 0 c4 ~ OC0C 4 4 0 0 2 XXZ ooR 0 0 H F zz 0 0

Cc! 4 ICOol à C%4 "w w 0 W CCIP 0 O FD R 0I W W g Ccl OI P C CV CVR W W g g Pl 4 W CVN

AA A WQASAAWA WM88 0 nlrb ccl Awlmmgc 0 0 ll! O O O W O O W O W O O O O CV Q Q CCICt Ct O Q Q Q Q Q Q Q Q CV N Q 0 P .G : R. cc c06 0 4> 0 Qcd .0 x 0 0ccl 0 C kj Ice CCI C j 0 $ U0 0z 0 c p 0 ccI 0 0 0 ~ 0 p 0,4 cII x 0 ~ c 0 0 c 0 0 0 0 b. 4 0 0 III III C v . 0 CUB C cd ~ ~ 8 tip ip ilia IX E ~QXZ ~~ i5 ~ Ltl Ctl W u5 u.pPz P o 0 g! o h

~ZZRRXA

LPj 3 boK Cj 0 P w wCj I cO I lcl N Q I I w w w w R Q K crI K 0 EAM A I crI cq cq A OQ W Q Q Q OQ O OONQQ O QQQQQ LCIQ Q Q Q CVCV CI Q PV Q Q Q CI 0

0 .0 0 Q Icl 74 06 8 0 6 0 ccj tl 2 0 '0 o 'IO c I-'0 4 4 V ~ 0 I-'0 ;j5 c 0 U CII 0mm Cco CU6 0 cg 0 0 Icl Q g~ c 0 0 0 OQO 0. 0J o..c 0 I0 0 04Qc 0 0 4 c 0 0 naa 0, ccI 0, 0, 0 0 0 0 g ccl ccl4 4 8' g F0 U UUUU UV v8~ QvvGBa88i5i58a Ue4IZc 'a U W Q O $P~QRPSRR!RB C0g CI 0

Design, Operationand Training ...47

or Concentra tions of Selected events in Seawater a tS alintiy t with Standardized Nutrient

ncentr ation Reference' ngfkg! 330 A B 10 40 2 480 120 390 CEF ED 170 70 6 1 GH ki an 983! 979!; B, Landing and Bruland 1982!; D. Knauer et al., 982! E. s and Burton 980!; G. Mukherji ule and Patterson 98'I! 48 ... Treeceand Fox

I DDT ,1 ppb 28 days mortality P. duorarum F ; Diazinon 4.8 ppb 96 hr. 50% mortality Mysidopsi bahia G 2 ppb 96 hr. low growth Mysidopsi bahia G 8.0 ppb 96 hr. 50% mortality P. aztecus G 0 ppb 48 hr. 50% mortality P. aztecus PLs H 5 ppb 48 hr. 50% mortality P. aztecus adult H 9 ppb 96 hr. 50%mortality P. aztecus l 11 ppb 96 hr, 50% mortality P, durorarum 4,0 ppb 48 hr, mortality penaeids K 0.0 ng/l 24 hr. 50% mortality P. stylirostris nauplii L 0 ppb 7 days 25% mortality P. duorarum M,N,O 2ppb 48 hr. mortality P. duorarum K 0 ppb 96 hr. 25% mortaility Crangonseptimspinosa P 0 ppb 96 hr. 25%mortality Palaemonetesvulgaris P 1974; B. C octal.,19 IVI. Lowe

Appendix J. Example of soil investigations at twopotentiai hatchery sites in Indonesia. These soil analyses were conducted by a reputable laboratory and excerpts of the results can be seen in this appendix. Soil Physical Sampling Soil sampling for physical tests was taken on the excavation hole at the depth of 0.5 m and 1.0 m through a 150mm thinw all and treated as undisturbed sample. The thinwall was put in a plasticcan and sealedby a plasticband so the moisturecontent was kept constant. Thecrust soil at site 1 showeda layerof soft silty clay with a traceof shell.The ground waterlevel rangedbetween 0.7- 0.9 m below the surface. At site 2 the crust soil was a very soft silty clay with a trace of shell and inundated with water. The results of the soil laboratory tests could be summarized as follows: Physical Characteristics Site 1 Site 2 Natural M.C. %! 40.00-54.00 54,30-65,9 Specific gravity 2.52 -2.57 2.50-2.53 Bulkdensity gr/cm3! 1.57-1.75 1.46-1.59 Natural void ratio 1.06-1.47 1.46-1.84 Degree of saturation 92.50-97,70 89.50-94.20 Liquid limit %! 98.70-101.70 102.5-105.0 . Plastic limit %! 37.80-41.50 40.0-41,5 ~ Plasticity index %! 59.0-61.7 62.5-64.1 Passingsieve ¹200 %! 97.0-99.0 94.0-99.0 Cohesion kg/cm ! 0.27 0.06 Design, Operationand Training ... 49

Selected Ref erences a successfulPenaeus monodort hatchery in Jepara,Indon l. Bruland, K.W. 1980.Oceanographic distributions of esa.World Aqua. Soc. 18th Annual Conf. p. 35. cadmium, zinc, nickel and copper in the north Pacific. 21. Treece,G.D. and M.E. Yates.1990. Laboratory Manual Earth Planet. Sci. Lett. 47: 176-198. for the Culture of Penaeid Shrimp l.arvae. Texas 2. Cranston, R,E. 1979. Chromium species in natural ARM University, TAMU-SG-88-202 r!.College Sta- waters.Ph.D. d issertation,University of Washington, tion, Tex., U.S.A. Seattle, Wash., U.S.A. 22. Treece,G.D. and M.E.Yates. 1992. Unpublished feasi- 3, Cruz, C.R. 1983.Fishpond Engineering: a technical bility study for Zanzibar,East Africa. manual for small and medium scale coastal fish 23. Yuan,Y.X., C.N. Gaoand D.X. Zhang. 1992. Metamor- farms in S,E. Asia. South China Seas Fisheries Dev. phosisto postlarvaeofP. chinensis Osbeckby removal and Coordinating Program, manual No, 5,, S.C.S., of heavymetals from rearingsystems with polymeric Manila, Philippines. absorbent.Journal World Aqua. Soc. 23!: 205-210. 4. Forst ner,U. and G.T.W. Wittmann. 1979.Metal pollu- tion in the aquaticenvironment. Springer-Verlag, Berlin,Germany. 5. Fujirnura,T. 1989.Mangement of a ShrimpFarzn in Malaysia. InProceedings of the Southeast Asia Shrimp Farm Management Workshop. Ed. Dean Akiyama, AmericanSoybean Assn., pp. 22-41. 6. Grasshoff,KM. Ehrhardt and K. Kremling. 1983. Methods of seawater analysis. Verlag Chernie, Weinheim,Federal Republic of Germany. 7, Knauer, G.A., J.H. Martin and R.M. Gordon. 1982. Iron in northeast Pacific waters. Nature. 297: 49-51. 8. Landing, W.M. and K.W. Bruland.1980. Manganese in the North Pacific. Earth Planet. Sci. Lett. 49: 45-56. 9. Liao, I.C. 1984.A brief review of the larval rearing techniques of penaeid prawns. Proceedingsof the First International Conference on Culture of Penaeid Prawns/Shrimp.Iloilo City, Philippines, p. 65-78. 10 Measures, C.I. and J.D. Burton, 1980. The vertical distribution and oxidation states of dissolved sele- niurn in the northeast Atlantic Ocean and their rela- tionship to biological processes.Earth Planet. Sci. Lett. 46: 385-396. 11 McLeese,D.W. 1974.Toxicity of copper at two tern- peraturesand threesalinities to the Americanlobster Homarus americanrts!.J. Fish. Res. Board Can. 31: 1949. 12 Mukherji, P.and D.R. Kester. 1979. Mercury distribu- tion in the Gulf Stream. Science. 204: 64-66. 13.Quimby-Hunt, M,S. and K.K. Turekian. 1983.Distribu- tion of elementsin seawater, The Oceanogr.Rep. Eos, Trans. Am, Geophys. Union. 64: 130-131. 14 Riley, J.P. and R, Chester.1971. Introduction to Ma- rine Chemistry.Academic Press, London, 465 pp. 15 Rosenberry,B. 1992.World ShrimpFarming. Aquac- ulture Digest, San Diego, Calif., U,S.A. 16 Spotte, S.H. 1970.Fish and invertebrateculture. Water management in closed systems. Wiley,!nterscience, New York, London-Sydney-Toronto.p. 90-91. 17 Strickland, J.D.H. and T.R. Parsons. 1972.A practical handbookof seawateranalysis. Bulletin of the Fish- eriesResearch Board of Canada,No. 167,Alger Press Ltd., Ottowa, Canada. 18 U.S. Environmental ProtectionAgency, 1986.Test Methods for Evaluating Solid Waste. EPASW-846. 19 U.S.Environmental Protection Agency. Methods for ChemicalAnalysisof Waterand Wastes.EPA600/4- 79-020. 20. Treece, G.D. and J.M. Fox. 1987. The establishment of 50 ... Treeceand Fox Design, Operationard Training ... 5l

Chapter 2 Maturation and Hatching

Although lack of a reliable supply of postlarvae is The choiceof speciesfor culture is dependenton a undoubtedly one of the biggest sourcesof uncertainty, number of factors induding local availability, ease of inefficiency and economic loss facing shrimp farmers maturation and reproduction in captivity, and growout worldwide along with the threatof diseases!,many farm- characteristics in a given culture situation such as nutri- ers still rely on wild postlarvaeas their "seed."Some rely tional requirements, stocking densities, survival and on ready-to-spawnadult femalesfrom the oceansas a growth!. As in other typesof aquacuIturc,species differ- source of seed. However, an increasing number of farms encesrequire testing of different culture regimes and are adopting technologyto control the reproductivepro- careful study of natural habitat. Of the speciestested thus cessand to producegeneration after generationof shrimp far, most attention in the Western Hemisphere has been without totally relying on the wild population.This tech- focused on P, styiirostrisand P. vannamei both Pacific nology offersindependence from the unpredictablefluc- Ocean speciesranging from Central Peru to Mexico!, tuationsin wild populations;accessibility to the superior while P,monodon eastern Africa to Japan!and Pjaponicus non-indigenousspecies; improvement in performance Mediterranean to Indo-West Pacific! have bccn the most through artificial selection;and some control over the popular candidates for mariculture outside the Western diseases found in wild stocks. Hemisphere. More recently, P.chinensis has played a large The technologyfor control of shrimp reproduction is role in the Eastern Hemisphere, and the Western Hemi- still young and under constantrefinement by commercial spherehas relied more heavily on P. vannameii. and academicgroups, The most important breakthroughs in this area occurred about 20 years ago. The market on Maturation technologywas implementedon a commercialscale by a few advancedfarms about ten yearsago, but it hasspread Research History slowly becauseof the reluctanceof leadingfarms to share The general life cycle is similar for most all of the "proprietary" information. Seventh and eighth genera- penaeids with spawning and early larval development in tion broods tock have nowbeen utilized by hatcheries with oceanicquality waters, and early postlarval stagesto good resul ts and the farms claim to have superior PLs in subadulthood in estuarine waters. A return to oceanic comparisonto PLs from other broodstocksources. Pond water precedes spawning. While the general life cycle of growout comparisonswere madein 1989with different penaeids is similar, the particular environmental and PL sources; therefore, there may be documented proof of nutritional parameters for each speciesmay be quite dif- theseclaims, but again,the resultsare "proprietary." The ferent. A large body of work remains to be undertaken to Oceanic Institute has recently reported disease-free define the optimal conditions for culture and in finding a broodstock lines as a result of work at its facilities in substitutefor thecommon practice of cycstalkablation for Hawaii Oceanic Institute, 1990!, but much more research captivespawn induction. is needed in this area. Specific Pathogen Free SPF! or The late Dr. Motosaku Fujinaga Hudinaga! made "high Health" animals have proven successfulin helping some of the most important contributions to the develop- domesticate the shrimp, ment of shrimp culture in 1934 when he first accorn- plished captive spawningoF mature P.japonicus Females Species Matured in Captivity and reared the resulting larvae to subadulthood Hud inaga, A number of species have been tested, at least on a 1942!.Thc capture of wild females with mature ovaries for preliminary basis, and ovarian maturation and spawning immediate spawning in captivity, which has become have been accomplished experimentally. However, the known as "sourcing," was the only method for inducing quality, frequency and size of spawns From captive fe- penaeid females to spawn in captivity for about40 years, males vary greatly and culture standards for nutritional, when induction of maturation and reproductionwas ap- physicaland hormonal factorsare only broadly defined. plied through unilateral cycstalk ablation scvcral species Of the species of marine shrimp of the family Penaeidae, were also matured without this hormonal manipulation!, genusPenaeus Holthius, 1980!,many havebeen matured The usc of sourcing is still practiced in Japan today, and spawned in captivity and viable eggs produced, with a total output of some 600 to 700 million postlarval including:P. aztecus, P. brasiliensis P. californiensis, P.indicus, shrimp annually. About 80 percent are used to restock P.japonicus, P. kerathurus,P. merguiensis,P. monodon,P. coastal fisheries and the rest arc used in commercial d uorarum,P,penici l fat us, P. plebej us, P. setiferus, P. styliros tris, cuI ture Liao and Chao,1983!. Sourcing has also been used P.vannamei, P. chinensis, P. merguiensis, P. canaliculatus, P. worldwide for experimentaland commercialgrowout of esculentus,P, latisulcafus,P. marginatus,P. Occidenlalis,P. numerous other species.This is particularly true for South- paulensis,P. schmitfi, P. semisulcatus, P.teraoi and P.subtilis, eastAsia, whcrca single sourced P. monodonfemale ready 52 , Treeee and Fox to spawn! sells for $500 or more. However, collection of passed through years of refinement and modifications by females that are ready to spawn limits culturists to indig- countless researchers and groups and is still being mocli- enous species that may or may not be the best or even a fied to meet the needs of individual hatcheries, Each suitable culture species, and is dependent on seasonal following group "carried the ball" or "carried the torch" availability, migratory movements, weather and natural magnificently, helping the causeand spreading the knowl- rhythms. Efforts to induce pcnaeid reproduction in cap- edge. The most logical and the most accurate way to trace tivity continued so that a consistent, reliable source of the refinement is through a published literature review. postlarval shrimp seedstock! could be obtained to sup- 1. Cook, H.L. 1965. Rearing and identifying shrixnp port commercial culture operations, and the basis for larvae, U,S, Fish and Wildlife Service U,S,F,W,S.! genetic selection to develop ideal domestic stock with Circular No. 230. strong growth and survival characteristics could be estab- 2, Cook, H,L. and M,A. Murphy, 1966,Rearing penacid lkshcd. shrimp from eggsto postlarvae.Proc. S.E, Assoc. of Other rcscarch of importance in maturation was done Game and Fish Comm. by Annie I.aubier-Bonichon and L. Laubier at the Centre 3. Cook, H.L. 1966.Identificatiort and culture of shrixnp Occanologiquc dc Bretagne, in Brest, France. Later called larvae. U,S.F.W.S, Circ.246. the "Laubicr method" of maturation, it involved matura- 4. Cook, H.L. 1966. A generic key to the protozoan, tion using temperature and photoperiod manipulation, xyySi, and postlarval stages of the Penaeidae. ~i ithout ablation of P, japonicus Laubicr-Bonichon and U,S.F.W,S. Bull, 65 !:437-447. Laubicr, 1976 and Laubicr, 1978!, The Laubicr method 5. Cook,H.L. 1967.Identification and culture of shrimp v. as not successful for commerical operation. The French larvae. U.S.F,W.S. Circ.268. also made other important advances in shrimp matura- 6. Cook, H.L. 1968. Taxonomy and culture of shrimp tion in the 1970s Aquacop, 1975;Aquacop 1977a,1977b, larvae. U.S.F,W.S, Circ. 295, 1979, 1984 among others!. Along with the French, the 7. Cook, H.L, 1969, Larval Culture. U.S.F.W.S. Circ. 325. SoutheastAsian Fish cries Devel opmc n tCenter made some 8. Cook,H.L. 1969.A methodof rearing penaeid shrimp very important contributions to our present knowledge of larvae. FAO Fish. Report No. 57, 3: 709-715. Food and maturation in the 1970s and 1980s Primavera, 1978, 1979, Agriculture Organization of the United Nations, Via 1985and Primavera et al., 1980!. A good literature review dcllc Tcrmc di Caracalla, 00100 Rome, Italy!. of maturation and reproduction in pcnacid shrimp ap- 9, Cook, H,L., A, Brown, C,R.Mock and M,A,Murphy. pcarcd in Chamberlainet al., 1985and Primavera,1985. 1970. L~al culture. U.S.F.W.S. Circ. 343. 10. Cook, H.L. and M.A. Murphy. 1969. The culture of Maturation-Hatchery ResearchHighlights in larval penaeid shrimp, Trans. Am. Fish. Soc. 98 !, the United States 11. Cook, H.L. and M.A, Murphy, 1971, Farjy develop- mental stages of thc brown shrimp reared in the Johnson and Fielding 956! reported the first success- NMFS GalvestonLaboratory. Fish. Bull. 69 !: 223- ful maturation and spawning with fertilized eggs! of P. 239. setiferusin the United States,but this was in ponds.A few 12. Mock, C.R. and M.A. Murphy. 1971. Techniques for years later, Cummings 961! described maturation and raising penaeid shrimp from the egg to postlarvae. spawning in the pink shrimp, P. dworarum., Proc. World Maricul. Soc. The most significant contribution toward pcnacid Many other publicationsfollowed and can be found in shrimp culture was made by the National Marine Fisher- the reference sections of this manual or in List of NMFS ies Laboratory in Galveston, Texas. Researchon the cul- GalvestonLaboratory Publications and ReportsRelated ture of larval shrimp started at the NMFS Galveston to Marine Shrimp Penaeusspp.! Aquaculture compiled I aboratory in 1959as part of an investigation into the life by Maurice L, Renaud, Ph,D,, Charles W. Caillouet, Ph.D., history of commercial shrimp in the Gulf of Mexico. NMFS-SEFCGalvestonLaboratory,4700Avc U. Galveston, Samples of plankton were taken in the Gulf to study the Tcx. 77550, or in Sindcrmann, C.J. Ed,!, 1987. NOAA seasonal abundance of shrimp larvae of the commercial Tcchnical Report NMFS 47, Feb. 1987entitled Reproduc- spccics, Thcrc was little information available about lar- tion, maturation and seedproduction of cultured species, vae of the different species and it was not possible to Proceedingsof the 12th U.S,-Japanxneeting on Aquacul- differentiate the commercial species from the non-com- ture, Baton Rouge, La., October 1983. mercial species. A project was started to collect grand There were also other groups working on pcnacid females of the various species, spawn them and culture shrimp in addition to NMFS in the state of Texas, Thc the larvae so that specimens could bc obtained for usc in Texas Parks and Wildlife Department and some of the idcntificahon of larvae collected in the plankton samples, universities published works on this subject in the 1960s. The researchprogram on larval culture was successful Oneexample is J.J.Ewald's The laboratory rearing of pink and the Director of the Laboratory, Milton J, Lindncr, was shrimp, P.duorarum. 965. Bull.Mar. Sc,Gulf and Caribb. then instrumental in obtaining the funding necessary to 15!:436-449!. develop the methodologyinto a prototypehatchery sys- Others who should be recognized for their work on tern. penaeid shrimp in connection or in coordination wr'th the This was the actualbeginning of the developmentof NMFS Lab, either directly or indirectly!, in a historical the cleanvatcr hatchery intcnsive culture technique!, account arc: Dave Aldrich, C, E. Wood, Neal Baxter, D, M. called the Galveston technique by some. From this point it Allen, T,J.Costello,W,W, Anderson,J.E.King, W.C.Renfro, Design, Operationand Training ... 53

R.H. Rigdon,C. Hanna,G.L. Beardsley,R.J, Berry, M.G. 1970s and served as an important demonstration and Kleve, D. Patlan, W.H. Clark, P. Talbot, B.R. Salser, F.S. training center for maturation-hatchery biologists world- Conte, M.S. Duronslet, J.C. Parker, J.D. Corliss, Z.P. Zein- wide. The methods utilized by the NMFS researchersare Eld in, J.M. Lyon, F. Marullo, A.I. Yudin, R.S.Wheeler, J.L, still widely known as the intensive method or "the Fenucci, C. T, Fontaine, R.G. Bruss, I.A. Sandcrson, S.E.P. GalvestonLaboratory Technique" Klima, 1978;Mock et GisIason, W,L, Trent, R.A. Neal, D.B. Revera, R.A. Gould, al,, 1980;McVey, 1983!. The methodsused and described D. Grajcer, G.W, Griffith, L.A. Ross, E.F. Klima, J.H, in this manual a rebasically modifications of this intensive Kutkuhn, L.M. Lansford, C.W. Caillouct, K.T, Marvin, method of culturing larvae. Researchat Texas A8rM Uni- A.L. Lawrence, D. Ward, S. Missler, J. McVey, B.S. versity continued along theselines in the 1980s Lawrence Middleditch, J.K. Leong, D.H. Lewis, K. Hanks, R.R. et al., 1980; Chamberlain and Gervais, 1984; Robertson et Procter,A.K, Sparks, J.R. Adams, A.M.Heirnpel, F. Marullo, al., 1987a;among others!, as well as similar researchat R,C, Benton, M. Hines, E.S. Chang, J.L. Munro, D. other institutions such as thcUnivcrsity of Miami Yang, Dimitriou, A,C, Jones, M.L. Parrack, J.C. Pearson, R.R. 1975!and the OceanicInstitute Wyban etal.,1987, Wyban Proctor, R.C.Benton, R.H. Ridgon, R.D. Ringo, G. Zamora, et al., 1988and Oyarna et al., 1988among others!. M.A. Solangi, A.K. Sparks, D. Tave, R.F. Temple, F.W. Weyrnouth, J,M, Fox, J. Wilkenfeld, Linda Smith, and Important Parameters for Maturation numerous others. The most important parameters for successfulma tura- Selectedexamples from thc literature are: tion of penacid shrimp are constant temperature, salinity, 1. Sparks,A,K. and E,F,Klima. 1965.Effects of injected pH, light, and good nutrition. The object is to rcproducc biological stains on oxygen uptake by shrimp. Trans. the near constant conditions found in the ~r oceans. Am. Fish. Soc. 94!: 277-278. Constancy is a must. Clear pristine, oceanic quality sea 2. Brown, A. and G. Patlan. 1974.Color changes in the water is the key to successful maturation. ovaries of penaeid shrimp as a determinant of their maturity. Marine FisheriesReview, Vol. 36:7p.23-26. Salinity Temperature pH Light D.O. Klirna,E.F. 1978. Aquaculture research in theGa Ives ton 28-36ppt 27-29'C 8.0-8.214L,10 D 5pprn+ Laboratory, WMS, 9, Also NMFS Contrib. 478-156!. Allowable ranges/24 hours 3, Duronslet, M., A.I. Yudin, R.S. Wheeler and W.H. +/-05ppt/Day +/4.5'C/Day +/-02/Day Clark, Jr., 1975. Light and fine structural studies of natural and artificially induce egggrowth of penaeid 1. Temperature optimums are 27 to 29' C for most shrimp. Proc. World Maricul. Soc., 6:105-122. warm water species. The senior author feels that a 4. Brown, A., J. McVey,et al,,1979. Maturation of white minimum of 28' C isrequired for P.vannarrrei, but 26' shrimp P. setiferrrs!in captivity. WMS 10:435-444. Cissufficientfor P, stylirostris. P.monodort willdobcst 5. Brown, A., J, McVey, et al,,1980,The maturation and at 28' C. A 0.5' C temperature fluctuation over a 24- spawning of P.stytirostris under controlled laboratory hour period is allowable. As much as+/-2 'C has been conditions, WMS 11: 488-499. experienced under commercial conditions by senior 6, Lawrence, A.L., D. Ward, S. Misslcr, A. Brown, J. author with P, rrrorrodon with no serious noticeable McVey and B.S,Midd leditch, 1979.Organ indices and effects. biochemical levels o fova from penaeid shrimp main- 2, Salinity optimum isoceanic<35ppt!.Althoughmatu- tainedin captivity versusthose captured in thc wild. ration may occur at a lower or higher salinity 8-36!, WMS 10:453-463, normal oceanic salinitics are considered to be 35ppt. 7. Middleditch, B.S.,S.R.Missler, D.G. Ward,J.P.McVey, Again constancy is important. If the salinity is low in A, Brown, and A.L. Lawrence. 1979. Maturation of the hatchery area it is generally not a good idea to add penaeid shrimp: deitary fatty acids, WMS 10:472-488. salt and trace metals if the difference is more than 8. McVey, J.P.1980. Current developments in the penaeid 5ppt. If artificial sea salts are used, then thc best shrimp culture industry. Aquaculture Magazine artificial sea salt brand is Hawaiian Marine. One can 6!:20-25. save costs by using Morton's Salt table salt quality; 9. Middleditch, B.S.,S.R. Missler, M. Hines, J.P.McVcy, rock salt is cheaper but has many impurities! and A. Brown, D.G, Ward and A,L, Lawrence, 1980. Meta- buying trace metals separate. Fritz "Supersalts" is bolic profiles of penaied shrimp: dietary lipids and another brand of trace metals. Salinities below 30ppt ovariona maturation. J. Chromatography 195:359- would be unlikely treatable for maturation use. Like- 368, wise, salinities above 40ppt should be avoided be- 10. Middleditch, B.S.,S.R. Missler, M. Hines, J.P.McVey, cause fresh water dilution may also interfere with A. Brown, and A.L. Lawrence, 1980. Maturation of trace metal balancesessential for maturation and high pcnaeid shrimp: ! ipids in the marine food web. WMS a nirna1 health. 11:463-470. 3. pH optimum 8,0-8,2,Normal sea water or average 11. McVey, J.P.,1983. Handbook of Maricuiture, Vol. I. seawater pH is consideredto be8.0. This canvary+/ CrustaceanAquacultlure, CRC Presslnc., BocaRaton, 0.2 depending upon the location. If thc pH is lower FL. 442 pages. Update is expected to be published m than 7.8 or higher than 8,2, the site should be avoided. 1993!. Any addition of buffers or acids may interferewith TheGalveston Laboratory continued to rcfincmatura- the trace metal balance and chemical reactions in sea tion, hatching and larval-rearing methodsthroughout the water and should be avoided unless absolutely nec- 54 ... Treeceand Fox

Photo27. Maturation room note loro light leoel!. Photo 28. Maturation tank 00 cm in diameter constructed of Ferrocement, painted black on inside!.

essary.Some adjustments havebeen reported to main- but do in closed or semi-closedsystems. Food by- tain pH/alkalinity levelsby using sodiumhydroxide products, feces,eggs, etc., all provide substratefor and/or calcium carbonate, bacteria,fungi, and protozoansto thrive. Brown flde 4. Light dim light, with approximately14 hours light and/or red tide canalso become an occasionalprob- and 10 hours dark is sufficient. The light cycle should lem for the hatchery. If it is not economically practical belongerthan the dark. Ablatedanimals reproduce in to use carbon filters to remove these unwanted a wide variety of light regimes Photo27!. dinoflagulates,then chlorinationmay becomeneces- 5, Nutrition one of the most important aspects in sary.Depending upon the organicload, normally 2 to sustaining a good maturation program is nutrifion. Sppmchlorine treatment overnight is sufficientto kill Most everyoneagrees that a combinationdiet works dinoflagulatesand evenbacteria in the seawater. The best. Some believe that bloodworms are essential for treated sea water should be vigorously aerated to P . vannamei.There is no question that the animals neutralize the chlorine, Extreme caution should be prefer this food over othersand becomevery excited exercised if chlorine is used. Chlorine can form when worms are placedin the tank. Thesource of the chromines and/or other by-product in seawater as a food needs to be carefully considered. Squid is most result of chemical reactions with trace metals. The often used forboth P. vannamei and P. rnonodon matu- chromines could cause problems if not toxic then ration programs.Some say that Gulf of Mexico-caught theycould causestress to the animalsand stop matu- squid carry Rickettsia a microbe with similarity to ration!. Chlorine can also be neutralized by sodium bothvirusesandbacteria! and would infect theshrimp thiosulfatein a 1:3to 1:6ratio part thiosulfateto 6 if used for feed. Squid is rela tively easy to obtain and partschlorine! but thiosulfatehasbeenfound tocause inexpensivein comparisonto bloodworms. Blood- deformitiesin shrimp larvaeand it would be reason- worms cannot be imported into the United States able to assume maturation and mating could be af- without having been processedin some manner. For fected by a tracemetal i~balance caused by the addi- example,if they are mixed with oystersand called tion of yet another chemical such as thiosulfate. Thio- maturationfood thenthey are considered a processed sulfate additions have caused P. rnonodon broodstock product and canbepurchased from Panama.The best deathsin India personalcommunication with Mike source for bloodworms in the United States Eastern Yates and Josh Wilkcnfeld!. Bait Co. Stetson Everett! Box 55, Hancock, Maine, U.S,A. 04640, telephone 07! 422-6822.They can be Additional Considerationsfor Optimum Maturation purchasedlive or frozen I 1/4 lb. bag = $US30. a. Tank Color Black, because animals see it better and seem to be more comfortable and at rest in a darker Other Parameters to Consider environment Photo 28!. a. Nitrogen ammonia levels should be non-existent. b. Noise-level shouldbe kept low. All machmery,large air Normal seawater nitrogenis 0.02-0.02mg/I NHI-N; bubbles or any human activity which would stressor 0.01-0.04mg/l NO>N and 0.1mg/l NO>N disturb the shrimp should not be allowed. b. Water should be on a flow-through systemor good C. Obstructions in the maturationtank should be kept to recirculated system to keep metabolic wastes and a minimum stand pipes, hoses,tubes, water inlets, air food by-products from building up in the tank. lifes, etc.!. Theseinterfere with swimming and mating C. Total suspendedsolids, organics, brown or red tide, and with capturingthe matedshrimp with a net.Ob- bacteria and other items in the incoming water. Most structions outside the tank pipes on the floor, etc.! suspendedsolids and organicsshould be removed should also be avoided for the safety of the workers. during settlingand slow sandfiltration. Organicsdo d. Nets soft with small meshso as not to damagethe notappearta poseaproblem in flow-throughsystems animals during handling. Design,Operation and Training ., S5 e. Vacuumhead a large swimming pool vacuumhead advancements include the use of portable aeration de- should be used with 1 I/2" to 2" flexible hose when vices and frequent exchangeof water in the holding vacuuming the tank. Most vacuum heads ride on buckets. Fishermen who sell broodstock have quickly rollers a set distance from the bottom and have a learned to associate themselves with a small group of strong vacuum capability to clean the tank rapidly hatcheriesand to identify which facilities are in needof without injuring the animals,Larger piecesof squid, broodstock. In 1987, the average price of large males molts etc.can be removedduring the cleaning routine. greaterthan 200g! was about Rp. 2@00 000 rupiah = U.S. f. Eye tags - somehatcheries use bird bands from the $1.17!.Females command a higher price, the price varying National Band 8z Tag Company or rubber tubing according to size and whether the shrimp is mated or not. placed over the shrimp eye on the eye stalks to mark The averageprice for an average-sizemated femalewas the shrimp!. Nostril expanderscan be usedto stretch about Rp. 8,000. U.S,$9.36!. This contrastssharply with the rubber bands.Other hatcheriessimply cut a por- reports from Taiwan where mated femalescan bring as tion of the shrimp's uropod off or knotch it to mark much as U.S. $600.Upon arrival at the hatchery, the the shrimp when marking is needed!. broodstock that have been bought from the fishermen are quarantinedfor diagnosisof MBV! mortodonbaculovirus. Sourcesof Broodstock Bray et al., 1991! If they are cIearof the virus, then the animalsare ablated There are three ways to spawn penaeidshrimp: ac- if female! and stocked into maturation tanks. quirematedfemales sperm-bearing!with matureovaries External Characteristicsof Adult I'enaeid Shrimp from the wild for immediate spawning in captivity; ac- quire adults from the wild to induce gonadaldevelop- Treece and Yates, 1990! ment, mating and spawning in captivity; and induce A penaeid shrimp is covered with a protective exosk- gonadal development,mating and spawning in adults eleton and has jointed appendages. Most organs are lo- that havebeen reared completely in captivity in pondsor cated in the anterior end head!, sometimes called thc other culture vessels, The first method, collection of wild- cephalothorax.The musclesare found in the tail or abdo- matured, mated females in nature, produces high quality men. The external body parts listed in both Figures 21and eggs and Iarvae. This is probably due to adults having 22 are apparent upon examination of an adult shrimp and beenexposed only to their naturaldiet and environmental are most often referred to in a shrimp identification key. conditions. However, this method has a number of disad- References on shrimp identification are Farfante, 1969; vantages,including the inability to work with non-indig- Bielsa et al., 1983; Holthius, 1980; Farfante, 1988; Yu and enous species,seasonal availability of mature adults, ex- Chan,1986; Dore and Frimodt; 1987; Cordover and Brick, penseof operating an offshore vessel,inconsistency of 1972;Dali, 1957;Kow, 1968;Racck, 1970;and Tirmizi and weather,migratory movements of the populations,and Bashir, 1973. variations in reproductive activity within the breeding season.Thismethodofobtainingmatureadultsforimme- General Reproductive Characteristics diate spawning incaptivity hasbeen widely used for years Penaeid shrimpcan be grouped into two broad catego- in Japan,where the acquisitionof live adult femaleswas ries, the open-thelycum or white shrimp! and closed- greatly simplified by existence of a commercial fishery for thelycum or brown shrimp! species, There are a few live adult shrimp. In most fisheries,shrimp are trawled exceptions to this e.g. P. indicus is considered a white and headed on board the vessel, and the vessels have no shrimp but has closed-thclycum!.The opcn-thelycum holding systemsfor live transportation.The method of speciesare five members of the genus Penaeus,subgenus wild collection of gravid females has been practiced com- Litopenaeus,indigenous only to the Western Hemisphere mercially in Southeast Asia with P. mortodon,on a limited P. setiferusand P. schmitti in the Atlantic Ocean; P, basis in the United States with P. setiferus,and in Central stylirostris, P. vannamei and P, occidenfalisin the Pacific!. and South America with P, vannarneiand P. sfylirostris. While the open-thelycum shrimp follow the sequenceof Sourcinghas also beenused for experimentalpurposes molt-mature-mate-spawn,the closed-thelycumshrimp with numerous speciesworldwide. follow the sequenceof molt-matc-mature-spawn, Sourcing P. monodon in Indonesia Male Reproductive System Motoh, 1981! Hatchery experience in Central Java has indicated that The male genital system consists of internal organs: during the monthsof April through Septemberit is quite paired testes,paired vas deferens,paired terminal am- easy to obtain not only mature-sized females and males, poules, and external organs: a petasma and a pair of but also mated females from local waters Java Sea!. appendix mascul ina, Broodstockare usually sold to hatcheriesby local bagan The testes,an unpigmentedand translucentorgan, is fishing platform! fishermen who bring their catch di- composedof an anterior and five lateral lobeslocated in rectly to the YayasanDian Desa Hatchery. In the past, the cardiac region dorsal to the hepatopancreasunder the transportmethodology was quite crudeand on numerous carapace.The lobesare connectedto eachother at their occasionsbroodstock had to be rejecteddue to handling inner ends and lead to the next organ, the vas deferens. stressincurred in holding and transferof theanimals from The vasdeferens arises from the posteriormargins of the the oceanto the hatchery.Typically, no aerationis used main axis of the testes and opens to the exterior through and the shrimp are sometimeskept for severalhours in genital poreslocated medially on the coxopodof thc fifth shallow plastic bucketsin the fishing boat. More recent 56 ... Treeeeand For

Figure 21. Lateral View of Adult Female Penaetjs setiferus From Young, 1959!

eye dorsal hepatic postrostral carapace 1stabdominal 2nd abdominal 3rd abdominal antennal spine segment

ventra bdominal rostral spin segment

rostrum antennular flage bdomina segment

antennae- al carina

bdominal antennal seal segment

3rd maxillipe tel son

exopodite 2nd rnaxillip

1st pereiop uropods

Figure 22. Dorsal View of Adult Shrimp From Young, 1959!

dorsal rostral spine ereiopods I carina

eye

antenn tefson

antenn podite of flag Uropod

ante

ante odite ol s uropod

d bdominal eye brush spine 2nd rnaxilliped 3rd maxi liped spine carina segments Design,Operation and Training ... 57

Figure 23 Details of Male and Female Reproductive System open thelycum type or non-grooved shrimp! 1King. Figure24.948!DetailsofMaeReproductiveSystem shrimp! Shrimpvvhite !King, 1948j

Diagramol male,lateral view, dissected iO Show reproduCtive organs T-testis VD vas deferens TA-terminalampoule «05 Diagramof terna!e.laterai view. diSSeCted toshOw relationship of ovary and oviduct 0-ovary OV-oviduct, «0 5 Diagramol snrirnpspermatozoan «9000 approx! Diagramal venlrafview Of Spermataphare aS in attaChedpaS lianl GM.gelatinousDiagramOilateraimaterial viewOi «2Spermataphare 75 «275 Diagramafmale repraduCtive Syatarh T-teatiS,PVD-prOximai vaS

deferenS,MVD-rhedial vaS deferenS, DVD-diatal vaS delerenS. TA-terminalampaule «1 75 Diagramof ventralsurface af maturemale P-peiasma,D-opening oi vas deferens. «1 75 DiagramOf peiaSmaOl maturemale Spreadopen tOShOW interiOr arran gerhent Of faldS "2 9. Diagramof secondpair afpleopadsof ma' AM-appenriixhiusciiaita «1 75 58 ... TTeecerand Fox

Figure 25. Male Reproductive System Figure 26 Details of Male and Female Reproductive System Penaeus monodon! open thelycum type or non-grooved shrimp! lxing, 1948!

TA

I Malerepioduelive Syaiemai P moiladpn T lesiia PVD 2 Spermsioaoaol P moroiriin proxmal vas delerens, MVD, medial vas delerens DVD Saeleiepreaenla 9 m CranS ceelalvaa deleiena, TA lermmal ampule

2 PelaamaOiP mOnOdOn AppenmxmaSCulina Olmale Sealsrepresents O2mm. P monodan.Scale represents I mm l as4 ascribedbyMoron l 99l i pereiopod Figures 23 and 24!. Each vas deferens consists of four distinct portions:a short,narrow, proximal medial portion having a double fixture medial vas deferens!; a relatively long narrow tube distal vas deferens!; and a DiagramOf lnule. dOraal WeW diaaeCted tO ShOW teataa and POrtian Of muscular portion terminal ampoule!. vasedelerentia T-testisl VD-vas deferens xo5 The terminal ampoule, a bulbous structure, possesses Diagramol female,dorsal view, dissected to showovaries 0 ovary x05 a thick muscular wall lined with extremely talI columnar Diagramol ventralsurtace Ol CephalOlharax of female. OP-Opening of epithelial cells. It has two internal chambers; one contains Ovidpol,TH-Ihelyoum '25. Diagramof ventralsurface of cephaloiharaxof femalewith the SperTnatophoresand the Other COntainSCalcareOuS sperrnatOphoreattaChed S-Spermatnphcre. x2.5. material of a slightly gray color. The paired terminal ampoules open at the base of the coxopod of the fifth Female Reproductive System Motoh, 1981! pereiopods,The spermatozoan,a minuteglobular body, The t'emalereproductive system consists of pairedova- iscomposedof two parts: headand tail Figures23-3and ries,paired oviducts and a singlethelycum; the first two are 25-2!.The head is large, almost circular in outline and internal and the last is an external organ. The ovaries are about 3 micronsin diameter,while the tail is relatively partly fused,bilaterally symmetrical bodies extending in the thick and short. Although it is logical to assume that the maturefemale for almostits entirelength from the cardiac spermatozoan is capable of movement, it has never been regionof the stomachto the anterior portion of the telson obscr vcd. Figure26-2!. In the cephalothoracicregion the organbears Thepetasmaisa pairof endopodsofthe first pleopods, a slenderanterior lobe and five to six finger-like lateral It is formedby the interlockingof minute hook-likestruc- projections.A pair of lobes,one from eachovary, extends tures Figures 24-3 and 25-3!. over the length of the abdomen, The anterior lobes lie close The shape of the appendix masculina Figure 24-4!, to the esophagus and cardiac region of the stomach. The which is locatedon the endopodof the secondpleopod, is lateral lobesare locateddorsally in the large massof the generallyoval in shapefor P. morfodon Figure 25-4!. hepatopancreasand ventrally in the pericardiac chamber. The spermatophores, one from eachterminal ampoule, The abdominal extensions lie dorso-lateral to the intestine becomefixed togetherlongitudinally at time of extrusion and ventro-lateralto the dorsalabdominal artery. and are then referred to as the "compound spermato- The oviducts originate at the tips of the sixth lateral phore." lobesand descend to the externalgenital apertures hidden Spermatophorestructures vary considerablyamong in the ear-like lobesof the coxopodsof the third pair of closed-thelycumand open-thelycumshrimp, but mating pereiopods seeFigure 26-3for openingof oviduct!. and courtshiprituals aresimilar within eachgroup. Sper- The thelycum is located between a pair of the fifth matophores among the open-thelycum shrimp are quite pleopodsand consistsof an anterior and a pair of lateral complicated Figures23-4 and 23-5!. plates Figure 26-3!, Note the variation of the thelycum on DeSign, Operafipnfsrtd Training ... 59

Figure 27, "QuiCk" IdentificatiOn Of SeleCted SpeCieS

Closed Teethon Shape of Rostrumand 1St Thelycum Petasma OtherCharacteristics P ENAEID T hei yourn Rostrum Locationof Teeth Antennae SPEC ES Mode! Rostrumcurves upward and spetpes 9-12 is easilyconfused with P sfyarosfris P occidenfa/is no 9'4! ft hasonly one spot on ventral portion 3 --5 of lail "WesleinWhite Shiirnp" BlueishpereiopodS walkmg legs! 2 7-8 spctaOn tail oneventral. one dOraall ~ 0 iyi4 . 5i P sfytiios ris 3--8 "BlueShrimp" Surfaceol carapaceis notas smooth 8 -9 asthe othersp whenyoung Red P vannamef no 8'2! antennaeNo coloration on telson 1 -2 uropodregion "Whiteleg" BlaCkCOIOiatiOn OntelSOn, urOpcda 4 -10 Rostrumlong slender, sometimes P. setiierus no Bi2! turningup Ventralteeth apart 0-3 "NorthernWhile" Petasmawithout diagonal creation 7- 10 inrierS r~ ace ol distalpart of lateral no i8 2! P sctimitfi 1 3 lobe Thelycumhas parallel create "SoulhernWhite" Dorsolateralgrooves ol siklh 7- 10 Brz! abdominalsomile narrow P duorarum yes 1 3 "Northernpink" Mediangroove ol postrostrafcarina 5 10 longand deep Dorsolal*ralgrooveS broad."Northern Brown" P aztecus yes 0 -3 lai2! Relearnswith lung prO eClicnaSnd . 11 distalfolds which penetrate deeply 8721 intopelasma "Red Spotted" P.Drasifiensis yes 0 -3 Saddle.kkestructure on posterior portionof 3rdabdominal segment and 8 -9 anteriorperl of 4thsegment rOStrum P. californiensrs yes straightEvtenor cof lee colored dorsal 2 Oclericrand ventrall "Yeilcwle " astventral tooth anterior lo or 0~ 8- 10 samelevel as first dorsal tooth P. breyfrostris yes NOiungiiudinal Carina On thelyCum 2--3 Darkpink "CryatalShnmp" GrOovefiostrat! iS Sharter than 7 in P.semisuicafus "Giant Tiger' r 2! P. monodon yes 2- 3 Mufticolored shrimp blue,red 7 yeilowon swimmerelts Banded P. Semisufcatue yes 2.3 antenna dark, fight! "GreenTiger"

Gastrorbitslcarina postenoi 5 -- 7 2/3 hepaticSpine and orbital angle P indicus yes 0 1 RostralcreSt high "IndianWhile"

differentspecies in Figure27 " Quick" Identification of Althoughthey are capable of full ovarianmaturation SelectedSpecies!. only when they reachabout 90g body weight,wild P. mortodortfemales actually mate when they are smaller. Sizeat FirstMaturity and Mating Primavera,1985! Spermhas been identified in the dissectedthelyca of wild In a male P. Trtonodotf,the presenceof identifiable femaleswith a minimum body weight of 63g. More inter- spcrmbymicroscopic examination of dissected spermato- esdngare observationson pond-rearedstock: a female phoresindicates sexual maturity. Both pond and wild P. weighingless than 40g was found to be positive for sperm, mortodortof 40gbody weight arealready sexually mature. Thefact that femaleswith very littlechance of full matu- Femalesare mature when they developStage III or IV ration of the ovariesand later of spawning in the ponds ovaries seeFigure 28!. However,unlike the males,they materegularly proves that maturation and matingare two rarely attain maturityin captivity unless they undergo independentevents in P.mortodort, Moreover, the smaller eyestalkablation. Individual records of wild gravidfe- size of the pond-matedfemales compared to the wild malescaught in thePhilippines and subsequently spawned stock could be due to a higher stocking density in the in SEAFDEChatcheries show an averagebody weight of pondsaffording greater opportunities for malesand fe- 142gwith minimumof 87gand a maximumof 206g. malesto mateor stuntedgrowth of mature-agedfemales. 60 ... Treeeeand For

ovaries. Dissected ovaries are yellowish but become more and more white as regression continues. Several observations can be made concerning staging seeFigure 28!. The final flush of color or rapid changein color associated with spawning seems to occur within hours of spawning. Also, there is considerable difference in ovarian color among species for example, a bronze color in P,stylirostris as opposed to yellow-orangeto drab in P.setiferus!. Additionally, the closed-thelycumspecies develop as light-green and then olive-green ovaries. Under captive conditions, particularly over time, dif- ferent ovary colors are frequently seen. The fullness of ovaries at Stage IV is often only a percentage of the Full ovaries of wild animals. There is frequently a separation between ova-filled lobes in the carapace and abdomen, and in somespecies e.g. P. vannamei!,only the anterior lobes may develop in captivity. Diet and light have been suggestedas explana tions for ovarian color differences in captivity. Unablated P. indicus females in white and black tanks generally took on a color more similar to the back- ground tank color, as did their ovaries, eggs and nauplii. White-tank femalesdeveloped pale-green to cream-col- ored ovaries, while black tank females developed dark- olive ovaries. Ovary color, although varying from the wild state,can befairly dependableasastaging determinant in P.vannamei and P.stylirostris. The size of thegonad in the first thoracic segment, compression of the posterior cephalic lobes ob- served at the thoracic abdominal junction, and a "granu- lar" texture, must be used as determinants of immediate spawning in P. rnonodon,P, merguiensisand P.japonicus.Of course, in open-thelycum species,the presence of a sper- matophore, spermatophoreportion, or sperm mass,is also an obvious indication of readiness to spawn, All of Ovarian Maturation/Staging Mature Shrimp thesejudgments, except the latter, are subjectivejudg- Primavera, I 985! ments guided by experience. The different maturation stagesfor both ablatedand The Shrimp Egg Chamberlain et al., 1985! wild femalesare the following: Oogonia are continually produced mitotically from StageI Immature! Ovaries are thin, transparent germinal epi thelycum throughout the reproductive life of and not visible through the dorsal exoskeleton. On dissec- the female. Oogonia enter microsis, differentiate into oo- tion they appear as colorless strands of tissue, devoid of cytes,begin yolk synthesisand becomesurrounded by visible eggs. follicular cells. Yolk deposition proceedsslowly during Stage II Early maturing! Ovaries observed as thin, linear band through the exoskcletonas they start to in- primary vitellogenesis but acceleratesduring secondary vitellogenesiswhen yolk material apparently is pinocy- crease in size, particularly in the anterior and middle totically drawn from the hemolymph through the blanket lobes. Color of dissectedovaries ranges from cloudy- of follicular cellsinto theoocyte wi thout anymajor change. white to light-brown and grayish-green. The chief componentof crustaceanegg yolk is a high Stage III Late maturing! Ovaries visible through density lipoglicoprotein, termed crustacean lipovitellin, the exoskeleton as a thick, solid, linear band as they which contains 27 to 35 percent lipid, a small carbohydrate considerably expand from the anterior thoracic to the componentand no protein-boundphosphorus. The lipid posterior aMominal region. Dissected ovaries are mostly component consistslargely of phospholipids usually con- light olive-green and firm and granular in texture with taining high-levels of medium length, monounsaturated clumps of eggs that can be seen. fatty acids!,a carotenoidand small quantities of choles- Stage IV Mature or ripe! Ovaries exhibit a dia- terol and triglycerides. Most of the nutrients are drawn mond-shapedexpansion a tthe forward abdominalregion from immediate food intake. and the linear band is thicker, Upon dissection, they Development of different egg types of P. rnonodoncan appear dark olive green and are so distended they fill up be seenin Figure 29, The types of development can be used all available space in the body cavity, to set criteria for "save or discard" decisions in the com- StageV Spent! Completely spent ovaries are limp, mercialhatchery. thin and outwardly appearsimilar to StageI immature! Design, Operationand Training , 6!

Figure 29. DAY1 DAY2 It 0 00p m-2'00 a m! i900 a m-10.00 a.m.!

RLTAIfsf If 40tftyor Greater!

R>alii Separeeonol COmplenOn Ol Fenilizedwn-Cell Moule Slag* EarlyNaupliue dermalDevelppreeni AherSpawning Exer*al Saembrane EeleinalMernprane Stage!40min I 11ni ggriiin,f lg min1 fig min,' 120m ni

1' I A' Aunpmal Deva Opn enl No

DISCARD I" egula0 y:op~aemio fIf E1gfyor Greater! r ormanone

NoDe e oorreni

Develpprren'.Ol different egg types Ol Penaeus n onodonfrom immeditate OOhl-spawning it:e!ween CylOolayrn'-vened Ov 1000 p m ard 2 00a m' to the followingmern ng Ig 001O1000 a mi Dantea mOdhed Irp p iinevera! 999,'

Mating of Open-Thelycum Shrimp occursand sperm undergo the acrosomereaction that facilit- atesssecondary sperm binding and initiates the cortical rod In the open-thelycum species, a male with hardened reaction and subsequentnudear fusion. exoskeleton mates with a mature female also hardened! Spermatophores are easily dislodged from open- only late in the day. The male deposits a complex sperm thelycum shrimp, and in somespecies may break down packet Figures 23-4 and 23-5! onto the ventral surface of sequentially prior to spawning. In offshore collections as the female between the third and fifth periopods Figure well asin the laboratory!, threespermatophoreconditions 26-4!. The sperm is extruded from the spermatophore are commonly seen in mated P. setiferus:! the full com- prior to spawning, and eggs are fertilized during the actual several minute spawning period. pound spermatophore; ! the wings only; and ! a sperm Mating in open-thelycum shrimp is also characterized mass only is present, obscured from vision when legs are in a normal position, but visible when the third percio- bymaleattraCtinn tOa fernal,a courtShipritual ofparallel pods are folded posteriorly. swimming Figure 30-1!and placement of spermatophores The attraction of males to femalesis thought to be after achieving a 90-degree rotation Figure 30-4! that placesthe male below and perpendicularto the female. through the sexually dimorphic flagella of the males "Chasing," the initial phase of sexual behavior, occurs Young, 1959!. The mating behavior in P. vannameihas been described by Yano et al., 988!. P. vannameimating somewhat randomly, occasionally involving females that are not ready for spawning. The elicitation of sexual wasd i vided into four phases:! approach! crawling! chasing and ! mating. In P,japonicrts, P, monodonand P. activity seems to be related to the intensity of light and with P, vannarneiand P, stylirostris occurs much earlier pattlensis,the male swims parallel and below the female and turns ventral side up and attaches himself to the when the sky is overcast. Aquacop, 1977b!. Mating in captivity begins late in the afternoon and continues until female Hudinaga,1942, Primavera, 1979,De Saint-Brisson, 1985!, These processes are the same as observed in P. around nightfall. This same sequence seems to be fol- lowed in all the litopenaeids. vannamei,and thereforeappear tobe a commonpattern for the mating behavior in penaeid shrimp. Several workers In open-thclycum penaeids that have no spermatheca, including P. vannamei, P. stylirostris, P, setiferus and P. have noted that the male flicks his head and tail and rotates 90 in relation to the female Figure 30-4!, assum- occidentalis,the spermatophore is attached to the exterior of ing a "hooked"position during mating in P.monodon and the thelycum of a hard shelled, fully mature female only P. partlensis.However, in P. vannameithis complexposi- hoursbefore spawning. The reproduction sequenceis: molt- tion has never been observed during mating. Only ventral mature-mate-spawn.Spawning, the monocular dischargeof side up attachment Figure 30-2! has been observed for P. recently ovulated oocytes,is proceededby releaseof sperm from the spermatophoreof open-thelycumspecies. As the vannarnei, usually face-to-face, but occasionally an in- verted position is observed Figure 30-5!, Two different oocytesare discharged into the sea,they brush past sperm- sex attractants may be produced and released by mature coatedsetae surrounding the gonopores.Primary binding females for signalling sexual receptivity, chasing and 62 ... Treece and Fox

noted that mating in closed-thelycum shrimp is indepen- dent of ovarian maturation and begins in rather young shrimp.Eldred 958! found P.duorarufn females as small as 91 mm total length mated about 18 mm carapace length!. The molting-mating sequencewas first observed by Hudinaga 942! in P. jfgponicus, and the sequence appears universal among the subgenera other than f 4t,g Li topenaeus. ! Thecourtship and mating processis startedwhen the newly molted'female attracts from one to three hard- shelled males who follow her as she makes brief upward swimming movementsover distancesof 50 to SOcm, Eventually, one male is able to position himself directly below the female. The pair engagesin parallel swimming movements, during which the male turns upside down and tries to attach his underside to that of the female. If successful,the male quickly turns around from this direct alignment to a perpendicular position, curves his body in a U-shapearound her and flicks his head and tail simulta- neously.It is probablyduring this time that thesperm sacs are inserted inside the thelycum, Among adult P.monodon, molting occurs about every three to five weeks; it is synchronous and is spread over a period of five days within a given broodstock tank. Copulation generally takes place at night following molting, which is primarily Figure3Q a nocturnal event. Courshipand mshng behavior of penaeidshrimp t! Femaleabove-male Molting of the female is a prerequisite to mating since belowin parallelswimming, 2! Maleturns ventral side up and attaches to P. monodonbelongs to the group of penaeids possessinga female,1! 8 2l obsenredin bothP, monodon and P vannamei3! P.monodon closed thelycum in which the sperm sacs have to be maleturns perpenrscular lo female; 4! P.monodon male curves body in L!.shapearound female and flicks head and tail simultaneously after inserted into receptacles within the thelycum. Insertion Primavera,f979! end 5! Inaddition to I! 8 2!Yano et al.988! observed can only take place when the thelycum is soft, i,e., the invertedposition in P vannamei. femaIe has just molted. The sperm remain within the thelycum until spawning when the female releases the mating; namely ! chasing-stimulating pheromone CSP!, sperm simultaneously with the eggs. If she does not and ! mating-stimulating pheromone MSP!. Many other undergo spawning becauseher ovaries remain immature, authors have deduced the presenceof sex pheromones in the sperm sacsare expelled with the shell during the next decapodcrustaceans for review, Dunham, 1978!. molting, shortly after which fresh sperm sacsare inserted oncemating takesplace. Mating doesnot accelerateova- Mating of Closed-Thelycum Shrimp Primavera, rian maturation directly, according to Yano 987!. 1985! Spawning and Rematuration Primavera, 1985! In the closed-thelycum penaeids, mating usually oc- Spawning generally takes place between 8:00 p.m.. curs at night between a hardened exoskeleton male and a and 6:00 a.m., although most females spawn between female just after molting Figure 30!. The spermatophore midnight and 4:00 a.m. Normally at rest or slow-moving is inserted into an opening in the exoskeleton correspond- on the bottom, a female about to spawn becomes restless ing to the same area where it is attached superficially on and starts swimming upwards in circles. The eggs are the open-thelycum shrimp. A position of the spermato- released,often Forcefully, as she swims and maycontinue phore is visible protruding from the thelycal opening, or even as she rests on the bottom, Spawning lasts from two seminal receptable, for about 24 hours until the exoskel- eton rehardensand, in a few species,such as P.j aponicus, to seven minutes. Of a givenba tch of StageIII or IV females,only 50to SO a "stopper" remains on the surface thoroughout the percent wilI actually spawn. Of all females that spawn, intermoltasevidenceofmating. Thus,thespermiscarried roughly 50 percent are partial spawners while the rest internally until the female either spawns or molts. Con- completely spawn all the eggs. Partial spawning or non- trary to the scenario with open-thelycum shrimp, multiple spawningmay be associatedwith stressdue to transport, spawnscan occurutilizing the samesperm masswithin handling, crowding, etc.Fully developedfemales or par- the same intermolt period. At the next molt, the old tial spawners will either spawn or continue spawning in spermatophoreis shedwith the exoskeletonand mating the next two to three days or regresstheir ovaries.The proceedsagain. Mating seemsto follow molting closelyin nature of a spawningcan be determinedby holding the time sequence. Primavera 979! observed a female P. female to a bright hght some of the anterior or posterior monodon in the laboratory that molted and then began portions of the ovaries remain in partial spawners while courtshipbehavior to rematewithin an hour, It shouldbe completespawners have no tracesof the ovaries. Design, Operationand Training ... 63

In nature, P. rnonodonfemales probably spawn more Hormonal Control Chamberlain et at., 1985! than once in a spawning season.Tagging experiments Sourcesof hormones involved in reproduction are the show that of a given number of ablated females that medulla terminalis X organ of the optic ganglia, sinus spawn once, 14 percent will spawn a second time, 3 gland, brain and thoracic ganglion, androgenic gland, percent a third time, and 0.8 percent a fourth time. A ovary and Y organ. The X organ and sinus gland are subsequentspawning may takeplace as quickly as three juxtaposedwi thin the eyestaIksof penaeids Van Herp et to five days after the precedingone. aI., 1977!. The sinus gland is a neurohemal organ corn- The act of spawning itself takes severalminutes. A prised of axon terminal that arises primarily from the fernaleinitially is resting on the bottom of a spawning neurosecretoryX organ of the central nervous system tank. Shebegins to swim upward just beforespawning, Andrew and Saleuddin, 1979!. Biochemical and struc- sometimeswith rapid flexing of the aMomen. The swim- tural examination of the X organ/sinus gland complex ming slows,and eggsare expelled from the oviductsinto indicatesthat precursorpolypeptides are synthesizedin the water, the pleopods moving rapidly. Spawning is the cell bodies of the medulla terminalis X organ, pack- frequently observed at the water surfacein spawning aged in neurosecretory vessicles, and transported tanks,and occursabout midnight or soonafterward, intraxonally to the sinus gland where they are stored and Fecundity Primavera, 1985! releasedas smallerpeptide cleavageproducts Fingerman, 1970; Andrew and Saleuddin, 1979; Bellon-Humbert et al., Penaeids areextremely fecund, and may produce from 1981!.The medulla externaX organmay regulatethe rate 100,000to 1,000,000eggs per spawning. The larger species of gonad-inhibiting-hormone GIH! production Faureet and the largerfemales within a speciesgenerally produce aI., 1981!. The X organ/sinus gland complex has been larger numbersof eggs. In captivity, spawns generally implicated in hormonal control of numerous processes rangefrom 50,000to 300,000ova. including sugar, respiratory and calcium metabolism, Evidenceindicates multiple spawningof unablatedP. reproduction,molting, osmoregulation, seasonalthermal setiferus five spawnsperlifetime! and at leasttwo spawns acclimation,retinal pigmentmigration and colorchanges per seasonfor P. setiferus,P. duorarum,P.faponieuss and Kamernoto et al., 1966; Adiyodi and Adiyodi, 1970; Metapenaeusaffinis. Sil ver thorn,1975; Kleinholz,1978; Kulakovskii and Baturin, In captivity,mulhple spawning of unablated P faporticus 1979!. hasbeen shown. In onecase,an unablated female spawned Panouse943! was the first to recognizethat removal of 19 times in seven months. Unablated P. merguiensfshave theX organ/sinusgland complex by eyestalkablation often been noted to spawn each 2.6 months in captivity com- results in premature or nonseasonalgonadal hypertrophy. pared with an average2.8 for P.fapanicus. Thiseffecthas beenattributed to removal of gonad inhibiting Viable eggs hatch within about 12 to 14 hours of hormone GIH!, which is neither sex- nor species-specific spawning at 28'C. After hatching, the larvae develop Otsu, 1963; Bomirski et aI., 1981!. The amount of GIH throughthe non-feeding stages nauplius1 through5 or6! produced within the eyestalk varies with seasonand matu- in about 30 hours. With transition to Zoea 1, the larvae rationstage. Experiments from femaleCrangon erangan show require specialized diets. that GIH activity is high after the breeding season. Accordingto Primavera,for average-sizeP. rrtonodon, G!H is presumed to inhibit release of gonad-stimulat- fecundity or number of eggs produced by one female in a ing hormone GSH! from the brain and thoracic ganglion. completespawning ranges from 100,000to 400,000with GSH, in turn, is thought to stimulatesecondary vitellogcn- an averageof 200,000for ablatedfemales, and 200,000to esis in females, and precocious spermatogenesis, hyper- 1 million with an average of 500,000for wild females.The trophy of the vas deferens and hypersecretionof the eggsand nauplii counts from spawnsat the Dian Desa androgenic gland in males. The action of GSH may be Hatchery were above-average 84,000 to 600,000-per- mediated through steroid hormones. spawn-averageof 22 spawns!and were probably due to The role of the Y organ analogous to insect pro thoracic above-average females 93g to 450 grams!. and ring glands! in reproduction is not well understood. A spawnermay producegood or bad eggs.To avoid This endocrinegl and produces ecdysane from cholesterol wasting time and effort in rearing inherently weak larvae, at rates regulated by molt inhibiting hormone MIH!, the quality of eggs from a single spawning should be which is released from the X organ/sinus gland complex. determined as early as possible. Preliminary observations Circulating ecdysone is converted to the more active show that P. mortodoneggs can be classified into various molting hormone, B-ecdysone, through the hydroxylase grades. Good eggs undergo normal cleavage and devel- activityof peripheral tissues suchas the testesand hepato- opmentof the nauplii and variousabnormal types

percent at premolt, but none occurred during late premolt growth, Anilkumar and Adiyodi 980! found that eye- or carly postmolt. During premolt, mature ovaries were stalkablation of thecrab Paratelphusa hydrodromous during rcabsorbed, presumably to provide nutrients for molting. the prebreeding season resulted in precocious ovarian This alternating hormonal dominance explains why eye- growth. However, in comparison to normal mature ova- stalk ablation sometimes accelerates reproduction and ries, the ovaries from ablated females were smaller, higher other times acceleratesmolting and somatic growth, de- in lipid composition, and more variable in distribution of pending upon the processthat would have normally yolk among oocytes. These differences presumably are transpired next Bliss, 1966;Highnam, 1978!. Researchin consequencesof hormonalinsensitivity of ablatedshrimp this area continues with a number of groups publishing to physiological or environmental limitations such as their results Quackenbush and Herrnkind, 19S3, improper oocyte differentiation, nutrient storage, food Quackenbushand Keeley, I 986;Rankin etal, 1989;Rankin supply or temperature, et al. in press and Bradfield et al., 1989!. After spawning, unilaterally ablated females immedi- ately reinitiate ovarian maturation and consequently Eyestalk Ablation Background and Research spawn more frequently under non-optimal conditions The stimulating effect of eyestalk ablation on repro- than unablated females. This fast pace can deplete fernale duction of decapod crustacea Panouse, 1943! was not nutrient stores and is probably partly responsible for the usedfor shrimp aquacultureuntil the early 1970swhen lower survival rates of ablated to unablatcd females, bilateral eyestalk ablation was first attempted. This was Emmerson 980! reported that ablated P, indicus that found to stimulate rapid ovarian maturation, but ablated spawned repeatedly every five days lost condition and females suffered high mortality probably dueto MIH and died, but suggestedthat this problemcould be alleviated GIH dcsynchronization!, and their eggs were usually by proper nutrition. rcabsorbedrather than spawned Caillouet,1972;Aquacop, Eyestalk ablation of male shrimp has rarely been con- 1975;Duronslct et al., 1975!.These problems were allevi- sidered necessary.Alikunhi et al., 975! reported that ated with thc usc of unilateral eyestalk ablation, which maleablationcaused precociousmaturationof P, morrodon provides moderate stimulus without reabsorption of ova and P. merguiensis.Chamberlain and Lawrence 981! or cxccssive mortali ty Aqua cop, 1975;Arnstein and Beard, found that male eyestalk ablation increased gonad size 1975;Wear and Santiago, 1976!.Consequently, unilateral and doubled mating frequency of undersized 5-30 g! P. eyestalk ablation rapidly emerged worldwide as a simple vannamei in comparison to unablated control shrimp, procedure for inducing reproductionof numerousspecies Ablation is not considered necessaryfor adult males. of captive penaeid shrimp and some researchers used ablation to increase the growth rate of shrimp Hameed Maturation Feeds and Dwivcdi, 1977!. A combination diet is most often used and gives the Eyestalkablation hasbeen performed using a variety best results, usually one consisting of squid and other of methodsincluding severingwith scissors Caillouct, products such as shrimp and shellfish clams, oysters, 1972!, cautery Duronslet et al,, 1975!, enucleation etc.!.Aneffectivedietarysupplement formaturingshrimp Primavera,'1978!,and ligation Schade and Shivers,'19SO!. is the bloodworm, which is very expensive. Bloodworms Eachof thesemethods has been effectivein removing or are thought to provide long-chain fatty acids essential in destroying the X organ/sinus gland complex, but enucle- maturation. The bloodworms Glycrm dibrachiata, com- ation squeezingout thecontents of theeyestalk through monly referred to in the United Statesas the Maine blood- thedistal end of theeyebaII! hastheadvantagesof simplic- worm becauseof its geographicsource, is also found in ity and rapid clotting of hemolymph within the empty other geographic areas, Panama,for example,has blood- cyestalk. Stress can be reduced and tossesminimized if worms rich in polyunsaturated fatty acids PUFA!, but it shrimp are held in chilled water before and after ablation is illegal to export the worm unless it is mixed with the Ca illouet, 1972!. oysters and sold as "maturation food." Middleditch et al,, The effects of cyestalk ablation vary with the seasonof 980! showedthat P. van namei grown in captivi ty reached the year and the stage in the molt cycle. Shrimp that are sexualmaturity when fed diets similar in fatty acid pro- ablatedas they prepareto enter their reproductivepeak files to that of the Maine bloodworm. Blood worms have a are morc conditioned to yield a reproductive asopposed high n-3 to n-6 PUFAratio and this variableis thought by to molting! responsethan thoseentering a reproductively someto be the key factor necessaryfor a maturationdict. dormant period Bliss, 1966;Adiyodi and Adiyodi, 1970!, Lytle and Lytle 989! looked at the fatty acid composition Within a molt cycle, ablation performed during prcmolt and variations in individual bloodworms. Dechan and leadsto molting; ablation immediatelyaftermolting causes Chen975! describedthe processrequired for theculture death; and ablationduring intermolt leads to maturation of a similarLugworm specieswhich could bc modified for Aquacop, 1977!. bloodworm, Resultsindicate squid, oysters and a diet The fecundity and viability of spawns from ablated supplementmade from Artemiacalled Marilla appearto females have been inferior to spawns from females ma- bestmatch the fatty acid profile of bloodworms. A large tured in the wild Aquacop, 1977; Beard and Wickins, hatcheryin Panamareported that Marilla, when addedto 1980; Emmerson, 1980; Lumare, 1981!. Furthermore, com- the maturation diet, saved approximately $27,000U.S./ mercial producers prefer postlarvae produced from wild year by increasing the frequency of females spawning, the rather than captive spawns, which suggests that embry- total number of viable eggs spawned and the survival rate onic characteristicsmay influence juvenile survival and of the nauplii produced WAS Confcrcncc, Guayaquil, Design,Operation and Training ... 65

Photo 29. Acclimating broodshrimpfor temperatureand Photo30, Measuring broodshri m pto get length to roeightconoersion salinity beforeplacing in maturation tank. jar tankbiomass estimate. This female zoas 355 mm long and zoeighed 450grams onepound!!. 1987!.Magarelli 981! alsofound sex-specificnutritional requirementsfor crude protein and fat in cultured P. and is acclimated to hatchery conditions Photo 29! with stylirostrt'sbroodstock. He found that the femaleshrimp the following guidlines: requireda higherprotein level, a lowerfat level, a higher ~ No moze than 2 C per hour temperature change protein/calorie ratio and a muchhigher protein/fat ratio ~ No more than 2 ppt salinity changeper hour than males.Again, a combinationdiet is most often used The animals are usually stressedand care must be so that all of the essential requireznents are znet for both taken to seethat the stzessfulconditions are improved or males and females. stopped,otherwise mortality will be high, Distribution into the maturation production tanks is BroodstockOperations on an as-needed basis or new animals can be held in a separatetank until needed.It is bestto wait until all stress Penaeusrnonodon, obtained principally from local fish- is gonebefore ablating the fernale. ermen,is the speciesbeing maturatedand spawnedon a The first choice of the hatchery is to obtain wild- regular basis in the Dian Desa Hatchery. P. tnonodon caught, mated feznales in late stages of developznent! appearsto be seasonallyavailable from fishermenat a from fishermen and hold the females until they spawn. reasonableprice $2,000Rp for a 50-to 80-grammale and Being that this is only seasonal,the hatchery must be $6,000ta $10,000Rp [$6.00to $10.00US j! for matedfeznale equipped to sustain production on a year-round basis; shrimp, dependingupon their size,stage of maturation, therefore, intensive maturation techniques are practiced health, etc. Of the three speciesobtainable from local during the periods in which mated females are not readily fishermen P. merguiensis,P. sernisulcatusand P. monodon!, available. P. rnonodonis the only species that can be obtained in Refer to Robertson et al., 1987b W.A.S. 18!:45-56 for sufficient numbers from fishermen! to support or sustain broodstock shipping recommendations. a maturation prograzn for a hatchery of this size. The larval-rearing section of Unit 1 is designedto BroodstockHolding Ponds producethree million postlazvaeper month. P. rnonodon Animalsbeingheld inbroodstock holding tanks should alsoappears tobe the speciesof choicebecause it is hearty, be tzeated the same way as in broodstock production very productive, matuzes and spawns easily, survives tanks same water supply and flow rate, same tempera- well in larval rearingwith minimal feed levels, and grows ture range, photoperiod, feeding regime, aeration, etc.!. out well in high densitiesin the ponds.It is the preferred Maintenance of braodstock ponds has been described by speciesof most postlarval buyers. The following is an a number of sources. One method is as follows: accountof the procedurespresently being used at the 1. Stocked 7,000 to 10,000/ha ,800 to 4,000/ac,! Yayasan Dian Desa Hatchery to produce larvae success- 2. Prior to filling, the pond is dried -7 days!,and lizned fully for the adjoining larval-rearing facility. if soil pH is lessthan 5 at 500kg/ha. It is thenfertihd BroodstockReceiving and Shipping with 16-20-0inorganic fertilizer at 100 kg/ha and chicken manure at 350 kg to 500 kg/ha and filled Fishermenusually bring the brood animals to the slowly. Benthic growth is encouraged during slow fill hatcheryduring thelate afternoon to early eveninghours. to 10 days!. Water exchange varies from 3 to 6 The animals range in size from 75 grams to 400 grams percent minimum! to 25 percent, depending on dis- with the average weight being 193 grams!. At that tizne solvedoxygen. the aniznals are inspected for stress, abrasions, cuts, or 3. Food consists of pellets containing a high-percent other damagethat might eventually causethem to die. protein given two or three days per week a t3 percent When a shrimp passesthis initial inspectionand is re- body weight per day. The animals also are given fresh ceived,it is placedinto a plasticcontainer with areation food fish, scraps from maturation tanks, etc.! at 5 66 ... Treece and Fox

Figure 31. Pertaeus vattnamei and mottodort length to Weight Conversion

Total length mm! Weight of Whole Shrimp grams! 135 19.10 140 2'1.25 145 23.56 150 26.02 155 28.65 160 31.46 165 34.43 170 37,59 175 40,93 180 44.46 185 48.19 190 52.12 195 56.25 200 60.59 205 65,15 210 69.93 Photo 31. Squid, purchasedfrom localfisher- men,arekeptfrozen until fed asmaturation food 215 7494 squid makesup 50 to 75 percentof the diet!, 220 80.17 225 85.65 230 91.36 percent body weight per day for four or five days per 235 98.74 week care must be taken not to overfeed fresh foods!. 240 105,91 245 113,60 Broodstock Distribution and Feeding 250 121.85 Upon receiving brood animals a length measurement 255 130.70 is taken Photo30! and later convertedinto weight using 260 140.19 the chart shown in Figure 31. 265 150.38 The length/weight is recordedon the data sheet Fig- 270 161.30 ure 32!.This methodis muchquicker and lessstressful on 275 173,01 both the animal and the operator obtaining an accurate 280 185.58 weight without stressing the animal is nearly impossible!. 285 199.05 The total biomass is then determined for the tank and 290 213.51 a feeding regime is established. A 1:1 sex ratio is desirable 295 229.01 in the maturation tank equal number of males and fe- 300 245.65 males!, stocked at a density of no more than five animals 305 263.49 per squaremeter or, in the caseof very largeanimals 73 310 282.62 grams to 400 grams!, no more than 7,000grams total 315 303.15 biomass or total body weight in one tank 0 males, 20 320 323,15 femaleswith an averageweight of 173grams!. Otherwise, too much food will have to be placed in the tank at one time 325 343 to keepthe animalsfed and it would be almostimpossible 330 363 to maintainoceanic cond i tions.Generally, brood animals 335 383 gain 5 grams total body weight per month for 55 to 80 400 grams total body weight. Feed rate is best determined in 420 a productionmode by trial and error or ad libitum feeding 440 feeding just enough so that there is a small amount 450 remaining before the next feeding! or feed to satiation all they want to eat plus a little!. Brood animals like fresh or freshly frozen food and they alsolike a variety.A variety or combinationdiet is also best for them. They will eat more if fed smallerquantities more frequently they will consumemore food if fed the sameamount, for example, 100grams x 4 times a day,than they will if fed them 400 grams once a day!. Subsequently,the more food they consume,the healthierthey are, the more active they are Design, Operation and Training ... 67

Photo 33. Afafura- tion food is weighed so overfeedingdoes Photo32, Small shrimpare also purchased locally and kept frozen until not begin to degrade neededfo supplementthe maturation diet theyare thawed and fed oceanicquality wa- whole!, fer an inexpensive plasticscaleis used!.

Photo34. All of thematuration food is spread Photo35, E'xcessfood and debris is siphoned evenlyarou nd thetank at four differentfeeding from maturationtanks tuncea day to maintain intervalsduring a 24-hourperiod. oceanicquahty water,

Photo 37, Healthy broodshrimp about one weekafter stock- Photo36. Broodshrimp sett ling in andeating squid; some are ing ready for abla- morest ressed than othersu ponarrival andappear red in color tion and to beplaced and swim at the surfaceuntil they too settlein. in production. 68 , Treeeearaf Fox

Figure 32. Length and Weight Data Sheet

Tank or Pond ¹ Date: Batch ¹ Person:

' Number Shrimp Measured: Average Length:

l Standard Deviation:

Average Weight: Mean Average Weight

Section below refers to length in mm of shrimp sampled/wt, g! i 01, 26. 51. 76. ' 02. 27. 52. 03. 28. 53. 78. , 04. 29. 54. 79. 05. 30, 55. 80. 06. 31, 56. 81. 07. 32. 57. 82. 08. 33. 58. 83. 09. 59. 10. 35. 85. 11, 36. 61. 86. 12 37. 62. 87. 13. 38. 63. 88. 14. 39. 89 15. 40. 65, 90. 16. 41. 91. 17, 42. 67. 92. 18. 43. 93. ' 19. 69. 94. 20. 45. 70. 95. 21. 46. 71, 96. ; 22. 47. 72. 97. 23. 48. 73. 98. 24. 49. 74. 99 25, 50, 75. 100. 101. 102.

sexually,and the moreeggs they produce. Refer to Figure33 Ablation of Female Broodstock at Dian Desa for an objective step-by-stepmethod of determining Hatchery broodstock feeding rates. Seven teen percent of thetotal body weight per day is a good initial percentage;make adjust- Ablation or unilateral eye content removal stimulates mentsfrom there.Maturation animals are fed a highprotein ovarianor eggproduction.After removingthe contentsof d ietconsisting of squid cumi-cumi! Photo31!, small shrimp one eye, egg development can be seen as soon as three Photo32!, and small fish and dams when available. Feeding days,and thefirst spawnshould be observedwithin six to amountsare divided equallyand fed at 0730,1200,1600and sevendays of ablation. Ablation should take placeonly 2100hours. Food must be thawed completely if frozen! in after the animals have recovered from the stress of transfer seawater, cut into small pieces, weighed and distributed and appearto be healthy no red appendagesor red gills, evenly around the tank so all shrimp can eat generous show active feeding habits, swimming, etc., Photo 36!, portions with little movement required on their part Photo Ablation shouldbe conductedduring the morning hours 34!. Excess food is removed from the tank at 0700 and 1530 when the water temperature is slightly cooler. It is not hours by siphoning Photo 35!. necessary to chill the water, but ablation in 29 to 30 C water will surely causesome stressand mortality. The Design,Operation and Training ... 69

Figure 33. Feed Rate Determination

1. Takeweight lengthand convert!samples of at least ten 0! animals chosenrandomly. , 2. Find the mean average!weight of thoseanimals. : 3. Multiply the mean weight by the number of animals in thetank. This gives the Total BodyWeight TBW!. 4. Determinewhat percentageof thatTBW is to be fcd to the animals, [Example: 3.2 percent; 4.50percent; 5 percent;etc. start at 3,75percent/day!] 5. Multiply the TBW by the chosenpercentage. This gj ves the Dry Weight Basis D WB}. 6. Determinewhat percentageof thedifferent types of foodwill be fed during a particularperiod Example: 20 percentof DWB will be whole shrimp or 30percent Photo38. Author demonstratingproper inspection and han- of DWB will be marine worms!. Figure what percent- dling techniquesfor broodslock. age of the DWB a given food represents. 7. In the case of all foods containing water worms, contentsof one eye are removedby holding the female squid, shrimp, etc.!,multiply the figure found in Step gentlybut firmly underwater with onehand and pinching 6 by the Wet Weight Factor WWF!.In all the foods the eye stalk and rolling the eye contents outward away we plan to useat this time,the WWF equals5, In the from thebody! betweenthe index finger and thumb of the caseof dry combination feeds,do not multiply. other hand. The contents of the eye are expelled into the ,' 8. Determine how many feedings the above will be water column and the female is gently released. Signs of divided into and divide accordingly Example: 50 shock are not abnormal. The hemolymph should clot percent of the MW in the morning and 50 percent in immediately and form a light blue sphere where the the evening!. contents of the eye were previously located. Do not rc- 9. Determine the above 8! for all feeds for the morning, ablate females. Do not ablate on both sides this will noon and evening feedings. eventually kill the animaI!. The unilateral ablation process facilitates sex recognition for the remainder of time the Example: broodstock are being held in a production mode. ~ The meanweight of ten 0! animals is 50 grams. Shrimp should be ablated only when hard-shelled ~ The number of animals in the tank is 30. Photo 37!, never when in post-molt newly molted or soft- ~ Therefore, 50g x 30 animals = 1500 TBW. shelled! or premolt ready-to-molt, with white spots on ~ The percentage of the TBW to be fed is 3.25percent. shell! stages.The procedure is as follows: ~ Therefore, 1500g x 3.25 percent = 48.75gDWB. 1. Hold the shrimp gently but firmIy with one hand, ~ 30 percent of the DWB will be rrerine worms. preferably in the water Photo 38!. Only females arc ~ Therefore, 30 percent of 48.75g = 14,63g. abIated. ~ The wet weight factor of MW is 5. 2. Ablation is performed on either the left or right eye. ~ Therefore, 14.63gx 5WWF = 73.15gor 73g of MW However,an alreadyinfected or otherwisedamaged will be fed to that tank/day, eye should be ablated to leave one unablated, healthy ~ 50percent of the MW will be fed in the morningand eye. 50 percent will be fed in the evening. 3. Ablation may be performed in any of the following ~ Therefore, 50 percent of 73g = 36.5g MW morning ways: feeding and 36.5g MW For the evening feeding. a. Pinching Grasp the eyestalk, just behind the Theeasiest way to feedis to startwith 10percent body eyeball,between the thumb and index finger.Squeeze weight pcr day. If there is food remaining before the next hard and roll the thumb and finger outward, thus feeding, then cut back slightly or if there is no food, then crushing the eyestalk and squeezing out the contents increasethe amount slightly. The idea is to feed them all of both it and the eye. The objective is to squeeze the they want, leaving just a lit tieta remove daily. Feed four contents outwards and not let it follow the cycstalk times a day,with the largestquantity being fed at night 5 back into the head region, An incision on the front of percent at 0900hours after siphoning out excessfrom the the eye with a sharpblade may be made to aid this night before,then 10percent at noon and 10percent in the process. Some people prefer this method, but it is afternoonat 1600hours, with optimum feedingtime at merely a matter of preference.We have found the night aftermated females are pulled out, feed55 percent incision to be unnecessary,and prefer to hold the of the daily ration!. Amounts eaten will vary daily. shrimp underwater and complete the process as fast as possible to minimize stress. Note: They will consumemore food if fed in smaller b. Ligation A piece of string is tied around the quantities, more times per day. Be very careful baseof the eyestalk,close to the carapace.The eye not to over-feed. Oceanic conditions are most should fall off in a few days Primavera, 1985!. important, if maturation is desired. c. Cautery The eyestalk is ablated either by electrocautery or with a silver nitrate bar. 70 ... Treece and Fox

4, To minimize stressand damageto the animal, the abla- tion should be performed as quickly as possibleand can be done under chilled water. Ablation in commcreial hatcheriesis usually done in the early morning when temperaturesare lowest Femalemortality due toabla- tion shouldbe very low, but somemortality shouldbe expected.A softnylon net is used Photo39!. Pinching is the preferred method of ablation. It can be done by one person and the wound will heal rapidly without requiring antibiotics. Ligation requires two per- sons, one to hold the shrimp and the other to tie the eyestalk.Cautery requires either a cauterizeror silver nitrate bar, neither being easily available.Egg develop- ment in some females should become visible three days a fterablation and the first spawn should occur approxi- matelyone week after ablation.The tank should be in full Photo39, A so/ nylon netis usedto capture broodfor ablationor just production three weeksafter ablation.If ablatedduring for inspection, the inter-molt stage, the females will mature and spawn immediately. However, if ablated during early premolt they will molt first, beforematuring. Someresearchers have been able to obtain egg devel- opment, mating and spawning without ablation, utilizing temperature and photoperiod manipulation, but no one has yet been able to base a long-lasting, profitable, highly productive commercialoperation on this approach,Re- searchers Chan et al.1988; Bradfield et al.1989; Rankin et al., 1989;Chan et al. in press; Rankin and Davis, 1990; and Rankinet al.1990! arenow a ttemphngto character- ize and isolate the hormones involved in maturation in hope of eliminating some of the side effects fewer fertile eggs and larvae per spawn with ablation and brood ani- mals "burning ou t" a fterthree months!. Until ablation can be eliminated successfully and replaced with a superior method, hatcheries must depend on it. to sustain produc- tion Treece and Yates, 1990!.

Tank Volume Determination ' In order to set a specific flow rate one must first Photo 40. Constant water level maintained in maturation tank by this dual standpipe water calculate the volume of the tank in which the flow is to be beirrgdrawn from fhe bottomof the tank at this applied,The formula for volume is asfollows: point, but by removing the outer standpipe Volume = ~3.142 d 2 x h water canbe "skimmed"from the uppersurface in casethere is excessfood floating!, 4 where d = diameter inside! of tank in centimeters and h = water depth in centimeters. The result should read in cubic centimeters cc!. I cc is about equal to 1 ml, 1000ml = 1 liter!. Example: Maturation tank Volume=~3.142 d xx h V=3~142 3962x74cm 4 4 d = 396cm V = 123,100 x 74 cm h = 74cm V = 9,109,441 cu. cm V = 9,109 liters

Maturation Unit

A maturation unit is considered to be one maturation tank that consists of the following: Circular, ferro-cement tank meters in diameter or 13fcct!,black interior, with water inflow, aeration and stocked with approximately five shrimp per square Photo4I. Effluentflow rateis checkeddaily at thematuration meter. tank to maintai n a constant, stable environment. Design, Operation and Training ... 71

2. Water level maintained at 74 cm or 29 inches depth givesa volumeof approximately9,000 liters. If visibil- ity becomespoor, then oceanicquality water is nat being maintained and steps must be taken ta keep water quality high Photo 40!. 3. 200to 250percent water exchangeper day is optimum Photo 41!. If exchangingthis volume of water be- comesa problem,lower the water level to 40 cm 6 inches! and continue to turn over water in the tank at 200 percent per day. This would be more desirable than maintaining the samewater level and decreasing the flaw rate, which would invite protozoan contarni- nations and cause rapid deterioration of oceanic con- ditions. P. monodon will mate in shallow water but temperature stability is better maintained with a greater volume of water, If flow is interrupted for Photo 42. Fresh molt on the bottom of maturation tank, some reason, then increase the flow rate so that the same overall turnover is achieved, An example might be a casewhere a fisherman's boat hits the intake pipe below the water level and puts a hole in the pipe, This means pumps must be shut off at law tide for ten hours each day until the pipe is repaired. The flow rate is increasedfrom 16 liters/ minute to 24 liters/minute for 14 hours a day, and temperature is monitored after the flow rate change. 4. Light over the maturation tank is dim and no direct sunlight is allowed to enter. The shrimp and food should be spread out evenly on the bottom if light, water flow and aerationare all adjustedproperly. If not, shrimp will be clustered together in the darker areas af the tank, such as around the sides, etc. 5. Constancy is of primary importance in the maturation unit. Temperature optimum is 28 C+ 1 C over a 24- Photo 43. Salinity readingsare taken routinely oncea week hour period. If temperature fluctuates more than this, unlessduring therainy season then refractomefer readings such as 26 C to 31 C during a 24-haur period, maybe required more often!. production may be affected.Salinity should also be stabIc for best results.The range for optimum matura- count and body weight in tank and make adjustments tion of P. manodon is 28 to 33 ppt but results are still in feeding. Record all data on maturation tank log obtainable with an even wider range 4 to 36 ppt! as sheet, Figure 35!. long as the change is gradual and not sudden. 2. 0700 hours. Check excess feed in tanks and record Maturation/Hatching Schedule of Activities excessfood or no excess;vaccum or siphon thebottom of the tank completely; siphon the first part through a This daily procedure list is important ta maintain a screen to check for eggs fram missed spawns or nau- maturation production room for open-thelycum or closed- plii if a spawn was missed the night before. Record a thelycum shrimp. It is absolutelyrequired if sustainable missedspawn and assigna batchnumber to it, so the maturation results are to be achieved Figure 34!. total spawns coming from ablated females and the Daily Procedure percentage of females spawning each evening can be This procedure should be followed everyday in the monitored. When this number begins to taper off or maturation facility. Any failure ta perform these duties egg-nauplii quality begins to deteriorate, it is time to cauld result in an interruption in the maturation process, initiate the production in a new maturation tank, with 1. 0600 hours. Lights on. Check maturation tanks for fresh animals from the wild. It will becomevery aeration,flows, etc. Checkspawning tanksfor flows, apparent when production is tapering off if these aeration, etc. Return females in spawning tanks to records are kept. proper maturation tanks and mark the female by 3. 0730 hours. Feed animals in maturation tanks. Start with clipping the tip of one of the uropods tail!. Checkall 17percent of total body weight per day, Feedfour times maturation tanks for molts Photo 42! and record. a day. If there is tao much excessfood after three hours Leave the malts in the tank until late that afternoon for or before the next feeding period, reduce the quantity the shrimp to consume.Check maturation tank for until there is a small amount af food remaining, but not mortalities and remove them, Measure or weigh the enough to causewater quality problems. dead shrimp and placethem in the freezerto be used 4, Takewater temperatures and record twicedaily ance later as Feed.Subtract the mortality from the total in the morning and once in the afternoon!. 72 ... Treece and For I Figure34. WeeklySchedule ofMaturation Activities

WEEK OF

DO: Initial when done Day / Month! 1. 0600 hours. Lights on. Check all tanks air, flow, etc.!. , 2 Return females to proper tanks. '3 Check to seeif spawn is fertile in S.T. Count eggs later,! 4. Removeany mortalities and note an 1ogsheet. 5. With a siphon,check maturation tanks for eggs. 6, Remove excess food. 7. Feed, check temperatures, check flow rates 730!, i 8. Count number of eggsS, T. and record on Egg-Nauplii Data Sheet. , '9. Assignbatch 0 ta eachnew fertile spawn. I 10. Count nauplii in S.T.'s, record. Fill out State of Health and transfer or discard. i 11, 200! Feed, check temperatures. i 12. 530! Vacuum tanks. Fastvacuum. once each day just before feeding!. i 13. 600! Feed, temps. 14, Setup S.T.'sfor eveningshift. i 15. Generalclean up empty garbage,etc.!. i 16. On Wednesday,clean tank sides,plumbing, etc. l 17, 800!Place late stage developed females in S.T.'s. Closed thelycum or brownshrimp: P. rrtonodort! If working with whiteshrimp apen- thelycum! then pull mated out 1900-2100. l 18. 830! Lightsout over maturation tanks. 19. 100! CheckS.T.'s to seeif any femaleshave spawned early and return them. ' 20. 100! Feed maturation units. 21. 330! Checkfemales in S.T.'s.If theyhave spawned eggs, return to proper tanks. 22. Lights off for the night.

5. Takesalinity ance a week unlessduring the rainy 100 ml x 8,000 because there are 8,000 100 mls in an season, when it should be taken more often! Photo 800-liter volume! = 848,000eggs or nauplii in the 43!. spawning tank. 6. If thereisa spawnin a spawningtank, stir the tankto Recordthis numberon the Egg-NaupliiData Sheet suspend the eggs just turning aeration up is not Figures 36 and 37! with the batch number and num- enough to get the eggs off the bottom>. A 2-inch PVC berof larval-rearingtanks to whichthey were trans- pipe works well to stir, taking care not to hit the ferred.Eggs will hatchin 14hours after the spawn at bottomwith thepaddle or pipe.Obtain an eggcount 28 C. bycounting the eggs from three 100ml beaker samples Recordhatching rate and d iscardany hatches that are and averaging.The beaker sample is partially poured below40 percent. For training,do a nauphistate-of- intoa petridish so it isnot necessary tocount too many health record Figure 38! until the observerbecomes eggsat one time. Counted eggsare returned to the familiarwith importantcharacteristics of nauplii spawningtank and allowed to hatch.The nauplii are healthy,strong swimming nauplii, no debrisattached, countedthe same way either that afternoon if stocking no deformities,etc,!, This objectivefarm canbe disre- in larval rearingis required,or the next morning gardedas long as these criteria are still usedto judge before transferring N5-N6 ta the rearing tanks. Ex- if spawnsshould be savedor discarded. ample of an egg or nauplii count: 109/100 ml, 90/100 7. Removenauplii from spawning-hatchingtanks by ml, 120/100ml. Averagecount is 106eggs or nauplii/ siphoningor by draininginto speciallydesigned nau- Design, Oprratian and Training ... 73

TRANSFERRED FROM AVERAGE WEIGHT AT STOCKING

START UP NOTES: 74 ... Treeeeand Fox

Figure 36. Actual Egg-Nauplii Data Log

Date Time Stage Presently in ¹ Source S.T. or I.M. 17/7 0700 Egg - MT6 MT6 18/7 0700 Eggs MT6 MT6 18/7 1100 Nl ST2 MT6 18/7 1300 Nl ST3 MT6 18/7 1300 Nl ST4 MT6 18/7 1300 Nl ST5 MT6 18/7 1300 Nl ST7 MT5 I 19/7 1300 Nl MT5 MT5 I 9/7 1300 Nl MT6 MT6 9 Discarded 20/7 0930 Eggs MT6 MT6 10 Discarded 20/7 1500 Nl ST3 MT6 872,000 ll 4,889,186 Nl to date 21/7 1200 Eggs ST1 12 not counted 21/'F 1200 ggs ST2 MT6 136,000 13 screen leak 21/7 1200 Eggs MT6 MT6 14 Discarded 2I /7 1200 Nl ST8 464,000 15 5,489,186 Nl to date 21/7 1200 Eggs MT5 MT5 16 Discarded 21/7 1200 Nl ST7 MT5 408,000 17 21/7 1200 Nl ST4 MT6 408,000 18 ~ 21/7 1200 Nl ST6 MT6 712+00 19 22/7 0900 Egg ST3 MT5 24,000 20 22/7 1030 E-Nl ST5 MT6 1,120+00 .16x106! 21 Big Marnoo Gave to BondoHatchery 22/7 1030 Eggs ST9 MT6 65,600 22 23/7 0730 Eggs MT6 MT6 23 Discarded 23/7 0730 Eggs ST9 MT5 896,000 840,000 24 23/7 0730 Eggs STI MT6 688,000 944,000 25 23/7 0730 Eggs ST7 MT6 88,000 512,000 26 17% hatch discarded ' 23/7 0730 Eggs ST5 MT6 760,000 808,000 27 24/7 1200 Eggs ST3 MT5 176,000 28 24/7 0730 Eggs MT6 MT6 29 Discarded 24/7 0730 Eggs MT5 MT5 30 Discarded 24/7 0730 Eggs MT3 MT3 31 Discarded 24/7 0730 Eggs ST8 MT6 792,000 648,000 32 24/7 0730 Eggs ST10 MT6 364,000 752,000 33 ' 25/7 0730 Eggs ST1 MT6 473,000 34 , '25/7 0730 Eggs ST4 MT6 896,000 35 6&7 . NOTE: 13,042,786nauplii counted in eight days of production! Design,Operation and Training ... 75

Figure 37. Maturation/Hatchery Egg-Nauplii Data Eog Comments

pliicatchers.Discard thosenaupliithatarenotneeded flow, Use goodjudgment in feeding and siphoning to in larval rearing or cannot be sold. The best stage to maintain good water quality. The second feeding is at move nauplii is at N5. Feed must be ready and avail- 1200hours, the third feeding at 1600hours if it does able to the larvae by the zoea stage or high mortality not interrupt mating period! and the fourth at 2100 will occur, hours, 8. Once a week, brush the sides and bottom of the 10. Clean spawning tanks daily whether there was a maturation tank to keep wall growth barnacles, etc.! spawn or not. Once a week, the spawning tanks to a minimum. should be cleaned with chlorine and allowed to air 9. Beforetwo or more of the maturation feeding periods dry. each day, siphon all excess food, debris and rnolts 11. Remove late-stage females from ~aturation tanks Photo 44! froTn the tank. Do not feed if there is no Photos 45 and 46! a t 1800hours for closed-thelycurn

Photo44. 1Vlolt s areremoved after they are no longer a goodfood source Photo45, Usingine~ensim underwaterlight attachedto usuallywithin 24 hoursand they have turned yelloro-gold!. PVC pole! to locatelate stagefemale shrimp. 76 ... Trees' and Foz

Figure 38.

Larval-State of Health

Score Ratings; ¹3. Excellent,Highest, Most. ¹2. Fair, Mid-Range, Average. ¹1. Poor, Lowest, Least. Design, Operationand Training ... 77

Photo46. Flashlight can be placed such that light beamsshow through Photo47. Recordsare keptfor the day on a formicaboard hung near eroskeletonand stageof developingfemale is determined. each tank.

Photo49. Late stage females are placed individually in a seriesof 800- liter spawningtanks, each equipped unth waterinflow and screen standpipe. shrimp such as P. rnonodon;between 1900 and 2100 hours if working with open-thelycum such as P. vannamei!and place in clean spawning tanks with gentleaeration and gentleflow-through no antibiot- icsor EDTAarenecessaryunless thereisnowaterflow for some reason. Spawning tank turnover should be around 100to 200percent to ensurethat temperatures are the same as in the maturation tank and to flush out placentaor after-birth when the femalespawns. Al- ways use fresh seawaterfor the spawning tank. Do not use excesswater or more than one-david water. 12. 1830 hours. Lights go out over maturation tanks. 13. 2100 hours. Last feeding. Check to see if any of the late-stagefemales have spawned m the spawning tanks. If so, return them to the maturation tanks. 14. 2330hours. Make sureall lights are out for the night in the maturation area.Check late-stagefemales in spawning tanks again. Return them to maturation tanksif they have spawned. Record Keeping Photo50, Counting eggsand nauplii out of a In addition to the previously mentioneddata sheets, 100ml beakersubsampletaken from the spawn- other important recordsto keepare thosethat pertain to ing tank note inflow and effluent screenwith individual maturation tanks: air collar!, ~ Maturation Tank Board Photo 47! ~ Egg-Nauplii Data Log Figures36, 37 and Photo48! 7S ... Treeceaiid Fox

It also is a goad idea to clip a small portion of the to a larval-rearing tank. There should not be many dead uropod fram ablatedfemales each time they spawn,Some onesand they should be from a spawn in which at least 40 females,even though ablated, will never spawn. If a percentof the eggshatched. If they do not haveall these fernalehas not spawnedafter threeweeks doesnot have characteristics, discard the batch and obtain a good one. a portionof theuropod missing!,she should be replaced. For training purposes,an objectivetest is provided Figure 38! to familiarize trainees with important charac- Spawning and Hatching teristics of nauplii. In the nauplii test, ten larvae are A spawning unit Figure 15! is consideredto be one sampledand rated excellent!, fair ! and poor ! for spawning tank that consist ofthe following; eachimportant characteristic. All of the scoresare added 1. Cylindrical, cementtanks with smoothinterior sur- up and averaged to give a 3, 2 or 1 final score. Nauplii face black in color! with slightly sloped bottom with batches with 2 pr 1 averagescores are discarded if nauplii central standpipe. The tank measurementsare 110cm arein abundantsupply and the luxury of selectingspawns insided iameter! by approximatelythe samedepth, A can be afforded. Inferior nauplii could also be sold rather ccnfral standpipewith an effluentscreen maintains a than discarded. water level at 85 cm depth or a tank volume of 802 Disinfection Procedures liters Photo 49!. 2. Thespawning tank is equippedwith a constantflow- No disinfection treatmentshavebeen found necessary through setat 1 liter/minute or 180percent exchange at the YDDhatchery. A24-hour flowismainfained to flus per day. This fiow keepstemperatures between the out unwanted protozoans, ciliates, Zoothamnium and maturation and spawning tanks the same and pre- Epistytis. Frequent molting also assisted the animals in vents any temperatureshock fa the stage-3female shedding off the black chitinaverous bacteria that is com- when she is transferred. !t also maintains oceanic- mon worldwide on shrimp exaskelefons.No bacteria quality water, which helps spawning and hatching, infection was noted after ablation and mortality was very and flushs out the placentaor after-birth associated low, so no prophylactic treatment was deemed necessary. with the act of spawning. A gentle aeration helps If such problems are encounted during a different season maintain a high dissolved-oxygen rate and helps keep of the year, they should be diagnosed using Johnson's eggsfrom layering on the bottom of the spawning Handbook of Shrimp Diseases989!, This can generally tank.Excellent results have been obtained in compari- be done by closeobservation of the infected area of the son with the stagnated condition with only aeration shrimp gills, etc.! using a dissecting or a compound and EDTA-antibiotics added. No chemical additions microscope. The best treatment and preventive is to main- are necessarywifh a constant flow system, thus sav- tain good oceanic water quality in the maturation and ing time and money, spawning tanks. In the event that problems do occur, a Spawning generally occurs between 2100 hours and regime of antibiotic and formalin treatments sometimes 0200 hours, so females in the spawning tank can be helps stop high br'oodstock mortality. Use one to fwa checked and rcfurned to a maturation tank either antibiotic treatments Furanaceat 2.5 ppm! and ane to immediately after spawning or the first thing in the three formalin treatments 0 ppm!. An antibiotic or for- morning. Eggs will hatch 12 to 14 hours after they are malin is added to the tank, water exchangeis stopped, and spawned at 28 C. Egg counts Pha fo 50! are made in aeration is increased. After one hour, water exchange is each spawning tank during the morning following resumedat the normalrate. These treatments should only the steps outlined in the Schedule of Activities sec- beusedasa lastresortbecause the formalintreatmentmay tion!. Visual observation using a binocular dissecting stress the animals and have a negative effect on produc- scope! of fhe eggsshould indicate at least a 40percent tion. or greater fertile egg rate or, if preferred, a comparison Aprelirninary study on disinfection methods ofpenaeid of the nu~ber of total eggs spawned count in morn- shrimp hatcheries contaminated with Bacutoviruspenaei ing! to the number of nauplii hatched should also was conducted by Akamine and Moares and reported at indicate at least a 40 percent hatch rate or spawn is the 1989 W.A,S, meeting, They reported that sodium discarded. Whichever method is preferred, one should hydroxide proved to be the mosf efficient of the common be implemented to maintain quality control. materials investigated to disinfect shrimp hatcheries. A method of spraying this chemical was reported with good Transferring Nauplii results. Routine examination procedures are discussedat Nauplii may be transferred to a larval-rearing tank the end of Chapter 6 andvarious treatment methods are in eifheranthefirstafternoon NI-N2! ifneeded,orleftinthe Chapter 7. spawning tank overnight and transferred as N5-N6 the fallowing day, whichever is more convenient for hatchery Selected Maturation References operations coordinated between maturation and larval rearingneeds!. Nauplii may eitherbe siphanedout of the 1. Adiyodi, R.G.and T. Subrarnoniam,1983.Arthrapoda- spawningtank or collectedin a nauplii harvesteras the crus tacea.In: K.G. and R.G. Adiyadi Editors!, Repro- ductive Biology of Invertebrates. Vol. I: Oogenesis, tank is drained. Nauplii shouldbe active,photopositive swim to the Ovipasition, and Oosarption. JohnWiley & Sons light!, freeaf debrisor bacteria,and setaear legsshould be LtdChichester,N.Y., pp. 443-495. well developedwith no deformities,before moving them 2. Adiyadi, K.G. and R.G. Adiyodi, 1970, Endocrine Design, Operationand Training ... 79

control of reproduction in decapodcrustacea. Biol. Thebiosynthesis of a andb ecdysone:In vivo,in vitro, Rev., 45:121-165. and ceII free approaches. In: P.J. Gallare and Boer, Akamine, Y. and J.L. Moores, 1989. A preliminary H.H. Eds.!, Comparative Endocrinology. Eisevier/ study on disinfection methods of penaeid shrimp North-Holland Biomedical Press, Amsterdam, pp. hatcheries contaminated by Bacuioviruspenaei. Ab- 491-494. stract. WAS 1989.p.55. 18. Bomirski, A., M. Arendarczyk, E. Kawinska and L.H Alikunhi, K.H., A. Poernorno, S. Adisufresno, M. Kleinholz, 1981. Partial characterization of crustacean Budiono and S. Busman, 1975.Preliminary observa- gonad-inhibiting hormone. Int. J. Invert. Reprod., tions on induction of maturity and spawning in Penaeus 3:213-219. rnonodon Fabricius! and Penaeusrnerquiensis de Man! 19. Bradfield,J.Y.,R.L. Berlin,S.M. Rankin and I..L. Keeley, byeyestalk extirpation. Bull. Shrimp Cult. Res.Cent., 1989. Cloned cDNA and antibody for an ovarian Jepara,1 l!:1-11. corticalgranulepolypeptideof theshrimp P. vannanrei. Andrew, R.D. and A.S,M. Saleuddin, 1979. Two-di- Biol. Bull. 177:344-349. mensional electrophoresisof neurosecretory polypep- 20. Bray, W.A., Jr., A.L. Lawrence 1991.Reproduction of tides in crustacean eyestalk. J.Comp. Physiol. B, Penaeusspecies in captivity, Pages93-170 in A. Fast 134!:303-314. and L.J. Lester Eds.!, Culture of Marine Shrimp: Anilkumar, G. and K,G, Adiyodi, 1980. Ovarian Principles and Practices, Elsevier Scientific Publish- growth, induced by eyestalk ablation during the ing Company, Amsterdam, The Netherlands. prebreeding season is not normal in the crab 21. Brown, A., J.P.,McVey, B.M. Scott, T.D. Williams B.S Paratelphusahydrodromous Herbst!. Int. J. Invertebr. Middleditch and A.L. Lawrence, 1980. The matura- Reprod., 2:95-105. tion and spawning of P. stylirostris under controlled Aquacop, 1984. Observations surla naturation et la laboratory conditions, WMS 11:488499, reproductionen captivite des crevettesPeneides en 22. Brown, A., Jr, J. McVey, B.S, Middleditch, and A,L milieu tropical, In: Aquaculture en Milieu Tropical, Lawrence, 1979.The ~aturation of white shrimp P. IFREMER, Brest, p. 132-152. setij'erus!in captivity. WMS 10:435-444. Aquacop, 1979.Penaeid reared broodstock: closing 23, Brown, A. and D. Patlan,1974. Color changesin the the cycle of P. monodon,P. stylirostris and P. vannarnei. ovaries of penaeid shrimp as a determinant of their Proc. World Maricul. Soc., 10:445-452. maturity. Marine Fisheries Review, Vol. 36:7 p. 23- Aquacop,1977a.Reproduction in captivity and growth 26. of P. monodon Fabricius! in Polynesia. Proc. World 24. Caillouet,C.W.,1972.0varianmaturationinducedby Maricul. Soc., 8:927-945. eyestalk ablation in pink shrimp, Penaeusduorarum 10. Aqua cop, 1977b.Observations on the maturation and Burkenroad!. Proc. World Maricul. Soc., 3:205-225. reproduction of penaeid shrimp in captivity in a tropi- 25. Chamberlain, G.W., M.G. Haby and R.J.Miget, 1985 cal medium. Aquaculture Workshop, ICES,May 1977. Texas Shrimp Farming Manual. Texas Agricultural Brest, France. Work done in Taravao, Tahiti; CNEXO- Extension Service publication of invited papers pre- COP, P.O.B. 7004!. sented at the TexasShrimp Farming Workshop on19- 1 l, Aquacop, 1975.Maturation and spawning in captiv- 20 Nov. 1985,Corpus Christi, Texas. ity of penaeid shrimp: Penaeustnerguiensis de Man!, 26. Chamberlain, G.W. and N.F. Gervais,1984. Compari- P.japonicus Bate!, P. aztecus Ives!, Metapenaeusensis son of unilateral eyestalk ablation with environmen- de Haan! and P. snnisulcatus de Haan!. Proc, World tal control for ovarian maturation of P. stylirostris. Maricul. Soc., 6:123-132. WlVIS. 15:29-30. 12. Arnstein, D.R. and T.W. Beard, 1975. Induced matura- 27. Chamberlain, G.W.and A.L. Lawrence, 1981. Matura- tion of prawn Penaeusorientalis Kishinouyi! in the tion, reproduction, and growth of Penaeusvannarnei laboratory by means of eyestalk removal. Aqrracul- and P. stylirostris fed natural diets, J. World Maricul. ture, 5:411-412. Soc. 12!:209-224. 13. Beard,T.W. and J.F. Wickins,1980. Breeding of Penaeus 28. Chang,E S.and J.D. O' Connor, 1978. In vitro secretio~ monodon Fabricius! in laboratory recirculationsys- and hydroxylation of b ecdysone as a function of the terns. Aquaculture, 20:79-89. crustaceanmolt cycle. Gen. Comp. Endocrin., 36:151- 14. Bellon-Humbert, C.,F.Van Herp, G,E.C.M,Strolenberg 160. and J.M. Denuce, 198l. Histlogical and physiological 29. Chan,S, S.M, Rankin and L.L. Keeley,in press,Effects aspectsof the medulla externa X organ, a neuro secre- of 20-hydroxyecdysone injection and eyestalk abla- tory cell group in the eyestalk of Palaernonserratus tion on the molting cycle of the shrimp, P. vannamei. Crustacea, Decapoda!. Natantia. Biol. Bull. Woods Comp, Biochem. Physiol. Hole!, 160:11-30. 30. Chan, S., S.M. Rankin and L.L. Keeley, 1988.Charac- 15. Bielsa, L.M,, W,H. Murdich and R.F. Labisky, 1983 terization of the molt stages in P, vannamei: Speciesprofiles: life histories and environmental re- setogenenesisand hemolymph levels of total protein, quirements of pink shrimp. U.S. Fish 8c Wildl. Ser. ecdysteroids, and glucose. Biol. Bull. 175:185-192, 82/11.17. 31. Cordover R.D. and R.W. Brick, 1972. Guide to the 16. Bliss,D,E., 1966. Relation between reproduction and identification of commerciallyimportant shrimpsof growth in decapod crustaceans.Am. Zool6:231-233. Southeast Asian inshore waters. Unpublished report 17. Bollenbacher, W,E., S.I.. Smith and L.I. Gilbert, 1978 prepared for the Meekong Committee/Giant Prawn 80 ... Treeceand Fox

Culture Training Program, University of Hawaii, 50. Kamemoto, F. I., K.N. Kato, and L.E. Tucker, 1966 Honolulu, Hawaii. Neurosecretion and salt and water balance in the 32. Cummings,W.C., 1961. Maturation and spawnmgof Annelida and Crustacea. Am. Zoologist, 6:213-219. the pink shrimp,P. d uorarum.Trans. Amer. Fish. Soc. 51. King, J.E,,1948.A studyof the reproductiveorgans of 90:462-468. the common marine shrimp, Penaeus setiferus 33. Dali, W., 1957.A revision of the Australian speciesof Linnaeus!. Biol. Bull, Wood's Hole, 94!:244-262. Pcnaeinae,Australian Journal of Marine and Fresh- 52, Kleinholz,L.H.,1978, Crustacean neurosecretory hor- water Resources. Volume 8, Number 2, Pages 136- rnonesand their specificity.In: P.J.Gallard and H.H. 231. Boer Eds!, ComparativeEndocrinology, Elsevier/ 34. Dunham,P.J., 1978. Sex pheromone in crustacea.Biol North-Holland Biomedical Press, Amsterdam, pp. Rev. 53:555-583, 397400. 35. Dechan, C. and C. Chen, 1975,Bait worm culture of 53. Klima, E.F., 1978. An overview of aquaculture re- Aristicus crit. Simpson!.Sea grant Report¹16. search in the Galveston Laboratory. WMS 9, 1978. Florida Sea Grant College, University of Florida, NMFS Contrib. ¹78-156. Gainesville, Florida 32611,.USA. 54. Kow, T A., 1968.Unit stocks of shrimp and prawns in 36. De Saint-Brisson,S.C.,1985. The matingbehavior of P IPFCregion and unit fisheriesexploiting them. FAO paulensis,.Crustaceana50:108-110. Fisheries Reports, Volume 57, Number 2, Pages207- 37. Dore,I. and C. Frimodt, 1987.An illustratedguide to 218. shrimp of the world. 0 Sprey Books, Huntington, 55, Kulakovskii, E.E. and Y.A. Baturin, 1979. The sinus N,Y, 229 pages. gland of the shrimp Eualusgainrardi and its state 38, Duronslet, M., A.I. Yudin, RS. Wheller and W.H. related to environmental salinity variations, Clark, Jr., 1975. Light and fine structural studies of Tsitologiya, 21:1200-1203. natural andartificially inducedegg growth o fpenaeid 56. Laubier-Bonichon, A,, 1978. Ecophsiologie de la re- shrimp. Proc. World Maricul. Soc., 6:105-122. productionchez la crevette P j aponicus Trois! annees 39. Emmerson, W.D., 1980.Induced maturation of pra wn d'experienceen milieu controle'.Contrib. ¹564. P. indicus. Mar. Ecol. Prog. Ser., 2:121-131. 57 Laubier-Bonichon, A and L. Laubier, 1976.Reproduc- 40. Farfante,P., 1988.Illus. key to penaeidshrimps of tioncontrolwith the shrimp PjaponicusF.A.O. Tech. commerce in the Americas. NOAA Tech. Report Conf. Aqua. Kyoto, JapanFIR: AQ/Conf./76/E.38. 6 NMFS, 64, 32 pages, pages. 41. Farfante, P., 1969. Western Atlantic shrimps of the 58. Lawrence, A,L,, Y. Akamine, B.S. Middleditch, G genusPenaeus. U.S. Fish & Wildlife Serv,Fish Bull. Chamberlain and D. Hutchins, 1980. Maturation and 67:461-591. reproduciton of P. setiferusin captivity. WMS 11:481- 42. Faure, Y., C. Bellan-Humbert, and H. Charniaux- 487. Cotton, l981. Folliculogeneseet vitellogenese second- 59. Liao, I.C. and N.H. Chao, 1983.Hatchery and grow- aries chez la Crevette Palaernon serratus Pennant!: out: Penaeidprawns. In: McVey, JP. Ed.! CRC Hand- controle par les pedoncules oculaires et I'organe X de book of Mariculture. Vol. I. pp. 161-167. la medulla externa MEX!. C.R. Acad. Sc. Paris, 60. Lumare, F., 1981. Artificial reproduction of Penaeus 293!:461-466. japoriicus Bate! as a basis for the mass production of 43. Fingerman, M., 1970. Perspectives in crustacean en- eggsand larvae.J.World Mari cul. Soc,,12!:335-344. docrinology. Scientia, 105:1-23. 61. Lytle, J,S,and T,F, Lytle, 1989.Fatty acid composition 44. Fox, J.M., 1987 unpublished manuscript!, "Shrimp and variations in individual bloodworms. Gulf Coast culture in Indonesia." distributed by C.J.E.D.P. ResearchLaboratory, P.O. Box 7000, Ocean Springs, SEMARANG, JAVA. In the Indonesian Language. Mississippi 39564,USA. 45. Hameed, A.K. and S.N. Dwivedi, 1977. Acceleration 62, Magarelli, P.C.,1981.Nutritionalandbehavioralcom- of prawn growth by cauterizationof eye stalksand ponents of reproduction in the blue shrimp, P, using Actes indicus as supplementary feed. J. India stylirostris,reared under controlledenvironment con- Fish, Assoc. Bombay, 3-4 -2!:136-38, ditions. Ph.D.dissertation, University of Arizona, 46. Highnam, K.C., 1978.Comparative aspects of endo- 63. McVey, J,P., 1983, CRC Handbook of Mariculture, crine control of reproduction in invertebrates. In: P.J. Vol. I. Crustacean Aquaculture, J,P. McVey, Eds,!, Gaillard and H.H, Boer eds.!, Comparative Endocri- CRC Press Inc., Boca Raton, Florida, 442 pages. nology. Proceedings of the 8th Intl. Symp. on 64. Middleditch, B.S.,S.R. Missler, H.B. Hines, J.B.McVey, Comparative Endocrinology held in Amsterdam, The A. Brown, D,G. Ward, and A,L, Lawrence,1980. Meta- Netherlands, June 19-23, 1978. Elsevier/North-Hol- bolic profiles of penaeidshrimp: Dietary lipids and land Biomedical Press,Amsterdam, pp. 3-12. ovarian maturation, Journal of Chromatography 195: 47. Holthius, L.B. ed.!, 1980.Shrimps and prawnsof the 359-368. world anannotated catalogue of speciesof interestto 65. Motoh,H.,1981.Studiesonthefisheriesbiologyofthe fisheries. FAO Species Cat. FAO. Rome. giant tiger prawn, P. rnonodon,in the Philippines. 48. Hudinaga,M., 1942.Reproduction, development and Tech. Report No. 7, SEAFDEC,Philippines. 128pages. rearing of P.j aponicus,Jap. J. Zool., 10!:305-393. 66. Oceanic Institute, 1990. Newsline. Newsletter! March 49. Johnson,M,C. and J.R.Fielding, 1956. Propagation of 1990. Vol, 3 No. 1, Disease free shrimp population the whiteshrimp P.setiferusincaptivity. Tulane Stud. established in Hawaii. Zool. 4:175-190. Design, Operationand Training . t11

67. Otsu, T,, 1963.Bihormonal control of sexual cycle in electronmicroscope protein-tracer study. J. Morphol., the freshwater crab, Potarnondehaani. Embryologia, 163:13-26. 8:1-20. 84, Simon,C.M., 1982.Large scale,commercial applica- 68. Oyama,R., R. Deupree,M. Edralin, and J. Wyban, tion of penaeidshrimp maturation technology,Jnl. 1988,Operation and performanceof theOceanicInsti- W.M.S. 13:301-312. tute shrimp maturation facility. O.I. Tech. Report 85. Silver thorn, S.U., 1975. Hormonal involvement in ther- it 88-1, mal acclimation in the fiddlercrab Ucapugr'iator Bose! 69. Panouse, J,B., 1943. Influence de I'ablation du -I. Effectofeyestalkextractson wholeanimal respira- pedonculeoculaire sur la croissancede I'ovaire chez tion. Comp. Biochem, Physiol., 50A:281-283. la crevette Leander serratus, C.R. Acad. Sci., Paris, T. 86. Tirrnizi, N.M. and Q. Bashir, 1973. Shore and offshore 217:553-555. penaeidprawns of Northern Arabian Sea,Dept. of 70. Primavera, J.H., 1985. A review of maturation and Publ., Univ. of Karachi, Karachi, Pakistan. reproductionin closed-thelycurnpenaeids. Inr Proc. 87. Treece,C.D. and M.E. Yates,1990. Laboratory manual of 1st Int. Conf. on the culture of penaeid shrimp for the culture of penaeid shrimp larvae. ablation 984!, SEAFDEC Aqu. Dept, section!. Texas A&M Univ., Sca Grant College Pro- 71. Primavera, J.H., T. Young and C. de los Reyes, 1980 gram, TAMU-SG-88-202 R!. 95 pages, Survival, maturation,fecundity and hatchingrate of 88. Tseng, W,, 1987,Shrimp Mariculture, a Practical unablated and ablated P. indicus. Contrib. No. 59 Manual. Chien Cheng,Pub. Rep. of China. SEAFDEC Aquaculture Dept. 14 pp. 89. Van Herp, F.,C. Bellon-Humbert,J.T.M. Luub, and A 72. Primavera, J,H,, 1979, Notes on the courtship and Van Wormhoudt,1977. A histophysiological study of matingbehaviorin P.rnonodon Fabricius! Decapoda, the eyestalkof Palaemonserratus Pennant! with spe- Natantia!. Crustaceana, 37!:287-292. cial referenceto the impact of light and darkness. 73. Primavera,J.H.,1978,lnducedmaturationandspawn- Arch. Biol. Bruxelles!, 88:257-278. ing in five-month-old P. rnonodon Fabricius! by eye- 90. Wear, R.G. and A. Santiago, Jr., 1976. Induction of stalk ablation. Aquaculture, 13:355-359. maturity and spawning in Penaeusmonodon Fabri- 74. Quackenbush,L,S. and L.L. Keeley,1986. Vitellogen- cius!, 1978,by unila terai eyestalkablation Decapoda: esisin the shrimp, P. vannamei.American Zoologist Natantia!. Crustaceana, 31!:218-220. Abs tract. 91. Wyban, J.A., J.N, Sweeney and W.K. Richards, 1988. 75. Quackenbush, L,S,and W.F. Herrnkind, 1983.Partial Design of the Oceanic Institute shrimp maturation characterization of eyestalk hormones controlling molt facility. OI Tech. Report It88-1. andgonadal developmentin thespiny lobster Panulirus 92. Wyban, J.A.C.S.Lee, J,N, Sweeneyand W.R. Richards, argus.J. Crust. Biol3:34-44. 1987.Observations and development of a maturation 76. Racek, A.A., 1970. Indo-west pacific penaeid prawn systemfor P. vannarnei.WAS 18!:198-200. speciesof commercialimportance, Indo-Pacific Fish- 93. Yang, W.T.,1975. A manual for large-tank culture of eries Council Proceedings No. C70/Sym 3, Pages1- panaeid shrimp to the postlarva1s tages,University of 31. Miami Sea Grant Technical Bulletin, 31: 94 77. Rankin, S.M. and R.W. Davis, 1990. Tissue and Cell 94. Yano, I,, 1987, Maturation of kuruma prawn P. Journal!. Ultrastructure of ooctyes of the shrimp P. japo nicus!cultured in earthen ponds. Proc. U,S. Japan vannarneiwith emphasis on cortical granule forrna- Aqua, Panel Syrnp. on Reproduction, Maturation and tion. SeedProduction of Culture Species,Baton Rouge,LA. 78. Rankin, S.M,, J.Y. Bradfield and L.L. Keeley, 1990 Oct. 1983.U.S. Dept. of Comm. NOAA Tech.Report NOAA Tech. Report, UNJR!, Ovarian development NMFS 47:3-7. in the South American white shrimp, P. vannarnei. 95, Yano, I., R.A., Karma, R.N. Oyarna and J.A. Wyban, 79. Rankin, S.M., J.Y. Bradfield, and L.L. Keeley, 1989. 1988. Mating behavior in the penaeid shrimp P. Ovarian protein synthesis in the South American vannamei,. Marine Biology 97, 171-175. white shrimp, P. vannanrei,during the reproductive 96, Young, J.H., 1959.Morphology of the white shrimp P cycle,Inter, Jnl, of Invert. Reproductionand Devel- setiferus.U.S. Fish & Wildlife Serv., Fish Bull. 59:1- opment. 68. 80. Robertson, L., B. Bray, L. Trujillo and A,L. Lawrence, 97. Yu,HP. andT Y,Chan,1986,The illustrated penaeoid 1987a. Practical molt staging of P. setiferus and P prawns of Taiwan. Southern Materials Center, Inc. stylirostris, WAS 18!:180-185. Taipei, 183pages. 81, Robertson, L., W.A. Bray and A.L. Lawrence, 1987b Shipping of penaeid broodstock: water quality limita- tions and control during 24 hour shipments.WAS 18!:45-56. 82, Santiago,A.C., Jr., 1977.Successful spawning of cul- tured Penaeusrnonodon Fabricius! after eyestalk abla- tion. Aquaculture, 11!:185-196, 83. Schade, M.L, and R.R. Shivers, 1980.Structural modu- lation of the surface and cytoplasm of oocytes during vitellogenesisin the lobster,Hornarus americanus. An 82 ... Treeceand Fox Design, Operationand Trainirtg ... 83

Chapter3 Larva1 Rearing

Introduction and Background 1981; Simon, 1981; Kanazawa et al., 1982; Kungvankis, 1982; Harneed et al., 1982; Gabasa and Sunaz, 1983; Liao, Penaeidshrimp larviculture techniquesseem well es- 1984;and Tacon, 1986!. These are only a fraction of the tablishedbutwhen compared to age-oldagricultural tech- research papers that contributed to our present knowl- niques, they must be consideredan art rather than a edge of penaeid shrimp larviculture. The modifications science. Many of the present day techniques used in and refinements of the original techniques of larviculture shrimp larviculture are the direct result of studies dating have continued through the late 1980's with numerous back to the early 1930's.Credit should be given to the groups making further contributions in larviculturc c,g,, Japaneseas well as countless other groups such as the Wilkenfeld et al,, 1983;Sorgeloos et al., 1983;Kuban et al., "Galvestongroup" for establishingthe techniquesfor the 1985;Al-Ha ji etal., 1985;Ro thlisbcrg etal,, 1985;Prima vera, successfulproduction of postlarvalshrimp. These contri- 1985; Aranyakanada, 1985; Taki et al., 1985; Trcccc and butionshavebeen ixnportant to thedevelopment of shrixnp Fox, 1987;Biedenbach et al., 1987and 1989a,1989b; Tseng, rnaricultureand haveenabled the traditional shrimpfish- 1987;Chamberlain, 1988; Lc Moullac et al., 1987;Leger et eries to advance from catch fisheries to farm fisheries now al,, 1987; Yates et al., 1987; Liao, 1988; Trccce and Yates, producingat least26 percentof the shrimp consumedon 1988; and Chen et al., 1989!, the world market!. Cold-tolerant speciesof penaeid shrimp were the topic The origins of penaeidlarviculture canbe tracedback of a recent Asian-U.S, workshop, The proceedings, "The to Japanwhen Dr. MotosakuFujinaga thcn M, Hudinaga!, Culture of Cold- Tolerant Shrimp: Proceedingsof an Asian- now recognizedas the "Fatherof Shrimp Culture," suc- U.S. Workshop on Shrimp Culture" K. Main and W. cessfully spawned and at tempted to rear Pexxaeusjaporticus Fulks,editors, April 1990!,are ava ilablc through the Oce- larvae in the labora tory Hudinaga, 1935!.Successful rear- anic Institute. Larval-rearing techniques for P. chinensis, P. ing on a large scale was first achieved in indoor tanks, pexticillatus,and P.japorticus are outlined in this book. using cultures of the diatom Sketetonemacostatuxrt as food There are many methods ot culturing pcnacid larvae Forthe zoealstages and Artetttianauplii for the rnysisand successfully and the beginning hatchery rnanagcr should postlarval stages Hudinaga, 1942!. not be intimidated by the numerous modifications and There have been xnany successful modifications publicationsin this area.Penaeid larvae are truly easyto through the years, but the samebasic techniques are still culture if one is aware of the important paraxnetcrs.This in practice today. One of those successful modifications chapter describes one method of culturing P, monodon came as a result of the National Marine Fisheries Service larvae that worked well at the Yayasan Dian DesaHatch- Laboratory in Galveston, Texas,U.S.A. Ewald, 1965;Cook ery, Please refer to the drawings and description of the and Murphy, 1966,1969 and 1971;Cook, 1969; Mock and YDD facility in Chapter 1. Murphy,1970;Salser and Mock,1974;Fontaine and Revera, 1980; Heinen, 1976; Mock et al., 1980; Fontaine et aL, 1981 Daily Larval and Postlarval Rearing Schedule and McVey, 1983! all resulting in what is still called "the YDD! Galveston Laboratory Technique." The major difference 0700-0800 Checkaeration in tanks;checkdrains for leaks in the early technique and this modified method was that the larval feed algae! was prepared outside the rearing in standpipe, etc.; take temperature in tanks and record on data sheets; take samples of container and was fed as needed. Refer to Sinderxnan three larvae from each tank and stage them 987! for a more detailed literature review. discard them later and record stage on lar- At the same tixne these modifications were being de- scribed in the United States,others were making sixnilar val-rearingtank log!; recordcoxnmcnts about health of larvae full guts, no debris, some contributions Hudinaga and Kittaka, 1967;Maeda, 1968; debris, healthy, poor!; observe color and be- Liao et al., 1969; Skokita, 1970; Liao and Huang, 1972; havior, appearance, presence of molts, and Shigueno, 1972,1975; Imamura and Sugita, 1972;Kurcha look at the shrimp under 4x then 10x power and Nakenishi, 1972; Tabb et al., 1972; V illaluz et al,, 1972; compound scopeto look for prcscncc of fungi, San Feliu et al., 1973; Pinto and Edward, 1974;Fielder et Zoothatttxxium,Lagextidium or other unwanted al,,1975;Hirata et al., 1975;Yang, 1975; Gopalakrishnan, 1976; Aquacop,1977; Liao, 1977;Tang, 1977;Ting et al., organisms on the exoskclcton. Take samples from tanks and run algal cell counts on each, 1977; Millamena and Aujero, 1978; Paton, 1978; Adjustccllcount tominimumof t00,000cclls/ Sudhakarao,1978; Yellow Sea Fisheries Research Inst., ml Chaetoceros,and, at the zocal 2 substagc, 1978; Jones et al,1979; Motoh, 1979; Exnmerson, 1980; beginmaintaining 20,000 cclls/ml Tetraselmis. Emrncrson and Andrews l981; Gabasa, 1981; SEAFDEC, 84 ... Treece artd Fox

Figure 39

Naupla Score: Larval Rearing Tank Data Log Batch Number: LRT Number, ¹ PLs Harvested LRT Volume: -'rDSurvival N-PL ¹ Naupla Stocked val P'1 -PL P' Pop Estimate

Do not allow cell counts to fall below 40,000 1700 End of forrnal work day unless additional cells/ml at least until the second mysis sub- work is required in harvesting, transferring stage. Take Artemia counts in larval rearing naupIii, cleaning tanks, refilling etc.!. tanks LRTs!, but do not feed Artemia until 2200 FeedPLs in raceways this canbed oneby one 1600 hours. Record all data on the LRT data person and can be done on a rotating basis log sheet Figure 39!. since it is after the formal work day is over!. The procedure is similar for postlarval rearing. Take samplesof shrimp from eachtank and look at them under a Larval-RearingTank Preparation Before Use 10xcompound scope, Look for presenceof disease,fungi, After harvesting,the larval-rearingtank Figure 19!is etc, Look for full or empty guts. Take Arferrtia counts if scrubbed thoroughly and then refilled with seawater. feedingArtemia and recordon the postlarvalrearing data Add chlorineto therearing tank tobring the concentration sheet.Siphon debris from the bottomof thc racewaytanks to 10ppm 0 gramsof chlorinefor the 4,000-literrearing attd then feed PLs in racewaysaccording to the schedulein tank!. Airstones, tubing and standpipe with screen are Table I. Checkflow ratesand resetif necessaryto 100percent also scrubbed and placed in the chlorine solution to kill exchange.Record water temperature of tank, bacteria and other unwanted organisms.The chlorine 0900 Breakfast for the stafo. solution is allowed to sit in the tank overnight or at least 0930 Check raceway effluent or siphon effluent for several hours if there is a rush to get the tank started again. dead PLs/escaping PLs. The tank is then drained and thoroughly rinsed with 1000 Harvest PLs or move PLs from LRT to PL filtered seawater and allowed to air dry before rcfilling raceways. Feed PLs in raceways before staff and restocking. lunch break. The seawater used to refill the larvalrearing tank is 1200-1400 Lunch and rest for the staff!. only asgood as the level of treatment filtration/disinfec- 1400 Harvest Arterrtia and restart new Artemr'a for tion! applied to it, It must be filtered down to one micron the next day. for bestresults. Neglect in this areacan prove critical to a 1600 Take temperatures, check aeration and make batch'ssurvival, Protozoansand other unwantedorgan- adjustments if necessary.Feed Arterrtia to all isms that can cornpeie with the larvae tor air, space and rearing tanks see feeding schedule for food will take over the tank if filtration is not properly amoun ts to feed each stage!.Give pellet feeds undertaken or there are containinants of this nature in the to raceways scheduled for this type of feed algae culture fed to the tank. This final filtration in the seeTable 1 for percentageof feed and corre- Dian Desa Hatchery was accomplished by using a car- sponding age!. tridge-typefilter with a onemicron filter element. DeSign, OperatiOnand Training ... Ss

ostlarval Feedi

FEEDING TREATMENTS Tetra. Art. Pellet Wa ter Treflan Chlor."' cells/rnl! ¹/ml! % % ml/T! m 200

20 30 20,000 030 0 5 LRT 3.2 Z2 100,000 20,000 40 6 LRT 3.5 Z3 ]00,000 20,000 40 7 LRT 3.8 Z3-Ml 50,000 20,000 1 50 40 8 LRT 3.8 Ml 50,000 20,000 1 50 40 9 LRT 3.8 M2 Trace 20,000 3 50 50 10 LRT 3.8 M3 Trace 20,000 6 50 50 ]] LRT 3.8 M3-PL 20,000 6 50 50 l2 LRT 3.8 PLl 20,000 6 50 50 13 LRT 3.8 PL2 Tr'ace 6 50 50 14 LRT 3.8 PL3 Trace 6 50 50 15 LRT 3.8 PL4 6 Flush 15 RWY 7-12 PL4 6 100 50 16 RWY 7-12 PL5 Trace 6 100 50 17 RWY 7-12 PL6 Trace 5 100% 100 50 ' 18 RWY 7-12 PL7 Trace 4 200% 100 50 ' 19 RWY 7-12 PL8 Trace 3 200% ]00 50 ~ 20 RWY 7-12 PL9 TI'ace 2 200% ] 00 50 ' 21 RWY 7-] 2 PL10 1 200% 100 50 ' 22 RWY 7-] 2 PL11 0 200% 100 50 ' 23 RWY 7-12 PL]2 0 200% 100 50 ~ 24 RWY 7-12 PL]3 0 200% 100 50 " 25 RWY 7-12 PL14 0 200% 100 50 ' 26 RWY 7-12 PL]5 0 200% 100 50 " 27 RWY 7-12 PL16 0 200% 100 50 " 28 RWY 7-12 PL17 0 200% 100 50 ~ 29 RWY 7-] 2 PL18 0 200% 100 50 ~ Use if needed Also, use Malachite Green ppm! if Zoothanrniumis noted "Chloramphenicol anhbiotic! ST spawning tank

The tank is then half filled. A solid, 2-inch PVC lt would be a good idea to let the tank sit for several standpipe no screen necessary until water exchange is hours with continuous aeration in case there are chlorine necessary! is placed in the center of the tank. The drains byproducts present, which should be neutralized by the arechecked for leaks. Teflon tape maybe required around aeration. This will also give you a chanceto seeif the tank the standpipe to stop leaks. Airstones are selectedso that will hold water beforeyou put larvae in it. The tank is they will be heavy enough to remain on the very bottom maintained on a batch feed basis until the late zoeal stage of the tank with air turned on, but the stone must put out or until a problem arises high pH ammonia! that re- a very fine bubble to flow evenly and smoothly to the quiresswater exchange.Initially, the tank is only half filled surfaceand to the edgeof the tank beforebursting, Large with filtered seawaterduring the naupliar stocking phase. bubblesshould be avoidedas they do not help muchwith As feed is added, the volumeis increased Photo 51!. By the thetransfer of oxygento the waterin therearing tank. It is time thelarvae are at thelate zoeal substage or earlymysis, important to havethe airstoneon the bottom so a "dead" the rearing tank is at its maxirnurn holding and carrying area does not form with HzS or debris which can be capacity. Less algae is required using this method. Main- deadly to larvae or may provide a mediumfor bacterial taining higher concentrations of algae during the zoeal growth!. stagesis critical to the successof the batch, A volume 86 ... Treeceand For

EGG PLEOPOOD FOLLrDErELDPED PL

'DGl

LEQPDDS rrSEDFrrrrrED

rrppprroDDE

p, mpnpdprr P. irtdfDDS p.japp Orcus

Photo 51. Larval rearing tank approximately one-halffull micronfilter cartridgeon inflow; central, screenstandpipe with air collar and 100,000cells/ml algae!. Figure40 ShrimpLarval Stages tatterMatoh. 1979!

recommendation can be found in Table 1 Nauplii, 1,000 liters volume; Z3-Ml, 3,800 liters, etc.!. The goal at the Dian DesaHatchery was to maintain a Chaetoceros feed level for Penaeus rnonodon larvae sozne- where around 100,000 cells/znl and to not let the cell concentrationsfall below 40,000cells/ml, especiallydur- ing thezoeal substages Table 1!.Maintaining these higher celldensities in theznysis substages is not necessary since Artentianauplii are being fed and a 50 percentper day exchangeof water is taking place.Approximately 20,000 cells/znl Tefraselrnisshould be maintainedin the rearing tank while feeding Artemia. The are many reasons for znaintainingalgae levels, even up to the PL stage the presenceof algae helps maintain aznmonialevels within Photo52. Seriesof larval rearingtanks; some covered with plastic acceptableranges; algae are still being consumedby the during emning hours to keeptemperature up. shrimpwhile znetamorphosingfrom algae-eatingto zoop- lankton-eating; and Artemia make a betterfood source if they havebeen feeding upon algae,as opposedto being starved!.It was not possibleto znonitoraznmonia levels with a simple, inexpensive test kit at Dian Desa. Thus, becauseof high magnesiumand iron levels in the water, the maintenanceof somealgae in the LRT becazneeven znore important. Much researchhas been done on the brine shrimp Ar ternia and most studies have shown that the In star I is an excellentfood source. However, when the yolk is de- pleted,the Arterniahas developed mouth parts, after 24 hours! and they znusthave somethingnutritious in the water column to feed upon e.g. algae! or they reach a "starved" state.The animal itself is no longer a goodfood sourcefor penaeidswhen in this condition, See Chapter 3, Larval Staging, Feeding and Observations for more information!. Photo53. 4,000-liter rearing tank filled to capacity. The temperature should be stabilized in the tank be- Design, Operationand Training , 87

fore nauplii are transferredto it. The recommendedtem- decompositionof proteins and other nitrogenouscom- peratureis 28' C +1'C ! for optimum growth and steady pounds within uneaten feed and waste, Ammonia is an metaporphosisof the larvae through the various stages end-productofprotein catabolism,and it comprises40 to Figure40!. The object is to get themthrough the stagesas 90 percent of the nitrogenous excretion in crustaceans quickly as possible without stressing them Photos 52 and Chen and Chin, 1988b!.High concentrations of ammonia 53!. A temperature of 26' C is too low. The larvae will and nitrite canbe detrimentalin the hatchery.Ammonia continue to metamorphosebut at a slower rate. Many and nitrite arecommon in hatcheriesand attain levelsup bacteria grow better at 30 C. One should consider all of to 0.808 mg/L ammonia-N un-ionized and ionized am- these factors. monia! and 0.118mg/L nitrite-N even with 30 percent water replacement per day Chen et al., 1986!. Ammonia Other Environmental Conditions of importance releasedby shrimpexcretionand bacterial decomposition Salinityis notas critical as temperature. Salinity ranges is either absorbedas a nutrientby algaeor oxidized,first of 25to 36ppt areprobably optimum for larval rearing,but to nitrite NOz!and then to nitrate NO>!by nitrifying should be kept from fluctuating as much as possible. bacteria,Nitrosomonas and Nitrobacter.In intensiveshrimp Salinity readings should be taken once or twice a week larval-rearingtanks, however, ammonia and nitrite can unlessduring a rainy period when fluctuation is likely. accumulate to toxiclevels periodically. Nitrate is not toxic Acidity pH! shouldbe read daily in the rearingtank. at levels usually encountered in LRTs. Two recent refer- A pH of 7,8to 8.4is acceptable,but theoptimum pH is 8.0. enceson this topic are Chin and Chen, 1987,and Chen and A pH of 8.4and abovewill begin to stressthe larvae.The Chin, 1988a. pH wasmonitored at Dian Desathrough a completelarval Total ammonia and nitrite can be monitored with rearingcycle. The readings remained be tween 8.0 and 8.13 inexpensive testkits unless there is interferencedue to the in the tanks until Mysis III and then dropped, due to presenceof magnesiumhardness! and can be regulated incoming seawaterdropping below 8.0 to 7.75.Some with water exchange.Ammonia exists in both a toxic un- mortality wasnoted but was not easilyattributable to the ionized form NH,!, predominantlyat higher pHs, and a drop in pH below the optimum range. Again, mainte- non-toxic,ionized form NH4+!,primarily at low pH. The nanceof algaein the LRTevenduring the Ml to PL4stages ionizedform is not consideredtoxic sinceits ionic charge has proven valuable. When pH readings of incoming preventsit from passingacross the cell membraneof the seawater were below 7.8, the addition of Tetraselmis cul- gills. tureswith pH readingsof 9.0helped to offsetthese low pH To determine the concentration of un-ionized ammo- levels in the LRT, A pH of less than 7 8 has been a ttributed nia,measure total ammonia and multiply thatvalueby the or associatedwith C,H metabolismproblems, i.e. molting, percentage that exists in the toxic un-ionized form see exoskeleton development. chart below!. Recommended safe levels of un-ionized In most hatcheries around the world, ammonia nitro- ammonia NH>-N!, nitrite NOz-N!and nitrate NOs-N! genis alsoan important parameterto watchin therearing are 0,1, 0.1 and 200 mg/1, respectively Chamberlain, tank.Ammonia and nitrite maybecome critical and lethal 1988!. in the late zoealstages and rnysis-to-postlarvalstages when Artemiaare being fed, Lethal ammonialevels are PercentageUn-ionized Ammonia in Aqueous So- thoughttobe dependent on thepH, salinity and tempera- lution at Different pH Values and Temperatures ture levels, The 96-h LC50 of ammonia and nitrite on P. Boyd, 1988! rnonodonpostlarvae PL6!are considered to be 11.51mg/ L ammonia-N Chin and Chen, 1987!and 13.55mg/L Temperature C! nitrite-N Chen and Chin, 1988b!. Larval penaeidsare pH 26 28 30 very sensitiveto nitrite, P. rnonodonhas a progressive increasein nitrite toleranceas thelarvae metamorphose 7.8 3.68 4.24 4.88 from the nauplius to the postlarvalstage. The LC 50sfor 8,0 5.71 6.55 7.52 nitrite on nauplii, zoea,mysis and postlarvaeare 5.0, 13.2, 20.6and 61.9mg/L NO2-N,respectively. A safelevel of 8.2 8.75 10.00 11.41 nitrite is estimatedat 1.36rng/L for PLsand 0.11 mg/L for 8.4 13.20 14.98 16,96 nauplii Chenand Chin, 1988a!.Generally, it is hopedthat ammonia nitrogen levels can be contained below 25 mi- If someform of oxidant gas,such as ozone,is main- crogramatoms per liter a small fraction of 1 ppm!.Am- tainedat a lowresidual level in the larval-rearingtank i,e. monia and nitrite levels should be monitored at least once introducedthrough aeration!,it canhelp to maintainNH eachday in the hatchery.Nitrogenous waste products 3 un-ionizedammonia! and nitrite with acceptableranges fromprotein digestion can accumulate to dangerouslev- Reid,1980!. Ozone is directlytoxic to aquaticorganisms, elsin thelarval rearing tanks. Like other animals, shrimp therefore the residual level must be considered if used. use the nitrogen component of digested proteins the Ozoneis discussedfurther in this chapter under Filtration, amino group, NH~! to build their own proteins,but they Sterilization and Disinfection. cannotmetabolize the nitrogen componentfor energy. High pH readingsor ammoniareadings can be dealt Whenproteins are metabolized for energy,the amino with by drainingand replacingenough of theculture water group is cleaved off and is directly excreted as ammonia and algaeto bring the readingsback within acceptable NH3!. A similar processalso occurs during bacterial ranges,High pH levels were not a problemat the Dian Dcsa 88 ... Tyeeeeytd Fox

Tetraselmis sp. 0- t 5pm!

I

tsochrysts sp. -5pm! r

Figure41 kaupliarsubslages olPenaeus duorarum a, iimt ahenna az- second anlenna, en endopod, ex exOppd.Ir- eomal Organa, lu- furca, im labrum,ml- mandarleriu1- firstmaxsla met - uuxindInaxala. fnxpI - firsti1ulx llioed, no p2- secondmaxiilipd o -esca!lux, sc ~nettles liornDobk ri, 'l96II 4! kaupbrsI Body pear-shaped. b! NonplusII 1IOng, I rroderale andI Shorttermmal Xmae On lxt antennae c! Nmlpais nl 2 dolindfurcal processes, eachwah 3sprres. d!Hauplius IV Each luroal procesx with6 Xpciex I!isegmeraatmn olapperxtsgex apparent Zl,1st and 2nd maxrlae aremaxilkpeds presek ! e!Nauplyus V Body inure or lexx depressed. swoaenknebleae xlrudurexat Oases olmandaziss prefers !. froraal organs prxsenr !

Ch!aetoceros sp. -6iZm!

Figure 43a. Some Commonly Cultured Mtcroalgae

!V

0 13 0 0si xi 0 0 0

CultureAge

Figure43 Typical Phytop!anktonCulture Grawth Figure42 ZoeaIsubstages ofPenaeus duoramnr ab abdomen, c carapace, dl drgestim trad,e eye, la bbxrnxmo marx!Sax,mxl I ratmax!as, mx2 - ascend manse mxpI bst max!aped; mxp2 accord maxaliped I roclrumi su supraorbxal Spore,Ih thorax, u uropod from Dobyrn, 1961! x! ZoeaI bi Zoeaa Eyesstaked I !. rostrum present !;supraorhlal lonred spmes present! C]Zcea uh PairOl bxarroux dsubly branohed! urofxx!x devekazed !i spines appearonabdommal sornrle ! Design,Operation and Training . 89

Table 2. Classesand Speciesof Microalgae Presently Under Culture tiW Class Species BaciHariophyceae S keletonemacostatum, Thalassiosirapseudomonas, T. fl uviatilis, Phaeodactylum tricornutum, Chaetoceros calcitrans, C. curvisetus, C. neogracile.,C.simplex, Ditylum brightwelli, Scenedesmussp. Haptophyceae lsochrysisgal hana, lsochrysis sp. Tahiti!, Dicrateriainornata, Cricospaeracarterae, Coccolithus huxley ! Chrysophyceae Monochrysissp. Prasinophyceae Pyramimonasgrossii, Tetraselmis t suecica, T. chuii, Micrornonas pusilla Figure44 Ssysesand Postlahraf subslagee OfPenaeue duoranrrrs pt pleopod from Do erin, Chlorophyceae Dunaliella tertiolecta, Chlorella 196!! autotrophica,Chlorococcum sp,, @lysoI ShrkTIP ferebedy S'lruCture Nannochloris a tomus, b! lysisII Pleopodbuds apparent bul unsegmented C! SrfystsII I Pleopodselongsred and segmented Chlamydomonascoccoides, d! Post anteI PL,!. SwimmingSetae present on pleopods Brachiomonas submari na Hatchery, at leastduring Julyand August. Low pH readings Chryp tophyceae Chroomonas salina below 7.8!posed a problemonly for a fewdays, after which Cya nophyceae S pirulinasp. theinflow pH readingsreturned to 8.0to 8.2,Water quality managementat Dian Desashould include pH and be moni- tored daily. Readingsbelow 7.8could causeperiods of stress area of major concern in most penaeid larval-rearing and even mortality in cultured larvae. systems is the provision of a suitable food source, Unicel- lular rnicroalgae has proven appropriate for a variety of pH Reading culture situations. Microencapsulated dried diets, such as STRESS 7.8 8.0 8.4 STRESS the one being used at the Dian Desa Hatchery have also been fed as a supplemental food! with some success,but Below OK optimu C' OK Above these are not widely used by commercial hatcheries. Fur- ther testingand comparativestudies need to be madeat 7.8 8.4 this specificlocation before thc practiceis adopted or used on a routinebasis. Dried diets are meantto be fed along Larval Staging,Feeding and Observations with algae, not in place of algae, The fats in the diet easily 1'cnaeid shrimp pass through three larval stages Fig- becomerancid if not kept refrigerated.Also, adding dried urc 40!, nauplii, zoeal and rnysis, before reaching the or inert diets means "dead"material is being placed in the postlarvalstage. As nauplii Figure 41!, they haveunde- tank; this providesa goodsubstrate for bacterialgrowth, veloped mouth parts and feed upon their yolk within the asopposed to the addition of live algae.Thcsc diets should body. After passingthrough the six naupliar stages,Penaeus be fed only if there is a shortage of algae and there is no monodonmetamorphoses to the zoeal stage Figure 42!, It other alternative. is a dramatic change and it is not at all unusual to lose 24 The Dian Desa Hatchery feeds Chaetoceros a small, percent of the larvae during this transition. The nauplii brown diatom! and Tetraselmis a larger, green, flagellated substagestake approximately 48 hours ranging from36 algae! on a routine basis through the zoeal and mysis to 51 hoursdepending on thetemperature!. Time periods stages Figure 43a!. This combination of a centric diatom of larval stagesmay be seenin Table 1 Suggested Larval and greenflagellate is widely used in the industry with Culture Sequences!as well as feeding information. good results. Chaetocerosranges in size between 4 and 6 Daily observations of the larvae should include look- rnicrons, while Tetraselmis is 10 to 15 micrometcrs in ing at the following characteristics while staging. Use the diameter, Classesand speciesof microalgaepresently microscope,not the naked eye, for theseobservations. under culture are listed in Table 2. Culture techniques are Look for debris on the setae, fecal threads, deformities, well establishedfor thesespecies and shouldnot be devi- bacteria and protozoans, as well as for other problems ated from to any great extent, such as fungi and ciliates. Zoea feed indiscriminately, but must have food small Zoeafeed on phytoplankton microscopicplants!; see enoughto enterthe mouth when theyencounter it while Figure 43afor somecommonly cultured microalgae.An constantlyswimming. Food levels must be maintainedin 90 ... Treeceand For

Figure 45a Stages of Artemia Development ArrSupply Drawn From Photo by Sorgeloos, 1979! ClassPiper C learPi astic HatchingCane LightTube

l-latchingCane PlasiiaValve Stand 300prn Pre-nauplius tn E-1 stage

Figure45b ArtemraHatohing COnea

swim backwardsand flip occasionally. Mysislarvae are capable of morevigorous swimming and can seek out, capture, grasp and hold food consisting of a variety of zooplankton as well as Artemia nauplii. Mysis substageslast 24 hours eachwhen held at 28 C. Pre-nauplius in E-2 stage Twenty-fourhours also determine the differencebetween PL1and PL2,etc, Artentia are fed to the shrimpas soonas the shrimp are able to grasp and consume large food particles.This larger food should be fed at late zoealor C. early mysis. Large algae cells alone are not sufficient food for mysis larvae. Shrimp larvae in the late mysis stage have been known to eat up to 50 Artemia nauplii per day, and larval shrimp at Dian Desa have shown these same feeding habits, Brine shrimp Artemia! are collected in cyst form from salinas located worMwide and sold commercially. They Freshly hatched instar I nauplius come in a sealedtin can. Once the can is opened, the cysts should be kept dry and free of any exposure to humidity. The cyst hatch will be greatly affected if not kept in a dessicator or other dry container. Most of the time the can will come with a plastic, resealable lid. This is probably adequate if the can is kept in a cool, dry place and the contents areused rapidly. Do not place Arterniacystsin the algae lab. SeeFigure 45 for the stagesof Artemiadevelop- ment.! Refer to Treece and Yates, 1988, for a review on Artemia used as food in hatcheries. Other organismshave been used to feed the mysis larvae, but none have been quite as effective or as easy to Instar V larva culture and feed as the Artemianauplius. Arternia meetsall the requirements of a good,live food source.!t is sufficient sufficient quantihes to keep them satisfied.During the nutritionally, although variations in the different strains later zoealsubstages,theuseofa srnalleralgalcellmaynot ofbrineshrimphavebeen shown, Copepadsandnematods satisfy their increasing appetite. The hatchery needs to have also been used,but have not proven to be a consistent feed a larger algal cell at the same cell densities or quanti- econotnical successfor hatchery operations. Micro- ties for more efficiency. The food also must be kept in encapsulated diets are being pushed by producers, but suspensionfor it to be availableto the larvae;therefore, actual, proven, long-range hatchery successesare very gentle aeration is used. few. These diets are to be avoided and used only as a Larvaemetamorphose through threezoeal substages researchand development project until they are shown to in approximately120 hours, averaging 36 to 48 hours in be economicallyand otherwise justified, Some results eachstage, before molting to the rnysisstage. In the mysis have shown that postlarvae are larger when stagethe larvae Figure 44! look more like adult shrimp supplementally fed micmencapsulated feed than larvae and onecan stage or tell the differencebetween the zoeal fed only algaeand Artemia. stageand the mysis stagewithout the use of the micro- Artemia cysts may have much bacteria on the outer scope.Mysis larvae will begin to stayin one placeor even shell and should be disinfected or hydrated in a chlorine solution to 10 ppm chlorine and either freshwateror Design, Operationand Training ... 91

Figure 46 CountingArtemia when concentrated

Artemia ¹ Artemia in 0.1 ml = H!

1 ml Pipet 1 ml Pipet

G! :100 dilution of original concentration!

G! = H! x 1,000 H x 10 = ¹ Artemia in 1 rnl!

Pipet

St

Interchangable Rubber "0" RingsTrap either 1 ml, 2 mls, 5 mls or 10 ml sample

seawater!.Aeration should be applied in the hydrating at the bottom. Freshlyhatched nauplii sink and can be containerso that all the cystsare exposedto the chlorine drained out of the container by opening the valve or solution.The cystsshould be hydrated or disinfectedfor stopcock Figure 45b!. approximatelyone hour, then rinsedthoroughly until all the chlorine is gone. Thecysts should then be placedin full-strength, filtered seawaterand aerated near a strong or intense light sourcefor 24 hours. For best hatching results, place 5 grams of cysts for every liter of seawater. Dependingon the strain of cysts,one should expectbe- tween 200,000to 300A0 nauplii fiom eachgram of cysts. A 1 liter cone can be used for small numbers of Arternia SeeFigure 45b!.For larger requirements, a 5-gallon or 19- liter clear glass or plastic drinking water bottle may be used to hatch the Artemia. Harvestingworks best if the aerationis removedand the solution is allowed to sit for approximatelyten min- utes. The unhatched cysts should float and the freshly hatched nauplii will sink to thebottom of the container or will swim toward the light source,and can be collected by siphoning Photo54!. One of theeasiest ways to hatchand Photo54,Siphoningofffreshlyhatched Artemia nauphiaftersettling harvestis in a conicalcontainer clearplastic!, with a valve note the stratified layers!. 92 ... Treeeeand Fox

After harvesting and concentrating nauplii, a Freshtubes inoculated sLrbsampiecanbe taken and the numberofnauplii counted eery tveveeks GTOCKCULTURE ModhonGulttrd's F /G between two marks on a,0l-ml graduated pipette, or 50ml test tube Temperatcue: 2GoC someother simple method of counting canbe used.Ex- Photoperiod1 2/1 2 light/dark trapolation will give the total number of nauplii concen- trated in the harvest container see Figure 46 for other methods of counting Arterrria!. Freshly hatched nauplii should be aerated and fed to the shrimp larvae as soon as Transferredevery tvo days possib1e.If too manynauplii havebeen hatched, they can be saveduntil thenext day by placingthem in a refrigera- WORKINGCULTURE Medosn.'Guittrd's F/2 ~ s 5Oml test babe Temperattre:ambient f24 - 28'C! tor at 4'C, with little or no nutrifional loss or mortality, Photepertod:24hr s light some Arterrgianauplii can be frozen or stored in a brine solution and held as back-up feed when a poor hatch occurs. The frozen nauplii should be fed only in an emer- gency, They are dead and will be quickly attacked by Transterredevery 92vo days bacteriaand protozoansin the rearing tank, Their extra WORKINGCULTURE MeehanGulgrd's F/2o weight alsorequires vigorous aerationto keepthem sus- e 2GOml to 2 htelflask Temperature:ambient pended so that they will be accessibleto the larvae. Photoperiod:24br c light The recommend ed Ar terriia naupIii feeding regime for the Dian Desa Hatchery is as follows:

LARVAL STAGE ARTEMIA DENSITYlml Transferredeery tvo days Fed by Volume!

La te Z3-Mysis I 1 WORKINGCULTURE ModkunGuillard's F/2 Temperatur ~: amb4nt Mysis I-Mysis II 3 Photoperiod:24hrs light Mysis II-Mysis III 6 horstion Const mt Mysis III-Postlarvae 6 One way to determine the amount of Arterrtia to be added to a rearing tank is by following thesesix easysteps: Feedlarval reruog tank as needed upto e+t-day-otd culture 1. Number of Arterniajml required = A 2, Presentdensity in larval-rearingtank ¹ Arterrtia/ml! NOTE;The etcher Gmllk d's F /Imedhon has tvice the amotstt =B o ef achnutrient per liter of seavater 3. A-B=C 4. C x Volume of larval-rearing tank in ml! = D Figure 47. Typical Cycle Used in Algae Culture 5. Number of Artemia/liter in hatching container = E Formula 6. D/E x 1000 = ¹ mls of Artemia to be added to LRT Decapsulation of the cysts is a standard procedure for Ittgrediattts the Dian Desa Hatchery. This is the best way to feed ~ 800 ml deionized water Artemia to penaeids. The technique for decapsulation has ~ 160ml cod liver oil, or other high omegafish oil beendescribed in many placesin literature and will notbe ~ 4 egg yolks described here SeeSorgeloos et at,, 1983;Sorgeloos, 1986, ~ 30 gr unflavored gelatin and Treece and Yates, 1988!. ~ 10 gr Vitamin premix, including E, C and B complex A small amount of algae is maintained in the rearing ~ 1 gr 8-Carotene tank even during the rnysis or Artemia t'ceding period, so that second-day-old Artemia will be a good food source Proced tdre when eaten by penaeid larvae. 1. Dissolve gelatin in 800ml boiled deionized water and let cool to 40'C. Artemia Enrichment 2. Mix the oil in a blender on the highest setting for 30 Arterrria Straina differ in Size and nutritianal quality, secondswhile adding B-carotene. particularly in content of HUFA. In 1986Leger et al., found 3. Whileblender is still on, add vitaminsand eggyolks. that strainscontaining greater than 4 percentof fatty acidsat Finally, add gelatin and blend for 90 seconds. 205 n3 yield significantly better growth than Artemia with 4. Store the product covered in the refrigerator. Iessthan3percent205n3.ThepremiumqualityArtnnia with high hatching rates,small size and greater HUFA levels are For Use with Hatching Artemia relatively expensive and sometimesdifficult to find in quan- Use about.5 ml enrichment diet per liter of incuba tion tity. As an alternative,a hatcherycan purchase less expen- water assuming2 gr dry cysts/liter incubation water! sive Artemia with little or no HUFAs and enrich them. The after24 hours hatching time, and another.5ml enrichment following is a simplified formula for Arterrtiaenrichment, diet per liter two hoursbefore harvesting. Harvest before provided by Paul Olin of the University of Hawaii at Manoa, Ar temiabecome too largefor predator larvae. Coconut Island Shrimp Hatchery. Design, Operation and Training ... 93

For Use with Hatched SeparatedArtemia in The classesand speciesof microalgaepresently cul- seawater! tured for aquaculture purposes are listed in Table 2. The algalspeciesmostoftenused incoznznercialhatch- Use about .5znl enrichment diet per liter of separated eries are the naked flagellates both green andbrown! and Artemia assuming a density of 100to 150 Artemia per rnl! a diatom of the genus Chaetoceros.One common strain for not less than four hours. Aerate the Artemia/diet mix used is the Tahitian strain of Isochrysis which tolerates during the enrichment process.Cooling the water with ice high temperatures up to 30' C! and strong light. It is a may slow the rate at which the Arternia grow during the small spherical shapedflagellate, approximately 3 mi- enrichment process if size is critical to predator larvae. crometers in diameter, without a thick cellular wall, ca- The drawbackto this procedureis that Artemiagrow pable of self-locornation by means of flagella!. The ab- very rapidly and metamorphose to second instar senceof a heavycell waB makes it a suitablefood for larval rnetanauplii and can become too large for late zoea and herbivores, but Isochrysisis low in HUFA content. Thebest early mysis penaeid shrimp to catch and consuzne.The coznbination of algal specieshas been shown to consist of recommended solution is to use premium quality, newly Chaeioceros and Tetraselmis. Thediatorn Chaetoceras has the hatched Artemia for penaeid zoeal and mysis stagesand qualities of rapid growth, high HUFA content and be- then switch to enriched rnetanauplii Artemia as food for causeit is largerthan Isochrysis, has a widerapplication in the postlarval penaeids. For further information, see Penaeid larviculture, Sorgelooset al., 1986 Manual for the Culture and Useof Tetraselmisis a green flagellate ranging from 10-15 Brine Shrimp Artemia in Aquaculture!. micrometers in diameter. It is good to feed this species later in the penaeid larval cycle to help fuMII their cver- Algae Culture in Support of Larval Rearing! increasing appetites. Fed in combination with the brown The purpose of the algal mass-culture system is to flagellate, it provides a "well rounded" diet for larvae provide a dense"starter" culture for the inoculation! of small-cell-brown and larg~II-green!. Some strains of the desired speciesof algae that are known to satisfy the Tetraselmishave a tendencyto settleout or grow on the nutritional requirements of penaeid shrimp larvae. This is walls of the culture vessel and should be discarded if this accomplished by producing "pure" algal cultures in the begins to happen with one of the cultures. laboratory in enri ched seawatermedium under controlled Growth Medium environmental conditions, and using these cultures to inoculatestill larger cultures,which are eventually used For simplicity, Guillard's F/2 medium can be pur- asfeed Photos55 and 56,and Figure47!. chased in a pre-mixed, concentrated form from a local The algal cultures are produced daily on a production- aquaculture supply and chemical dealer-distributor. See line basis by transferring small cultures at intervals into Table3a for the make-upof Guillard's F/2 algal culture larger growth vesselscontaining new media Figure47!. znedium, The transfers are made according to a schedule of inocu- Guillard's methodis mostoften used with thegreatest lations that provide alternate food organisms or a rnix- success in hatcheries. Some other methods used to culture ture! to the shrimp larvae. The next stagein the algal- algae are: Allen's Medium, Artificial Seawater Medium, culture systemis the carboy stage.Commercial shrimp AS100 Medium, Bold's 3N, Bristol's and Bold's 1NV hatcheriesgenerally usemuch larger culture vessels,tanks Bristol's, Bristol'sSolution, Chu's Medium, Desmidagar, or evenpools a stheir last stagein thealgal-culture system "Double Strength Seawater," Erdschreiber Solution, ES beforefeeding to penaeids. Enrichment for Seawater, Soil-water Medium, Tres-Buff- ered Medium, See Starr, 1978, for descriptionsof eachof thesemeth- ods!. Fritz Chemical also sells an "Algae Food." Examples of other growth me- dium methods used at various loca- tions around the world can be seen in Table3b ThePhilippine's Method of Algae Culture! and Table 3c In- donesianMethod of AlgaeCulture!. Another growth medium example used in a commercial hatchery,a "community" method of algae pro- duction used by Continental Fisher- ies, Ltd. PanamaCity, Fla., USA, is described as follows. Obtaining Cultures One place to obta in the algaecul- Photo55. Laboratory roork bench, zoith balance Photo56, Algae lab behind dosed doors in air- tures Chaetoceros,Tetraselmis chuii, and chemicalstorage. conditioned room. Isochrysissp. Tahiti! and others is 94 ... Trsscr atrd Fox

ble 3a. Guillard's F/2 Algal Culture Medium Composibonper liter of seawater!.

jar Nutrients Final Concentration Stock Solution mg/I seawater! Note ¹1 Preparation aN03 sodium nitrate! 75 mg Nitrate/Phosphate Solution aH PO . H 0 sodium phosphate! 5mg Working stock:add 75 g NaNOs + 5 g NaH>PO4to 1 literDI Na~SiOs,9H20 sodium silicate! 30 mg Silicate Solution See Note «2! Working stock add 30 g Na>SIosto 1 liter Dl

EDTA!-see Note ¹3 4.36 mg Trace Metal/EDTA Solution Chloride! 0.01 mg Primary stocks:make 5 separatestocks in 1-liter volumes sulphate! 0.01 mg 10,0 CoC12/1DI,9.8 CuSos/1DI,180 g MNC12/IDi hloride! 3.15 mg 6.3g Na~Mo04/IDI, 22.0g ZnS04/1DI nese Chloride! 0.'l8 mg Working stock:add 1 ml of eachprimary stocksolution um MolybdenumOxide! 0.006 mg + 4.35gf Na2CrpH,sOsN2+ 3.15 g FeClqto 'I liter DI lphate! 0.022 mg

0.1mg Vitamin Solution 0.5Ijg microgram! Primary stock:add 20 g thiaminHCl+0.1 g 0.5Irg microgram! biotin+ 0.1 g B12to 1 literDI Working stock add 5 ml primary stockto 1 liter DI he final f/2 enrichment,add 1 ml eachof thefour working stocksolutions per liter of seawater. be omitted for speciesother than diatoms. ylenediaminctetraaceticacid a white crystalineacid usedas a chelatingagent!. umsalt powder!whichiseasierto dissolveintodistilled water. ThisformofEDTA= EIHYLENEDINITRILO! CID = Cl0 H1408 N2 Na2~ 2H20, analyticalreagent grade is preferred for usein the chelate in marinemedia, and is readily metabolizedby mic robes. The free acid is insolu on containing2 molarequivalents of NaOH.The disodium salte Na2EDTA.2H20! is re is marketed asVersene Fisher Scientific Co.,711Forbes Ave., Pittsburgh, Pa.,USA,152 Quebec,Canada! or asSequestrene Carolina Biological Supply Co., 2700 York Rd., ein 973! for further information on EDTA. ppines Method of Algae Culture culture of selected algae red seawater in culture tank h 100g of 46-0-0 N-P-K! or urea and 10g of 16-200inorganic fertilizer per ton of seawa ter I algal starterper ton of seawaterand aerate aeafter one or two dayswhen blooming occurs;this is indicatedby a brownishcolor for diatoms or Chaetoceros!and greenish color for Tetrasetmis. culture of mixed diatoms ms Chaetoceros,Rhizosolenia, That!asiosira ! are found in seawater. Mass culture of these can be done, ethod: red seawater in algal tank h 100 g to 46-0-0and 10 g of 16-20-0 eave for 2 or3daysuntil water turns feeding or as starter for next culture. m species,however, are available onl

n Method of Algae Culture

I Concentration Add g! l. UREA 100 ppm 0.1 I 2. TSP, 50 ppm 0.05 Trisodium phosphate! '3. ZA 25ppm 0.025 Zinc acetate! i 4. FeCls 3.5 pp rn 0.0035 Ferric Chloride! 5. EDTA 0.5 pp rn 0.0005 Design, Operationand Training ... 95

from the culturecollection at The University of Texasat Two metric tons of the phytoplankton bloom from the Austin Dr. Jeff Zeicus, Department of Botany, Austin, outdoor tank were pumped into the larval-rearing tank Texas,U.S.A. 78713-7640 512/471-4019!. The chargeper when shrimp larvae were in thenauplius stage.Fertiliz- culture is $10.00U.S. for University-affiliatedprograms ers, at the same dosageused in the outdoor tank, were and $20,00U.S. for private companies. Commercial algae addedand continuous lighhng provided. The phytoplank- culture suppliers, local government-run hatcheries or ton bloom in the rearing tank occurredabout the same university researchfacilities can also supply cultures. timethat the nauplii molted to theprotozoeal stage. The Other supplies are; the Center for Culture of Marme phytoplanktonbloom from outdoortanks was added Phytoplankton CCMP! in Maine, U,S.A. tel. 07! 633- periodically.tothe rearing tank to maintainthe bloom. 2173. A two-week notice is required, Chaetocerosclone The phytoplanktonbloom in the outdoor tank lasts CCMP 1318 and Terraselmr's clone CCMP 882 are available onlya fewdays. The neighboring outdoor tanks are inocu- for $45/research and $U.S. 90/commercial; and Carolina lateddand fertilized to produce new phytoplank ton blooms Biological Supply, N.C., U.S.A. and providea continuous supply of plankton for the One may contact a nearby hatchery and obtain a cul- rearing tank. tureor one may choose to isolate a articularspecies from T. chuiz,I. aff. galbanaand Chlorellasp. are each nearbywaters and use it for culture. Local specieshave monoculturedin a 20-liter carboyto provide a supple- advantagesof being resistant to conditions specific to the mental inoculum. Stockcultures are put into 2800-ml areaand thereshould be an endless supply in casefurther pyrex flasks, which contain2000 ml of sterilized, boiled, 1 isolationis requiredin thefuture. The technique for isolat- micron-filtered seawater. Guillard's F/2 medium isadded ing species has been thoroughly described in literature at the same time of the inoculation but silicate is omitted. and is not discussed here See Fox, J.M., 1983 CRC Manual!. Thedosage of majornutrients and tracemetals is 1 ml per Figure 43a in the sectionon larval tearing! depicb liter of seawaterand thevitamin dosageis 1/2 ml per liter someof the more commonly cultured microalgaeand of seawater.Strong aeration is providedthrough an their relative sizes. airstone.The 2,800-ml flask isplaced 50 cm in front of four 40 watt, coolwhite fluorescent bulbs. In approximately CommercialApplication and Community four days, it is ready tobeused as an inoculumfor a 20-liter Method of Algae Production used by Continental carboy. Eighteen liters of sterilized, boiled, I micron- Fisheries,Ltd., Panama City, Florida filtered seawa ter and Guillard's F/2 medium are added to A phytoplanktonculture is necessaryto provide the the carboyat thetime of the inoculation.Lighting and protozoeawith a suitablefood. The community method moderateaeration are provided. The bloom occurs in the is used to produce a phytoplankton culture in the larval- carboyin approxunatelytwo to threedays and is usedto rearing tank. There are three factorsthat determine the inoculateother carboys.The blooms from six carboysare effectivenessof this method: the phytoplankton inocu- usedto inoculatethe larval-rearingtank. Silicateis omit- lum, fertilizers and lighting. The phytoplankton in the ted whenthe rearing tankis fertilized, naturalseawater in thelarval-rearing tank is not sufficient Growth Characteristics tobeusedas an inoculum; instead, phytoplankton blooms from theoutdoor tank and carboys are used as an inocu- Given the right conditions nutrients, light, tempera- lum, Ferhlizersare composedof commercialgrade feed ture,etc.! algal cultures will generallyexibit the growth urea 6 percentnitrogen! Archadian Corp.,Memphis, pattern shown in Figure 43b. Tenn.,USA!, laboratory grade sodiuzn phospha te dibasic The objectis to keep the culturein the exponential FisherScienhfic Company, Fair Lawn, N.J.!and labora- phaseor phaseof rapidcell division. The best way to do tory grade sodiumsilicate Fisher Scientific Company!. thisis to dilute out theculture constantly with new media Lighting is provided by 16fluorescent bulbs 0-watt!. or betterstill, move the existing culture into a larger,clean The community method is also used to produce a vesselwith new media.Algal growth dynamicsare cov- phytoplankton bloom in outdoor tanks. Two znetric tons eredin greatdetail in a paperby J.M. Fox on Intensive of fresh seawaterfrom St.Andrews Bay, Fla. were added Algae Culture in CRC Manua 1,1983. to the outdoor tank and 300grams of urea, 15 gramsof sodium phosphatedibasic and 15 ml of sodiuznsilicate Culture Techniques were added at the same time. An airstone was used to Stockcultures, obtained from another hatchery, a gov- provide strongaeration and circulation in the tank. Under ernment aquaculture researchfacility or other culture normal weather conditions, the color of seawater in the suppliersuch as The University of TexasDepartment of tank turned yellow-brown within a day or two. This Botanyandotherslisted in AquacultureMagazine Buyer' s indicatedan increaseof phytoplanktonin thetank Eight Guide, are used as starter cultures. Stock cultures can be tons of fresh seawater were added after the color of the maintainedin smallscrew-cap test tubes, with thetops left culture medium was yellow-brown, The color of the me- loose see Figure 47!. The algaecan be kept in a dormant diuzn turned dark brown within two days afteradding stageby dilutingout the nutrient medium to F/8 which seawater,which indicated a phytoplankton bloom. S. is Guillard's F medium, diluted four times more than the costaturrrwas found to be the dominant speciesof the standardF/2 medium! and maintainingthe algae at 24'C bloom. The phytoplankton bloom in the outdoor tank was in low light. Thesecondihons will restrict growth to a thenready tobe usedas an inoculum for thelarval-rearing maintenancelevel and there will not be a build-up of tank and neighboring outdoor tanks. metabolic wastes. You should not handle stock cultures 96 ... Treeceand Fox exceptwhen transferringon a monthlybasis. It is bestto AirSupply keepthexn in a separateroom andtransfer sxnall amounts, Aeraton Filter under sterile ar aseptic conditions, to a new enriched ICOaontilledl ugnLoose Cap medium tube every other week or on a xnonthlybasis dependingon the preferredschedule. Figure 47 depictsa typicalalgal transfer routine and production-line sched- TestTube Ino bottom! ule. GlassTube Small flasks Photo 57! are inoculated from the tubes andheld in a highlight area.Once the cultures in theflasks are a few days old and their respectivecell countshave nirBubbtea increasedconsiderably, they can be transferredfirst to largeflasks and, after a sixnilarwaiting period,to a 19-liter Mediumand Algae carboy Figure 48!. The objective is to keep the algal speciesthe dominant speciesin the culture. This is done by adding a pure inoculum culture to a relativelyclean andsterilized seawater growth xnedium.Carboys are then placedon analgae rack Figure49! or arrangedsimilarly to the ones in Photos 58, 59 and 60!, which are photo- FigureAB GlassCarboy graphssof a typicalalgae room. Filtration, Sterilization, Disinfection Seawater is filtered down to one micron and disin- fected, sterilized or treated before it is used in algae culture. The axnount of filtration required is largely de- StockCult ~ ie T ~bes pendenton characteristics ofthe incoming seawater. There- Sc~eenWiie Base fore, the amount of filtration is largely site-specific.In SmaltFlasks ScreeriWire Base soxneareas, extensive filtration is required and in others, AllowsF II~ Benebl only minimal stepsare required to clean up the water. AirSupply OlI igltt According to Wheaton 987! sterilization is the total destructionof all living organisms,but disinfectionis the destructionof all or nearly all organismsharxnful to the LignlTubes userof that water.Only disinfectionxnaybe necessary and --Carboys will be cheaper than sterilization. Therequirement for or level of sterilizationis alsosite- specificto someextent. Standard sterilization conditions are described as 121' C and 20 psi, with sterihza tion tixne dependentupon the voluxneof water being treated5 xninutes for liqu idsless than one liter,45 minutes for larger volumes such as a 19-liter carboy!. Sterilization or disinfection! as the treatment of sea- waterrbefore being used in the algaerooxn can be accom- Figuredg TypicalAlgae Culture Aeok plished using heat, ultraviolet UV! radiation and/or chexnicals.

Photo57. Algae production in smallflasks, Photo5S. Algae production in glassjars widemouth allows easyaccess for cleaningjar!. Design,Operation and Training ... 97

3. Pasteurization Heating water can effectively sterilizeor disinfectif it is heatedto a sufficientlyhigh temperatureand held at this temperaturefor a cer- tain time interval. Bacterial kill is a function of tem- peratureand time as weil asthe species targeted for elminatian. For example,pasteurization of milk is recommended at 60 C for 30 zninutes or 71.1'C for 15 seconds.The ha tchery manager may adjust the extent of kill necessaryfor his or her site by varying the temperatureand jor holding time at the elevated temperature.For mostareas, the authors have found that the following stepsare necessaryfor pasteuriza- tion of seawater to be used in the algae room: Heat a carboyor other containerof seawaterto 73'C and hold 5 minutes media included!, then let it cool overnight. The following day reheat the en- riched seawater to 73'C and let it cool as before. The media is then ready to inoculate. The idea is to eliminateanycontaminantsthatmayhavebeen forced into a sporestage as a resultof thefirst heattreatment while not heatingthe waterhigh enoughto causethe tracemetals in the seawater and enrichment znedia to precipitateout asthey do when autoclaved.Heating can be done with an immersion heater or with some type of flame or other heat source.This is a time- consumingmethod of sterilizing and cantaminants seemto appear much soonerthan when using the autoclaveto sterilize,but again, the primary disad- vantageof using heat for disinfecting water is the energy requirement of the process. Note: Microwave ovens may also be used to pas- teurize seawater in tubes and small flasks. See Keller et al., 1988 for details!. 4. Ultraviolet Trea tment Design Summary Wheaton,1987! The designof an ultraviolet UV! Photos59 and 60. Typical algaeroom arrangnnen t. treatment system is summarized as follows. ~ Determine dosage of UV radiation necessary to 1. Autoclaving This systeznis the most effectiveand kill the target organisms. the most preferred,but is also very expensive.The ~ Adjust dosagefor water turbidity. It is easierand glasswarewill last longer using this method, com- more cast efficient to eliminate turbidity than to pared to direct heating methods.Carboy size auto- purchasemore equipment i.e. higher dosage!. clavescost approximately $3,000,and the smaller, Determine flow rate needed. pressurecooker size autoclave,for steriTizingtubes ~ Usinglamp mtensitydata from the manufacturer and small flasks, costs approximately $300. This and the zeflector characteristics for suspended method is most often used in research labs, and is not systems determine the following. widely usedby commercialoperations where a znore Water depth needed toget 90 percentor greater cost effective method mustbeused. Autoclavescanbe UV absorption. eitherelectrically powered or gasfired,but theenergy Number and spacing for a suspended system! requirementis the primary disadvantagefor this pro- and type of UV lamps to be used to provide cess. required dosageto treat required flow rate, 2. Chlorination Bleach, with a 5.25 percent activity, Locateba ffles to achieve required turbulence and 2.5-pprn solution in seawater has been found tobe an water depth ta get 90 percent UV absorption. To effective treatment znethod, The treated seawater is minimize absorption by baffles, the devices are lek for 24 hours and then aeration is added to remove best placed parallel to the lamp axis. However, remaining chlorine or at least neutralize its effects. turbulent flow requirements may dictate other The useof sodium thiosulfatehas been used by some baffle designs.! hatcheries to neutralize the chlorine, but with the 5, Ozone as a Disinfectant Summarized from Wheaton, delicate trace metal balance found in seawater, it 1987! Ozone is a triatomic form of oxygen that is a comesas no surpriseto find out that the addition of bluish gas of pungent odor, is naturally in the upper this chemical has caused side-effects of its own at atmosphere although apparently in less and less some of these hatcheries. quantities thesedays! and is formed by a photochemi- 98 ... Treeceand Fox

cal reaction with solar ultraviolet radiation. Accord- disinfection.In most cases,aquacultural systems are ing to Wheaton 987! ozone hasbeen recognized as a ableto reducethese values because incoming water is "purifying agent" since 1782;the first commercial use of better quality. The organiccontent and inorganic of ozone was in 1906. chemicalozone demand should be lower for aquacul- Ozone is formed when oxygen rnolecules are excited tural watersthan for full strengthsewage. Blogoslawski to decomposeinto atomicoxygen, Collisions of these et al. 975! found undetectable bacterial survival in atomic oxygen atoms cause the formation of ozone. seawaterat 056 mg/liter ozoneconcentration when Nearly all commercialozone generatorsuse a high using the water in an aquaculturallaboratory. Con- voltage corona discharge system described by tact time was not specified. Based on kill time of Wheaton987, p. 633!. variousbacteria and virus speciesreported in litera- The effectiveness of ozone as a disinfectant is a func- ture Katzenelsonand Shuva1, 1975; Kelly, 1974;Smith tion as is UV radiation treatment! of contact time and and Bodkin, 1944;Sproul and Majumdar, 1975!,it dosage,Ozone reacts very quickly cornpared to com- would appear that 1-to 5-minute contact time and a pounds such as chlorine because ozone is toxic on dosageof 0.56to 1.0mg/liter arereasonable values for contactwhereas chlorine acquits toxicity.Compared ozonetreatment of most aquaculturesystems. How- to chlorine,ozone is twice as powerful asan oxidant. ever, on-site tests are the only positive methods for Pavoni ef at.,975! and Nebel et al.,975! demon- determiningrequued ozone concentrations and con- strated that ozone reduced both bacterial and viral tact times. It should also be noted that measuring organismsvery rapidly. Many other investigators ozone concentration is not an exact science in that haveshown similar results e.g.Kelly, 1974!. different measurementsystems give different con- Organic matter exhibits an ozone demand, In fact, centrationsin the samesample. ozoneis usedfor removalof color,odor and turbidity in somemunicipal water systems.Inorganic chemi- A Commercial Application of Ozone cals in the water also exhibit an ozone demand i.e. From commercial experiencein Ecuador990 to 1991!, iron and magnesium canbe oxidized to the insoluable Maria Gonzales personal communication, 1992!achieved oxide forms by ozone!. an increment in production from 20 million to 40 million Ozoneis directly toxic to aquatic organismsand ta postlarvaeper month using ozoneas a disinfectant.She people.Ozone is fatal to people when they are ex- reportedthe following advantageswith ozone:decrease posed to 11,000ppm by volume for 0.1 minute or in overallbacterial levels; almost no application of chemi- about20 pprn by volumefor 1+00minutes. Toxicity calsand antibiotics;achieved postlarval 10 production in startsto occurfor peopleat exposuresof 3,000ppm by 17days from the beginning of cultivation, which normally volume for 0,1 minute or 4 pprn for 1,000minutes took20 to 22days without ozone;animals were larger and Nebel et a1.,1975!. Toxicity data for aquaticorgan- more active than thosecultivated by usual means;and isms is not available exceptas very scatteredindi- reduced bacterial levels in Arternia washed with ozone vidual pointsthat cannotbe put into a meaningful water. picture. For example, Arthur and Mount 975! found The ozonesystem offers greatadvantages, but it can that 0.2to 0.3ppm ozoneis lethalto fatheadminnows. also be very dangerousif correct levels are not used. Luckily,ozone is quiteunstable and rapidly decom- Figure 50 shows the recommended amount of ozone that posesto molecularoxygen. Providing a Sewminutes shouldbe used for eachshrimp larval stage and for algae between ozone treatment and introduction of the andArtetniacysts disinfection, and gives the formula for ozonatedwater into a culture systemmay be suffi- checking the ozone residual in seawater. cient to avoid problems. Designofozone water-treatment systems requires the establishmentof intimatecontact between the target organismsand theozone gas. The mixing of theozone and water is of primary importance. Selectionof the appropriate contactingsystem de- pendson theapplication,flow ratesand other param- etersof a specificdesign. The objective is to provideas muchcontact between target organisms and ozoneas possible.This means having the largest possible ex- changesurface between gas and liquid, The appropri- ate contact time must also be maintained to achieve disinfection.When pure oxygen isused in theozonator, it usually is economicallyattractive to recycle the oxygen given off in the contactor. Thenecessary contact and concentrationvary with target organisms and water quality. However for municipal sewageit appearsthat residualozonecon- centrationsat the contactoroutlet of 0.5mg/liter and a 5-to 10-minu te contact time give 99 percent or better Design, Operationand Training ... 99

Procedure to Determine Residual Ozone in through the entire distribution system on a continous Seawater and FormuIa to Calculate It cleaning basis. Ultraviolet light is an effective disinfectant as long as Procedure: turbidities are kept below reasonablelimits. Moreover, Step1. Two solutionsmustbe prepared.To preparesolu- UV doesnot produceresidues in water and is simple to tions: operateand install.Economic methods for large-scaleuse SolutionA: Mix 20 gramsIodine potassium IK! per arenot ptesentlyavailable.In laboratoryand aquacultural liter distilled water. operations,UV systemsprobably constitute the best dis in- Solution B: Standard Solution sodium thio sulfate = fection method available.They can be purchasedfrom NazS20sx5H20, Aconstantof0.025isusedinthe several manufacturers. Maintenance of these units can be formula in addition to added amount!. cumbersome the quartz sleevesmust be cleanedperiodi- Step2. Add equal amountsof SolutionA IK at 20g/liter cally and are very breakable.The half-life of the bulbs is dist. H20! and measured in weeks instead of months. Periodic bulb ie- water sampleto be testedfor ozoneresiduaL Example: placementis a continuingexpense, as is electricity.How- 300ml Sol, A added to 300 mls of water sample. ever, theseunits must be comparedwith the price of the Step3. To themixture madein Step2 equalam ts. of IK and chemical when using chlori~e, or electrical requirements watersample! drop in Solutio~B Na2Sz03 x5 H20! for ozone production. until the mixture changes color. When the color changes,stop and seehow many mls of SolutionB it Other Disinfection Methods took to make the color change this number of mls is substituted for A in the formula. Centrifugation Washing, Antibiotics or other Step4. Using thefollowing formula calculatethe residual Chemicals ozonein mg/l. Filtration and disinfection or sterilization techniques Ml ~ IMPER. = Residual ozone in aresite-specific and the specificmethod used at onefacil- B sample mg/l! ity may not be suitable at another. One of thesethzee methods heat, ultraviolet radiation or chemicals! or a A= Amount mls. of Solution B to change color of combination of these should meet the needs of most sample hatcheries. Contamination of algae cultures is gootg to 0,025 = constant assigned for sodium thio sulfate happeneventually no matter which methodis used,but 24,000= constant the operator must experimentand find which method 8= Amount mls. in water sampleto be tested only works best for the location, budget constraints, etc.,and be water sample!. prepared to restart the culture routine from scratch if a NOTE: Ozone can be hazardous to humans and must be used contamination does occur somewhere in the assembly- with extreme caution with the safety of the workers in mind! line. New stock cultures should be initiated as a matter of typical management. Comparison of Disinfection Methods Summa- rized from Wheaton, 1987! Counting Algal Cells and Determining Heat is effective as a dismfectant and does not leave Amount to Feed Larvae harmful or toxic residue. However, it is so expensive however that it is not economically feasible under most A. To determinethe numberof cellsper ml of sample,do circumstances. the following: Chlorineand two other halogens,bromine and iodine, 1. Count number of cells per 1/10 cubic mm in are effective disinfectants. They work well on bacteria but hemacytometer chamber i.e. the number of cells require considerable contact time 0 to 30 minutes or in 1 squaremm in hemacytometerchamber!. znore!for virus kill. Chlorine combineswith organicma- 2. Multiply this number by 10,000 See Figute 51, terials to form chloramines. Both chloramines and chlo- depicting the hemacytometerparts!. rine gas are highly toxic to most aquatic organisms. Chlo- 3. See"Preparation of Sampleand Hemacytometer rine gas can be removed by aeration, but chloramines "and "The Use of the Hemacytomete r." cannot be removed easily using this means. B, Todeterminethenumberofcellstobeaddedtolarval- Ozone is highly effective as a disinfectant, having rearing tank LRT! in ml: about twice the oxidizing capabilities of chlorine. The 1. Totalnumberofcellsrequired in LRT=Volumeof oxidation potentialof chlorineis 1.65E V, while ozoneis LRT, in ml OR x number of ceUsrequired per ml. 2.05E V!.Ozone kills both bacteria ance virus with equal 2. Number of cells at present, per ml. effectiveness and speed, Dosage and contact time are 3. Number of cells to be added to LRT = Total normally lower for ozone than for chlorine to produce number of cells required in I.RT number of cells equal results, yielding some capital investment savings at present in LRT. for contact tanks. Ozone must be manufactured on site = Vol. of LRT, in ml x no. of cells req'd,per ml! becauseit is too unstableto withstandshipping. Ozone is Vol. of LRT, in ml x no. of cells at present! presentlymoreexpensive thanchlorine, Themajoradvan- = Vok of LRT, in ml! x No. of cells req'd no. of tageof gaseousoxidant disinfection is the productionof a cellsat present! residual that is hydraulically entrained. Thus, it travels C. To determine volume of algal culture to LRT: 100 ... Tmm ard Fox

1. Volume to be added, in ml x no. of cells per ml in algalculture tank = no,of cellsto beadded to LRT, THEREFORE: Volume tobe added, in ml = no. of cells tobe added to LRT Alternate Explanation of Algae Cell Counting and Determining Amount of Feed A. How to count algae Refer to Aquaculture Extension Manual No. 9, SoutheastAsian FisheriesDevelop- znent Center!. B. Determining amount of feed: The amount of algal food to be fed to the larvae is com- puted as follows: Without previous feeding in larval-rearing tank LRT! Vol. of water Desiredalgal to be added Algal Density in Culture Tank EXAMPLE: MAGNIFICATIONOF COUNTING AREA 000 I x 5 000 cells al ae ml Vol. of algae = 1,000,000cells algae/ml 15 liters With previous feeding in larval-rearing tank LRT! Vol. of water Desired algal Algal density Vol.ofalgae =in LRT X d nsi inLRT - inLR to be added Algal density in culture tank Example:

Vol. of algae = I X 2 lls ml-1 ml sHOULD ER to be added Algal density in culture tank = 15 liters The Use of the Hemacytometer ss Note:Not all heznacytometersare the same.Only one variety is describedhere.! The authors prefer the "New Improved Neubauer"hemacytometer because there is a Figure 51. Hemacytorneter depressionon the bottom of the hemacytometerin the correspondingarea of the counting chamberthat helps keepscratches from occurringon theglass.These scratches vided into 400,0.05-mm squares. The dimensionsof the can eventually cause confusion to the operator when the coverglass are very important to achievingreplication in scratchesappear similar to the scoreson the upper sideof counts. the counting chamber. The authors also find that the Preparation of Sample and Hemacytometer scoresare easier to seeon the 'New Improved Neubauer." The hemacytorneter Figure 51! consistsof two parts. 1. Beforestarting, the hemacytometerand coverglass The major element is formed from a slab of thermal and shouldbe cleanedand dried.A pieceof lenspaper shock-resistantglass. An H-shapedtrough hasbeen cut dampened with distilled water should be used. into this,forming two raisedcounting areas, Raised shoul- 2. A sampleof algaeis obtainedfrom the algaeculture derson eitherside of the H areprecision-milled to exactly tank or from the larval-rearingtank. This is placedin 0.1 mm abovethe counting area.The cover glass,a 0.4- a clean test tube and, if necessary,2 to 3 drops of mm,highly polishedpiece of glass,Tests on the shoulders, Lugol's stain are added and the tube is shaken to kill forming the top of the counting chamber. and immobilize the cells. The counting areasare coveredwith a thin, metallic 3. The tube is agitated again to mix the cells in the film that givesthem a slightlydarkenedappearanceunder mediumthoroughly. A cleaneyedropper or pipetis the microscope.Lines are scribedinto this Mm with great usedto removeapproximately 0,5 ml of the mixture, precision seeFigure 51!. The "tic-tac-toe" pattern has nine 4. A single drop is introduced to the V-groove on the squares,each 1 mmby 1 mm. Theseare divided into 25 sideof the hemacytometer,This will be drawn under smaller squares,and the center squareis further subdi- thecover glass, into thecounting chamber, by capil- Design, Operationand Training ... 101

lary action. Care should be taken to introduce just 4. To maintain algal cultures at desireddensities and enoughto fill the countingchamber. If thetroughs are log-phasegrowth bestnutritional value!, it is advis- filled and shoulderswet, the coverglass will lift and able to harvest or cull about 50 percent of Chaetoceros thecounting chamber will enlarge.Should this occur, and about 33 percentof Tetraselmison a daily basis the hemacytometershould be cleanedand theprocess oncethe desired feeding densitites havebeen reached. started again. Thus, the total volume of each speciesbeing fed daily 5. The cells will take about a minute to settle. The shouldrepzesent 50 percent and 33 percent of thetotal hemacytometeris now placed on the stageof the volume of eachspecies, respectively, that is available compound microscope.Using the lowest power of for feeding on that day. Thus, there should be the magnification,the microscopeis focused. following: 6. For accuracy,a minimum of 100 cells should be Cttsrtorrres:2 4ttl liters =4,80tt liters ot counted.Depending on the concentrationof thecells, Chaetocerosavailable on a daily basis this znightsquire counting the entire scribedcount- 0,500 percent! ing area, counting just the four larger corner squares round to 5,000litezs/day! or counting all or someof the center square.This Tetrasetmis: Mg~lit = 9g91 liters of should be done systematically, counting the same Tetraselmisavailable on a daily basis blocks each time, thus eliminating bias. 0.33 3 percent! 7. The calculations should then be carried outas follows: round to 10,000liters/day! a. Count number of cells X! 5, Algae massculture tank allocation: b. Divide X! by the nuznber of squarescounted Y! a. One 5,000-liter tank of Chaetocerosand one 10,000- c. Multiply that numberby the numberof squazes liter tank of Tetrasebnis should be available at per square mm Z! feedingdensitites on a daily basis. d. Multiply that number by 10,000to obtain the b. Two additional identical mass culture tanks for number of cells in each ml of medium N! eachspecies should also be in productionstarting at five-day intervals behind the first pair. Y! 6. Six 750-liter intermediate algal znassculture tanks to be used as the inoculate source for the mass culture How to Calculate Algae Culture Requirements tanks will be kept m a separateclimate-controlled Based on 60,000Liter Larviculture Capacity room. These will be run as three pairs one tank per l. Assuznptions species!on a strict five-day crop and removalsched- a. Desired algal species: ule, coinciding with an operating scheduleof 5,000 Chaetocerosgracilis neogracite! and 10,000-liter algae mass culture tanks. Tetraselmis chttii 7. Intermediatealgae mass culture tanks50 liters! will Thesetwo specieshave proven themselvesto be be supportedby appropriately scheduledflask and very reliable in termsof larval performanceand carboyor hangingbag cultures, ease of culture. 8. Two 5,000 liter reservoirs will store cooled seawater b. Projected maximum algal feeding densities for for use in the intermedidate algae culture lab 1 to larval-rearingtanks LRTs!: 23'C!. Chaetoceros: 120,000 cells/ml Tetraselmis: 35,000 cells/ml Alternate Larval Diets c. Expectedcelldensities in algaemassculture tanks at peak log-phase growth: Egg Diet Preparation for Penaeid Larvae Mysis I Chaetoceros:3x10s cells/ml or older! Tetraselmis: 7xl0 cells/ml 1. With a grinding implement,grind up two tabletsof 2, Maximuzn number of cells required for a complete one/day feeding of total larviculture volume com- vitamin B-12 and one tablet of vitaznm C. Put contents puted as znaximunfeeding density! x larviculture mto 100 ml plastic beaker. capacity!: 2. Add 25 mls ofbrewer's yeast to 100znl plastic beaker. Chaetoceros:120,000 cells/ml x 6.0x10 ml = 7.2x10' 3. Set up blender without lid. Add 11 large raw eggs cells without shells. Tetraselmis: 35,000 cells/ml x 6.0x107 ml = 2.1xl0'2 4. Add contents from 100 ml beaker mto blender and cells mix at high speedfor 30 seconds.Stop blender, 3. Total volume of algae mass culture needed for maxi- 5. Add 270ml cod liver oil and start mixing again a thigh mum daily feedingat standardexpected mass culture speed. densities: 6. As soonas possible measure out 150ml of white flour Chaetoceros: 7~2xl g~ll Zl = 2+00,000 ml = and add to blender. 2,400liters/day 7. Mix contents until all flour has been put into solution 3.0x106 cells/ml in mass culture about 30 seconds!. Tetraselmis: ~2.1xl ' ~lls ~ttal = 3,000,000ml = 8. Add 80ml of liquid lecithinand mix for 11/2 minutes. 3,000liters/day Now it is ready to cook. 7.0x105 cells/ml in mass culture 102 ... Tmecr, and Fm

Cooking Directions potentialuse of singlecell protein suchas brewer's yeast! 1. Pour one third of the egg mixture into squareglass asa larvalshrimp feed substitute Furukawa,et al., 1973, container and put into microwave on high for two and andAujero, et aL, 1985!, but yeastproducts are typically one half minutes. deficientm essentiallong chain, highly-unsaturated fatty 2. Repeatstep one for remainderof the eggdiet. adds HUFA!. Yeastproducts, which have been artifi- 3. Put into plastic bags labeled Egg Diet and date. ciaHyenriched with essentiallong chain,highly-unsatur- ated atty acids HUFA! of the omega3 n3! family, have FeedingDirections and Application Rates alsobeen used to replacephytoplankton with varying degreesof success,Yeast has a tendencyto adhereto the 1. Forceegg diet through stainlesssteel mesh, 200-250 appendagesof the larvaeand causeproblems similar to rnm 0 mesh! or smaHer. externalbacteria and debrisbuild-up on the larvae inter- 2. Feed at least four times daily at even intervals by suspendingtheamount tobe fedintoanequalamount fering with swimming,feeding and metamorphosing!. of water beforefeeding the slurry. Theaddition of a chelatingcompound such as EDTA may 3. Feedis adjusted adlibitum to ensure that a build-upof help this problem. uneaten feed does not occur. Miscellaneous Sources Used as Feed 4, Generally, for a 9300-liter tank, stocked with 100 larvaeper liter, 100ml. of slurry 0 percentwater by The following is a listof other sourcesused as feedfor volume! is fed per feeding initiaHy. The amount is larval penaeidshrimp: Rotifers, Mytilus eggs,frozen adjusteddaily. Artemia,brine shrimp flakes,Moina sp., Copepoda, Gammarussp., Baianussp. Nematoda, Annelida, clam Microencapsulated Diets meat,shrimp meat, fish meat, miHed feed, sprayed dried feed,and freeze-driedalgae. Microparticulateand microencapsulateddiets have beenused to provide chemicaHyknown compoundsto A widevariety of potentialalgal substituteshavebeen utilized including the oneslisted above boiled chicken decapodlarvae Joneset al., 1979; ViHegas and Kanazawa, eggyolk, microencapsulateddiets, microparticulate diets, 1980; Kanazawa et al., 1982; Levine et al., 1983; Teshima yeasts! in addition to fermentedsoybean cake, but eachof and Kanazawa, 1983;Levine and Sulkin, 1984,Scura et al., 1985,Jones, 1985 and 1987!.Among the most promising thesecan only offer a minor component less than 50 types of microcapsulesare the calcium alginate percent! of the diet of zoea because none has a nutrient composition equal to that of phytoplankton. Another microcapsulesdeveloped by Levine.Many companies substitutediet, calledthe "CrustaceanTissue Suspen- now offer microencapsulated diets such as FRIPPAKS, sion," is used in India Tacon, 1986!. Alma, OceanStar International,Inc., Zeigler Bros.,Inc., Biokyowa,Inc., and Nippai ShrimpFeed, Inc.!, each with varying results.They aHremain quite expensive.Further Signs of Larval Stress, Diseasesand refinementsare required before these diets can be used in Treatments at Dian Desa place of live foods on a commercial scale. OnereFinement, reported by Dr.D.A. Jonesat the 1987 Usually,the first signof stressis debriscollecting on .World Aquaculture Society conference, was a new the animal.If mortalitiesare seenthen, larval samples microcapsulewith a thinerwall designedto breakdown shouldbe taken and observed under the compound scope in thegutofzoeainonlyfourminutes!.Thisparticulardiet to determinethe cause.It maybelow food levels,bacteria, wasbeing commercially manufactured by FrippakFeeds Zoothamnircm,Lagenidiutti, or any number of otherprob- in England, which was sold recently to a Frenchgroup!. lemsthat need tobe identified and treated. It isimportant Personal experiences with these diets have shown to look at the larvaeand postlarvaein eachtank under the themto be a goodsupplement to live food but unsatisfac- microscopeeveryday.More frequentexamination maybe tory to usealone as a completelarval diet. Someof these requiredif thereis a problem,The problems encountered dietshave oils andcreate a "slimy"or "oil slick"surface during two runsof larvaeat theDian Desa Hatchery in andthe water level in thetankrnustbelowered daily. The July and August 1987were as follows: oils canthen be wipedoff the tankwaH with a drycloth PROBLEMS TREATMENTS beforerefiHing the tank, Most of thesediets contain fats andneed to be frozenor keptrefrigerated after the con- 1. Empty guts 1. Feed more taineris openedto keepthe diet frombecoming rancid. Realistically,bacterialcontamination isprobably the great- 2. Luminous bacteria 2. See Control of Luminous estthreat in a larval-rearingtank. Adding live foodto the Bacteria in Disease section tank helpscut down on this threatand helpskeep the 3. Zootharnniam 3. MalachiteGreen ppm! bacteriaat manageablelevels. When finely powdered diets deadfoods! areadded, even though they arecoated 4. Iagenidium 4. Trefian ppm,50ml/ton! with a cross-linkednylon-protein wall to helpstop nutri- Thesewere the mainproblems encountered. Low tem- entleaching and contamination, there is stiH a highprob- peratureswere a problem 5' C to 26' C! until heaters ability of bacterial contamination. weretemporarily placed in the tanksto offsetthe early Omega Yeast morning temperaturedrop. The heaterswere not needed during the afternoon. BothJapanese and Filipino researchers recognize the Design,Operation and Training ... 103

More Detail of Treatments fected effluent to aquatic life. InternationalOzone Institute, Waterburg,Conn. SymposiumProceed- Fungicide Contamination of larvae by fungi ings, pp. 778-786. &gent'diernsp.! is frequentand a continuouspreventive 2. Blogoslawski,W.J., C. Brown, E.W. Rhodes,and M. treatmentis necessary. Treflan, diluted to 5 ppmsolution, Broadhurst, 1975. Ozone disinfection of a seawater is distributed constantlyat 50ml/ton. supply system,Irt FirstInternational Symposium on Antibiotics Antibiotic treatments are used to control Ozone in Water and Wastewater Treatment. Interna- bacterial contamination, which results in necrosis of the tional Ozone Institute, Waterbury, Conn., pp. 674- larvae and often numerous mortalities. Chloramphenicol 687. has been found to be most effective and is used as a 3. Harris, Weldon C. 1972.Ozone disinfection. Journal preventivewith 2 to 6 pprn every two days or curatively of the American Water Works Association. 64!: with dosesof 2 to 10 ppm. 182-183. EDTA May alsobe neededat 2 pprnas a chelator if 4. Jones,J.R. Erichsen.1964. Fish and River Pollution. some debris appearson larvae or particles seemto be Butterworths, London. adhering to the larvae's setaeor appendages!.EDTA 5. Kelly, C.B., 1974.The toxicity of chlorinated waste increasesthe availability of trace metals,and a 2 ppm effluents to fish and considerations of alternative pro- solution m the LRTs assiststhe larvae metamorphose on cesses.Virginia State Water Control Board. to the next stageif they appearto be having a problem. 6. Kelly, C.B.1965. Disinfection of seawaterby ultravio- let radiation. SecondReproduction. ResearchDepart- Harvesting Larval-Rearing Tanks at Dian Desa ment, Salt Water Fisheries Division, Florida Board of Conservation, Tallahassee.12 pp. Whenlarvae staging shows that 100percent of thetank's 7. Kelly, C.B.1961. Disinfection of seawaterby ultravio- contentsare in the PL stage,and three to four days have let radiahon. American Journal of Public Health, passed larvae are at PL3or PL4!,then theLRT is gravity 511!: 1670-1680. drainedinto the PI. racewaybelow. The Arterrtiaand algae 8. Kjos, D.J.,R.R, Furgason,and L.L. Edwards. 1975. from the LRT act as a food source, but are diluted out when Ozonetreatment of potablewater to removeiron and transferred to the 20,000-liter,maximum-capacity raceway manganese:preliminary pilot results and economic which is usuallyonly half full at stockingtime!. Therefore, evaluation. In First International Symposium on additional Arternia must be added as soon as the PLs are Ozone for Water and Wastewater Treatment. Inter- transferred.Fast siphoning PLs into a small bucket for national Ozone Institute, Waterbury, Conn., pp. 194- transferto the racewayshould be avoided.This may actto 203. cleanup thewater,but wiii causea 50-percentlossofanimals 9. Leiguarda,R.H., O.A. Pesoand Z.R. Palazzola,1949. dueto handling.A gravitydrain or a slowsiphon directly to Bactericidal action of ozone. Abstracted in Water Pol- theraceways below appears tobe thebest method. Another lution Abstracts 222!:268. method is to flush out the LRT with a continuous flo w prior 10. Nebel,C., P,C.Unangst, and R.D. Gottschling.1975. to harvestor siphonham the top down. Ozone disinfection of second effluents: laboratory Extreme care should be taken to see that conditions in studies.In First International Symposiumon Ozone the racewayare as closeto thosein the LRT as possible for Water and Wastewater Treatment. International temperature,pH, etc.!before transferbegins. Example: Ozone!nstitute, Waterbury, Conn., pp. 383400. Racewaywater tern perature on August3 700hours! was 11. Pavoni,J.L., M,E. Tittlebaum, H.T. Spencerand M.R. Fleishman, 1975. Ozonation as a viral disinfection 25.9'C and LRT water temperaturewas 28' C. Flushing technique.International Ozone Institute, Waterbury, the racewayto bring the temperatureup wasnot possible Conn,Symposium Proceedings, pp. 340-366. becausethe inflow temperaturewas 24 C, sotransfer was 12. Smith, W.W. and R.E. Bodkin, 1944. The influence of postponeduntil the afternoon.At 1200hours, the inflow hydrogenion concentrationon bactericidalaction of watertemperature was 28'C and the racewaywas at 26.9' ozone and chlorine. Jml. of Bacteriology47:445. C, A rapid flow wasgiven to bring the temperatureup, A 13. Sproul,O.J. and S.B.Majumbar, 1975. Virus inactiva- flow-through wasalso initiated in the larval-rearingtank tion with ozone in water, In Symposium Proceed- to acclimatethe PL4sto pH ,9 in LRT and 8.2 in the ings,International Ozone Institute, Waterbury, Conn. inflow or raceway!. pp. 288-295, Thesesteps must be taken to minimize transferloss. Keeprecords of everything that is done so that when a Algae Culture problem developsa solutioncan be found or an answer 1, Enright, C. 1984.Determination of the relativevalue can be discerned.Record temperatures,pH readings, of phytoplanktonfor feedingthe juvenile oyster, Ostrea feedingtimes and what was fed for eachbatch of larvae. edulis, Doctoral dissertation, Halifax, Canada. Usethe larval rearing data log Figure 39! to keep these 2. Fox, J. 1983.Intensive algal culture techniques. CRC records,Place the data log on a clipboardnear the tank Handbook of Mariculture, Vol. I. CRC Press, Inc., with a thermometer and pencil nearby. BocaRaton, Fla. p. 15-42. 3. Griffith, G.W., M.A. Murphy and L.A. Ross, 1973,A Selected References mass culture method for Tetraselmissp. W.M.S, 4:289- 294. Ozone 4. Guillard, R.R.L. 1975. Culture of phytoplankton for 1. Arthur, J.W.and D.l. Mount, 1975.Toxicity of a disin- feedingmarine invertebrates.In Cultureof Marine InvertebrateAnimals.W.L. Smith and M.H., Chanley, ed. Plenum Press,N.Y. pp. 29-60. cultural Experiment Station, P.Q.Box Q, Port Aransas, Hamilton, R. 1979.Sterilization. Handbook of Phyco- TX 78373. logicalMethods. CambridgeUniv. Press,Cambridge, Biedenbach, J.M., L.L. Smith, T.K. Thorn sen, and A.L. Mass. Lawrence, 1987. Optimal level of the nematode Keller, M.D., W.K.Beliews and R.L. Guillard, 1988, Panagrellusredivivus, in the larval penaeid diet. Jour- Microwavetreatment for sterilizationof phytoplank- nal WAS 18:13A.Aquaculture Communiques il41. ton culture media.J. Expo. Mar. Biol.Ecol. 117: 279- 10. Chamberlain, G. 1988.Coastal Aquaculture Mewslet- 283. ter. Vol. V, No. 2, page 9, and Vol. V, No. 1 Jan,1988; McLachlan, J. 1973. Growth media-marine. In Hand- entitled "Shrimp Ha tcheries." book of Phycological Methods, Culture Methods 11. Chen,J,C., P,C. Liu, andY.T. Lin, 1989 manuscriptin and Growth Measurements,J R. Stein,ed, Cambridge review, JournalWAS!, "Larviculture of P. monodonin University Press,London. a recirculation and a semi-static system." Dept. of Smith, L,L,, J,M. Fox and G.D. Treece. 1992.Intensive Aquaculture Nat, Taiwan College of Marine Science algae culture techniques, CRC Handbook of Maricul- and Tech. Kellung, Taiwan 20224,Rep, of China. ture CRC Press, Inc,, Boca Raton, FL 12. Chen, J.C, and T.S, Chin, 1988a. Acute toxicity of Simon, C.M. 1978. Thecultureofthediatom Chaetoceros nitrite to tiger prawn, P. monodon,larvae, Aquacul- gracilis and its use as a food for penaeid protozoel ture, 69:253-262. larvae. In Aquaculture 14:105. 13, Chen,J.C. and T.S. Chin, 1988b.Joint action of ammo- 10 S tarr,R.C., 1978.The culture collection of algae at the nia and nitrite on tiger prawn P. monodonpostlarvae. University of Texasat Austin, Journal of Phycology Journal.WAS 19!:143-148. Vol. 14, Dec. 1978 plus supplement, additions to 14. Chen, J,C., T,C. Chin, and C.K. Lee, 1986. Effects of UTEX Conection since 1978, dated 1985!. ammoniaandnitriteonIarvaldevelopmentofshrimp 11 Stein,J.R,1973. Handbook of PhycologicalMethods, P. monodon!.pages 657-662 in Maclean, Dizon and Culture Methods and Growth Measurement. Carn- Hosillos, ed. The first Asian fisheries forum, The bridge Univ. Press,London. Asian FisheriesSociety, Manila, Philippines. 12 Wheaton, F.W1987, Aquacultureengineering. Rob- 15. Chin, T,S. and J.C. Chen, 1987.Acute toxicity of am- ert Krieger Pub. Co., Malabar, Fla. 708 pp. monia tolarvaeof the tiger prawn, P,monodon. Aquac- ul ture, 66:247-253. Larval Rearing 16, Cook,H.L. and M.A. Murphy, 1971.Early develop- 1, Al-Haji, A.B., C.M. james, S. Al-Ablani, and A.S.D. mental stagesof the brown shrimp, P, aztecus Ives!, Farmer, 1985,Advances in hatchery production of P. reared in the laboratory. Fishery Bull. 69!:223-239, semisulcatus, Kuwait Institute Sci. Research, Bull. of 17, Cook, H.L. and A.M. Murphy, 1969. The culture of Mar. Sc. !: 143-154. English Abstract!. larval penaeid shrimp, Trans. Am. Fish. Soc., 98!: 2. Amjad,S. and D.A, Jones,1989.A comparisonof feeds 751-754.. used in penaeid larval culture. Presenteda t1989 WAS 18. Cook,H,L. 1969.A methodfor rearingpenaeid shrimp Conferencein Los Angeles, CA. USA. larvaefor experimental studies. FAOFish Rep.,7: 3. Aquacop, 1977,Larval rearing of penaeids in a tropi- 709. cal environment. Actes de Colloques du CNEXO, 19. Cook,H.L. and M,A. Murphy. 1966.Rearing penaeid 4:179-191 in Frenchwith Englishabstract!. shrimpfrom eggsto postlarvae.Proc. Conf.S.E. Assoc. 4, Aranyakananda, P, 1985.Larviculture of native white Game Comm. 19, 384. shrimp, P. sefiferusand exotic white shrimp, P, 20. Dobkin, S. 1961,Early developmentalstages of pink vannamei, at ContinentalFisheries, Ltd., PanamaCity, shrimp, P. duorarum,from Florida waters. Fish. Bull., Florida An internship report submitted to the De- 6190!:321-349. partment of Wildlife and Fisheries Sciences,Texas 21. Emrnerson, W,D. and B. Andrews, 1981. The effect of A&M University,! stocking density on the growth, developmentand 5. Aujero, J.,Tech. E., and Javellana,S. 1985,Nutritional survial of P. indicus larvae, Aqua, 23:45-47. value of marine yeast fed to larvae of P. monodonin 22. Emmerson, W.D., 'l980. Ingestion, growth and devel- combination with algae. ln Proc. of First Interna- opmentof P.indicus larvaeas a function of Thalassiosira tional Conf. on the Culture of Penaeid Prawns/ weisflogii cell concentration. Mar. Bio. 58:65-73. Shrimps. Taki, Primavera, and Llobrera Eds.!, 23. Ewald,J.J. 1965. The laboratory rearing of pink shrimp, SEAFDEC; Philippines. P, duorarum. Bull. Mar. Sc. Gulf Caribb., 15!;436- 6. Besbes,R. and J, Guillaume, 1989.Effect of protein 449. level and iron supplementationon the growth and 24. Furukawa, I., K. Hidaka, and K, Hirano,1973. Produc- survival of P.japonicus larvae fed microbound diets, tion of larvaeof P,japonicus with marineyeast. Bull. Presentedat 1989WAS Conferencein LosAngeles, Fac.Agr. Miyazaki Univ., 20 l!:93-110. CA, USA. 25. Fontaine, C.T., D.B. Revera, H.M. Morales, and C.R. 7. Biedenbach,J.M., L.L. Smith, T.K. Thomsen, and A.L. Mock,1981.Preliminary observations and procedures Lawrence,1989a. Use of the nematodePanagrellus for themass culture of thenematode Panagrellus redivius redivivusas an Artemiareplacementin a larvalpenaeid for use as food stuff in penaeidshrimp hatcheries. diet. Journal WAS 20!:61-71. Spec.Publ. Galveston Lab. NMFS, NOAA, Galveston, 8. Biedenbach, J.M., L.L. Smith, and A.L. Lawrence, Tx. 1989b. manuscript in review!. Preliminary findings 26, Fontaine, C.T. and D.B. Revera,1980. The mass cul- on the use of a new spray-dried algal productin ture of the rotifer, Brachionusplicatilis, for useas food penaeidlarviculture. TexasA&M University, Agri- stuff in aquaculture. Proc.WMS 11:211-218. Design,Operation and Training ... 103

27. Fielder, D.R., J.G. Greenwood, and J.C. Ryall, 1975 Res., 1!:41-46 in Japanese!. Larval development of the tiger prawn, P. escrtlentus 45. Leger, P. and P. Sorgeloos, 1987.I~proved hatchery Haswell!, reared in the lab. Aust. J. Mar. Freshwat. productionof postlarvalP. vannamei through applica- Res., 26:155-175. tion on innovative feeding strategies with an algae 28. Gabasa,P. 1981.Recent developments in design and substitute and enriched Artemia. Journal WAS managementof a small-scalehatchery for P.monodon 18!:17A. Aquaculture Communiques ¹56. in the Philippines. FAO/UNDPSCSP Working Party 46. Leger,P.1986. Theuseand nutritionalvalueof Artemia on small-scale shrimp hatcheries in S.E.Asia. as a food source. Oceanogr. Mar. Biol. Ann. Report. 29. Gabasa,P. and F. Sunaz, 19S1.Design and con truction Vol. 24, pp. 521-623.Aberdeen Univ. Press, of a low-cost hatchery system. Paper submitted to 47. LeMoullac, G.R. Leguedes, G. Cuzon, and J SEAFDEC,Philippines. Goguenheim,1987. Micro-particulatediets for penaeid 30. Gopalakrishnan, K.1976. Larvalrearingof red shrimp shrimp larvae, Journal WAS 18!:18A, Aquaculture P. rnarginatus.Aqua., 9:145-154. Cornrnuniques¹61. 31. Hameed, A.K., S.N. Dwivedi, and K.H. Alikunki, 4S. Levine, D.M., S.D.Sulkin, 1984.Ingestion and assimi- 1982.A new hatchery system for commercial rearing lation of microencapsulated diets by brachyuran crab of penaeidprawn larvae. Bull. Central Inst. Fish. larvae, Marine Biology Letters 5:147-154. Education, Bombay, 2-3:9. 49. Levine, D,M. S.D. Dulkin, and L. Van Heukelem, 1983. 32. Heinen, J.M. 1976. An introduction to culture meth- The design and development of microencapsulateddi- ods for larval and postlarval penaeid shrimp. Proc. etsfor the study ofnutritionalrequirementsofbrachyuran WMS, 7:333-343. crab larvae. Pages193-203 in C.J.Berg Jr. ed.!, Culture 33. Hirata, H., Y. Mori, and M. Watanabe,1975.Rearingof of Marine Invertebrates. Hutchinson RossPublishing prawn larvae, P.japonicus, fed soy-cake particles and Company,Stroudsburg, Pennsylvania. diatoms. Mar, Bio., 29:9-13. 50. Liao, I.C., 1988.Feasibility study for alternate culture 34. Hudinaga, M. and J. Kittaka, 1967. The large scale species in Taiwan-P. peniciliatus. WAS meeting Jan, production of the young kuruma prawn, P,j aponicrts 1988. Honolulu, Hawaii. Bate!. Bull. Plankt Soc. Japan. 35-67. 51. Liao, I. 1984. A brief review of the larval rearing 35. Hudinaga,M. 1942.Reproduction, development and techniques of penaeid prawns. In Proc. of First Inter. rearing of P.japonicus. Nat. Res.Council of Japan, Conf. on the culture of penaeid prawns/shrimps. Tokyo., 10!:305-393, Pub.by SEAFDEC.1985, pages 65-78. 36. Hudinaga,M. 1935,Studies on the developmentof P. 52. Liao, I.C., editor, 1977. Manual on propagation and j aponicus Ba te!. Rep. Haya torno Fish. INST.1!:1-51. cultivation of grass prawn, P. monodon.Tungkang 37. Imarnura, S, and T. Sugita, 1972.Penaeid shrimp Mar. Lab., Taiwan. seedling mass production technology development 53. Liao, I.C. and T.L. Huang, 1972,Experiments on the studies:early rearingof larvaeusing artifical organic propagationand culture of prawns in Taiwan.Pages suspendedmatter, Culture Tech,Res. 1!:35-46, in 328-354V,R. Pilag Ed.!.In CoastalAquaculture in the Japanese!. Indo-PacificRegion. Fishing News Books!Ltd. 497 38. Jones,D.A., K. Kurrnaly, and A. Arshard, 1987.Feed- PP. ing and assimilation of artificial feeds by crustacean 54. Liao, I.C., T.L. Huang, and K. Katsutani, 1969.A larvae.Journal WAS 18!:17A. Aquaculture Com- preliminary report on the artifical propagationof P. muniques¹58. monodon,J.C.R.R. Fish. Ser.,8: pages67-71, 39. Jones, D.A., 1985. Penaeid larval culture using 55. Maeda,Y., 1968.The seedlingproduction of penaeid microencapsulated diets. Abstract!First Inter. Conf, shrimp. Fish Culture, 5!:64-70 in Japanese!. on the CuIture of PenaeidPrawns/Shrimps. Pub- 56. McVey,J.P., 1983. Handbook of Mariculture, Vol. I. lishedby SEAFDEC,Iloilo, Philippines, Oct. 1985, CrustaceanAquaculture, CRC PressInc.. BocaRa ton, 40. Jones, D.A., A. Kanazawa, and K. Ono, 1979. Studies FL 442 pages. on the nutritional requirements of the larval stagesof 57. Millamena, O. and E.J.Aujero, 197S.Preserved algae P.japonicus Bate>using microencapsulateddiets. Mar. as food for P. monodonlarvae. SEAFDEC, Quarterly Biol54:261-267. Report 2:15-16, 41. Kanazawa, A., S. Teshima, H. Sasada, and S.A. 58. Mock, C.R., D.B. Revera, and C.T. Fontaine, 1980. The Rahman, 1982.Culture of prawn larvae with micro- larval culture of P. stt/hrostris using modifications of particle diets. Bull. of Jap. Soc. of Sci. Fish. 48:195- the Galveston Lab. Technique. Proc. of WAS 11:102- 199. 117. 42. Kuban, F.DJ,S. Wilkenfeld,and A.L. Lawrence,1985. 59. Mock, C.R. and M.A. Murphy, 1970. Techniques for Survival, metamorphosis and growth of four larval raising penaeid shrimp from the egg to postlarvae. penaeid shrimp species fed six food combinations, Proc. World Marie. Soc. 1:143-156. Aqua. 47:151-162. 60. Motoh, H. 1979. Larvae of decapod crustacea of the 43. Kungvankij, P,, 1982, The design and operation of Philippines- III, Larval development of the giant tiger shrimp hatcheries in Thailand. In Working party on prawn, P. monodonreared in the lab. Bull. Jap. Soc. small-scaleshrimp /prawn hatcheriesin South East Sci Fish., 450!:1201-1216. Asia, Semarang, Central Java, Indonesia, 16-21 Nov. 61. Pinto, L.G. and J.J.Edward, 1974. Desarrollo larval del 1981.II Tech.Report, SCSFD & C.P.,Manila, Philip- camaron blanco, P. schmitti. BoI. Cent Invest Biol,, pines, May 1982. Univ. Zulia, 2!:1-61. 44. Kureha,N.andT.Nakenishi,1972.0n theP, japonicus 62. Platon, R., 1978.Design, operation and economics of seedlingproduction in 2800-tontanks. Culture Tech. a small-scale hatchery for larval rearing of sugpo, P, I06 . Treeceand Fox

monodon Frabricius!. Extension Manual No, 1, monodonin combinationwith algae.In: Proceedings SEAFDEC, Philippines. 30 pp. of the First Inter. Conf. on the Culture of Penaeid Primavera, N.G. ed.! 1985. Prawn hatchery design Prawns!Shrimp, Dec. 1984.Iloilo city, Philippines. p. and operation.Aquaculture ExtensionManual No. 168. 9, SecondEdition, SEAFDEC,Iloilo, Philippines. 80. Tang,H.C., 1977.Use of the high-proteinblue-green Rothlisberg,P.D., B.J.Hill, and D.J, Staples, Eds.!, algae Spirulinasp. as feed for shrimp larvae. Chin. 1985, Second Australian National Prawn Seminar. Fish. Mon., 290:2-7. NPS2, Cleveland, Queensland, Australia. 81. Teshima, S. and A, Kanazawa, 1983.Effects of several San Felici, J.M., F. Munoz, and M. Alcaraz, 1973. factors and survival of the prawn larvae zeared with Techniquesof artifical rearing of crustaceans.Stud. microparticies.-Bulletin of the Japanese Society of Rev. GFCM, 2!:105-121. Scientific Fisheries, pages 1893-1896. SEAFDECAquaculture Dept,, 1981. Crustaceanhatch- 82. Ting,Y.Y., T:T. Lu, andM.N. Lin,1977.Experiment on ery. Annual Rep., pp. 13-17. propagationof P.occidentalis. Chin. Fish. Mon., 292:22- 67 Shigueno,K., 1975.Shrunp culture in Japan.Assoc. 28. for Int. Tech. Promotion, Tokyo, Japan, 153p. 83. Treece,G.D. and M,E, Yates,1988.Laboratozyznanual 68 Shigueno,K,, 1972.Problezns of prawn culture in for the culture of penaeid shrimp larvae. Sea Grant Japan.Overseas Tech. Coop. Agency Publ., 37p. College Program publication TAMU-SG-88-202, 69 Simon,C.M. 1981.Design and operationof a large- TexasA&M University, College Station, TX. 95 pages. scale,commercial penaeid shrimp hatchery,J. World Reprinted 1990!. Maricul. Soc. 12!: 322-334. 84. Treece,G.D. and J.M. Fox, 1987.The establishment of 70 Sindermann, C.J. Ed.!, 1987,NOAA Technical Report a successfulP. monodonhatchery in Japara,JAVA. NMFS 47, Feb. 1987:entitled Reproduction, znatura- JournalWAS 18 'I!:20A. Aquaculture Coznmuniques tion and seedproduction of culturedspecies; Proc. of ¹72. the 12th U.S.-Japan zneeting on Aquaculture. Baton 85. Treece, G.D. and N. Wohlschlag, 1987.Raising food Rouge,LA, Oct. 1983. organismsfor intensivelazval culture. Chamberlain et 71 Skokita,S., 1970. A note on the developznentof eggs al., Eds.! 1987; Manual on Red Drum Aquaculture and larvae of P. Iatisuleatus.Biol. Mag. Okinawa, 6:34- out of print!. 1990update available from TexasA&M 36. University SeaGrant College publication TAMU- 72. Smith, LL.J.M. Fox,G.D. Treeceand J.P.McVey. 1992 SG-90-603,entitled "Red Drum Aquaculture." p. 57- Intensive larviculture techniques. CRC Handbook of 76!. Mariculture. CRC Press, Inc., Boca Raton, FL 86. Treece,G.D., 1985.Larval Rearing Technology, Sect 73 Sorgeloos,P. et al., 1986.Manual for the culture and III pp.43-64In: Chamberlain et. al, 1985;Texas Shriznp useofbrine shrimp Artemiain aquaculture.Prepared FarmingManual outof print!. for the Belgian Administration for Development, 87, Tseng, W, 1987. Shrimp Mariculture, A Practical and FAO; State Univ. of Ghent. Artemia Reference Manual. Chien Cheng Publisher, Republic of China. Center!. 88. Yang, W.T., 1975.A manual for large-tank culture of 74 Sorgeloos, P., E. Bossuyt, P. Lavens, P. Leger, P. penaeid shrimp to the postlazval stages. Sea Grant Yanhaecke, and D. Versichele, 1983. The use of brine Technical Bulletin 31. 94 pp. shrimp Artemiain crustaceanhatcheries and nurser- 89. Yates,M.E., G.W. Chamberlain, and R.J.Miget, 1987. ies, pages 71-96 in J.P,McVey Ed.>, CRC Handbook Effectof long chainhighly unsaturatedfatty acidson of Mariculture Vol. 1. CRC Press, Inc., Boca Raton, penaeid larval nutrition. Journal WAS 18 l!:14A. Florida. Aquaculture Communiques ¹46. 75 Sorgeloos,P., 1979.The life history of the brine shrimp 90. Viiialuz, D.K., A. Viilauz, B. Ladera, M. Sheik, and A Artemia. In:Inter. Symposiuzn on the Brine Shrimp Gonzaga, 1972. Reproduction, larval development Artemia, Abstract and picture of Arternia egg and and cultivation of sugpo, P. monodon!.Philip.J. Sci., nauplii, 98:205-236. 76 Sudhakaro, R., 1978. Larval development of Indian 91. Villegas, C.T. and A. Kanazawa, 1980. Rearing the penaeid prawns. In: Coastal Aquaculture: Marine larval stages of the prawn, Penaeusjaponicus Bate!, Prawn Culture, CMFRI Bull. 28:60-64. using artificial diet. Memoirs of the Kagoshizna Uni- 77 Tabb, D,C,, W.T. Yang, Y. Hirono, and J. Heinen, 1972. versity Research Center for the South Pacific, Vol. A manualfor the cultureof pink shrimp, P.durorarum 1 I!:43-49. from eggs to postIarvaesuitable for stocking. Sea 92. Wilkenfeld, J.S.,A.L. Lawrence,and F.D. Kuban,1983 Grant Spec. Bull. No. 7, Univ. of Miami. 57 pages. A reliable small-scaleexperimental system for rearing 78 Tacon, A,G,J,, 1986, Larval shrimp feeding. Crusta- penaeid shrimp larvae in a synthetic sea-saltmedium. cean tissue suspension:A practical alternative for Crustaceana Leiden! 1:72-81. shrimpculture. Aquaculture Development and Coor- 93. Yellow Sea Fisheries Research Institute, 1978. Artifical dination Programme,United Nations Development cultivation of penaeidshrimp. Sci. Publ: pp, 77 in Programme,FAO Report ADCP/MR/86/23.Rome, Chinese!. Italy. 79 Taki, Y., J.H. Primavera, and J.A. Llobrera, Eds.! 1985. Nutritional value of znarineyeast fed to larvae of P.

108 ... Treece and Fox

Example:PL racewaytank Vol. = ~227 4 2 x h V = ~3.34500 2 x 65cm 4 4 d = 500 crn h = 65cm V = 12,756/50 cms V = 12,756 liters Flow rate in the raceway is set at 100percent exchange per 24hours. This meansa flow rateof 531liters per hour or 8.85liters per minute for a tankof this volume. Water inflow is spreadout and forcedin a circularflow. A 1-inch pipe is supported acrossthe diameter of the tank, just abovethe water level. Approximately ten small holes mm in diameter! are drilled in a row on half of one side of Photo63, Feedingpostlarvae a jinetyground pellet. the pipe. Ten small holesare alsoplaced on the opposite endand oppositeside of the secondhalf of the pipe.When a 100percent exchange of water is applied to the tank via the LRT contents cuts this by one third and gives a savings thedistribution pipe, theforce of the waterjets encourages of 128grams of Artentiacysts. If water quality is known to directional flow. Six 2-inch PVC airlifts are also positioned be very poor in the LRT, it may be necessaryto collect evenlyaround the outsidewalls of the tank and the flows larvae in a screenedbucket, but this should be done only from theseairlifts are also oriented to encourage flow and if needed.Concentrating PLs and handling them,even if maximize circular- tank design. Either an airstone or two slowly and gently, usually results in stressand some directionalairlift pipes shouldbe placedin the centerof mortality sometimesas high as 50 percent!. the tank to keeplarvae and debris from settling there. Postlarval Feeding As a Analpreparation before stocking PLs in the race- way,a fungicideis addedto preventthe larvae from being Photo 63! The postlarval feeding regimeconsists of contaminatedby fungi Lagenidium, etc.!. SeeTable 1, calculating100 percent of thebiomass in the tank assum- Chapter3 SuggestedLarval Culture Sequenceand Post- ing PLls weigh 0.0008g each,PL5s weigh 0.005g eachand larval Feeding,"for preventiveamounts and a schedule PL20sweigh 0.02g each and areapproximately 1 cmlong!. for treatment. Fifty ml Treflan/m3 seawateris added These calculations have worked well thus far and have from a 5-ppznsolution of Treflan.Treflan from the United maintained more than 200,000 larvae in Dian Desa's race- Statesis 44.5percent active, so a specialformula wasused ways with very little mortality. There must be a fairly to calculate the amount for a 5-ppzn solution .01 ml of accuratepopulation estimate when the animals are placed 44,5 percent active Tref1anwas placed in one liter of in the tank as accurate population estimates are question- distilled water to obtain the 5-ppm solution!. From that 5- able after the PLs are in the raceway. Estimation of mor- ppm solution, the 50 ml/Ton treatznent was taken. talities removed during siphoning and cleaning routines Once the seawater filtration and treatznent systems would be vezytizne-consuming, but possible.President's were fully operational at the Dian Desa Hatchery UV ¹ NEW 1 feedis given to the PLs at 100to 200percent/ syste~ was installed as planned, etc,!, thesepreventive body weight/day. The feeding times are divided into at treatznents were not necessary. least four times a day 700, 1200, 1600and 2200 hours!. President's¹ NEW1 isa finely ground,40-percentprotein Raceway Stocking level feed madein Taiwan and is an excellent pellet to start When all preparaflonsare complete,the racewayis feedingat PL6.Use of continuouswheel-type feeders is an ready to be stocked.Somewhere around 10,000-12,000 efficient way of offering the PLs a continuous versus PL4s/znz00 PL4s/ft ! should be stocked,or approxi- intermittent source of nutrition. matelyy150P00 PLs per raceway.When all 15raceways are The standard President's ¹ NEW 1 heed consists of the stocked at this density, the hatchery will have a carrying following ingrediants: capacity of more than 2 million PLs. Further testing with Protein 34.8 percent habitats may show that stocking densities can be in- Lipid 2.8 percent creasedwith proper management. Fiber 3.4 percent PL4s are siphoned directly from the larvae-rearing Ash 16.0 percent tanks to the raceways below them, With a 100 percent Moisture 13.0percent waterexchange per day, any residualcontaminatedwater Calcium 5.7 percent comingfrozn the LRT will be flushedout. Theadvantages Phosphorus 2.6percent of adding algaeand six Arterrtia/rnlfrom the LRTvolume Water stability 24-36 Hours offsetsany contaminationthreat and, if properconditions Arterrtiaare alsoused for food, startingat 6/ml at PL4 havebeen maintained in the LRT,it will help to maintain and taperingoff completelyby PL11 seeTable 1 forfeed a stability for the larvae during the transfer. To feed types and amounts for the various stagesand ages!.If aniznals in 12,756 liters would take more than 76 million temperatures have been maintained at 28'C, +I'C, a PL11 Arfemianauplii or about 383grams of cysts.Transferring should be large and strong enough to swim against a Design, Operationand Training ... 109 gentle current in the tank, It should be able to move fzeely to the bottom to feed and return to the net habitat to zest, If temperatureshave been lower than the optimum, then Arterrtiamay be required as feed until the animals have been observed swimming freely in the tank and not being constantlyswept around by the currents.In other words, an animal grown at 26 C would not be able to accept a pellet diet asits only food sourceas would the sameaged aniznal that had been held in 28'C. A diet that stays in the watercolumn is requiredif the animal is smalland weak. The guideline shown at the bottom of the page is recommended for feeding PLs at Dian Desa if the water teznperature is maintained at 28 C, +1 C: Table 4 gives an alternatepostlarval-feeding guide- line/feed requireznent scheme and estunated feed costs per month for the Dian Desa hatchery. Photo64, Siphonused to clem PI. holding tank a%ennecessary, It is recommended that the volume of the raceway be kept around 12to 13tons for greater temperature stability. A small-zneshed screenbucket can be placed to catch the Ar temiain the effluent. TheseArterrtia can be fed algae and returned to the tank as a good food source or a smaller sczeencanbe placed to retainthe Artentia,if smallamounts of algae are added to the raceway each day for them. This ensures a good food source, Excessfood and metabolic wastes are removed by siphoning necessary Photo 64>. Daily Inspection A daily inspection ofaniznals in eachtank is necessary to detect potential problezns.Samples of the animals should be observed under the microscope each morning, looking at the following characteristics: ~ Guts full or empty! ~ External debris ~ Diseases ~ Growth Photo 65. Postlaroalreanng and holding data board formicaboard ~ Eye surface loaated near the tank!. ~ Gills ~ Periopods ~ Pleopods ~ Telson spines ~ Uropod seatae ~ General pigmentation or color ~ Length of animal

Stage Arterrtia/Ml President's ¹ New I PL4 PL5 100percent body weight 5 percentx 4 times/day! PL6 200 percent body weight 0 percent x 4 times/day! PL7 200percent body weight 0 percentx 4 times/day! PL8 200 percent body weight 0 percent x 4 times/day! PL9 200percent body weight 0 percentx 4 times/day! PL10 200 percent body weight 0 percent x 4 times/day! PL11 200percent body weight 0 percentx 4 times/day! PL12 200percent body weight 0 percentx 4 times/day! PL13 200 percent body weight 0 percent x 4 times/day! PL14 200percent body weight 0 percentx 4 times/day! PL15 200percent body weight 0 percentx 4 times/day! PL16 200percent body weight 0 percentx 4 times/day! PL17 200percent body weight 0 percentx 4 times/day! PL18 200percent body weight 0 percentx 4 times/day! 110 ... Treeceand Fox

After this inspection,each tank shouldbe individually is used. Postlarvae with a 5:1 ratio have been found to treated in accordance with the findings. The following havethe most success, The best larvae may not be the observations should also be noted: most colorful or the most active. In fact, hyperactivity ~ Is waterfiow 100 percent has been associated with stress. ~ Are airlifts working ~ Fatty acid compositionof postlarvaehas also been ~ Are animalsspread out evenly usedto evaluate postlarvaequality E. Arellano,1990 ~ Is there excess food on the bottom of tank WASConference in Halifax!.A pH-stresstest.5-7.5! ~ Is there no food in tank wasapplied to animalsto evaluatethe physiological ~ Any mortalities condition of thelarvae. Higher resistancewas present ~ If Ar temiaare being fed, is thereenough and arethey in the postlarvaewhen the body compositionwas spreadout evenly or lying on the bottom. high in highly unsaturatedfatty acids, HUFAS!espe- All of these observations should be made by a person ciallyywith thelonger-chain polyenoic acids C:205W3 experiencedin postlarval-rearingwho is capableof react- andC:22:6W3!. Also,postlarvae are expected to have ingpositively to theobservations. It will not do any good a bettergrowth efficiencyin ponds study performed to make the observations and do nothing about them. at the Departmentof Marine Science,ESPOL, Ecua- dor!. Diseases and Treatments at Dian Desa The following are tips for buying and stocking healthy The diseases and treatments are almost the same for postlarvae postlarvaeas they are for larval rearingexcept that the Tail treatmentsarea little strongerin somecases with postlarvae A PLs tail should be noticeably open. The preferred than with zoea. Consult Chapter 3 on signs of stress, ageof PL at stockingis PL 18+for P.Morrodorr and PL diseasesand treatments in the larval-rearing section and 5-10 for P. Vannamei. consult Table 1 for treatment amounts and preventive Color levelsand Table7 in Chapter6 for a list of diseasesand Goodquality PLshave transparentbodies with deep their control methods.! rust-like, greyish or dark streaks along the body, Thefollowing is a listof diseasesand typeof treatment: Those with brown to black coloration are still accept- ~Bacteria Shows up as necrosis and red nerve- able. Postlarvae that perform poorly are those show- ending in the tail and head regions, ing signs of red or pink coloration which can be generaloverall weakenedcondition, directly related to rearing or transport stress. not eating,empty guts. Treat with Activity Chloramphenicol-10ppm!. PLsshould visibly be strong.They should be actively ~Zoothamnium Shows up on the exoskeleton almost swimming from side to side!. Active PLs swim against overnight. Treat with 'Ipprn malachite the current when the water is agitated or whirled. green. When in a basin they may not always be moving, but ~Lagerridium Show up on the exoskeletonalmost will reactto a tapon the side of the bowl, movement overnight, Treat with 50 ml/Ton, 5- in the water, or shadows. pprn solution of Treflan. NOTE: Ac- Size cording to Williams et aI., 1986,trefian At PL20,the averagelength of P. rnonodomnisabout 18 triflurain! is rapidly lost by aeration, mrn. To the trained technician or grower, the normal photodegradation light! and absorp- length at a certain age can be easily detected. The PL sizeshould be uniform and any significantvariation tion by algae and needs to be adminis- is indicative of different age levels. Growers can gen- tered drop by drop for 12 hours per erally acceptage variations so long assize differences day. are minimal, Each of these treatments should be done with static Appearance water conditions or should be done on a daily basis if a PLs are inspected for presence of infestations and flow is maintained. other physicaldefects or abnormalities, such asdebris Records adhering to the body and swimmerets,broken ros- All observations and data are kept on the postlarval trurn, crooked body, etc, Healthy PLs have a gener- holding and rearingdata log, which is kept on a clipboard ally clean appearance and have no physical abnor- or formica board, Photo 65! near the culture tank see malities, Table 5!. Feeding Observation of good feeding requires a field micro- Identification of Postlarvae and Recognizing scopefor exactdetermination. Healthy Fry feed ac- Healthy, Desirable or Undesirable Larvae tively and havea Fulldigestive tract, unless there is no The "rule of thumb" for selectionof good Perraeus food available,such as after a long shipment.If you rnonodonpostlarvae by buyers in Indonesiawas to "look observe Fry at a hatchery and notice that the gut is for thelong blackcigars." New studiesfrom Taiwanhave empty, beware. When animals are sick or stressed indicated that P. monodort postlarvae can be selected by they usually don't eat. using the following criteria: Clean shell ~ A ratioofhepatopancreas height to tail muscleheight A clean shell indicates that the animal is growing fast Design, Operationand Training... 111

RRRRRRRSRRSSSSRSRSSRRRSRRRRRSRRR

EA IXI H Ch W K N W tt 4 W H IA r.geV LA w 8 w 8 6 h, CD rF N 00 Y! + 4 X 8 IA EOX rh QOR re CV CV Y! 9 5 CD 8 ~ IA P g IA P W I PVM W W M P Ch Q g Q g CV $ $ Q W W Ch RrSCDrAIO.4hrCV>$AIf4f4CDY! M 8 00 VAICVAICV<VNAIAI

Nhl AI NCVCVAICVPICV&fV VAlfVAlhlhl 'a QI IDg th X f4QOggCDrp!~w 'IQ'w g P R w Ih 0~v4 4 ~ V!Ch I <+g ~I ' CDj~Ob.N+hchPV CD Ch 4 4 9 Y!w CD QO5 I @Y! IA PvUQ

0 WWWWWb h h WW h g w M C4 IQI

I $%CR~ O K ma" I $ I '4 '4 AI Al

'a

ID cn ID LA 'QDQO w rv CV A w N K w Y! P! PV IA w tentw w w h % Ch W W R % 00 QDQO QO QO P PKU I QO Ch CD U'U8 'AW< CaFt II gwA mINNIsBBeNWNNIINI~NIIsmNml--m tD, II U' M'4D'40N ih N lA AI IAM % N W MIAw Ih LAW ~ WP4 N h4 PQO CD l4 I QO uKVa QO CD

3 IQ 888888888g g888888888888888 5 VbO FmH RICED II 4 'CI M cD8 @CD'

,6 0Oy 0 0 PJ

~UUU

CD Al rh W LA N b. QO4 CDr hl K W LA CV hl AI Al AI N LLOLLLLO 112 ... greece arrd Fox

TabIe 5. Postlarval Holding and Rearing Data Log.

Raceway ¹ Date Stocked

~D Iime ~Ha/& ~Tm~ Arr/ml //.mtfal T~r~~n Design, Operationand Training ... 113

and molting frequently,Slow growth is indicatedby Harvesting Postlarvae the presenceof protozoans,other dirt and necrosis Whenpostlarvae are soldor readyto be stockedinto black spots! on the shell. ponds they may be drain-harvestedinto a harvestbasin Good muscle development suchas theone in Figure 14. Postlarvae may thenbechilled Usinga microscope,muscle development can be seen to 18'Cand boxed in styrofoamcoolers, with 1,000PL's/ in the sixthtail segment.The muscleshould com- liter of seawater,and shippedfor long distancesby air pletelyfill the shell from the gutdown to the under- freight.Usually 10liters of seawaterare placed into each side.In older fry PL22and up! thismay bepigmen ted box, which is equipped with a doubleplastic bag. The and hard to see. When the PL is stressed the muscle innerbag is then filled with pureoxygen and the two bags hasa "grainy"look muchlike thegrain in wood!and are sealed in some manner such as rubber banding or is greyishor brownin color.Healthy fry havea clear, otherbanding devices. smooth muscle. For transportingshorter distances, postlarvae may be In most hatcheries,muscle development is carefully concentrated after harvesting>into containers such as monitored. Muscles are highly susceptible to stress thosein Photo 66! 1,000liter plastic containers,aerated, such asrapid temperature and salinitychanges. Even and transported to the ponds for acclimation. Photo 67! when fry appearhealthy, active and are feeding, if the shows an example of a fiberglass postlarval transport tail muscleis grainy the fry are stressed.If themuscle tank. The tank is equipped with internal standpipe and is clear and thick, then the fry also meet the other screenedoverflow. When postlarvae arrive at the pond, a criteria: full gut, clean shell and good color. 12-volt submersible pump is placed into the pond. Many criteria havebeen adopted by fartnersfor judg- Pondwater is pumped into the tank until acclimationis ing postlarvalquality. Thesehave been discussedby complete.Then the standpipe is removed and postlarvae Motoh and Buri, 1981,Parado-Estapa, 1988 and more are releasedinto the pond through a large flexible hose. recentlyby Fegan,1992. The mostimportant of these The hatchery may also start some of the acclimation by is stageof developmentalong with size,age, stress lowering the salinitybefore the animalsleave. Postlarval tests and physical condition. The following physical transport,acclimation and receivingis coveredin Practi- features can be used to determine PL quality of P. cal Manual for Semi-Intensive Commercial Production monodon: of Marine Shrimp by JoseVillalon, 1991 SeaGrant Col- lege Program, Texas A&M University, Publication 91- Feature Unsuitable Suitable 501!. 6th abdominal Longerthan Shorter than segment carapacelength carapacelength Selected References

Rostral spine 1-5 dorsal 6-7 dorsal 1. Bagarinao,T.U., N.B. Solis,W.R. Villaver 1986.Im- formula 1-2 ventral 2-3 ventral portant fish and shrimp fry in Philippines' Coastal Waters:identification, collection and handling. Aqua. Gill system Incomplete Complete Ext. Manual No. 10. ISSN 0115-5369:52pp. 2. Baumann,R.G.H. and D. Jamandre1990. A practical Behavior Planktonic Benthic method for determing quality of Penaeusmonodon Fabricius!fry for stockingin grow out ponds.in M.B. New, H. de Saramand T. Singh eds.! Proceedingsof the Aquatechconference on Technicaland economic aspectsof shrimp farming. Publ,INFOFISH. Kuala

Photo66. 1~0-liter plasticcontainers asst to transportpostlarvae. Photo67. Postlaroaehauh'ng tank. 114 ... Treeoeand Fox

Lumpur. 3. Briggs,M. andJ.A. Brown 1991. Intensive rearing of postlarvalPenaeus rnonodon in a concretenursery tank system. Paper pres. at 22nd Ann. Meeting of the World AquacultureSociety, San Juan,Puerto Rico. 4. Charmantier, G., N. Bouaricha, M. Charmantier- Daures, P. Thuet and J-P.Trilles 1989.Salinity toler- anceand osmoregulatory capacity as indicators of the physiological state of penaeid shrimps, KAS Special Publ. No. 10:65-66. 5, Cook, H. 1965.A generic key to the protozoen,mysis and postlarval stagesof the littoral penaeidaeof the N.W. Gulf of Mexico. Fish. Bull. 65!:437-447. 6. Dalla Via, G.J. and C. Lederer 1989. Intermediary metabolism in Penaeus vannamei after acute locomo- tory stress. EAS Special Publ, No. 10;285. 7. Fegan,F,F, 1992.Recent developments and issues in the penaeid shrimp hatchery industry. In: Wyban, J., ed.! Proc.of Special Session,WAS. 1992.pp.55-70. 8, Loesch,H. and H, Avila 1965.Clavespara identificaion de camaronespeneidos de interes comercial en el Ecuador Identificationkeys for commercialEcuador- ian penaeid shrimp!. Institutionacional de PescaDel Ecuador. Boletin Cientifico Y Tecnico. Guayaquil, Ecuador. 9. Mallikarjuna, R. and V. Gopalkrishnan 1989.Identifi- cation of juveniles of the prawns P, rnonodonand P. indicus. IPFCfTech. 13:4p. 10 Maugle, P. 1988. Post-larvae sic! shrimp mortality study. Artemia Newsletter 8, Publ. Artemia Refer- enceCentre, Ghent, Belgium. 11 Mohamed, K,H,, P.V. Rao and M.J. George 1970. Postlarvae of penaeidprawns off S.W. Coastof India with key to identi fic tion. FAO Fish.Reports 57<2!:489- 504. 12 Motoh,H. andP. Buri 1981. Identification ofpostlarvae of the genus Penaeus appearing in inshore waters Res. Crust Noll;pp.86-94. 13 Nietes,A.D, 1990.The effectof feedtypes/forms on thegrowth and quality of Penaeusrnonodon postlarvae. Masters Thesis, Asian Institute of Technology, Bangkok, Thailand. 14 Parado-Estapa, F.D. 1988. Selection, transport and acclimation of prawn fry. In "Technical Consider- ationsfor the managementand Operationof Inten- sive Prawn Farms" Y.C. Chui, L.M, Santo, R,O. Juliano eds,! PubL U.P. Aquaculture Society, 172p. Iloilo City, Philippines. 15 PerezFarfante, I. 1970.Diagnostic characters of juve- niles of shrimps. P. azteus,P. duorarurn,and P. brasiliensis.U,S. Dept. Comm. NOAA Rep. 559:1-26. 16 Tackaert, W., P. Abelin, P, Dhert, P. Legere, D. Grymonpre,R. Bombeo,and P. Sorgeloos1989. Stress resistancein postlarval penaeid shrimp reared under different feeding procedures.Presented at Aquacul- ture '89, Los Angeles,U.S.A., Feb. 12-14,1989. 17 Yunker,M. 1990.Tips for buying and stockinghealthy fry. Pages102-103 in D.K. Akiyama ed.! Proceedings of the SoutheastAsia Shrimp Farm Management Workshop. Publ, American Soybean Association, Singapore. Design, Operationand Training ... 115

Chupter5 Alternate Production Strategies for the Yayasan Dian Desa Hatch exy

This supplementsthat in Chapter 1 under "Culture Alternate Production Strategies Tanks." The intent is to suggest that a new hatchery should produce at maximum capacity for a 24-month To produce 3 million PL18s per month, another pro- period until ideal capacity is determined. duction strategycan be used.It is a bit more coznplicated than just simply trying to keep all the tanks full,but if there Maturation is anefficient productionmanagerat the site,this method Two tanks with 20 males and 20 females m each would can be used. be enough if procedures are followed as described in Stocking ail 11 LRTs with 400,000larvae each 00/ Chapter 2 of this manual. Sell all excessnauphior flush the liter! requires1 millionnauplii perdayfor four days.Stock remainder. onday1,2,3and4.Harvest PL3s onday10, ll, 12and13 if tank temperatures have been maintained at 28'C. Re- Larval Rearing stockLRTs on the samedays that theyare harvested. Stock If a larval-rearing tank is stocked every ten days with either eleven raceways with the first stocking and then N6sat 28'C,plus or minus 1'C, it willbe possibleto obtain four raceways ten days later, or stock eight raceways in the threebatches of PLs per znonth. Stock with 400,000N6s or first stockingand sevenraceways ten days later. In both about100per liter. Expectat leasta50 percent survival rate casesproduction will work out to thirty racewaysper or 200,000PLs x 3 per month = 600,000PLs per LRT x 11 month at 100,000 PL18s each, or 3 znillion PLs. Each LRTs = 6,600/00 PL3s, raceway is stocked with 200+00 PL3s that are held for 15 Postlarval Rearing days to produce PL18s!.This production rate can be achievedat the Dian DesaHatchery with propermanage- Stock 15 racewaystwice a month with 220+00PL3s ment.Judging from the resultsof the trial runs conducted holding the PL3s for15 days at 28 C,plus or minus 1 and in July and August 1987,and from the maturationtrials in selling!. Expect at least 50 percent survival rate in PL July and August 1986,these aze conservative estimates. holding,or 3.3x 106PL18s per month. With optimal conditionsand proper diseasemanagement pzeventiveprogram! and/or a better water-treatment system,production could be pushedwell above3 million PLs per month. A schematic of this system follows. 116 ... Treeaeand Fox Design, Operationand Training ... 117

NOTE:The first month's production will notshow the 3 millionpostlarvae, but the following months will eachshow this production if tanksare harvested and restockedaccording to scheduleand there are no othercomplicating factors. 118 ... Thea end Fox Design,Operation and Training... 119

Chapter6 Shrimp Toxicology arcedShrimp Diseases

Introduction Polychlorinated Biphenyls The purposeof this chapter is to review information Polychlorinated biphenyls PCBs! are industrial pol- availableon the pollution ecologyand diseasesof repre- lutants that may strongly affect penaeid-shrimp ecology and aquatic ecology in general. Table 6a presents a sum- sentativeespecies of penaeidshrimp. Data and information mary of how PCBs affect penaeid shrimp. from both field and laboratorystudies, as well as informa- For many years these industrial chemicals have been tion reported in literature, are used.As our oceanwaters present in the aquatic environment as a result of waste continueto be degradedby human sources,shrimp toxi- effluents, disposal of dielectric fluids, and other industrial cology and diseaseswill become pressingissues, espe- ciallyfor thehatchery operator who must maintain pris- sources. It is a weIl-established fact that certain Fresh and marine bodies of water are contaminated with various tine-water conditions. compoundsof PCB.For example,PCBs have been found in water, sediments,and tissuesof animals including Shrimp Toxicology penaeid shrimp! from Escambia Bay near Pensacola, Couch, 1979,plus updates to 1990! Florida. The majority of availablereports regardingpollution Considerable research has been done on the effects of and penaeid shrimp e.g. Overstreet, 1988! concern stud- PCBson estuarinespecies with emphasison pink and ies involving the commercially-valuablepenaeid shrimp brown shrimp. Thesetwo penaeidspecies were killed by of the U.S. Atlantic states and Gulf Coast. Therefore, most a two-week exposure to 0.9, 1.4, and 4.0 pg/liter Aroclor of the information presented here is related to the follow- 1254 in fIowing seawater United States Environmental ing three species:Perrtrerts duorarrtrn pink shrimp!, Perraetts Protection Agency, Gulf Breeze, Florida!. The minimum azfecrts brown shrimp!, and Penaeussetiferrts white concentration causing mortality was 0 9 pg/liter. Penaeid shrimp!, all Atlantic and Gulfof Mexico species.Reference shrimp sufferedgreatest mortality when exposedduring to other speciesof penaeid and some nonpenaeid crusta- pre-molt just before molting! and during molt. Most cea are made when specific studies contribute signifi- exposedshrimp becamelethargic, stoppedfeeding, and cantly to our understanding of the pollution ecology of did not dig into the substrate digging is a normal activity shrimp. for penaeids!.Subtle to dramaticchromatophore changes This section covers the following pollutant categories in the cuticle of exposed shrimp were more frequent and and situations: orgaruc-chemicals other than petroleum obviousthan in unexposedcontrol shrimp. and related compounds, heavy metals, biological agents, On the light-microscopicallevel, no lesionswere con- and interactionsof pollutants and other factors.Under sistently found that were indicative of PCBexposure in each of thesedivisions all known toxicity and specific shrimp. However,several interesting cytophatic changes tissue,organismic, population and ecologicaleffects are were noted in exposed-shrimp studies with electron mi- reviewed.Further, the uptake,transport, and fate of pol- croscopy. lutants is discussed and how they may affect the ecology Pink shrimp wereexposed to 3 !rg/liter Aroclor 1254in of penaeid shrimp. For more details on references to flowing seawaterfor 30 to 52 days, During theseexpo- material in the Toxicologysection of this chapterrefer to suresup to 50percent of the animalsdied. Bothliving and Couch 1979,Overstreet 1988,and Johnson 1989, dead shrimp were analyzedby gaschromatography and from 33 to 40 p.g/gm Aroclor 1254 was found in their Organic Chemicals Other than Petroleum hepatopancreatictissues. Aroclor uptake in the hepato- pancreaswas linear with time. Hepatopancreasabsorp- tive cells Fromexposed shrimp revealed the following Industrial Organic Chemicals departuresfrom thoseof control shrimp: The last four decadeshave witnessed the unprec- ~ An increasedor proliferation of rough endoplasmic edented release of synthetic organic chemicals into the reticulum in 30-50 percent of ceIls, natural environment. These so-called xenobiotics have ~ Productionof membranewhorls with enclosedlipid numerous origins and usesand are rarely innocuou s upon droplets, entering natural waters. Becausepenaeid shrimp occupy ~ Nuclear degeneration characterized by the occurrence estuariesduring a significantportion of their life cycles, of vessiclesin the nucleoplasm 0-50nm and 100-700 they areexposed to pollutantscharacteristic of industries nm in diameter!. associatedwith the estuaries.Relatively few industrial The proliferation of smooth endoplasmicreticulum pollutants have been investigated specifically with regard ER! in hepatocytesof higher animalshas been described to the ecologyof penaeidshrimp. 120 ... Treece arid Fox

Table 6a. Polychlorinated Biphenyls and Penaeid Shrimp Couch, 1979!

PhysiologicaV Ecological/environ- References Compound Toxicity histological eHects mental implications in Couch, 1979 Aroclor 1254 3 pg/liter 30-day Lethargy;chromatophore Pink shrimp absorbed Duke et al. exposurecaused 50% changes;ultrastructurai 1254 from sediments from Nimmo et al. mortality, pink shrimp pathologyin polluted estuary, loss of hepatopancreascells burrowing activity-exposure to predators; shrimp could 0,9 p,g/liter caused not avoid contamination some mortality in 14 waters as some fish could. days,pink shrimp Long life of PCB'sin sedimentsmaintain pollution at chronic levels over severalyears,

Aroclor 1016 10 ling/liter, 96-hour None reported None reported Hansen et al. exposedcaused 43% mortality

0.9pg/liter, 96-hour exposure caused 8% mortality, brown shrimp.

Table 6b. Organochlorine Pesticides and Penaeid Shrimp Couch, 1979!

PhysiologicaV Ecological References Pesticide Toxicity histological eHects implications in Couch, 1979 Chio rd ane 96-hour LC 0,4 ling/liter, None reported Residues in estuaries from Parrish et al. pink shrimp five southern states; effect in nature unknown.

DDT 18 days LC 0.1 pg/liter, Loss of cations Na, K! Death of exposed shrimp Butler et al. No longer white shrimp in hepatopancieas; takes out possible step in Nimmo et al, used in U.S.! possible ATPase tropic accumulationin nature. inhibitor.

Dieldrin 96-hour LC 0.9 itg/liter, None reported Commonly found in estu- Parrish et al, pink shrimp aries; second most common Butler pesticide in mollusks

Endrin 96-hour LC 0.28 ji.g/liter, None reported None reported Schimmel et al, pink shrimp

HeptachIor 96-hourLC 0.11kg/liter None reported None reported Schimmel et al.

Hexachloro- Lethargy in 25 p.g/liter Hepatopancreas Accumulatedby estuarine Parrish et al. benzene and 33% death at 96 grossly, abnormally fishes.Exposed shrimp hours, pink shrimp, white. possibly unable to escape predation due to lethargy.

Lindane 24-96hour LC range, Nonereported None reported Chin and Allan 0,17-33iig/liter; pink, Schimmel et al. brown, white shrimp

Mirex 7-dayLC 1.0pg/liter, Delayedtoxicity to pink Mirex applied as a particle Lowe et al, pink shrimp shrimp 4 days after bait in nature may render Tagattz et al. removal from mirex. it unavailable to many Markin et al, organisms.

Toxaphene LC nauplius,22 ling/liter, Differential toxic Temperaturedissolved 02 Courtenay LC protozoea, 1.8 ug/ responsesof different variations affected and Roberts liter; LC mysis, 1.4 ug/ larval stages. survival in exposed mysis. liter, pink shrimp Design,O~ration andTraining ... 121 asindicative of toxic responseto drugs or chemicalssuch seawater.Exposure to a DDT concentrationgreater than asphenobarbitol, dilantin, dieldrin, and carbontetrachlo- 0,01 pg/liter was lethal to pink shrimp in 28 days. A ride. Proliferation of ER has been related to detoxication of physiologicaleffect of DDT exposurein pink andbrown poisonsand may,in shrimp, representan attemptan the shrimpwas lossof certaincations in thehepatopancreas. part ofhepatopancreaticcells to metabolizePCB absorbed Sodiumand po tassiumconcentrations in shrimp exposed from the lumen of hepatopancreaticducts, If this is the to0.05 pg/liter DDT for 20days were lower than in those case, cellular alterations at the ultrastructural level such as notexposed. Magnesium, however, was not significantly proliferationof ERmaybe valuable indicators of sublethal lowered.The significance of reduced cation in thehepato- effects of certain pollutantsin penaeid shrimp. pancreasof shrimpfor the pathophysiogicalbehavior of Another PCB, Aroclor 1016, has been introduced for shrimp is not known, but a lossof ATPascactivity in ion limited use in thc United States.This compound has been transportmaybe indicated. Blood-protein levels have also testedfor toxicityin brown shrimp. Aroclor 1016was been found to drop in shrimp exposed to DDT. In acute, foundto havenearly thesame toxicity forpenaeid shrimp high-concentrationlaboratory exposures, shrimp showed as Aroclor1254; 0,9 pg/liter Aroclor 1016 in flowing tumors, hyperkinetic behavior and paralysis, all classic seawaterkilled 8 percentof testshrimp in 96hours; 10 gg/ signs of DDT poisoning shrimp did not becomepara- liter Aroclor1016 killed 43 percentof testshrimp in 96 lyzedbut sankinto lethargy,refused food andthen died!. hours. Pink shrimp weremore sensi tive to dieldrin thanwere It is apparentthat PCBs as pollutants pose a threatto grassshrimp Palaemonidae!in testexposures. However, pcnaeidshrimp that show a high sensitivity to these both speciesdied when exposed to concentrationsof compounds.In thisregard, it hasbeen demonstrated that dieldrin in the low parts-per-billionrange. pink shrimp could absorb a PCB Aroclor1254! from Somejuvenile pink and brown shrimp died after labora- sedimentstaken from a PCB-pollutedestuary, Escambia tory exposureto low concentrationsof mirex Table6b!. Bay,Florida. It was found thatsome shrimp showed no However, all survivors from this test died after fourdays in avoidancereaction when given choicesof clean or PCB- mirex-free seawater,demonstrating a delayedtoxic effect of contaminatedwater. These and other data suggestthat mirex.Mircx poisoning in shrimpproduces loss of coordina- PCBs, as pollutants, could have influence on relative tion,equilibriumand finally,signsoflethargyand paralysis. survival and abundance of penaeid shrimp in natural Shrimpand blue crabswere exposedto low concentra- waters, tions of mirex for long periods 0 days or more! and examined for histopathologicaleffects. No pathological Pesticidal Chemicals effects at the tissue level were found in the animals that In the last 30 years, many kinds of chemical pesticides were examined. The organs studied were muscle, the havebeen inadvertently or intentionally rclcascdinto the hcpatopancreasand gonads. environment.Aquatic life is exposed to thesecompounds A detailed ecological-monitoringstudy of the distri- becausethe aquatic portion of thebiosphere often behaves butionof mirex in oysters,crabs, shrimp, and fishes from asa "sink"or receptaclefor thesecompounds because of estuarinewaters from the Gulf of Mexico to Delaware Bay runoff or fallout. Someof thesepesticides, such as certain was done. Sampling stationswere in MississippiSound, organochlorinesortheir metabolites are refractory tobreak- Mississippi;Mobile Bay, Alabama; Tampa Bay, Florida; down and thus tend to accumulatein various compart- Jacksonville,Florida; Savannah, Georgia; Charleston, ments of the aquatic environment, Shrimp have been South Carolina; Morehead City, North Carolina; Chesa- found to accumulatecertain pesticidal compounds in the peakeBay; and Delaware Bay. Only shrimp in the Savan- laboratory, and feral shrimp have possesseddetectable nah,Georgia, area had detectabIeconcentrations ofmircx levels of compoundswhen taken directly from contami- .007' g/gm in tissues!.The Savannah,Georgia, area has natedor apparently "clean"waters. The staff of the U,S, a longhistory of mirex usage.Mirex docsnot appearto be EnvironmentalProtection Agency Laboratory, Gulf Breeze, aswidespread in estuarineregions as are PCBs and DDT. Florida,has found that over severalyear of testing,pcnaeid Mirex, usuallyapplied as a particle-bait poison, would not shrimp generally are far more sensitiveto toxic and eco- be as directly available to many marine organisms as are logicaleffects of mostpesticidcs than are fish or mollusks. broadcastliquids or powderformulations of other pesti- The effectsof some of the well-known compounds are cides. reviewed below. Further work has been reported with The finding of 0.007ug/gm mirex in shrimpnear monitoringpesticides in aquacultureby Vogt 987!, Savannahprobably reflects a considerableaccumuIation of mircx by shrimpover a long period of time from Organochlorines possiblyextremely-Iow concentrations in the environ- The following organochlorine pesticidcshave been ment.It was found Table6b! that25 percentof thepink studiedin regardto organismicand/or ecologicaleffects shrimptested died in the laboratoryafter sevendays of on penaeidshrimp: chlordane, DDT, dieldrin,endrin, exposureto only 1.0pg/liter mirex.Therefore, the concen- heptachlor,hexachlorobenzene, lindane, mirex, and tox- trations of mirex must be assumed to be considerably aphene.Data on the effects are summarized in Table 6b for lowerthan 1.0Itg/liter in Georgiaestuarine waters. If the thesepesticides Couch 1979!. aqueousconcentrations were above 1.0 '/liter fewshrimp White shrimpthat died asa resultof DDT exposure would probably survive in that area. currentlybanned in the UnitedStates! accumulated up to Toxaphencdifferentially affects the early metarnor- 40.4pg/gm DDT and DDE by-product!in thehepatopan- phicstages of pink shrimp Table 6b!. The mysis stage of creasafter 18 daysof exposureto 0.20pg/liter in flowing 122 ... Treeoearrd Fox

pink shrimp is most susceptible to toxaphene, particularly munication!. The hatchery now operates under the name under various temperature and dissolved-oxygen condi- GMSB Joe Mountain and Jim Norris! under the same tions. This might suggest that fluctuations in abundance conditions. of juvenile and young-adult penaeid shrimp in toxaphene Both organophosphates and carbamates are reported contaminated waters could result from impact of the to be potent acetyl-cholinesterase Ache! inhibitors in the pesticide on an early life-cycle stage during periods of vertebrates. Little evidence of early presyndromic inhibi- changingtemperature and/or during periodsof dissolved tion of Ache activity in the ventral-nervecord of pink oxygen fluctuation. shrimpwas found,butinhibitionashighas75percent was found in moribund shrimp experimentally exposed to Organophosphatesand Carbamates malathion. Few organophosphatecompounds have been tested Carbamate pesticides e.g. sevin! have not been tested on speciesof crustaceans,However, those compounds extensively on penaeid shrimp, but sevin may be lethal to tested have been shown to be approximately 1000 times other crustacea when applied to field sites in the marine more toxic to shrimp than most other pesticides tested, environment. Carbary sevin! was found to be quite toxic with penaeid shrimp showing greater sensitivity than to penaeidshrimp in laboratorytests. Brown shrimp have fishes or mollusks. a 24to 96 hour LC50 of 2.5 Itg/liter, the lowest concentra- Baytex Bayer 29, 493! was toxic to penaeid shrimp in tion of sevin reported to kill any crustaceantested. the laboratory. Naled ,2-dibromo-2, 2-diehlorethyl phos- phate! had little effect in field tests on shrimp, Fast dilu- Petroleum tion and instability without persistence of compounds Annual spillageof oil and oil productsinto the ocean may contribute to the lack of mortality of shrimp in field is over3 million tons not including the 1989Alaskan spill testing of this organ ophosphate. In the laboratory, Dibrom by Exxon!. Most of these spills occur in estuaries or near is lethal to postlarvalbrown shrimp at 2.0ILg/liter, and at coastal regions of significant biological value. Of particu- 5.5 pg/liter = LC50 for 48 hours!. It is also lethal to adult lar importance is the fact that many coastal areas effected pink shrimp, Malathion, at 14i'/liter, causedhyperactiv- by oil spills including potentialspill areas!are in penaeid ity, paralysis, and death in penaeids. Parathion's lethal shrimp producing regions. A few detailed studies of eco- concentration for 48 hours in pink shrimp was 0.2 pg/liter. logical and physiologicaleffects of oil on penaeidshrimp No histopathogenesis has been reported for penaeids have been published Couch, Anderson!. exposed to organophosphates. It was found that when penaeidshrimp were experi- In the 1970s, Conte and Parker found that malathion mentally exposedto oil-contaminatedseawater, they ac- aerially applied to flooded marshes in Texas caused from cumulated hydrocarbonsin their tissues.Table 6c gives 14to 80percent mortality in brown and white shrimp held information on two forms of water-solublepetroleum in cages, They recommended that malathion not be ap- productsin relation to penaeidshrimp, Aromatic hydro- plied to flooded marshes that maintained shrimp. Caged carbonswere accumulated in greateramounts than were pjnk shrimp were not killed in small areas receiving alkane hydrocarbons. Bioaccumulation of aromatics in- malathion via thermal fogging for mosquito control in salt creased in proportion to the increase in their molecular rnarshesin northwestFlorida. Ralph Gouldy's hatcheryin weights up to, but not including, the heavier polycyclic Summerland Key, Florida did have problems with aerial aromatics four to five ring!. Brownshrimp Peraumaztecus! mosquito spraying with malathion and had to run a closed exposedto oil in seawaterand then depurated in clean systemhatchery during the early 1980's personalcom- seawater,released accumulated hydrocarbons more rap-

Table 6c. Petroleum and Penaeid Shrimp Couch, 1979!

EcologicaU References Physiological environmental in Component Toxicity implications implications Couch, 1979 Water-soluble 2.9 mg/liter 96-hour Differentialsusceptibilities Different susceptibilitiesof Neff et aL fraction of LC, brown shrimp of' postlarvae, early different speciesin samegenus, Aromatic juveniles; 1.0 mg/ juveniles, and late juveniles i,e., brown shrimp vs. white hyrocarbons liter 96-hour "LC of single species i.e., shrimp might lead to changesin white shrimp brown shrimp. Postlarvae speciescomposition in polluted juveniles are more tolerant than regions. juveniles.

Louisiana 19.8 mg/liter May show chronictoxicity Brown shrimp postlarvaeless Neff et al. crude oil 96-hour LC, brown but not as acutely toxic as sensitivethan polychaetesand water soluble shrimp post larvae refined product above. one fish Menidiasp.!; about fraction! equal in sensitivity with two fishes Furrdulusand Cyprirrodorri. Deign, Optation and Training ... 123 idly than ciams Rangiaeuneata! and oysters Crassostrea toxicity of heavymetals to aquaticanimals McLeeseand virginica!. Shrimp can metabolize the hydrocarbons Ray.,1986; Correa, 1977; Del Ramoet al., 1987;Holwerda whereasthe mollusks havea limited, if any, capabihtyto etal., 1988;Nugegoda and Rainbow,1988!. The biological do this. The higher molecular-weighthydrocarbons are effectsof metalsare complicatedby their synergisticand releasedfrom shrimp tissuesmore slowly than lower antagonisticinteractions with other metals Fosterand molecular-weighthydrocarbons. Carcinogenic polycyclic More!,1982! and by metal speciation Zamuda and Sunda, hydrocarbonssuch as benzo a! pyrene,therefore, would 1982!. For example, zinc influences the toxicity of cad- beretained longer, possibly to exertchronic effects or to be mium Dunlop and Chapman,1981! and chelatingagents shunted into the human food chain. such as Tris, NTA, and EDTA reduce the toxicity of metals Couch 979! presentedresults of testson the water- by sequesteringreactive species Engeland Sunda,1979; solublefractions and oil in water dispersionsof four oils Muramoto, 1980; Castille and Lawrence, 1981; Lawrence South Louisiana crude, Kuwait crude, refined No. 2 fuel et al., 1981!. and Bunker C residual! againstbrown shrimp postlarvae EDTA has been used for a variety of purposes Mark, for toxicity. The testsshowed that fractionsof refined oils et al,, 1965!; one of them as a chelator of heavy metals in weregenerally more toxic to penaeidshrimp and to other aquaculture hatcheries in order to avoid toxic effects of aquaticspecies than were the fractionsof crude oils. The thesemetals to crustaceansof commercial importance crustacean species tested, including brown-shrimp during their early stagesof life. postlarvae,were more sensitiveto oil fractionsthan fish Several studies have been carried out in order to deter- speciestested Cyprinodon,Menidia, and Fundulus!.The rnine the bioaccumula tion and toxicity of heavy metals to 24-hour median tolerance limit of juvenile brown shrimp aquaticorganisms Evans,1980; Kumaraguru et al., 1980; exposedto componentsof No. 2 fuel oil naphthalenes, Muramoto,1980; Correa, 1987, Del Ramo et al.,1987; Chen methyl naphthalenes,and dimethyl naphthalenes,ranged and I.iu, 1987!. Liu and Chen 987! found that there is a from 0.77to 2.51ml/liter. The naphthalenes were the most negative linear relationship between hatching rate of toxic componentsof fuel oils Andersonet al,! to shrimp. Arternia cysts in seawater and heavy metal concentration, Others have observed considerable interspecies varia- and Chen and Liu 987! found that heavy metal concen- tion in oxygen consumption between different speciesof tration in Arternia nauplii increased linearly with an in- marineanimals when they were exposedto the sameoil- creasein the heavy metal. seawater mixtures. Therefore, physiological responsesto The heavy metals that can be methylated, such as oil pollution may vary with the test speciesused, and one mercury, tin, palladium, platinum, gold and thallium, cannotpredict with certainty which specieswill be more pose special threats as environmental pollutants and some sensitive to oil. should be continuously monitored. Other metals, such as Studies of detailed pathologic mechanisms of oil toxic- cadmium, lead, and zinc, do not form stable alkyl-metals ity in shrimp havebeen rarely reported,There have been inaqueoussolutions, but may have different modesof reports of nonspecific lesions in the cuticular chitin, the toxic action than do the alkyl-metalssuch as methylmer- lining of the gastric mill and the mouth region, and the cury, a neurotoxicant. Cadmium, which does not persist proliferation of cells and necrosisin the basalportion of as an alkyl-metal in aquatic systems but does as an ion, is gill filaments of shrimp exposed to sonified crude oil a strong cytotoxicant to gill cells of crustacea. Couch 1979!. The ecological effects of an oil spill depend, to a large Cadmium Cd! extent, upon the environmental, meterological, and geo- Spotte 970! reported that natural seawater oceanic! graphical location. contains 0,00011ppm cadmium, Unusually-high levels of cadmium have been reported from certain estuaries in Heavy Metals which penaeid shrimp commonly occur i.e. Laguna A variety of heavy metals are found as both naturally Madre, Corpus Christi, Texas U.S.A.!. This metal is also a and anthropogenically- derived components of the estua- pollutant from several industrial effluents into aquatic rine environment.These metals may exist in severaloxi- systems. dation stateswith different reaction potentials depending It has been observed that in a significant number of on their specific chemistries. Certain heavy metals are pink shrimp exposed to approximately 760 pg/liter cad- pollutants generatedby industry. Some may be acted mium as CdC 12! for an additional nine days in flowing upon by estuarine microbes to produce alkyl-metallic seawater, an unusual blackening of gills occurred. Unex- compounds that can be accumulated by estuarine species posed control shrimp did not develop black gills. It was and are potent toxicants. found that the LC50 of cadmium in 30 days was 719 pg/ Although heavy metals occur naturally in the aquatic liter, and that during these tests many exposed shrimp environment as a result of weathering and land drainage, developed the black gill syndrome prior to death. in recentyears, the useof various metalscontaining pes- Couch 979! reported the uptake of cadmium in the ticides and fungicldes has added large quani ties of heavy tissue of pink shrimp between 1 and 10 pg/liter Cd in metals to the aquatic environment Kumaraguru et al., waterwlicited uptake, but below 1 Itg/liter Cd there was 1980; Chen et al., 1985!. Excessive additions of heavy no accumulation of the metal in shrimp tissue!, Castille metals to the aquatic environment could have an adverse and Lawrence 981! found that cadmium in a concentra- effect both on the animals and on people who eat these tion of 20 microme was lethal to P,stylirostris nauplii. animals as food, There are a number of reports on the Current values for cadmium concentrations in seawa- 124 ... Treece and Fox ter at 35ppt salinity with standardized nutrient levels is 70 Taiwan's coastal waters asbeing comparable to U,S.coastal ng/Kg according to Quinby-Hunt and Turekian 983>, or water criterion Clark, 1975!.They did however find some at 0.00011ppm Cd2+! by Spotte 970!. The level of Cad- higherconcentrations of iron in theseawater from Chyijin/ mr'um found in Fumba, Zanzibar, East Africa open ocean Kaohsiung City, which wascaused by industrial pollution site>seawater samples was 0.01 ppm see Appendix E in and was cause for concern. The iron appeared as divalent Chapter1 ofthis manual "SeawaterAnalyses from Other in reducing conditions but it was readily oxidized to the Areas of the World and Normal Seawater". As a compari- trivalent state as a colloid which may also block the gills of son, Fujimura 989! reported hatchery water samples shrimp larvae. from Sabah,Malaysia with Cadmium levelsof 0.05pprn. Chen981! reported thatcadmiumconcentrationshigher Copper Cu! than O.l ppm might cause mortality of shrimp larvae. Spotte 970! reported the copper level in normal DUnlopand Chapman 981! found that cadmiumtoxicity seawater to be 0.003 ppm. Couch 979! reported that a can be influenced by the presence of zinc, copperconcentration of 0.5Ilg/liter was lethal to nauplii, protozoea and mysis of P. azteeusand P. duorarum that Iron Fe! were exposed in a seawater-brine mixture similar to that According to S.K.Johnson personal communication!, derived from desalinationplants. The samelarval stages iron is not usually toxic to larval shrimp in the hatchery as were able to grow normally in seawater 5 ppt! contain- such, but it is the precipitant that causes problems. The ing 0.025 mg/liter copper. Kumaraguru et al., 9SO> re- suggested treatment for a high iron level in seawater is to ported a 96hLC50of 0.57ppm copper to Meretizcasta.The treat the water by: copperlevels found in water samplestaken from Fumba, ~ oxidizing iron! Zanzibar East Africa open ocean site! and from a man- ~ chlorinating grove swamp area on the same island were both 0.08 ~ aeration ppm see Appendix E in Chapter 1 ofthis manual!. Normal According to Bill Bray personal communication! if seawater has 0.04-0.1ppm copper according to another iron is found in seawater, he uses EDTA up to 15 ppm. source Forstner and Wittman, 1979!. Fujimura 989! Tests conducted by Treece 981! in St. Croix, U.S.V.!., reported 0.03ppm in hatchery water from Sabah,Malaysia indicated that 2 ppm EDTA was sufficient in stopping and Yuan et al 991> reported 0 3-2.1ppm levels of copper debris from adhering to appendages and assisted larvae from Qingdao, China water. metamorphoseto the next stage.Useful information can be found in a paper by Chen, Ting, Lin and Lian, 1985, Mercury Hg! "Heavy metal concentrations in seawater from grass Mercury hasbeen reported to exist in natural seawater shrimp hatcheries and the coastof Taiwan", WAS 16:316- at a level of 0.00000Sppm Spotte,1970!, Mercury as a 332>. metal has not been suspected to have toxic effects on Iron and manganese can also be oxidized to the in- organisms; however, mercuric salts and methylated mer- soluble-oxideforms by ozone.Kjos et at., 975! were abIe cury are extremely toxic with both short and long-term to reduce iron concentrations in freshwater from 9.54 to chronic effects. Mercuric chloride is used in some histo- 0,07mg/liter with ozone.Oxygen used in placeof ozone logical fixative fluids becauseof its protein-precipitating reduced the iron concentration to 3.99 mg/liter. Manga- effectsin tissues.Enzymes of liver and other tissuesmay nesewas reduced from 1.21to 0.71mg/I by oxygen and to be bound and their activity may be altered by ionic mer- 0.05 mg/I by ozone. Care must be taken with ozone use cury. Thesemercuric products were responsiblefor the especially in closed seawater systemsbecause some desir- 1988closing of the areaaround the Alcoa plant in Lavaca able salts may be oxidized too, thus removing them from Bay, Texas to fishermen, the systems,See section on disinfectionin chapter3 ofthis Couch979! reported studieson the uptake and dis- manual for more detail. tribution of mercuricchloride in brown shrimp P,aztecus!, Iron Fe! or Total Iron levels for Fumba, Zanzibargast and alsoexamined the effectsof mercuricchloride expo- Africa water sampleswere found to be 0.25and 0.26ppm. sure on the ability of brown shrimp to adjust to salinity Iron levels in water samples taken form a mangrove changes.They found that after two hours of exposureto swamp area of Zanzibar, East Africa had slightly higher 0.5 ljg/liter mercuric chloride in seawater, accumulation levelsof iron in them than the Fumbasamples an open of mercury in the shrimp was 285 mg/gm with only 9 oceansite!, These levels were found to beat 0.30ppm. see percentof themercury in themeat muscle!and 91percent AppendixE inChapter 1 ofthis manual"Seawater Analy- in the shell. This suggestedthat a surface absorptive ses from Other Areas of the World and Normal Seawater". processfor mercuryexisted in brown shrimp exposedfor As a comparison,Fujimura 989! reported hatcherywa- brief periods.Shrimp obtainedoff Louisiana'sSouthwest ter samples to contain 0.23 ppm iron from Sabah and 0.3 Passhad naturallevels of only 4.6ng/gm mercurydistrib- ppm from Guam water samples. Natural seawater has uted as 64 percent in the muscle and 36 percent in the been reported to have 0.02 ppm trace metal iron in it cuticle, suggesting that chronic exposure results in inter- Sverdrup,Johnson and Fleming,1970! and Spotte970! nal accumulationof mercury.17 ILg/liter ionic mercuryis reported the iron level at 0.01 ppm. Warnick and Bell theLC50 96hours> for postlarvalwhite shrimp P.setiferus!. 969! reported that the 96 hr. median tolerance limit of Chronic exposureof postlarval white shrimp to 1.0pg/ iron to aquatic insects,mayflies, stoneflies and caddesflies liter for 60 days did not affect respiration, growth, and was 0.32mg/I. Chenet al., 985! reported iron levelsin molting. Chinnayya 971! found that mercury did de- creaserespiration in freshwater shrimp, but did not state Design, Operation and Training ... l25 the levels present in the water. 972! considers seawater containing in excessof I ppm Brown shrimp are active regulatorsof blood chloride manganese unacceptable for fisheries. Li et al., 969! levels ion regulators!. Exposure of brown shrimp to mer- studied the distribution of manganese in both the sedi- cury and to salinity changesresulted in interferencewith mentsandtheinterstitial watersofan Arcticdeepseacore. the shrimp's ability to regulate their internal ion levels to They found that the manganese content of the interstitial compensate for external-salinity changes. Mercury could waters showed a constant increase with depth to about prove to be detrimentalto penaeidshrimp if the form and one meter from the top of the core. Many of the shrimp amount are sufficient enough to prevent them from ad- hatcheries in Japan and Taiwan have reported numerous justing to freshor high-salineconditions that result from black particles appearing during the larval phasesboth in rapid changesin estuariesor tidelandsdue to seasonalor the water and on the sides of the tanks. Those black meteorological extremes. particles when dissolved in sulfuric acid, were found to contain a large amount of manganese. Sano 979! re- Zinc Zn! ported that well water contains manganeseas manganese According to Chinnayya971! zinc in the water col- bicarbonate in its reduced condition!. Chen 983! re- umn caused a decreasein the rate of oxygen consumption ported that the higher the carbon dioxide concentration, of the freshwater shrimp. Correa 987! reported a dif fer- the greater the chancesare of the manganesebeing mobi- ential reduction in respiration and ammonia excretion in lized from the manganesecontaining sediments. The tox- shrimp with increasing concentrations of zinc reporting icity of manganese to shrimp has not been documented, static 96 LC50 valves for zinc at 0.2 mg/L. The Environ- but Chen et al., 985! reasoned that large amounts of znentalAgency of Japan972! set 0.1 ppm zinc as the manganese particles may block the gill thus inhibiting upper limit allowed in seawater for fisheries activities. respiration. Again, as suggested with other compounds, Dunlop and Chapman 981! found that zinc influenced EDTA can be used as a chelator to avoid toxic effects of cadmium toxicity. Normal seawater has 0 to 0.1ppm zinc these metals. in it accordingto Sverdrupet al., 970! and Forstnerand Wittman 979!, respectively. Spotte 970! reported zinc Magnesium Mg! to be 0.01 ppm in normal seawater.As a comparison, Magnesiumlevels in normal seawaterhave beenre- Fujimura 989! reported zinc levels in hatchery water ported to be 1,272ppm Sverdrup et al., 1970!,1,294 ppm from Sabah,Malaysia at 0.06ppm, 0 levels in Hawaii and Riley and Chester,1971>and 1,350 ppm Spotte, 1970!. Guam, and Yuan et al., 991! reported 0.9-3.2ppm in Fujimura 989! reported magnesium levels in seawater Qingdao, China water samples. SeeAppendix E in Chap- from Hawaii to be 1,250ppm and Guam1,175 ppm. Water ter 1 of this manual Seawater Analyses from Other Areas samples taken from Fumba, Zanzibar, East Africa open of the World and Normal Seawater! for a comparison. oceansite! rangedfrom 837-879pprn and samplesfrom Bumbwini a mangrove swamp area on the same island! Lead Pb! ranged from 900-940 ppm. See Appendix E, Chapter 1 Chinnayya971! reportedthat leaddecreased oxygen Seawater Analyses from other areas of the World and consumption in the shrimp, Caridina rajadhardi.Forstner Normal Seawater!for a comparison. and Wittman 979! reported the lead level in normal seawaterwas between0.005-.01 ppm, but Spotte 970! Aluminum Al! reported it in much lower levels .00003 ppm!, Seawater Spotte 970! reported that 0,01 ppm aluminum is analysis in Sabah,Malaysia made by Fujimura, 989! found asa tracemetal in normal seawater.Water samples listed lead levels of 0.14 ppm. Another report by Yuan et taken from Fumba, Zanzibar, East Africa contained from al., 991! from Qingdao, China, listed lead levels in sea- 0.76-0.78ppm aluminum and samplestaken from a man- water between 0.1-2.0.Seawater samples from Fumba, grove swamp area on the same island had a higher level of Zanzibar, East Africa contained 0.08-0,11ppm traces of 1.73ppm. EDTAcould be usedas a chelator at thehatchery lead and 0.08ppm from a mangroveswamp areaon the site nearFumba, Africa! if this sitewas ever developed, sameisland. When sampleswere taken at the Dispensary site near Fumba, fishermen had a number of boats with Treatment of Heavy Metals outboard motors anchored just offshore, upwind of the Hopefully the site selected for the hatchery does not sampling station, This is very likely the sourceof lead involve heavy metals, but if ever faced with the problem found in the slightly higher sampleat Fumba.See Appen- then there are several ways to deal with them. Charcoal dix E, Chapter 1 SeawaterAnalyses from Other Areas of filtration of wateris probably thebest treatment, followed the World and Normal Seawater! for a comparison. by: complexationor chelationof ionic metalsby natural substances like humic acids or by synthetic compounds Nickel Ni! like EDTA, Those substances decrease the metals environ- Spotte 970! reported the level of nickel in normal mental toxicity through a diminished rate of uptake as seawaterto be0.0054 ppm. TheEnvironmental Agency of compared with the free ion Holwerda et al., 1988!.Trace Japan972! placeda 0.1ppm upper limit on the element metalsin seawater are mainly associatedwith chlorides nickel allowed in seawater for fisheries activities. and other inorganic complexes but also may be associated with organic ligands Engel and Fowler, 1979!. Manganese Mn! EDTA has been extensively used in the culture media Spotte970! reportedmanganese in normal seawater of unicellular algae. Culture assays indicate that both to be 0.002ppm. The Environmental Agency of Japan EDTA and EDTA-chelated trace metals intensify the 126 ... Treece and Fox

growth of phytoplankton in sea water Johnston 1964!. egories that may influence penaeid shrimp are biochemi- Two mechanisms have been proposed for the enhance- cal agents arthropod-hormonal agents or their mimics! ment of algal growth by EDTA. Johnston 964! suggested and infectious-pathogenic agents for arthropods viruses, that EDTA increasesthe solubility and thus the availabil- bacteria, fungi, protozoa, helminths and parasitods!. ityyof trace metals that are necessaryfor growth. However, To date, there is no strong evidence that any of the Sunda and Guillard 976! suggested that the increment above agents are pollutant factors in penaeid shrimp on algae growth was due to reduced inhibition by toxic ecology, There is concern, however, for the future use and metals which are present in seawater, Several authors safetyof such agentsin coastalregions because of their have reported decreased heavy metal toxicity on aquatic specific rifles in arthropod ecology, physiology and pa- organisms in the presenceof EDTA, and even depuration thology. Biological-control agents must be tested and of accumulated and enhanced regulation of heavy metals. evaluated regarding their safety for nontarget species Nugegodaand Rainbow988! found that Palaemonelegans suchas valuablecrustacea and someaquatic insects. The was clearly able to regulate body zinc at higher external effectsof someinsect-growth regulators and insectpatho- concentrations in the presence of EDTA. A decrease in gens thatarebeingused ordeveloped asbiocontrol agents cadmium concentration in a cadmiumwontaminated have been tested in nontarget-crustacean speciessuch as Cyprinus carpio after a short exposure to EDTA was re- penaeid shrimp or related forms. The results of these ported by Muramoto 980!, Holwerda et al,, 1988! de- studiesare reviewed in the following sections. scribed the cadmium depuration of Anodontaanatina ex- posed to cadmium-EDTA complex. McLeese and Ray Biochemical Agents 986! demonstrated that cadmium and copper in che- Naturally-occurring insecticidal chemicals such as lated form are considerably less toxic to some marine pyrethrum, nicotine,rotenone, hellebore, ryania, and sa- invertebrates. Reduction of the toxicities of cadmium, badilla will not be discussed here because of lack of copper and manganeseto P. styli rostris larvae are re por ted evidencethat theyare potential pollutants, Insect-growth in the literature. regulators,however, have shown promise as synthetic EDTA has been proven to improve survival of crusta- insecticides and have the potential to be used on a wide ceanlarvae exposed to heavy metals by chelating free ions scale,possibly resulting in environmental exposureof and thus reducing their concentration. Since concentra- nontarget species.Rotenone is commonly used in ponds tion of heavy metals on natural waters varies over time, to kill fish by depleting the oxygen!. routine treatment of these waters by addition of chelating The major insect-growth regulator that has been tested agents could reduce the risk of heavy metal toxicity to a for effectson crustaceais methoprene.This compoundis certain extent. theonly registered U.S. Environmental ProtectionAgency! Chelating agents of the Versene family EDTA and commercially available insect-growth regulator, and is DTPA included! are toxic to fish. Batchelder et al., 1980, listed as a controlagent for flood-water mosquitoes.Per- and Lawrence, et al., 1981, demonstrated detrimental ef- sistenceof methoprenein the aquatic environmentis of fectsof EDTA to shrimp larvae at concentrations of 0.67mM short duration to 7 days or less!, and does not and lethal effect at 1.34 mM. Because of these adverse bioaccumulate.Methoprene is effectivein manydifferent effects,caution in the use of thesesubstances in shrimp insect speciesbut has not shown significant toxicity to hatcheriesis recommended.A very narrow rangeexists nontarget species.The LC50 of methoprene for white and between the concentrations needed and the concentra- pink shrimp penaeids!was 100ppm. Freshwater crayfish tions that produce adverse effects to shrimp larvae, Nei- alsorequired relatively-high concentrations LC50 = 100 ther metamorphosis nor survival were affected by EDTA ppm! for toxic effects to be evident, at concentrations of 0.3 mM. Thereare no reportsavailable on sublethalphysiologi- Stabilityconstants of metal-conplexaBon by EDTAare caleffects of an insect-growthregulator in penaeidshrimp. different for each metal Sillen and Martell, 1971!, and How the growth regulators interfere with insects'matura- while it could keep concentration of free ions of some tion and reproduction should be examinedas possible metalsat low levels,for other ions the efficiencyof com- mechanismsin Crustaceabecause of the relatively close plexation would be lower. This problem might be solved phylogenetic affinities of Crustacea and Insecta. by simultaneousutilization of another chelating agent Infectious Pathological Agents Discussed in more with large stability constants for the ions, which EDTA has detail in the second haIf of this chapter! a low affinity for c.g. DTPA!. Further studies are needed in relation to acute and long term toxicities of other chelat- The major group of potential bioinsecticides studied in ing agents to cultured shrimp larvae. pcnaeidshrimp havebeen the entomopathogenicviruses More recently Yuan et al., 992! have described a knownas nuclear palyhedrosis viruses Baculovirusgroup!, method of heavy metals removal from larval shrimp Theseinsect viruses are of particular interest because rearing systemswith polymeric absorbent, relatedviruses have been found tobe naturalpathogens in feral and cultured shrimp Table7! and in feral crabs. Biological Agents Discussionsconcerning the potential relationships This section includes a discussion and review of bio- betweeninsect baculoviruses being developedas bio- logical agents that are being considered or developed for Iogicalwontrolagents! and nontargetspecies from shrimp commercial, agricultural and health purposes as to humans have been going on since the 197 Ys.Couch biopesticidesor biological-controlagents. Two majorcat- 979! pointed out the need to better understand the Design, Op;ration arsdTraining ... 127 physicaland biochemical nature of theseviruses in differ- control shrimp exposed to salinity change only or PCB ent host systems.He suggested that new viral insecticides only. The 3.0 !tg/liter concentrationof PCB was previ- in nontarget crustacean species such as penaeid shrimp ously found to besuble thai in an eight-hourperiod. There- with an emphasison potentially susceptible larval stages fore, the deciding factor that induced mortality was the of shrimp>be evaluated and testedfrequently. Testsof combination of PCB exposure and salinity change, and insect baculoviruses in penaeid shrimp revealed no sus- that most major ions in the blood of dying shrimp became ceptibility of shrimp for the then-available viral-insecti- significantly less as the ambient salinity decreased.How- cidal agents. Postlarval, early, and late juvenile stages of ever, osmotic pressure overall showed no corresponding brown and white shrimp were inoculated with a freevirus loss in the PCB-salinity stressed shrimp, thus complicat- and were fed a diet containing a virus. Over a 30-day test ing any interpretation of physiological mechanism for the period,no mortality attributableto virus exposureoccured lethal ef feet. in the test shrimp. These types of cross-infection experi- Couch 979! reported that cadmium, zinc, and lead ments i.e., insect virus in shrimp! should include careful toxicity was enhanced in marine and estuarine isopods ultrastructural, biochemical, and genetic monitoring to under stressful salinity changes in experimental expo- ensure that sublethal infections establishing the virus in a sures. Hemolymph osmotic concentrations were altered new host do not occur Couch, 1979!. significantly in isopods exposed to cadmium, zinc and In the estuary,the modesof behaviorof viral, fungal, mercury. Since it is known that cadmium destroys the bacterial, and protozoan biological-control agents devel- soft-gill tissues of penaeid shrimp, it is probable that the oped as commercialproducts and for usein coastalagri- ion and water regulatory mechanism at the epithelial and culture are not known, More needs to be understood cell-membrane levels are structurally and functionally about the effects of novel agricultural agents in estuaries, affected in crustacea exposed to certain heavy metals. particularly regarding the developmentof integrated pest- Thus, when salinity changes occur, the osmoregulatory control methodsthat may utilize combinationsof chemi- systemsof metal-exposedcrustacea fail to respondor are cal and biological-contxol agents. Mutagens, in the formof inhibited. chemicals such as fertilizer components, pesticides, or Pollutant- Temperature Stress Interactions preservatives, may interact with biological-control agents in ways not presently anticipated, resulting in mutants Penaeidshrimp have definite upper and lower tem- with expansionistpotential. Viruses,bacteria and fungi perature tolerance limits; they are shrimp of the temper- should be studied to more fully understand this potential ate, subtropic, and tropic zones. Below water tempera- problem. tures of about 1 -46.4 F!, penaeidshrimp becomelist- less and at lower temperatures they die, Above tempera- Interactions of Pollutants, Pollutant Mixes turesof 91.4'-95'F!penaeids do not do well. Any pollut- ant that has a high-oxygen demand would probably, at and Other Factors in Penaeid Shrimp higher water temperatures in warm months, contribute to Perhaps the most important area for future researchin lower oxygen tension in water and thus to asphyxiation of pollution ecology is the study of interactions of specific penaeids that are sensitive to marginal or low ambient- pollutants and pollutant complexes with physical, chemi- oxygen concentrations. cal and biological factors in the environment. Estuaries are Pollutant-Pollutant Interactions prime examples of multivariate and complex ecosystems where no single variable is altered without influencing Most specificpollutants enter the estuaryas compo- some other component of the system. Penaeid shrimp are nents of complex mixtures of pollutant fallout, runoff or effluents.In many casesestuarine species are exposedto very dependent upon varying physical, chemical, and biological factors for their survival because of the wide several pollutants at once or to varying concentrations of range of factors that they must contend with in their life two or more pollutants. Therefore, it is important to kno w cycle ranging from the open ocean to the tidal estuary. what thecombined and singleeffects of pollutantsmay be Chief among the natural factors influencing penaeid in estuaries, and the effects of combinations of toxicants in shrimp are salinity, temperature, oxygen concentration experimentally-exposed shrimp to cadmium-malathion, Castille and Lawrence, 1981!, bottom types or substrate, cadmium-methoxychlor,and cadmium-methoxychlor- Aroclor 1254. The toxicities of the combinations reflected nutrition and infectious diseases Couch, 1978!. A severe or sudden perturbation of any of these factors, combined no dramatic interactions when compared to the toxicities of eachsingle component of the mixtures, They stated that with pollutant stress, may affect total stress or injury in penaeid shrimp. Review of some two-factor interactions the toxicity of each component was independent and additive and no synergism was detected. More studies follows. utilizing a broaderrange of definedpollutant mixesshould Pollutant-Salinity Stress Interactions be done to more fully explore the possibility of pollutant- A few reports havebeen published on the interaction pollutant interaction and possible synergism. of pollutant and salinity stress in penaeid shrimp, Couch Pollutant-Natural Disease Interaction 979! reports that when brown shrimp were exposed to 3.0 pg/liter of the polychlorinated biphenyl Aroclor 1254 Pollutant-disease interaction is one of the least-studied phenomena in penaeid shrimp larvae. There are numer- and to a gradual decrease in salinity over an eight-hour ous examples in people and o thervertebra tes of poilu tant period, mortality of exposed shrimp was greater than in stressleading to increased susceptibility to natural patho- 128 ... Treece and Fox gens such as viruses, bacteria, and noninfectious diseases 1974a, 1974b; Laramore, 1977; Couch, 1978; Overstreet, such as naoplasia and respiratory dysfunction. There is 197S,Bueno, et al., 1989!, and also baculoviral midgut growing evidence that some fish diseasesare exacerbated gland necrosis BMN! in P.japonicus Sanoet al., 1981!; by certain pollutants, Monodonbacuiovirus MBV! diseasein P.monod on Lightner Penaeid shrimp have a virus disease caused by a and Redman, 1981;Lightner, 1983a;Chen and Kou, 1989!; BacuIovirusthat is exacerbatedby a chemicalpollutant, infectious hypodermal and hematopoietic necrosis Aroclor 1254.This virus-shrimp system has been studied IHHNV! in P. stylirostris, P. monodon Lightner, 1983a!, in order to test its suitability as a model-indicator system and more recently in the late 19SO'shas been found to for determining the degrees of influence that sublethal cause problems in P. vartnamei;hepatopancreatic parvo- concentrations of pollutant chemicals may have on a like virus HPV! recognized in cultured P. merguiensis natural-virushostcomplex, Thepinkshrimp P,duorarum! Lightner and Rcdman, 1985! and in P, chinensis, P, has proven to be a natural host for the shrimp-specific semisulcatus, P. esculentus and P. monodon; a reo-like virus, Baculovirus originally described as a result of findings in P. japonicus Tsing and Bonami, 1987! in P. monodon made on experimentally-stressed penaeid shrimp. A cap- Nash et al., 1988!; and lastly Plebejusbaculovirus in P. tive population of pink shrimp with relatively low initial plebejus Lester et al., 1987!; have all proven to be a serious viru s prevalencewas stressedby 1-3pg j liter Aroclor 1254 threat to the shrimp-culture industry. Table 7 modified PCB! for up to 30 days and experienced a significantly from Liao 19S4 with updates to 1990! summarizes the more rapid spread and increased prevalence of the major diseasesin the larval and postlarval stagesof Baculovirus than did a control, nonstressed population penaeids and the corresponding treatments. The geo- with equal, initial viral prevalence. The mortality in the graphic ranges of these viruses are also increasing and Aroclor-Baculovirus-stressed population wasgreater than have been addressed by Lightner and Redman, in press; that of the Aroclor-stressed population and the control Li ghtncr etal., 1988,Lightner, Redman and Ruiz,1988; and population. At the end of the test, prevalence of viral Lightner et al., in press. infcchons in the Aroclor-stressed population was ap- It is becomingevident that diseasesare increasingin proximately 50 percent more than in the control popula- varietyand frequency, especially with respectto the virus- ti on and intensi ty of individual viral infections was greater causeddiseases, At the present992!, about eight viral in the Aroclor-stressed shrimp. diseaseshave been identified sixdifferent types! in penaeid The ecological implications in these findings suggest a larvae. The ultimate concern is obviously for the preven- phenomena in nature that to date has not been evaluated. tion of diseasesand how to reducethe devastatingeffect If population immunity or resistanceto natural pathogens on larvae so that great lossescan beavoided. MBV disease were reduced in dense populations of Crustaceaby suble- shows its lethal effect only when combined with the thal exposures to specific pollutants, then natural disease serious symptoms of other diseases. It is true that by induced by human-pollutant activity could cause subtle providing MBV-infected larvae with a suitable environ- but high mortality over longer periods of time and gradu- ment and food they are better protected from other dis- ally contribute to an alteration of the ecological balance easesand can attain normal growth. MBV is known to among certain susceptible species.Aquatic pollutionprob- existin Taiwan,Indonesia, The Philippines, Hawaii, China, lems have been further discussedby Overstreet988!, Malaysia, Singapore, Australia, South Africa, Israel, Ku- and a symposiumon humaninfluences on aquaticecosys- wait, Italy, Tahiti, Mexico, Ecuador, Brazil and Texas,but tems was held at the 1989World Aquaculture Society the extentof its rangein other areasis unknown. Beingan meeting in Los Angeles. enzootic virus, MBV can and should be eradicated. To avoid further spreading,strict quarantinesand burning of Larval Diseases Overview the infectedlarvae should be carried out Lightner et al., Liao, 1984,plus updates to 1990! 1983a!.Apparently, it was introduced into the Pacific and The Americas Lightner, et al., in press! by imported In the initial period of the development of the shrimp larvae. ind ustry, unsuitabl e andinsufficient food, which resulted The concept that prevention is more important and in substandardnutrition andstarvation, were major causes effective than cure in controlling a diseaseis absolutely of larval mortality. Occasionally, non-lethal or low-rnor- accurate.Reducing stress due to over-crowding and ex- tality diseases caused by protozoan infections occurred, ecuting properly-operatedquarantines are as necessary but no serious larval diseases or high mortality were as continuing researchon viruses and determining the encountered. In contrast, since penaeid culture has be- etiology of other diseases. come popular and profitable in recent years, hatcheries Generalknowledge of whata normalshrimp looks like are often overcrowded with larvae and this is generally canbe obtainedby consultingBell and Lightner 988!, If accompanied by the occurrence of diseases.White-turbid the shrimp appearto have a disease,it is recommended midgut gland disease was reported in P. japonicus that the diseasebe identified by microscopicexamination Shigueno, 19S5!, as well as lagenidium infection in all and comparedto oneof the publicationsthat havefigures penaeids Couch, 1942;Cook,1971; Lightner and Fontaine, or photographs of the diseases such as Johnson's Hand- 1973;Lightner 1977;Lightner and Redman, 1981;Lightner, book of Shrimp Diseases,Texas Sea Grant Program19S9, 1983a!. or Lightner'sDiseases of Cultured PenaeidShrimp, CRC, Baculooiruspenaei BP! diseasein P. aztecus,P. d uorarum, 1983!.After the diseaseis identified, either Lightner's P. setiferus,P. stylirostris, P.s ublitis,and P.van namei Couch recommended treatment or one of the treatments listed Design, 07aration and Training ... 129

Table '7. DiseasesFound in the Developmental Stagesof Penaeid Larvae and Their Contml Methods List from Liao, 1984, plus updates to 1990!.

Olsease Affected Symptoms Treatment Life References Parts Stages Affected*

Bacteria

Bacterial Appendages Appearing as localized necrosis or Furnace 11 ppm Z1 Tareen, 1982 necrosis discoloration on any appendage, causing Erythromycin 1 5 ppm M1 Lig htner 1983 high mortality of zoeaand mysis stages, Achromycin 1 2 ppm PL affects post-larva Io a lesser extent.

Vibl'lo Hemol ym ph, Initial stages of one form, some Furazolidone 20 ppm PL Nickelson and infection midgut larvae will show yellow-vermillion and Teramycin 450 mgikg Vanderzant, 1971 gland red color permeating entire nervous biomass Lewis, 1973 system. Another form exhibits "White- Furnace 13 pprn Shigueno, 1975 turbid liver', where the midgut gland of Lightner, 1977 the larvae becomesgenerally white-turbid Johnson, 1978 Turbidity becomes more apparent and well- Cipriani, et al. defined as the disease progresses 1980

Filament- Gills. Commonly found attached to the gill Cutrine plus 0.5 ppm PL Delves-Broughton ous pleopods filaments and the pleopods, turning Malachite and Poupard, 1976 bacteria blackish when bacteria mix with dirt. green 10ppm Streenbergen and If severly affected, the respiratory Potassium and Schapiro, function of the gill suffers damage permanganate 85 ppm 1976. Cuprous Johnson. 1978 chloride 1.0 ppm Solangi, et al, 1979 Tareen. 1982 Lightner, l983

Shel! Exoskeleton, If infected by chitinoverous bacteria, Malachite PL Cook. 1973 disease muse!es the exoskeleton will display eroded green 0.9 ppm Delves-Br o ugh ton blackened areas. Also bacteria can and and Poupard, 1976 rapidly enter the body through surface Formalin 22 ppm Johnson. 1978 breaks to cause internal damage combined Tareen, 1982 Light ner, 1983

Black Gills In initial stages, gill color turns Malachite PL Shigueno,1975 gill dull orange-yellow or light brown. green 3.0 ppm Tareen, f982 disease when advanced. the area darkens untii Methylene it is finally black blue 8-10 ppm

Fungi

Lagenidium Body Only thin-cuticled shrimp can be infected TreflanP 0.1 ppm Zl Hubscharnan and infection cavity, thus larval shrimp are highly sensitive. Malachite Schmitt 1969 appendages The hyphae appear inside the body of green 0,01 ppm Lightner and zoea and continue into mysis stage, Fontaine, 1973. resultingin massivemuscle destruction Lightner 1977 and heavy mortality of zoeaand mysis. Johnson 1978 Gopa an et al. 1980 Tareen 1982 Lightner, 1983 130 ... Treece and Fox

Table 7. DieasesFound in DevelopmentStages of PenaeidLarvae and Their ControlMethods continued!.

Olsesse Affected Symptoms Treatment Life References Parts Stages Affected'

Ectnccmmensalprotozoea

Ciliate Gills, Heavy infestation by Zoofhamnivm sp on Malachite Johnson, el al., 1973 infection eyes gills and eyes of larval shrimp results green 10 porn Overstreet, 1973 Zoofham- Exoskeletan in high mortality. Espisfyfis sp. seems and Oeves.Broughton and nium sp to prefer exoskeletonas attacment site Formalin 25 ppm Poupard, 1976 Episfybs and is less harmful. When abundant on combined PL Lightncr, 1977 sp! gill Surface,both can Causehypoxia Quinacrinc Liao. et al, 1977 and death Additionally, their hydrochl or id e 08 ppm Johnson, 1978 abundant presenceon general body Chloramine- 5.5 p pm Lightner, et al., 1980 surface of larvae may interfere with Methylene Tareen, 1982 locomotion, feeding. molting etc. blue 8.0 ppm Lightner, 1983 Parasite burden increases until ecdysis Saponin IIP%%d 5.0 ppm provides relief

Viruses

Penaeid Hepatopan- Penaeidbaculoviruses infect epithelial PL Johnson, 1978 bacula- creas, cells of the hepatopancreasand, less Sano, el al., 1981 1984.1985 VIruses Anterior commonly, anterior midgut, causing high Lightner. 1983,1987, 1988 fBP,IVIBV, midgut mortality in Ihe post-larval stage. Lightner 8 Redman, 1981 B ITIN! Lightner, el al, 1983c 19BB 1990 Couch, 1974a Lester et al, 1987 Mamaya rna, 19B3 Oversfreet,ct al, 1988 Chen 8, Kou, 1989 Bueno. et al, 1989

Infecti ous Hypodermin, Shrimp dying from acute IHNNshow massive PL Lightener.1983a, 1987, 1988 hypodermal Hematopoietic destruction of cuticular hypoderinis and Lightner 8 Bell, 1987 and hemato- organs often of the hematopoietic organs. of glia Lightner et al., 1983a. poietic cells in the nerve cord, and of loose 1983b,1983d, 1985, 1990 necrosis connectivetissues such as the subcutis and Brock et al.,1983 I HHN! gut serosa Only shrimp within a size range Bell 8 Lightner, 198.1987 of 0.005-1.0g have been observed to have these epizootics, resulting in massive mortalities often 80 to 90'%%dwithin 2 weeks! of onset!

Hepatapan- Hepatopancreas Nonspecificsigns including paor growth rate, Larvae may be Lighlner 8 Redmen,1985 crealic Parvo- anorexia, reducedpreening activity, increased infectedbut does Paynteret al, 1985 like virus surface fouling. and occasional opacity of tail not show up until Colorni ct al 1987 HPV! muscle luvchile stage. C hong 8 Loh, 1984 Tsing 8, Bonami, 1987 Reo-like Hepatapancreas Found in Pjaponicus and P monadonthus far. Nash et al., 1988 virus REO! R-cells! Nash 8, Nash in press"

Miscellaneous0iseases

Abnormal Appendages Occursas a result of poor quality of N Tareen, 1982 nauplii spawner.

Amoebiasis Subcutis, Invasion of muscles and subcuticular Z Laramare and Barkale, 1979 of larvae muscle tissues located in the abdomen, cephalothorax, antenna, and eye stalks. Lightner, 1983 by unclassified amoeba.

Larval Exoskeetan Brown to black encrusted deposits which Z, Lightner, 1983 encrusta- contained iron salts affect larval penaeids M, PL

'N=nauplius, Z=zaea,M=Mysis, PL=post-larva Design, Operationand Training ... 13I

here can be used. Details of the current diagnosticproce- larval and early postlarval stages of their principal host duresfor the viral diseasesof pcnaeids have been recently species Table 8! Couch 1981; Sano et at., 1981!, and BP published elsewhere Lightner, 1988 and Lightner and may cause disease and mortalities in juvenile and sub- Redman, in press!. For a more general background on the adult animals Couch 1981!. Epizootic disease outbreaks pathologyof animals in the marine aquacul ture industry, due to MBV in hatchery-rearedP. monodonare known to consultThe Fish Health BlueBook publishedby theFish occur from late postlarval PL 25 to PL 50! through the Health Section of the American FisheriesSociety; Amos, juvenile and adult-life stages, although the most serious 1985!;"Disease Diagnosis and Control in North American losseshave been observedin the late-postlarvalstages Marine Aquaculture" Sindermann and Lightner, 1988!; Lightncr et al., 1983!. "Synopsis of InvertebratePathology Exclusive of Insects" The geographic distributionof these baculoviruses in Spark,1985!;and Control of theSpread of MajorCommu- culturcdpcnaeidshrimp suggeststhat they are problems nicable Fish Diseases FAO, 1977!, to shrimp cul turists only in those areas where the virus is enzooticin local wild popu'lations.This appearsto be the Larval and Juvenile Shrimp DiseasesDetail case of BMN, which has thus far been observed in P. Lightner 1984;updated to 1990! japonicus in hatcheriesin Japan.However, MBV and BP have been documentedas having beenintroduced into In most semi-intensiveand intensive-culture systems, new geographic regions by the transfer of infected recognition, prevention, and treatment of disease is pos- postlarvae or broodstock to areas outside the normal sible. Whereas, in intensive and many semi-intensive range of the host species. According to Lightner et al., in culture systems,treatment of diseaseis impractical even if press, BP is widely distributed in cultured and wild they arediagnosed. Furthermore, except for certaintypes penaeidsin the Americas.BP has not yet beenobserved in of parasiticdiseases, it is the very nature of intensiveand wild, culturedor imported from the Americas!penacid semi-intensive culture systems i.e., high shrimp density shrimp outsideof the Americas.New information on the per unit volume of water used! that encouragesthe devel- host and geographic distribution of BP has come from opment and transmission of many shrimp diseases.The Brazil, Ecuador and Mexico. In Brazil and Ecuador, BP same economic incentives for using semi-intensive and infccts larvae and postlarvae of six pcnaeid spccics.!t is intensive-culturesystems dictate that disease can be un- significant that the imported Asian speciesP, rnonodon and derstood and controlled. P. peniciliat tts werc also found to be infected. BP was found Infectious Diseases in Mexicoin culturedlarval and postlarvalP. stylirostris at a facility near Guaymas,Sonora on the West Coast of Viral Diseases Knowledge of the diseasesof the Mexico Lightner et al., 1988!, penaeid shrimp have been reviewed a number of times Patent acuteBP and MBV infections may be readily within the past 16years Ovcrstrcct, 1973and 1982;Sano and diagnosed by demonstration of their characteristic occlu- Fukuda,1987; Sindcrmann, 1974and 1988;Sindcrmann and sion bodiesin either wet-mountsor histologicalprepara- Lightner, 1988; Johnson, 1975, 1978, 1989; Couch, 1978; tions of the hepatopancrcasand midgut Lightncr 1983; Lightncr, 1977,1983,1984 and 1989;Lightncref al.,1987; Liao, Lightner et at., 1989;Bonami et al., 1986!.BP occlusions are 1984!.This section indudes recentdevelopments and dis- distinctive tetrahedralbodies easily detectedby bright coveriesin shrimppathology through mid-1990. field or phase microscopyin unstained wct-mounts of A number of viral diseasesof cultured pcnaeids have tissuesquashes, while MBV occlusionsare sphericaland been reported Table 8! and several additional diseases thereforedifficult to distinguishfrom lipid droplets,secre- have been noted to be associated with the virus-like or tory granules, etc. The usc of a stain like 0.1 percent chlamydial-likestructures. Included among the docu- aqueousmalachite green in preparing wet-mounts for rnentedvirusescausingdiseasein pcnaeidsare: Baculavirus MBV diagnosis aids in demonstration of the occlusions. penaeior BP Couch 1974a!,baculoviral midgut gland Presumably,the protein making up thc occlusionabsorbs necrosis or BMN Sano etal., 1981!, and Monodon bacu1ovirus the stain more rapidly than doesmost of the host-tissue or MBV Chen and Kou, 1989;Lightner and Redman components,making them distinct within a few minutes. 1981!; the probable picornavirus infectious hypodermal BPand MBVocclusion bodies in histologicalpreparations and hcmatopoicticnecrosis virus or IHHNV Lightner ef al.,1983 and 1987!;the small DNA-containi ng vi rus named appear as prominent cosinophilic,usually multiple occlu- hepatopancreatie parvo-like virus or HPV Lightncr and sion bodies, within the hypcr-trophied nuclei of hcpatopancreatictubule or midgut-epithelial cells. Redman,1985!; a reo-likevirus in thehcpatopancreas of P. Unlike BPand MBV, which are Type-A baculoviruscs japonicus Tsing and Bonami, 1984!, a rco-like virus in P. becausethey produceocclusion bodies, BMN is a Type-C monodon Nash et al., 1988!and Ptebejusbaculovirus in P, baculovirusthat doesnot produce an occlusion body, plebejus Lesteret al,, 1987!. Hence,diagnosis of BMN infectionsis dependentupon Baculoviruses The knownpenaeid baculoviruscs the clinical signsof the disease,histopathology and trans- infect the epithelial cells of the hepatopancreasof mission electron microscopic TEM! dcmon-stration of protozoeal-through-adultlife stagesand the midgut epi- thebaculovirusinaffcctcd hepatopancreatocytcs ightner l thelium of larvae and postlarvae,Baculovirus infections etal., 1984!.Sano ef al., 983!, developeda rapid fluores- may result in disease in cultured pcnaeids that is accom- cent antibody test for BMN that reportedly simplifies the paniedby high-mortalityrates. ln hatcheriesBP and BMN diagnosis of BMN. often causeserious epizootic disease outbreaks in the Thc cytopathology of BP, MBV, and BMN is generally 132 ... Treece artd Fox

Table 8. The PenaeidViruses and Their KnownNatural and Experimentally infected Hosts Lightneret al., 1985 plusupdates to 1990k

Virus' Host Subgenus arrd Species" BP MBV IHHNV HFV REO Litopenaeus: P. van narnei +++ + P, stylirostris ++ +++ P. setiferus + + ta! f'. schmitti Penaeus: P,rnonodon ++ ++ ++ P. esculentus + + ++ P. sernisulcatus +++ Fenneropenaeus: P, rnerguensis +++ P. indicus + P, chinensis ++ = orientalis! P. penicillatus ++ Mar supenaeus P,j aponicus ++t P. plebejus ++ Farfantapenaeus P. aztecus +++ + @! P. d uo rar urn + @! P. brasiliensis P. paulensis ++ P. subti7is Meli cer tus: P. kerathrus P. marginatus +++ P, plebejus ++

"Abbreviations: BP = Bacatoviruspenaei MBV = P. mortodon baculovirus i BMN = Baculoviral midgut gland necrosis IHHN = Infectioushypodermal and hematopoieticnecrosis HPV = Enteric parvo-like virus RBO = Reo-like virus t = Infection observed in species,but without signs of disease. ++ = Infection may result tn moderate diseaseand mortalities. +++ = Infection usually results in seriousepizootic disease outbreaks with high mortality rate. @ = Experimentally infected; natural infections not yet observed. = Classification according to Holthuis, 1980, FAO SpeciesCatalog. similar when studied by light microscopy, differing prin- A TEM test of BPand MBV-infectedcells showed large cipally by thelack of occlusionbodies in BMN. Oftenthe numbers of rod-shaped baculovirus particles both free affected hepatopancreatocytenuclei have a peripherally and occludedwithin the proteinaceouscrystalline matrix displacedcompressed nucleolus and marginatedchro- of theocclusion body, but only free virus in thenuclei of matin,giving affectednuclei a "signet ring" appearance, BMN-infected hepatopancreatocytes. even before occlusion bodies become well developed. A more recent baculovirus Plebejus baculovirus! has Brown and Brenn histologic gram stain I.una, 1968!, been reportedin P. plebejus Lester et al., 1987!.Also although not specific for baculovirus occlusionbodies, Akamine and Moores 9S9 WAS meeting! describeda tends to stain occlusion more intensely than the surround- spraymethod, using sodium hydroxide to disinfectshrimp ing tissue, aiding in identifying their presencein low- hatcheries inflected with Bacuioviruspenaei. grade infections, BPis widely distributedin culturedand wild penaeids Design, Operation and Training ... 133 in the Americas, ranging from the Northern Gulf of Mexico tissue, and striated muscle!. Usually the midgut, midgut south through the Caribbeanand reaching atleast asfar as caeca and the hepatopancreas endoderm-derived tis- the state of Bahia in Central Brazil. On the Pacific Coast, BP sues! are unaffected, except in severe cases where ranges from Peru to Mexico, and it has been observed in hepatopancreatie involvement has been observed. These wild penaeid shrimp in Hawaii Lightner et al., in press!. inclusion bodies were described by Cowdry 934!. Baso- IHHN Virus This probable picornavirus, named philic chromatin strandsare occasionallyvisible by light IHHNV for infechous hypodermal and hematopoietic microscopywithi n IHHNV intra nuclearin elusion bodies. necrosis virus, was first recognized in 1981 in Hawaii in Thesechromatin strands area prominent feature of IHHNV populations of cultured P. stylirostris that had been im- intranuclear inclusionbodies shown by a TEMtest. IHHNV ported from a number of commercial penaeid hatcheries intranuclear inclusion bodies are common early in acute Lightner etal., 1983a!.Since its discoveryin P.styli rostris, infections, later decreasing in number, and are followed IHHNV hasbeen found to infect a variety of other penaeid by necrosis and inflammation of target tissues. Affected species either in natural infections or in experimentally- cells may also have highly vacuolated cytoplasm and induced infections Table 8!. small cytoplasmic basophilic inclusions. Although the IHHNV causesserious epizootic disease outbreaks in prominent intranuclear inclusions present in shrimp in- intensively or semi-intensively reared P. styli rostris, with fected with IHHNV are evidence of nuclear involvement, accumuIative mortalities typicallyexceeding90percent of assembly of the virus occurs in the cytoplasm of affected the affectedpopulations within 14 to 21 days of onsetin cells. The size of the virus 7 to 26 nm in tissue sections 0.05 to 2 g juveniles Lightner et al., 1983a!. IHHNV has and 20to 22nm in purified preparations!,its morphology, also beendocumented to causedisease and seriousepi- and its replication within the cytoplasmsupport the ten- zootic disease outbreaks in larger juvenile and adult P. tative classification of IHHNV with the picornaviruses. stylirostris Lightner and Bell, 1987!and in juvenile and Since 19S5, no new hosts for IHHNV have been dern- adult P. rnonodon reared in intensive or semi-intensive onstrated. However, the geographic distribution of the culture systems Brock et al., 19S3!, IHHNV has been virus in culture facilities hascontinued ta expand. shown to infect and to be carried asymptomaticallyby P. HPV This probable parvovirus named HPV, or vannarnei Lightner et al., 1983a;Bell and Lightner 1984!. hepatopancreatic parvo-like virus, was first recognized in Further study of IHHNV diseasein P. vannamei may show cultured P.merguiensis in Singaporeand Malaysiain 1983 that under stressful culture conditions some mortality Lightner and Redman,1985!. HPV or a very similar losses and or reduced-growth rates may occur, agent! was subsequently recognized in four additional IHHNV hasbeen detected in penaeidshrimp sampled penaeid species P. chinensLs,P. semisulcatus,P. esculentus from a number of shrimp-culture facilities located in and presumedP. monodon! in either captivewild popula- widely separatedgeographic locations Bell and Lightner, tions or in cultured populations,Individual shrimp with 1983 and Lightner et al., in press!. This suggeststhat HPV displayednonspecific signs including poor growth IHHNV has becomewidely distribu ted in penaeid culture rate, anorexia, reduced preening activity, increased sur- facilities Bell and Lightner, 1987 and Lightner et al., in face fouling, and occasionalopacity of tail musculature. press!probably as a result of the difficulty of detecting Mortalities accompaniedby thesesigns occuredduring viral infection in asymptomaticcarrier hosts such as P. the juvenile stages,after apparentlynormal development vannarneior becauselosses due to the virus in pond-reared through the larval and postlarval stages.The accumula- stocksare difficult to detect.IHHNV is a diseaseof juve- tive mortality rate in HPV epizootic outbreaks in P. nile or older shrimp; apparently it does not adversely merguiensisand P. sernisulcatusreached as high as 50 a ffectthe larval or postlarvalstages and thereforeIHHNV percentto 100percent, respectively, of the affectedpopu- does not occur in hatcherieswhere it would be readily lations within four to eight weeksof diseaseonset. detected.Instead, IHHNV producesits most seriousepi- The principal lesion in HPV disease, common to all zootic outbreaksin P. stylirostris,P. vannarneiand in P. affectedspecies, is a necrosisand atrophy of the hepato- monodonshrimp of 0,05g to 2 g, the size when shrimp pancreas, accompanied by the presence of Iarge promi- typically havebeen moved to nurseryor grow-out ponds. nent basophilic, PAS-negative, Fuelgen-positive Water turbidity and the shrimp's small size at this time in in tranuclear inclusion bodies in a ffec ted the life cycle makes detection of the disease in intensive hepatopancreatocytes.These inclusion bodies are diag- and semi-intensive culture systems difficult. Control of nosticfor HPV, and presumablydevelop from smalleosi- the diseaseis further complicatedby the fact that penaeid nophilic intranuclearbodies that are also presentin the shrimp surviving IHHNV infections become carriers of affected tissues. Electron microscopy of affected the virus for life and passthe virus on to their offspring hepatopancreatocytesrevealed aggregations of 22 to 24 Lightner et al., 19S3a!. nm diameterparticles within the electron-densegranular Diagnosisof infection by IHHNV is dependentupon inclusion-bodyground substance.The virus-like particle histological demonstration of prominent eosinophilic, size and morphology, the close association of the nucleo- Fuelgen-negative intranuclear inclusion bodies within lus with the developinginclusion body, and thepresence chromatin-marginated, hypertrophied nuclei of cells in of intranuclearbodies wi thin developinginclusion bodies tissuesof ectodermal epidermis,hypoder mal epithelium are similar to cytopathological features reported for of foregut and hindgut, nerve cord, and nerve ganglia! parvovirus infections in insects and vertebrates. and mesodermalorigin hematopoieticorgans, antennal HPV has a geographicrange in Asia and Australia gland tubule epithelium, mandibular organ, connective similar to that of MBV, and like MBV it has been intro- 134 ... Treece and Fox duced ta the Americas with imported penaeids. More served,Samples may be prepared for wet-mount micro- recently,HPV was foundfor the first time in dual infec- scopicexamination for BP and MBV, or preservedfor tions with MBV, It was found in postlarval and juvenile P. histologicalevaluation. The enhancement proccdurc is far monodonsampled from farms in the Ping Tung area of more sensitivethan the direct samplingprocedure for BP- Taiwan. In 1987this region experiencedserious disease and MBV-causcd diseases, and for IHHN disease in P. losses,and HPV is suspected as being a contributor ta the stylirostris Lightner et al,, 1987!.Paynter et al., 984!, 1987epizooticdisease outbreak. The geographic distribu- found that diagnosisof HPV in captive wild P, esculentus tion and known hosts of this virus have expanded. Paynter in Australiamay also lend itself to the enhancement etal., 1985reported the virus in P.esculentus from Austra- procedure.Enhancement is nat a suitableprocedure for lia; it hasbeen found in P. monodonimported to Israel fram demonstration of IHHNV in asymptamatic carriers. For Kenya Calarni et al., 1987!and lastly it was found in example, the enhancementprocedure will not readily captive-wild and hatchery-reared P. indicus and P. demonstrateIHHNV tobe present in subadultor adult P. merguiensisin Singapore Chang and Loh, 1984!.HPV has stybrostris that are IHHNV epizootic diseasesurvivors, or been observed in the Americas, mainly Brazil and Ecua- in speciessuch as P. vannameithat arereadily infected by dor Lightner et al., in press!. thevirusbut seldomshow diagnosable infections Lightner Re o-like Virus A rca-like virus was present i nlarge, et al., 1983a!. vital areasin thecytoplasm ofhepatopancreatic R-cells of In the bioassay prodccure, carriers of IHHNV may be discased laboratory-reared P.j aponicusfrom the Mediter- detectedby sensitive "indicator"shrimp. Indicator shrimp ranean city of Palavas in France Tsing and Bonami,1987!. in this procedure known IHHNV-free juvenile P. Purified virians were non-enveloped, icosahedral par- stylirostris of 0.05g to 4g body weight! may be exposedto ticles of about 60 nm in diameter. The disease was repro- samplesof suspectedcarrier shrimp by one or more of the duced in healthy P. japonicusby inoculation with purified following three methods: vi ru s and by feedinganimals pieces of the hepatapancreas ~ Injection with a cell-frcc filtrate prepared from a from infected shrimp. Disease developed slowly in ani- homogenate of suspected carrier shrimp the indica- mals exposed to the reo-like virus, requiring about 45 days tor shrimp will show signsof IHHNV discase wi thin to develop. Secondary infections by agents such asF. sola ni 5 to 15days if thesuspected shrimp were infectedwith were common in reo-like virus-infectedPjaponicus Tsing IHHNV!. and Bonami 1987!. Rco-I ikc virus has also been found in P. ~ Rearing suspected carrier shrimp with indicator rnonodon Nash et al., 1988and Nash and Nash,in press!. shrimp in the sametank the indicator shrimp will show signsof IHHNV disease within 15 to 60 days!. General Proceduresfor Virus Screening ~ Fccdingchoppcd carcassesof suspcctedcarrier shrimp The following three diagnosticprocedures have been to indicator shrimp the indicator shrimp will show developed for screening penaeid shrimp for viral infec- signs of IHHNV wi thin 15 to 60 days!. Lightner et al., tions: 1983a!. ~ Direct sampiings for microscopic wct-mount! exami- Actual diagnosisof infectionby BP,MBV, BMN, HPV, nationnand /or his topa thol ogy. and IHHNV is dependenton microscopicor histologic ~ Enhancement of infection followed by microscopic demonstrationof the particularcytopathology that is examination and/or histopathalogy. unique to each disease.Gross signs and behavior are ~ Biaassay of a suspected shrimp population with a usuallynot sufficientlyspecific in shrimpinfected by sensitive indicator speciesfollowed by sampling and these penacid viruses to be reliably used in diagnosing histopathalogy Lightncr et al., 1983a!. thesediseases. Also see methods for routinevirological In the direct sampj ing procedure, nonrandamsamples examinations, Johnson, 1989! discussed later in thischap- of shrimpare selectedfrom culture tanks, ponds, or cages ter. and examined directly for signs of BP or MBV in wet- Rickettsia Rickettsia infectionshave been reported mounts, ar they may be preserved in Davidson's Alcohol to causeshrimp disease Brock et al.1986; Brock, 1988; Formalin Acetic Acid AFA! or 10pcrccntbuffercd forma- and Colorni et al.1987!. Rickettsia are microbcs that are lin Humason, 1967! for histologicalevaluation. The sen- similar to both viruses and bacteria and have a size that is sitivityof this procedureislimited and it will only demon- normally somewhere in between the two. Cells of the strateshrimp with viral infections that are acute ar sub- digestivegland are damaged fram Rickettsiamicrabes. acuteein a population with a high-incidence rate. IHHNV, BP, MBV, and HPV have been diagnosed with direct Bacterial and Fungal Diseases samples, but such samples have also produced false- Bacteria A number of bacteriahavebeen implicated negative diagnoses on populations later shown on en- as causesof discaseand mortalityin culturedpenaeids, hancementar bioassay diagnostic procedures ta be posi- especiallyin the larval, postlarvaland juvenilestages tivecfor one af these viral diseases Lightncr et al., 1985!. Johnson,1978 and 1989;Lightner, 1983!. Bacterial infec- In the enhancement procedure, a quarantined popula- tions in shrimp take three general farms. erosionsaf the tion, is reared under relatively crowded and stressful cuticle covering the generalbody surface,gills, and ap- conditions. Postlarvac are best used for this test, which pendages bacterial necrosis and shell disease!, localized usually requires 30 to60days to complete. Random samples lesions within the body, and generalized septicemias. aretaken at inter vals throughout the test period, or nonran- Rcportson thcoccurrcnccafbacterialdiscascsinculturcd dam samples are selected as moribund animals are ob- penaeidsin Kuwait Tareen,1982!, and China Meng and Design, Operationarul Training ... 135

Summary of PenaeidViruses in the Americas and Their Status as of 1990 From Lightner et al.,in press!

Virus Status in the Americas and Hawaii IHHNV Widely distributedin culturedP. vartrtatnei and P.styli rosf ris;not recognizedin wild penaeids; enzoo tie in Southeast Asian wild penaeids; recently introduced into Western Mexico from Texas and Panama with postlarval P. vattnmnet'.

HPV Enzootic in Asia, Australia, and Africa; introduced to one or more sites in South America from Taiwan; can infect the American penaeid P, vartnamei.

BP Widely distributed in American penaeids; enzootie in wild penaeid son both Atlantic and Pacific sides of tropical and subtropicalAmerica.

MBV Enzootic in Asia, Australia, Africa, and the Mediterranean; introduced to several sites in Hawaii, North, Central, and South America; can infect P. vanrtatttei; contaminated stocks eradicated from Hawaii, Mexico, and Texas.

BMN Enzooticin Japan;not reportedoutside of Japan.

REO Enzooticin Japan;introduced to Hawaii from Japan;contaminated stocks eradicated in Hawaii.

Observed and Reported Occurrences of the Penaeid Viruses in Wild and Cultured Penaeids Indicating Their Probable Natural and Introduced Geographic Distributions From Lightner et al,, in press!

Virus Region/site where found Virus Status IHHNV Atlantic side: SE U.S. Caribbean, and Brazil introduced Pacific side: Ecuador, Peru, and Central America introduced? Pacific: Hawaii, Guam, Tahiti introduced Asia: Taiwan introduced Singapore, Malaysia and Philippines enzoo tie'? Middle East: Israel introduced

HPV IndoPacific: P.R. China, Taiwan, Philippines, Cul, CW, W enzootic Malaysia, Singapore and Australia Africa: Kenya W enzootic Middle East: Israel and Kuwait Cul, CW enzootic Americas: Brazil, Ecuador Cul introduced

BP Atlantic side: SE U.S., Caribbean and Brazil Cul, CW enzootic Pacific side: Ecuador, Peru and Central America Cul, CW, W enzootic Mexico Cul, CW enzootic Hawaii W enzootic

MBV IndoPacific:P.R. China, Malaysia, Cul, CW, W enzootic Singapore and Australia Africa: S. Africa W enzootic Middle East: Israel and Kuwait Cul, CW,W enzootic Mediterranean: Italy Cul, CW,W enzootic Pacific: Tahiti, Hawaii Cul introduced Americas: Mexico, Ecuador, Texas and Brazil Cul introduced

BMN Japan Cul, CW, W enzootic

REO Japan, Malaysia Cul enzootic Hawaii and France Cul introduced Cul = "cultured;" from cultured or captive-wild broodstock, CW = "captive-wild;" ftom wild-caughtseed or from single-spawnwild broodstock W = wild population. 136 ... Treece and Fox

Yu, 1980, 1982, 1983! are similar to previously reviewed viability of this bacterium is maintained in optimum reports from other shrimp-culture groups Lightner,1977, freshwater conditions for more than150days while viabil- 1983!. While bacterial diseases of a probable primary- i tyin brackish-watersituations is limited to only 50 days. bacterial etiology have been reported in penaeid shrimp Seawater medium is not tolerated by this bacterium. This Nickel son and V and erzant, 1971; Cook and Lof ton, 1973!, speciesis thus able to survive and thrive only in freshwa- the majority are of a secondary etiology, occurring as a ter and brackish-water culture systems under optimum result of syndromcs due to such things as ascorbic-acid temperature5 -30 C!." deficiency, toxins, wounds, extreme stress,etc. Lightner, 1983!.A number of reports in literature support this Luminous Bacteria observation. Many laboratory attempts have been made According to C. Pitogo, SEAFDEC Vol.X No. 1, p. 9 to completeKoch's postulateswith bacterialisolates ob- March 1988,"luminous bacteria have longbeen known to tained From penaeids, and in each study a relatively be associated with some crustacean mortalities in the massive inoculurnhad tobeadministered to overcome the wild, but such infections have not been reported in naturaI defenses of the host and to produce disease and penaeids. Death of the hosts due to luminous bacteria was death in the experimental animals Vanderzan t et al., 1970; always preceded by visible luminescence that can be Lewis, 1973,Lightner and Lewis, 1975;Corliss et al., 1977; observedat night! resulting from the large concentration Huang etal., 1981!.One study showedthat cell-freesolu- of bacteria in the body fluids of the affected animals." tions of crude extracts of endotoxins and exotoxins of In Penaeusmonodon hatcheries in many parts of Asia, Vibrio parahaemolyticuss and V, alginolyb'cus injected into P. larval mortalities due to luminous bacteria have become setiferusproduced significant mortalities with gross signs widespread and have caused the temporary shutdown of similar to those observed in actual bacterial infections many rearing operations. Leong and Hanrahan, 1980!. Lu minescence observed in infected larvae consisted of In everyreported bacterial infection in penaeidshrimp, a continuous emission of greenish light. Brightly lumi- motile, gram-negative,oxidase-positive, fermentative rods nescing weak larvae settled to the bottom and got en- have been isolated from lesions or host hemolymph. Most tangled with the sediments, sometimes forming lumines- isolates have been Vibrio ssp., usually V. alginolyticus, V. cent mats on the tank bottom. From the number of lumi- parahaemolyticus,or V. angui7larum.Certain other gram- nous larvae in the tank during the time of observation, it negative rods, including Pseudornonasspp.,andAerornonas is possible to roughly predict the resulting mortality. spp. may occasionally be involved in bacterial-disease Primary bacterial isolation of heavily-infected larvae re- syndromesin penaeid shrimp Lightner, 1983!. All of vealed that purely luminous colonies can be recovered on thesegenera and specieshave been reported to be among the agarmedia. Two speciesof vibrios, Vibrioharveyi and the normal microflora of penaeids Vanderzant et al., 1970, V. splendiduswere identified, with V, harveyibeing the 1971; Hood and Meyers, 1977; Yasuda and Kitao, 1980; more dominant species. Lewis et al,, 1982!. Although a variety of gram-positive The observed incidence of infection was highest at the cocci, including the etiological agent Aerococcusviridans! postlarval stages and lowest at the naupliar stages, al- that causes highly lethal Gaffkemia disease in Hornarus though this observation did not correlate with the occur- lobsters have been isolated from shrimp, none have been renceof mortalitiesin the respectiverearing facilities. linked with diseasein penaeids Stewart and Rabin, 1970; It was found that the bacteria infecting the larvae can Vanderzant et al., 1971; Vanderzant et al., 1972!. Hence, it also be readily isolated from natural seawater near the would appearthat shrimp haveonly opportunisticpatho- shore and the sediments. gens that are part of their normal microflora. A possible It is very likely that the water acts as the main carrier exception to this was the discovery of a gram-negative, of infectious agents.Infection in Penaeusrnonodon was acid-fast rod causing diseasein adult P.vannamei Lightner, detectedas early as the egg stage. The use of contaminated unpublished!. Shrimp infected with this microorganism spawning water may be the source of the bacteria. The were moribund when collected, but showed no externally possibility of transmission of the disease through apparent abnarmalihes. Histopathology, however, re- transovarian means remains to be verified. vealed that the acid-fast bacterium was present in very The number of luminous bacteriaper ml was many largenumbers either encapsulated in melanizedhemocy te times higher for the rearing water of infected stocks than nodules or in the tissuessurrounding such granulomatous theseawa ter source.This indicates that therearing system lesions in the host hepatopancreas, antennal gland, and provides a favorableenvironment for the multiplication mandibular organ. Additional sources of bacterial con- of this bacteria. Many reports have shown that bacteria tamination in penaeid shrimp hatcheries are discussed by proliferatein Fecalmaterial and excessfood accumulating Hasson 987! and Chen et al., in press. in the rearing tanks. According to Cecilia Baticados in SEAFDEC Vol. X, Fluorescent Bacteria No. 1, p, 9, March 1988!,"The con trol of luminousbacterial According to Duremdez and Lio-Po, SEAFDEC Asian infectionin shrimplarvae, as in othermicrobial infections, Aquaculture Vol, X, No. 2 page 9, June 1988,"Pseudomo- consistsprimarilyofenvironmental,biological,andchemi- nas fluorescens is a gram-negative, rod-shaped bacterial cal methods.It is always best to prevent the entry of the specieswith flagellar filaments for active motility. It pro- pathogenof diseaseagent into the hatchery. Applying duces greenish to yellowish diffusible pigments on selec- treatmentwhen the diseaseis alreadypresent is a difficult tive agar medium such as PseudosedAgar Growth and processand may not alwayssucceed, especially when the Design,Operation and Training... 137 shrimp larvae are already too weak. The following arc green,and mcthyleneblue Aquacop,1977; Tareen, 1982; someguidelines which may help preventand control the Lightner, 1983!.Vaccines against Vibrio sp. have been occurrence of luminous bacteria in shrimp hatcheries: reportedby Lewisand Lawrence, 1983! to bc potentially ~ Waterfor spawningmustbekeptclean. Remove scum effectivein preventinglosses due to Vibriospp. infections after spawningas thesemay attach to eggsand could in aquariumand pond-reared P. setiferus, but theeftica- encouragebacterial growth. Eggsmay bc disinfected cious useof this vaccinein penaeidsremains to be docu- with 20 ppm mg/1 or g/ton! Tide detergentfor 2 h mented. and rinsed thoroughly before hatching Li-Po and Luminousbacteria are nowcausing problems in hatch- Sanvictores, 1986! to remove surface bacteria, fungi, eries personalcommunication Fernando Carvaca-E SPOL, and debris. 1990!,and in grow-out ponds in Ecuadorand antibiotic ~ Stock healthy, clean and uninfected nauplii only. treatmentin feedsarcbeing considered personal commu- Healthy nauplii arephototactic, i.e., they are attracted nication with Jose Villalon, 1990!. to light. Water containing the nauplii must also be "The occurrence of luminescence in Penaeusrnonodon changedbefore stocking the larvae in rearing tanks. larval cultures followed by significant mortality of the ~ The rearing watermust alwaysbe kept cleanand free larvae has been observed for quite some time in the Aklan from sediments and debris. This may be done by Provinceon Panay Island, Philippines. It was not until using physical methods through sand filters, ultra- 1987, however, that the microorganisms responsible for violet sterilization, cartridge filters, filter bags, etc.! or this phenomenomwere isolated and identified as pre- chemical means disinfection with 10 ppm chlorine!, dominantlyVibrio harveyi and occasionally as V, sptendidus. ~ Siphon out and wipe off sediments/debris/algal This study was performed to determine:! the invitro growth and wastesfrom bottom and sidesof rearing sensitivity of the luminousbacterial isolates V. harveyi and tanks since these could serve as substrates for bacteria V.sptendidus to variousdrugs, ! the minimum inhibitory to grow on. concentrations MICs! and the minimum bactericidal con- ~ Always disinfectinfected batches of shrimp with 200 centrations MBCs! for drugs to which luminous vibrios ppm chlorine for 1 h before discarding these,It is are sensitive, and ! the tolerance of Penaeus rnonodon important that discardedwatcr/dirt/larvae from the larvae the bactericides." hatcherydo not drain directly into the sca to avoid The study's abstractstates: "Only chloramphenicol, polluting the sourceof rearing water as well as thc sodium nifurstyrenate and the nitrofurans furazolidonc, environment. nitrofurazone, nitrofurantoin and Prefuran! showed rela- ~ Provide the larvae with adequatefood, i.c., the right tively low MICs and MCBs <25ugmi'!. Thebacteria quantity of good quality food at the right time. The showed various responses to chloramphenicol, and ability of the shrimp to resist diseasedepends, to a Prefuran and low sensitivity to oxytetracycline. large part, on its nutritional state. Chloramphcnicol,oxytetracycline and Prcfuranare com- ~ Avoid the indiscriminate use of antibiotics/drugs in monly used in shrimp hatcheries.Shrimp larvae showed shrimpculture. Drug resistantstrains o fthe luminous high survival ratesand activeswimming movementafter bacteria have been isolated in areaswhere commonly 25-hour exposure to in vivo bactericidal dose of used antibiotics such as chloramphenicol, penicillins, chloramphenicol,Furicin, nitrofurantoin protozoeaonly!, erythromycin, kanamycin, oxytetracycline, poly- oxytctracycline nauplius only!, Prefuran mysis only! myxin, streptomycin, and sulfa drugs have been regu- and sodium nifurstyrcnate,but the drugs causedeforrni- larlyused as part of the rearing protocol. So far, some ties in the carapace,rostrum and setae.Chemical control degreeof successhas been mct only with nitrofurans, of luminous vibriosis among shrimp larvae appears lim- e.g., furazolidone, but recent personal conununica- ited, basedon the efficacy of existing and readily available tion 990! with Cecilia Baticados indicates that larval drugs, because of the passible development of resistant deformities may occur if thesedrugs are used!. In- shainsofbacteria and the limited tolerance of the shrimp fected larvae protozoeae, mysis! may be treated with larvae to the drugs." 10ppm pure furazolidonefor 12h for 4-5consecutive Source: Diseasesof Aquatic Organisms. Studies on the days. Treatment is best conducted at nighttime when chemical control of luminous bacteria V. harveyi and V. the temperature is cooler 8-29'C! with almost com- sptendidusisolated from discased P. rnonodonlarvae and plete water change 80-100 percent! after 12 h. In- rearingwater. M.C.L. Baticados,C,R. Lavilla-Pi togo, E.R. fectednauplii may bc exposedto the drug for only 6 Cruz-Lacierda, L.D. de la Pena and N.A. Sunaz Fish h, It is important that such protocol be strictly fol- HealthSection, Aquaculture Department, Southeast Asian lowed only in casesof infection; otherwise, regular Fisheries Development Center, Tigbauan, Illilo 5021,The exposureto the drug at low levelscould result again Philippines!,V-9, N-2, P-133,October 4, 1990. in the development of resistant strains of bacteria. Continuedexposure to the drug at the recommended Fungi level but beyond the recommendedperiod could Severalspecies of fungi infect pcnacidsand someare result in morphologicaldeformities in the larvae." major pathogensof theseanimals, Several reports have Severalgroups have reported effective therapy of these beenpublished that expandthe documentedgeographic diseasesusing antibiotics such as Furance, Furacin, and host range of 4tgenidiumsp., Sirolpidiumsp., and Terramycin,Aureomycin, and Chloramphenicol,and an- Fusariumsolani. Members of thesegenera werc reported to tibacterialchemo therapeutics such as formalin, malachite cause disease losses in cultured P. semisulcatus in Kuwait Tareen, 1982!, and in P. chinensis cultured in the Yellow Agmasoma =Thelohania!,and Pleistophora,are known to Sea region of China. Large-scale hatchery losses of eggs infect captive-wild and cultured penaeids, especially in and larvae to Lagenidium sp. and Sirolpidium sp. were ponds or in enclosed natural bodies of water Overstreet, reported Meng and Yu, 1980,1982!,and Fusarium sp. was 1982; Lightner, 1983!. Tissues infected by these parasites reported to infect juveniles in grow-out ponds in the same include striated muscle, smooth muscle and the gonads. area of China Meng and Yu, 1982,1983!. As was the case Infectionprevalences in penaeidculture ponds haveap- in most of the bacterial species reported from cultured- proached 10 percent Couch, 1978!. Severe infections in penaeid shrimp, the imperfect fungus Fusariurn sp, all cultured penaeids may cause chronic-disease mortality isolates that have been identified are F. solani>,the phyco- Couch, 1978; Lightner, 1983! and parasitic castration rnycetous fungi Lagenidiumsp. and Sirolpidium sp, appear Enriquez et al., 1980!,resulting in an unmarketable prod- to be present in virtually all shrimp-culture facilities uct. throughoutthe world, This is not surprisingbecause each Gregarines Gregarines Protozoa,Apicomplexa! of the fungi has a wide host range or can exist as a free- are common inhabitants in the guts of wild and pond- living saprophyte Johnson and Sparrow, 1961;Moss and reared penaeids Johnson, 1978, 1989; Overstreet, 1978; Smith,1984!. Whilenoeffectivechemotherapeutantshave Couch,1983!. Two genera,Nematopsis and Cephalolobus, been found for treatment of F. solaniin fections in penaeids are known in penaeids Lightner, 1983!.These organisms Hatai et al.1974; Lightner, 1981, 1983; Tareen, 1982!, a use a mollusk for completion of their life cycle and may be numberof effectivechemotherapeutants have been iden- excludedfrom tank and raceway-culturesystems John- tified and tested against Lagenidiumsp, Bland et al1976; son,1978!,Even when presentin suchlarge numbers as to Lio-Po et al., 1982!. occlude the midgut or hindgut lumen, gregarines do not Thehistopathology Bian and Egusal,1981!and patho- appear to cause significant disease in penaeids. genesis Hose et al., 1984!of F. solani infections in penaeids have been reported. Penaeids respond to invasion by F. Noninfectious Diseases solanihyphae with an intense hemocyticresponse that Diseases Caused by Epicommensals Among the includes hemocyte encapsulation, melanization, and the more serious diseases of cultured penaeids are those deposition of collagen fibers within a granulomatous le- causedby noninfectious-epicommensal organisms. These sion that surrounds and isolates the invading hyphae organisms are common and apparently ubiquitous in Bian and Egusa, 1981!.Studies of these lesions by a TEM shrimp-culture facilities. All life stagesmay be affected, test have shown that despitetheintensityof host response, but the most serious losses are encountered in juvenile a large percentageof hemocyte-encapsulatedF. solani and adult stageswhen the gills of the host become fouled hyphae remains viable within the granulornatous lesions resulting in various formsof gill disease!by heavyinfes- Lightner, 1981;Hose et al., 1984!.Contributing to the tations of epicommensal organisms such as filamentous pathogenesis of F. solani, in addition to its direct bacteria,peritrich protozoans,and pinnate diatoms.The invasiveness and destructive effect on host tissues, are following is a list of the more commonly observed and secondary bacterial infections and changes in the reported epicommensal organisms that alone, or with hernolymph content of the host. Hemolymph from se- other epicommensals, cause "gill disease" and surface verely F. solani-infected P. californiensis was fouling in cultured penaeids. hypoproteinemic,hemocytopic, and frequently failed to Themost significant of thesediseaseslisted in the chart coagulate Hose et al., 1984!. below are discussed here. TheChinese have recently made important contribu- Bacterial epicommensals tions to shrimp mariculture production on the world market producing 165,000MT of farm-raised shrimp in Leucothrixmucor is a very common ubiquitous estua- 1989!and have also made advancesin hatchery-disease rine marine bacterium,reported from every penaeidcul- diagnosis and cures Chen, 1988;Chen et al., in press; Wu turing area of the world McKee and Lightner, 1982; and Lu, in press, and Zheng, 1988!. Lightner, 1983!.Consistent with this are reportsof losses due to L. tnucor in penaeids cultured in Kuwait and China Protozoan Parasitic Diseases Meng and Yu 1980, 1982, 1983; Tareen 1982!. L, mucor Microsporidians Microsporidians Protozoa, attachesto livingand nonliving substratesand inpenaeid- Microspora! causea group of diseasesin penaeids tha tare culture systemsit readily attachesto the body surfacesof collectively called "cotton" or "milk shrimp disease." At shrimp. In juvenile and older penaeids, L. mucor favors least three genera of microsporidia, Ameson =Nosema!, attachmentto the gills and accessory-gillstructures. Lar-

Bacteria Ciliates Protozoa! Blue-Green Algae Leucoth rix muco r Zoothamniumspp. Spirulina subsalsa Thiothrix sp. Vorticella sp. Schizothrix calcicola Vibrio spp, lagenophrys sp. discussed later under Pseudomonas spp. Apostomeciliate toxic diseases! Flavobacteriasp. Suctora Protozoa! Diatorns Aeromonasformica ns Acineta spp. Amphorasp, Ni tzschiasp. Achanthessp. Design, O~ration and Training ... 139 val and postlarval penaeidsmay becomeso fouled by L. as the essential amino acids, cholestrol, linoleic acid, H- mucorfilaments that respiration,feeding, locomotion, and caroteneand potassiumare neededin penaeiddiets for molting may be seriouslyimpaired, resulting in mortali- optimum growth, survival and appearance New, 1976; ties.L.. mucor is noninvasive and it causes no demonstrable Kanazawa,1980!, only one nutritional-diseasesyndrome pathologyto the surfacesto which it attaches Lightncr et of cultured penaeidshas been describedin detail. This al.1975; Lightner, 1978!.Severity of diseasedue to L, disease,called black death or shrimp scurvy Lightner et mucorin shrimp is relatedto organicloading of the culture al,, 1977and 1979!occurs in penaeidsthat are rearedin system,to its oxygencontent, and to the added stressof culture systemsIacking algaeand receiving diets with molting. Mortalitiesdue to L, mucorsurface and gill infes- insufficient ascorbic acid. The disease has not been ob- tationsare due to hypoxia. served in shrimp cultured in systemswhere there is at Severalother speciesof bacteriahave been implicated least some algae. Shrimp with black-death disease possess in bacterial-gill diseaseand surface-foulingdisease of melanized hemocytic lesions in the epithelial and sup- culturedpenaeids. Included among the filamentousforms portive connectivetissues of the general-bodycuticle, the are Thiothrix sp,, Cytophagasp. and Flexibacteriasp. foregut and hindgut, the eyestalks,and the gills. The Lightner, 1983!.Unlike L. rnucor,inflammation and mela- lesions are most prominent in tissues with a high-collagen nization of the gills often accompanieshigh levels of content Hunter etat.,1979!. Addition of L-ascorbic acid to infestationby some of thesefilamentousbacteria Lightner, the shrimp's ration or rearing shrimp in the presenceof 1978!. Lewis et al., 982! reported aggregation of hatch- algae effectively prevents black death disease Lightner et ery-rearedP. stylirostrislarvae by surfacefouling due to al., 1979!. infestationsof Pseudomonaspiscicida, Aermonas forrnicans, Toxic Diseases and Flavobacteriasp. Hemocytic enteritis HE! Blooms of certain fila- Protozoart epicommensals mentous blue-greenalgae, all belonging to the family A numberof speciesof protozoahave been reported to Oscillatoriaceae, have been implicated as causing the HE causesurface fouling and/or gill diseasein all life stages disease syndrome in primarily young juvenile penacids, of cultured penaeids Overstreet, 1982; Couch, 1983; The occurence of HE seems to be ubiquitous, and ex- Lightner,1983!.The mostcommonly reported protozoans amplesof the diseaseexist from marine and freshwater include the peritrich ciliates Fpistylisspp., Zoothamnium shrimp aquaculture facilities in North America, Hawaii, spp., and Vorticellaspp.; the loricate ciliate Lagenophryss Brazil, The Philippines, and Israel. One speciesof blue- sp.; an undescribedapostome ciliate; and the suctorian green algae, shown experimentally to cause this syn- Acinetaspp. Couch, 1978,1983; Overstreet, 1978, 1982; drome, is Schizothrix calcicola McKec, 1981!. S. catcicola Mengand Yu, 1980,1983; Lightner, 19S3!. Johnson 978 p. occurs in both freshwater and seawater, and has been 14!and 989! showsan easyway to differentiatebetween reportedto possessa potentendotoxin Keleti etal., 1979!. Zoothamniumand Epistyliss.Zoothamnium has a myoneme While HE is most commonly observed in early juvenile or muscle fiber visible in the stalk whereas Epistytis does penaeids,it has been observedin subadult penaeidsas not. As is the case with bacterial epicommensals, when well. abundant on the body surfaces,appendages, or gills, these The principal lesion of HE, which occurs as the result protozoanscan causedifficulties to the host in locomo- of algalendotoxin released in the gut from ingestedalgae, tion, feeding, molting and respiration, Like L. mucor,most is a necrosisand marked hcmocyticinflammation of the of the protozoans causeno appreciable internal damage to mucosal epithelium of the midgut and its caeca Lightner, the host surfaces or gills. The exception to this is the 1978!, accompanied by necrosis and degeneration of the unidentified apostomeciliate which causedmelanized hepatopancreas Lightner and Redman, 1984!, The cause hemocytic lesions in the gills of P. aztecus Lightncr, 1975; of death in shrimp with HE may be due to osmotic imbal- Overstreet, 197S, 1982!. ances, poor absorption of nutrients, or to secondary-bac- terial infections.Species of Vibrio, usuallyV. alginol yticus, Algae are the organisms most commonly isolated from the septic A number of species of blue-green algae and diatoms hemolymph of shrimp with HE Lightner 1983!,Mortality havebeen reported tobe amongthe epicommensalorgan- ratesin raceway-rearedP. stylirostris with HE have reached isms causing surface fouling and gill disease in cultured 85 percent Lightner, 1978!, but usually are less than 20 and captive penaeids Lightner, 1983!,Amphora sp, has percent.Runting of shrimp affectedwith HE is apparently even beenobserved growing internally in the gills of P. due to midgut disfunction and to the length of time setiferusreared in a shallow, nonturbid, well-lighted tank required for the midgut mucosa to regenerate and is a Overstreet and Safford, 1980!. chronic effect in animals that survive the disease. Dinoflagellate Poisoning Dinoflagellate blooms Nutritional, Toxic and Environmental red tides! have been circumstantially linked to serious Diseases Detail mor tali ties of cultured-penaeid shrimp in Mexico Lightner Lightner, 19S4;updated to 1990! etal,, 19SO!,but a cause-and-effectrelationship of mortal- ity to the suspectspecies of dinoflagellateshas not been Nutritional Diseases experimentally demonstrated Lightner, 1983!. The occurencc of a toxicity syndrome called "blue shrimp Although a numberof the nutritional requirementsof syndrome unknown" BSX! in P. califorrtiertsisand P. culturedpenaeids have been identified and suchnutrients 140 ... Treece and Fox

stylirostrts cultured in Mexico Lightner 1983! has been companiesthe degenerativechanges in the mandibular correlated to thc occurcnce of red tides. Shrimp with BSX organ Lightncr et al., 1982!. Further information on die during molting or following handling stress,and in an aflatoxicosis disease diagnosis and control measures are affectedpopulation a largepercentage of the shrimp has discussed by Lightner 98S!. been observed to develop "blunt heads." This condition Red Disease Red disease, or red discoloration, was was thought to develop from damage to the head append- first noted in Taiwan Liao et al., 1977; Liao, 1977! in ages from the convulsive behavior pattern that occurs in cultured P. rnonodon. The disease hasalso been observed in this syndrome Lightner, 1983!. Dinoflagellate toxins are captive wild-adult P, monadonand in juvenile and adult- thought to be nontoxic to crustaceans Sievers 1969!, but cultured P. manadonin the Philippines by, J.F. LeBitoux only short-term toxicity tests have been run on shrimp. and C. Emerson, Filipinas Aquaculture Corp., 19S2!and in However, during those tests the few shrimp that molted pond-rearedP,stylirostrisinHawaii LightnerandRedman, aIso died. That observation and the circumstantial asso- 1985!. Liao 977! noted that in some years red diseasein ciation of red tides and the BSX syndrome in Mexico Taiwan was "quite serious," especially in cultured-adult indicates that the importance of red-tide toxins to penaeids P, manodon,The hepatopancreasof normal decapod crus- may be significant. During the red-tide outbreak in Texas taceans contains a variety of carotenoid pigments, with in 1986, it appeared that the rcd-tide Ptychadiscusbrevis most of the total-body content of beta-carotene being did kill penaeidshrimp larvae.Information gatheredfor stored in the hepatopancreas Goodwin, 1960!.Atrophy a larval-rearing run for a course in Port Aransas, Texas and necrosis of the hepatopancreas result in release of Treece, 1986! indicated that it was necessaryto charcoal stored beta-carotene and other carotenoids into the filter all water to keep the red tide from killing larvae. hemolymph, Distribution and deposition of Mcdlyn980! also discussed thesusceptibilityof Artemia hepatopancreatic carotenoids by the hemolymph into the to Ptychadiscusbrevis toxin s!. tissues explains the rcd-tissuc discoloration that charac- Aflatoxicosis and Red Disease Aflatoxicosis and terizes this disease. red disease are discussed together here because of the Liao's et al., 977! observations of P. rnonadon with red close similarity of their histopathology Lightner et al., disease indicated the development of red disease to be 1982;Lightner and Redman,1985!, However, the etiology subacute or chronic, with no evidence of an infectious of red disease is unknown, and while it may have a toxic etiology, An infectious etiology was considered unlikely cause,the possible role o fan infectious agent in its etiology because the disease was refractory to antibiotic therapy, hasnot beencompletely explored. Both of thesediseases the diseasewas observedonly in P. monodon even when have, as their principal feature, a necrosis of the hepato- polycultured with P. penecillatusand P. semisulcatusand pancreas that is accompanied by marked intertubular becauseattempts to transmit the discase to unaffected P. hemocytic inflammation, tubule encapsulation and mela- monodan failed.! nization. Toxic constituents of other plant-food stuffs are Liaoet aL, suggested a linkbetween feeding rancid fish discussed by Liener 980!. to the shrimp and red diseasebecause thc diseasewas not Aflatoxicosis Necrosis and inflammation of the observed when only fresh fish was fed. However, Emerson hepatopancreas, mandibular organ, and hematopoietic noted that red diseasein the Philippines occured in pond- organs are the principal features of arhficially-induced reared and captive wild P. monadonfed exclusively artifi- aflatoxicosis Lightner et al., 19S2!.Although aflatoxicosis cial diets, However, Emerson 982! indicated that in his has not been proven to be an important diseasein cultured experience red disease was most common in manure- pcnaeids, the mechanism for it becoming an important fertilized ponds with thick anaerobic detritus deposits. disease is in place. Penaeids reared in semi-intensive or Liao et al., 977! describedthe sequentialdevelop- intensive systems are fed artificial diets that may contain ment of red diseasein P. monodon:affected shrimp passed ingredients which, on occasion, can contain aflatoxin in through four stageswith the earliestdetectable signs of sufficient amounts to result in aflatoxicosis Arafa et al., the diseasebeing a yellowish-greendiscoloration of the I 979;Wiseman et al.,1982!. Aflatoxin canalso be produced shrimp's body. Otherwise, affected shrimp remained ac- "in situ" in penaeid feeds improperly stored under warm tive and displayednormal behavior.During the next two and humid conditionstypical of penaeid-cultureregions to four days, affected shrimp became reddish with the Wiseman et ai., 1982!, This could include microen- normally white gills and the pleopods also becoming capsulatcddiets for hatcheryuse and/or postlarvalfeeds, reddish,Finally, after five to sevendays affectedshrimp The principal lesions of aflatoxicosis in penaeids became distinctly red and totally lost their normal brown Lightner etal,, 1982!occur in the hepatopancreas and the and tan pigment banded! pattern. Shrimp in the final mandibular organ. In the hepatopancreas acute and sub- stagesof the diseasewere lethargic, anorexic and showed acute aflatoxicosis is expressed as necrosis of the a tendencyto excessivesurface fouling by epicomensal hepatopancreatic tubule epithelium that proceeds from organisms.The amount of body fluid in thecephalothorax the proximal portion of the tubules to the peripheral- increasedover that of normal shrimp and had a foulodor, tubule tips. A marked intertubular-hemocytic inflamma- Thehepatopancreas was reported to be yellow or pale. tion, followed by encapsulation and fibrosis of affected Histologicalexamination of P.monodan and P. stylirostris tubules, follows in subacute and chronic aflatoxicosis that with red diseaserevealed a markedatrophy of thehepato- displays a necrosis of the peripheral epithelial cells of pancreas and the presence of numerous melanizcd in- cords within the gland that progresses proximally to the fl ammatorylesions in thehepa topancreas, antennal gland, central vein. Only a slight hemocytic inflammation ac- mandibular organ, gonads, midgut and gills Lightner Design, Operationand Training .141 and Redman, 1985!. Hepatopancreatic inflammatory le- this review and are also frequently a sign of toxic syn- sions were the most consistently observed lesion type. The dromes caused by chemical irritants including certain affectedhepatopancreata were atrophied reducedby as heavy metals, oil, ammonia, nitrite, and ozone. much as 50 percent of expectednormal size!, usually Gas-Bubble Disease Gas-bubble disease has been contained multiple hemocyte-encapsulatedhepatopan- reported to occur in penaeidshrimp as a resultof super- creas tubulcs with necrotic or sloughed epithelial linings, saturation of atmospheric gases and oxygen Lightner, and possesseda marked hemocytic infiltration in the 1983!.Shrimp are similar to fish in their sensitivity to intcrtubular spaces.Brown and Brenn gram-stainingof supersaturation of atmospheric gases.Although thc level affectedhepatopancrcata of P. monodonand P. stylirostris of nitrogen or atmospheric-gassupersaturation required showed the tubule lesions to contain masses of gram- to cause gas-bubble disease in penaeids has not been negative rod-shappedbacteria and in P. monodonocca- formally studied, a threshold of about 118 percent satura- sionalprominent clustersof large.0 to 1.5u mdiameter! tion is assumed Lightner, 1983!.Oxygen causedgas- gram-positive cocci or short rods. Unlike the gram-nega- bubble disease in pcnacids was reported to occur when tive rods that were always present in the tissue debris in oxygen reached or exceeded250percentof normal satura- the lumen of hemocyte-encapsulated tubules, the gram- tion in seawater Supplcc and Lightner, 1976!.Regardless positive organisms in P. monodonwere observed in clus- of the gas causing gas-bubble disease in shrimp, the clini- ters within cytoplasmicvacuolcs of tubule epithelialcells cal signs are the same. The most obvious sign of gas- as well as in the lumen debris. bubble discase is that affected shrimp float in all other The significance of the relatively large numbers of diseases, dead or dying shrimp sink!. Examination of gram-positivebacteria present in many of the P, rnonodon fresh preparations of gills or whole tissue by microscopy hepatopancreatawith red diseaseis not known. Gram- reveals the presence of gas bubbles. positive bacteriado not typically make up a significant Cramped Tail Condition This occasionally-ob- part of the normal microflora of pcnacid shrimp servedcondition of penaeidshrimp hasbeen reported to Vanderzant et al,, 1970; Vanderzant ef al., 1972; Lewis, occur in the summer months when both air and water 1973;and Yasuda and Kitao, 1980! or the known patho- temperatures are high Llghtner, 1977; Liao ef al,, 1977; gens of penaeids Lightner, 1983!,The absenceof these Johnson,1975;Mengand Yu,1980!. Penaeidswith cramped gram-positivebacteria in the Hawaii-rearedP. styh'rostris tails while stillalive! have a dorsal flexure of the abdomen with red disease suggests either that they are not the that cannot be straightened. This condition typically fol- etiologicalagent of rcd diseaseor that rcd diseasemay be lows handling,although shrimp havebeen observed with a generalizedsyndrome with more than one cause,resulti- cramped tails in undisturbed ponds Johnson, 1975!,The nggfrom a necrosisof the hepatopancreasand releaseof its cause of cramped-tail condition is unknown, but its content of carotenoid pigments into the hcmolymph occurenceonly during the summer suggests that elevated Lightner and Rcdman, 1985!.This diseasehas not been as water and air temperatures, thc handling of shrimp in air much of a problem in hatcheries as it has usually been that is warmer than the culture-system's wa ter, and o ther found in the grow-out phase. stressesmay contribute to the causeof the condition. This Gut and Nerve Syndrome This idiopathic, prolif- is usually not noticed until the grow-out phase. erative condition affecting the midgut and ventral-nerve Muscle Necrosis Spontaneous Necrosis! Muscle cord has only been observed in populations of postlarvae necrosis is the name given to a condition in ail penaeid and juvenile Penaerrsjaponicusreared in ponds, tanks, and species that is characterized by whitish, opaque areas in raceways in Hawaii Lightner et al., 1984!. It has appar- the striated musculature, especially in the distal-abdomi- ently not been observed in P.j aponicusreared in Japan or nal segments Rigdon and Baxter, 1970!. The condition elsewhere, The disease was named gut and nerve syn- follows periods of severe stress from lo w oxygen, sudden drome GNS! to reflect its idiopathic nature and the prin- temperature or salinity changes, severe gill fouling, etc.! cipal organs affected. The severity and high prevalence of Lakshmi ef al., 1978;Lightner, 1983!. It is reversible in i ts GNS in virtually all populations of cultured P.japonicus initial stages,but it maybe lethal if large areasareaffcctcd. studied since 1980in Hawaii had precluded the successful "Tail rot" is the name given to the chronic and usually rearing of this species in Hawaii, particularly in high septic form of the disease when the distal portion of the densityculture Lightncr etal., 1984!.Although thereis no abdomen or appendages! becomes necrotic, turns rcd, evidence to support the hypothesis, GNS is thought to be and begins to slough. causedby a toxin, possibly an algae toxin, that is unique to Hawaii Lightner et al., 1984!. The principal lesions ob- Methods for Routine Examination of Larval Shrimp served in P,japonicus with GNS are a hypertrophy of the J ohrtsort 1989! anteriormidgutmucosal epithe!ium-basement membrane Larval stagesand young postlarvae are small enough BM! and a hypcrplasia of thc cpincurium that covers the to examine whole as wet mounts with a compound or ventral-nerve cord and segmental ganglia in the stereoscopic microscope. Upon examination look exter- gna tho thorax. nally for damaged or deformed structures. Opaquenessor Black-Gill Disease A number of diseasesyndromes other discoloration of the musculature should be recorded of cultured pcnacidsare accompaniedby the presenceof as to location and distribution. Color and spatial distribu- black mela nized!, inflammed lesionsin the gills Lightncr, tion of chromatophorcs are characteristic to stage or spc- 1977;Ligh tner and Redman,1977!. In fact,black gills may ciesand abnormalitiesshouldbenotcd, Prcsenceof sessile accompanymany of the syndromesdescribed earlier in protozoa and bacteria may be noted by microscopical 142 .Treeee and Fox scanningof thebody surface.The blood hcmolymph!can Methods for Routine Examination of Juvenile and be easily observedthrough the transparentcxoskcleton, Adult Size Shrimp particularly at the extremities.Observe for fungi, viruses, When a shrimp is dissected,the species,length and protozoaand motile bacteriausing a compoundmicro- living condition active, sluggish, type of unusual scope.In the largelarval stages,the digestivetract and its behavior! should be described. contentsmaybe easily observed through the exoskeleton. Estimation of the freshness in dead shrimp is made Heart,digestive gland and other organscan be examined when a non-living sample is examined.Criteria for in similar manner. Mashing a whole animal between this estimation are: smell, appearance of darkened coverslip and slide will allow more detailedexamination fringes of carapace,gills and internal organs, Espe- of individual tissues, cially intergrity of digestivegland! and presenceand Vital stains may be used to highlight tissuedamage. gradeof postmortal stiffness rigor mortis!. Only re- Larvae are placed in stain solution for 10 to 30 minutes. centlydead shrimp aresuitable for examination.If the Aqueoussolutions O.lpercent!of methyl green,neutral gills or cuticle have browned, smell is intensive, mus- red, Janusgreen or trypan blue will mark lesions. Sodium culature has lost translucency or become pink and chloride crystals added to staining compartment will internal organs have begun to decompose, the shrimp convert staining solution for marine species, is not suitable. The presence of postmortal stiffness Particular Examinations indicates that the carcass is still fresh enough for dissection. Bacteriological Take larval shrimp and place in dcprcssionslide. Using a compoundmicroscope prefer- Examine visually: ably phasemicroscopy! focus at 400Xon an extremity of Ezoskeleton or cuticle for erosive areas, scars, whit- thcbody.Motilebacteria within thebloodshould beeasily ish or blackish areas,ulcers, deep wounds, changesin pigmentation, knots or nodes, parasites, fungus or discerned. Add 100 ml of distilled water ar chlorinated tap water defects! Moufh to each of three or four clean beakers, Arrange beakers in Carapace external or internal, as exoskeleton! a row. With a pipette or dropper add three shrimp larvae Antennae incomplete, broken! to the first beaker. Swirl water with shrimp. Rinse drop- per, Removeshrimp to next beakerrepeating procedure Eyes wounds, blindness! Anus color and appearanceof the fecespressed out so as to wash shrimp clean of external microbes, Arrange by slight prcssure on the sides of the body! another four or five beakers in a row. To the first beaker Gorurds for proper developmentin adults,swelling add 0.002% w/v! iodine as iodophore i.e, Wescodyne, Ciba-Ceigy!or 1 mg/I quartinary ammonia compound and darkening of vas deferens! Hold shrimp to light and try to detect parasites i.e. benzalkonium chloride! for 10 minutes. Rinse by passingthrough other beakers.Remove shrimp from last embedded in body. This examination should not last too long becausecuticle and gills must still be wet for beaker with a loop and place on agar surface of prepared the following steps. plate. Disrupt structure of shrimp with the loop and BIood; If the shrimp is still alive, insert a capillary tube spread about on part of the plate, Do the samefor the other through the membrane that separates the carapace shrimp. The intent of this procedure is to isolate internal bacteria onto bacteriological media. It does not differenti- and the abdome. This may be easily done if the tube is held in one hand, the shrimp in the other with the ate between bacteria of tissue fluids and those of the cephalothorax bent forward. Take care not to insert digestive tract except in the caseof nauplii, tube into the intestines. Alternate: Cut off one-third of Fungal Fungi are easily seen by light microscopy antenna and utilize the drop that forms on the newly but isolahon may be desired for identification or other cut tip. Examine for ability of blood to clot, protozoa purposes. Place infected larvae in sterile chamber with 1 in fluid using stereoscopic microscope and bacteria mI of sterile water seawatcr if marine shrimp! that con- tains 10 mg/1 of chloromycetin.Two hours later loop using compound scope.Take some fluid from shrimp transfer zoospores check for presence with microscope! and quickly spread on a slide for staining purposes. Examination of cuticular surface for parasites of mi- by streaking onto savouraud dextrose algae plates. croscopic size and bacteris: Adapted from: M.S. Gorttel and M,K. Toohey, 1983, J, Prepare two slides with a drop of wateron them. Cut Invert. Pathol. 41:1-7!. Virological Viral examination of larvae is similar to portions off using scissors and place the samples in that describedfor larger shrimps./ Routineexamination the water drops, The samples should be taken from parts where parasites or bacteria appear more abun- of tissues for occlusion viruses, however, applies squash of whole animal's mass. In larger larvae postlarvac! it is dant, i.e., from visibly changed areas,particularly advantageousto removea portionof the tail beforemak- edges of segments or tips of appendages. ing the wet-mount squash,This can be done by severing Cover the preparation with a coverslip and examine the tail at mid-point of the first tail segment with a scapel for mo hie organisms. It bigger parasites are expected, examine first under stereoscopic microscope. With while viewing through a disscctinig microscope,This examination of small shrimp it is most convenient to procedure will causethe digestive gland and anterior cut off whole appendages, especially uropods. intestine to wash free of other body portions. Certain When lesionsare present,stained smearsshould be stains e.g.0.1% aquous malachite green! in tissuesqua shes, preparedfrom the changedarea: The margin of the Design,Operation and Training ... 143

lesion and the tissue where the changesoccur should other parts. be usedfor smearing.Material shouldbe takenwith a ~ Musculature: Examine musculature for unusual sterile loop or scalpel and evenly not too thick! opaqueness;rcmove unusual tissueand placea small smeared on at least two clean slides. portion on a slide in a drop of water. Mash with ~ Examination of gills: Gills are first examined in position coverslip and examine for fungi, bacteria, by removingthegill cover!for color,amountof detritus, microsporidian and metazoan parasites, Remove a presenceof parasites,cysts, necrosisor other changes. segment of a leg and tease out the internal parts for AHgills on both sidesshould be examined. Then one or observation on a slide. two gill filaments ar takenout by meansof scissorsand Particular Examinations forceps. They are put on a glass slide and observed under a stereoscopicor compound microscope. With Bacterial isolation Visibly examine the shrimp for biggershrimp, several portions of gill filamentsshould lesions,record iFpresent. Wash shrimp in tap water soas be cut off with scissors choosing those which look to rinse the specimen of excesssurface bacteria. Hamc an abnormal!put a dropof wateron the slide,cover with inoculating loop and set it aside. Wet a cotton ball with coverslip and examine under a compound microscope alcohol and swab the surface and the area around the for presence of parasites, cysts, fungus, bacteria and membrane that dorsally separates the carapace and the changesin the structute. Look at normal and abnormal abdome. Take the inoculating loop and press it through gills to compare their morphology. the membrane into the haemocoel. Do not cut intestine ~ Cephalothorax: The adult or juvenile shrimp should with loop. Streakthe materialon the loop to surfaceof the be openedwith scissorsby making four cuts. The First plated bacterial medium. Mark plate with the date and removes the rostrumat its base. The second cut starts your name. Place in incubator for growth. at the rostral base and proceeds posteriorly along the An alternate method of obtaining blood is to sterilize side at the level that the gill cavity begins and stops at the surface of an antenna using an alcohol soaked cotton the end of the carapace.The third cut is the same as the ball. The distal third is then cut off with a sterile blade. The second,but on the other side., The fourth cut proceeds drop that forms, upon bleeding may be touched directly to posteriorly along the dorsum to the end of carapace. the plate and then streaked with a loop. Blood may be Care must be taken in all cuts to avoid damaging obtained from larger animals with a sterile syringe which underlying organs. After cuts are made the detached is inserted into hcmolymph space after swabbing dorso- exoskeleton parts arc teased away from underlying lateral surface of carapace. Blood may also be used in tissue. This exposesorgans. Observe the organ posi- making a hanging drop slide Fordirect bacterial cxarnina- tion, hemolymph, and any free parasites, Huid color tion. and turbidity should be recorded.Note cystson sur- After careful surface disinfection and dorsal carapace face of digestive gland or in surrounding tissues. removal, a portion of digestive gland tissue may also be Mesenteries are normally transparent, smooth, glis- removed with watchmaker's forceps and streaked di- tening and moist. Any changes should be recorded. rectly onto plated media. Although the gland's tubules Check them for parasites, turbidity, thickening, etc. feeddirectly into the digestivetube, bacteria of particular Similarly examine heart. Remove heart with forceps type and magnitude will be isolated from this gland in and examinefurther with microscopenoting para- moribund shrimp. sites or abnormal tissue, Identification of isolated bacteria Various manu- ~ Digestive system: Examine digestive gland for inter- als are available that describe methods for bacterial iden- nal and external color, size, texture and presence of tification. The key that follows is helpful in identifying parasites. Take a small piece of the tissue, put it on a isolatesto level of genus.Also provided is a list of media slide, press it with a coverslip and examine under a and test procedures of special importance in presumptive microscope. Look for mohle bacteria, viral occlusion, identification of bacterialisolates, It is basedon facility or parasites.Also note presenceand amount of any expectations of a field lab. necrotic tissue in the gland, Media and test procedures of special importance Prepare shrimp for removal of digestive system: cut inpresumptive identification of bacteria. off tail at the anterior of the sixth abdominal segment and save. Make cuts on already partially dissected 1. Brain heart infusion agar shrimp! along either side of the dorsal midline and dehydrated BHI agar ...... 26.0 gm remove cxoskcleton and tissues to exposeintestine. distilled water ...... 500.0 ml Then find and cut esophagus at the bottom of the Heat to boiling to dissolve the medium completely. cephalothorax. Remove digestive system taking care Sterilize. to release it from the thoracic partitions. Rcmove Plates: Pour sterilized media into sterile petri dishes digestive gland. Examine esophagus, stomach, stom- one third full. Let stand for ten minutes with lids off achfilter, anterior intestine,mid-intcstinc and poste- center so as to make an outlet for steam. Cover, lct rior intestine separately by placing portions on slide stand until it hardens. Invert, identify media and in a drop of water and teasing apart with dissecting refrigerate. needles. Observe for protozoan and mctazaoan para- Tubes: Fill tubes to desired amount; place caps on sitesand damagedtissue. Take the tail portion cut off loosely and sterilize. Tighten caps after autoclaving. earlier and dissect out the rectum and examine as for Lay tubesat an angle to solidify leaving a butt at the cnd of tube. 144 .. Treeceand Fox

Method of sterilization: Autoclave for 15 minutesat 15 1.0% ---.-- "-- "--"arginine HCl pounds of pressure121'C. water to 100% 2 Brain heart infusion agar with salt. Tritrate with .1 n NaOH until pH is 6.8. The color Add NaCl to the above medium to make 3 percentsalt should be orange. Autoclave. The final pH should be mdium. Prepareas indicated for BHI. 7.2. Salt should be added to media for isolation proce- Dispenseinto tubes and cover with one half inch of oil. dures that involve animals from distinctly marine Procedure: inoculate as for the 0 F basal medium. water, Incubate and read at 24 hours. Tryp tie soy agar Results: positive, intense pink color develops; nega- dehydrated T S agar ...... 20.0gm tive, no change. distilled water ...... ,.......... 500.0 ml 8 Cytochrome oxidase test Heat to boiling to dissolve the medium completely. Procedure: Smear a large amount of bacteria on Sterilize. Prepare as for BHI. For marine shimp work Pathotec-CO test paper General Diagnostic Division, make up 3 percent salt medium. Warner-Lambert Co., Morris Plains, N.J. 07950!. 4 Azide dextrose broth and dextrose agar with sodium Results: positive, bright blue color develops wi thin 30 azide seconds; negative, no change in color. for gram positive bacteria!, 9 Catalase Azidedextrose broth: Procedure: Drop five drops of 3% hydrogen peroxide beef extract, .........4.5 gm on a white spot plate, Scoop a moderate amount of tryptone, ....,....,....,.......,.15,0gm bacteria from a colony and place in the peroxide. dex trose 7 5 gm Results: positive, strong bubbling in three to five sodium chloride...... 7.5gm seconds; negative, no bubbling, sodium azide......,.....,....,...0.2gm 10. Flagella stain Bacto Flagella Stain, Difco Laborato- distilled water.....,...... ...l.0 liter ries, Detroit, Michigan, U.S.A. Azidc dextrose broth is availablc from Difco Labora- Stain preparation tories. Streptococcuscan be separated from Staphylo- dehydrated Bacto Flagella Stain ... 1.9 gm coccus and Micrococcus with catalasc test. For dex- 95% ethanol . 33,0 ml troseagar add 0.2gm sodium azide/liter medium. distilled water 67.0 ml 5. Nutrient broth Mix water and ethanol then add stain, Shake for 10 beef extract...... ,...... 3,0gm minutes. pcptonc,.......5.0 gm Procedure: distilled water...... l.Q liter a. Grow the bacteria in 5 ml of half-strength nutrient Prepare as above in tubes but do not slant since this is broth for about l8 hours. liquid media. Make with and without NaCI, b. Add 1 ml of 5-10 percent formalin and centrifuge 6. 0 F basal medium Hugh Leifson! 000 rpm, 10 minutes!. Preparation: c. Remove the supernatant Auid by aspiration. dehydrated 0 F basal medium...... 4.7 gm d. Add 10 ml distilled water and resuspcnd sedi- distilled water...... 500.0 ml ment, Heat to boiling to dissolve the medium completely. e, Centrifuge at 3,000 rpm for ten minutes Dispense 4.5 ml into each tube, f. Repeat wash. dextrose .10.0gm g, Resuspendbacteria in 10 ml distilled water. distilled water...... ,.....100.0 ml h, Using precleaned slides Corning! pour a drop of Add 0.5 ml of dextrose solution to each tube. suspension onto slide. Parafin oil, i. Tilt slide and allow to run down to end of slide. Overlay half of tubes with oil for a depth of 1 onehalf j. Air dry the film do not heat!, inch. k, Place slide on staining rack and add 1 ml of the Procedure: stain solution, Stain for ten minutes. Inoculate two tubes, one with oil and one without, Flood off the stain with water. Place loop well below surface in medium and swish. m. Drain and allow to air dry. Read at 24 and 48 hours. n, Examine slide with microscope searching for bac- Results: tests oxidation and fermentation of carbohy- teria with flagella. Many slides may have to be drate. Positive, acid A! is yellow color: negative, examined before flagella arc found. alkaline Alk! remainsgreen. 11. Susceptibility testing 7. NH3 a. Froman isolatedbacteria colony rernovcaloopof Preparation: bacteria and place on BHI plate, Add the folowing together: b. Using a sterile swab cotton on wooden stick! or 0.1% , peptone inoculating loop,distribute bacteria to all parts of 0.5% ...,...,, NaCI the media surface. 0.3% agar c. If media surface is not particularly moist, the 0.3% . KzHPO4 addition of several ml of sterile water will facili- 0.001% -- --" -" -" " "phenol red tate distribution of bacteria. Desigvr,Operation arrd Trairring ... 145

d. Add sensitivitydiscs to theplace, spacing so that 14. Spore Stain Wirtz-Conklin! overlaps of diffusion will not cause confusion Prepare smear on slide as above. Flood entire slide when reading. with 5 percentaqueous malachitegreen. e. Susceptiblebacteria will not grow on area where Steam for three to six minutes and rinse under run- antibacterialaffects them and a ringof no growth ning tap water. Counterstain with 0,5 percentaque- is formed. ous safranine for 30 seconds. f. Readwhether the microorganismis very sensi- Results:Spores are seenas green spherulesin red stain tive, moderately sensitive, slightly sensrtive, or rods or with red stained debris resistant. 15. Gelmsa stain g, If the sensibilitydiscs are not marked it will be Removeblood or other tissueand quickly make smear necessary to mark name on plate surface just on slide. Air dry. Fix the film with methyl alcohol for under disc. 30 seconds. Apply dilute stain for ten minutes. Rinse 12. Inhibition by vibriostat with distilled water and air dry. Make a 5 percent solution of 2,4-D amino -6, 7- 16. Acid-faststain Gugol adaptationof Ziehl-Neelsen disopropyl pteridine phosphaate known as /129! technique! in distilled water of chloroform.Dip blank antibiotic Materials sensitivity discs in solution and allow preparation to Stain impregnatead paperstrips The GugolStain Co., dry. Use asfor preparedantibiotic discs by the method 43-50 11th St., Long Island City, NY 11101! described above under susceptibility testing.Inhibi- 20 percent and 100percent solutions of reagentgrade tionof growth by thiscompound is characteristicof methanol. most vibrios compound. 0/129 is available from: Procedure: Gallard SchlesingerChemical Mfg. Corp.,854Mineola a. Heat fix smears Ave, Carle Place, LI,, NY 11514 and BDR Chemicals b. Dip Carbol-Fuchsin strip in 20 percentmethanol. Ltd., Poole,England. c. Place on top of specimenfor three minutes, re- 13. Gram stain move, rinse with water. Solutions: d. Decolorize and counterstain with second strip A. Modified Hucker's Crystal Violet dipped in 100 percentmethanol by covering for Solution A two minutes. ethyl alcohol 95% ...... ,....,...... ...20,0ml e. Rinse,air dry, examine. crystal violet certified!...... ,...... 2,0g 17. Hanging drop Solution B a. Apply vaselineor water around the depression Ammonium oxalate ...... 0.8g of the slide. distilled water,....,....,....,...,....,....,.80,0 ml b. Using the inoculating loop, aseptically transfer Mix solutions A and B one drop of bacteriato the centerof the coverslip, B. Iodine .1.0 g If bacteria are removed from solid media, mix a potassiumiodide 2,0 g loop full in a ml of water and then transfera drop distilled water ...... 300.0 ml of mixtureto coverslip. C. Decolorizer c. Invert the hanging drop slide and centerit well ethyl alcohol . 95.0 ml over the drop of bacteria.Press down on the edges acetone 5.0 ml of the coverslip so the vase]inc or water makes a D. Counterstain seal. safranine 0.85% . .6.0 g d. Quickly and carefully turn the slide right side up ethyl alcohol . . 20.0 ml so the hanging drop is suspended in the depres- distilled water......,...... ,..........,. 200.0 ml sion. Stainingprocedure: e. Observethe organism for motility and morphol- a. Air dry smearabout 15 minutes. ogy b, Hood with methyl alcohol for two minutes. Motility: motile, non-motile don't confuse Brownian c. Rinse with dishlled water. Movement with motility!. d. Floodwith crystalviolet solution and let standfor Morphology;Long rods,short rods, cocci, tetrads, etc. one minute. 18. Anaerobic growth environment for anaerobicbac- e. Wash smear briefly with tap water and drain off teria excess water. Materials include the following: f. Flood smear with iodine solution GASPAK anaerobicjar BBL CockeysvilleMaryland g. Wash with tap water and decolorizeuntil solvent 21030! flows colorless from the slide. Gaspakpacket h. Wash briefly with tap water. catalyst i, Counterstain with safranine for 20 seconds. methene blue indicator j. Wash briefly with tap water, blot dry and exam- Procedure: ine. Add platesor tube to jar. Result:Gram-positive organisms are blue; gram- Place on Gaspak envelope one anaerobic indicator negative, red. and a catalyst in the jar. 146 ... Treeceand Fox

Add 10 ml of water to Gaspak envelope and clamp on known to be produced by viruses. A common sign is the lid. occurrence of "occlusions" observed in tissue cells. In Hydrogen producedby thc packetreacts in the pres- contrast to the distinct viral units of occluded viruses enceof the catalyst to produce an anaerobic atmo- mentioned above, theseocclusions are unusual structures sphcrc.CO> is producedby the packetalso. of and within the host cell. Occlusions may occur within Incubate for 48 hours. the nucleus or cytoplasmof affected cells and possess Bactcrio]ogical media available trom: particularstaining characteristics. Ifocclusions are present BBL Division of Bioqucst, Box 243, Cockysvillc, in a susceptibleanimal then this signis takenas presump- MD 21030 tive evidenceif virus presence.The proceduresof this Difco Laboratories, Detroit, Michigan techniqueare briefly as follows..Tissues of suspectani- Other techniquesare employedin DiagnosticLabora- malsarepreserved, embedded, sectioned with microtome tories. Gram and other staining is used in conjunction and appropriately stained.A positive result consistsof with histological preparation. Equipment that mea- finding occlusionswith light microscope.Embedding, suresfatty acid profiles is alsoused to quickly differ- sectioningand stainingfollow standardprocedures. Pres- entiate pure cultures of bacteria. ervation fixation! proceduresrequires special consider- ation. Samples should be fixed in a manner that the Virological fixative can readily penetrate important organs.Fixative I.ight microscopy. Many of thc known crustacean may be injected or better, bodies may be cut open or virusesbelnngto thcoccludcdtype, their variouslyshaped halved. If cut portions are divided, subsequent handling occlusinnsbeing visible with aid of a light microscope. should be conducted so as to avoid confusion nf identity Several staining procedures maybe used to enhance vis- betweenshrimps. Two commonly usedfixatives are: ibility if the occlusinnsbut in acuteinfection, this isunncc- Davidson's Solution cssarytn the trainedeye. In the caseof carrieranimals, the 3 parts ethyl alcohol chances for detection are related to thc time utilized in 2 parts formalin examination and proper selection of tissues expected to 1 part glacial acetic acid contain ~~ral uni ts. add acid last or at time of fixation The advantages of light microscopy are that direct Buffered neutral formalin solution observation nf viruses is possible and a result is obtained 1 part formalin 7 percent formaldehyde solution! quickly. l'hc disadvantagesare that viruses cannot be 9 parts water detected beyond limits of magnification and one can 4 gramssodium phosphate, monobasic, monohydrate easilyoverlook very light infectionsin large animals. 6.5 grams sodium phosphate, dibasic, anhydrous PrOCedureCnnSiStS Of mak1'ngWet mOuntSOf SuSpeCt For marine forms useseawater from sourceof capture. tissuesand subjecting them to microscopial examination. In caseswhere animals are inapparent carriers of the Open shrimp so as to expose the digestive gland and virus, a virus may be demonstrated by exposing i,e., with cleanforceps tear back the coveringtissue capsule! feeding or injection! appropriate life stages of highly of gland, exposing internal tissues. Remove several small susceptible animals and waiting i.e., a month! for devel- internal portions nf thc digcsitvc gland and intestine and opment of histological signs. If inclusions are then found, place each on a slide. Quickly place a coverslip onto tissue the test is considered positive for virus presence. If no fragment. With a clean blunt instrument press the cover- inclusions are noted, animals are considered either virus- slip until the tissue is thin enough under the cover slip for free or else the test failed to detect the presenceof virus. In detailed cellular observation. Examine for occlusions. Thc the later case additional tests could be conducted, The nccIusions of Bacutoviruspenaei are tetrahedral or pyrami- advantages of this test are that it serves to demonstrate dal in three dimensional form. Other occcludcd virsus viral pathologyof virusestoo small for light microscopy may have other shapes.Use high-dry magnificationn 00 and are in lieu of tissue culture techniques which are yet tn 600X! nr nil immersion 00 to 1000X! and look for to be developed, Another advantage is that it has the characteristic shapes. potential for detection in carriers. Disadvantages are that lt should be maintained that viruses infecting the for carriers, a result takes a long time to produce, suscep- hcpatnpancreasare passedout of the animals via the feces. tible live animals of proper size are required, and if not Occl usions can be dctcctcd sometimes by examination of confirmedby electronmicroscopy then the resultis based fecesacquired directly from the animal or from the water on only signs.The applicaiton of feeding/histopathology in the form nf fecal casts, viral enhancement! method is well suited for attempts at Serology. Flunrcsccntantibodyproccdurcshavebecn establishing virus free stocks. It is less functional in rou- developed for detection of baculnvirus presence in in- tine diagnosis. sects.The sam«cnmmcrcially availablclabeled antibodies Electron Microscopy with Negative Staining. The will also react with crustacean bacutoxiruses. This tech- main advantage of this technique is the capability to niqueis likely tn assume widespread practical application directly observe even the smallest virus. Another advan- in labnratorics with accessto a fluorescent microscope. tage is that i t provides a quick resul t. A limita tion is that i t I.abnratnry rcs arch with crustacca has also developed is best used for animals with acute virus disease rather techniques for fluorescent antibody and ELISA. than carrier animals. The procedure demands the occur- Histological changes in host. Detection of the pres- rence of advanced viral damage so as to give a threshold ence nf viruses is sometimes based on histological signs presence of one million virus particles per cubic Design, Operationand Trairring ... 147 centimeter.The general procedure is as follows: Grind 200u/mlpenicillin G for inhibition ofbacterial growth. tissue into a suspendable mass, Suspend in three parts water/one part tissue ta a total volume of approximately Protozoological 15 nulliliters. Centrifuge at 1000G for 30 minutes =3000 Use scissorsta clip off portions of the body thatarc thin rpm!. Removefive milliliters of supernatantand ultracen- and transparent pleopods, edges, of uropods, etc.!. Place trifuge at 70,000 G for one hour. The resulting pellet is the clipped portion on the slide and cover with a drop of resuspended in 0.1 milliliter water two drops!. A mixture holding water or water with appropriate salinity!. Pre- is then made of one milliliter water, 0.2 milliliter faur pare a wet mount by touching the edge of the drop with a drops! of phosphotungstic acid, one drop bovine serum cavcrslipand lowering it onto the dissectedspecimen in a albumin, and one drop sample.This mixture is then put manner that prevents entrapment of bubbles. Examine into an all glassnebulizer and atomizedon a coatedgrid. with a microscope for protozoa such as Zootharnniurn, Material is viewed directly on the grid with electron Acineta, Lagenophrys,and Epistyhs. Note relative abun- microscope. Transmission electron microscopy after tixa- dance of protozoa. tion, embedding and scctioning will also demonstrate Usescissors to clip off portion of carapacethat covers virus particles but thc procedure is too involved and the gills. Clip off a small portion of gill and prepare for costly for routine diagnosis. examination as described above. Note kinds and abun- Cell Tissue! Culture. Cell cu1tu re basically consistsof dance of protozoa. Also remove a portion of the gill near isolating living cells from an animal and placing them into the bailer and check for apostome cysts. a sterile medium that will support their growth. Virus Removeblood as described above and check for proto- detection in routine cell culture work consists of adding zoa. Also examine blood in situ or as it exudes fram virus suspect material ta the cell culturcand watching for removed appendages or gills. Special staining or irnpreg- damage.Such virological examination relies on estab- nation methodsare required to properly identify species lished "ce11lines" which are known ta be susceptible to a af most protozaaaffixed to cuticular surfaceor invading particular virus. Established cell lines are cell cultures that the blood. have been passed from growth vessel to growth vessel50 Open the specimen a describedabove and examine the times, a process that takes at least two years ta complete. digestive tract. Note abundanceof gregarinesand loca- Some fish cell lines are naw 20 years old. The initial tion of peculiar concentrations., successfulattempts at passingcells from vesselta vessel Dissectspecimen as ind icated above paying particular are called "primary cell cultures," Primary cultures may attention to peculiar coloration in tissues. Remove por- be of some benefit in detection of virus even though their tions of such tissueswith sharp forcepsand preparewet life span is highly unpredictible. mount. Apply gentle pressureta expose spores.Note Cell culture is the basis for detection of mammal and number of spores in spore groupings, structure of fish viruses. Procedures are nat well established for crus- induvidual spores and tissue from which they were re- taceancell culture.Na cell lines havebeen developed but moved. there has been some successin starting primary cultures. Helminthological Flukes, Acanthocephalans, and Chen, S,N, and G,H. Kou 1989, Journal of Fish Disease. Tapeworm s! 12:73-76!. Cell lines from nan-crustacean animals have been challenged with crustacean viruses but have not Checkparticular body locationsthat handbooksgive proved usable. as preferred by worms. No te abundance of worms or their cysts.Identification will oftenrcquircmountingand stain- Rickettsia/Chlamidia detection ing. Locatea worm and releaseit by rupturing the cyst Thereis limited knowledgeaf this group of organisms carefully with fine needles.Prepare a wet mount af the or their importance. Members of the group are sometimes worm so as ta catch it in an extended position. Light found in histologicalsections of tissues.They form obvi- pressure on the coverglass will ensure that tapcworrn ous masses in tissues especially hepatapancreas!, Be- scoleccs and acanthocephalan proboscises are extended cause of their small individual size observation by elec- and that flukes are flattened.Acanthocephala kept in tap tron microscopy is needed for confirmation. water for a fcw hours ta several days will retain everted proboscises. With a paper Kleenex! remove most ot' the Fungal water fram under the cover-glass. Immediately flood with Fungus samples may be scraped or cut fram foci of a hot but not boiling 5 percent formalin. After a minute invasion. Wetmauntsare madeand structureand fruiting release the pressure. Place slide with worm into a dish of bodies examined. Identification can often bc made on this alcoholso as ta cover the entire preparation.Cover dish basis. The use at special stains such as Cracott's is useful and wait until thenext day beforeproceeding. By the next bath in tissuesquashes and on histological sections.For day the worm will be adheredto either the coverglassor further investigationfungus may bc isolatedsimilarly as the slide. Whicheverthe case,conduct staining thereon. bacteriaand grown an petri dishes containing suitable The worm will often detachcompletely. If so,remount at mycological medium. end of staining process, Sabouraud dextrose agar, Stainingmay be bv proceduresgiven in variouspara- dehydratedsabouraud dextrose agar ...... 37.0gm si tology manuals. distilled water. 500.0 ml The one that follows is commonly used: pius salt if marine! 1. Add formalin-preserved worms warms only into 35 Option: the above plus 200mg/I streptomycin sulfate and percent ethyl alcohol-ETOH!. 'I 48 ... Treeoeand Fox

2. After about five minutes draw off 35 percent ETOH anic Institute.. These animals were only supplied to certi- and replace with 50 percent ETOH. fied U.S. producers. Now that the shrimp both brood 3. After five minutes draw off 50 percentalcohol and shrimp andlarvae! are availablefrom certifiedcommer- replacewith stain that is diluted withat leastan equal cialsuppliers such as AmorientAquafarm Inc., Hawaii volume of 50 percent alcohol. and Harlingen Shrimp Farm, Texas! any producer may 4, About 15 minutes is usually enough time to slightly obtain SPF animals, SPF animals should assist in the ovcrstain. control of the IHHN, HPV and B-P type viruses as well as 5. Draw off stain and replace with 1 percent HCL in 70 other pa thogens.Obtaining SPFanimals does not guaran- percentalcohol to destain. teedisease-free animals. The producermust also practice 6. Examine frequently and destain until light pink. good managementprocedures and employ strict precau- 7, Draw off acid 70 percentand replace with 100percent tions againstdisease introduction to SPF animals. Good ETOH for five minutes. nutrition is important in maintaining shrimp health, The H. Repeat procedure to remove trace acid ETOH. producer must try to keep the environmentalconditions 9. Draw off about 1/2 of the 100 percent and replace it optimum, keep stressmimi nized, continually monitorthe with clearingagent terpineol!. shrimp's health,and take immediateaction if a health 'IO. Draw off and replace with pure clearing agent. problem is detected. 11, Af ter a fewminutes removeclearing agent and mount in Permount on slide and cover with coverglass. Acknowledgments Stain Scmichon's carmine - add one volume of glacialacetic acid slowly with mixing to an equalamount Material for this chapterwas provided through publi- of distilled water. Add carminepowder in excessof that cationsof Dr. Don Lightnerand others and isused with his with dissolves immediately and heat to 95 to 100degrees permission, Lightncr acknowledges R.M. Redman, W. C for 15 minutes. Cool and filter. The filtrate is the stock Randall and T.A. Bell for their assistancein assemblingthe stain which is diluted with at least an equal amount of 70 review, which was publishedby the Aquaculture Depart- percent alcohol before usc, ment SoutheastAsian FisheriesDevelopment Center in Acanthoccphalanshave a resistant cuticle. A small the Proceedingsof the First International Conferenceon puncture made with a fine needleprior to staining will the Culture of Penaeid Prawns/Shrimp. Parts of it are allow stain to reach the internal organs. used with the written permission of the Center,and many Ncmatodes are difficult to stain but can be identified thanks are extendedfor this cooperation, without staining. Clearing is required, however, because Dr. Lightner also acknowledges grants from the Ha- the cu ticle usually interferes wi th examination of in ternal waii Aquaculture Development Program and the Na- structures, Place hot 70 percent alcohol preserved speci- tionalSea Grant Program, and thanks TexasA& M Univer- men on a slide and remove most of the liquid with a sity, National Marine Fisheries Center, Continental Fish- Kleenex. Examine in Hoyer's solutionor lacto-phenol. To eries Ltd., The Marine Research Division of the Alabama prevent specimen collapse add lacto-phenol slowly by Department of Conservationand Natural Resources,as portions. Hoyer's dries to give a permanent mount. well as others too numerous to list for submission of preservedsamples. Updates and the methodsof routine Hoyer's Solution: Lacto-phenol: examinationwere provided by Dr. S.K,Johnson, Depart- Distilled water 50.ml Glycerine 2 parts ment of Wildlife and Fisheries Sciences, Texas A&M Uni- Gum Arabic flakes 30 ml Distilled water 1 part versity. Material compiled for the Shrimp Toxicology Chloral hydrate 300 gm Phenol, melted 1 part section was provided by Couch 979!, with written per- crystals mission from Tod Rupp, Academic Press,permissions Glycerine 30 ml Lactic acid 1 part department, Orlando, Florida. and the Academic Press, Mix in order at room temperature. New York. Specific PathogenFree SPF! Shrimp SPF shrimp were developed in 1989through the U.S. SelectedRecent Shrimp Toxicology and Marine Shrimp Farming Program Thc Gulf Coast Re- Disease References searchLaboratory Consortium USDA! and the Oceanic Institute, Hawaii! efforts. Commercial suppliers who 1. Abu-Hakima, R., 1984. Preliminary observations of mustbcccr tidied by thc USDA's Shrimp Consortium! now theeffects of Epipemaeanelegans Isopoda: Bopyridae! off'er SPFshrimp. on the reproduction of Penaeussemisulcatus de Haan! Test results at Harlingen Shrimp Farm in Texas, Pre- Decapoda: Penaeidae!,Int.J. Invertebr. Repro.,7:51- sentation at Texas Aquaculture Association meeting, 1992 62. by Dr. Frank Castilleof Texas A&M University!have been 2. Akamine,Y. and J.L. Moores,19S9. A preliminary very favorable with SPFanimals. Growth and survival of study on disinfectionmethods of penaeid-shrimp hatcheries contaminated by Baculovirus penaei.Ab- SPFP. vannamei werebetter than non-SPFanirnals. Results from Ecuador personal communication with Mr.A. Perez! stractWAS Conference.Feb. 1989,Los Angeles, CA, also indicate increasedgrowth and survival with SPF 3. Akiyama, D.M., J.A. Brockand S.R.Haley, 1982.Idio- shrimp obtained from a Texas supplier. pathic muscle necrosis in the cultured freshwater prawn, Macrobrachium rosenbergii. VM/SAC, 1119- Until recently, SPF animals adults! were available only through the U.S. Shrimp Consortium and the Oce- 1121. Design, Operationarat Training ... 149

Alcaraz, M. and F. Sanda, 1981.Oxygen consumption H. Van Campen, 1986. Hepatopancreatic rickettsial by Nephrops noroegicus L.! Crustacea,Decapoda! in infection of the penaeidshrimp, Perraeusmarginatus relationship to its moulting cycle. J, Exp. Mar, BioL Randall!, from Hawaii. J. Fish Disease. 9:353-355. EcoL, 54:113-118. 2l. Brock, J.A., D.V. Lightner and T.A. Bell, 1983. A re- Amos, K.H., 1985. Procedures for the detection and view of four viral BP, MBV, BMN and IHHNV! identification of certain fish pathogens TheFish Health diseasesof penaeid shrimp with particular reference Blue Book!. Third Edition. Spec. PubL Amer. Fish. to clinical significance, Council for the Exploration of Soc. 114p. the SeaC.M. 1983/Gen:10/Mini-Syrnposiurn. Anderson, I.G., M. Shariff and G, Nash, 1989, A 22, Bueno, S.L,, R. Nascimento and I. Nascimento, 1989 hepatopancreatic microsporidian in pond-reared ti- Baculoviruspenaei infetion in P. sublitis: A new host ger shrimp, Penaeusmonodon, from Malaysia.J. In- and a new geographical range of the disease, pre- vert. Pathol., 53..278-280, sented WAS meeting, Feb. 1989, Los Angeles, CA., Batchelder,T.L., H.C. Alexanderand W.M. McCarty, USA. 1980. Acute fish toxicity of the Versene family of 23. Castille, F.L., and A.L. Lawrence, 1981. The effects of chelating agents, Bull. Environmental Contam. EDTA ethylenedinitrotetracetic acid! on the survival Toxicol. 24: 543-549. anddevelopment of shrimpnauplii Penaeusstyli rostris Baticados, C.L., R.M. Coloso and R.C. Duremidez, Stimpson! and the interactions of EDTA with the 1986.Studies on the chronic soft-shell syndrome in the toxicities of cadmium, calcium and phenol. J. World tiger prawn, Penaeusmonodon Fabricius!, from brack- of Mariculture. Soc. 12: 292-304, ishwater ponds. Aquaculture, 56:271-285. 24. Castille, F,L. and A,L. Lawrence, 1986.The toxicity of Bautista,M.N.,1986. The responseof Penaeusmonodon erythromycin,minocycline, malachite green and for- juveniles to varying protein/energy ratios in test di- malin to nauplii of the shrimp P. stybrostris. WAS ets, Aquaculture, 53:229-242 17:4!: p. 13-18. 10. Bell, T.A. and D.V. Lightner, 1988. A handbook of 25. Castille,F.L. and A.L.Lawrence, 1981. A comparison normal penaeid shrimp histology. World Aquacul- of the osmotic sodium and chloride concentrations ture Society, Baton Rouge, Louisiana, 114 pages, between the urine and hemolymph of P. setiferusand 11. Bell, T.A. and D.V, Lightner, 1987. IHHN disease of P. stylirostris. Comparitive Bio. Chem. and Physio. Penaeusstylirostris: Effects of shrimp size on disease 70 A:525-528. expression. J. Fish Diseases, 10:165-170. 26. Chen,D,,X.Liu, Y, Yu,Z, Yang,T.Xiao and Q. Wang, 12. Bell, T.A, and D,V. Lightner,1983. The penaeid shrimp in press.The Vibrio diseaseof penaeidshrimp in the species affected and known geographic distribution larval rearingsystems and its comprehensivepreven- of IHHN virus. First Bieannial Conference on Warm tions, Ann. Rept. Open Lab, Acad. Sinica Inst. Water Aquaculture- Crustacea.Brigham Young Univ. Oceanol. Exper. Mar. Biol., 1. Hawaii Campus!,AVI Press,Westport, CT. 27. Chen,H.C., 19S1,Some abnormal aspectsof the prawn 13. Bland, C.E., D.G. Ruch, B.R. Salserand D.V. Lightner, Palaemonelegans exposed to heavy metals, Proc.R.O.C.- 1976.Chemical control of Lagenidium,a fungal patho- U.S. Coop. Sci. Seminar on Fish Disease 3:75-83. gen of marine crustacea.University of North Caro- National Sci Council, R.O,C, lina SeaGrant Publication, UNC-SG-76-02, 38pp. 28. Chen,J.C. 1983. Management of waterquality, Chieu- 14. Bland, C.E,, 1975. Fungal diseases of marine crusta- Ta Book Co., Taipei, Taiwan, cea, Proceedings of the Third US-JapanMeeting on 29. Chen,J,C.,andPC.Liu,1987.Accumulationofheavy Aquaculture at Tokyo, October, 1974,41-48. metals in the nauplii of Artemia.J. World Marculture 15. Bonami,J,R., M. Brehelinand M. Weppe,1986. Obser- Soc. 18: 84-93. vations sur la pathogenicite, La transmission et la 30. Chen, J.C.,Y.Y, Ting, H. Lin and T.C. Lian, 1985 resistance du MBV monodon baculovirus!. Proc. Ind. Heavy metal concentrations in sea water from grass- International Colloquium on Pathology in Marine prawn hatcheries and the coast of Taiwan. J. World Aquaculture,Porto, Portugal, 7-11 Sept.,1986, p. 119. Maricul, Soc., 16:316-332. 16. Bonami, J.R., 1976. Viruses from crustaceans and an- 31. Chen, L., 1988.The effect and applicaton of Wu Bei Zi nelids: Our state of knowledge. Proc. Int. Collo. to cure shrimp fungus disease Dui Xia!. China Fish- Invertebr. Pathol., 1:20-23. eries, 6:12 in Chinese!. 17. Bovo, G., G. Ceshcia,G. Giorgetti and M. Vanelli, 32. Chen, S.N. and G.H. Kou, l989. Infection of cultured 1984. Isolationof anIPN-like virus fromadult kuruma cells from the lymphoid organ of P. rnorrodorby shrimpPenaeusj aponicus. Bull. Eur. Assn.Fish Pathol., monodon-type baculovirus MBV!. J. Fish Diseases, 4:21. 12:73-76. 18. Brisson,S.,1985.Gas-bubblediseaseobservedinpink33. Cheng,W.W. and W.Y. Tseng,l982. A parasite, shrimps,Penaeus brasiliensis and Penaeuspaulensis. Epipenaeoningens Nobili! of the commercialshrimp. Aquaculture, 47:97-99, Penaeussemisulcatus de Haan! in Hong Kong.Intro- 19, Brock, J.A., 1988. Rickettsial infection of penaeid duction of Taxonomy, B, Morton ed.!, I:393-399. shrimp. ln: C. Sindermannand D. Lightner eds.! 34, Chinnayra,B.,1971. Effect of heavymetals on oxygen Disease diagnosis and control in North American consumption by the shrimp Caridina rajadhardi Bou- aquaculture.Elsevier, Amsterdam, pgs. 3841. vier!.Indian J. Exp,Biol. 9; 277- 278. 20, Brock,J.A., L.K. Nakagawa, T. Hayashi,S. Teruya and 35 Chang, Y.C. and H. Loh, 1984. Hepatopancreas 150 ... Treecearrd Fox

chlamydialand parvoviralinfections of farmedma- Nunez, 1987. Effectsof tcmperatiure on the acute rine shrimpin Singapore.Singapore Vet. Jrnl.9:51- toxicityof heavy metals Cr, Cd and Hg! to the 56. freshwatercrayfish, Procam bar us clarkii Girard!. Bull. 36 Chu, S.H., 1976. Note on the fungus diseaseof the Environm. Contam. ToxicoL 38: 736 - 741. pond-culturedgrass prawn Penaeusrnonodon. Jour. 53. Deardorff, T.L. and R.M. Overstreet, 1981. Larval Fish. Soc. Taiwan, 4:31-35. Hysterothylacium Thynnascaris! Nematoda; 37 Cipriani,G.R., R.S. Wheeler and R.K. Sizemore, 1980. Anisakidae! from fishes and invertebrates in the Characterizationof brawn-spotdisease of GulfCoast Gulfof Me>chico.Proc.Helminthol. Soc. Wash.,48:113- shrimp.J. Invertebr, Pathol., 36:255. 126. 38 Clark, J.V., 1986. Inhibition of molting in Penaeus 54. Deardroff,T.L. and R,M.Overstreet,1981.Review of semisulcatus de Haan! by long-term hypoxia.Aquac- Hysterothylaciurnand Iheringascaris both previously ulture, 52:253-254. Thynnascaris! Nematoda: Anisakidac! from the 39 Colorni, A,, T. Samocha and B, Colorni, 1987. Patho- northern Gulf of Mexico. Proc, BioL Soc. Wash., genicviruses introduced into Israeli mariculture sys- 93:1035-1079. temsby importedpcnaeid shrimp. Barnidgeh, 39:21- 55. Diamant, A., A. Colorni, 1987. Viral infections in 28. pcnacidshrimp in Israeland use of acridineorange 40 Corkern, C.C,, 1978. A larval cyclophyllidean fluorescenscas a rapiddiagnostic method. E.A.F3'. Cestoda:Trypanorhyncha!, J. Parasitol, 45:490. 3rd Inter. Conference, Bergen Norway!, Aug. 31- 41 Corkern, C.C., 1977, Protozoan and metazoan Sept. 3, 1987 Abstract!. symbiotsof naturaland mariculturedpopulations 56. DoughtieD.G. and K.R. Rao,1983, Ultrastructural ot'commercial shrimp from thc northwesterncoast andhistological studyof degenerativechanges lead- of the Gulf of Mexico. Dissertation Texas A&M Uni- ingtoblackgills in grassshrimp exposed to a dithio- versityy,167 pages. carbamatebiocide. J. Invert Pathol. 41:33-50. 42 Correa,M., 1987,Physiological effects of metaltox- 57. Doughtie,D.G. and K.R.Rao, 1983. Ultrastructural icityonthe tropical freshwatershrimpMacrobrachium and histologicalstudy of degenerativechanges in carcinus Linnco, 1758! Environmental Pollution, theantennal glands, hcpatopancreas, and midgut of 45: 149 - I 55. grassshrimp exposed to two dithiocarbamatebio- 43 Couch,J.A., 1983. Diseases caused by protozoa.In: cides.J. Invertebr. Pathol., 41:281-300. Provenzano,Jr,, A,J, cd.!, Pathobiology.The Biol- 58. Doughtie,D.GP.J. Conklin and K.R. Rao, 1983 ogy of Crustacea,p. 79-111, Cuticular lesionsinduced in grassshrimp exposed 44 Couch, J.A., 1981. Viral diseases of invcrtcbratcs to hexavalentchromium.J. Invertebr, Pathol,,42:249- other than insects.In: Pathogencsisof invertebrate 258. microbial diseases E.W. Davidson, ed.!, pp. 127- 59. Dunlop,S., and G. Chapman,1981. Detoxication of 160. Allanheld Osmum Publ.! Totowa, New Jer- zinc and cadmium by the freshwater protozoon sey. Tetrahymenapyriformis . Growth experimentsand 45 Couch, J.A., 1979. Shrimps Arthropoda:Crustacea: ultrastructural studies on sequestrationof heavy Penaeidae!.Chapter 7 Inpollution ecology of esturine metals. Environ. Res. 24: 264 274. invertebrates, L.W. Hart and S.H. Fuller, 60. Dykova, I. and J. Lom, 1988. A new haplosporean eds!.AcademicPress. N.Y. 1979,406 pages. infectingthe hcpato-pancreas in thepenaeid shrimp, 46 Couch, J,A., 1978. Diseases,parasites and toxic re- Penaeusvannamei. J, Fish Diseases,11:15-22. sponsesof commercial pcnacid shrimp of theGulf of 61 Egusa,S., Y, Takahashi,T. Itami and K. Mornovama, Mexico and South Atlantic. Fish. Bull, 76:1-44, 1988. Histopathology of vibriosis in the Kuruma 47 Couch, J.A. and L, Courtney, 1977. Interaction of prawn, Penaeusjaponicus Bate!. Fish Pathology, chemical pollutants and virus in a crustacean:A 23:59-65, novelbioassay system. Ann.N.Y. Acad. Sci.,298:497- 62 El-Musa, M.M. Al-Hossaimi and A,R. Abdul-Ghaffar, 504. 1981, Infestationof the shrimp Penaeussemisulcatus 48 Couch, J,A,, M,D, Summersand L. Courtney, 1975. DeHaan! by thebopyrid Fpipenaeon elegans Chopra! Environmentalsignificance of Baculovirus infections in Kuwait. Kuwait Inst. Sci. Res. Tech. Rep., 369:1- in cstuarinc marine shrimp. Ann. N.Y. Acad. Sci., 22. 266:528-536. 63 Engel,D.W., and B.A. Fowler, 1979. Factors 49. Couch, J.A., 1974a. Free and occluded virus, similar inffluencingcadmium accumulationand its toxicity to Baculovirus,in hepatopancreasof pink shrimp. to marineorganisms. Environ, Health Prespectives Nature 247:229-231. 28: 81-88. 50. Couch,J, A., 1974b,An enzooticnuclear polyhadroxia 64 EngelD,W,, and W.G. Sunca1979. Toxicity of cupric virusof pink shrimp:ultra structure, prevalence, and ion to eggsof thespot Leiostomus xanthurus and the enhancement.Jrnl. Invert. Path. 24:311-331. Atlantic silverside Menidia menidia. Mar. Biok 50; 51. Dali, W. and D,M. Smith, 1986. Oxygen consump- 121 126. tion and ammonia-N excretion in fcd and starved 65 EnvironmentalAgency ot Japan.1972. Water pollu- tigerprawns, Penaeus esculentus Haswell!. Aquac- tion control in Japan.1:54. Printing bureau, Min of ulturee, 55:23-33, Finance.Tokyo, Japan 52. Del Ramo,J,Diaz-Mayans, A. Torrcblanca,and A. 66 Evans,M.L. l980. Copper accumulationin the cray- Design, Operationand Training ... 131

fish Orconectes rusticus!. Bull. Environm. Contam. of shrimp from Texas rearing ponds, Texas A&M Toxicol. 24: 916 - 920. UniversitySea Grant College Publica tin n No,TAMU- 67. FAO., 1977,Control of the spread of major commu- SG-74-207,19 pages. nicable fish diseases. FAO Fisheries Report, No. 84. Johnston, R., 1964.Sea water the natural medium <>f 192. FID/R192 EN!. 36p. phytoplanktnn.II. Tracemetals and eh«lotion,and Foster, C.A., C.A. Farley and P.T. Johnson, 1981. general discussion. J. Mar. Biol. Ass. U.K.. 44: 87 Virus-like particles in cardiac cells of thc brown 109. shrimp,Penaeus aztecus. Ives!.J. Submicrosc. Cytol, 85. Kjos, D.J.,R.R. Furgcason and L.L. Edwards,1975 13:723-726. Ozone treatment of potable water to remove iron 69. Foster, P.L,, and F.M.M. Morel 1982, Reversal and and magan«sc. ntcr. Ozone Institute, Wa erburv, cadmium toxicity in a diatom: an interaction be- Conn. Symposium Proceedings pages 194-203!. tween cadmium activity and iron. Limmol. 86. Kumaraguru, A, E., D. Se!vi ar>dV.k. Venugnpalan, Oceanogr.. 27: 745- 752. 1980.Copper toxocity to an estuarine clam Mere!ix 70. Fusco, A C. ar>d R M. Overstreet, 1978. casta!. Bull. Envirorun. Contam. Toxicol. 24: 853 S pirocamallanuscricotus sp.n. and S.halitrophus sp. n, 857. Nematoda: Camallanidea! from fishes in the north- 87. Lawrence, A.L., J. Fox ar>d F..L. Castille, 1981. De- ern Gulf of Mexico.J. Parasitol., 64:239-244. creased toxicity of copper and manganese tons to 71. Gardiner, J., 1976.Complexation of tracemetals by shrimp nauplii Penaeusstylirostris, Stimpsnn! in the cthylenediaminetetraceticacid EDTA! in natural presenceof EDTA.J. World Maricu lture Soc.12!: waters. Water Research 10: 507 - 514. 271-280. 72. Halcrow, K,, 1988. Absence of cpicuticle from the SS. Lenng,J.K. and C.T. Fnntair>c,1979. Experimental repair cuticleproduced by four malacostracancrus- assessmentof the virul«ncc of four speciesof Vif>n'o taceans.J. Crustacean BioL, 8:346-354. bacteria in pcnaeid shrimp. pp. 109-132.In: Lewis, 73. Hassnn, D.W., 1987. Sources of bacterial contamina- D.L. and J.K, Leong eds.!. Proceedings of the Sec- tion in a penaeid shrimp hatchery, Jnl. WAS ond Biennial Crustacean Health Workshop. Texas 18!:34A. AquacultureCommuniqe's ¹134. A&M University Sea Grant Program Publication 74, Hatai, K., B.Z. Bian, C.A. Baticadns and S. Egusa, Nn. TAMU-SG-79-114. 1980. Studies on fungal diseases in crustaceans. II. 89. Lester, R.J.G., A. Dnubrovsky, J.L. Payntcr, S,K Hatiphthorosphitippensis sp, nov. isolated from culti- Sambhi, and J.G. Athcrtnn,1987, Light ar>dclcctrnn- vated larvae of the jumbo-tiger prawn Penaeus microscopic evidence ofbaculnvirus infcc inn in thc nronodon.Transactions of the Mycological Society prawn P, pfebeIus,Diseases of Aquatic Organisms of Japan, 21:47-55. 3:159-'I 65. 75, Hol werda, D.A., J. Hemelraad, P.R. Vecnhof and D. 90. Li, Y.H., Bischnff, J. and G. Maathieu. 1969. Earth I. Zandee, 19S8. Cadmium accumulation and Plan Sci. Letter 7: 265. depuration in Anodontaanatinaexposedto cadmium 91. Lightner, D.V. and R.M. Redman, in press, Hnst, chlorideorcadmium-EDTA complex. Bull. Environ. geographic range and diagnostic procedures for thc Contam. Toxicol. 40: 373- 380. penaeid-viru s disea s«of concern tn shrimp cul tu ri sts 76. Hose, J.E.,D.V. Lightner, R,M, Redmanand D.A. in the Americas. In: W. Dnughcrty cd.!, Proc. of a Danald, 1984.Observations on the pathngcncsis of Symposium on thc Frontiers of Shrimp R>.search, the imperfect fungus, Fusariumsolani, in the Califor- Elscvicr. New York, nia brown shrimp,Penaeus caiiforniensis.J. Invertebr. 92, Lightner, D.V., R.M. Rcdman, T.A. Bell, ar>d R,B Pathol., 44:292-303. Thurman, in press. Geographic dispersion of the 77, Johnson,P.T., 1984.Viral diseasesnf marine inverte- viruses IHHN, MBV and HPV as a consequenceof brates.Helgolander Meeresunters, 37:65-98. transfers and introductions of penaeid shrimp tn 78. Johnson,P.T., 1983,Diseases caused by viruses, rick- new regions for aquaculture purpnses. In: A sympo- ettsiae, bacteria, and fungi, In: Provenzano, A.J. ed.! sium on human influences on aquatic ccosystcms The biology of Crustacea, Vol, 6, Academic Press, held at the 1989'world Aquaculture Society meeting New York, p. 1-78. in Los Angeles, CA. 79. Johnson, S.K., 1989.Handbook of shrimp diseases. 93. Lightncr, D.V., R.M. Redman, and E,A. Almada TexasA&M UniversitySea Grant Publication TAM U- Ruiz, 1988. BP in P. stylirostrfs cultured in Mexico: SG-90-601. uniquecytopathology and a newgeographic record. 80. Johnson, S.K., 1989.Guidelines for the examination Jrnl. Invert, Path. 51:137-139. of biological disease agents in marine and freshwa- 94. Lightner, D.V., 1988. Diseasesof cultured pcnac>d ter shrimp unpublishedhandout!, shrimp and prawns, pp. 8-l27. In: C.J. Sindcrmann 81. Johnson,S.K., 1978.Handbook of Shrimp Diseases. and D.V. Lightr>cr, cds.!, Disease diagnosis and TexasA&M UniversitySea Grant Publication TAMU- control in North American aquaculture. Elscvicr, SG-75-603,23 pages, Amsterdam. 82. Johnson,S,K,, 1975.Cramped condition in pond- 95. Lightner, D,V. and T.A. Bell, 1987.lHHN discase of reared shrimp. Texas A&M University Fish Disease Penaeusstylirostris: effects of shrimp size nn disease Diagnostic Laboratory Leaflet No. FDDL-S6. expression. J. Fish Diseases,, 10:165-170. 83. Johnson,S,K1974. Ectocommensalsand parasites 96. Lightner, D,V,, R,M, Redmanand T.A. Hell, 1987, 352 ... Treece and Fox

Diagnosticmethodscurrently in usefor thepenaeid- tional Shellfisheries Assn., June 25-28, Tampa, virus diseases of potential concern to shrimp Florida, page 40. culturists in Hawaii. Jnl, WAS 18!:34A. Aquacul- 111. Mark, H.F., J.J. McKetta and D. F. Othmer, 1965. ture Communique's ¹135. Kirk-Othmer Encyclopedia of Chemical Technol- 97 Lightner, D.V. and R.M. Redman,1986. A probable ogy, Vol. 6, pp. 21 24.Interscience, N.Y. Mycobacteriumsp. infection of the marine shrimp 112. Mathews, C.P., M. El-Musa, M. AI-Hossaini, M Penaeusvannamei Crustacea: Decapoda!. J, Fish Samuel and A.R. Abdul Ghaffar, 1988. Infestations Diseases, 9:357-359. of Epipenaenonelegans on Penaeussemisulcatus and 98 Lightner, D.V., R.P.Hedrick, J.L. Fryer, S.N.Chen, their use asbioligical tags.J. Crust. Biol8:53-62. I.C, Liao, and G.H. Kou, 1987.A survey of cultured 113. McKenney,C,L,, and J.M. Neff, 1979,Individual penaeidshrimp in Taiwanfor viral and otherimpor- effectsand interactionsof salinity, temperature,and tant diseases. Fish Path. 22:127-140. zinc on larval development of the grass shrimp 99 Lightner,D.V. and R.M.Redman,1985. A parvo-like Palaemonetespugio.. I. Survival and developmental virus diseaseof penaeid shrimp.J.Invertebr. Pathol., duration through metamorphosis. Marine Biology 45:47-53. 52: 177 - 188. 100. Lightner, D.V., R.M. Redman, T.A. Bell and J.A 114. McLeese,D.W., and S. Ray, 1988,Toxicity of CdC12, Brock, 1984.An idiopathic proliferative-disease syn- CdEDTA, CuCI 2, and CuEDTA to marine inverte- dromeof themidgut andventral nerve in theKuruma brates.Bull Environ. Contam. Toxicol. 36: 749-755. prawn, Penaeusjaponicus Bate!, cultured in Hawaii. 115. Medlyn, R.A.,1980. Susceptibility of four geographi- J. Fish Diseases, 7:183-191. cal strains of adult Artemia to Ptychodiscusbrevis 101 Lightner, D.V., 1983.Diseases of cultured penaeid toxin s!. Pages225-231, in the brine shrimp Ar temia, shrimp pp. 289-320.In: J.P.McVey ed.!,CRC Hand- Volume I, Morphology, Genetics, Radiobiology, book of Mariculture. Vol. 1 Crustacean Aquacul- Toxicology.G. Persoone,P. Sorgeloos,O. Roels and ture, CRC Press. Boca Raton, FL. E.Jaspers eds.!. Universal Press,Wet tern, Belgium, 102 Lightner, D.V., R,M. Redmanand T.A. Bell, 1983a. 345. pages. Histopathologyand diagnosticmethods for IHHN 116. Momoyama, K. and T. Sano, 1988.A method of and MBV disease in cultured penaeid shrimp. Proc. experimental infection of Kuruma shrimp larvae, Symposium on Warm Water Aquaculture - Crusta- Penaeusjaponicus Bate!, with baculoviral midgut- cea. Brigham Young University. Laic, Hawaii, Feb. gland necrosis BMN! virus.J. Fish Dis., 11:105-111. 9-11, 1983. 117 Momoyama, K. and T. Matsuzato, 1987.Muscle ne- 103 Lightner, D.V,, R,M, Redman and T.A. Bell, 1983b. crosis of cultured Kuruma shrimp Penaeusjaponicus!. Infectious hypodermal and hematopoietic necrosis Fish Pathology, 22:69-75, IHHN!, a newlyrecognized virus diseaseof penaeid 118 Mornoyama, K., 1983. Studies on baculoviral rnid- shrimp, J. Invertebr. Pathol., 42:62-70. gut-gland necrosis of Kuruma shrimp Penaeus 104 Lightner, D.V., R.M. Redman and T.A. BelI, 1983c. japonicus! - III, presumptive diagnostic techniques. Observations on the geographic distribution, patho- in Japanese!,Fish Pathology, 17:263-268. genesis and morphology of the baculovirus from 119 Momoyama, K., 1981.Studies on infectious midgut- Penaeusmonodon Fabricius!. Aquaculture,32:209-233. gland necrosis of Kuruma shrimp Penaeusjaponicus 105 Lightner, D,V., R.M. Redman, T.A. Bell and J.A. Bate! - I, occurrence and symptoms. in Japanese!, Brock, 1983d. Detection of IHHN virus in Penaeus Bulletin Yamaguchi Prefectural Naikai Fisheries stylirostris and P. vannameiimported into Hawaii. J. Experimental Station, 8.1-11. World Marie. Soc., 14:212-225. 120 Muramoto, S., 1980. Decrease in cadmium concen- 106 Lightner, D.V., D. Moore and D.A. Donald, 1979,A tration in a Cd- contaminatedfish by short term mycotic disease of cultured penaeid shrimp caused exposure to EDTA, Bull. Environm. Contam. by the fungusFusarium solani. In Proc.2nd Biennial Toxi col. 25: 828 831. Crustacean Health Workshop, D.H. Lewis and J.K. 121 Nash, G., I.G. Anderson and M. Shari ff, 1988. Patho- Leong, eds.!, Texas A&M University SeaGrant Pub- logical changes in the tiger prawn, Penaeusmonodon lication No. TAMU-SG-79-114, pp. 137-158. Fabricius!, associated with culture in brackish-wa- 107 Lightner, D.V., R,M, Redman,R.R. Williams, L.L. ter ponds developed from potentially acid sulfate Mohney, J.P.M,Clerx, T,A, Bell and J.A. Brock, 1985. soils, J. of Fish Diseases, 11:113-123. Recentad vancesin penaeid virus disease investiga- 122. Nash, M. and G. Nash, in press. A reo-like virus tions. J. World Aquaculture Soc., 16:267-274. observed in the tiger prawn, P. monodon,from Ma- 108 Liu, P.C. and J.C.Chen, 1987.Effects of heavy metals laysia. Jrnl. of Fish Diseases. on the hatchingrates of brine shrimp Artemiasalina 123. Nash, M., G. Nash,. Anderson and M. Shariff, 1988. cysts.J. World Maricul. Soc. 18: 78 - 83. A reo-like virus observed in the tiger prawn P. 109 Lovell, R.T., 1984. Microbial toxins in fish feeds. monodonfrom Malaysia, J. Fish Diseases 11:531-535. Aquacult. Mag., 10:34. 124. Nash, G., S. Chinabut and C. Limsuwan, 1987. Idio- 110 Manthe, D., R. Malone and H. Perry, 1984, Factors pathic-musclenecrosis in the freshwater prawn, affectingmolting successof the blue crabCallinectes Macrobrachiumroscnbergii de Man!, cultured in Thai- sapidus Rathburn! held in closed, recirculating sea- land. J. of Fish Diseases, 10:109-120. water systems. Abstracts, 1984 Ann. Meeting, Na- 125, Natarajan, P,, 1979. On the occurrence of Design,0~ation andTraining ... I 53

trypanorhynchanplerocercoid in the marineprawn '141. Rosenberry,B,, 1989,1990. World Shrimp Farming Penaeusindicus. Curr. Res. India!, 8:63-64. 19S8and 1989.Both supplementsto Aquaculture 126 Nearhos, S,P.and R.J.G.Lester,1984. New records of Digest.9434Kearny Mesa Rd., San Diego, CA 92126. Bopyridae Crustaceas:Isopoda: Epicaridea! from Dated Jan. 1989and Jan. 1990 respectively, Queensland waters, Mem. QueensL Mus., 21:257- 142. Sano,K. 1979,Aquaculture and water. ScientistInc 259. Tokyo. 127 Nearhos, S.P., 1980. Aspects of parasitization of 143. Sano, T. and H. Fukuda, 1987. Principal microbial Queenslandpenaeid prawns by two membersof the diseasesofmaricultureinJapan, Aquaculture,6759- family Bopyridae Isopoda:Epicaridea!. M, Sc.QuaL 69, Thesis, University of Queensland. 144. Sano, T., T. Nishimura, H. Fukuda, T. Hayashida, 128. Nishimura, S,, T., H. Fukuda and T. Hayashida, and K. Momoyama, 1985. Baculoviral infectivity 1985.Baculoviral infectivity trials on Kurumashrimp trails on Kuruma shrimp larvae of different ages,p, larvae, Penaeusj aponicus, of different ages.In: A,E, 397-403.In: Fish and Shellfish Pathology, Academic Ellis ed.! Fish and Shellfish Pathology. Pages397- Press, N.Y. 403, Academic Press, London. 145. Sano, T., T. Nishimura, H. Fukuda, T. Hayashida 129. Nugegoda,D., and P.S.Rainbow, 19SS.Effect of a andK. Momoyama,1984.Baculoviral midgut-gland chelatingagent EDTA! on zinc uptakeand regula- necrosis BMN! of Kuruma shrimp Penaeusjaponicus! tion by Palaemonelegans Crustacea: Decapoda!. J. larvae in Japanese intensive-culture systems. Mar. Biol. Asso. U.K. 68: 25 - 40. Helgolander Meeresunters,37:255-264. 130. Nyhlen, L. and T. Unestam,19SO. Wound reactions 146. Sano, T,, T, Nishimura, K. Oguma, K. Monoyama and Apahnvmycesastaci growth in crayfishcuticle. J and N. Takeno,1981. Baculoviral infection of Kuruma Invertebr. Pathol., 36 187-197. shrimp, Penaeusjaponicus in Japan.Fish Pathol., 131. Overstreet,R.M., 1988. Aquatic pollution problems, 15:185-191. Southeastern U.S. coasts: histopathological indica- 147 Sawyer,T.K. and S.A. MacLean,1978. Some proto- tors. Aquatic Toxicology, 11:213-239. zoan diseasesof decapod crustaceans.Marine Fish- 132. Overstreet R.M., K.C. Stuck, R.A. Krol and W.E eries Review Paper, 1342:32-35. Hawkins, 1988. Experimental infections with 148 Shaughnessy,M. 1983.EDTA removes formation Baculoviruspenaei in the white shrimp Penaeus damageat Prudhoe Bay. J. Petr. Tech. pp. 1783 vannamei Crustacea: Decapoda! as a bioassay. J. 1791. World Aquaculture Soc., 19:175-187, 149 Sillen, L., and A.E, Martell, 1971.Supplement No. 1 133. Overstreet,R.M., 1983. Metazoan symbionts of crus- to stability constants of metal-ion complexes. Spe- taceans,In: A.J.Provenzano ed,!,Pathobiology. The cial Publication No. 25. The Chemical Society, Lon- Biology of Crustacea, p. 155-250.Academic Press, don. N.Y, 150 Simpson,H.J., H.W. Ducklow, B. Deck and H.L. 134. Overstreet, R.M. and S. Safford,19SO. Diatoms in the Cook, 1983. Brackish-water aquaculture in pyrite- gills of the commercialwhite shrimp.Gulf Research bearingtropical soils. Aquaculture, 34:333-350. Reports, 6:421-422. 151 Sindermann,C,J., 1988. Disease problems caused by 135. Overstreet, R,M, and T. Van Devender, 1978.Impli- introduced species.pp, 394-398,In: C.J.Sindermann cation of anenviron-mentally-induced hematomain and D.V. Lightner eds,!, Disease diagnosis and commercialshrimp.J. Invertebr. Pa thol.,31:234-238. control in North American marine aqua. Elsevier, 136. Owens,L., 1987.A checklistof metazoanparasites Amsterdam. from Natantia excludingthecrustacean parasites of 152. Sindermann,C.J. and D.V. Lightner eds.!, 1988. the Caridea!. J. Shellfish Research, 6:117-124. Disease diagnosis and control in North American 137, Owens, L., 1985. Polypocephalus sp. Cestoda: marine aquaculture. Elsevier, Amsterdam. 431 p. I.ecanicephalidae!as a biologicalmarker for banana 153. Soderhall, K., L. Hall, T. Unestam and L. Nyhlen, prawns,Penaeus merguiensis de Man!, in the Gulf of 1979. Attachment of phenoloxidase to fungal cell Carpentaria.Aust. J. Mar. Freshw.Res., 36:291-299. walls in arthropod i mmuni ty, J. Inve t. P athol.,34:285- 13S. Owens, L, and J.S.Glazebrook, 1985.The biology of 294. bopyrid isopods parasitic on commercialpenaeid 154 Solangi, M.A. and D.V. Lightner, 1976.Cellular in- prawns in northern Australia. In P.C. Rothlisberg, flammatoryresponseof Penaeusaztecusand P.setiferus B.J. Hill and D.J. Staples, eds.!, Second Aust. Nat. to thepathogenicfungus,Fusarium sp. isolated from Prawn Sem. NPS2, Cleveland, Australia, 105-113. the California brown shrimp P. californiensis.Jour- 139. Owens,L., 1983.Bopyrid parasiteEpipenaeon ingens nal of Invertebrate Pathology. 27!. 77-86. Nobili! as a biologicalmarker for bananaprawns 155. Sparks,A.K., 1985.Synopsis of InvertebratePathol- Penaeusmerguiensis de Man!. Aust. J. Mar. Fresh. ogy Exclusiveof Insects,Elsevier, Amsterdam. pp. Res., 34:477-481. 423. 140. Paynter,J.L, D.V. Lightner and R,J,G.Lester, 1985 156, Sparks, A.K., 1981, Bacterial diseasesof inverte- Prawn virus from juvenile Penaeusesculentus pp. 61- brates other than insects, In: Pathogenesis of inver- 64 In. P.C. Rothhsberg,B.J. Hill and D,J. Staples tebrate microbial diseases, Davidson E,W. ed.! eds.!, Second Australian National Prawn Seminar AllanheM, Osmum,Totowa, New Jersey,323. NPS2 Cleveland, Australia. 157 Sparks,A.K., 1980. Multiple granulomas in themid- 154 ... Treece and Fox

gut of the dungenesscrab, Cancerrnagisfer. J. of Invertebrate P athol., 35:323-324. 158. Spotts,D.G, and P.L. Lutz, 1981.L-lactic acid accu- mulahon during activity stressin Macrobrachiurn rosenbergiiand Penaeusduorarurn.J. World Mar. Soc 12.244-249. 159. Stern, S. and D. Cohen, 1982. Oxygen consumption and ammonia excretion during the molt cycle of the freshwater prawn, Macrobrachiurnrosenbergii de Man!. Comp. Biochem. Fhysiol, A,, 73:417-419. 160. Sunda, W. and.R,L, Guillard, 1976.The relationship between cupric ian activity and the toxicity of cap- per to phytoplankton.J. Mar. Res.34: 511 - 529. 161. Tsing,A, and J.R.Bonami, 1987. A newviral disease of the tiger shrimp Penaeusjaponicus Bate!. J. Fish Diseases, 10:139-141. 162. Tsing, A., D,V. Lightner, J.R. Banami and R.M Redman,1985. Is gut and nervesyndrome GNS!in the tiger shrimp Penaeusjaponicus Bate! of viral origin? 2nd Internationl Conf. of the European Association of FishPathologists, Mon tpellier, France abstract!. 163. Vogt,G., 19S7.Monitoring of environmentalpollut- ants such as pesticides in prawn aquaculture by histological diagnosis, Aquaculture, 67:157-164 164, Vogt, G., V. Storch,E.T. Quinitio and F,P.Pascual, 1985.Midgut-gland as monitor organ for the nutri- tional value of diets in Penaeusrnonodon Decapoda!. Aquaculture, 48:1-12. 165. Wheaton, F.W., 1987. Aquaculture Engineering Robert E. Krieger Publishing Co, Malabar, Fla. 708 pages.Original Copyright 1977by John Wiley & Sans, Inc. 166. Wickins, J.F.,1984. The effect of hypercapnic seawa- ter on growth and mineralization in penaeid prawns Aquaculture, 41:37-48. 167, Wiseman, M.O., R.L. Price, D.V. Lightner and R.R Williams, 1982,Toxicity of aflatoxin B, ta penaeid shrimp. Appl. MicrobiaL, 44:1479-1481. 168, Wu, Y. and M. Lu, in press. Electron microscopic studieson Vibriored leg diseaseof penaeidshrimp. Proc. Fifth Cong. Chinese Soc. Oceanol. Limrol. 169. Yasuda, K. and T. Kitao, 1980. Bacterial flora in the digestive tract of prawns, Penaeusjaponicus Bate!, Aquaculture, 19:229. 170. Yuan, Y.X., C,N. Gao and D.X, Zhang, 1992. Meta- marphosista postlarvaeof Penaeuschi nensis Osbeck by removal af heavy metals from rearing systems with polymeric absorbent, J. World Aquaculture Soc., 23: 205-210. 171. Zheng,G.,1988.Thesuccessoftheseparateinfection of pathogenic bacteria of the "Red Leg" disease of Uui Xia. Marine Fisheries, 1:34 in Chinese!. 172. Zamuda, C.D., and W.G. Sunda, 19S2,Bioavailabili ty of dissolved copper ta the americanoyster Crassostrea virginica I. Importance of chemical speciation, Mar. Biol, 66: 77- 82.

156 ... Treeceand Fox

Furazolidone Table 9. Routine Prophylactic Treatments for ~ Used to clean up "slime" or routineIy for three days Shrimp Larvae. Method used by Clyde from PLI lppm!. Simon of Aquatic FarmsLtd.!. 1989 Tref lan Larvae:Treflan: Days 1,2 artd 3-0.05 ppm eachday ~ Used routinely for three days after stockinglarvae .05 ppm!. Stage Treatment ~ Used if Lagenidirrmis evident on dead animals .05 N6 1.0 ppm Prefuran ppm!. Z2 4,0 ppm Oxytetracycline Malachite Green Mt 4.0 ppm Chloramphenicol ~ "May" help to control gregarines daily between M2 1.0 ppm Prefuran secondday of Zl and PL3 .0075 ppm!. PLr 4.0 ppm Erythromycin PL2 2.0 ppm Erythromycin Oxytetracycline ppm! & Erythromycin ppm! PL3 2,0 ppm Erythromycin ~ Used if all other drugs fail not well thought of. after transfer to PL section! Erythromycin"might" cause deformities of setae. PL< 4.0 ppm Oxytetracycline PLg 1.0 ppm Prefuran NOTE: PL,2 4.0 ppm Erythromycin ~ None of theabove drugs are administered during Z3/ PLrs 1.0 ppm Prefuran Ml transitionbecause larvae are prone to deformities PL 4.0 ppm Chloramphenicol and mortality. ~ When starting-upa hatchery,"routine" prophylaxis should be used sparingly at first. When no longer quences,Postlarval Feeding Regime and Dis- effective they should be switched to another in the ease Preventative Schedule used by Josh hopesof prolongingdurations between forced shut- Wilkenfeld, 1989. downs, etc. Figure52. Chemicaland AntibioticTreatments used in ~ Many of thesedrugs listed above have not been ap- Philippine Ha tcheries. proved for usein the United States seeexplanation in Figure 53, FormsUsed for the Calculation of Drug and thefollowing section,"Chemotherapeutantsin United chemicalRequirements for each tank, morn- StatesShrimp Hatcheries"!, ing and evening. Diligent management procedures have been devel- Chemotherapeutants in United States Shrimp opedthrough the years.Examples of theseroutines can be seenin the following tablesand figures: Hatcheries Table 9. Routine ProphylacticTreatments for Shrimp "Potentialinvestors in penaeid-shrimpculture in the Larvae Methodused by ClydeSimon, 1989!, United Statesface a dilemma. Expertise,materials and Table 10. Alternate Method of Larval Culture Se- proximity to a largedomestic market favor investmentin

Figure 52. Chemical and Antibiotic Treatments Used in Philippine Hatcheries

Thefollowing is a listof chemicaland antibiotictreatments that may be helpfulin treatinglarvae where disease has been observed: Agent Dosage Egg and Larval Treatment PL PL Bacteria Larval Bacteria Parasites Parasites Prefu ran 0.7ppm +++ + + Arythromycin 4ppm ++ + ++ Malachite Green 0.007ppm ? ++ Treflan 0,05ppm ? ++ ++ ++ Furazu lido ne 0.5ppm + + Streptomycin 4ppm + Tetracycline 4ppm + Chloromphenicol + + 3ppm + Neomycin 4ppm + Formaline 2-5ppm g + ? EDTAat 10ppmmay help hatching of eggsand help purify water!water stocking nauplii. +++ - 1st choice or most effective ++ - 2nd choice + - 3rd choice ? - uncertain as an effective treatment Design,Operation and Training ... l57

E I-CD I-CD I-CD C3 CD LO CD E CL IA lO 0 lO CL lA0! O o: 0 0 LCJ lA 0 o C3 PJ oCD Cb CD C 0 Oa C3 K U 0 0 aa lCJ o O 0 oo C D IV 0 0 GO Q. ICO I 0V! 0 O GCI 4 CD CD CE- CIl O CJ E oJ lO c5 0

E E E E E E LO CU LO lO 0 IA LO O O c IL I-E ' O O O 0 0 0 0 0 0 C> Ul 00 0 0 0 0 o OJ I0! O 0 -0 0 0 O 0 0 Z 0000 C3 00 CD LO E E o Q. C3 E I CO LA O D C3 VJ C! CCI CD C Cl E jz! 03 K 0 alL. 0

0 0 0 0 0 1 T C E 0C> 0 CB IJl E E E CLl 0 Ul LL lO CJ 0V!

0CO C P LO CU CU CV h 4l C 0E 0 O EC0 0

O I- CD CD CO 0 0 0 O 0 O 0 0 0 0 0 0 0 LJ ill 1 M 1 T T 1 ~ M T 1

e+NN+NX>222gpgp 0EC2 0 0C 0 I-0! <0 CU W M lO CD P 00 Q! <0 0I CL EE 0 0.E C0t4CLECL a0 O. CCI C3 C> C! Cfg E I Q. N 0 N 0 II LJ II ~o" o CI Cl N O NLL I- V 0 158 ... Treece artd Fox

Figure 53. Forms Used for the Calculation of Drug and Chemical Requirements for EachTank, Morning and Evening.

Drug and chemicalrequirements: Morning Drug and chemicalrequirements: Evening Date: Time: Date: Tinle'

TAN VOL CP FZ PREF OXY MG' TREF" FORM TANK VOL CP FZ PREF OXY MG' TREF" FORM" NO. MT! GM! GM! GM! GM! SOL! SOL! ML! NO. MT! GM! GM! GM! GM! SOL! SOL! ML!

10 10

12 12

13 13

14 14

15 15

16 16 TOTALS TOTALS

TOTALS CP FZ PREF OXY MG TREF FORM TOTALS CP FZ PREF OXY MG TREF FORM GM OR ML GM OR ML CHEM CHEM ML WATER ML WATER

To dispense.When total GM or ML of compoundis known, mix with water at rateof 50 ML per unit of compound. Then dispenseat rate of 50 ML of solution per GM or ML of chemicalrequired For any given tank.

Definitionsand normaldosages: MG' = Malachite green; 0.0075ppm = 1 ML/MT of solution madefrom 5.625GM malachitegreen in 750ML water CP = Chloramphenicol; 4ppm = 4 GM/MT TREF = Treflan;0 05ppm = 1ML/MT of solutionmade from FZ = Furazolidone;1 pprn= 1GM/MT 10 ML Treflan in 200 ML water PREF = Prefuran; 0.05-0.10ppm = 0.5-1,0GM/MT 0'Fo active! FORM ' = Formalin;50 ppm for one hour = 50ML/MT OXY = Oxytetracycline; 5 ppm = 5 GM/MT Design,Operafion and Training ... 159

the developing United States shrimp-culture industry, penaeidshrimp. Work on approval of oxytetracyclineis while climatic factors, operating costs and regulatory approximately50 percentcomplete, In 1986,FDA, Pfizer, constraintsoppose it, To compete,a United Statespro- Inc., and the University of Arizona reviewed the data ducer must be more cost effective and have a more inten- generatedunder theINAD. Fivestudies were iden ti fiedas sive-culture system. The use of chemotherapeutants may yet to be completed.They included ! a feed storage be required to maintain costeffectiveness in an intensive stability study, ! a fieldtrial to completeefficacydata, ! culture to reduce the diseaseproblems. The classesof an addi honal residue-depletion study to reduce the with- neededagents range from the antibioticsand antifungals drawal time from seven to three or four days, ! a residue ~o disinfectants and water-quality agents. These depletion study in broodstock,and ! a study to deter- chemotherapeutantsmust be both reasonablypriced and mine the presenceof the drug in tank or pond effluent readily available.In the United States,availability and to water under field conditions." some extent cost, is dependent on clearanceby the United Trifluralin Tref lan! States Environmental Protection Agency EPA! and the United StatesFood and Drug Administration FDA!. The "Trifluralin is an herbicide that is effective in the preven- EPAis responsible,for the mostpart, for thosepesticides, tion of larval mycosis. It is applied as a water treatment to disinfectants, algicides and water sani tizers with no claim inhibit the growth of the phycomycetousfungi Iagenidiurn for disease control, while the FDA regulates substances sp. and Sirolpidiumsp. in the seawaterof hatching facilities. used as therapeutants and food additives." This compoundappears to be a goodsubstitute for malachite "Only Aquatrine also known as Cutrine-Plus! has green for this particular use. The registration process for beenapproved for use in penaeid-shrimpculture in the trifluralin is in its early phases.A sponsor for trifluralin has United States.Several other compounds used by shrimp not beenidentified, and without a sponsor,registration is not culturists are on the FDA Generally Recognized As Safe possible.Although IR-4may sponsora compoundfor ap- GRAS! list. These are: sodium bicarbonate, acetic acid, proval or registration, the manufacturer of thc compound calcium oxide lime! and calcium hypochlorite [chlori- must permi t the use of data in its Master File and either the nated lime], Most other compounds are being used with- manufacturer or a companywith distribution rights mustbe out approval, and thus illegally. Several compounds are willing to add the new usage to its label. Discussions~~ th well into the approval process. These are formalin, FDA indicate that EPA will be responsiblefor regulating this oxytetracycline and trifluralin," compound.Several efficacy studies are in the literatureas "Formalin is used in controlling peri trichousciliate gill well as a studydocumentingrela tively low toxicity to penaeid and surfacefouling organismsof thegenera Zootharnniurn, larve compared to i tseffective dose. A residue study and a Episfylis and Vorticella,as well as other fouling organisms study on tri fluralin dynamics under hatchery conditions has suchas algae and filamentousbacteria. An Investigational been completed. This data indicated na detectable tissue New Animal Drug INAD! permit was issued to the residuesin smalljuvenile shrimp that had beenexposed to University of Arizona in 1983for formalin use in penaeid- trifluralin as larvae and conhrmcd the rcport from Aquacop shrimp culture. The University of Arizona has worked in 977! that trifluralin is rapidly lost from hatchery water. cooperation with the United StatesFish and Wildlife Possibly 50 percent of the required studies for registration Service and with the sponsor Natchez Animal Supply have been completed. A copy of a draft master file was Co., Natchez, MS, and with another sponsor if Natchez submitted to IR-4 for review in 1984 and 1988," elects not to pursue approval of formalin!. Since then, the One study indicated that treflan trifluralin!, which is studies specified by the FDA have been completed. The commonly used in hatcheries to treat fungal diseasesdue FDA has reviewed the data from these studies and ac- to btgeniduirn and Sirolpidium, is rapidly lost by aeration, cepted them as supporting approval for this use. Only photodegradation, and absorption by unicellular algae. revision of the Master File, Freedom of Information Sum- This substantiates the information presented by LeBitoux mary and writing the draft label remains for completion of and the Aquacop team in 1977. They recommended that the approval process. Thus, it is estimated that the ap- Treflanbe administereddrop by drop for 12hours a day. proval process for formalin is about 95 percent complete." According to a morc recentstudy, the estimatedhalf-life, under varying conditions of aeration and light, ranged from Oxytetracycline 30 to 138 minutes. When diatoms were added to the seawa- "Oxytetracycline is used to treat bacterial disease in ter, Trcflan levels dropped to 4 percent of theoretical levels shrimp grow-ou t facilities. This drug, becauseof its reac- within two to three minutes. These results conhrm the need tion with thecalcium in seawater,is mosteffective if given for continuous administration of Treflan to maintain an in feed and is not efficient as a water-borne trcatmcnt. effective dosage for treatment of larval mycoscs, Source: Oxytetracycline has been approved by FDA as a feed Williams, R.R.,T.A. Bell, and D.V. Lightner., 1986.Degrada- additive for use in lobster-holding facilities to control tion of trifluralin in seawater when used to control larval gaffkemia and in Japanin medicatedfeeds for treating mycosis in pcnacid shrimp culture. Journal of the World vibriosis in penaeidshrimp. It is effectiveagainst a large AquacultureSociety 17:8-12!. percentageof the bacteria,primarily Vibrio species,iso- lated from discased penaeids, In 1984,the FDA issued an Compounds to be Replaced INAD permit to Pfizer, Inc., that allowed cooperative "Several chcmothcrapcutants in common usc in the work with Marine Culture Enterprisesand the University penaeid culture industry have a very poor chanceof of Arizona for the approvalof the uscof oxytctracyclinein approval either by FDA or EPA. The first of these is 160 ... Treeceand Fox malachitegreen. Malachite green is an extremelyuseful Acknowledsements and References compoundin treating and preventing larval mycosisof the variety noted above as well as treating gill fouling of Many thanks to Mike Yatcs,josh Wilkenfeld, Clyde peritrichousciliates, algae and filamentous bacteria. How- Simon and Andy Kuljis for a portion of the information ever, it has been shown to be a potential teratogen and given in Chapter 7. It is very generous and commendable carcinogen in laboratory animals." of thesegentlemen toshareinformation of thisnature with "The nitrofuran class of antibactcrials used in shrimp others. Also, the section on United Statesshrimp hatcher- culture include nitrofurazone Furacin!, furazolidone and ies and the use of chemothcrapeutants was taken from nifurpirinol Furance!. Nirtofurazone and furazolidone "Regulatory Status of Therapeutants for Penaeid Shrimp have been used to treat external bacterial and fungal Culture in the United S tates" Rodney Williams and Donald infections in fish and shrimp. Both nitrofurazone and Lightner, Vol. 19, No. 4, JrnL of World Aquaculture furazolidonc have been implicated as potential carcino- Society Dec. 1988! and a portion of the March 1989issue gens. Nifurpirinol is absorbed from the water and is of Aquaculture Digest, and some of the antibiotic com- effective against a wide range of bacteria that causeprob- ments were cited from Coastal Aquaculture, Chamber- lems in both fish and shrimp, as well as Saproiegniasp. lain, 1988. infections in fish. Nifurpirinol was registered for usc on aquarium fish, but is no longer available." "Chloramphcnicol Chloromycctin! has been used primarily to control bacterial disease primarily vibriosis! in hatcheryoperations because of its activity againstthis group of gram negative bacteria. However, thisdrug may produce blood dyscrasias such as aplastic anemia in hu- mans. In anofficial statement, FDA noted that the'drugis potentially harmful even to people who come into inci- dental contact with it. This drug may not be used for any purpose in the aquaculture and exotic fish industries.' Therefore, approval of chloramphcnicol is not likely," Needed Chernotherapeutants "Bacterial disease syndromes are a significant prob- lem in penaeid shrimp culture, affecting primarily larval, postlarval and juvenile stages, Replacements for chloramphenicol and the nitrofuransareneeded, particu- larly in the hatchery phase. Primary candidates for replac- ing chloramphcnicol include oxolinic acid, nalidixic acid, or other 4-quinolone antibiotics; the various potentiated sulfonamides of which Romet-30 sulfadimcthoxine + ormetoprin! and Septra sulfisoxazole + trimethoprim! are examples, and the tetracyclines tetracycline, oxytetracycline and chlortetracycline!. Other less likely antibiotics needing further investigation are the aminoglycosides streptomycin, kanamycin, neomycin and gcntamycin!, erythromycin, lincomycin and some non- potentiated sulfonamides such as Dimeton su 1famonome thox inc!. Formalin has been used a sa d isin- fcctant and water pretreatment chemical to reduce bacte- rial loading of larval rearing tank water. For use with larval stagesprior to the mysis stage,an ideal antibacterial drug or chcmotherapeutant would need to be soluble in water and not appreciably toxic to the speciesof algae fed to the larvae, Once the larvae are at the stage where zooplankton is given as food, water-insoluble antibiotics may be provided by orally incorporating the drug into Artemianauplii or otherorganisms prior to their being fed to the larvae," The May 1989 issue of Fish Farming International reports that formalin, oxytheracycline and trifluralin are nearing approval by the U S, FDA and EPA, but malachite green is expected to be turned down. Design, Operationand Training ... 161

Chapter 8 Results of Larval Rearing and Postlarval Rearing Trial Runs at Yayasan Dian Desa Hatchery

Larval and postlarval trial runs were made during July PL2 to 59 percent at PL4!. This is still an above-average and August 1987 at the Yayasan Dian Desa Hatchery in survival rate for P, rnonodonwhich usually runs around 48 Jepara,Indonesia. percent in the hatchery according to Aquacop, 1983!. The intensive-culture system was used with both lar- Artetnia became a problem as soon as the PL4s went into vae and postlarvae with very good results. The results of the 13-ton holding and rearing circular raceway. Artemia the trial runs are as follows. instarswere settling out or weredead when introducedto One spawn from a Penaeusntonodon, wild-caught ab- the tank as food. The problem was traced back to the lated female produced 720,000nauplii. This spawn was decapsulation process, when it was found that the wrong the second spawn from the female after ablation. This amount of chemicalswere being used.Once this problem spawn was split into two equal parts 60,000 nauplii was solved, the Artentia became a good food source. each! and placed in two conical larval-rearing tanks with Temperature in the raceways was continuously low a 4,000liter capacity.Only 1,000liters of water was used 5.1 degreesC to 27 degreesC!; therefore,growth was to start.Each day new seawaterwas added filtered to one very slow. Lagenidiuntsp. was a constant problem in micron, 28 ppt salinity! and then feed was added accord- Raceway 7 with 212,940PLs! and occasionally bacterial ing to the scheduleon Table l. Water volumes of the tank necrosisdevelopedin Raceway8 with174,800PLs!. Treflan kept onadailybasiscanalsobefoundon this table. Adaily 5-ppm solution was administered for the Iagenidittm at a record was kept and temperatures, feeding practices, dose of 50 ml/ton or 635 ml for Raceway 7 and disease treatments etc. were all recorded on a larval rear- chloramphenicolwas given for thebacteria in 2-ppmto 10- ing data Iog. Datalogs for LRT 1 andLRT 2 arein Tables ppm doses,depending on theseverity of thebacterial attack, 11 and 12. There was an estimated 95 percent or greater survival in Raceway 7 stocked with 212,940PL4s! and an estimated Results of Larval Rearing Runs: 80 to 85 percentsurvival in Raceway8 stockedoriginally Survival Percentage with 174,800!. Mortality in Raceway 8 came when the Tank No Dates of Run PL1 PL2 PL3 PL4 habitat screenswere not placedbelow the water surface 1 July 22-Aug. 3 82 61.5 59 and the water flow and airlift bubbIesleft many PLshigh 2 July 22-Aug. 4 73 53 48.5 and dry on the screen a hard lesson learned!. Table 13 Numerical population estimates were as follows: shows the actual tank log for Raceway 7. Raceway 8 was first held at 7,000liters instead of 12,65 Number of Shrimp Surviving tons, as in Raceway 7, in an attempt to save Artemia Tank ¹ PL1 PL2 PL3 PL4 feeding a smaller volume tank!. This proved to be a 1 296,400 221,666 212,940 mistake because of temperature and water circulation 2 264,100 191,000 174,800 problems,so the volume in Raceway8 wasbrought up to Note that LRT I wasonestage ahead of LRT2. This was the same level as Raceway 7. Table14 gives the actual tank directly relatedto temperature;LRT1 had two heatersand log for Raceway 8. LRT 2 had one heater. Also, survival was lower in LRT 2 In addition to Artentia, a pellet diet wasgiven to the PLs and is thought to havea directrelationship with disease starting at PL6. The feeding rate is outlined on Table 1. treatments. Only when diseasewas obvious and mortali- Weights of the animals were very difficult to obtain accu- ties appeared was disease treatment administered. This rately with the equipment at the laboratory. Previous approachhad an obvious bearing on the survival as no feeding studies at Dian Desa in 1990 showed that a PL2 treatment was given in tank 2 until the Mysis III substage, weighed 0.000238grams. Previous experienceby Mr. eventhough somemortality wasnoted as early as theZ3- Bagiyo indicated that a PLl should weight 0.085 grams. M1 substages.Mortalities always appearin thesestages Therefore,attempts weremade to obtain thosePL weights even under opttimum conditions. It is also common to see of the shrimp in Raceways7 and8. One attempt showed mortalities at the N to Z transition and the Z3 to M1 stage that 27 PL8sweighed 0,01 grams or about 0.0003grams transition. each.Anotherattempt with the samescale indicated that The appearance of Zootharnniumin tank 1 at PL4 com- the average weight of 38 PL8s was 0.005 grams each. plicated the transfer. The problem was first treated with Therefore,an average weight of 0.3to 5 mgeach should be 0.1 ppm of malachitegreen m.g.! with no success.The within anacceptablerange, Previously feed amounts were next day 1 ppm m,g, was used to get rid of the problem, calculated using the weight from Mr. Bagiyo .085 g! x With the combinedproblems of treatingand transferring numberof PLsin tank x 200percent body weight per day, at the sametime, thesurvival ratedeclined 1.5 percentat This 200percent feed President's¹ New 1! was split into 162 ... Treeeeand Fox

Table ll. Actual Larval Rearing Tank Log for Tank Number l.

Tankg d. Date Stoeked~pgz~Saupl I i Stookedgooooogpt's'I'.I" SSur.r, -'. PoSei Algae Date Time Stage Health Temp. Species Cells/ml Amt. IArtemial Comme'.t ~IPIAQ Io i r.tar,k f lsd l/rnl./ !I fed ~z~, gogo,'~V ,'orzoZ~r'Ig,'Zpd ,' p 22. I ~4gO I ~/V-~ i 29 0 i i i i i i H 9'03 I i ~2.2 i Chgzcr, i < I~I I I 3 CPIIRSofg ~Pss ~ooD C4~e, i ~~z+O~~i 0 I i i TOO COLb!! I I I ~03 I I I I i~ma i N~ I "~8"<~V.o I I Egljfl ~/p i f 500 I w~ i 6< I 2v.2 I i g5 ooomgi 2 adrIAs ~Z> i ZIPPOI 1~2, I I I I I ~// F IF 2 i ~ i ~g i ~n i ~>4i 'I i~>l igiBIITlld5I I I gad>PSE+~pfdI I I I I ~>Z i ~OO I id'/STffL2 IIyR7 i~SOD i .0 i $20+oo6I O I I I lg FOTAIP +Iopd I I I I I 5 i~@I Z~ Iop/C I ~ I I 6 eioo Ib~fioo i Z ad'TTLFS I I 2 1~00 iZ80i gI 4p ooo IA 60O SdTTLF Q ~+2S-'~/,eO I I 2Q'51 I I2 aoWsi i ~I/ 8'.t% opoo ~8z-8 o ', z~z , 'zr ooo l soaoo I 5 E~T I I I I I I /zoo8~3 I W+ooo'I te,o I' Ioi vgrorwgok~zr ,got+'jaI I I I !oo i d~e i fifr i 2Q.o i I I I I I I c '~coo l ~o' si Ir '~l o'~cl. 7ergpii5% 4 25k.Tef: I I I ~ZIO ~ ~Z.O I I I iV I I g ~Ps i goo ~sr-SS tuau~ X9.O I rf. ~+~ i ~ooo i I~84 i I +rod+i'j'3wW~e~g i 0~45 i I~i 1~%2 I I 8 i /63O i ~hf2I I ~71 I I ~ZH i 2 I DOi IH2 29,0 i ~+2'f i O~aO i I ~o i If2 ad.g>i I ~O I 2Q,O i rg ' ~IF040 Q !. ~+9 i ~WOO i g't.o i I I I I i ++00 i i~bi I i Cups.gal '/D'OR,PSeagzDO. p SOI 6 IbO i 2$5 I gp I aptodz9O I ~l ~rru 0 0 $00 i /l/l '5 I~~idrl 29,di i Terr, oi~eI I ~~ 6 I I I I I I 6 i XOOI I T= Tqdl, i KZPo6 @+8, I I I i ZOO TRZ- /~ ~ I +766 i Pf.f ,'+Op o =erg Vg/sI I I >os I I X9,Q, 7errlS , 'lo doO 'Arear,'l% ?3oO i lp'tf 9 r !;$ I COM< EN TS: opt~Voelf 6/OciIIRca gc=e~f-~ ++ nfl 7fy j /3 i PP'Ti o Te rf Dosigrl, OPerutiollElnd Trebling ... 163

Table 11 continued!.

Tank f 2 Date StOCked~>2~fNauplii Stocked~3apooOfPL'Sal

I I I I I I

I I I

coMMENTs:Ip p~aesT «~~i .~>~4 =eieeeo I/g- IVVEMIx 9ie37=49M = 293eeopir ~V7 ~<

3 C a

Design, Operationand Training ... 165

Table 12 continued!.

Tank 4 2 Date Stocked~>z2.f Nauplii Stocked' .000'f PL's~26'f/at/SSur.g3 IPeyZ. ef P-! Algae Date Time Stage Health Temp. Spec i es Ce l l s/ml Amt. ',Artemia', Comment i~tank fed tf lml,/ ff fed /3f I ~IO TeT . ' ~Ok l zek JI30 ~hh I 20k' ' ,Wrco-c33'dk~gg I +~i +3oo I ~H-,Q I ~ R9,O I I I ~r+ ~ '~k-PI l ek 29'g I Ter< . I 3 lerif+r ~f i 0~7 I I L I fkoa ~PI »k Pf'.5' I I I W~e.!. ' z. I I I 8 I I f400 I 2 F.fp' I ~8 I 223O I I ~f, I I I I ',g+30I I QZ.O I I I I I & 2 I~>obi I ~I.0 I I I 'I I I a '~3o '~PI. I 2f'.Q i ev I I I ~I.O I I 8 I 3|333 l~» i »I%5 I O ~2~ I M~ I P'4 Z./ I I I I I I I 3~ C 3rd, I ~83 I ~/SO I ~L I I ~' m /,~~ I I ' L~?ment I I I I S~TWCC AS//Py'4 I I I I /OOO I PL~I I I CI I C I I I @~ 3 j2do I i ~LS~ I I IIOeO l~P< I I 73tAPPSÃaÃra I I I I I I I I I I I /flCf4/ 166 ... Trees@and Fax

Table 13. ActualPostlarval Rearing Tank Logfor RacewayNumber 7.

Rtacevayf7 Date Stocked ~83 tPLPs Stocked 2~l2.8&%Survival

Date Time Hea 1 th Temp. Ar t. /ml Amtofed Treatment Comments ~// 8' ,ll 80' ,2~. Ose/,'8%.5' ,D ,'~l/2 /. 8// l ~PI I ', O. PP>m~J,.Cr.I T ~S ~8 l >3oaI jg~i 0700i~"~~~i i.XC.S ' ,g35M TrB/@~IL~a. ~Bred 8 4 I J bDOI PL+ Blf%k Q t.o I I 4 I 8 4 I tb30 I R. z .8~4r ~p.o / o I s/'/*/Be~sor~e / I I ~84 I ~4b / I l O.S22/I/J./.l 8 + ~o',z~ BQI ' OD00' /BTLILP 8~15. I ro8202 ~B.r/ ~08 I I 030 I XL ~ / I ~84 I a~~ I P~L~ I > 5'.Ot 8~C I OR>csi P'L%< / ~JS' TW/~ 8~WC ~ I I 5 010 ~LO ; Ci e.rrir! I ~B ~B PLB " ~0~ I I ~HQ I O/~45I PILS I I So7T~rcELeg@ a ~ ' /sob / rLe " ~2C. I I 2Pot+~"l '~ol PLB / a PPtnIE' bT~ I 8 I ~odI ~T-ban% RBB+I I ~aS~ 7~P~ // l l>00 l PLR.O 0/~0.2. I I .8 I ~80 ' 0+Do ' /OLIO " ' ~5 S2S~T //oo d~sP ~BT l 2» 0 l ~Puo 000B~~ ' ,j PP/BICH/ORO. I ~AT/<' Fum I ~lo ~10 0 ~PL I WOO /.'O/.b

g/Ij 23O< 8<5. 578OT/'C~COBB/ 7gEgT ~~tB, / / peB/erfoesP ds Tds/o/ e +psT/dsos. + T' 1 0/oy sdoP0$ fs +!

/ 8! TTL0 C/TOETO FOR Resdo ieo sBRTI so /I re//OT es/-' 8 T 0/Doe /Dooso 8'k8. "T/odsj Wor~X~ ~ eZP ~s< .~4 <4P ogr ~k.

168 ... Treeaeand Fox

4 feedingsper 24hours 700, 1200,1700 and 2200hours!. The leftover feed was vacuumed or siphoned out of the tank the next morning. Other Information about the Raceways Waterexchange was maintainedat 100percent per 24 hours unless chemical treatment for disease required an overnight static condition. A 500ljm Nitex-screened internal standpipe kept PLs in the tank but allowed Artemia and other smaller ma teri- als to pass through. The level was set by an external standpipe.A small-screenedbucket was placed under this externalstandpipe to catchthe Artemia.The Artemia were occasionallyreturned to the racewaywhere they were fed onebot tie of algaeper day to try to makethem abetter food source. A smallscreen was tried on the internal standpipebut it cloggedoccasionally and raised the water level in the tank so that the airlifts did not properly circulate the water.

Recommendations: 1. Use 5004m Nitex as described above. 2. Place habitats below water surface keep in mind that the more habitats there are, the less circulation there will be unless more airlifts are added>. 3. FeedArtemia longer if temperaturesare below 2S'C, plus or minus 1'C, becausethe animalsare smaller and cannot swim to the bottom against the current and feed solely on pellets.If temperatureshave been held at 28'C, the animals should be strong enough by PL11, as indicated by the feeding schedule, to stop feeding Artemia.

170 ... Treece ami Fox

field with prospectivebuyers for oneweek per month. and/or investoras a returnon investment.Accounting is ~ Costs sales trips for marketingwill be coordinated a tool to chart this flow of investment. The arrows repre- with saleMelivery trips, businessmanager will seek sentthe flow of capital. volume discounts or long-term contract discounts on supplies and materials. Hatchery Capital Flow ~ Personnel institute monthly staff meetings during which managersbrief workers on the hatchery'sover- all situation, soliciting the workers comments, com- THE HATCHERY plaints and suggestions. Fixed Inputs: ~ Plant and equipment schedule painting interior Land and exterior! for hatchery's vacation month and in- THE OWNER/ r i Buildings ~ ~ OUTPUTS clude costin annualbudget; plant managerwill out- INVESTORS ' r Machinery~r< SALES! line detailed preventive-maintenance program for Equipment generator,pumps and water intake system. Variable Inputs, Quarterlyreviews of anannual business planallow for Stock adjusting goals due to altered expectationsor problems Feed that develop as well as keeping the goals evident and Chemicals active.It is important to seethe businessplan asa guide, Fuel a map for the hatchery operations to follow; it allows all Office Supplies decision-makers at the hatchery to have common goals Repairs 8zMaintenance and a planon how to attain them. Transport The annual budget is a process that sets out revenue and costprojections for the upcomingyear. It setsout the The Purpose of Accounting bestassumptions for the upcomingyear's revenues, costs and profits. In order to do this, the manager mustaccumu- Goodrecords provide revenue and costinformation to late and evaluateinformation from the following areas: managers,letting them seewhere the hatchery'smoney is ~ Production capacity What is the hatchery'stop beingspent. This knowledgethen enablesthe managerto capacity,i,e, how many post-larvaecan the hatchery make better decisionsregarding the control of thoseex- produce in a given month7 Judging on the basis of the penses.Internally, cost accountingallows for controls, condition of the equipment and the dependabiIityof pricing analyses,and establishesoperational records for supplies, what is the targeted expectation of "full historically-basedprojections. Externally, financial ac- capacity7" Is it in fact maximizing profit to run at full countinggenerates financial statements, helping thehatch- capacity? ery to gain credibiIity with investorsand lendexs. ~ Productionschedule at what percentageof capac- The Monthly Profit and Loss Statement ity wiII the hatcherychoose to operateduring given The monthly profit and loss statementis used by the months, i.e. will the hatchery slow down production hatcheryto monitor its business.This is doneby noting the during slackmonths? Will the hatcherytemporarily revenuesgained through salesand then subtractingex- expand its full capacity during peak demand months pensesto show the hatchery'smonthly profit or loss!.A by adding temporarysupplemental raceways7 ~ Salesprojections how good is the ability of the sample monthly profit and loss statement is shown. Like hatcheryto market the post-larvae7Will new buyers all examplesused, it is merely illustrative and not meant be solicited during the slackMemand season, and if to be indicative of the performanceof any particular hatchery. so, what level of successis expected?Or, will lower salesbe acceptedduring the periodsof low demand? Notes on Operating Expenses ~ Cost estimates what wi11be the hatchery'scosts Labor expensesinclude salaries, bonuses and benefits during the upcoming year?Will costsavings be real- given to the employeesin payment for their services izedthrough improved technology?Are feedand fuel include paid-out medicalexpenses the empIoyees!.This prices expected to rise7 Have any major capital pur- line item is usedto evaluatehow high or low! a percent- chasessuch as machinery or equipmentbeen planned? age of sales must be to cover labor costs. It is also used to A budget analysisfor a shrimp hatcheryunit and a compare labor costs with those of other hatcheries. While maturation facility was written by Johnset aI., 1981 it is important to pay fair wagesto attract and keepgood WAS 12! 305-321 and WAS 12 l! 104-189. employees,it is alsoimportant not to unilaterally escalate Accounting/Bookkeeping the pay scale, Office expensesinclude office supplies,communica- The following flow chart explains hatchery capital flow. It showsthat the owner/investor is responsiblefor tion costs and marketing efforts. This line item tends to widely fluctuate.The main concernis to balancerestric- the initial capital expenditure and additional capital in- tions and limitations on spending with the flexibility of vestmentsthat the hatcheryis unableto coverfrom sales. operationand the easeof getting the job done.While the The hatcheryuses fixed and variable inputs to produce office supplies componentof this line item will remain outputs sales!,Sales revenues are reinvested in the hatch- more or fess constant, communication and marketing ery, covering variable inputs or returned to the owner costsmay havelarger seasonalvariances. Design,Opemtion and Training ... 171

Production expensesinclude such items as the mated The Annual 8alance Sheet females, feeds, fuel for the generator and other supplies The annual balance sheet is the second major annual directly related to production. This includes laboratory financial statement. It is routinely produced at year's end, costs as well. at the same time as the profit and loss statement, Unlike Maintenance covers upkeep on the buildings and re- the profit and loss statement,which illustrates perfor- pairsof machineryand equipment,Include such expenses mance over a period of time, the balance sheet illustrates as cleaning materials, paint and lubricants, repair costs the state of the business at a moment in time. It is as if at and spare parts for machinery. Older machinery will midnight on the evening of December31, all accounts require more repairs; at some point it will become more were frozen and their status recorded. A sample annual costefficient to replaceit with new equipment. balance sheet is on page 172. Transportation includes all public-transport costs as well asany vehicleexpenses such as fuel, spareparts and Marketing repairs.This categorywill rise if the hatcherydelivers its Marketing is the label given to the effort of selling a product to the tambak farmers or does marketing out- product. It brings to mind advertising campaignswith reach. radio and newspaper announcements, but marketing is The misceilaneous category is for representation, chari- not limited to advertising;it encompassessuch efforts as table contributions, bank charges, entertainment and packaging,delivering and customer relations. The es- whatever other "miscellaneous costs" the hatchery incurs senceof marketing is knowing one's customer and con- during the month. vincing him/her that it is in their bestinterest to buy your The YearlyProfit and LossStatement product. Theyearly profit and lossstatement is a compilationof Packaging themonthly profit and lossstatements with an adjustment A product that is effectively packagedis a goodmar- madefor taxesthat havebeen paid. This statementshows keting tool. A good appearancecan be presentedby how well

Hatchery Cash Flow Example Development Costsfor a Maturation/Hatchery Facility to Produce 10 Million PL/mo. in Ecuador Cash Outflow for Constructionand Design 1 Year Economic Sum Quantity Price US$! Total Life years! Life SHOP EQUIPMENT Table Saw 10 580 580 10 Drill Press 200 200 10 Grinder 200 200 10 Welder 300 300 10 Torch 190 190 10 Vise 65 65 1535 Generator large! 10000 10000 Air Compressor 450 450 BatteryCharger 50 50 Jack 12 Ton 175 175 Wheelbarrow 50 50 Hand Tools 2000 2000 Gen.Supplies 300 300 Generator small! 500 1000 Hand Drill 3/8 120 120 10873 73 Drill 1/2 100 100 Jigsaw 100 100 Circular 150 150 Ladder 50 Miscellaneous 2000 2000 2520 SUBTOTAL 18080 OFFICE Desk k Chair 300 12 Conference Table 400 400 12 Conference Chairs 30 120 12 Blackboard 70 70 12 Bookshelves 150 0 12 FilingCabinet 100 990 Computer 5000 5000 Typewriter 300 300 1265 65 5300 Radio 3000 3000 Slide Projector 200 0 Copy Machine 2500 2500 Calculator 75 75 Air Conditioning 500 1000 5 5 Miscellaneous 901 901 7476 SUBTOTAL 13766

LAND AND BUILDINGS Land Ha! 2 20000 40000 Buildings Mat/Hatch! 20000 9 180000 20 StaffHousing 2 25000 50000 Miscellaneous 2250 2250 20 SUBTOTAL 272250

MATURATION EQUIPMENT Tanks MT! 2200 22000 15 PVC Pipe 101 1 3500 3500 15 2550 Food Processor 90 90 10 Spectrophotometer 1100 1100 10 Pipets 75 150 10 Air Collar 21 1 1MO 1100 10 Freezer 1500 1500 10 Design, ~tian and Training... 175

CashOutflow for Constructionand Design cont!

Misc. Lab I 8000 8000 11940 Lighting 18 350 6300 SmallPumps Utility 3 100 300 02 Meters I 1100 1100 Refractometer 2 300 600 Triple BeamBalance 125 125 Balance K I 1600 1600 107 7 Microscopes 2 900 1800 Biofilter 22 500 11000 Hemacytometer 2 90 180 Miscellaneous I 4231 4231 27236 SUBTOTAL 64676

WATER SYSTEM Pipe Line I 8000 8000 Tanks settling! 5000 10000 Tank storage! 2I I 10000 10000 WaterHeater Exchanger 2000 2000 10 Solar Panels 15 90 1350 UV Light 4 I 300 1200 4550 Pumps 400 400 Filters 150 4500 109 9 PlasticTubing 30I I 2000 2000 Miscellaneous 2762 2762 9662 SUBTOTAL 42212

HATCHERY EQUIPMENT Tanks I OMT! 12 1000 12000 15 Algae Tanks 180 2700 15 PVC Pipe 15I I 3500 3500 18200 Air Conditioner 300 300 Carboy Rack 150 150 Aeration Blower 150 600 1575 5 4I Refrigerator 600 600 Ph Meter 300 300 CarboysPlastic 20 60 1200 CarboysGlass 10I 100 1000 5 5 Miscellaneous 1565 1565 5265 SUBTOTAL 23915

TRANSPORTATION Truck I ton! 30000 5 Hauling Tanks 1000 10 SUBTOTAL 31000 ANNUAL TOTAL 465898 Monthly Cash Flow Mat/Hat facility producing 10 mil/mo in 13 runs/mo.

Jan Feb Mar Apr May June July Aug Sept Oct Nov Dec

BEGINNING CASH 200000 334031 250596 164486 81051 10000 10001 4929 20957 12917 29299 84792 BALANCE INCOME Sale PL $12.00/1000! 24000 48000 74400 74400 74400 744M TOTAL CASH INFLOW 200000 334031 250596 164486 81051 I QDDG 34QDI 52929 95357 87317 103699 159192 CAPITAL OUTLAY Land 40000 0 0 0 0 0 0 0 Shop Equipment 3013 3013 3013 3013 3013 3013 0 0 Ofhce Equipment 2294 2294 2294 2294 2294 2294 0 0 Bldg. Construction 38708 38708 38708 38708 3870S 3870S 0 0 Maturation Equipment 10779 10779 10779 10779 10779 10779 0 0 Water System 7G35 7035 7035 7035 7035 7035 G 0 Hatch Equipment 3986 3986 3986 3986 39S6 3986 G 0 TOTAL 105816 65816 65816 65816 65816 65816 0 0

OPERATING EXPENSES Fuel 200 200 200 200 200 200 200 200 200 200 200 200 Cons Repair 13'l6 1316 1316 1316 1316 1316 0 0 0 0 0 0 Insur Machines 5000 0 0 G G 0 0 0 0 0 0 0 Labor $350./mo/labor! 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 3500 Utilities $0.24/kwh! 2180 2180 2180 2180 2180 2180 2180 2180 2180 2180 2180 2180 Telephone 200 200 200 200 200 200 200 200 200 200 200 200 Supplies 500 500 500 500 500 500 500 500 500 500 500 500 Payroll Taxes 5%! 525 525 525 525 525 525 525 525 525 525 525 525 Social Security %! 245 245 245 245 245 245 245 245 245 245 245 245 Travel 300 300 300 300 300 300 300 300 300 300 300 300 Broodstock $10/animal! 0 0 0 0 0 0 9500 0 0 9500 0 0 Feed Broodstock 7.2/yr! 0 0 00 11400 1140 1140 1140 1140 1140 SupplementalFeed 0 100 100 100 100 100 100 Arfemia 0 I BO 180 180 180 180 180 Chemicals 0 0 00 00 0 200 200 200 200 200 200 Antibiotics 0 0 00 6000 600 600 6DG 600 600 Other 0 300 300 300 300 300 300 TOTAL OPERATING EXP 13966 8966 8966 8966 8966 8966 19670 10170 10170 19670 10170 10170

RXED COSTS Permits 2000 0 0 0 0 0 0 0 0 0 0 0 M/H Manager 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 Biologists ! 30QQ 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 3000 Fringe Benefits: 25% 1500 1500 1500 1500 1500 1500 1500 1500 1500 1500 1500 1500 Property Tax 1%! 880 0 0 0 0 0 0 0 0 0 0 Legal & Accounting 0 0 2500 2500 0 0 0 02500 0 0 0 2500 Miscellaneous Expenses 1704 1153 1328 1153 1153 1328 1902 1237 1412 1902 1237 1412 TVTAL FIXED COST 12084 8653 11328 8653 8653 1]328 9402 8737 11412 9402 8737 11412

SCHEDULED DEBT PAYMENTS Intermediate-Principal 0 0 34356 -Interest 0 0 58497 Long Term -Principal 0 0 0 -Interest 0 0 0

TOTAL CASH OUT 131867 S3435 86110 83435 83435 86110 29072 18907 21582 29072 18907 21582 TOTAL CASH AVAIL 68133 250596 164486 81051 -2384 -76109 4929 34022 73775 58245 84792 137610 Design, Operationand Training ... 177

Monthly CashHow cont!

NEW BORROWING Short Term 0 0 12384 86110 0 0 0 Intermediate 265898 0 0 0 0 0 0 Long Term 0 0 0 0 0 0 0 Total New Borrowing 265898 0 12384 86110 0 0 0

PAYMENTS SHORT TERM DEBT Principal 0 0 0 0 0 0 12384 57685 28425 Interest 0 0 0 0 0 0 681 3173 521 TOTAL SHORT TERM PA 0 0 0 0 0 0 13065 60858 28946

ENDING CASH 334031 250596 164486 81051 10000 I 0001 4929 20957 12917 29299 84792 137610 BALANCE

SUMMARY DEBT OUTSTANDING Short Term 0 0 0 0 12384 102260 102260 86110 28425 0 0 0 Intermediate 265898 265898 265898 265898 265898 265898 265898 265898 265898 265898 265898 231542 Long Term 0 0 0 0 0 0 0 0 0 0 0 0

INCOME STATEMENT SUMMARY OF INCOME FOR 1988 - 1991

CASH INCOME FIRST YEAR 1990 1991 PL Sales 2/1000! 369600 892800 1152000 1152000 1152000 TOTAL CASH INCOME 369600 892800 1152000 1152000 115200

CASH EXPENSES VARIABLE EXPENSES Hatchery 16916 14232 14232 14232 14232 Brood stock 19000 38000 380M 38000 38000 Feed 8520 18180 18180 18I80 18180 Fuel 2400 2400 2400 2400 2400 Maintenance repairs! 7898 3000 3000 3000 3000 Utilities tel; elect.! 28560 28560 28560 28560 28560 Labor 42000 42000 42000 42000 42000 Other 1800 3600 3600 3600 3600 TQI'AL VARIABLE EXPENSES 127094 149972 149972 149972 149972

FIXED EXPENSES Property taxes 880 880 880 880 880 Insurance 5000 5000 5000 5000 5000 Interest on Debt 58497 50939 41718 38469 16744 Manager 36000 36000 36000 36000 36000 AssistantBiologists 36000 36000 36000 36000 36000 TOTAL FIXED EXPENSES 136377 12%19 119598 116349 94624

TOTAL CASH EXPENSES 263471 278791 269570 266321 244596 NET INCOME 106129 614009 882430 885679 907404

NONCASH ADJUSTMENTS. DEPRECIATION Mach,and Equip. 18062 18062 18062 18062 18062 Buildings 6500 6500 6500 6500 6500 TOTAL DEPRECIATION 24562 24562 24562 24562 24562

INVENTORY CHANGE Supplies NET INVENTORY CHANGE 17& ... Treeceand Fox

SUMMARY OF INCOME FOR 1988 - 1991 I I I I TOTAL TOTAL TOTAL TOTAL TOTAL 137610 137610 661961 1445512 2229063

369600 892800 1152000 1152000 1152000 1572659 1030410 1813961 2597512 3381063

40000 180&0 13766 232248 64676 42212 23915 434896

2400 2400 2400 2400 2400 7898 3000 3000 3000 3000 5000 5000 5000 5000 5000 42000 42000 42000 42000 42000 26160 26160 26160 26160 26160 2400 2400 2400 2400 2400 6000 6000 6000 6000 6000 6300 3156 3156 3156 3156 2940 1476 1476 1476 1476 3600 3600 3600 3600 3600 19000 38000 38000 38000 3&000 6840 13680 13680 136&0 136&0 600 2000 2000 2000 2000 1080 2500 2500 2500 2500 1200 2400 2400 2400 2400 3600 7200 7200 7200 7200 1800 3600 3600 3600 3600 13&818 164572 164572 164572 164572

2000 0 0 0 0 36000 36000 36000 36000 36000 36000 36000 36000 36000 36000 18000 1&000 1&000 18000 18000 880 880 880 8&0 &80 10000 5000 5000 S000 5000 16919 15144 15144 l5144 IS144 119799 111024 111024 II1024 111024

34356 41914 51135 62384 76109 58497 50939 41718 30469 16744 0 0 0 0 0 0 0 0 0 0

693513 368449 36&449 368449 368449 879146 661961 1445512 2229063 3012614

98494 265898 0 364392

98494 4375 102&69

137610 661961 1445512 3012614

0 0 0 0 231542 189628 138493 76109 0 0 0 0 Design,Operation and Training ... 179

The following sectionincludes a description,produc- had beengradually increasedwhile managementexperi- tion summaryand proforma incomestatement for a large mented with variations in pond size and design, stocking hatchery from 1980and projected through 1995. density and strategy as well as various techniquesof feedingand distribution, water-exchangerates, and me- Large United StatesHatchery Description chanical aeration. The primary businessobjective is the production of Brood shrimp were selectedfrom the ponds in 1983 shrimp for sale as food. The speciesexdusively used is the and wintered for 1984 production. Late in 1983,a 30,000 Pacific white shrimp P. vanntnnei!.The current market is square feet hatchery complex was constructed that in- entirely in the continental United States.They also sell cluded water treatment, maturation and 165 metric-ton nauplii and postlarval shrimp to other shrimp farms in the larval-rearing areas. In late 1984 the maturation capacity United States, Central and South America. was doubled in size and a brood-holding facility was The hatchery also supports 450 acres of grow-out constructed. This expansion increased the facility to its pond. These range in size from 0.25 acre experimental present size of 40,000square feet. ponds to the largest pond of 43 acres. There are ten In 1985the larval-rearing tank capacity was increased to nurseryjgrow-out pondcombinations. The nursery ponds 230tons. In 1986the larval-rearing capacity was increasedto range in size from six to nine acres and the adjacent grow- 270 tons. In 1987 another expansion increased the larval- out ponds range from 26 to 43 acres.There are two large rearing capacityto 310tons. In 1990theyincreased thelarval- grow-out ponds without adjacent nurseries. rearing capacity to 370 tons. PL8-10production at this rear- The current maturation complex consistsof 24 circular ing volume was 25 million per month. In late 1988a research tanks supported by extensivebiofflter and water-treatment greenhousefacilitywasbuiltto testhead-startingpostlarvae facilities. There also is an indoor-raceway system capableof in 1989.As detailedbelow in postlarvaldisposition, the end holding1,000brood. Using one 12-tank room for production production of the hatchery has progressed from PL2-3sin with the secondroom asbackup,the current nauplii produc- 1983to PL8-10sin 1987and 1988.The hatchery was sold to a tion capacityis 2.5million nauplii per day. new group in 1990. The larval-rearing section has 29 fiberglass tanks with a total water capacity of 310tons. These range in size from Large Hatchery Postlarval Disposition 9,000 liters to 20,000 liters. In 1989 they were able to 1982 No records increase their survival rate from Nauplii to PL8 to an 1983 10 million postlarvae2-3s produced average of 70 percent. This has been accomplished simul- 10million postlarvae 2-3s stocked taneouslywith a decreasein the use of antibiotics. Al- 1984 26 million postlarvae4s produced though the hatchery was not run at full capacity in 1989, 20 million postlarvae4s stocked full production was 25 million postlarvae per month in 6 million postlarvae4s sold 1990. Full production was 20 million per month in 1988 1985 35 million postlarvae4-5s produced when average nauplii survival to PL8 was 45 percent. By 23 million postlarvae 4-5s stocked adding tanks in existing space and adding vessels to the 11million postlarvae4-5s sold existingalgae-production room, the PL productioncapac- 1 million postlarvae 4-5s research ity was increased by another 5 million per month without 1986 39 million postlarvae 5-6s produced a substantial capital investment. 34 million postlarvae 5-6s stocked The hatchery also has an comprehensive water-treat- 3 million postlarvae 5-6s sold ment facility. This allows the facility to handle all but the 2 million postlarvae 5-6s research most severe water quality variations. 1987 122million postlarvae8-10s produced The maturation, larval-rearing,and water-treatment 54 million postlarvae 8-10s stocked facilities, as well as the administrative offices, are housed 27 million postlarvae 8-10s sold in a 48,000square foot concretebuilding. Thebuilding was 41 million postlarvae 8-10s research constructed in 1985, is postioned 18 feet above sea level 1988 112 million postlarvae 8-10s produced and satisfies all coastal-building codes. 58 million postlarvae 8-10s stocked This will be the fourthyear of operation for the450acre 10million postlarvae8-10s sold grow-outcomplex. Prior to 1986the totalacreage of ponds 44 million postlarvae 8-10s research Large Hatchery Production Summary Year Nauplii Stocked PLs Harvested Overall Percent Pond Stocking Survival Duration millions! 1983 40.0 10.0 23% Apr-Sep 26.4 23% Mar-Aug 35.1 6% Feb-Aug 39.3 12% Mar-Aug 21.7 45% Mar-June 12,0 36% Mar-June 37.0 70% Apr-June O.O estimate sales Feb-Sept 1990! Diseasesencountered in the hatcheryincluded bacte- rial infestations,fungus and filamentous-bacterialprob- lems and a viral infection. Most of these diseases became problemswhen there were stresses on the animals caused by water,food, systems, or managerialproblems. Correc- tion of thesestress-producting problems resulted in re- duced incidence of disease. Antibiotics and water sterilization are used to keep bacterialproblems under control. Further investigations are underway to combatviral infections. Design, Operation and Training , 181

Actual artd Projected Large Hatchery Operating Costs 1989-1995

19S9 1990 1991 1992 1993 1994 1995 Personnel Biologists ! @ 21,000 18,000 42,000 44,100 46@05 48+20 51,051 53,064 Biologist 22,050 23,153 24/10 25326 26+02 Algae Technicians ! @ 16,000 16,000 16400 17,640 18/22 19,448 20,421 21,442 Worker/Technicians 0! IN 5.75/HR 28,700 30,135 31,642 33/24 34/85 36,629 38,461 Worker/Technicians ! 45/03 47,463 49/36 52@28 54,944 57,691 Worker/Technicians ! 31,642 33,224 34485 61,061 64,115 Maturation Specialist ! @ 5.75/HR 15,000 15,750 16/38 17/64 18+33 19,144 20,101 Maturation Spec 15,750 16+38 17@64 IS/33 19,144 20,101 Water Supervisor I 19,000 19 000 19 950 20 948 21 995 23 095 24 249 2~562

96 700 248 559 260 987 274 036 327 778

Feeds Nauplii 30,000 75/00 86,625 90,956 95~ 100,279 105,293 Post Larvae 7 000 ~17 00 22 969 24 117 2~523 ~26 89 27 919

37 000 3~850 109 594 115 073 120 827 126 868 133 212

Utilities Water 21,000 22,050 28,941 30@38 31,907 33302 35,178 Nauplii 25300 26360 34,729 36,465 38+88 40/03 42,213 Post Larvae ~11 50 12 128 15 917 16 713 ~17 9 18 426 19 348

57 750 6~0 ~79 87 8~3 87 744 92 132 96 738 Supplies Water 20,000 50,000 65,265 68,906 72+52 75,969 79,768 Nauplii 3,000 7' 9/44 10+36 10553 11395 11,965 Post Larvae ~1 19 688 20 672 21 705 22 791 23 930

2~9 ~00 95 156 99 914 104 910 110 155 115 663

Maintenance Water 7+00 15,000 17/25 18,191 19,10'1 20,056 21+59 Nauplii 6,000 12,000 13.860 14/53 15,281 16,045 16+47 Post Larvae 4~ 9 000 ~10 9 10 915 ~11 60 12344 12 635

J111Q 18 900 4~180 4~3 4~52 4~1 ~l

Total Yearly OperatingCosts 238 450 334+43+ 603 199 633 359 6II~94 9 723 932

Pls to Ponds $5/1,000! 3/GO! 87~! 30,475! 83,523! 43,000! 43,000! 43,000! Pl Sales Central America $7/1,000! 8500! 85,000! Nauplii Sales $1/1,000! 90,000! 35,000! 80,000! 25,000! 25,000! Pl SalesOutside $7/1,000! ~14400/} pi~47 g} ~647 Q} $6~47 0~0 Nauplii SalesOutside Unallocated Hatchery Casts l'17 5~50 ~885 182 ... Treeeeand Fox

1990-91Equipment Price List for Central American Shrimp Hatchery On U.S. $! with a ProductionCapacity of 10 Million Postlarvaeper Month

UNIT EXTENDED DESCRIPTION PRICE US$! UNITS PRICE US$!

SEAWATER TREATMENT SYSTEM TREATMENT EQUIP MENT! Intake Screens 250,00 2.00 500.00 Hatchery Pumps '1000.00 3,00 3000.00 Hydrocyclone Separators 1500.00 2.00 3000.00 Rapid SandFilters 40 gpm! 831.00 3.00 2493.00 Pall Filters, 1.0 um 5000.00 1.00 5000,00 Contact Columns, 8.0 MT 2000.00 4.00 8000.00 UV Sterilizers 4500.00 2,00 9000.00 Ozone Generator, PH490 11315.00 1.00 11315.00 Spare Lamps 500,00 6.00 3000.00 SpareQuartz Sleeves 400.00 2.00 800.00 Spare Ballasts 500.00 4.00 2000.00 Compressor, Thomas 580.00 2.00 1160.00 Hydraulic Howmeters 7.00 455.00 Pneumatic Flowmeters 40.00 4.00 160.00 Ozone Dif fusors 250,00 4.00 1000,00

TOTAL 50883.00

I SEAWATER TREATMENT PVC, VALVES, FITTINGS, ETC.! 2" PVC Flange 7.26 13.00 94.38 2" PVC Pipe $73/100ft 288.00 210,24 2" PVC Ell 90 1.02 33.00 33.66 2" PVC Ball Valve 23.62 20,00 472.40 442 Red Tee 10.69 17.00 181,73 4" PVC Pipe $213/100ft 127.80 4" PVC Tee 11.22 4.00 4" PVC Caps 4,16 8.00 33.28 4" PVC Ball Valve 155.22 4.00 620.88 4" PVC Ell 90 7.21 2.00 14.42 2 "PVC Tee 1.26 4.00 5.04 2" PVC Caps 0.52 11.00 5.72 I/2" PVC Flanges 3.07 4.00 12.28 Ozone Tubing $254 /100ft 100.00 254.00 Ozone Diffusors 256.00 4,00 1024.00 1/2" PVC Valves 6.98 4,00 27.92 I/2" PVC Pipe $25 /100ft 100.00 25.00 Condensation Trap 100.00 2.00 200.00

TOTAL 3387.63

HATCHERY SEAWATER DISTRIBUTION 12.0 MT Header Tank 2000.00 1.00 2000.00 4" PVC Pipe $213/100ft 310.00 660.30 4" PVC Ell90 6,66 13.00 86.58 4" PVC Flanges 11.06 8.00 88.48 441 Red Tee 9.88 7.00 69.16 4XI Red Bushing 5.00 2,00 10.00 4" PVC Cap 4.16 1.00 4.16 442 Red Tee 9.88 5.00 49.40 2" PVC Pipe $73/100ft 460,00 335.80 221 Red Tee 1.35 45.00 60.75 Design,Operation and Training... 183

UNIT EXTENDED DESCRIPTION PRICE US$! UNITS PRICE US$! HATCHERY SEAWATER DISTRIBUTION con't. 112 Red Tee 2.78 2.00 5.56 2" PVC Tee 1.26 9.00 1134 2" PVC E1190 1.02 15.00 15.30 1" PVC Pipe $35.33/100ft 450.00 158.99 1" PVC E1190 0.35 12.60 1" PVC Cap 0.29 32.00 9.28 1" PVC Ball Valve 10.40 520.00 1" PVC Tee 0.46 6.00 2,76 Hydraulic Flowmeters 6.00 360.00

TOTAL 4460.46

HATCHERY AERATION MAIN BLOWER! Main Air Blowers 1500.00 2.00 3000.00 3" PVC Ball Valves 75.60 3.00 226.80 3" PVC Pipe $152/100ft 500.00 760.00 3" PVC Tee 5.94 6.00 35.64 3" PVC Ell 90 3.72 6.00 22.32 332 Red Tee 5,94 6.00 35.64 2" PVC Tee 1.26 14.00 17.64 2" PVC Ell 90 1.02 18.00 18.36 221 Red Tee 1.35 12.00 16.20 2" PVC Pipe $73/100ft 250.00 182.50 2" PVC BalI Valves 23.62 4.00 94.48 2" PVC Caps 0.52 8.00 4.16 1 "PVCPipe $35.33/100ft 150.00 60,00 1" PVC Ball Valves 10.40 16.00 166.40 1" PVC E1190 0.35 16.00 5.60 2.5" Plastipore $41.12/3ft 16S.OO 2302.72 1/2" Thr Hosebarb 0.25 22.00 5.50 1/2" id Tygon Tubing $197/200ft 200.00 197.00 1/2" PVC Ball Valve 6.98 22.00 153.56 1 "X3" Airstones 2.80 40.00 112.00 3/16" Tygon Tubing $67.88/100ft 200.00 135.76 3/16" Thr Hosebarb 0.25 30.00 7.50 3/16" In-Line Valves 0.50 30.00 15.00 Blower Air Filter 100.00 2.00 200.00 Sidestream Injection Arr. 200.00 1.00 200.00

TOTAL 7974.78

HATCHERY BLOWER AERATION ALGAE CULTURE FACILITY, LAB! Air Blowers 1000,00 2.00 2000.00 Blower Filters 100.00 2,00 200.00 2" PVC Pipe $73/100ft 1000.00 730,00 2" PVC Tees 1.26 26.00 32.76 2" PVC Ell 90 1.02 20.00 20.40 3/16" Mipt 0,25 100.00 25.00 3/16" Tygon Tubing $67.88100ft 1000.00 678.80 3/16" Valves 0.50 200.00 100.00 1 "X2" Airstones 2.00 300.00 600.00 2" PVCCaps 0.52 1.00 032 3/16" PVC Four-Way 0.63 40.00 25.20 3/16" Ell 90 0.32 80,00 25.60

TOTAL 4438.28 184 ... Tree

EXTENDED PRICE US$! UNITS PRICE US$! ILITY MAT ROOM, LAB, SPAWNING! 1000.00 6.00 100.00 6.00 600,00 16.84 6.00 101.04 20.00 6.00 120.00 100.00 2.00 200.00 40.00 6.00 240.00 63.62 4.00 254.48 150,00 1,00 150,00 167.51 2.00 335.02 Assorted Brushes NA NA 100.00 Salinometer/Refractometer 360.00 1.00 360.00 Thermometers, Assorted NA NA 100.00 DissectingMicroscope 610.00 1.00 610.00 DissectingKit 48.50 2.00 97.00 HACH Ammonia Test Kit 100.00 1.00 100,00 Top LoadingBalance 1195.00 1.00 1195.00 TripleBeam Balance 181,65 1.00 181.65 Refrigerator 750.00 1.00 750.00 Box Freezer 600.00 1.00 600.00 1.0qt. Containers,Plastic 1.14 120.00 136.80 Assorted Glassware NA NA 300.00 Magnetic Stirrer 1.00 172.50 SpawningTanks, Fiberg, 150.00 16.00 2400.00 PlasticBuckets, 30 qt. 5,70 5.00 28.50 Nauplii Harvesters 20.00 7.00 140.00 Wooden Shelves 1000.00 1.00 1000.00 Flashlight,Charg. Port, 40,00 1.00 40.00 Utility Cart, Rubberized 124.90 1.00 124,90 Portable Aerator 50.00 1.00 50.00 AssortedCleaning Utensils NA NA 100.00

TOTAL 16586.89

ALGAE CULTURE FACILITY AlgaeCulture Tanks, '1.0 MT 215.15 20.00 4303.00 Algae Culture Tanks, 500 ltr 125,00 7.00 875.00 GlassCarboys, 20 ltr 43.82 10.00 438.20 Aspirator Bottles,2.0 ltr 9.47 20.00 189.40 ErlenmeyerFlasks, 250 ml 3.03 20.00 ErIenmeyerFlasks, 125 ml 1,94 40.00 Stock Culture Tubes NA 48.00 50.00 Glass Petri Dishes 3.79 24.00 Bunsen Burner 27,00 2.00 4-Bulb Light Banks,160 watt 100.00 35.00 3500.00 InoculatingLoops 22.13 2.00 44.26 Hand Counters 41,20 2.00 82.40 DisposablePipet, 1.0 ml 0.14 500.00 71.40 DisposablePipet, 2.0 ml 0.18 250.00 44.23 Test Tube Racks 19,30 2.00 PipetCannister, Autodave 19.64 2.00 39.28 Hemacytometer 66.00 4.00 264.00 CompoundMicroscope 1408.00 1.00 1408,00 Salinometer 360.00 1.00 Air Conditioners 1500.00 4.00 CarboyCulture Shelf 400.00/Unit 3,00 1200,00 Zksign,Operation arrd Traipsing ... 185

UNIT EXTENDED DESCRIPTION PRICE US$! PRICE US$! ALGAE CULTURE FACILITY con't. Autoclave,Upright 5000.00 1.00 5000.00 Pressure Cooker 230.00 2.00 460.00 Utility Cart, Rubberized 124.90 1.00 124.90 Jerrican,w/spigot 26.75 10.00 267.50 Round Nutrient Bottles 1.24 12.00 14.84 Wooden Transfer Hood 50.00 1.00 50.00 Refrigeratorw/freezer 600.00 1.00 600.00 Submersible Pump 124,00 2.00 248.00 Miscellaneous Glassware NA NA 200.00 Miscellaneous Plasticware NA NA 200.00 Miscellaneous Cleaning NA NA 100.00 Miscellaneous Nutrients/Chemicals NA NA 750.00 TOTAL 27206.17

GENERAL HATCHERY LABORATORY pH Meter,Stationary 1570.00 1.00 1570.00 pH Meter, Portable 495.00 1.00 495.00 ORP Probe for pH Meter 135.00 1.00 135.00 ORP Test Solution 25.00 1.00 25.00 Assorted pH Buffers NA NA OxygenMeter, Portable 1195.00 1.00 1195.00 Salinometer 360.00 1.00 360.00 HACH Drei 7 Kit 2500,00 1.00 2500.00 Compound Microscope 1408.00 1.00 1408.00 Bacteriological Test Kit 595.00 1.00 595,00 AquacultureTest Kit 350.00 1.00 350,00 Magnetic Stirrer 172.50 2.00 345,00 Refrigerator 600.00 1.00 600,00 Top Loading Balance 1895.00 1.00 1895.00 Miscellaneous Glassware NA NA 200.00 Miscellaneous Plasticware NA NA 200.00 Miscellaneous Chem/Nutrients NA NA 300.00 TOTAL 12223.00

LARVICULTURE 6.0 MT Larval Rearing Tanks 1000.00 16.00 16000.00 Center Screens 16.00 1200.00 Postlarvae Harvesters 150,00 4.00 600.00 Submersible Heaters 250.00 16,00 4000.00 Protein Skimmers 45,00 16.00 720.00 Artemia Tanks, 100 ltr. 16.00 1200.00 Magnetic Stirplate 172.50 1,00 172.50 Triple Beam Balance 181.65 1.00 181.65 Blenders 2.00 100,00 Various Sieves NA NA 200,00 Miscellaneous Plasticware NA NA 200.00 Miscellaneous Cleaning NA NA 100,00 Additional Nitex Screen NA NA 200.00 TOTAL 24874.15

QUAIL'~E/DISINFECI'ION 4.0 MT Quarantine Tanks 4.00 1600.00 Ozone Gen., Portable PH190 3551.00 1.00 3551.00 Small ThomasCompressor 350.00 1.00 350.00 Aeration Inj. System 300,00 1.00 Center Screens 4.00 200.00 186 ... Treece and Fox

EXTENDED DESCRIPTION PRICE US/! PRICE US$! QUARANTINE DISINFE N con't. Chlorine Drain Dispensor 150.00 1.00 150.00 Dissecting Microscope 610.00 1.00 610.00 Triple Beam Balance 181.65 1.00 181.65 Miscellaneous NA NA 300.00

TOTAL 7242.65

OFFICE FURNITURE office, other areas! Small 640K Computer, etc, 1000.00 1.00 1000.00 CitizensBand Set-up 750,00 1.00 75G.GG Of fice Furniture NA NA 1500.00 Notebooks, Records NA NA Whiteboard 75.GG 1.00 Overhead Projector 350.00 1.00 Miscellaneous NA NA

TOTAL 4475.00

STORAGE AREA StorageShelves, Hand-built NA NA 1000.00 Plastic Nesting Bins NA NA 500.00 PVC Inventory NA NA 2000.00 Artemia Cysts, Cases 480.00 5.00 2400.00 Counter Top 250.00 1.0G 25G.GG Dollies, Carts, etc. NA NA 200.00 Miscellaneous Hard ware NA NA 2000.00 glue,hammers, PVC cement, crowbars, hand saws, nails, skill saws,screws, drills, bolts, levels, silicon, screw drivers, tap and dyekit, fiberglass,tape measures, chlorine Alconox, nitex scree, paint, paint brushes, sandpaper, wrenches, ratchet set, files, woodrasps, wood, cutting torch, bottled 02, regulators,batteries various!, clamps, chisal voltmeter, wire, solder gun, solder, pliers, power cords TOTAL 8350.00

WORKSHOP FIXED ITEMS, NON-REMOVABLE! Work table, heavy-duty 1.00 300,00 Vises heavy! 100.00 3.00 300,00 Table Saw 1.00 400,00 Drill Press 400.00 1.00 400.GO Brooms 25.00 2.00 50.0G Compressor,Cart-Type 400.00 1,00 400,00 Miscellaneous NA NA 20G.GG

TOTAL 2050.00

SUPPORT AND ADMINISTRATION Light-Duty Truck 10000.00 1.00 10000.00 Automotive Supplies NA NA 200.00 30 kva Gen-Set 10000.00 1.00 10000.00 Fuel StorageTank 500.00 1.00 500.00 Trash Bins 100.00 4.00 400.00 Incinerator 1.00 250.00

TOI'AL 21350.00

GRAND TOTAL 195502.01

Aquaculture Equipment Suppliers New Research Developments of 1993 A listof equipmentsuppliers to theaquacultureindus- The Addition of Paprika Nature's Highest Source try can be found in Aquaculture Magazine's "Buyer's of the Carotenoid, Astaxathin! Guide" which comesout yearly, or in other publications Researchers,at the Oceanic Institute in Hawaii, have suchas "The North AmericanDirectory of Aquaculture" noticed that after broodstock have been in a maturation published in Canada. Design,Operation arat Trai nieg ... I87 systemfor severalmonths, the female ovaries begin to bleach from a bright red color, which produces high quality larvae,to a whitishcolor, with 10-20percent lower survival to secondstage zoea. In addition, the females' shell softened and turned a bluish color. In other shrimp, a bluecolor hasbeen diagnosed as a carotenoid deficiency, suggesting that there is not enough pigment in the broodstockdiet. Carotenoid was incorporated into the broodstock diet with the addition of paprika, nature' s highestsource of carotenoid,astaxanthis. This is doneby marinating squid in paprika beforefeeding. As a result, larval quality and survival, as well as broodstockovary color and shell hardness have improved. Adding Enteromorphato BroodstockDiet Theadditionof Enteromorphato the feedingregimeis recommendedby somehatcheries. Enteromorpha green aIgae!can be collectedfresh from rocks below the tide level. Microwaving is suggestedto pasteurizethe algae before feeding it.