PREFACE
TheUnited States and Japanese counterpart panels onaquaculture wereformed in 1969 under the UnitedStates-Japan Cooperative Program inNatural Resources UJNR! The panels currently include specialistsdrawnfrom the government andacadeinic departinents mostconcerned withaquaculture- Chargedwith exploring anddeveloping bilateral cooperation, thepanels have focused their efforts on exchanginginformation related to aquaculture thatcould be of benefittoboth countries. TheUJNR was begun during theThird Cabinet Level Meeting ofthe Joint United States-Japan CommitteeonTrade and Economic Affairsin January 1964,ln addition toaquaculture, currentsubjects in theprogram include toxicmicroorganisms, airpollution, energy, forage crops, national parkmanagement, rnycoplasmosis,windand seismic effects, protein resources, forestry, andseveral joint panels and corninitteesin marine resolve research, development, andutilization. Accomplishmentsiticlude:increased communication andcooperation amongtechnical specialists; exchangesofinformation, data,and research findings; annual meetings ofthe panel, a policy-coordinating body;administrative staffmeetings; exchanges ofequipment, materials, andsamples; several major technicalconferences; andbeneficial effects of internationalrelations. The26th U.S.-Japan Aquaculture PanelSyinposium washeld in Durham, New Hampshire, from16- 18September ] 997. Following thesyrnposiuin, fieldtrips during a seven-day period included theareas of Portsmouth,NewHainpshire; andBar Harbor, Eastport, Camden, and Boothbay Harbor, Maine. T' he syrnposiurnwasorganized byprogram chair Anne Bucklin, Sea Grant Director; Hunt Howell, Professor of Zoology;and Roll ie Barnaby, Sea Grant Extension Officer, atthe University of New Hampshire.
Panel Chairmen:
JamesP. McVey, UnitedStates
Yukio Uekita,Japan Participantsin the 26th UJXR AquaculturePanel Symposium, held in Durham,New Hampshire,U.S.A., September16-18, 1997. CONTENTS
/. GeneralAquaculture
G. Nardi Research in Flatfish Culture in the Northern United States
T. Seikai JapaneseHounder Seed Production from Quantity to Quality
W. I., Rickards SustainableHounder Culture and Fisheries: A Regional 17 ApproachInvolving Rhode Island, New Hampshire,Virginia, North Carolina, and South Carolina
T. I, J. Smith Tank andPond Nursery Productionof JuvenileSouthern Flounder, 21 W. E. Jenkins Paralichthyslethostigma M. R. Denson
III. Hea th Management
G. Birnbaum Licensingand Regulation of VeterinaryBiologics for Fishin theUnited States 33
N. Iwata Effectsof RearingConditions on BlindSide Hypermelanosis in K. Kikuchi JapaneseFlounder
S. H, Jones Microbiologyof Early Larval Stagesof SurnrnerFlounder Paralichthys 45 B. Summer-Brason dentatasGrowth in a RecirculatingWater System G. Nardi
II/, Nutrition I
H. Furuita NutritionalRequireinents in BroodstockMarine Fishes
N. Ohkubo SequentialUtilization of FreeAmino Acids, Yolk Protein, and Lipids by 61 T. Matsubara DevelopingEmbryos and Larvae in BarfinHounder Verasper moseri
N, J. King Ef'fectsof Microalgaeand Live Diet Type on the Growth of First-Feeding 67 W. H, Howell Winter Flounder, Pieuronectes americanus
T. Kurokawa DevelopmentalProcess of DigestiveOrgans and their Functions in Japanese 79 T. Suzuki Flounder, Parali chthys oli vaceus
I. Oohara A/E RatioProfiles of the EssentialAmino Acid Requirements 85 T. Akiyama Among VariousFinfish Species T. Yamamoto IV. AlarineStock Fnhranc H. Fushimi DevelopingaStock Enhancement Program Based onArtificial Seedlings; 95 Activitiesof the Japanese Sea-Farming Association JASFA! in the est Decade Y, Ohsaka Effectsof Coveringa Tidal Flat with Sand for Stock Enhancement of 105 Y. Koshiishi To>guefish:A FeasibihtyStudy at AriakeSound in Kyushu,Japan T. Murai ~spec s in StockEnhancement of Japanese Flounder Y. Koshiishi M, N. Wilder Reproductive Mechanismsin Macrobrachiumrosenbergii and Penaeus 125 J~rricns: Endocrino!ogicalResearch and Potential Applications in Aquaculture K. Adachi Primary ProductivityofSandy Shores 137 K. Kirnoto J. Higano T. Nakasone Nutrient Concentrationsin Groundwater Through Sandy Beaches 149 K Adachi T. Takeuchi J, Higano H. Yagi V. Nu rition II M. Yokoyama C'ysteineMetabolism in Rainbow Trout 159 N, King Effect of StockingDensity on the Growth and Survival of 173 H. Howell Larval andJuvenile Sununer Flounder, Paralichrhys dentafns E. Fairchild A, Kanazawa 1trtportanceof DietaryLipids in Flatfish 181 H. V. Daniels Effectsof LowSalinity on Growth and Survival of SouthernFlounder, 187 R. Borski ~~ M. B. Rust An 1 tItageAnalysis Approach toDetermine Microparticulate F~ 193 F. T. Barrows Acceptabilityby LarvalFish VI. Engineeringfor Open OceanAqttacttlture M, R. Swift FishCage Physical Modeling for SoftwareDevelopment M. Palczynski andDesign Applications K. Kestler D. Michelin B. Cclikkol M. Gosz N. Takagi Creationof OffshoreAquaculture Gmund by FloatingBreakwater 207 R. W. Dudley AWATS: A Net-Pen AquacultureWaste Transport Simulator 215 V. Panchang for Management Purposes C. R. Newell S. Kawamata EngineeringTechniques for Enhancetnentof NearshoreRocky Habitats 229 for Sea Urchinand Abalone Aquaculture B, Bragittton-Smith DesignCottcepts for Integrationof OpenOcean Aquaculture 239 R. H. Messier andOSPREY~ Technology K. Takayanagi WaterQuality Guidelines for Aquaculture:An Examplein Japan 247 K. C. Baldwin Marine Mammal Gear Interactions; Problems, Acoustic Mitigation 255 S. Krauss Strategies,Open Water Aquaculture J. S. Goldstein North AmericanLobster Culture Hontarusamericanus!, Hatchery 263 Methods,and Techniques: A Toolfor Marine Stock Enhancement? K. Kikuchi BlueMussels in theDiet of JuvenileJapanese Flounder VII Sttmrnaryof Panel Discussion D.Bengston Summary ofthe Panel Discussion onCulture Held at the 275 ConclusionoftheUJNR Aquaculture Panel's Scientific Symposium in Durham,New Hampshire, USA, 1 8September 1997 VIII. Acknowledgments IX. Appendix Conference Attendees 278 RESEARCH IN FLATFISH CULTURE IN THE NORTHEASTERN UNITED STATES GeorgeC. Nardi GreatBayAquafarms, lnc. 153 Gosling Road Portsmouth, NH 03801 e-tnai1:GAquafartn@ao].corn ABSTRACT Althoughflatfish have been commercially cultured for oversdecade in Europeand Asia, theu culture in North Americahas only recentlybeen cornmercitdized. The commercialization of Atlantic halibut Hippoglossus hippogirrssasand summer flounder Paraii chrhys dear artss culture followed years of collaborativeeffort between industryand university researchers. Other important flatfish species are being evaluated for commercialculture throughoutthe region, including winter flounder Pteuronecres americanas, witch flounder Glyprocephaias cynogiosstrs,snd yellowtai! flounder Pieuronecres ferrrrginea, Juvenfle production continues to beao impedi- tnentto commercialization.Commercial on-growing strategies include both net pen and land-based rank culture systems, INTRODUCTION 10, 20, 30, and60 larvaeJL,and weaning diet proteincontents of 45, 50,and 55%, Muchof this Stunmer flounder PandicIsthygdeggtatug NRAC-sponsoredresearch is beingundertaken at Researchis underwayin New Hampshire, GBA. Massachusetts,Rhode!sland, Connecticut,and GreatBayAquafanns, the New Hampshire New York. Cotnmercialproduction was begun in Industrial ResearchCenter NHIRC!, Sea Grant, New Hampshirein 1995,Massachusetts and New NRAC, and the Electric Power Research Insti- York in 1997, and is slated for 1998 in Rhode Is- tute EPRI! havecooperatively funded a number land. Much of the researchin New Hampshire of researchprojects. The NHIRC research in- has taken place at the University of New cludesmicrobiology and veterinary diagnostics, Hampshire's UNH! CoastalMarine Laboratory wastewater characterization and effluent treatment andat GreatBayAquafarms, Inc. GBA!, a corn- design,«nd thermal engineering to capturethe mercial hatchery. UNH researchhas beensup- waste heat of a utility for heating the seawater portedby both the U.S. Department of Conurterce's andair. UNH SeaGrant and GBA are workingto NationalOceanic and Atmospheric Adxninistratiotr identifyand develop a probioticapproach for early NOAA!/National Marine Fisheries Service larvalrearing in order to increase survival and limit NMFS! Saltonstall-KennedyIndustry Grants Pro- Pathogenhabitation of theculture environment or gram S-K! andthe U.S. Departmentof Agricul- larvalgut, In addition,UNH SeaGrant research- ture Northeast Regional Aquaculture Center ers are workingwith GBA to identifygenetically NRAC! The S- K work investigatedsubstrate superiorbroodstock by trackingthe performance colorprefcrcnces and effect on pigmentation, ju- of individual families raised in a cotnmonenviron- venile stockingdensities as a percentageof bot- rnent. The EPRI fundingassisted in the develop- totncoverage 00, 150, and 200%!, feed prefer- rnentof a commercialscale grow-out dernonstra- ences,growth performance, and use of a recircu- tion systemwhere research is beingundertaken to latingseawater system. The NRAC researchin- evaluatethe perfonnanceof alternaterecirculat- vestigatedthe efficacy of natural spawningvs. inglife support systems, specifically the biofilters, hormonalinducement, larval stocking densities at One bioft!ter is a fluidize sandbed, the other is a 92:jÃRTccbnical Report 'bio. 24 plasticmedia submcrgcd inan aerated tank, This moneon larval deve]opmcnt andsurvival, hormonal researchis alsoassisting in thcengineering of a influencesondeveloping embryos, optimal culture land-ha»cdtank farm heing planned for GBA. This environmentconditions, marketing, economics, and demonstrationfarm will allow GBA to compare outreach. URI is also the lead institutionin a variablessuch as stocking density on biofil ter per- inulti-instttutioneffort currently funded by NRAC formance.The operatingcosts of the two sys- Thisresearch is investigating larval stocking den- tem»will alsobc cotnpated. sitiesin greenwater,natural vs. hormonal induc- In Massachusetts,Aqua Future,lnc a tionof broodstock,comparison of survival, growth, hybridstriped bass grow-out operation, isbuilding health,and behavior of fish rearedin recirculation a hatcheryfor thcproduction of surnrnerflounder systemsvs. openocean net pens. Vk G Seafarms, juvenilesand, as thc recipient of a NOAAFishing in Quonset,Rhode Island, has constructed a coin- IndustryGrant FIG, is providingoversight and mercialscale demonstration grow-out site as part designinput for twogrow-out demonstration sites, of theFIG programadministered by NMFS. This onc in New Bedford, Massachusetts,and one in facilityutilizes recirculating technology with mini- Quonset,Rhode Island. The objectives of thiswork mal new wateraddition and a shallow raceway are to dcmonstratc thc commercial culture of sum- tanksystem. The. tank system is stackedtwo lay- mer flounder, thc utilizauon of underutilized sea- ersdeep. Commercialproduction is plannedfor foodprocessing space as culture sites, and the vi- early1998. ahilityof aquacultureas an alternativemeans of TheUniversity of Connecticutis conduct- cinploymcntfor displacedfishermen and plant ingresearch as part of theNRAC projectbeing workers.Trio AlgarvioSeafoods, Inc. harvests coordinatedby URI. The objectivesof this re- andprocesses groundfish in New Bedford,The searchare to identifyprotein and lipid levels in companyowns a processingfacility that is cur- grow-outdiets and an optimumcalorie to protein rentlyundcrutilized because of government-rnan- ratio, datedreduced fishing effort which has, in turn,made MaricultureTechnologies, Inc. in New it diflicultfor the company tosupply local fish. Trio Yorkis a pri vate commercial production farm which hasinstalled into its facility the first phase of a is presentlyculturing suinmer f!ounder in open giow~utfarm utilizing raceways and a recirculat- oceannet pens. The company supports its net pen ingseawater system, Trio is stocking this system operationsfrom land-based nursery operations, suutingin late1997 with juvenile summer floun- Juvenilesin excess of 100g arestocked in thc der, springand fed through November, when they are TheUniversity of RhodeIs!and URI! has typicallyharvested. As this isthe first seasonsurn- bccninvolved with flatfish culture for many years 92 merflounder have ever been stocked in thesenet mostrecently investigating coinmeicial culture for pens,it is unknownif thefish will be ableto suc- stockenhanccmcnt and grow-out. URI's flatfish cessfullyoverwinter in thislocation. In an effort aquacultureresearch has been funded in part toderermine their thermal tolerance, soinc fish will throughS-K, Sea Grant, and NRAC a~aids. The beheld in thenet pens throughout the winter of S-Kresearch was done in collaborationwith the 1997-98.Under a NOMdNMFSFIG, Maricul- Universitiesof Massachusetts andNew H ew amp- tureis testing a number ofnet pen systems, includ- sshire. irc.thc New England Fisheries Deve!opment As- ingones manufactured byBridgestone, Northern sociation NEFDA!, Northeast Organ ics, Inc., and PIIastics,Ocean Spar Technologies, andAtlantic GBA, TheS-K research supported efforts for takingthe cuhure ofsummer flounder tocoinmer- AquaCage. Finally, as a commercialparticipant cialscale by demonstrating theability to re onthe NRAC project coordinated byURI, Mari- edl s cultureismonitoring fish growth, survival, health y spawnsummer flounder in captivity,toim- andbehavior. These are being compared tofish provesurvival rates through larval and weaning raisedin land-based systems. stages,and to further elucidate larval nutritional rcquiremcnts.SeaGiant research hasinvestigated causesoflarval mortality, influence ofthyroid hor- Wiatcrflounder PIclrowcctes uincrscersus Winterflounder supports a strong recre- mardi 3 ationaland comtncrcial fishery fromthe Atlantic Atlantichalibut Hippoglossus hippoglossus Maritiines throughthe rnid-Atlanticstates, Like Atlantichalibut, the largestof ourflatfish sutntnerflounder, commerciallandings for winter withindividuals exceeding 200 kg notunheard of flounderare at historiclows, There is strongin- in the commercialfishery, are beingcommerc i ally terestin aquacultureresearch for purposesof both culturedin Europeto betwccn5 and l0 kg. At stock enhancement and commercial culture. Re- presentthe harvest is small,about 50 tons;how- search in New Brunswick, Maine, New Hamp- ever,with capacity expanding this number is sure shire and Rhode Island, and a commercial opera- to increase,particularly as NorthAmerica starts tionin NovaScotia, support the developtnentof tobring both academic and industrial resources into thispromising culture candidate. thedevelopment of a hahbutcuhure industry, These In Maine, the Departmentof Marine Re- fish are active swiminers and could be cultured in sources DMR! hasdevoted considerable time and bothtanks and net pens. Research programs have resourcesto evaluatingthc potential of stocken- beendeveloped at a numberof NotthAmerican hancementfor groundfish,particularly cod. Re- locations.At the Memorial Universityof New- cently,DMR hasgiven consideration to winter foundland,investigators are seekingto develop flounderfor stockenhancement. DMR personnel broodstock managetnentprotocols, improve aretearing small numbers of winterflounderjuve- live-feed enrichmentdiets, refine start-feeding pro- nilesfrom captive broodstock. tocols,and develop juvenile-formulated diets, Also In New Hampshire,researchers at the in Newfoundland,thc privatelyowned company UNH CoastalMarine Laboratoryare also investi- MaritimeMaricultute is working closely with gov- gatingthe potential of stockenhancement aswell ernmentand academicresearchers to developa as the commercial cultureof winter flounder. They commercialhatchery and grow-outfarm for At- have been successfulin batch rearingjuvenile lantichalibut. Maritime hasincorporated proven flounderto over 500 g. Graduatestudents are Norwegianculture technology itito its rearing strat- workingon methodsto itnprovelarval survival egy, In New Brunswick,the Canadian Depart- throughmetamorphosis, and are comparing tnent of Fisheries and Oceans in St Andrews is hatchery-rearedand wild-caught juvenilesas part workingon broodstock management, juvenile pro- of a programto determinethe efficacyof stock duction,and the improvementof culture systems enhancetnent. The stock enhancement research for rearinghalibut. Researchers atthe University is beingdone in collaboration with the personnel at of Maineare working on methods to refine spawn- the Maine DMR, the MassachusettsDivision of ing,fertilization, and larval rearing. They are also Marine Fisheries, and URI. workingon sexdetermination methodology and Winter flounder researchis also underway larvaldevelopment issues. Future work at the in the maritimeprovinces of Canada. Universitywill seekto optimize culture techniques, TheUniversity of NewBrunswick is workingon andto improve larval and juvenile nutrition, developingconunercial culture techniques and the optimizationof feeding.In NovaScotia, research YellowtaII Sounder Pklrorrectesferruginea anddevelopment are being undertaken by Sambro For decades,yellowtail flounder has been FisheriesLimited, whichis a commercialproces- a inajorcomponent ofthe cotnmercial fishery land- sor,wholesaler, and distributor of groundfishspe- ingsfor groundftshfrom New Bedford,Massa- cies.Recognizing that its supply was being limited chusetts,north through Atlantic Canada. Land- by a combinationof overfishing and government ingshave declined drainatically, and if thisflatftsh regulation,Satnbro has begu.n to investigatethe can be shown to be a viable cold water culture commercialculture of winter flounder. It began candidateit will enjoya readyinarket throughout ona pilotscale in 1994-96,maintaining a popula- the Northeast, tion of captivebroodstock and rearingseveral Researchon this speciesis occurringat batchesof juveniles Basedon thisexperience, thcMetnorial University of Newfoundland,where Sambmis planning large scale production systems investigatorsare looking closely at the potential of startingin 1997. yellowtailas a conunercialculture candidate. Their 4 t:JNR TechulrsiReport iS0. 25 initialefforts are focused onbroodstock manage- rnent,improving juvenile on-growing protocols, determininganoptimal culture environment, and needsfor diet formulation, WitchQouttder 61yprocephaCus cyisoglosses Thewitch flounder isconsidered bymany thepremier flounder in theNorthwest Atlantic. Thisstatement isborne out in the prices paid for thisfish by commercial dealers, with boat prices oftenin excessof $8/kg. Becauseit is sucha prizedfish, there is interest in its commercial cul- ture,but the interest istempered somewhat bythe factthat witch flounder appear tobe a relatively slowgrowing, difTicult-to-rear species.The only researchbeing conducted on witchflounder is througha joint project between the Meinorial Uni- versityof Newfoundland andUNH. At these two institutions,researchers areinvestigating protocols forbroodstock development andmanagement, as wellas larval rearing pmtocols 1Ite larval research seekstodetertnine theoptiinal incubation tempera- turefor eggs and early larvae, to ascertain the ne- cessityofgreenwatcr forlarval culture, and to com- paregrowth rates of first-feeding larvae fed wild, cultured,and enriched cultured diets. They have beensuccessful in rearing small numbers of fish throughthe juveniles stage. ACKNOWLEDGMENT S I amgrateful to the following individuals whoprovided inforination for the compilation of thisreview: John Batt, Sambro Fisheries; Dave Bengtson,University ofRhode Island; Joe Brown, MetnorialUni versity ofNewfoundland; Kathy Downey,Trio Algarvio; Hunt Howell, University ofNew Hampshire; Greg Huba, V&G Seafarms; LindaKling, University ofMame; Bob Link, Mari- cultureTechnology, Inc.;Matt I itvak,University of NewBrunswick; and Dave Raymond, Mat'i- tirneMariculture, JAPANESE FLOUNDER SEED PRODUCTION FROM QUANTITY TO QUALITY Tadahisa Seikai KyotoUniversity, Faculty of Agriculture,Fisheries Research Station Maizuru, Kyoto 625-0086, Japan Einai1;[email protected] ABSTRACT Japaneseflounder Paralictuhys ofiivsceus,distributed widely from the coast of Hokkaido to Kyushu, is one of the most importanttarge speciesfor stockenhancement uials and aquaculnnein Japan. lvlore than 2tl milnon juveniles were released on thc coast of Japanm 1994. Studies nf seedproduction in this species are dividedinto threephases: the challengeof seedproduction, the quantitativeexpansion, and thequalitative iinprovement.The first phase,started at the end of the l9th century,involved egg collec iousfrom wild- caughtfish, artificial fertilization, and hatchingexperiments. During the period of thc 195 lsio 1960s, suitablefood organismswere foundfor the feedingof marinefish larvae.Then, seed production of Japanese flounder becamesuccessful following the successof red sea bream Pagrus mojvr seed production. During this period, matured eggs were taken from wild-caught fish in thc spavrningseason and fertilized artiticially. Fish larvae and juveniles were fed rotifers, Arremia. minced meat. snd then chopped sand eels as they grew. Duringthis phase,the quality andthe quantityof fertilizedeggs fluctuated, because stripping and artificial fertilizationwere conducted froin the wild-caughtfish. Severalyears after the first success.fertilized eggs were taken from the spawnerswhich grew in captivity from newly hatched larvae. We then could enter the secondphase when brood stockskept in land-basedtanks inatured and continuously spawned large numbers of eggs with high quality. Tank spawning expandedthe quantiues of the J~ flounder seedproduction very quickly, for we could transfer the basic mhnologies of red sea bream seed production. Moreover, accordingto themattuaiion control. we could acquirefertilized eggsalinost year round. Sincethe larvaeof this speciescould accept formula feed inure readily than other species, formula thets were actively introduced to reducecosts together with the nutritional improvement in producedfish. ln the third phase,we have been trying to improvethe quality of prixlucedjuveniles. We have succeededin severalaspects: dramatic ieducbon in the percentageoccurrence of albinism by nutritional improvementduring the larval stage,behavioral iinprovementby trainingin semi-naturalconditions before releasing, and labor savingsand autonuzatlonin the seed production process. We soll face many probleins that include egg quality, abnormal blind side piginentation, vertebral abnormaliiy, disease control, and genetic diversity. Jn addition, hormonal control during larval metanmqkosi s, bi otechnological trials.and rnechanisinof sex determination shoukl be elucidated in relaiion to the seedproduction of this species. INTRODUCTION technologieshorn biology to engineering.Japanese flounderseed production has expandedquickly Japanese flounder, distributed from within the past20 yr Fig. l!. As for the quantitative Hokkaidoto Kyusyu,is oneof themost important expansionof seedproduction, related technologies fish for Japanesecooking, along with redsea bream. needto developconcurrently and harmoniously. We Annual production fmm aquacultureis more than Japaneseare now facing new situationsin the 6000tons, exceeding landings from boatfishing in transitionfrom the pioneertrials to the enterprise l994. Moreover,Japanese flounder is one of the of stockenhancement to producehigher quahty most important target species for the stock juveniles. enhancement project. Thirty-four prefectures I presenta historicalreview of studiesin releasedmore than20 nullion juveniles in coastal Japarleseflounder seedproduction, severaltrials areasas part of governmentalactivities in 1994 for the transition from quantity to quality, and the Furusawal 997!. Thisbecame possible based on futureissues and remaining problems. the mass seedproduction that integratesvarious l tJ statTechtdeal Report Vo gg I5 120 gt 100 ~gg ~ L gal 8 m C 60 0 0 40 0 20 1980 f982 1984 198ti 1988 1998 1992 1994 198] 1983 'f985 1987 l 989 199} 1993 1995 Year Ftgttpe1. Seedproducuon of marine finifsh ln Japan, I980-I 995, Daysaaer katettgag I Challengetyf larvtetttlture and seedprndtgt onferttgeeeeggI et sao~ rnite ln Japaneseflounder, artificial fertili mgegete andhatching trial s usingwild fi shbegan at t. AlsA4$aaeptg of the 19thcentury Fujita 1903!. Howe' scalyaelegsl ttsl tense trials endedat the completionof yolk abso gre serac~ becaureof thelack of initial foodorganism tivegate eaeppegltsa From the 1950s to the first half peedlagaeltegaks orthe first seeress H Klaki Ualeerstty 1960s,the feeding scheme for marinefinfish was nearly developed, and larval reari Japaneseflounder froin hatchingto juvenih Artrogea septa e egoreg Aaeogs successfulin 1965 Hara' et al. 1966!. 4 taoppellrts ~ time,eggs were taken from wild fishcaught egoptaehg aost spawningseason, and were fertilized artifi Earlyiaodel ef footling seiedok fer mass prndaeuoa Unfertilizedeggs of seaurchins copepods, At nauplii, newly hatchedfish larvae, live shrirt gobi, andchopped fish meatwere fed succes asthe floundergrew Fig. 2!. Survivalrat pormstefae4 0.7% duringthe first 63 days from hatcht Jteeeatfeeding segogales at Mlyalte ate doe of JASFh juvenilesat 3.03 cm TL Fig. 3! Fromthat seriouscannibalism and abnormal piglnentat the ocular side were noticed as characte phenomenaof this species. Ftgttte> Cotnpaiisonof feeding schedule inJapanese Itoun Establishment of basic technologies derseed production from the first success atKith tlttantitativeeytyansion Universitytothe recent data at Miyako station of the ln 1969, cultured flounder raised JapanSea-Farnung Assocation, hatchingmatured in captivity,and fertilized were takenfrom these fish Harada 1980!. During the 1970s, the Japan Fisheries Agency JFA! supportedthe basic studies for themass production o f Japanese flounder in se vera1 pre feetu ra I experimentalstation s, During the first half of the 1970s, the quantityand the quality of eggswere unstable, as eggswere stripped andfertilized artificially from wild fish. Then,cultured fish startedto spawnlarge amountsof highquality eggsin land-basedtanks. Sincethe basictechnologies of seedproduction in 0 II SP SP % II PI 70 red sea breamcould be transferredto Japanese IIP flounder,spawning became readily possible and the seedproduction expanded very rapidly Takahashi et al, 1980, Hiramoto et al, 1981a!. Moreover, the conditioning of maturation by the control of photoperiodsand tetnperature permitted fertilized eggsto betaken year round ijima et al. 1986! Fig. 4! Generalpmcedures at the presenttime in Japan are as follows: spawnersare kept in captivity throughoutthe year; inaturationis controlledby g~~ II1 SPI10% 00v0 00 50 photoperiodsand temperature; fertilized eggs are nuya atterEtta&lug takenfrom natural spawnings in land-basedtanks; newly hatchedlarvae are stocked in several 10-rn' Figure 3. Comparison of growtharid sttrvivaibetween the concretetanks at a densityof 10,000-50,000/m', first successand the recent results at. Miyako station of they are raised in several tanks until juveniles the JapanSea-Famting Association ctmespondittgto the become 3 cm TL Fig, 5!, and fed on rotifers, developmentof technology. Artetttianauplii, anda formuladiet asthey grow Fig. 2!, Foodorganistus are enriched with special oils containing highly unsaturatedfatty acids and To reducethe mortality by cannibalism,it fat-solublevitamins Torii et al. 1994!. isnecessary to maintainreasonable stocking density In the first phase of seed production, and feedingto reducesize divergency,and to do mincedor choppedfish meat was generallyused sizeselection thoroughly during ihe seedproduction as juvenile feed Hiramotoet al. 1981b! Fig. 2!, process. Because these diets lose their nutrients to water and deterioratethe water quality,they were replaced TrialSfOr quahtativeimprIyvetnent with a formula diet. Since the larvae can feed on a. Fgg quality irttprrsvetrtetitand genetic diversity formula diet with relative ease Tange and Japaneseflounder spawn frequently during Nagahama1986!, and the formula diet could reduce a long spawningseason Hirano and Yamamoto the appearanceof albinismin thejuveniles, many 1992!. Evenin theland-based tanks, the egg quality hatcheries are active in the use of formula feed from natural spawning is not always high. whichreduce labor and cost in the seedproduction However,the most important factors affecting egg Takahashi1990! Fig, 2!, quahty might be spawner'sfeed and the mating Serious cannibalism should be mentioned environment, but these factors have not been as oneof the mainfactors of tnortalityduring the analyzedthoroughly in manyspecies Moritnoto seedproduction of thisspecies Harada et al. 1966!, 1994!, At the present time, frozen fish especiallywhen juveniles have a largesize variance supplementedwith several nutrients, such as of 13 to 2.0. Bitingand cannibalism frequently vitamins, are usedfor spawners Torii et al, 1994!. occurredin the rearingtanks Torii et al. 1994!. Specialformula diets for spawnerswere examined 0 UJNR TecbniearRepcet Va, R 25 4 I 20 " $J J5 l5 he g l0 10g 500 7c 200 IOO 0 1984Feb, 3 Mar.sop 4OO o 500 I o 200 P! 100 0 Mar. Apr. May Jun. 1984 llgure4. Enhancedtnarttratioa and spawning ol'Japanese flounder with pbotapeiiodcontrol. Phntoperitid control ivas started ai 42 daysbefore first spawnitig in espe6ttientaltank. itttodifted from Jijitiia et al., 1986! to obtain higher quality eggs, but the feed Since commerciallyavailable formula diets and compositionisstill under investigation Table 1!. enrichment materials contain many industrial As to the matingenvironment, stocking secrets,hatcheries are choosirtg brands according ratiosbetween females and males,and stocking totheir experiences. Fomrula diets have better and densitiesin spawningtanks an. maintained at 1:1-3, morestable nutritional qualities than live food or and 3 kg/m', respectively, to improve the choppedmeat. Therefore,formula diets can fertilizationrate and fecundity Y, Hondo,Miyazu improvethe tolerance ability to stressand handling Station,JASFA, personal communication!. duringthc seedproduction. For example,the A recentconcern in seedproduction is the MiyakoStation of JASFAbegan to produce inore geneticproblem. Continuoususe of limited than2 millionjuveniles crn TL} at a survival spawnersfrom the hatchery-reared fish results in rate higherthan 80% fram hatchingwith the themass release of juveniles with surtpli6edgenetic effective use af formula diets Torii et al. 1994}. information.Therefore, it hasbecome popular to replacethe spawners from artificially produced fish Studieson abnortnalpigtnertration arid practical to locallycaught wild fishto keepgenetic diversity prevention intactwith localcharacters in producedjuveniles ln associationwith thedevelopment of seed Tanakaet al. 1997!. productionin thisspecies, typical abnormal pigmentationhas been studied seriously. There are lVrttriti onal stay anddevelopment o jfornruladiet twotypes of abnormalpigmentation in flatfish A largenumber of nutritionalstudies have albinism hypomelanosis onthe ocularsid.e! and been conductedfrom the viewpomtsof growth, ambicoloration hypermelanosis onthe blind side! survival,vitality, pigmentation, and vertebral Seikai1985, Seikai et al. 1987!. However,both deformation Kanazawa 1990, Takeuchi 1997!. albinism and ambicoloration appeared in These studieshave helped to improve the hatchery-rearedflounder at extremely high compositionand processing of formuladiets and percentages,andthe former phenomenon attracted enrichmentprocedures of live foodorganisrrts. biologistsfrom the outset of theseed production. Figure 5. Sequenceof separationand harvestduring Japanesefiouuder seedproduction at Miyako stationof thc JapanSea- Farming Association Modified from Torii et al. 1994!. The factors were analyzed mainly from the factor for hyperinelanosis staining type! during nutritional viewpoint during the larval stage. It juvenile and young periods Iwata and Kikuchi, was revealed that the percentage in appearanceof 1998!. Many unresolvedfactors remain in relation albinism was highly dependent on the feed during to other types of hypermelanosis true and spotting the larval stage Seikai 1985, Seikai et al. 1987, types!. At present,hypermelanosis is usedas the Kanazawa 1993!. In the practical seedproduction, effective inarker, which nearly all artificially enrichmentof live food organisins with vitamin A produced fish have without any treatments, to and docosahexaenoicacid DHA! Miki et al. 1988, distinguish releasedfish froin wild fish Furusawa 1989!, and early feedingof formuladiets for larvae 1997!. Sinceheavy pigmentation on the blind side Kitajima et al, 1985!could reducethe percentage decreasesthe price of recaptured flounder at the in occurrences to negligible levels. However, the fishinarket, this isbecoming a probleinfor the cost percentage appearance of albinism increased performance of the stock enhancement program because of the outbreak of larval diseases Furusawa 1997!. Takahashi 1992! or genetic background of spawners Tabata 1991!. There are still unresolved Morphological abnormalities and prevention factors in relation to this abnormality. Japaneseflounder never show lordosislike red sea breambecause this speciesdoes not have On the other hand, nearly all cultured fish an air bladder. Artificially produced juvenile show hypermelanosis on the blind side. Stocking flounder showa high percentage in occurrence of density and the type of feeds during larval stage, vertebral abnormalitiessuch asfusion, deformation, light irradiationduring juvenile stage,effects of and compression Iseda et al. 1978, Seikai 1979!. ocular sidepigmentation abnormality, and source Recently, the percentage of occurrence has of examined fish wild or hatchery-reared! were increasedfrom the past. Onereason for this was suggestedas the factors for this abnorinality Seikai suggestedto be thee5ect of enrichmentto live food 1991, Takahashi 1992!. Recently, it was revealed organisms with fat-soluble vitamins to reduce thatthe effect of substratuin wasthe most important albinism. It was revealed that the excessive la UJNR ~ ReportNo. Js Exp. lots Feed Control +Lecithin 1.5% +VE G 1% Female MeanBW' no.! 1,388 0! 1,389 0! 1,391 0! Male MeanBW no,! 807 80! 814 80! 813 80! Spawning 990! Duration 13Mar-8 Aug 13Mar-2Aug 20Mar-2Aug Frequency 93 90 88 Total eggs x1,000! 35,721 69,620 59,038 Floatmgrate %! 46.7 49.8 48.7 Fertilizationrate %! 54,0 48.G 38.0 Eggdiameter rnm! 0.88 0.88 Hatchingrate lo! 81.4 88.3 86.7 Deformationrate %! 41.4 42.9 50.3 50.6 35,1 35.3 'Bodyweight g! ASSAI survival activity index! = ~ N-hi!i/N i=1 to k!; N =initial number of larvae,hi = accumulatedmortaHty atday i, k = dayof no survivor modifiedRom Wakui and Otaki 1991!, Tab4 J. Kxpaiazatoffomula kal farspaimers administrationof vitamin A throughenriched live abilities.Earlier seed production which can realize foodduring the specialperiod when ossification earlierreleasing to avoidpredator and crush of vertebraeadvances induced the high percentage of mysidsin the nursery ground is conducted.At the occurrenceof this abnormality Dedi et al, 1995, sametime, hatcheries aretrying to produce juveniles Takeuchiet al. 1995,Dedi et al, 1997!, Other whichhave a competitiveedge over wild fish. For factorssuch as genetic effects in relationto this thispurpose, juveniles were raised to the releasing abnorinalityare expected Y. HondoMiyazu size to acclimateto the wild environmentin the Station,JASFA, personal communication!. enclosures Furuta 1993!. Under such environment, juvenileflounder approximate their behavior and Laborand cost savings chemicalcompositions closely to that of wildfish. Simplificationof operationand labor savingsare essential factors for the expansionof Outbreakof diseases and countermeasures Table quantiticsin production.ln many cases, 2! simplificationof thefeeding schedule, active use Diseasesoflarvae, juveniles, and spawners of formulafeed, introduction of autoinatic feeders, occurduring the seed production process Muroga andcleaning robots could bring success to some 1992!, Bacterialdiseases for juvenilesinclude extent.Labor savings with automization sotnetimes infectionswith Flexibacterrnaritirnus, Vibrio backfireto increase the running cost and the risk anguillarurn,and Kdwardsiellatarda. The latter whichproduce poor qua1ity juveniles because of twocan infect the young and spawners. Protozoan disregardfor speciesand developmental stage- diseasesare caused by infections with Ichthyobodo specificcharacters. There is a conceptthat large sp, Cryptocaryon irritans, and Scuticociliatida rearingtanks are considered assmall ecosystetns, gen,sp, Bacterialdiseases of larvaeand ju veniles whereself-discipline oforganistns intearing tanks are bacterial enteritis infected by Vibrio isattained and reduction indaily work is possible, ichthyoenteri.Viral diseasesare epidermal called"Hottoke-Siiku" Hottoke means 'let' and hyperplasiaand viral nervousnecrosis VNN!. Shiiku'grow'! Takahashi1990!. Thesediseases during the larva1stage cause extretnely high tnortality of over 90%, The guality improvementfor releasing safeguardsagainst diseases ate ! noinlroduction Mostof the juveniles produced in public of pathogens, ! maintenance clean environments, hatcheriesarereleased inthe coastal areas of Japan. ! enhancementof live food activities, ! Thceffects which are extremely different between sterilizationof tanksand tools, and ! utilization areasare dependenton timingand location of of UV-treatedseawater for rearing.Frequent releasing,and the qualityof juveniles.Different outbreaksof viraldiseases during seed production behavioralpatterns and chemical compositions in causedserious pmblems for thestock enhancement theartificially ptoduced juveniles froin wild fish project. As viral-infectedfish e.g.,VNN! are areexpected as the reasons for higherpredation impossibletotreat, we inn st destroy all juvenilesif mortality Furuta 1996!. lt was revealedthat the producedfish have had an attack of viral releasedsizes larger than 8 cmTL couldpromise disease. goodsurvival and recapture because flounder juvenileshave a relativelyhigher hierarchy in the Biotechnologyin seed production nurseryground ecosystem Yamashta et al. 1994!. Japaneseflounder is one of the most As the cost for the productionincreases advancedspecies in relationto biotechnological logarithmicallyas fish grow larger in land-based studies Tabata 1991, Yamamoto1995!. As tanks,it is desirableto producejuveniles with femalescan grow faster than inales in this species, strongerpotential for survival at smaller sizesthan chromosomemanipulation was attempted to 8 cm, Theconcept ofthe quality of juveniles for produceall fetnales by gynogenetic diploid. During releasingis well accepted Tsukamoto 1993!, but suchattempts, phenotypic expression ofsex in this the standardfor this conceptis now under specieswas found to be determined by genetic and investigationusing feeding behavior, and burrowing environmentalfactors e.g., temperature, diet, and l2 UJNR TechnicalRepast No. 26 Disease Pathogen Note Viral Epidermal hyperplasia Flounderherpesvirus 10-25days after hatching FHV! high mortality Viral nervousnecrosis Stripedjack nervous larvaeand necrosisvirus juveniles, SMA'! high Inortality Birnaviraldisease Hiraxnebimavirus 1-2.4g juveniles Hiralne rhabdoviral Hirame rhabdovirus disease MRRV! Bacterial Streptococcicosis Streptococcusiniae juveniles,adult Vibriosis Vibrio arrgtcilIarum juveniles Bacterialenteritis Vibrioichthyoertteri larvae Gliding bacterial Flexibactermari timus juveniles disease Edvrardsiellosis Ekeardsiellatarda juveniles,adult ProtozoanIchthyobodosis Ichthyobodosp. Whitespot disease Cryptocaryortirritans juveniles scuticociliatidosis Scltticoriliatida gen. sp. ParasiticSkin fluke disease Neoberledeniagirellae juveniles Tabte2. Di.casesofJapanese flounder during seed production andculture stockingdensity!. Several cloned progeny which Fujiiand Nishida 1996!. To resolve this problem, couldbe produced by usingthese technologies have we shouldreconsider egg-taking methods, for strongpossibilities to produceeffective strainsfor instancestripping and artificia] fertilization of ma]es aquaculture purposesfor shorter than usual andfemales. Recently, therole of hormones in eggs breeding procedures Yamamoto ] 997!. has attracted attention, and the effects of maternal Biotechologicallytreated fish are impossib]e to use hormoneson thc survival after hatching have been for releasing;however, such strains with high revealedin differentspecies. The relationship growth potentialor resistanceability to diseases betweenegg qualities including matcrna] hormones are usefulfor aquaculture Yarnamoto ] 997!. andsurvival and development should be one of the topicsto concentrate on in futureefforts Tagawa CONCLUSION AND REMAINING ] 997!. PROBLEMS In thecase of Japaneseflounder, relatively largeamounts of biologicalin formation have been Juvenilesof Japanesef]ounder are now accumu]ated.Recently, a wel] organizedreview producedin massscale at many public hatcheries. on biologyand stock enhancement of Japanese Suchrapid expansion in quantitypresents many flounderwas published from the viewpointsof relatedproblems, and if we cannotresolve these ecologicalaspects Minarni 1997, Nishidact al. problemstogether, it will havea negativeimpact 1997,Noichi 1997, Tanaka et al, ]997!,ecology on the trials of the Japaneseflounder stock and physiologyof metamorphosis Seikai ] 997, enhancementproject. Tanaka 1997, Yamano ]997!, and stock Firstof al], it is necessaryto stabilize enhancement Takeuchi ]997, Yarnamoto1997, furtherthe mass st production Furusawa ] 997!. Yamashita]997!. Recenttopics on the biology of If theobject of production isnot achieved, problem Japaneseflounder include the role of thyroid situationscould occur frequently, such as the hormonesregulating metamorphosis Yamano introductionof fertilized eggs or produced juveniles ]997!, and sex determination mechanisms froin otherareas. Thesebring more serious dependingon geneticsand environments Tabata concernstofish disease and genetic problems. The 1991,Yatruunoto 1995!. Basicinforination on the concernsinclude: ]! eggqua]ity improvement, ! bio]ogycan solidify the foundationof the seed clarificationofnutritional demand, ! development productiontechnology and stock enhancement, offormula feed, ! automizationandlabor savings, ! reconsiderationofbiological funcnon including LITERATURE CITED foodorganisms, and ! preventionof fishdisease. Regardingdisease, the amounts of'restocking to Dedi, J T. Takeuchi,T. Seikai,and T. Watanabe. thecoastal area have already attained the levelsof 1995 Hypervitaminosisand safe levels naturalresources in Japan,and we shouldestablish of vitamin A for larval flounder a bettersystetn to protectthe diversity of carrier Paralichrhys olivaceus! fed Arremia fish to the wi]d environment. nauplii. Aquaculture133: 135-146. Thenext step is to improvethe quality of Dedi, J., T Takeuchi,T. Seikai,T. %atanabe,and juvenileswhich ba]ance with the stock enhancement K. Hosoya 1997. Hypervitaminosisa re]easeseconoinicaJl y Tsukamoto 1993!. For this, duringvertebral morphogenesis in larval the tnost important is the feedback from the Japaneseflounder. Fish. Sci. 63:466-473. ecologicaland fisheries resource surveys after Fujii, T. and M, Nishida. 1996, Heriditical releasing.In addition,produced juveniles for stock diversityin hatchery-rearedjuvenile enhancementshould maintain the local genetic flounderanalyzedfrom the basic sequence informationandtheir diversity Nishida etal. 1997!, in D-roop regionof mt-DNA. Abstract. Recent mt-DNA analyses revealed that Autumnmeet, Jpn. Fish. Sci. Soc, 92 p. unexpectedlyfewma]es and females compare with Pa Japanese], thenumber of stockedspawners inthe spawning Fujita,T. 1903.Artificial fertihzation and hatching tankto the mating behavior of 1-dayegg batches of Japaneseflounder. Zool. Mag. 15f ]79!: 14 UJNRTectudad Report No. 26 316-322, [In Japanese], S Tabata.1978 Experimentof Japanese Furusawa,T. 1997. Key problemsof sea-farming flounderseed production, II. Therehition associatedwith its perspective,pp. 117- betweenfeed and albinism,and ossification 126. In. T, Minaini, and M. Tanaka eds,!, stageand the deformation. Annu. Rep, Biology and Stock Enhancementof Showa 51st KumamotoPref. Fish, Exp. JapaneseFlounder. Koseishakoseikaku, Sta., pp. 257-261. [In Japanese]. Tokyo. [In Japanese]. Iwata,N., and K. Kikuchi,1998. Effectsof sandy Furuta,S. 1993. Releasingtechniques and fry substrateand light on hypcrinelanosisof quality,pp. 94-101, In: C. Kitajima ed.!, thcblind side in culturedJapanese flounder Healthy Fry for Release, and Their Paralichthys oli vaceas. Environmental Production Tech n iques. Biology of Fishes.52; 291-297. Koseishakoseikaku,Tokyo. [In Japanese], Kanazawa, A. 1990. Nutrition and feed in Hirarne FurutaS. 1996. Predationon juvenile Japanese culture. Suisannzousyoku 38: 398-399.[Iii flounder Paralichthys olivacetts! by Japanese] diurnalpisci vorous fish: fieldobservations Kanazawa, A. 1993. Nutritional mechanism andlaboratory experiments, pp. 285-294. involved in the occurrence of abnormal In: Y. Watanabe, Y. Yamashita, and Y. piginentationin hatchery-iearedflatfish. J, Ozeki eds.!,Survival Strategiesin Early World Aquacult.Soc. 24: 162-166. Life Stagesof Marine Resources.A,A. Kitajiina,C., G. Hayashida,M. Shimozaki,and T. Balkema, Rotterdam. Watanabe 1985. Reductive effect of Harada,T. 1980. Presentstate and problems in micro-binded diet on occurrence of color Japaneseflounder culture. Yoshoku 17 !: anomaly in hatchery-rearedflounder, 48-53. [In Japanese]. Paralichthysoli vaceus, Bull. Nagasaki Harada, T., S. Umeda, O. Murata, H. Kumai, and Pref. Inst, Fish. 11: 29-35. [In Japanese K. Mizuno. 1966. Rearingand growth of with Englishsummary]. Japaneseflounder larvae froin artificially Miki, N T. Taniguchi,and H. Hainakawa,1988. fertilizedeggs. Rep. Fish. Res. Sta., Kinki Effect of rotifer enriched with fat-soluble Univ. I! 28: 9-303. [In Japanese]. vitamins on occurrence of albinism in Hirarnoto,Y., N. Miki, andK. Kobayashi.1981a. hatchery-reared"Hirame" Paraii chchys Studieson the Japaneseflounder seed oli v accus preliminary report!. Suisan production-lll, Rearing on fish meat Zoshoku36: 91-96. [In Japanese]. during juvenile period TL 15-50min!. Miki, N., T. Taniguchi,and H. Hamakawa,1989. SaibaiGiken N !: 89-103. [In Japanese]. Adequatevitamin level for reductionof Hiramoto,Y., N. Miki, andK. Kobayashi,1981b. albinism in hatchery-reared Hirame Eggcollection from naturalspawning and Paralichthysolivaceous fed on rotifer larvaLhatching in Japaneseflounder. Rep. enrichedwith fat-soluble vitamins. Suisan Tottori Pref. Fish. Sta. 23: 7-12 . [In Zoshoku37: 109-114. [In Japanese]. Japanese]. Minaini, T. 1997. Life history,pp.9-24. In: T. Hirano, R. and E. Yanarnoto. 1992. Spawning Minarni andM. Tanaka eds,!,Biology and rhythinand eggnumber at the iridividual StockEnhancement of JapaneseFlounder. femalespawning experiment in the hiraine Koseishakoseikaku,Tokyo. [In Japanese]. flounder,Paralichthys oli vaceas, Rep. Morimoto,H. 1994 Eggquality, pp, 83-96. In: Tottori Pref. Sta, Fish, 33: 18-28. [In M . Tanaka and Y. Watanabe eds.!, Studies Japanesewith Englishsummary ]. on Early Life Mortality of Fishes. Ijima, T., T. Abe, R. Hirakawa,and Y. Torishima. Koseishakoseikaku,Tokyo. [In Japanese]. 1986. Inducedspawning with long day Muroga,K. 1992,Hatchery diseases of marine treatmentin Japaneseflounder, Saibai fish in Japan,pp. 215-222. In- I. M. Giken 15 !: 57-62. [In Japanese!, Shariff,R, P.Subasinghe, and J, R Arthur Iseda, H., S. Sumida, M. Ishihara, M. Owaki, and eds.!,Diseases in AsianAquaculture. SdiLai la Fish Health Section, Asian Fisheries hormonesin carlydcvclopmeni of tclcosis Society,Manila, phil ippincs. NiPPonSuisan Gakkaishi 63 ! 498 501 Nishida,M., T. Ohkawa,and T. Fujii. 1997. [ln Japanese], populationstructure, pp. 41-51. In: T. Takahashi, V. 1990, Reduction of thc amount of Minarniand M. Tanaka eds.!, Biology and foodorganisms supplied and simplification StockEnhancement of JapaneseFlounder. of rearingoperations in thcseedling of thc Koseishakoseikaku,Tokyo, [In Japanese]. 3apaneseflounder. Suisan Zoshooku Noichi, T. 1997. Early life history,pp 25-40. In: 38 l!: 23-33. [In Japanesewith English T. Minami andM. Tanaka eds,!,Biology ~ummary]. and Stock Enhancementof Japanese Takahashi,Y. 1992. Appearanceand prevention Flounder,Koseishakoseikaku, Tokyo. [In of abnormallypigmented fish in Japanese Japanese]. flounder seedproduction. JapanSca- Seikai, T. 1979. Studies on the abnormality of FarmingAssoc. Spec. Rep, 3: 1-58, [ln vertebraeand scalesin companywith the J apanese]. occurrence of abnormal coloration in the Takahashi,K Y. Hayakawa,D. Ogura.and H. juvenile and young of hatchery-reared Nakanishi, 1980. Egg collectionfrom flounder,Paralichrhys olivaceus. Bull. natural tank spawning in 3apanesc NagasakiPref. Inst, Fish. !: 19-25. [h flounder. Saibai Giken 9: 41-46, [ln 3apanesewith Englishsummary]. Japanese!. Seikai,T. 1985. Influenceof feedingperiods of Takeuchi,T. 1997. Nutritionalrequirement for Brazilian A rrernia during larval improvementof rearingseed production, developmentof hatchery-rearedHounder pp.96-106,In; T. Minamiand M. Tanaka ParalichtIiys oli vaceuson the appearance eds.!,Biology and Stock Fnhancement of ofalbinism. Nippon Suisan Gakkaishi 51: JapaneseHounder. Koseishakoseikaku, 521-527. Tokyo. [In Japanese]. Seikai,T. 1991. Influenceof fluorescentlight Takeuchi,T., J. Dedi, C. Ebisawa.T. Watanabe, irradiation,ocular side pigmentation, and T. Seikai, K.Hosoya, and J. Nakazoc, source of fishes on the blind side 1995. The effect of 8-carotene «nd vitamin pigmentationin the youngJapanese A enriched Arremia nauplii on the flounder, Paralichrhys ali vaceus. malformation and color abnormality of Suisanzoshoku 39 !; 173-180. [In larvalJapanese flounder. Fish. Sci. 61 I !: Japanesew ithEnglish sununary]. 141-148. Seikai, T, 1997. Mechanismof abnormal Tanaka,M, 1997. Ecologicalsignificance of pigmentation,pp.63-73. IrL T. Minami metamorphosis,pp,52-62, In: T. Minami and M. Tanaka eds.!,Biology and Stock andM, Tanaka eds.!,Biology and Stock Enhancement of JapaneseFlounder. Enhancementof 3apaneseFlounder. Koseishakoseikaku,Tokyo, [In Japanese]. Koseishakoseikaku,Tokyo. [In Japanese]. Seikai,T., T. Watanabe,and M. Shimozaki.1987, Tanaka, M., T. Minanu, and Y, Koshiisi. 1997. Influenceof threegeographically different Researchdirection, pp 127-130. In: T. strainsof Artemia naupliion occurrence Minamiand M. Tanaka eds,!. Biology and of albinismin hatchery-rearedflounder StockEnhancement of JapaneseFlounder. ParaIichthysoii vaceus. Nippon Suisan Koseishakoseikaku,Tokyo. [In Japanese]. Gakkaishi 53: 195-200. Tange,K. andK. NagahatruL1986. Secondary Tahata,K, 1991.AppIication of chromosomal phaseof flounderjuvenile rearing fed on manipulationin aquaculture of hirame formula feed, Saibai Giken 15 I !: 63-71, Paralichrhysolivaceas. Bull. Hyogo Pref. [In Japanese]. Fish.Exp. Sta. 28: 1-134.[In Japanese Torii, S., Y. Shiokawa,Y. Hayakawa,S, Siota,K. withEnglish suinmary]. Igarashi,Y, Uji, K Sato,A,lto, H. Wakui, Tagawa,M, 1997.Involvement of thyroid M. Kawanobe, A. Iwamoto, and T. Fukunaga.1994. Corpusof hirarneseed productiontechnologies in Notth Pacific Area.Japan Sea-Farming Assoc Tokyo, pp. 87. [In Japanese]. Tsukarnoto,K, 1993. Fry quality,pp. 102-113. In. C. Kitajima ed.!, HealthyFry for Release,and Their Production Techniques. Koseishakoseikaku,Tokyo, [In Japanese]. Wakui, H. and K, Otaki. 1991. Resultsof the experimentfor thedevelopment of formula diet for Hirarnebrood stock, Heisei3rd Annu. Rep. SeedlingInst. Fukushiina Pref.,pp. 57-79. [In Japanese]. Yarnarnoto,E. 1995, Studies on sex-manipulation andproduction of clonedpopulations in Hirameflounder, Paralichthys ohvaceus Temminckand Schlegel!, Bulk Tottori Pref. Fish,Exp. Sta. 4!: 1-145. [In Japanesewith English abstract]. Yamarnoto,E, 1997.Biotechnology, pp.83-95, In: T. Minami and M. Tanaka eds.!, Biology and Stock Enhancement of JapaneseHounder. Koseishakoseikaku, Tokyo. [In Japanese]. Yamano,K. 1997.Mechanism ofmetamorphosis, pp. 74-82. In: T. Minarni and M. Tanaka eds.!,Biology and Stock Enhancement of JapaneseHounder. Koseishakoseikaku, Tokyo.[In Japanese]. Yamashita,Y. 1997. Ecologyand releasing techniques,pp. 107-116.Irt: T. Minami andM. Tanaka eds,!, Biology and Stock Enhancementof JapaneseFlounder. Koseishakoseikaku,Tokyo, [In Japanese]. Yamashita,Y S. Nagahora,H. Yarnada,and D, Kitagawa 1994. Effectsof releasesize on survivaland growthof Japanese fl ounder Parali chthys oli vacms in coastal watersoff I watePrefecture, northeastern Japan.Mar, Ecol, Pmg.Ser, 105:269- 276. Rtrkards SUSTAINABLEFLOUNDER CULTURE AND FISHERIES: A REGIONAL APPROACHINVOLVING RHODE ISLANDs NEW HAMPSHIRE, VIR- GINIA,NORTH CAROLINA, AND SOUTH CAROLINA William L, Rickards VirginiaSea Grant College Program 170Rugby Road Charlottesville,VA 22903 c-mail:[email protected]! A8STRACT !nrerestincu!turing flounders along the east coast of thet!orred States has increased gready due to reduced fisherylandings andincreased cvnsumer demand. Three flounder spec!es arebeing evaluated foraquaculture developmentbyresearch teams from several states. Through region-wrde meeungs, theseresearchers have sharedinformation regarding progress andprovided mput to a regiona!p!ancontainmg future research needs andprioriues re!ated toflounder cu!lure and stock enhancement. Theresulting strategy wi!! contmue toser~e asblueprint for an integrated approach toresearch andoutreach activities su~ bySea Grant in rhe comingyears. INTRODUCTION federalgovernment, primarily through the National Oceanicand Atmospheric Admiru strati on NOAA! This paperpresents a brief overviewof withinthe U.S, Department of Conunerce. Within recentplanning and research developrnen!.s concem- eachlocal Sea Grant program are research, educa- ing flounderson the Atlanticcoast of the United tion,and outreach/extension projects related to States.The author is notdirectly involved in any variousaspects of marineand coastal resources, of theresearch described; rather, he is oneof thc includingaquaculture, that are important to that SeaGrant program administrators who has facili- state, tated the interactions and collaborationsthat are Of particularrelevance to thetopic of this beingdeveloped, The following, then, is a descrip- paperare the flounder aquacuhure projects sup- tionof theemergence and content of a multi-state portedthrough the local Sea Grant programs in complexof SeaGrant-supported research in ovari- New Hampshire,Rhode Island, Virginia, North ousaspects of flounderaquaculture focusing on Carolina, and SouthCarolina, threedifferent species The regional plan Sea Grant and the national network In mid-1 996,a groupof flounderculture Within the structure of the National Sea researchers,extension leaders, industry represen- GrantCollege Program, there are state-based pro- tatives,and Sea Grant program administrators met gratnadtninistrative units that are usually located to discussthe statusof flounder culture activities at universities.Together, these so-called local Sea in thestates noted above and to producea docu- Grantprograms form the nationalSea Grant net- rnentsetting forth a strategyfor researchand ex- work,and there is a localprogram in eachcoastal tensionactivities that will provide the technical foun- andGreat Lakes state, including Hawaii and Puerto dationupon which comnerciai flounder culture will Rico. The local SeaGrant progratns provide the beable to furtherdevelop and prosper along the focal points for developing partnerships be- U.S.eas coast. The resulting strategic plan for tween the local universities, state agenciesand "SustainableFlounder Culture and Fisheries" Wa- ters1996! provides an agenda for focused, inte- ductingstudies on larval survival and growth in the gratedpublic and private investment in research, winterflounde Pleuronectesarrtericanuswith the facilitydevelopment, andeducation byrecommend- ultiinategoal of assistingefforts in wild stocken- ingpriority actions in six key areas: hancernent, 1. Hatcheryand reproduction technology Proceedingsouthward along the coast, the 2. Productionand culture systems nextstate which is participatingin this regional 3. Stock enhancement activityis Virginia. Discussion of flounder culture 4, Economicfeasibility and marketing activitiesin Virginiais presented in a latersection 5, Policyand regulation of thispaper. 6. Education and outreach In both North Carolinaand South Caro- Spacedoes not permit going into the specific pri- lina,the species ofprimary interest isthe southern oritiesidentified and discussed in thedocument, flounderParali ch rhys lethosti gma, Later sessions buta copycan be obtained by writingto North of themeeting included a presentation on South CarolinaSea Grant, Box 8605, North Carolina State Carolinastudies ofpond ploduction, including con- University,Raleigh, NC 27695-8605, USA, trolledreproduction, tank, and pond nursery sys- Thisstrategy is now serving as the basic ternsas well as grow-out tomarket size in this spe- justificationfor researchers from the states noted, cies.The tri-state research team for these studies aswell as prospective researchers from other Sea includescollaborators froln Rhode Island and North Grantprograms, todevelop project proposals for Carolina, supportwithin their respective states and for such Studiesin NorthCarolina are focusing on emergingprojects tobe linked and integrated with southernflounder reproduction, in collaboration otherflounder culture research within the overall withinvestigators from South Carolina, upon nurs- SeaGrant network. It is the intentionof the Sea eryrearing of larvaeand flngerlings, and upon pond Grantnetwork todevelop aninter-related sequence productionof food-sizedfish. In addition,research ofresearch and education projects that will leadto isunderway todeternune the ecological dynamics commercial flounder cu1.ture. of stockenhancement using southern flounder throughassessment of food habits, survival rates, Currentflounder culture within Sea Grant releasetiming optimization, and habitat availabil- Severalflounder culture studies are on- ity. Thenext generation of Sea Grant projects in goinginthe New England states, butthese will only NorthCarolina will examine the possibility of cre- bementioned here because they were presented in atingan all female population offingerlings, con- detaiiin other parts of the UJNR meeting agenda trollingegg quality in broodstock, rearing condi- In themeeting's first presentation, George tionsfor larvae, pond pmduction offlingerlings, and Nardidescribed the commercial success that his optimizedgrow-out conditions forproducing food company,GreatBay Aquafarrns, ishaving with the fish.Several of thcstudies noted thus far are sup- production of juvenile summer flounder portedwith fundingfrom sources in additionto Sea Paralichrhysderttarus. Much of the technology Grant,and there are non-Sea Grant flounder cul- usedhas been derived from New Hampshire Sea tureprojects that are not included inthis summary Grantstudies conducted several years ago. becausetheir justification forfunding isnot tied to In a latersession ofthis meeting, speakers theSea Grant strategic plan noted previously. describedrecent studies atthe University of New However,the results of all studies,regardless of Hampshireonthe effects of stockingdensity on fundingsupport, will beintegrated through outreach/ extensionactivities to assistwith thecomrnercial- larvaland juvenile growth and survival in sununer ization of flounder culture. flounder. In addition,researchers at the University Flonjtderculture in Virginia of RhodeIsland presented production economics Virginiais a relativelyrecent entrant to the resultingfrom their studies of summerflounder fieldof flounder culture, but the reduced landings grow-out. The sameresearch team is also con- ofwild-harvest flounder, increasing market demand, Richards Iii andrecognition ofthe potential toapply recirculat- 1315East West Highway, Silver Spring, MD 209l 0- ingculture system technology toHounder has gen- 3282.USA e-mail:Jim,[email protected]!. eratedconsiderable interest in ouracademic and businesscommunities. LITERATURE CITED In 1996,researchers atthe Vtrginia Insti- tuteof MarineScience constructed recirculating Waters,E,B. 1996. Sustainableflounder culture systemsand purchased summer Hounder juveniles and fisheries. North Carolina Sca Grani fromGreatBay Aquafarms, Initial activity focused Publ.UNC-SG-96-14. I pp. North onsystem optimization and preliminary grow-out, CarolinaState Univ Raleigh, NC.! feeding,survival, and water quality rnaintenancc inthe recirculating systems. In addition,these stud- iesencountered significant disease outbreaks that resultedinconsiderable mortality among the juve- niles in culture. In 1997,the development ofgrow-out pro- tocolsfor usein recirculatingsystems has contin- ued;and, as a resultof the 1996work, an inter- institutionaland inter-disciplinary team has been forinedto investigatediseases that occur in cul- turedsummer flounder, This group includes ex- pertisein parasitic and non-infectious disease pa- thology,bacteriology, virology, irnrnunology and therapeutantapplication, and water quality rnoni- toring.Through Sea Grant project seed money in 1997,this group is developinga fundamental un- derstandingof the water quality needs of summer flounder,the variety of pathogenicconditions that affectthe speciesin culture,and the infornMtion neededfor rapid and accurate diagnosis of disease conditions. Our flounderdiscase group is also func- tioningas a focalpoint for diseaseissues that arise at otherflounder culture projects within the Sea Grantnetwork. In otherwords, this is etnergingas a cross-cuttingproject notonly within Virginia's flounderresearch, but it alsoprovides the same function in concert with other flounder research within the Sea Grant network. This flounderdisease research and diag- nosticsactivity will continueas a seed effort through1999 and is expectedto develop into a se- quenceof f'ully-supportedSea Grant projects as of 2000. In themeantime, others encountering floun- derdisease problems are invited to establishcol- laborationand cooperation with the Virginia Sea Grant research team. Names and addresses of the team membersare readilyavailable through eitherthe Virginia Sea Grant College Program or theNational Sea Grant College Program, NOAA, Smith tt TANK AND PONDNURSERY PRODUCTION OF JUVENILE SOUTHERNFLOUNDER PARALICHTHYS I ETHOSTIGMA! Theodore I J. Smith SC Department of Natural Resources P. O. Box 12559 Charleston, SC 29422 843762-5047 phone! 843 762-5110 FAX! e-Ittail: [email protected],ug and Wa!laceE. Jenkins,and Michael R. Denson SouthCaro! ina Departmentof NaturalResources P,O. Box 12559 Charleston, SC 29422 USA ABSTRACT InSouth Carnhnx, studies have been conducted todevelop nursery techniques forsouthern flounder Parrdictuhys Iet/tosrigmawhich could be used for aquaculture developine m andforstock enhancemem programs Wild caught adultswere tank-conditioned andspawned using hormone iinplants. Larvae were stocked intanks at6 daysof ageand used ina two-pait study. Three diet treatments weretested which included feeding rotifers and Art rmia naupliiwith and without a commercial larval dier suppleinent, The second part of thestudy exanuned theetfect ofrearing larvae under two light intensities low -457 lux, high 1362lux}. At 24'C,metamorphosis began on day23 and wss completed byday 30. Analysisof survival,size. and pigmentation data at compleuonof metamorphosisindicated there were no significant diffetences among feed ~ts orhght treatments. Overall survivalwas 33,8% and mean length was 11.5 mm TL . However,only 30% of thelarvae were normaliv pigmented, Developmentof pond nursery systetns was examined during l995-l997. Ttuee O. I-ha ponds at the Waddeli MaricuttureCenter were stocked with 3 to 5-day-oldlarvae. Stocking densities ranged from to 2g4,760 to 740,000fry/ha and fish were harvested after 2'/i to 7 months.Survival was low and ranged from 3S to 6.I % incan 4.5%!. However,approximately 99% of the fish hadnormal pigmentation, Results indicated that southernflounder are tolerant of a rangeof environmentalconditions and that pond systems may be useful in seasonalproduction ofjuveniles. Two short-terin salinity tolerance tests were conducted. In a 72-bstudy, fish whichhad recently metamorphosed I 3.7 mm TL, 50 days old! exhbited low survival ai0 g/L salinity 6-20%! whitethose exposed to 5-30g/L had a meansurvi val of 99.1%.A 2-wkstudy using older juveniles 95 2 rnmTL 220days old! showed that they could tolerate salinities of 0-10 g/L 00% survival!. Thus, salinity tolerance increaseswith ay.. Twoweaning studies were conducted with pond-produced juvenfles. Younger juvemies 7 mm TL, 77 daysold! were uansfcrredto dry dietsover a 2-wk periodwith over 80%survivaI while older juveniles 95 mmTL, 220 daysold! required l06 daysto weanand exhibited a 58% survivaLResults of thc variousnursery trials and related studies suggest that mass production of southern IIounder juveniles should be possible with refinements to current techniques, INTRODUCTION Stnigielski 1975,Arnold et al. 1977,Brisbaj and Bengston ! 993,Daniels et al. 1996!. !n 1996,the Along theeast and gulf coastsof the United NationalSea Grant College Program convened a States, there is growing interest in producing 'Task Force on Hounder Culture and Stock flatfishes for food and for stock enhancement Enhancement,' The resulting report, which t'JNR TeebnieatReport Nts. Xs includedinputs from Sea Grant directors, scientists, adultsinay remain within the estuaryall year,most extensionspecialists, and industry representatives, adultsmigrate offshore to spawnin late fall and suininarizedthe research,outreach and policy winter Ginsberg 1952!. Additionalinformation needsfor successfulflounder culture and possible onthe life historyand ecology of southernflounder stock enhancement of U.S. flounder stocks throughoutits range is providedby Powefl and Waters 1996!. Schwartz 977, 1979!, Stokes 977!, Music and The SouthCarol ina Department of Natural Pafford 984!, and Wenner ct al, 990!. The Resources SCDNR! initiated culture researchon southernflounder can attain a sizeof 9 kg, making the locally occurring southern flounder it thelargest bothid inhabiting inshore waters along ParaIicItrhys Ierhosrigmain 1994. This is a the southAtlantic and Gulf of Mexico. As such, it euryhalinespecies well knownin seafoodmarkets has substantial commercial and recreational and in anglers'creels Wenneret al. 1990!, The importance. Landings data for the southern southern flounder inhabits coastal waters from flounderare difficult to obtain as this speciesis Albemarle Sound, North Carolina, throughthe commonlylanded and reported with the sympatric southAtlantic states to CorpusChristi Pass, Texas suminerflounder P.dentartts, and the gulf flounder Ginsburg 1952!. From spring through faH, P. albigurra. In South Carolina, there are no southernflounder typicafly inhabitcoastal bays, focusedflounder fisheries.Instead, landings occur sounds,and river systems Ginsburg 1952, Gutherz priinarily associatedwith shrimp trawlers as 1967! and are most abundant in the rnid- to upper bycatchwith mostof the fish providedto the vessel's estuarine areas with occasional movement imo crew a.spart of their wages. Thesefish are then freshwater Dahlberg 1972!. They prefer silt and sold or consumedby thecrew. In SouthCarolina, organic mud substrates Powell and Schwartz flatfishlandings are relatively low, but arealmost 1977!. In South Carolina, southern floundcr are exclusively southernflounder J. Moran, SCDNR commonlylocated in shallowtidal flats, shellbanks, Fisheries Statistics Section, personal and aroundpi lings Bearden 1961!, Although some communication!. During 1991-1996, landings 30000 20000 0 19781980 1982 1984 1986 l988 1990 1992 1994 1996 Hgure 1. Reportedlandings of flounder primarily southern flounder! in SouthCarolina during 1 978-1996, selia z! averaged6,238 kg which is substantiagy lower than overall survivalwas hiw and there was a high in previousyears Fig, I!. Thismay be ducto percentageof larvae that did not eornplcte severalfactors including data col lection difficulties. metamorphosis,Thus. additional rc»earchis increa~ein the legalminimum size 0 cmTL!, heededto developthc culture tcchilologv nccc»sary and a decline in abundance. for massproduction of juvenile». Similarly, Market pricesfor cast coastflounders informationis lackingon weaningtechniques io reflectseasonal abundance, with wholesale prices transferjuveniles to dry rations far rearingin market inthe range of $3.85-5.50/kg NMFS 1995!, Retail s iTA'.. pricesin theorder of $22.00/kgare received for Basedin parton the aboveinformation. filleted products. Due to their benthic and thc southernflounder appear» to havedesirahlc gregariousbehavior, and resistance to handling aquaculturecharacteristics, Reported growth rate» stress,flounders appear to beexcellent candidates of wild fish in South Carolina indicate that fcniale» for live sales, In fact, live east coastflounders are attaina meansize of about0,9 kg at 2 yr of age regularlysold to upper-scaleJapanese restaurants Wenncret al, l 990!, However,»mall juvenile» in theNortheast where they command a premium recruitedto coastalimpoundments containing price -$15/kg! andarc also shipped to Tokyo abundantfood may suhstantiall y exceed this growth wherethey are valuedat $45-60/kg Ackerman rate. Fisheriesdata suggestthat this specie»i» 1997!. In additionto the wild fisheriesstudies, regularly captured in freshwater and nearly there has been some culture research. Much of freshwaterenvironments and, thus, there iriay bc this researchhas been focused on identifying opportunityto grow this speciesin inland and spawning techniques, Arnold et al, 977! coastalsites as is donewith theeuryhaline hybrid successfullytank-spawned 3 of 6 femalesusing stripedbass Morone saxatiiisx M. chrvsops! photothermalconditioning «nd produced 120,000 Smith and Jenkins 1996, Smith ei al, 19961. An eggswith fertilizationranging from 30-50%. In overview of rcscarchis provided focused on the anothertrial using carp pituitary extract.only developmentof suitablenursery systems; results 25.000eggswith 80%fertility were produced from to dateon tank studiesand pond trials as well as 14 females Lasswetl et ai. 1978!. Tcnyears later, informationon techniquesfor weaningfish to a similar attempt using luteinizing hormone- formulated rations. In addition, results of short- releasinghormone analog LHRHa! implanlswas tcrm studiesfacused on elucidatingthc saliniiy unsuccessful,apparently due to lack of male tolerancesof smalljuveniles are included. participation Henderson-Arzapalo et al. 1988!. However, in 1995 successwas obtainedusing MATERlALS AND METHODS gonadotropichormone-releasing hormone analog GnRHa irnplantsto inducefinal maturationin 12 General photothermallyconditioned females with oocytes Larvae usedduring the variousnursery %00 pm Bcrlinskyet al. 1996!, Thesefish were trialswere obtained from captive wild adultswhich manuallystripped and a total of 1.6 x I0' eggs hadbeen photothcrmally conditioned for at least6 batchfertility 7-95%! was obtained. During 1997, monthsprior to spawning.Spawning was delayed repetitivetank-spawnings were accomplished over until Marchby holdingbrood»tock at a constant a 3-monthperiod Smithet al. unpublished!.Thus, temperatureof 17'C,and photoperiod of 12h light. the spawningtechnology for wild broodstock This allowed zooplanktonblooms consisting appearsto be reasonablydeveloped and fry may primarilyof rotifers -2,200/L! to bedeveloped in beavailable thmughou the year using photothertnal theoutdoor ponds. Females were induced to spawn manipulationaf broodstock. usingGnRHa irnplants, For each study, eggs werc The next stepin the cultureprocess is obtained from a minimum of' two females and development of suitablenursery techniques. fertilizedwith mih from several males. Egg s werc Daniels et al, 996! provided some basic obtainedby either strippingor tank-spawningand mformationon the effects of density,light intensity, incubatedin 30-348/L salinityand at a teniperature andsalinity on larval growth «nd survivaL However, of 16-2~. Hatchingoccurred in 36-40h and the z4 UJNR TechoicaiReport No. 2s fry were held for 3-6 days,until the eyeswere sledand then with a seineas they grew larger. At pigmtntedand thc mouthparts fully formed, before harvest,fish werc dip-netted from the harvest basin stockingin thenursery trials. At thistime, larvae andplaced into tailks corltainirig clean seawater, were a mean size of 2-3 mm TI.. Survival was basedon an individual count of the As appropriate,data were statistically lish harvestedwhile a subsarnplewas used to analyzedusing parametric ANOVA andT- Test! determine fish size. arKInon-pararnxnric tests Kruskall-Wallis One Way Analysisof Ranks,Mann-Whitney Rank Sum Test!, Tank Nursery Dunn's methodwas usedto identify specific A tanknursery study was conducted during 1996 differencesamong more than two groups. Percent to evaluate the effect of three larval diets and two data were normalized using thc arcsin light intensities on growth, survival, and transformationbefore analysis, Significance was pigmentation Denson and Smith 1997!. acceptedat Ps 0.05, Cylindrical70-L blackfiberglass tanks containing centerstandpipcs and filled with 1-pmfiltered water PondNursery at 23"C and 34-35 g/L salinity{combination of During1995-1997, three O. 1 -haponds at CharlestonHarbor water and evaporated sea salts! theSCDNR's Waddcll Mariculture Center WMC! servedas the experimental culture units. Tanks were werestocked with fry. Theponds were lined with filled with 16I. of waterinitially, with the volume 30mil high-density polyethylene and the bottoms increasedto40 L whenA rtemianauplii were added covered with 20 cm of native soils. Pondswere as food. The diettreatments consisted of: treannent slopedfrom a minimumdepth of 1 m toa maximum 1 - rotifers Brac/rionirs plicati/is! fed at a depthof 2 m. Theponds contained a 4.9-m-long x concentrationof 10/ml duringdays 1-9, and 1.5-m-wide slopedharvest basin to assist in Artemia{3/ml! fed days 7 throughmetamorphosis; harvestingfish. Water,either saline or brackish, treatment2 - rotifers fed days 1 through wassupplied at theshallow end of thepond with metamorphosiswithArtemia added bcgmrung day anadditional water val ve located above the harvest 7; treatment3 rotifers fed days 1-9, and Arremia basin.During the studies, ponds were filled with andan artificial larval feed Larva "Z' Plus,Zeigler filtered 00 lrm!saltwater from the adjacent BrothersInc., Gardeners, PA! fed days 7 through ColletonRiver. At harvest,water of lowoxygen metamorphosis.Rotifers were fed a dietof algae contentand containing a highsilt load occurred in {Isochrysisrahiri! and a commercialhighly thecatch basin near the end of pond drainage. To unsaturatedfatty acid HUFA!enriched feeding reducestress and mortalities, clean and highly product Culture Sclco, Artemia Systems Inc., oxygenatedwater was added to the basm during Baasrode,Belgium! before being placed in the harvesting.Fish were stocked near the cnd of' larvalrearing tanks. Lighting was continuous using Marchduring tach trial and harvested 2' and7 overheadfluorescent lights 15 W coolwhite high monthslater. Stockingdensity ranged from outputbulbs!. Light intensity was measured atthe 284,760-740,000larvae/ha based on volumetric surfaceof eachtank and thc lowest and highest estimation.Water quality was measured regularly lightreadings were averaged toprovide a combined duringtrial 1 andless frequently during the other tankvalue. Two deatments were examined: high trials,To reduce risk of low oxygen concentrations light- 1362lux, and low light- 457 lux. At and temperaturestratification, a 0,75-kw initiationof thestudy, each tank received 200 paddlewheelaerator was run condnuously. After indivi~lycounted larvae and four replicates per approximately1 month, supplemental feed treatmentwere utilized in theexperimental design. consistingofsinking salmon starter 8% protein! wasadded 6 days/wkduring trial 1. Feedsize was Thestudy was completed on day 30 andall graduallyincreased, andbeginning on5 July a 24- post-metamorphicfish were hand-counted, measured, and pigmentation characteristics mrnpellet 8% protein!was fed Jcnkinset al, categorizedbased on a subjectivekeymodified from 1997!.In trials 2and3, no supplemental feedwas Seikai 985a! Denson and Smith 1997!, Water provided.Fishwere first sampled using a plankton qualitywas monitored every two days m eachtank Smith 25 andadjustinents made as necessary, In-tank sponge fiberglasstanks 5 cm diameterx 45 cm deep filters were used to providebiological and with roundedbottom! fitted with centerstandpipcs, particu]atcfi]tration. All three replicates of each treatment were connected to a recirculating system which Salinity Tolerance oontinuouslyrecirculated filtered water to thetanks. Two short-termsalinity to]erance studies Tankswere filled with 28 g/L CharlestonHarbor wereconducted. The first study utilized recently seawaterand thejuveniles acclimated to one of metamorphosedjuveniles produced from the tank foursalinity treatments {0, I, 5, and 10g/L! using study while the second study examined the dechlorinatcdtap water. Juvenileswere fed live toleranceof larger,6-month-old juveni]es, These mosquitofishGambrtsia sp. to satiationdaily. larger fish were obtainedfrom the ]995 pond nursery trial, During both studies, fish were Transition Diets inspectedseveral tiines daily anddead fish were Two studies were conducted to wean fish removedwhen observed. Lighting was continuous from five feeds to commercially available dry usingoverhead fluorescent lights, Waterquality rations. Fish used in the trials were obtained from wasmonitored at thebeginning and end of studyI, thepond nursery studies, After harvesting, fish were anddai]y duringstudy 2. Dechlorinatedtap water placedin cleanwater and transported to theMarine g/L salinity;mean alka]inity 45 mg/L;hardness, Resources Research Institute. Fish were measuied, 66 mg/L CaCO, equivalent!;pH 7.5! wasadded counted,and p]acedin 1.8-m-diameterx 0.8-m- several times during the studiesto adjust for deepcylindrical tanks fitted with centerdrains and evaporative loss and to maintain salinitiesat equippedwith screenedstandpipes which drew appropriatetreatment levels. waterfrctm throughout the waterco]umn. Water In the first study, the recent]y was recirculatedthrough a biologica]filter and metamorphosedjuveniles 3.7 ~ 2.6 mmTL, 50 suppliedto the umksat a rateof 20 tank volumes/ daysold! werestocked into I -L glassbowls filled day.Water was aerated using four air stonesspaced with 30 g/L seawater. Each treatment was aroundthe perimeterof the tank. Basedon data replicatedin three bowls eachcontaining ]0-]4 collectedftem thesalinity tolerancestudie~ and the juvenileswhich were acclimated to sevendifferent desireto reducethe potentialfor infestationwith salinity levels: 0, 5, 10, 15, 20, 25, and 30 8/L the dinoflagellateAmy/oodinittm sp., fish were control!. Fish were acclimatedto treatment rearedat 3-10 g/L salinityusing a combinationof salinities by diluting the Charleston Harbor dechlorinatedtap water{freshwater!, low salinity seawaterwith dechlorinatedtap waterat a rateof well water,and settledseawater fmm the Charleston 5 g/L/h. In the 0 g/L treatment, water was Harbor estuary. complete]yreplaced with dechlorinatedtap water Water temperaturewas recordeddaily after acclimationto near 0 8/L usingdilutions of whileother water quality parameters were measured seawater. Total acclimation time for the 30 to 0 8/ week]y. Fish wereweighed several times during L treatment was 6 h. Additionally, as a separate trial 1 studyduration 103 days!and at beginning treatment,flounder in threebowls were converted andend of trial 2 7 days duration!. Fish were to 0 g/L withoutacclimation to examinethe fed dry feeds sa]mon startercrumbles t2-t/3, influenceof acclimationtime. Juveni]esin al] Zeigler Brothers,Gardeners, PA! at 1 1/2-h treatmentswere fed newlyhatched to 24-h-old intervals24 h/dayusing overhead automatic feeders Artettrianauplii {400 pm! stocked ata densityof SweeneyEnterprises Inc., Boeme,TX!. The live 5/ml, The studywas conducted for 72 h after feedsand previously Irozen feeds were provided 2- acclimationwas completed. 3 tiines/dayduring norma]working hours {0830- Study2 eva]uatedthe effects of four ] 700h!. When feedinga mixturecontaining dry salinitylevels on surviva] and feeding behavior over feeds, the amount of natural feed was gradually a 2-wkperiod, Five juveniles, mean size 95.2 s reducedduring each feeding period, while the 14,0 ruinTL, 7,4 ~ 3.6 g, 220 daysold, were amountof dry feedwas increased.Once fish had stockedinto three replicate 70-1 black cylindrical beenconditioned to exclusivelyaccept dry feeds UJNtr TtchaieatReport No. 26 theweaning experiments were terminated. normallypigmented. Intrial I 995!,200 fish mean size 8.3 g and96 rnm TL,220days old! were stocked ineach Tank Nursery oftwo replicate tanks at a densityof 77 fish/mi of Nosignificant differences inwater quality tankbottom. Fish were fed as follows: live and weredetected among tanks. Mean water quality deadgrass shrimp Palaerrroneres sp.during days valueswere as follows:temperature 22.9'C; 0-15;chopped mullet hfugil cep/raiiis and spot salinity35,7 g/L; dissolved oxygen 6.6 mg/L; pH Leiostomusxanthurus during days 16-52; ¹2 7.6;and total ammonia nitrogen 0,8 mg/L. Larvae salmonstarter 0% protein!mixed with refined beganactively feeding on rotifcrs at age6 days rrmnhadenr/revoorriarvrannrrsoil and chopped fish post-hatch dph! and on Ariemia nauplii at age 14 duringdays 53-78; ¹3 salmonstarter and refined dph.On study day 17 3 dph!,mean size was 8.2 menhadenoilduring days 79-105; and dry feed only 0.6 minTL. At that ti me, coinpletc beginningday 106. metamorphosisanda variety of transition stages In trial2 997!,400 fish meansize 1,2 wereobserved among fish in alltreatments. By g,46.7 mm TL, 77 days old! were stocked ineach day30, 98%of theanimals had completed ofthe two replicate tanks at a densityof 154fish/ metamorphosis,There were no significant m', Fishwere fed as follows: ground Atlantic differencesd~ amongsurvival levels between mackerelScomber scombrrrs, frozen blood worms feedtreatments mean 37,5 x 15.5%!or light Tubifexsp., and frozen adult Arrernia, days 0-8; treatments mean 26.4a 12.8%! Denson and Smith ¹ I and¹2 salmonstarter and emulsified menhaden, 1997!,Similarly, there were no differences in total days9-13; and dry feed only, day 14, length overall mean 11.5 + 1.3inrn! among fish in anytreatments Denson and Smith 1997!. RESULTS Bodypigmentation washigMy variable. On day30, no significant differences inpigmentation PondNursery weredetected among the three feed treatments, Duringharvesting oftrial I, thepond was buton average only 29,7% of the population had drainedrapidly asis done with other fish species. normalpigmentation. Recheck ingpigmentation UnfortunatiJy,most of the juvenile flounder became patternsa weeklater indicatedthat there hadnot stranded,burrowed into the soft pond bottotn, and beenany significant change. On day30, there hadto be individually harvested byhand, During wasno difference inthe degree of albinism among trials2 and3, pondswere rapidly drained to near fishreared in high and low light conditions, butagain exposureof the pond bottoin. Then, during the therewas only a lowpercentage of normally night,the remaining water was very slowly ~ pigrnentedfish, Subsequent tothe study, the light whichallowed the small flounders to movewith intensityin thelow light treatment was increased thewater and become concentrated inthe harvest tohigh light conditions onday 30. Onday 37, there basins.Nighttime draining eliminated birdpredanon wasstatistically more normally pigmented 4.5% problemsand water temperature increases due to vs. 13.3%!animals than on day 30. Nochange in solarheating. Collection of thefish in thebasin pigmentationwasobserved among fish in the high madeharvesting more efficient although removing lighttreatments which were held at highlight fishfrom the bottom of thebasin was still difficult. intensityfor an additional week 4.5% on day 30 Waterquality recorded during the trials vs23,0% on day 37! Densonand Smith 1997!. appearedsatisfactory although temperatures on sotnedays in July and August during trial 1 SahlnityToleranm exceeded31'C Table I!. Fish rearedfor 2'6 In study1, no differenceswere observed months trials 2 and3! wereabout half thc length in water quality betweentreatments. Mean ofthose reared for 7 months Table 2!. Survival water temperaturewas 25.2'C, pH was 7.5, and waslow tnean4.5, range 3.5-6,1%! during each totalammoma nitrogen was within acceptable trial Table 2!. Examinationofpigmentation pattern limits 2 mg/L!. Some fish in each of the inthcatedthat about 99% of theharvested fish were treatments,including the non-acclimated0 g/L 8mit b 27 Tetap. C! Oaygea atgrL! Mean March1995 19.0 17.420 2 7.9 7.2-9 0 8 7 8.6-8.8 22.0 20.0-24.0 April 1995 20.8 15.8-25.0 6.8 5.6-8 6 8S 8.2 9.0 24.7 23.0-26.0 May 1995 24.8 21,4-2'7.4 5 3 4.24.7 8,5 8.24.8 27.7 26 0-30.0 June1995 26.8 24.5-29. 9 4 9 3.4-6 2 8.2 8.1-8.3 25 0 24 0-260 July 1995 29 7 27.2-31.3 4 4 3.4.5.8 8,4 8.2-8.5 25.0 August1995 28.8 22.7-31. 9 4 4 3.5W.O September1995 25.4 30.9-28.2 5.1 6.26,2 October1995 24.7 23.8-25. 7 6.2 5.6-6.7 March1997 21.0 7.2 30.0 Ayril 1997 20.5 18.4 22.5 7.3 6.2-9.9 87 33.0 bray1997 23.8 22.6-26.0 6.2 5.7-7.1 26.0 June1997 21.0 7.1 28.0 btareb1997 20.9 7.0 30.0 April 1997 20.4 18.2-22.3 6.9 5.6-9.1 8.5 33.0 May 1997 23.9 22.5-26.0 6.1 5.5-6.9 26.0 Jure1997 21.0 7.1 %.0 Tsthte1. Waterquality parametersmonitored during pond nurserytrails with southern flounder. Data withoutranges indicate oofy one observationwas made. Trial Date Densitg EisLaze lhttattoa d/rn/y! mt.!ha! fTL tata! d! TL rtnm! wt. g! tMt.aha! %! M03/95 284,760 2.0ZO. 1 217 96. 3Z 13.5 8 3 ~,4 t7,460 6. 1 2 28/03I97 740,000 2.0*0. 1 74 55.5+8.0 1.9+0.9 3',ttR 4.0 3 28J03/97 'J40000 2.0g0. 1 74 36,6 +6.8 0.5 +0.2 25, 850 3. 5 %aMe2. Stocking and production data for pond nurserytrials with ~ flounder. Table3!. Meanoverall survival in 5-30 g/L salinity treatment, were observed feeding on Arrerrsirr treatments was 99.1% and no differences were immediately after they were addedto the culture detected Table 3!. containers.Within 24 h, somejuvenile flounder In study 2, no mortalities were recorded began to die 5%! in the non-acclimated0 g/L during the 2-wk study penod. No significant salinity treatment. At this time, only one fish in differenceswere detected between fish total length one replicate had died in the acclimated 0 g/L mean l02.5 s l4.6 mrn TL! Table 4!. treatment. At the conclusion of the 72-h study, therewas significantly higher mortality in the0 g/ Trans%iota Diets L acclimated 80%! and non-acclimated 84%! In trial l, fish would not consumepelleted treatments p=0.007! than in the higher salinities feeds initially even though large amountsof feed had beenprovided to the nurserypond for 6 months UJWR Ieednica! Rcport No. 26 Sarvival onday 53 Table5!. Chopped fish was complctcly %! removedfrom the.diet on day 79, The fishwere .7 h not weaned froin the inenhaden oil and saltnon startercombination until day ] 06 ata sizeof 28.6g 20.0h and140 tiun TL, Survivalduring this l06-day 97.0B weaningperiod averaged 5S,2%. As in the io 100.0B previoustria], fish wou]d not consume dry feeds initia]ly in nial 2. However,no live feeds were l00.0B providedin trial 2, Instead,fish were provided a combinationdiet of ground mackerel, adult 4rtemta, 100.0B andblood worms which they readily consumed. Onday 9, this combination dietwas replaced with 30 cmsrol! 100.0B a mixture of pelleted feed and emulsified menhaden, Fish atc this diet and were weaned to 'pdaawe sad ~ iad pbccdddaadr adaa Sa g/L adrdf add 0 g/Ldccrdoraaad tap thedry salmon starter diet by day 14, At sampling onday 27, survival averaged I]0.4% with fish more thandoubling in sizeto 2.6g and64 tnmTL Table ']!able3. Meansurvival of recentlyrnetarnorphosed 5!. southernflounder 3.7 5 2.6nnn TL! in a 72-hsalinity tolerancetest. Valuesfollowed by the sameletter are not significantly different, DISCUSSION Intensivelyinanaged tank nursery systeins are typically usedto produceflatfishes in Japan priorto harvest.Popu]ation sampling on day 15 andEurope Fukusho etal. 1985,Ho 1 mefjord et al. indicatedthat very little gniwth had occurred while 1993,Minkhoff and Broadburst]994!, Such fishwere being fed grass shrimp Table 5!, During systeinshave a numberof advantagesincluding thenext period, fish readily consumed chopped fish the abilityto strictlymanage water quality and andincreased toa meanweight of 11.9g byday feedingregimes and to producejuveniles year 36. Thistrend continued during the period with around Table 6!. However,they are more costly fishattaining a mean size of 15g and116 nun TL to constructand require more skilled labor. A] so, fishproduced in suchsystems may have varying Salinity treatment Fish size mtt TL! Survival g!L! Initial Final %! 91.1 + 12.4A 96.5 + 13.1A 96.5 + ]5,8A 103.4 + 17.5A 96.4 + 13.8A 104.0 J 14.7A 96.6 g ]4.6A 106 1 4 12 3A t]able4. Meantotal length + SD!of southernflounder juvenfles in 14-daysalimty study. Values in columns followed by tbe sameletter are not significantly different. Smi tb 29 Day f Wr. Sltrvlvsl 1! 8.3 live anddead grass shrimp RO.O 16 8.7 chopped mullet + spot 52 15.0 116 choppedsaul les + spot + menhadenoB + f2 sahnor.starter 79 133 rtaabaden oil + N salmon starter 28.6 140 S4 sahnonstarter 58.2 47 dead bloodsvortns+ athth 100.0 Arfemcz + grsatnd mackerel eanMfted nenhadett + fl and tf2 sahnm starts 14 H aahnonstarter 2.6 '1hble5. Data for weaningtrials with juvenile southernflounder. Table&, Chiastic s of intensivelank and extensive pond nursery systems for pmductionof juvenilesouthern flounder. 3O t.'JRR TcchnlcatReport No. 26 degreesof pigmentauonabnormalities which make juveniles 0 daysold! appearless tolerantto themless desirable for saleas a live product.In freshwaterconditions than older 20-day-old! contrast,pond nursery systexns are common in the juvenileswhich exhibited 100% survival at salinities southernUmted States for a numberof species rangingfrom 0 to10 g/L, Similarly,Daniels et al. includingchannel catfish lcrafurvx puncraxus 996!reported 0% survivalfor larvae undergoing Tuckerand Robinson 1990!, stxiped bass and its metamorphosisa 0 g/Lbut 59%survival at 20 g/ hybrids Harrell et al. 1990!, and red drum L. Longerterm studies will berequired to further $ciaenopsoce/latus McCartyet al. ]986!. Pond definethe salinity requirements of different life systemsallow less control of the rearing stagesand especially thc effectson growthand environmentbut are cheaperto developand survivalrates, Morta]ity of southernflounder due manageHowever, production is seasonallylimited to infestationsof Amy/oodinium sp.and Argidus andfish inust be converted odry feedsafter harvest sp.have been noted in ourlahoratory inhigh salinity Table 6!, culturetanks, Rearing of flounderin lowsalinity Developmentof nurserysystems for tofreshwater conditions may avoid simi]ar pmblems southernflounder is still in theearly stages but withthese parasites. Additionally, if this species resultsto date indicatethat juveniles can be canbe successfullygrowxi on non-coastalsites, pmducedin tankand pond systems. The tank study substantialsaving in land costswill occur. showedthat they can be reared on a rotifer/Artemia Resultsof our weaning studies suggest that diet with a survival level of about 37%. This is an it maybe easier to train young juveniles 7 days improveinentover that reported by Danielsct al. old!to acceptdry diets, than older ju veniles 20 996!. However,a highincidence of pigmentation daysold!. The olderfish required106 days to abnorma]itiesoccurred inour study -70%! as we]1 transferto drydiets as compared to 14days for the asamong juveniles 0-40%! producedby Daniels youngfish. Additionalresearch should focus on et al. 996!. In contrastto ourtank culture results, ixnprovementof weaning techniquesand productionof juveni]esin pondswas low mean identificationof practicaldiets suitablefor survival 4.5%! but so was the incidence of producingfood-size fish andcultured broodstock. pigmentationabnormalities -1%!. Albinismin In summaxy,studies to date indicatethat intensive]yreared f]atfishes is a commonproblem. the southern f]ounder is a suitable candidate for The causeappears to be complexand may be culture. However, additional researchis neededto related to environmenta] and nutritional factors identifythc performance of variouslife stagesin Seikai]985 a.b,Stsittxup and Attrarnadal 1992, differentrearing systems and to identifyoptimal Seikaiand Matsumoto 1994, Spedicato etaL 1994!. and acceptableenvironmental coxxditions and Refinementsto pondand tank nursery satisfactorydiets. Such researchwill needto be systemsover the next severa]years should result coupledwith marketingstudies to determinethe in improvedproduction levels and production of economic potential for southern flounder morenormally pigmented fish. Pondnursery aquaculture. researchfocused on improvedmanagexnent of zooplanktonpopulations should improve survival ACKNOWLEDGMKNTS levels,Better c!eaning of theharvest basin prior to harvestand incorporation of a differenthaxvesting We thank the aquaculturestaff of the technique e.g., fish pump! xnay increase efficiency MarineResources Research Institute including: of fish removaland xninixnizestress and injury. LouisHeyward, Charles Bridghaxn, Pxentiss Lund, Tanknursery research on identification of suitable andLisa Carter for their assistance in conducting environmentaland nutritiona]parameters should rearing experiments, We also thank the pond result in higher productionlevels of normally managementstaff of the Wadde]l Mariculture pigmentedfish. Center including: John Holloway, Richard Resultsof studieson thesa]inity tolerance Haxnilton, JoeHosts, JacobRichardson, and Peter of southernflounder indicated that thisspecies is Hami]tonfor assisrance with the extensive nursery euryhaline. However, recently xnetamoxphosed system experixnents. This work was funded in part by the U,S. Department of Commerce, Tetraselmis retrathele for larvae of a NationalOceanic and Atmospheric Administration, flounderParalichthys oil vaceus. Bull. Office of Sea Grant contract numbers NA90AA- Natl. Res. Inst. Aquacult.No. 7: 29-36. D-S 6790 and NA46RG0484!. This is Contribution Ginsburg, I. 1952. Flounders of the genus No. 402 from the South Carolina Marine Resources Paralichthys and related genera in Center.Reference to tradenames does not imply American waters. U.S. Fish. Wildh Serv., endorsement. Fish. Bull. 71: 267-351. Gutherz,E. J. 1967. Field guide to the flatfishes LITERATURE CITED of the familyBothidae in thewestern North Atlantic, U.S. Fish. Wildl. Serv., Bur. Ackerman,J. 1997. Recreatingthe bountyof the Commer.Fish Circ, 263, 17 p, sea. Boston Sunday Globe, March 6, Harrell, R.M., J.H. Kerby, and R.V. Minton, 1990, 1997, section F, pp, 1, 4. Boston, MA. Culture and propagation of striped bass Arnold, C.R., W.H. Bailey, T.D. Williams, J. andits hybrids. Striped Bass Committee, Johnson, and J.L. Lasswell. 1977. SouthernDiv., AmericanFisheries Society, Laboratoryspawning and larval rearing of Bethesda,MD, USA, 323 p, red druin and southern flounder. Southeast, Henderson-Arzapalo,A., R,L, Colura, and A.F. Assoc.Fish Wildl. Agencies31: 437-440. Maciorowski, 1988. Temperature and Bearden, C.M. 1961. Commonmarine fishes of photoperiod induced maturation of South Carolina Contrib. Bears Bluff Lab southernflounder. Management Data Ser. No. 34, 47 p. No, 154,Texas Parks and Wildlife Dept.. Berlinsky, D.L W, King, T.I.J. Smith, R.D, Austin, TX, Hamilton, J. Holloway, Jr., and C,V, Hohnefjord,L, J. Gulbrandson,I. Lein, T, Refstie, Sullivan. 1996, Induced ovulation of P.Leger, T. Harboe,L Huse,P. Sorgeloos, southern flounder Paralichthys S. Boola, Y. Olsen, K.l. Reitan, O. lethostigmausing gonadotropin releasing Vadstein,G. Oiem, and A. Danielsberg. hormoneanalogue implants. J. World 1993. An intensiveapproach to Atlantic Aquacult.Soc, 27 !: 143-152 halibutfry production.J. World Aquacult. Brisbal,G.A. andD.A, Bengston.1993 Reversed Soc. 24 !: 275-284. asymmetry in laboratory reared summer Jenkins,W.E., T.I J, Smith, C.V. Sullivan, and D.L. flounder. Prog. Fish-Cult. 55: 106-108. Berlinsky, 1997. Productionof southern Dablberg,M.D. 1972. An ecologicalstudy of flounder Paralichthys lethostigma! Georgia coast fishes. Fish, Bull U.S, juvenilesin an outdoornursery pond. J. 70:323-353. WorldAquacult. Soc. 28 !: 211-214. Daniels,H.V D.L. Berlinsky,R.G. Hodson,and Lassweli,J.L., B.W. Lyons, and W.H, Bailey. 1978. C.V. Sullivan. 1996. Effects of stocking Hormone-inducedspawning of southern density, salinity, and light intensity on flounder.Prog, Fish-Cult, 40: 154. growthand survival of southernflounder McCaity, C.E., J,G Geiger, L.N. Sturmer, B.A. Paralichthys lethosrigma'!larvae, J. Gregg,and W.P. Rutledge. 1986. Marine WorldAquacult, Soc. 27 !: 153-159 finfish culture in Texas: a model for the Denson, M.R, and T,I,J. Smith, 1997, Effects of future,pp 249-262, lrr: R.H. Stroud ed.!, dietand light intensityon survival,growth Fish Culture in FisheriesManagement. and pigmentationof southernflounder American FisheriesSociety, Bethesda, Paralichthys Iethosti gma!. J. World MD. Aquacult.Soc. 28 !: 366-373. Minkhoff, G, and A.P. Broadhurst. 1994, Intensive Fukusho, W., 1VI, Okauchi, H. Tanaka, S,I, production of turbot, Scophthalrrius Wahyun i, P Kraisingdech a, and T. maximus,fry, pp, 14-31, Irt: P. Lavens Watanabe, 1985, Food value of a rotifer and R.A.M, Remmerswaal eds.!, Turbot Brachionus plicatilis, cultured with Culture: Problems and Prospects. nJ-'iR rccbaical Report No. 26 EuropeanAquaculture Society, Oostende, A.D. Stokes, 1996, A guide to pond Belgium. culture of hybrid striped bass. South Music, J.L. andJ.M. Pafford. 1984. Population CarolinaSea Grant ExtensionProgram, dynamicsand life historyaspects of major Charleston,SC. 41 p. inarine sportfishesin Georgia'scoastal Spedicato, M.T., G. Lembo, and P. Trotta. 1994, waters.Ga. Dept.Natur. Resour., Contrib, Larval and post larval rearing of Ser.No. 38, 382 p. Paralichthys oli vaceus Temrninck and National Marine Fisheries Service. l995. New Schlegel!:the first experience in Italy,pp. YorkFulton fi shmarket primary wholesale 292-301. In; P, Lavens and R.A.M. selling prices, Fishery Market News, Retnmerswaal eds.!, Turbot Culture: Januaryto October,New York,NY, Problems and Prospects European Powell, A.B. andF J, Schwartz, 1977, Distribution AquacultureSociety, Oostende, Bel gi uin. of paralichthid flounders Stokes,G.M. ! 977. Life historystudies of southern Bothidae:Parali chthys!in North Carolina flounder Paralichthyslethostigtna! and estuaries.Chesapeake Sci. 18;334-339. gulfflounder P. albigutta! in theAransas Powell, A.B. and FJ. Schwartz. 1979, Food of Bay area of Texas. TexasParks Wildl. ParalicIttItysdentattts and P. lethostigma Dept.,Tech. Ser. No. 25, 37 p. Pisces:Bothidae! in North Carolina Stettrup, J.G. and Y. Attramadal. 1992. The estuaries. Estuaries 2: 276-279, influence of different rotifer and Artemia Seikai,T. 1985a. Influenceaf feedingperiods of enricheddiets on growth, survival and Brazilian Artemia during larval pigtnentationin turbot Scophthalrnus developmentof hatchery-rearedflounder nttuimttsL.! larvae. J World Aquacult. ParalicItthysolivaceus. Bull. Jpn. Soc. Soc. 23 !: 307-316, Tucker, C,S. and Sci. Fish. ! 521-527. E.W. Robinson. 1990. Channel Catfish Seikai, T. 1985b. Reduction in occurrence of Farming Handbook, Van Nostrand albinismin juvenileflounder Paralichthys Reinhold,New York,NY. 454 p. alivacetts hatchery reared on wild Waters, E.B. 1996. Sustainable flounder culture zooplankton.Bull. Jpn, Soc. Sci. Fish. and fisheries. North Carolina Sea Grant 51 8!: 1261-1267, Publ.UN-SG-96-}4, Raleigh,NC, 12 p. Seikai, T. and J. Matsurnoto. 1994, Mechanism Wenner, C.A., W.A. Roumillat, J.E. Moran, Jr., of pseudoalbinistn in flatfish: an M,B. Maddox, L,B, Daniel, III, and J,W. associationbetween pigment cells and skin Smith. 1990. Investigations on the life differentiation,J, World Aquacult.Soc. historyand population dynamics of tnarine 25 !: 78-85, recreationalfishes in SouthCarolina: part Smigielski, A.S. 1975 Horinone-induced I, southern flounder Paralichthys spawningof the summerflounder and lethostigma.Report to USFWS, Atlanta, rearingof larvaein thelaboratory. Prog. GA, for ProjectF-37, pp. 21-35. Fish-Cult 37 !: 3-8. Sinith,T.LJ, and W.E. Jenkins. 1996. Regional development of hybrid striped bass aquaculturein the southeasternUnited States, pp. 175-179. Irt; T, G, Heggbergett,J. G. Woiwode,and R J, Wolotira Jr. eds.!, The Role of Aquaculturein WorldFisheries Theme 6! . ProceedingsWorld Fisheries Conference. Oxford & IBH Publ Co. Pvt. Ltd., New Delhi, India. Smith, T.IJ., W.E. Jenkins,J.M, Whetstone,and Bi rub num 33 LICENSING AND REGULATION OI VETERINARY BIOLOGICS FOR FISH IN THE UNITED STATES Nathan G. Birnbaum U.S. Departmentof Agriculture Animal andPlant Health inspectionService VeterinaryServices Centerfor VeterinaryBiologics Licensingand Policy Development 4700 River Road, Unit 148 Riverrlale, MD 20737-1231 e-mail:nbirnbaum@'aphis.usdagov ABSTRACT TheU.S. Department of Agricutture USDA! regulates biotogics for fish.including vaccines. bacierins, and diagnostictest kits, produced in, imparted into, or exportedfrom the United States. The regulatory process is designedto ensurethat biotogics under USDA jurisdiction are not containinated,worthless, dangerous, or harmful.The Animal and Plant Health Inspection Service APHlS!, an agencywithin the USDA, licenses and inspectsbiologics production facilities, and licenses and tests veterinary biotogtcat products. Veterinary biologi- calproducts should be pure, safe, potent, and efficacious. A biologics-producmgfirm locatedin theUnited Statesmay seH its productsprovided the firin possessesa valid U.S. Veterinary Biologicat Product License for eachproduct produced for sale, as well as a valid U.S. Veterinary Biologics Establishment License. A permittee i.e.,the !egal representative inthe- United States of a biologics-producing fum located outside the United States! mayiinport biologics into the United States provided the permittee possesses a valid U.S. Veterinary Biological ProductPermit. Biologics available in theUnited States lor fish aremanufactured by AlpharinaNW Inc.,Aqua HealthLld., andDisgXotics. Monovalent and multi-fnction bacterins i.e., antigemcsuspensions of inactivated bacterialorganisms! are available for the vaccination of fishto aidin thcprevention of furunculosiscaused by Aeromonassolmonicirkr, enteric septicemia of catfishcaused by Edwardsieflnicrahrri, colunmnris disease caused byI'lavohacre ruun cohunnare, vibriosi s causedby Vihrio anguif4 runt and V. o rdalii, cold water vibiiosis caused by V.saknoninuiurn. snd enteric redmouth diseases caused by Yersiniaruckerii. Qualitativeand quanntati ve test kitsto diagnose the presence ofthe bacterial kidney disease antigen ftenibacrcrr'rim sofntoiunarunt infish are also available in the United States A bacteriarecommended as an aid in theprevennon of winter ulcerscaused by V. viscosnris producedin theUnited States for exportonly. INTRODUCTION DISCUSSION The U.S, Department of Agriculture Biologicscurrently available for fish in the USDA! regulatesbiologics for fish producedin, UnitesStates include bacterins and diagnostic test importedinto, transported through, or exportedfrom kits. Bacterins i,e.. an antigenicsuspension of theUnited States,Veterinary biologics include vi- inactivatedbacterial organisms! are usedfor the ruses,serums, toxins, and analogous products e.g., vaccinationof fish as an aid in the preventionof vaccines,bacternts, allergens, antibodies, antitox- furunculosiscaused by Aerorrionassalrrrorricida, ins, toxoids, immunostimulants,cytokines, etc,! enteric septicemia of catfish caused by which act through an immune mechanismto pre- EdrvarrLriella iciatrirr, columnarisdisease caused vent, diagnose,manage, or cure diseaseof ani- by Flavobacterirrrricolrsinnure, vibriosis caused mals. by Vibrioangrsillanuri and Vibrioordalii, cold water vibriosiscaused by Vibriosalrnoninarism, 34 UJNR TechtticslReport Ne. 2S andenteric redmouth diseases caused by Yersinia Furthersources of informationand guidance in- rrrckerii.Qualitative and quantitative test kits to clude:the semi-annual Veterinary Biological Prod- diagnosethe presence of thebacterial kidney dis- uctspublication listing the licensees, permittees, and easeantigen Rerribacreri ttnz sairnorrinarum in fish veterinarybiologics produced, the CVB internet are also available in the United States. A bacterin homepage, and APHIS-sponsored public meet- recommendedas an aid in theprevention of win- ings, ter ulcerscaused by Vibrioviscosus is produced Veterinarybiologics e]igiblc for distribution inthe United States for export only. Depending on and salein the UnitedStates may be rnanufac- the specificbiological product being considered, tured in facilities located either in the United States bacterinsmay contain a singlefraction or multiple or abroad.In orderto sella veterinarybiologic in fractions,and may be recommendedfor adminis- theUnited States, a biologicsmanufacturer located trationto fish by immersion, by injection,or by in- in the UnitedStates inust possess two typesof gestion. Federallicenses: a U.S. Veterinary Biologics Es- Theregulatory process is designedto en- tablishmentLicense for the production facility, and surethat all biologicsunder USDA jurisdiction ate a separateU,S, VeterinaryBiologica] Product Li- pure,safe, potent, and efficacious,and not worth- censefor each biological product. In orderto im- less,contaminated, dangerous, or harmful.Thc portfrom abroad and sell a veterinarybiological Anima] and Plant Health InspectionService productin theUnited States, a foreignveterinary APHIS!, an agencywithin the USDA, licenses biologicsmanufacturer's legalrepresentative per- andinspects biologics production facilities, and li- mittee!in theUnited States must possess a U,S. censesand tests biologics produced in licensed VeterinaryBiological Product Permit for thc biologics-manufacturing facilities. bio]ogic s!to bc imported. With only nunor differ- The Centerfor VeterinaryBiologics ences,the licensing process for domestically pro- CVB! is theveterinary bio]ogics regulatory pro- ducedveterinary biologics is the same as the per- gramwithin APHIS and is composed of three uiuts mittingprocess for veterinarybio]ogics imported withdefined functions. The Licensing and Policy from abroad. Development CVB-LPD! unitestablishes licens- Theapplicant for anestablishment license ingstandards and policy; reviews prelicense docu- or a productpermit should submit the following mentation;reviews test methods, outlines of pro- documents to the CVB; ductionand labels; and issues, suspends, orrevokes l. Applicationfor U.S.Veterinary Bio]ogics Es- licensesand permits. The Inspection and Comp]]- tablishmentLicense APHIS Form 2001!; a ance CVB-IC! unit inspects production facilities, one-pagedocuinent indicating general infor- methods,and records;rc]eases serials lots or rnationregarding the domestic bio]ogics-rnanu- batches!of biologicsfor distribution inthe rnarket- facturingestablishment. place;performs post-release product surveillance; 2. App]icationfor U.S. VeterinaryBiological andinvestigates suspected law vio]atorsand con- Product Permit APHIS Form 2005!: a sumercomplaints. The Laboratory CVB-L! unit one-pagedocument comp]eted by thcperrnit- developstest methods, standards, and reagents; tee of a foreign biologics-manufacturinges- performsprelicense, surveillance, and field prob- tablishmentindicating general information re- lemtesting; and trains personnel from other labo- gardingthe pemiittee, the foreign biologics pro- ratories. ducer,and the biological product s!. The authoritiesand procedures for the regulationof biologicsare defined in a varietyof 3. Articlesof Incorporation:a legal document published documents, including the indicatingthe businessoperating status of the Virus-Serum-Toxin Act of 1992 amended3 in I 985!, manufacturingestablishment. Title9 ofthe Code of FederalRegulations, Veteri- 4. WaterQuality Statement: a documentrequited naryBiologics Menwnundums, Veterirmty Bio]ogics for domesticveterinary biologics manufactur- Notices,Veterinary Biologics General Licensing ers only indicating the manufacturing Considerations,andSupplemental Assay Methods. establishment'sstatus regarding applicable Birsbsnm 35 U.S. effluent waterquality control standards. preventionof a specificfish disease are typi- 5, Applicationfor U.S. VeterinaryBiological cally e valuated for efficacy by Product License APHIS Form 2003!: a vaccination-challengestudies. The vaccine one-pagedocument I'or domesticallyproduced producedwith the lowestantigen level and at veterinarybiologics indicating general biologi- thehighest passage level from Master Seed cal productinformation. approvedin the filed Outlineof Prtxiuction! shouldbe administeredaccording to labeldi- 6, Qualificationsof VeterinaryBiologics Person- nel APHIS Form 2007!: a one-pagedocu- rections e.g., injection,immersion, or oral!to mentindicating specifi~ information regarding theyoungest age or smallestsize fish for which the educationaland work backgroundof em- the productshall be recommended.After an ployeesinvolved in biologicsproduction, appropriatepost-vaccination observation pe- riod, the vaccinated fish and other 7. FacilitiesDocuments: blueprints, plot plans, non-vaccinated control fish are challenged and legendsdescribing the biologicsproduc- with a virulent strain of microorganism for tion facilities. which protectionis recommended,and all In supportof theproduct license or permitap- post-challengefinriings are accurately re- plications,the applicantshould prepare in an ac- corded, The precisechallenge method and ceptablemanner and submit to Licensingand Policy thecriteria for detertruning protection vary with Developmentthe following items somevariation the irnrnunizingagent. Forproducts with two may existdepending on the particularveterinary or tnore fractions, data should be subrnittcdto biologicalproduct being considered!: evaluateany in vivoor in vitrointerference of l. Outline of Production and Related Special the various fractions. Outlines:documents describing the protocol 6. SerialPurity, Safety, and Potency Test Report: for manufacturingand testmg of a particular the VeterinaryBiologics Production and Test biologic. Reportform APHIS Form2008! indicating MasterSeed Purity andIdentity Test Report: all requiredtest resultsfor each of at least the VeterinaryBiologics Production and Test threeconsecutively produced prelicense seri- Reportform APHIS Form 2008! indicating als batchesor lots! of finishedproduct: the testresults for the organismselected and a. Puritytest resuhs indicate if extrane- permanentlystored at a specifiedpassage level ous viable bacteria and fungi are from which all additionalpassages are derived. presentin the finishedproduct. The 3. Master Cell Stock Purity, Stability, and permittee of imported veterinary Non-tumorigenicQuality Test Report: the biologicsis chargeda monetaryfee if VeterinaryBiologics Production and Test Re- APHIS conductsadditional testing of port form APHIS Form 2008! indicatingthe thefinished biological product for ex- testresults for the cellswithin a specific pas- otic viruses. sagelevel range used to grow seedorganisms b. Laboratorysafety test results indicate for biologicsproduction, if thereare any adversereactions at- 4. BackpassageTest Report: resultsof rever- tributableto the vaccinationof suscep- sion to virulence studies for conventional modi- tible fish with the biological product fied live or live recombinant-derived vaccines duringthe pre-challengeobservation indicatingthe MasterSeed's genetic stability period. andreversion to virulencepotential following c. Potency test results indicate ihe rela- administration to the host animal. tivestrength of thebiological product. 5. EfficacyReport: studyresults indicating the andare designed to correlate e ith ih» effectivenessof theveterinary biological prod- approved host an i m,ii uct to performas indicatedon the pmductla- vaccination-challengeefficacy study'. bel. Vaccines recommended as an aid in the Potency tests for killed viral or killed 36 UJNR Techohsl Report %o. 26 bacteriaIproducts typically utilize labo- 8. ProductStability Report: results of studies ratoryanimal or host animalevalua- validatingproduct shelf life i.e.,expiration tionsor else quantitative invitro meth- dating!. ods. The potencyof live virusand bacterialvaccines is typicallyinea- 9. Label:the insert, container label, and carton suredby meansof bacterialcounts or labelindicating thetrue pnxluct name, the name virustitrations. The bacterialcount andaddress of the producer and also the irn- of a livebacterial vaccine must bc suf- porterfor importedproducts!, the establish- ficientlygreater than that shown to be mentlicense or permitteenumber, the recom- protectiveinthe immunogenicity ef- mendedstorage temperature, the full instruc- ficacy!test to ensure that at any time tionsfor use, the withdrawal time if thebio- priorto theexpiration date the count logicis administered tofood animals, the expi- wi11be at leasttwice that used in the rationdate, the serial identification number, the recoverablequantity and number of doses,the irnmunogenicitytest. Thevirus titer ofa live viral vaccine at release should presenceof any antibiotic used as a preserva- beat least I,2 logarithmsgreater than tive,the indication to use the entire contents of thatshown to be protective inthe im- a multi-dosecontainer when the container is munogenicitytestto ensure that at any firstopened, the recommendation toburn the timeprior to theexpiration date the containerand unused contents of all live or- titerwill beat least0.7 logarithms ganismproducts, and any special restrictions, Thelabel may not contain any information greaterthan that used in theirnmuno- genicitytest. whichis false or misleading, All labelclaims mustbe supported by datasubmitted and filed d. OtherOutline of Pmductionfinished byLicensing and Policy Development asac- producttest results indicate specific ceptable. requiredinformation, e.g., microorgan- ismidentity, residual-free forrnalde- Beforeissuing an establishment license or hyde,viricidal activity, etc, permit forgeneral distribution and sale, APHIS will conductanon-site inspection ofthe biologics pro- 7. FieldSafety Report. study results indicating ductionfacilities and equipment todeterrrune that the level of unsuspectedadverse theseare acceptable forproducing, testing, and product-relatedreactions that may not have distributingveterinary biologics using good rnanu- beenobserved during pmduct development. facturingprocedures and good laboratory tech- Two or moreprelicense serials are tested at niques.The permittee for a foreignbiologics manu- threeor more distinct geographic locations on factureris charged a monetary fee to payfor the a largenumber of appropriatelysized fish that on-siteinspection ofa foreignbiologics pmduction donot belong to the manufacturer. The manu- facility,Biologics manufacturers should use good facturerreceives authorization toconduct field sanitarymeasures incompliance withFederal regu- safetystudies only after submission ofaccept- lationsand the Outline of production. Atthe preli- ableefficacy data and satisfactory testing re- censeinspection, APHIS reviews all aspectsof sultsof three consecutively produced prelicense the manufacturingprocess, including accurate serials,The field study is approved only if the recordkeeping and product sampling. Following testconditions are adequate to preventthe submissionbythe finn to APHIS of satisfactory spreadof disease,and the firm hasobtained resultsfor all requiredprelicense serial release permissionfrom the proper animal health au- tests,APHIS will conductconfirmatory prelicense thoritiesfor each state where the tests will take testingof representativesamples of three consecu- place,Before beginning the field safety test, tively pmducedprelicense serials at the CVB-L. thefirm should submit to Licensing and PoHcy APHISwill issue the appmpriate establishment and Developmentfor reviewa detailedprotocol producthcenses orpermit only after all preHcensing indicatingthe proposed observation and record- requirements have been satisfied. ing methods. There arc severaltypes of U.S. Veteri- naryBiological ProductLicenses and U.S. Veteri- naryBiological Product Permits. A regularbiologics productlicense authorizes the distribution of a vet- erinary biological product manufacturedin the UnitedStates, with or withoutrestrictions e,g., for useby or underthe supervisionof a veterinar- ianonly, intra-state distribution limited to authorized recipientsor approvedlaboratories, use only on premiseshaving a history of the disease,for ex- port only, etc,!. A conditionalproduct license is issuedin an expeditedprocedure to make a bio- logicneeded e.g, in anemergency or limitedrnar- ket situation! availablefollowing the demonstra- tionof productpurity andsafety eventhough prod- uct efficacy and potency studies remain in progress!, A permit for generaldistribution and saleallows for the importationinto the United S tates anddistribution with or withoutrestrictions! of a specifiedbiologic or biologics.Permits may also beissued to allowthe importation of biologicsinto theUnited States for research and evaluation pur- posesor for transitshipment only. EPILOGUE Biologicalproducts for vaccinatingfish are currentlyavailable for sale in the UnitedStates fromtwo companies: Alpharma NW Inc.,Bellevue, Washington,telephone 06! SS2-0448,and Aqua Health U.S.A., Buhl, Idaho, telephone 08! 543-5369.Test kits for thediagnosis of bacterial kidneydisease antigen in fish are availablefrom DiagXotics,Inc Wilton, Connecticut, telephone 03! 762-0279, Qualifiedpersonnel at APHIS' CVB are availableto assist biologics manufacturers andper- mitteesin theapplication process. For further in- formationregarding the regulationof veterinary biologics,contact: U.S. Departtnentof Agricul- ture,Annual and Plant Health Inspection Service, Veterinary Services, Center for Veterinary Biologics,Licensing and Policy Development,4700 River Road, Unit 14S, Riverdale, Maryland 20737-1231;telephone: 01! 734-8245,fax 01! 743-8910,Information regarding the CVB is avail- able from the Internet web site EFFECTS OF REARING CONDITIONS ON BLIND SIDE HYPERMELANOSIS IN JAPANESE FLOUNDER Nakahiro Iwata and Kotaro Kikuchi CentralResearch Institute of Electric Power Industry 1646Abiko, Abiko, Chiba 270-1194, Japan Phone: 0 l 1 -81-471-82-1181 Fax: 81-47 l -82-7922 e-mail:[email protected] ABSTRACT Theeffects of light direction, intensity, and bottom substrate onthe hypermel anosis inJapanese flounder were cxatnined.Hatchery-produced juvenile or youngflounder were kept in aquariawith transparent iopr and bottomsto illuminate the fish with upward and downward light. When the bottom of theaquarium was a transparentplastic plate, hypermelanosis occurred inall the fish tested regardless oflight directio~ or intensity downward/upwardillumination: 1300/1100, 1300/60, and 150/7 lux!. However. only 14% of the fish showed hypcnnclanosisinthe aquarium with a sandybottom and no upward light. Percentagetxxurrcnce of fishwith hypcrmelanosisdecreased drastically when the bottom was covered with glasssand, even if thc fish were exposedtoa highintensity ofupward light 400 lux!.Similar trends werc observed in the entargemcnt of the blindside pigmcnted area. Jv'one ofthe fish showed visible expansion ofthe pi gmented area in thcaq uarium with sandor glass sand on the bottom; however, the pigmented area was enlarged in half of the 0 shin theaquarium witha transparentplastic piste bottom. From these results, it is considered that, not hght, hut the presence of sandon the aquariuin bottoin is theprimary cause of blindside hypcrmelanosis in Japanese flotmdcr. INTRODUCTION manystudies not ou]yon Japanese flounder Seikai 1991!but alsoon otherflatfish species which The Japaneseflounder Parttlichthys showed a relationship between light and olivacetts is oneof themost important mariculture hypermelanosis Cunningham 1891, 1893, 1895, fishspecies inJapan. Thc wild fish of thisspecies Osbom1940, 1941, Stickney and White 1975!. generallyhas a whiteb! ind side,while almostall However, because most of the studies were thecultured flounder show a darkpigrnented area conductedm aquariumin whichthe bottoinwas ontheir blind side ambico!oration orhyperrnelanosis coveredwith sandto preventupward light, the onthe blind side!.This color anomaly is a serious sandysubstrate may have affectedtheir results. probleinin flounderculture, because it usually In thisstudy, rearing experiments were conducted decreasesthe marketprice of the fish. todetermine if light directioii and intensity or bottom Norman 934! dividedhypertnelanosis of substrateare the most importantfactors for flatfishesinto three types by its characteristics: hypertnelanosisin cultured Japanese flounder. staining,spotting, and true arnbicoloration.The stainingtype is the most common in cultured MATERI/V 'S AND METHODS Japaneseflounder Yarnamoto and Oda 1991! Somefactors such as illumination on theblind side, Fish of about lg body weight were food,and stocking density are considered to affect obtainedfroin commercial hatcheries, andwere kept thistype of hypermelanosisin Japanese flounder in a tankwith a recirculatingseawater system until Seikai 1991, Suzuki 1994,Takahashi 1994!. the startof experiments. Amongthese factors, illumination to theblind side Experimentswere carried out in aquaria seemsto be thc mostplausible, because there are equippedwith a closedrecirculating system Fig, 40 UJNR Technical Report 'No, 26 ' ~ w vevwilvnwww>a>vY451v, v4 Figure 1. Experimental setup. Light intensity lux! Percentage of Survival rate Specific growth Bottom material of aqttarium t!ownward Upward byperrnelanosisf'sb %! %! rate %! Transparent plate 150 100 93 2.1 1300 60 100 80 2.3 1300 1100 100 93 2.2 Sand lcm thickness! 200 80 1900 14 1.4 Table L Effects of downward and upward light intensity on the blind side bypermelanosis of Japaneseflounder reared with or without sand on the bottom of the aquarium, expcritnent 1. 1!. The aquaria with transparent acrylic bottoms bottoms of two aquaria were covered with a 1-crn were placed on transparent acrylic plates. The layer of coarse sand particle size 0,5 1.0 mm!. insides of aquarium walls were made of matted Fifteen fish of about 6 g body weight without visible black vinyl chloride plates, and the top was covered pigmentation on the blind side were reared for 16 with a transparent acrylic plate. Downward and wk. At the end of the experiment, all surviving upward illumination was provided with fluorescent fish were anesthetized, and photographs of their lights installed above and below the aquaria. These blind sides were taken individually to examine the lights were turned on for 12 h/day. Fish were fed pigmentation. with a commercial pelleted diet twice a day for 5 [n experiments2 and3, three aquariawith days/wk during thc experimental period. upward light and different bottom conditions were Temperature was maintained at 23 'C. prepared as follows Tables 2 and 3!: ! a Experiment 1 was designed to examine transparent plastic plate and strong upward light, the effect of light intensity and direction on the ! a white opaque plate and weak upward light, hypermelanosis of the flounder in transparent ! similar light conditions as the first, but the plastic bottotn aquaria with or without sand. bottom was covered with a 1-cm layer of Experimental conditions are shown in Table 1. The transparent glass sand particle size 0.5 1.0 mm!. Iwata and Kiknchi 41 Light intensity lux! Percentageof Survival rate Specificgrowth Bottom material of aquarium Downward U ward hypernielanosisfish %! %! rate %! Transparentplate 88 2.9 White opaqueplate 1800 10 2.9 Glass sand 1900 1400 27 3.0 Table 2. Effectsof upward light andthc presenceof sandysubstrate on the bottom of the aquariuroon the blind side hypermclanosisof Japaneseflounder, experiment 2. Light intensity lnx! Percentageof Survivalrate Specificgrowth Bottom material of aquaritnn hypermelanostsSsh %! %! rate %! Transparentplate 1800 88 85 3.3 Wtuteopaque plate 1800 3.4 Glass sand 1400 18 85 3.4 Table3. Effectsof upwardlight andthe presence of sandysubstrate on thebottom of the aquariumon theblind side hypermelanosisof Japaneseflounder, experiment 3. Twenty-five fish of about 4 g body weight were and the changesin pigmcnted area were compared. reared for 12 wk in experiment 2 and 20 fish of about 1 g were rearedfor 16 wk in experiment3. RESULTS AND DISCUSSION None of the fish usedin the experiments had visible pigmentationat the start. Fishwith hypermelanosis The results of experiment 1 areshown in weredescribed in experimentl. Table l. In the aquarium without sand on the Experiment 4 was carried out to examine bottom, all fish showed hypermelanosison the blind theeffect of bottomconditions on the enlargement sideat theend of theexperiment, regardless of light of the dark pigtnentedarea on the blind side with intensity, However, only 7 and 14% of the fish three aquaria prepared as follows: ! bottom of showed hypermelanosis in the aquaria with sand transparentplate with strong upward light, ! on the bottom Fig. 2!. Specific growth rate of the bottoin coveredwith a 1-crnlayer of transparent fish was lower in the aquaria with a sand bottom glass sand and strong upward light, ! bottom than those without sand. covered with a 1-crnlayer of coarsesand to prevent The results of experiments 2 and 3 were upwardlight Table4!. Fish of about 25 g body similar to eachother Tables2, 3!. Namely, 100% weight,all of which hadpartial dark piginentation of fish had hypermelanosis in the aquarium with on their blind side, were reared for 4 wk. the white opaque plate bottom, and 88% in the Photographsof the blind side of individual fish were aquariuin with the transparent plate bottom. In takenat the startand at the end of the experiment contrast to these, less than 30% of fish showed 42 VJVR Teehnieel Report Vn, 2' dark pigmentationon the blind sidewith glasssand on thc bottom in spite of thc strong upward light. Specific growth rate was almost thc same among treatmentsin both experiinents. Sand on thc bottom ot thc aquarium was also effective for preventing enlargement of the pigmcntcd area on the blind side Table 4!. No fish showed cnlargcrncnt of their pigmcnted area in the aquaria with sand or glass sand on the bottom, while half of the fish enlarged their pigmented area in the aquarium with a transparent plate bottom. None of thc fish died during the experimentalperiod under any conditions, and thc specific growth rate varied with treatment. Previous papers concern ing the pigment.ation on the blind side in flatfish species suggested that light is the primary factor for such an abnormal coloration. However, from the results of this study, it is bcttcr to consider that, not light, but the presenceof sandy substrate on the bottom of the culture tank has an important role in this phenomenon. ln this study, sandy substrateon the bottom preventedthe occurrenceof hypermelanosis as well as its enlargement, As there is no other information that supports our results, more research Figure Z. Photographs of the blind side of Japauese is needed to determine how inuch the occurrence of flounder at the end of experiment l: a - Fish showing typical hypermelattosis, reared in an aquarium with a hypcrmclanosisdepends on sandy substrateor light. transparent acrylic plate on the bottom. b - Fish without Furthermore, sand on thc bottom of culture tanks visible pigmentation ou the blind side, reared in aquarium is not consideredto be practical, because it will with coarse sand ou bottom, easily cause deterioration of the culture environment by producing anaerobic areas. Therefore, alternative substrates will be required Ught intensity gnx! Percentageof fish Bottomnsaterisl of aquarium with bypermelanosisarea enlargement %! Transparentplate 1300 400 0.5 Glass sand 1500 300 0.1 1300 0 0.6 Table 4. Effects of bottom substrate,light intensity iluxl, aud direction on the ettlargemeatof blind sidehypermelanosis of Japaneseflounder, experiment 4. for practicalusage. Ambico!oration ia tank cultured Most of thepigmentation on the blind side f!ounder,Paralichthys deritatus. of the Hounderwas shown at a marginof thetrunk, Trans. Am. Fish. Soc. 104: ! 58- I 60. caudalpedunc!e, and basement of pectoralfin in Suzuki, N. 1994, Ultrastructure of the skin on thisstudy l'Fig. 2!, and its location wasthe sameas reverse side of hatchery-reared generallyseen in culturedJapanese flounder Seikai Japaneseflounder, Parali chthys 1991, Yainamotoand Oda 1991!. olivaceus, with reference to ihe pigmentation. Bull. Nansei Natl. Fish. ACKNOWLEDGMENT Res.Inst. 27: 1 -128 in Japanese!, Takahashi,Y, 1994. Influenceof stocking We wish to thankDr. Harry V. Daniels, densityand food at latephase of larval North CarolinaState Uni versity, for criticalreview periodon hyperrne! anosis on the blind of the manuscript, bodyside in juvenile Japanese Hounder. NipponSuisan Gakkaishi 60: 593-598 LITERATURE CITKD in Japanese!. Yamarnoto, S. and T Oda. 1991. Thc effects Cunningham,J. T, 1891. An experi ment of severaldiets on survival, growth aud concerningthe absenceof color from the color anomaliesin juvenilefounders lower sides of flat-fishes. Zoo!. Anz, 14: Paralichthysolivaceus. Bull. Fish, 27-32. Exp. Sta. OkayarnaPref. 6: 0- 8 Cunningham,J. T. 1893, Researcheson the in Japanese!. coloration of the skins of flat-fisbes. J. Mar. Biol, Assoc.U. K. 3 N. S,!: 111-118. Cunningham,J. T. 1895. Additionalevidence on theinfluence of light in producingpigments on thc lower sides of flat fishes. J. Mar. Biol. Assoc.U.K. 4 N. S.!: 53-59. Norman,J. R. 1934,A systematicmonograph of the f!atfishes Heterosomata!.Vo!urne 1. Brit.Mus., London. 459 p. Osborn,C.M. 1940.The experimental production of melaninpigment on the ! owersurface of surnrnerflounders Paralidithys detitattts!. Proc. Natl, Acad. Sci. U.S.A. 26: 155-161. Osborn,C. M. 1941. Studieson the growthof integuinentarypigment in the lower vertebrates.I. The originof artificial!y developedme!anophores onthe norma!!y unpigmentedventral surface of thesummer flounder Parali chthys dentatus!. Biol. Bull WoodsHole! 81: 341-351. Seikai,T. 1991.Influences of fluoresce light irradiation,ocular side pigmentation, and source of fishes on the blind side pigmentationin theyoung Japanese flounder,Paralichthys oli v acetous. Suisanzoshoku39: ! 73-180 in Japanese!. Stickney, R. R. and D, B. White, !975. Jones et ot. 4S MICROBIOLOGY OF EARLY LARVAL STAGES OF SUMMER FLOUNDER PAR4LICHTHYS DK1VTATUS GROWTH IN A RECIRCULATING WATER SYSTEM StephenH. Jones,Beata Summer-Brason Universityof New Hampshire JacksonEstuarine Laboratory Durham, NH 03824 e-mail:shjQchri sta.unh,edu and GeorgeNard i Great Bay AquaFarms,Inc. Portsmouth,NH 03801 c-mail:GAquafarm gaol.corn ABSTRACT Finfish in early larval stagesof growth can suffer high mortality in aquacultural facihties becauseof diseases and nutritionalproblems. Recent studies suggestthat bacteria associatedwith the live feed and hatchery environmentsthat colonize finfish can have beneficial or detriinentaleffects on fish health. A localcommerci aI facility that growssummer flounder in a recirculatmg water system has been the subject of microbiological studies for their f estfour production runs. The culiiue of summer flounde is in its infancy arutthe microbiology of thesefish is not well characterized.Samples of fish,tank water. and feedcollected at times of changein feting regime,metamorphosis and episodic high mortality aad diseaseevents were analyzed for different bacteria, Growth media targetmgtotaI heterotrophs, total vibrios and Vibrio anguiiluruin were usedto enumerateand isolate bacteria. Isolates were identified to species and/or genus. Differences and similarities in nucrobial communitydiversity and abundance at differentlife stagesand feedingregimes were noted. 'Ibe resultsprovide an initial databasefor determimng tbe role of bacteria in the onset of diseaseand the health of early stagesof sunuuerflounder growth. INTRODUCTION predictingthc onsc.t of disease.Prophylactic and direct treatment of diseases often invo/ves use of Aquacultureis becomingwidespread and antibioticsand vaccination CahiH 1990, Joostenet growingrapidly in northernNew England,USA, nl, 1995!. There are inanydisadvantages to using and thmughoutthe world. Among manyuncer- antibiotics,including the potentialfor evolutionof tainties,one of the biggestis theincidence of dis- drug resistantstrains Kapetanakiet al. 1995!, easein thef ishbeing cultured. Diseases can cause harmfuleffects on fish eggs Munro et al. 1995!, significantfish mortalities,especially in early life negativeeffects of seawater Barnes et al. 1995!, stages,and such events are obviously catastrophic and complexgovermnental regulations. In recir- to any industry. culating aquaculturesystems RAS!, the use of Bacterialpathogens that cause diseases in antibiotics is even more limited because of the need fishoften enter the host with ingested food or feces for establishingstable microbial cosnmunities on and colonize the intestinal tract Romalde et al. biofiltersneeded for removingwastes. An alter- 1996!. The bacterialdiversity is enormousin fish nativeapproach to diseasemanagement is theuse tissueand hatcheryenvironments Muroga et al. of probioticbacteria. This approachemploys use 1987,Nicolas et al. 1989,Sorgeloos 1994!, mak- of the beneficialor benign~ microflora asso- ing it di%cultto identifypathogens or monitorfor ciatedwith healthyf ishto establishand maintain a UJsiR TechnicalReperr hlo. 26 microflorathatcan suppress potential pathogens fishwere transferred to12 weaning tanks and fcd andpromote fish growth. Inhibition can be ac- artificialfeed weaning diets. complishedbyproduction of toxins Fouz et al. Samplesfor microbiologicalanalyses were 1995!,siderophore production Pybus etal. 1994! takenfrom different tanks on a weeklybasi s. The prby competitive exclusion of pathogens Smith justificationfor notsampling specific tanks in a andDavey I 993!. consistentfashion was that fish reared in specific GreatBay AquaFarms GBA! is a land- larvaltanks were mixed into different weaning basedRAS facility located in Portsmouth,New tanks,and some weaned fish were remixed between Hampshire,that is uniquein NorthAmerica for weaningtanks. These factors made it difficultto thecombination of system and the cultivation of conductanalyses under controlled experimental summonerflounder Paralichrhysdenaius. Different conditions,sosampling was eventually focused on aspectsof the cultureand diseases of twoother tankswith clearly distinguishable healthy and un- biologicallysimilar flatfish species Japanese healthyfish. Samplingfor sick and healthy fish flounderand turbot have beenstudied. How- involvedpaired fish sainplesfrom the same tank ever,little is known about the microbiology ofsmn- onany given sample date. Tank water tetnperature merflounder, especially inan RAS. An early study remainedrelatively constant, ranging from I 6,4to identifiedVibrio anguiliarurn asa common.patho- 19.9'C.Salinity ranged from I 8 to32 ppt. genassociated with kidney tissue in deadfish, both Accurateestiinates of fish densitiesin all feral«nd cultured, from the coasts ofNew Harnp- rearingtanks were not available, so percent sur- shimand Maine Strout et al. 1978!.More recent vivallcould not be calculated, The densities in tanks workin New Hampshire hasfocused largely on rangedfrom 100,000to 200,000fish in tanksnot theincidence andecology ofhuman pathogenic affectedby disease, and substantially lower in tanks @briosp. in theGreat Bay estuary Jones et al. wheredisease had been present, Assessment of 1991,O'Neili et al. 1992,Jones et al. 1997!, the thedegree of mortality offish was based on quan- sourceofwater for GBA culture tanks. The pur- tifyingdead fish on adailybasisin eachtank. Sick poseof thisstudy was to determinethe effects of fishwere identified by altered pigmentation and intestinalrnicmflora and the culture environrnen- feedingbehavior, talconditions onthe health and survival of larval Fish,feed, and water samples were col- summer flounder. lectedusing sterile containers and transported on iceto the Jackson Estuarine Laboratory for analy- METHODS sis,Samples were processed within 2 hof collec- tion,The fish were anesthetized, measured, sur- GreatBay AquaFarrns,Inc is a comtner- facesterilized, and ground with a mortarand pestle. cialhatchery dedicated to theculture of summer Tissue,water and feed samples were diluted in flounder.Young larvae are grown from fertilized sterilebuffered peptone water and aliquots from a eggs,provided by broodfish on site, in recirculat- rangeof dilutionswere collected onto membrane ingcultum tanks until the juveniles reach a sizeof filtersand placed on different agar media. Total 5-10g -8crn!, at which time they are transferred heterotrophswere cultured from 2216E medium, toon-growing operations. The focus of this study total vibrioswere cultured from thiosulfate-citrate- wasthe fourth production run sincethe startof bile salts-sucrose TCBS! medium, and V. GBAin 1996,which began on 22 March1997. anguillanrmwas cultured from VA1Vf agar Alsina Theconditions inthe rearing tanks were subject to etal. 1994!,all incubatedatroom temperature 8- manychanges during the 100-day study, including 22'C!. Focuswas placed on vibrios because they feedingregime, tank disinfection andcleaning, and havebeen shown to be important inother aquacul- movementof fish between tanks. Larvae were fed turalsettings both as agents of diseaseand as ben- algaeand rotifers ineight larval rearing tanks for eficiaI'probiotic' organisms, and are the dominant thefirst 20 day,then Artemia nauplii followed by bacteriain theintestines of larval and juvenile enrichedAnemia for the next -20 daysprior to marinefish Murogaet al, 1987!.In addition,the metamorphosis.After35-40 days, metamorphosed sourcewater from the Great Bay estuary is known Jotieset al. 47 to have abundantvibrios Joneset al, 1997!,par- tality in all of thelarval-rearing and weaning tanks. ticularlyyduring the time of the productionrun un- The first spike in fish mortalityoccurred within a der study. week after feedingon Arremiathat beganon day Dominantand unique colonies on all plates 2l. The salinityin thetanks dropped from - 25 ppt wereidentified using an identificationscheme simi- to 18 ppt betweendays 27 and29, when9.7 cmof lar to Murogaet al. 987!. Colonymorphology rainfell in 48 h, droppingthe salinity of thesource and color plus carbohydrateutilization reactions estuarine water in the process. A consistent, me- were noted, isolated colonies were re-streaked onto dium level of mortality persistedin someof the TSA medium andthe colony morphology and color tanksduring the first 3 wk of weaningdiet, fol- plus pigmentproduction were noted for re-grown lowedby a shghtdrop in mortalityrate, The per- isolates.Cell morphology,motility, oxidasereac- sistentmortality in tanksafter day 60 wasnearly tion, andgram reaction were determined. Gas pro- all associatedwith delayedmortality in tanksthat duction,growth, and acid productionwith single hadshown good survival early in theweaning pe- carbonsources were determined along with amino riod. acid decarboxylasereactions. Growth at different Total vibrio concentrations increased dra- salinitiesand temperatures were also used to iden- maticallyin the rearingtanks after day 20 when tify bacterial isolates. A rremiafeeding began Fig. 2!. The highestcon- centrationof vibriosoccurred during the time when RESULTS the first heavy inortality occurred. Total het- ettstrophconcentrations increased only afterday Figure 1 illustratesthe dynamicsof rnor- 30, followingthe spike in vibrionumbers. A sinall i r 0.9 4 o.e- 0.7 i e 0.6 I- y ~0 0.5t = .'-041. 0.3- 0.2- o.t -' 04 rv n m m t n 0 r, e Ash ttge dtty! Figure1. Degreeof fish mortalitygati larval ~ days!attd weatiiitg 2-98 days!tanks. UJNR TechnicalReport No. 26 ~ Tacat hcrrrrrrnrphr 50066 e R P. 4tOXO a 300000 ~ 3000tn i2 20 26 33 41 48 53 62 69 77 K3 90 95 Hahace rtay! Ftgrtra2. Bacterial concetratiorrs inlarval anti weaning tanlrs. cfu= colony-forming units. peakin concentrationsof putative yellow colo- nieSOn VAM agar! V. arrgrrilfr2raurs cOinCided with thetotal vibrio peak data not shown!. Relativelylow concentrationsof all bacte- riawere apparent inthe tank water from day 45 to day69, followed immediately bya secondlarge peakin totalvibrio concentrations Fig. 2!. This ! mum secondvibrio peak occurred at thebeginning of June,when estuarine temperatures began toincrease a g aboveIS'C andmicrobial coinmunities dramati- IsrrooD callychange, typically characterized bysignificant increasesin the diversity and population sizes of Vibriosp, O' Neill et al, ]992!. The salinity in the culturetanks also increased from the low of l 8ppt I orrMo onday 29 to 32ppton day 70. The second peak in totalvibrios also corresponded with theonset of anotherincidence ofelevated mortality, nearly all of whichoccurred in tanks that had relatively healthyfish early in the weaning phase. Thus, the microbialdynamics in thefish tankshad soine ie- 0 httionshiptothe occurrerice ofdiseaMmortality m a thefish. Peaks intotal heterotrophs, totalvibrios, rrlrhrari&r! andV. rrrtgtriHarrrrrr alsooccurred on day 90 po«- Figure 3. Bacterial concentrations in "beatthy" iisb. cfu = hatch. colony frumingunits; DW = dry weigbL J0000 et Ol. 49 Thedelay in onset of increases in total het- IIXRxxo crotrophpopulations relative to totalvibrios was notseen in thehealthy f ish.Simultaneous peaks in IIMNXO concentrationsfor both tota1heterotiophs and to- tal vibriosoccurred on days33, 77, and95 Fig. 3!, a three-peakpattern similar to themicrobial. I IXXXO dynamicsinthe rearing tanks. A comparisonof total vibriosin healthyand unhealthy fish taken 3o0 fromthe sametanks on five samp1edates from day I 0000 3 41thrtnrgh day 83 showed unhealthy fish had higher ii concentrationsof totalvibrios than healthyfish on fourof thcfive sample dates, with overall average concentrationsin unhealthy fish >10 timeshigher thanin unhealthy fish Fig.4!. In contrast,tottd heterotrophconcentrations werehigher in healthy xa fish in the first four of the five satnples Fig. 5!. Concentrationsof total heterotrophs in unhealthy fishwere inuch higher in the fifth sample and the overallaverages for unhealthy and healthy fish were similar,The TCBS mediumfor recoveryof total 1 0 10 ro 30 lo 10 00 10 ol BraOsr *yi 10000M Agttre5. Totalhetrotrophic bacteria concentrations ia "healthy"Ond "SiCk" fiSh. Cfu = colonyfarming unitS; IOton DW = dry weight. v ibriosrecovered higher numbers of bacteriathan 10100 the 221 6E medium. ta Predominantbacterial isolates froxn live 0 feedand tank water wereidentified to speciesand/ orgenus Table 1!. Theresults are biased toward 1$KI isolationof vibriosbecause of theisolation media used. The feedhad a inorepredoini nant presence Ii of Vibriosp. although vibrios occurred inboth the 'Z 100 feed and the water. Numerous bacteria were presentin the rotifers and the Arremia. The tank watercontained many spec ies, with major changes incomposition accompanying changes inthe feed and tank environrtienL Moraxella sp, was the mostconsistently prevalent organisin. Otherwise, therewere few similaritiesbetween isolates froin the tank water and the feeds. 0 10 ro ai 00 xI 00 i0 lO 00 0100000 Ftlttta 4. Totalvibrio concentrations in "healthy" oad Bacterialnumbers and species composi- "sick"fish. cfu = colonyfunning units;DW = ttry tionvaried widely during the early stages of sum- weight, stt UjlVR TecttttieatRcport No, sd 1-20 days 21-33 days Rotifcrs/algae Artois Morrmlla sp, Morme rasp. Vibrio sp.I Viibriosp. I Vibrio sp.111 Vibrio sp. 111 V. algittolyrictts V, damsela Ea~eriaceae Eatetobactetiaceae Ftavobacterittiit sp, V.partthaemolytictts Aeroatonarsp. Tank waiter Acineiobacrersp, Argobacrerivrrtsp. Aerorrtarias sp. Enteaobacteriacctte MoroxeNasp. Moivtteliasp. Psettdcueonassp, INIV Psetrdoatonas sp. MI V,Jlvvialis Vibrio sp. 1 V. angttillarstttt V.algmolyticttr V.paruhaeittoiyiicve 'Ihbtet, Bacterialspecies inlive feed and tank water for summer flounder atGreat Bay Attuafarrns. mer floundergrowth at GBA. Factorsthat could summer flounder. However, fish considered have affected the abundance and succession of healthywere present in tanksthat also contained bacterialspecies include the microflora of thelive unhealthyfish, making cross contamination highly feed, nutritionaldifferences in feeds,fish growth probable.The simplepresence of vibriosis appar- and changesin physiology,seasonal changes in entlynot a clearindication of diseasepotential in source water, the transfer of fish between tanks, summerflounder. However, the general trend of andenvironmental conditions in the culturesys- highernumbers of total vibriosin unhealthycom- tem. Othershave reported similar microbial com- paredto healthyfish suggests that total vibrio counts rnunitydynamics and species composition in a va- maypro vide a betterreflection of diseasethan to- riety of culturedfinfish Campbelland Buswell tal heterotrophcounts. The earlieroccurrence of 1983, Murogaet al, 1981, Nicolas et al. 1989, a peakin totalvibrios compared to total heterotro- Sorge!oos1994!. The similarityin speciesdiver- phsin tankwater just prior to thefirst episode of sityand abundance of differentbacteria with other highmortality suggests that monitoring total vibrio studiessuggests that thereare no uniqueinicro- concentrationsin tank watermay be usefulin pre- biologicalcharacteristics of summerflounder or dictingdisease. northernNew Englandculture conditions. The microflora of the feed and cttlture en- Therewere peaks in bothtotal hetcrotro- vironmentwas dominated by Vitbriosp. 'Ihe useof phsand total vibrios that corresponded toughly with traditionalculture methods provides results that are elevatedmortality episodes. These peaks were stronglyinfluenced by thecornpositi on of the iso- observed in both the tank water and the fish tis- lation media and the isolation conditions used. sue. Munroet al. 995! clearlydemonstrated that Becauseheterottophs other than vibrios were also V.angttillarurn is a pathogenof larval turbot,and present, the lower numbers of bacteria recovered Rico]aset al. 989! reporteda dominationof the on2216E medium compared tn TOMBS suggests that rotifermicroflora by ttrtbrio sp. associated with high a bettertnediurn for recoveryof total heterotrophs ~ity of larvalturbot. In thisstudy, vibrios were isneeded. Others have reported that Vibriosp. are associatedwith unhealthy fish, but also with healthy the dominant bacteria in the intestines of larval and fishand their tank water, even in highnumbers at juvenilemarine fish Murogaet al. 1987!. The use certaintimes during the early growth stages of the of TCBS andVAM agarsin thisstudy anticipated this,biasing the resultsin orderto provideisolates 169. thattnay be usefulin futurestudies on probiotic Barnes,A.C., T.S. Hastings, and S.G,B. Amyes, bacteriaandpathogens. The phylogeny of thefish 1995. Aquacultureantibacteria!s are an- pathogensand general microflora would be better tagonizedbyseawater cations. J,Fish Dis. determinedusing molecular methods Amann et al. 18: 463465, 1995!,although this was clearly not the purpose of Cahi!l,M,M. 1990, Bacterialflora of fishes:a review. Microb. Ecol. 19: 21-41. this study. There were a few apparentdifferences Campbell,A.C. and J.A. Buswell. 1983. The in- betweenthe fish andtank water microflora, as well testinalinicroflora of farmedDover sole as in the abundanceof bacteriain healthyand un- Soleaso ea!at differentstages of fish hea!thyfish. These pre!iininary observations sug- development.J.App!. Bacterio!, 55: 215- gestthat detection ofthe selection ofbacterial spe- 223. ciesin fish both during colonization ofthe fish in- Fouz,B., B. Novoa,A.E, Toranzo, and A. Figueras. testinefrom live feed andduring disease episodes 1995.Histopathological lesions caused by maybe diffiicult using the methods in thisstudy, Vibrio damsela in cultured turbot, Moredetailed data on abundance of bacterial spe- $cophxhalrnusmaximus L.!: inoculations ciesand speciation of isolates from healthy and with live cellsand extracellular products, unhea!thyfish during the targeted production run J. Fish Dis. 18: 357-364. andother runs at GBA arecurrent!y being ana- Jones,S,H., K R.O' Neill, and T.L. Howe . ! 991. lyzed,and the results will hopefully provide clearer Differentialelimination of indicatorbac- resultsfor a futurepublication. Further work and teriaand pathogenic Vibri osp. from Maine refinementof inethodsare needed to tnoreclear! y oysters Crassos trna virg riii ca! in a coin- identifyprobiotic and pathogenic bacteria. This inercialcontrolled purification facility. J. studyalso suggests thatfurther work should bedone Shellfish Res. 10: 105-112. to betterunderstand the relationship between the Jones,S.H., R, Langan, W.H. McDowe!l, and B.W. inicrofloraofthe live feed and the eventual co!oni- Summer-Brason.1997. Influenceof or- ganicand nutrient pollution onfecal-home zationof larvaland juvenile fish. andindigenous bacteria inestuarine envi- ronments,Final report, National Estua- ACKNOWLEDGMENTS rineResearch Reserve System, Sanctuar- Theauthors gratefully acknowledge Steve ies and ResearchDiv., OCRM, NOAA, Eddy,Chris Duffy, and Gaston Gingues fortech- SilverSpring, MD. nicalexpertise and laboratory support, The re- Joosten,P.H.M,, M, Aviles-Trigueios, P.Sorgeloos, searchwas supported by UNH/UMSea Grant andJ.H.W.M. Rombout.1995. Oral vac- Co!!egeProgram Project No. RIF!AD-! 44, and a cinationofjuveni!e carp Cypririuscarpio! matchinggrant from the New Hampshire Indus- andgilthead seabream Sparus auraxa! trialResearch Center and Great Bay AquaFarms, withbioencapsulated Vibrio angui Kzrum bacterin.Fish She!Fish Imxnunol. 5: 289- Inc, 299, Kapetanaki,M., J. Kerry,M. Hiney,C O'Brien. LITERATURE CITED R.Coyne, and P. Smith. ! 995,Emergence, Alsina,M., J. Martinez-Picado, J.Jofre, «nd A.R. inoxytelxacycline-fiee marine mesocosins, Blanch.1994. A mediuinfor presump- ofmicroorganisins capable ofcolony for- tiveidentification of Vibrioangui lkirum, inationon oxytetracycline-containing me- Appl.Environ, Microbiol. 60: 1681-1683. dia.Aquaculture 134: 227-236 Amann,R, I.W.Ludwig, and K.H. Schleifer. 1995. Munro,P.D A. Barbour,and T.H. Birkbeck. Phylogeneticidentification andin situ de- 1995.Comparison ofthe growth and sur- tectionof individual microbial cells with- vivalof larvalturbot in the absenceof outcu]tivation. Microbiol. Rev. 59: 143- culturablebacteria with thosein thepres- s2 UJ'va TechnicalRcport bio. Js enceof Vibrioanguillaruiir, Vibrio algirrolyricus,ora marine Aeromonas sp. Appl.Environ, MicrOiio. 61; 4425-4428, Muroga,K., M. Higashi,and H, Keitoku.1987. Theisolation of intestinalmicroflora of farmedred seabream and black seabream Acarrrhopagrussclrlegeli! at larvaland juvenilestages. Aquaculture 65:79-88. Nicolas,J.L., E. Robic, and D. Ansquer. 1989. Bacterialflora associated witha trophic chainconsisting ofmicmalgae, rotifers and turbotlarvae: influence ofbacteria on lar- valsurvival. Aquaculture 83 237-248. O'Neill, K.R., S.H, Jones, and D.J. Grimes. 1992. Seasonalincidence ofVibrio vubrifrcus in theGreat Bay estuary ofNew Hampshire andMaine. Apph Environ. Microbiol. 58: 3257-3262, Pybus,V., M,W. Loutit, LL, Lamont,and J.R. Tagg. 1994. Growthinhibition of the salmonpathogen Vibrio ordalii by a siderophoreproduced by Vibrio anguilloruirrstrain VL4355, J. Fish Dis. 17' 311-324. Romalde,J.L B, Margarinos, S.Nunez, J,L. Barja,and A,E. Toranzo. 1996. Host rangesusceptibility of Earerococcus sp, strainsisolated from diseased turbot: pos- sibleroutes of infection.Appl. Environ. Microbiol.62; 607-611. Smith,P. and S. Davey. 1993. Evidence forthe competitiveexclusion of Aeromonas salmorricidafrom fish with stress-induced furunculosisby a fluorescent pseudornonad,J, Fish Dis. 16;521-524, Sorgeloos,P. 1994 Stateof theart in marinefish larviculture, World Aquacult. 25; 34-37 S trout,R.G., E.S. Sawyer, andB.A, Couterrnarsh. 1978,Pathogenic vibrios in confmement- rearedand feral fishes of theMaine-New Hampshirecoast. J. Fish. Res. Board Can. 35: 403-408, i uruita 53 NUTRITIONAL REQUIREMENTS IN BROODSTOCKOF MARINE FISHES Hirofurni Furuita NationalResearch Institute of Aquaculture Tarnaki,Mie 519-0423,Japan e-mail:furuitaCnria-tmk.affrc.go.jp ABSTRACT Thepresent work reviews the relationship between broodstock outrition and quality of egg and larvae in marinefish. Nutrientsin thediets have profound effects on gonadal development in fish. Eggproduction, hatchingrate, and larval survival are negatively affected bydeficiency in nutrients such as n-3 highly unsaturated fattyacids and a fewvitamins in the diet. protein quahty and quantity also have an effect on egg quahty. Effectivebroodstock diet, however, cannot be developed aslong as the nutritional requirements of broodstock remainobscure. Supplementation ofcomponents tothe diet for growth may bc requited for further enhance ment of thenutritional quality of broodstockdiets. More research effort is neededon broodstock nutrition and reproductivephysiology for theimprovement of seed production. INTRODUCTION andn-3 highly unsaturated fatty acids n-3 HUFA!, in particulardocosahexaenoic acid DHA!, are Gonadaldevelopment in several species of essentialfor larval development Watanabe 1993, fish is greatlyaffected by broodstocknutrition. Furuita er al. 1996a, b!. When red sea bream Duringthe last decade, inneming attention has been brotxisttockwere fed a dietcontaining a highcontent paidto therole of individualnutrie.nt components of cornoil anEFA-deficient diet! before and during in broodstockdiets Bromage 1995!. Nutritional spawning,the percentage of viable eggs, hatching studies in broodstock of marine fish have been rate, and normallarvae were significantlylower conductedmainly on seabreams red seabream thanthose of thecontrol Fig. 1! Watanabeer al. Pagrusmajor and gilthead seabream Sparus 1984a!.There was also a directcorrelation between aurora. Little is known about the nutritional thelevel of broodstock dietary n-3 HUFA and larval requirementsof broodstock in other species such growthin gilthead sea bream Tandler etal. 1995!. as flotinderin spiteof the importanceof these Larvae from broodstock fed a diet in which n-3 speciesin aquaculture.It is importantto review HUFAwas completely excluded had a 34%growth data and currentproblems affecting bmodstock retardationcotnpared tolarvae from broodstock fed nutritionfor futureresearch. The majorgroups of a highn-3 HUFA diet 5 rng/gdiet!. While there feed componentswhich have been previously wasno significant effect of dietary n-3 HUFA leveLs studiedare essentialfatty acids EFA!, proteins, on32-day stsrvi val, the s wirnbladder inflation r atc and several vitamins. Table 1 shows effects of the was affected significantly. The fatty acid feedcotnponents on egg and larval quality in marine compositionof eggs is directly affected by the fatty fish. The purposeof thispaper is to summarize acidcomposition of the broodstock Mourente and and discuss current information on the nutritional Odriozola1990!. Some fatty acids affect the egg requirementsof broodstockand to suggestareas qualityof theJapanese flounder Paralichthvs for further research. olivaceus Fig. 2! and the Atlantic halibut Hippoglossushippoglossus Pamsh er a1.1994!. Essential fatty acids However,it is often observedthat the n-3 HUFA Lipids play a major role as merubrane contentof red seabream eggs hasno relationto constituentsand energy reserves in fishembryos, eggquality Watanahe 1985!. 54 tJJNRTecbaicel Report Ko. 2' sl 0 g -g8 R g +ZAN + + + + +,, +3++ ++ + + + + I I I + + < > +++ ++ + + + ~ ~ + + ~ ' + H + + +f + + +++ + 4++ 4+ + 0 Faruita ss High Cuttlefish Raw Com oil" protein meal krtll' Eggproduction 149.5 12 l.6 202.1 58.7 x 10 /fish! Viable eggs %! 49.1 68,6 82,1 18.2 Hatchmgrate %! 83.] 93.7 90.3 27.3 Normal larvae 'Ya! 51.6 B2.2 91,2 24,0 Finalproductivity 21.1 52.8 681 12 of larvaefrom totalcgg produced able 2. Effect of broodstock.diet on spawningand egg quality in red seabream. Tandlerer at, 1995!. In conclusion,the nutritional value of lipids in the broodstock diet has a considerable effect on e eggand larval quality.Mobilization of bodystores of EFA during spawning can probably only 75 compensatefor tninordeficiencies in thediet. For CSI C optimumlarval growth,survival, andswimbladder C ~o inflationrate in giltheadsea bream, the broodstock 50 dietmust include at least15 rng/g diet of n-3 MFA, with 50-60% DHA Tandler et at, 1995!. Femandez-Palacioserat. 995! also suggestedthat eggquality in giltheadsea bream can be improved >8 by increasingthe n-3 HUFA levelto 1,6%. Thisis similarto the levels reportedfor red seabream by Watanabeand co-workers. However, a highlevel EFA~ietrrt of dietaryn-3 HUFA is likely to havea negative Fignrn t. Etreet of dieuny EFA on egg quality of red sea effect on larval survivalof gilthead sea bream bream drawnfrom datain Watanabeei al. 1984a!. Fernandez-Palacios er at. 1995!. Furthermore, recentstudies suggest the importance of the ratio of n-3 series ton-6 series HUFA in broodstock diet In fish like the red sea bream which feed and that efforts should be directed toward duringspawning, egg quality is affectedby diets establishing the optimum ratio of DHA/ given shortlybefore spawning Watanabeer al. eicosapentanoicacid/ arachidonic acid in thediet 1984c!. 'Theegg qualityof red seabream fed a Bell er at. 1997!. fishmeal diet is improvedby feeding them raw kril] andthe egg quality of broodstockfed a cuttlefish Prratein mealdiet was reduced by feedingan KFA-deficient The protein level and quality in diets for diet Table 2!, Changesin egg compositionand broodfish affect the repnxlttctive ~ormance. Egg egg and larval quality after a change in the production is reduced both in rcd sea bream broodstockdiet occurredwithin 15 days Fig. 3! Watanabe er at 1984a! and sea bass s1 UJNRTechuteal Report lva. 2' Docosahexaenoic acid Arachidonic acid 60 br 60 60 e c 40 40 |t 20 0 0 2324 25 26 27 26 1.4 1 6 1.6 2 22 Fattyacids in polarlipida %! Figure2. Fattyacid levels ineggs aud hatching rates ofJapanese flounder U Viable atty aa 8 Atttttarinategg 40 6 Hatetttrsgrata 10 ~ 20 10 Dieticaller dietary change Figure 4. Spawnproduction of giltheadsea bream broodstocit redrawn froin Fernandez-Palarios etai, 1997!. Ftgure3. Effect ofchanges indietary hpid ou egg viability FM denotesfish meal; DA4 = defattedfish meal; SM = ofgilthead sea bream redrawn from Tandlei etal. 1995!. squid meat;DSM = defattedsquid meal. Dicerttrarchttslabrax Cerda et ai. 1994!by evaiuatedthe impOrtanceof squid meal protein loweringthe protein level from 50 to 35%. An extractfor gilthead sea bream by replacing it with optimumprotein level for broodstockdiet was equalamounts of caseinor wheatgluten, The estimatedto be around45% for red sea bream resultssuggest that the positive effect of squid Wataruabeetal. 1984a!. Ctittlefish meal and squid proteincould be attributedto its similarityin tnealare superior to fishmeal as protein sources essentialamino acid EAA! cotnposition tothe egg forred sea boun Watanabeet al. 1984b!and protein.Based on this information, it was possible gi!theadseabream Fernandez-Palacios etal, 1997! toimprove the wheat gluten diet by supplementing Fig.4! . Watanabeetal. 991a! showed that the it withan EAA profile which resembles that of the effectivecomponent ofcuttlefish meat is contained egg. Suchdiets resulted in a doublingof survival innonfat-soluble fraction. Tandler etal 995! at 15 dayscompared with the wheat ghtten diet Fu mits 57 30 theAsA contentin eggsbefore spawning is critical act fornorinaJ development of thenewly hatched larvae 25 in seedproduction Ikeda 1985! A dietcontaining >a very low levels of AsA hasnegative effects on the 20 Japanese parrotfish Oplegnathtts fasciatus Ishibashi et al, 1994! and sardine Sardirtops at ntelanosticta Akiyama et aL 1990!. Ishibashiet al. 994! showedthat the gonadosoinaticindex 10 GSI! of fernale parrotfishwas coiTelatedto the AsA level in the diet, although the AsA content in the goriadwas not cotre!atedto the dietary AsA level. The numberof eggsspawned by sardine broodstockfed a dietcontaining 8 mg/100g AsA Dietary treat tnent was sigiuficantlylower coinparedto those fed a dietcontaining 320 tng/100g AsA Akiyarnaet al. Figure5. Effectof dietaryprotein on larvalsurvival at day 1990!. In the cod Gadus rttorhtta, differences in 15after hatciting, During 15 days of spawning,broodstock free aminoacid profile,egg strength,and neutral wtue fed diets containing a full squidprotein extract or a buoyancy were found between treatmentsof wheatgluten-based diet or a wheatgluten-based diet supplemented with essential anrino acids EAA! which different levelsof AsA in thedict, whereasno effects resembled thosenf the gilthead seabream egg redrawn On vital parameterssuch aS the fertilization rate from Tandler et al. 1995!. and survival rate were observed Mangor-Jensen et al, 1994!. Vitatrtin E Fig.5!. However,this was still lowerthan the full VitaminE VE! is knownto be essentialfor squid meal diet, The effect of protein reproductionof freshwaterfishes such as ayu supplementationof bmodstock diets on egg quality Ptecoglossstsaltivelis, commoncarp Cyprirttts wasnot found with changes in atninoacid profiles carpio, and rainbowtrout Oncorhynchttsrrtykiss Tandler et al. 1995!. Moreover, changes in the Watanabe1985!. The viabilityand hatchability aminoacid composition of eggswere sinai 1 despite of red seabream eggs Watanabeet aL 1991b! Fig. markedchanges in proteinquality of thebroodstock 6! andgilthead sea bream eggs Fernandez-Palacios diet Tandler et al. 1995!. Tandler et aL 995! et al. 1996!was improved by raisingdietary VE suggestedthat reduction in egg quality from levels. In Japaneseflounder, rates of fertilization broodstock fed an imbalanced EAA diet resulted andhatching were not affectedby the addition of from a change of the concentration at the VE to the diet althoughegg production increased vitellogenin Vg! bindingsites. For improvement comparedto the control Takeuchi1997!, It is not of egg andlarval quality, the protein should have a dear if thisphenoinenon resulted from differences similar EAA compositionto the egg proteinand in VE requirementbetween flounder and sea breams broodstockdiet shouldcontain 45-50% protein. or other factors. However, VE is suggestedto B~ dietaryprotein promotes Vg synthesisand playan importantrole in eggsand larvae of flounder uptake,which lead to highfecundity and egg quality sinceVE contentin eggsis usually high but quickly Tandler et aL 1995!. decreasesafter hatching Takeuchi 1997!. Further studiesare necessaryto clarifythe io1eof dietary Vitamin C ascorbic acid! VE in broodstocknutrition and egg production in Ascorbicacid AsA! is importantin the theflounder and other species. processof sexualinaturation as it playsa part in the biosynthesisprocess of gonadal steroid Astastiaathin hormones Sandnes 1984!, Since AsA is essential Feeding red sea breamfrozen raw krill for the biosynthesisof collagenin connectivetissue, shortlybefore spawning is knownto irnprovcthe SS UJNRTechaical Rcport No, 2$ astaxanthincontained in kri]]rnea] may have negativelyaffected spawning performance. Y5 Verakunpiriyaer af. 997b! examinedthe supplementationeffect of astaxanthinon the spawningperformance of yellowtai1 byfeeding diets containing various]eve]s of synthesized astaxanthin.The results indicated that optimum astaxanthinlevel in broodstock diet for yellowtai] is around30 ppm. Thenutritional value of thebroodstock diet considerab]yaffects cgg and larval quality. However,effective broodstock diet cannotbe Caatral Vltatnlaa pltaaphotlpttt Iataaattthla developedsolong as the nutritional requirements 6 vialseega 8 Hatdinttrata6 taamal larvae ofbroodstock remain obscure. In particular,the effectof micronutrientssuch as AsA,VE, and Ftttttre6. Effectsof t]tetary supptetnenatton withvitamin E, astaxanthinon spawttingperformance and egg phospholipid,andastasaotltin oncttg and larval quality in qualityisdifferent among species. Studies on other redsea bream drawn from data in Watanabe etal. 199 I b!. nutrients,such as vitaminA, are not available despiteits importantrole in deve]opment.When littleis known of the specific broodstock nutritional requirements,a practical composition ofbrnodstock dietcould be based on the general requirement of eggquality. The effective components inkrill were eachspecies. Supplementation ofcomponents such foundin both the polar and nonpolar]ipid fractions as vitaminsmay be requiredfor further Watanabeeral, 199]a,b!. Thekrill oilcontains enhancementofthe nutritional quality of brnodstock po]arand nonpolar fractions, mainly composed of diets. More researcheffort is requiredon phosphatidylcho]ineandtriglyceride, respectively, broodstocknutrition, oocyte maturation, and larval This]ed to thepostulation that the attributive deve]opmentfor the improvementof mass componentsmight be phosphatidylcholine inthe productiontechnology. polarand astaxanthin inthe nonpolar fractions Fig. 6!, Theegg qua]ity ofred sea breatn was impmved LITERATVRK CITED by a suppletnentof synthesizedastaxanthin Watanabeand Kiron ]995!. Akiyama,T., M. Shiraishi,T. Yamamoto,and K. Astaxanthin,along with other camtenoids, Hirose.1990. Effects of dietaryascorbic vitaminE, and phospholipids arethought toact as acidat the maturation and spawning time in quenchersorscavengers ofsinglet oxygens orother sardineSardinops mefanosricta. Abstract. freeradica]s, i e., they absorb the energy of these Meet.Jpn. Fish, Sci., Tokyo, p,l]0. [In JapaneseJ. cotnpoundswithin their extensive double bond structure,effective]y preventing reactive damage Bell, J. G, B. M. 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Importanceof docosahexaenoicacid in inarinelarval fish. J.World Aquacult. Soe. 24; 152-I 61. Watanabe,T. and V, Kiron. I995. Red sea bream Pagrusmajor!, pp, 398-4I3. In: N. R. Bromageand R. J. Roberts eds,!, BroodstockManagement andEgg and LarvalQuality, Blackwell Science, Oxford, Watanabe,T.,T. Arakawa, C.Kitajima, andS. Fujita.1984a. Effect ofnutritional qua!ity ofbroodstock diets on reproduction ofted seabream. Bull. Jpn. Soc. Sci. Fish. 50: 495-50]. Watanabe,T.,A. Itoh, C.Kitajima, andS.Fujita, 19841.EfFect of dietaryprotein levels on reproductionofred sea bream. Bull. Jpn, Soc.Sci. Fish. 50: 10I 5-1022. Watanabe,T.,A. Itoh,A. Murakami,Y. Tsukashima,C.Kitajima, andS. Fujita. 1984c,Effect ofnutritional qualityof diets giventobroodstock onthe verge ofspawning onreproduction ofred sea bream, Bulk Jpn. Soc.Sci. Fish, 50; I 023-l028. Watanabe,T.,M.-J, Lee, J.Mixutani, T.Yamada, S.Satoh, T.Takeuchi, N.Yoshida, T.Kitada, andT, Arakawa.1991a, Effective componentsincuttlefish meal and raw krill forimprovement ofquality of red seabream Pagrasmajor eggs. Nippon Suisan Gakkaishi57: 681-694. Watanabe.T.,T,Fujimura, M.-J. Lee,K. Fukusho, S.Satoh, andT. Takeuchi. I991b. Effect ofpolar and nonpolar lipidsfrom kriII on qualityofeggs ofred seabream Pagrus major. Nippon Suisan Gakkaishi 57:695- 698. Obkuba and Mslsnbars 6t SEQUENTIAL UTILIZATION OF FREE AMINO ACIDS, YOLK PROTEIN, AND LIPIDS BY DEVELOPING EMBRYOS AND LARVAE IN BARFIN FLOUNDER VERASPER MOSERI NobuyukiOhkubo and Takahiro Matsubara Hokkaido National Fisheries Research Institute 116Katsurakoi, Kushiro, Hokkaido 085-0802, Japan e-mail:[email protected],jp ABSTRACT Changesin contentsof freeamino acids FAA!, lipovitellin Lv! whichis the major yolk proteron in ovulated egg, and liplds were examined in developing embryos and larvae of barfin flounder trerospermoserr to elucidate the sequentialutilizatioo of these nutrientiaocks before first feeding. Hatchingtakes place on the 10th day after fertilization at a water temperature of 8'C, and the hatchedtarvae almost absorb their entire yoik sacs within 11 days after hatching. Total FAA content showedno change during the first 4 days, then decreasedto about 13% of the initial level by the 13th day after fertilization. During the process, non-essentialamino acids tendedto ~ faster thanessential amino ac:ids. Tbe lipovitellin content, measuredby quantitative imrnunodiff'usiimusing antiserumagainst 170 kDa Lv of ovulatedeggs, wasapproximately stable during the 13 days after fertilization, thea decreasedrapid! y until the end of yolk sac absorption. Phospholipids, which seemedto be boundwith Lv apo-proteins.decreased gradually after hatching,coinciding with the decreaseof Lv. From theseresults, we considerthe following four periodsfor the sequentialnutrient uhlization m barfin flounderembryos and larvae: 1! before FAA utilizationperiod, 0-4th day; ! FAA utilizationperiod, 4-10th day; ! switching period, 10-13th dsy; and ! Lv and phospbohpid utilization period, 13-21stday. INTRODUCTION are consideredto he the main substratefor energy metabolism. However, it is difficult to directly Yolk nutrient stocks of a teleost egg are determinethe proteinutilization by biochemical utilized as a sourcefor energy metabolisrnand for measurementsof wholeeggs and larvae, because it embryonicbody construction as in otheroviparous determinesonly the net sumof a decliningyolk animals.Generally in fish,carbohydrate, lipid, and protein and an increasingbody tissue protein pmteinare consutned prior to hatchingfor energy Heming andBuddington 1988!. production,while lipid andprotein catabolism Matsubaraand Koya 997! demonstrated predominates after hatching Heming and the occurrenceof yolk proteolysisduring oocyte Buddington1988!. Especiallyneutral lipids, such mattrrationin barfinflounder that spawn pelagic eggs astriglyceride TG! andwax ester, are considered having no visible oil globule. The to be the mostimportant energy reserves in fish ntaturatirm-associatedyolkproteolysis provides FAA eggs Vetter and Hodson1983, Hemingand and monomericlipovitellin Lv, molecularmass: Buddington1988!. 170kDa! in maturaleggs. In the presentstudy, we Recently, in some marine pelagic egg analyzedquantitative change of FAA andLv during spawners,free amino acids FAA! aresuggested developmentm barfinflounder to clarifythe pattern to be consumedas an importantfuel duringthe of thesenutrient stocks. Furthermore, wc analyzed energymetabolism of developingembryos and quantitativechange of phospholipids PL! andTG, larvae for reviewsee R snnestadand Fyhn, 1993!. which ate suggestedto be the major hpid classes In Atlantic cod Finn et al. 1995a! andAtlantic beingcatabolized by embryos and larvae in Atlantic halibut Finn et aL 1995c!,whose eggs have no halibut RainuzzoetaL 1992!and Atlantic cod Fraser visibleoil globules, amino acids FAA and protein! et aL ] 988, Finn et aL 1995b!. 62 UJNit TechnicalReport Sro. 26 MATERIALS AND METHODS procedures Phospholipid B-Test Kit and TriglycerideG-Test Kit, Wako!. Theadult male and female barfin flounders Statisticalanalysis was carried out by usedin the present study were kept in a 40-kL Duncan'smultiple range test. Significance was aquariaatAkkeshi Station, Japan Sea-Fartning acceptedat p < 0.05. Association JASFA!, inHokkaido. A totalof five groupsof fertilizedeggs A to E! wereobtained fromdifferent females during the April spawning RESULTSAND DISCUSSION seasonin ] 995and 1996, Eggs were artificially fertilizedandincubated ina flow-through hatching Thetotal FAA contentin an ovulatedbut cylinderat a temperatureof 8"C. Under these unfertilizedegg of barfmf]ounder was 267 nrnoV conditions,hatching occurred onthe 10th day after egg,corresponding to about 35 pg/egg Fig. 1!, fertilization, and the hatched larvae absorbed their TheFAA in an egg is suggested tobe provided by yo]ksacs within ]1 days after hatching, Eggs and degradationsofyolk proteins during thefind oocyte unfedlarvae were sainpled atthe time just before maturation Matsubara and Koya 1997!. The ferti]ization,andon the 2nd, 4th, 6th, 8th, 10th, increasedFAA seems toplay an important role in acquiringthe buoyancyof eggsas anosmotic 13th,16th, and ]9th dayafter ferti]ization, effecterfor oocyte hydration. The FAA content of MeasurementofLv, ammonia, and lipid contents eggsshowed norhangc during the first 4 daysafter werecarried out on all fiveseries A toE!, while fertilization,then decreased rapid! y to about 13% FAAcontent was measured onthree series A to C!. ofthe initial level by the 13th day. The 4th-day embryoalmost completed epiboly and was at the Forthe analyses of FAAand ammonia stageof early somite formation, andthe ] 3th-day contents,samples of 10 eggsor larvaewere larvawas at thestage of appearance ofpectoral homogenizedin0,25 ml of 6% trichloroacetic acid. fins. Aftercentrifugation at10,000 rpm for 10 min, the Theammonia content inan unfertilized egg supernatantswere col]ected and mixed with 0 5 ml was6,4 ninoVegg Fig. 2!. lt increasedrapidly ofdiethyl ether. The mixture wasthen centrifuged fromthe 4th to 8th dayafter ferti]ization and againat 2500 rpm for 3 minand the lower phase reacheda peak of 44 nmoVegg,coinciding with wasused for FAA and ainrnonia analyses. Amino acidswere ana]yzed using a ShimadzuLC-]OA analyzingsystem as described byMatsubara and Koya 997!. Ammoniaconcentration was determinedby thepheno]-hypochlorite method AmmoniaTest Kit, Wako!. For the measurementof Lv content, samplesofeggs and larvae were homogenized at C ~ 200 10 individua]s/m]in 0,9% NaC] solution. After 0 E centrifugationat 10,000rpm for 10 rni, the supernatantswere co]]ected. The Lv concentrations 100 inegg and larvae homogenates weredeterntined by st themethod ofMane ini et al. 965! using antiserunt against170 kDa Lv of ovulated eggs as described by Matsubaraand Koya 997!. 6 8 to 12 14 tB 18 20 Lipidswere extractedwith 0.3 rnl of Oaya altar fertltlzatlori ethanol-diethylether :1! fromthe samples of 10 eggsor larvae,The supematantswere collected Flgtsral. Changesin the totalcontest of l 6 faceamino aftercentrifugation at 6000 rpm for 10 min. acids FAA!of developingeggs and larvae in barfin Aounder,Data are pnesented asmean of threesamples Phospho]ipid PL! andtriglyceride TG! in the seriesA-C!. Hatchingis representedby a vertical supernatantswere quantified using enzymatic shaded bar, Ohbobo and Matsnbara 63 et al. 993!. However,no selectiveutilization of NEAA was observed in sotne inarine fish such as 50 Atlantic cod Rtsnnestadand Fyhn 1993, Finn et al. 1995a!. 40 ln thepresent study, we «lso measuredLv contentsin eggs and larvae using quantitative 30 immunodiffusion Fig. 4!. The Lv content of C 20 unfertilizedegg was 82lig/eggand approximately stable duringthe 13 days after fertilization. The Lv contentthen began to d~ significantlyuntil theendof yolk sac absorption. Thus, utilization of Z0 10 20 64 10812 14 16 18 20 Lv for bodyprotein synthesis and substratcs for Oaye altar fertllzation energysupply is suggested to occur during the late stageof developmentin barfin flounder.The Fttptre2. Amnonia contentof developingeggs and beginning of Lv utilizationfrom the 1 3th day after larVae m barfin nonnder. Data are preSentedaS incan Of fertil izationcoincides well with the end of total FAA five satnp!es series A-E!. Hatching is representedby a decrease, Therefore, it is suggested that the source vertical shadedbar. of aminoacid supply of barfinflounder larva shifts fromthc FAA pool to Lv atthe time of exhaustion of theFAA poolas rnentioncd by Rennestadet al 993!. thedecrease of total FAA content Fig. 2!. Frotn Generally, TG and wax ester neutral these results, FAA seems to be utilized as lipids'!are the mostimportant energy reserves of substratesin energy metabolism with the production developingfish on a caloricbasis. In contrast, some of ammonia as other pelagic eggs of marine marinespecies, such as Atlantic codand Atlantic teleosts see review by Rtsnnestadand Fyhn 1993!. halibutwhich have PL-rich eggs and relatively low After hatching,the larval ammoniacontent levelsof total lipid, appearto use PL as a tnajor decreasedrapidly. The decreaseof atnmonia lipidsubstrate in developingembryos and larvae contentafter hatchingis suggestedto be due to Fraseret al. 1988, Rainuzzoet al, 1992, Finn et excretion Risnnestadand Fyhn1993!. al, 1995b, c!. The contentsof PL and TG in an Figure3 showschanges in thecontents of unfertilizedegg of barfinflounder were 14.9 pg/ individualFAA of developingeggs and larvae, eggand 2.0 p.g/egg, respectively Fig. 5!, ThePL Amongthese, leucine, alanine, lysine, and serine contentwas approximately stable before hatching, were quantitativelydominant. We classified thengradually decrcmed until the 19thday. The leucine, threonine, lysine, valine, isoleucine, contentof PL on the 19th day was significantly arginine,histidine, methionine, phenylalanine, and lower pc0.05!than those of eggstages, On the tyrosineas essential arrtuto acids EAA! according otherhand, no significantchange occurred in TG to the classificationof Wilson 985!. Although contentduring the 19 days, Thus,the barfin the contentof all FAA decreasedfollowing the flounder larvae also use PL as a major lipid pmgressionof developinent,decline of eachFAA substrate.Nakagawa and Tsuchiya 97 l, 1972! was not equally shared between EAA and describetwo statesof majorlipid in eggsof rainbow non-essentialamino acids NEAA!. The contents trout:one is free lipidsactxunulated in oil globules, of all NEAA showedrapid decrease compared with andthe other is bound lipids binding to lipoproteins. EAA, anddecreased to lessthan 10% of initiallevel However,barfin flounder eggs have no oil globule, byhatching, On, the other hand, most EAA seemed and Lv contains about 15% of PL and 4% of TG to decem at a slowerrate than NEAA, especially Matsubaraand Sawano 1995!, In addition,the in tyrosineand phenyl alanine. Mere likely seems decease of PL coincides well with the d~ to be selectiveutilization of FAA duringthe egg of Lv afterhatching Figs,4, 5!. Therefore,most stagesin barfinflounder as mentioned by Risnnced of the PL in barfinflounder egg is thoughtto bind aa t'J!eg TecbaiCalReport Ne. 24 C 3 E2 C ~24 18 0 E1Z t 6 ~ 0 ~~ 10 8 l5 E $ z ~ 0 G 2 4 6 8 1G12141618200 2 4 6 8 101214161820 ~10 8 Tyrosine E O t: 4 $ z Q 0 2 4 6 8 101214161820 ~ 4Q ct C x 30 i5 E zo C q 10 tI Q ~12 21O Gycine Asparticacid ProHne O E6 C 2 tt. Q 02 4 6 8 1012141618200 2 4 6 8 1012141618200 2 4 6 8 101214161820 Oaysafter fertilization Days after fertilization Days after fertilization Figttse3. Changesin tbecontettt of individualfree amino acids FAA! of developingeggs and larvae in barfmflounder. Hatchingis representedby verticalshaded bars. This fractionwas a mixtureof threonme,ast:stragine, and glutamute. measuredthe content of this fractionby usingthreonine as standard. Oblsabo aad Matstsbara 65 100 stocksare suggested tobe supplied by Lv andother yolk proteins,These findings provide useful 80 information for improvement in nutrition of embryosand larvae beforefirst feedingin 60 aquaculture,as wellas for researchin clarifying 40 the nutritionalrequirement in developinglarvae. 20 ACKNOWLEDGMENTS 0 0 2 4 6 8 1012 14 16 18 20 The authorsthank S. Imarnura, A. Nisiki, K, Watanabe, and other staff of the Japan Days atter fertilization Sea-FarmingAssociation, Akkeshi Station, Hokkaido,for their kind help and supplyof the Ftgnre4. Changesin lipovitellin Lv! contentof barf in flounder. developingeggs and larvae in barfin flounder. Hatching is representedby a vertical shadedbar. LITERATURE CITED Finn, R.N., H.J. Fyhn, and M.S. Evjen. 1995a. Physiologicalenergetics of developing 20 embryosand yolk-sac larvae of Atlantic cod Gadtssrrtorhsas!. l. Respirationand ~16 nitrogenmetabolism Mar. Biol. 124: 355-369. ~ 10 Finn,R.N., J.R.Henderson, and H3. Fyhn. 1995b. Physiologicalenergetics of developing C embryosand yolk-sac larvae of Atlantic cod Gadtssmorhtsrr!. 2. Lipid metabolism andenthalpy balance, Mar. Biol. 124: 0 0 2 4 6 8 10 12 14 16 18 20 371-379. Finn, R.N., I. R@nnestad,and H.J. Fyhn. 1995c. Days atter tertllizatlon Respiration, nitrogen and energy metabolismof developingyolk-sac larvae Figtsre5. Changesin the.contents of phospholopid PL! of Atlantic halibut Hippoglossus and triglyceride TG! of developing eggs and larvae in hippoglossusL.!. Comp. Biochern. barfin flounder, Hatching is represented by a vertical Physiol.111 A: 647-671. shaded bar. Fraser,A.J., J.C.Gamble, and J.R. Sargent. 1988. Changesin lipid content,lipid class compositionand fatty acid cotnposition of to Lv apoproteinand to becomeavailable after Lv developingeggs and unfed larvae of cod degradation, GadtssmotAtsa!. Mar. Biol. 99: 307-313. From these results on the analysis of Heming,T.A. and R.K. Buddington.1988. Yolk biochemicalcoinposition of eggs and larvae in absorptionin embryonic and larval fishes, barfin flounder, wc consider the following four pp. 407-446. In: W.S.Hoar. and D.J. periodsfor the sequentialnutrient utilization in RaadaH, eds.!, Fish Physiology. Vol. 11A. barfin flounderembryos and larvae: ! before Academic Press, New York. FAA utilizationperiod. 0-4th day; ! FAA tilization Mancini,G., A.O. Carbonara,and J F. Helemans, period,4-10th day; ! switchingperiod, 10-13th 1965. Imtnunochemicalquantitation of day; and ! Lv andPL utilizationperiod, 13-21st antigensby single radial immunodiffusion, day, Furthermore,tnany partsof thesenutrient Innttunochemistry2: 235-254. ~ s UJvirt Techaicalttepars No. 26 Matsubara,T. and Y. Koya. 1997. Courseof proteolyticcleavage inthree classes ofyolk proteinsduring oocyte maturation in barfin flounderVer asper raoseri, a marineteleost spawningpelagic eggs. J, Exp.Zool. 278: 189-200. Matsubara,T. andK. Sa.wano.1995. Proteolytic cleavageof vitcllogeninand yolk proteins during vitellogeninuptake and oocyte maturationin barfin flounder Verasper rnoseri!.J. Exp. Zool. 272; 34-45, Nakagawa,H, andY, Tsuchiya,1971. Studieson rainbowtrout egg. III. Determinationof lipid composition of oil globule and lipoprotein,J. Fac. Fish. Anim, Husb. Hiroshim. Univ. 10: 11-19, Nakagawa,H. andY. Tsuchiya.1972. S tudieson rainbowtrout egg, IV. Changesof yolk contentduring embryogenesis. J. Fac. Fish. Anim. Husb. Hiroshim. Univ, 11: 111-118. Rainuzzo,J.R., K.l. Reitan,and L. Jtargensen, 1992.Comparative study on the fatty acid andlipid composition of fourmarine fish larvae,Comp, Biochem Physiol. 1038: 21-26, Rtsnnestad,I. and H,J, Fyhn. 1993. Metabolic. aspectof Free amino acids in developing marinefish eggs and larvae. Rev. Fish. Sci, 1; 239-259. Rtsnnestad,l., E.P, Groot, and H.J. Fyhn. 1993. Compartmentaldistribution of freeamino acidsand protein in developing larvae of Atlantic halibut Hippoglossus hippoglossus!.Mar. Biol. 116:349-354. Vetter,R.D. and R.E. Hodson. 1983, Energy metabolisrninrapidly developing marine fish egg,the red drum Sciaeaops ocellara!.Can. J, Fish.Aquat, Sci. 40: 627-634, Wilson,R.P. 1985. Amino acids and protein requirementof fish, pp. 1-16. Irr;C 8, Cowey,A. Mackie,and J,B. Bell. eds.!, Nutritionand Feeding inFish. Academic Press,London King and Bowel! 67 EFFECTS OF MICROALGAE AND LIVE DIET TYPE ON THK GROWTH OF FIRST-FEEDING WINTER FLOUNDER PLEURONECTFS AMERICAN US! NicholasJ. King and W. Huntting Howells Departmentof Zoology Universityof Ncw Hampshire Durham,New Hatnpshire03824, USA *Authorto whom correspondenceshould be addressed Phone 03! 862-2109 Fax 03! 862-3784 email: [email protected] ABSTRACT Tbeaddition of inicroalgaeto larvalrearing systetns "green water" treaiinent!,bas been shove to enhancethe growthand survival of cemuninarine fish species. Along with the presence orabsence of microalgae,diet type affectslarval growth, and several studies have demonstrated that cultured live food organisms e.g. rotifers and brine shritnp!are nutritious! ty inferior to wild zooplanktonas a first foodfor marinefinfish larvae. ln a 2x 2 factorialdesign experiment that lasted for iveweeks from first feeding, we examined the effects of greenwater, clearwater, wild zooplanktonand cultured roofers Bractuonus p//car les!on thegroisth of winter flounder Pleuronectesninericunus! larvae. Resultsfrom thetwo way analysisof varianceindicated that therewas no significantinteraction ~.80! betweenthe two factors presence/absenceof microal gae. wild/cultured prey!. Therefore,we consideredthe two factorsindependently of eachother. At any tiine, fish in thegreen water treatmentswere significant!y longer pep,05!than those in dear watertreatments. There were no differences Po0,05!in larvallengths between food types within eilher green or clearwater, The incan mstantanmus growth rates lengthincreases per week! were 15.4, 14.2, 1 2.2,and 9.6% for greenwater/wi!d zooplankton, green water/ rotifers,clear water/roofers, and clear water/wild zooplankton, respectively. Results of thisstudy indicate that greenwater enhances the growth of winter flounderlarvae, and there is little,if any,difference between wild zooplanktonaod rotifers as a first feeding diet, INTRODUCTION niquesfor culturing this species have been devel- oped. Theseinclude the techniquefor the artifi- Domesticand overseas demand for high cialspawning of captive broodstock Smigielski and quality flatfish, combined with the declining har- Arnold 1972!, larval rearing Smigielski 1975; vest from wild populations,has greatly increased Rogers1976; Laurence 1977; Klein-MacPhee et interest in the culture of various Rounder species ai. 1982, 1993!, and the successfulweaning of ju- Waters 1996!. Thosebeing consideredfor corn- veniles onto formulated diets Lee and Litvak mercial aquaculturealong the Atlanticcoast of the 1996!. United States include summer flounder The problemsthat have impeded cornrner- Paralichthys derttatrss!, ycllowtai 1 Rounder cial cultureof manyfinfish speciescenter around Ptetsmnectesferrtsgirtea!, and southernfloun- low larval survival and growth, particularly at the der Paralichthys lethostigma!. In addition to time of first feeding. Theuse of live food in the these,winter flounder Pletrronectesamerica/siss! culturingof theearly life stages of marinefish lar- is alsobeing consideredbecause many of thetech- vae,including winter flounder, is currentlyconsid- UJFiR TechnicalReport No. Z6 Tablel. Analysisof algalpresence and food type on winter flounder growth, P-va!ues derived from unpaued T-tests, 1hble2. Meanlenghts mm! aod tnstantsneous growth rates %hvk! for replicates ofgreen water, clear water, rottfer, and wild rooplankton treatments.sd = standarddeviation. 'ihMe 3. lVleanlenghts rmn! andinstantaneous growrh rates %/wk! for all fourtreatments. sd = standard deviation. * = no sd due ro lossof a replicate eredobligatory, The most widely used live food theinechanis m s!by whichrhe microalgae improve organisms are cultured rotifers, such as growthand survival remains unclear, and may dif- Brachionus pli carilis, andbrine shrimp Arremia fer amongboth rnicroalgaland fish species,sev- salina! nauplii. While the use of thesetwo prey eralhypotheses have been proposed to explaintheir speciesis coinmon,they are relativelyexpensive positiveeffects at first-feeding. They may pro- becauseof the labor involvedin their production vide nutritionalbenefits either directly via inges- Ehrlichand Rust 1989!, Le Ruyet et al. 993!, tionand absorption Moffatt 1981!,or indirectlyby for example,have calculated that live prey feeding increasingthe ainountsof essentialfatty acids in niainly Anemixi!represented 79% of thetotal pro- the rotifersbeing fed to the fish larvae Reitanet ductioncost of a 45 day old seabass. A second, aL 1993!. Microalgae may also trigger digestion and critical disadvantageof culturedlive foods processesin the larvae Hjelinelandet al. 1988!. Brachionusand Arteniia!, is thatthey do notpro- In additionto nutritionalbenefits, microalgae may videoptimal larval nutrition, largely due to low lev- inhibitpathogenic bacteria Austin et al. 1992!,in- els of essentialfatty acids{Watanabe ei al. 19S3a, fluence the establishment of intestinal microflora Wittet al, 19S4,Leger et al. 1986,van der Meeren Skjermo & Vadstein 1993!, and stabilize water ct aL 1993!, For this reason,fish larvae reared on quality Houde 1975, 1978!, It hasalso been sug- culturedfoods often exhibitabnorinal development, gestedby Naas et al. 992! that micmalgaecan poor growth, and low survival Watanabeet al, changeambient light conditionsin thelarval tanks, 1980,Fujita et al. 1980,van Ballaer et al. 1985, whichmay, in turn, leadto an increasein the con- Izquierdoet aL 1989, Kovenet aL 1990, van der surnptionof zooplanktonat first-feeding. Meeren 1991c!. Methodsof improvingthe nutri- Becausefirst-feeding diet {live food type! tionalquality of culturedfoods, via enrichinentwith and the additionof microalgaehave both been highlyunsaturated fatty acids{HUFA!, are avail- shown to influence larval growth and survival,and able {Watanabeet al. 1983b!.but they add to the becausewe are unawareof any publishedwork costof live foodproduction. Natural wild! live that has examined these variables for winter floun- foods{primarily copepods! have beenused with der, we setout to determineif live foodtype and greatersuccess {Naas et al, 1987, Ellertsenet al. the presenceor absenceof microalgaceffected 1981,van der Meeren 1991b, Le Ruyetet al. 1993!, thegrowth and survival of winterflounder larvae. becausethey are generallyricher in essentialfatty In thisresearch, the following null hypotheses were acids Pedersen1993!. In Atlantic cod for ex- tested: I ! the additionof microalgaeto larvalrear- ample, larvae fed diverseassemblages of wild ing systemshas no effect on the gmwthof winter zooplanktonin bothseini-intensive van der Meeren floundex;and ! there is no differenc in thcgrowih 199t h, Otters 1993, van der Meeren and Naess betweenfirst feeding winterflounder larvae fed 1993! andextensive systems {9iestad et al. 1985, live, laboratorycultured rotifers, and thosefed a Skjolddalet al, 1990,Bliam et al, 1991!have gen- dietof wild zooplankton. erallydisplayed good survival and growth, In addition to thc use of live larval food MATERIALS AND METHODS organisms,whether cultured or wild, the addition of microalgaespecies to larval rearing tanks {"green A 2 X 2 factorialdesign experiment with water"Ixeatment! has been widely acceptedas a two replicatesper treatmentwas initiated in May techniquefor commercialmarine finfish produc- 1996 and lasted for five weeks fnom first feeding tion. The additionof inicroalgaehas enhanced to metamorphosis.Two hundred,five day post- larvalgrowth and survival of a nuinberof species, hatch winter flounder larvae were stocked into each includingturbot {Howell 1979;Scott & Middleton of eight20 liter, tapered. round, gray, plastic aquaria 1979;Jones et aL 1981;Brornley & Howell 1983; measuring32 c mhigh, with a 43 cm diametertop Reitanet al. 1993!,halibut {Naas et al. 1992;Bergh and35 cm diaxneterbottom, A greenwater star- et al. 1994!,summer flounder {Al veset al. 1997!, vation treatment served as thc contr>>l. Treatments cod Pedersenet al, 19S9; van der Meeren 199 I a!, were staticwith 50% waterchange {10 liters!ev- andgrunion Vasquez- Yeomans et al. 1990!.While erythird day Replacementseawater was filtered 70 UJNR TechutcstReport >o. 26 8 3 Week FigureL Winterflounder growth in thepresence or absenceof microaigae.Vertical bars represent +/- i standarddeviation. to removeparticles greater than 5 pm and treated at an averagedaily rateof 2100/l. All treatments with UV lightprior to additionto theexperimental werevisually inspectedprior to feeding,and it was aquaria.Aquaria were submerged in a l 3 cm deep determined that larvae were fed to satiation based flow throughwater table to maintainambient sea- upon the presenceof residualplanktors. Three water temperature.Temperature ranged from 8 liters of Isochrysisgatbrrrta were addedto treat- to 15'C, with a daily average of 10'C over the mentsreceiving green water every third day with courseof theexperiment. Salinity wasmaintained waterchanges! at a densityof 200,000cells/ml. at 30-32 ppt. Larvaewere exposedto 24 hours A randotnsample of tenlarvae from each light usinga 35 Watt fluorescentlight suspended replicatein eachtreatment were measuredto the 76 cm abovethe aquaria. nearest0.5 rnm total length!each week. Mortali- Serru-continuouscultures of themicroalga ties werenot replacedthroughout the durationof Isochrysisgalbana Tahitian strain! were main- the experunent. Mean lengthper replicatewas tainedin 80 L fiberglasscylinders, and were pro- usedas the response variable. A two-wayanaly- videdwith f> media Guillard and Ryther 1962!. sis of variance ANOVA! was usedto determine Rotifers L-type! werecultured in identical cylin- if therewas any interactionbetween the two fac- derson a dietof Isochrysisgalbarra and dry yeast tors preytype, presence/ absence of microalgae!, at 1g/milhon rotifers/d!. Cultured rotifers were fed Wherepossible, unpaired t-tests were used to com- to respectivelarval fish treatmentsat an average parelengths of larvaeraised in differentcombina- daily rateof 260M. Wild zooplanktonwere har- tions of the two factors. Instantaneousgrowth vestedfrom Portsmouth Harbor, New Hampshire rate, G %/wk! was calculatedusing Ricker's by towingan 80 micmnplankton net through the 979! formula:G = In Y;+t- lnY !/ t;+> t;!, where top two meters. Collectedplankton were sieved Y;is length at time t;, Survivalbetween treatments througha 200inn and a 48 pm screen,and counted. wasco~ usingone-way analysis of variance Wildzooplankton, consisting ofapproximately 90% ANOVA!. copepodnauplii, were fed to respectivetreatments JQna and Hnvrell 71 to Table2. Effectsof foodtype on witt ter flounde growth. Vertical bars represent +/-t standarddeviation. RESULTS larvae that were reared on cultured rotifers and wild zooplankton,Larvae in treatmentsfed roti- Resultsfrom the two way analysis of vari- fers grew to a meanlength of 7,5 a I.I mm at a anceindicated that there was no significant interac- meaninstantaneous growth rate of 11%/week,while tion P=0,80!between thc two factors presence/ thosefed wild zooplankton grew to a meanlength absenceofmicroalgae, wild/cultured prey!. Becmm of g.1 ~ 1,5min at a meaninstantaneous growth of thislack of interaction,we wereable to consider rate of 13%/week Table 2!, thetwo factors independently of one another. Lar- Theloss of a replicatein thegreen water/ vaein tnmtrnentsreceiving micmalgae green wa- rotifer treatment precluded us from making stati s- ter! werecompared to tntheir absence clear wa- ticalcomparisons between this treatment and oth- ter!. ln this comparison,which disregarded food ers. We werehowever, able to comparefinal mean type,a highlysignificant difference Pc8.01! in total lengthsof larvaereared in two combinationsof lengthwas found in eachweek of theexperiment foodtype and the presence/absence of microalgae. TableI!. FigureI shows the txends m meanlength Final meanlength of larvae raised in the clear overthe five weeks for larvae reared in the pres- water/rotifercoinbination .0 mm!was not sig- enceand abSence of tnicraalga. Larvae in green nificantlydifferent PA!.05! from that of larvae watertreatments grew to a meanlength of 9.I s raisedin theclear water/wild prey combination .0 0.64 nunat a meaninstantaneous gmwth rate of min! Table3!. Thefinal mean length of larvae 15%/week,while final incan length of those in clear raisedin the greenwater/wild prey combination water was 7 0 ~ 0.60 mm, with a meaninstanta- 9.3 txun!was significantly longer P<0.05! than neousgrowth rate of 10%/week{Table 2!. thatof larvaein the clearwater/wild prey coinbi- No significantdifference P>0 05! in nation .0 mm! Table 3!, lengthwas found between larvae in replicates re- 'Hmewas no significant difference gb6.05! ceivinggcultured roti fers and those which were fed betweenfinal mean survival values which ranged wildzooplankton, regardless of algal presence from 135 to 22.0% Figure 3!. All larvae in thecon- TableI!. Figure2 illustratestrends in length of tro] greenwater with no food!died by weektwo, 72 UJA}t TecbniratRepa-rr No. ZS 35 30 25 20 V! t 1S 10 %rbte3. The effects offood type and algal presence onwinter flounder survival afterfive weeks. Vertical barsrepresent+/- 1 s~ deviarion, DISCUSSION 1979;Scott &. Middleton1979; Jones et al. 1981; Brornley& Howell 1983; Pedersenet al. 1989; Growthof winterflounder larvae has been Vasquez-Yeomanset al 1990; van der Meeren studiedextensively, and has been found to vary 1991a;Naas et al. 1992;Reitan et al. 1993;Bergh withboth temperature Laurence 1975! and prey et al. 1994;Alves et al. 1997!. As seenin Table 1 density Laurence 1977!, In general,growth in andFigure 1, larvaereared in greenwater were lengthof bothlaboratory-reared andwild-caught significantlylonger than those in clearwater in each larvaeis curvilinear Pearcy 1962a; Bertrarn et al. week of theexperiment. Differences in instanta- 1996;jerald et al, 1993!, with growth being quick- neousgrowth rates were most prunounced at the estduring the first weeks after hatching, and then endof thefirst week of theexperiment 7% vs, slowingas the fish approach inetarnorphosis, Al- 5% for greenwater and clear water treatments, thoughthe weeldymean sizes we found in this respectively!.This suggests that the presence of studyvaried between treatments, all of our larvae microalgae enhatbmi larval growth within the first grew at ratesgeneral]y comparable to those re- weekof exogenousfeeding, Although not quanti- portedfor winter flounder larvae in nature Pearcy fied in this experiment,we notedthat larvae re- 1962a!,in thelaboratory Laurence 1975, 1977; ceivingmicroalgae initiated feeding sooner than Chambersand Leggett 1987; Jerald et ah 1993; larvae in clear water. A similar observation was Bertramet al, 1996!, and in in-situ mesocosms inadef' or halibut larvae reared in green water Naas Laurenceet al, 1979!. et al. 1992!,where enhanced first-feeding was Resultsfrom this study demonstrated that ascribedtothe microalgae effecting ambient light thepresence ofmicnMlgae significantly improved levelsin the culture tank, which in turnimproved thegrowth of larval winterflounders. Similar re- larvalfeeding efficiency. It hasalso been sug- sultshave been found for the larvae of otherspe- gestedthat microalgae may stimulate enzymatic cies,including turbot Scophrhalmrrsmaximus!, activityof thelarva's gut during first feeding halibut Hippoglossushippoglossus!, summer Hjelrnelandet al. 1988!,or supplyexogenous en- flounder Parahchrhys dertrates!, cod Gadsrs zymesthat assist tbe larvaein their digestionof rrrorhua!,and grunion Lerrresresterruis! Howell zooplankton Bromage and Roberts1995!. If, as we expect,our winterflounder larvaein green ispossible that the microalgae was responsible for waterinitiated feeding earlier than those in clear thetendency not statistically significant! for sur- water,and if themicroalgac triggered the diges- vivalto behigher in greenwater treatments than tionprocesses, it could account for the significant inclear water treatments Figure 3!. Survivales- differencein length and growth rate! we observed timatesfrom this study ranged from about 13,5- at the end of wcck 1. 22% at theend of 5 weekswhich arc lowerthan Thebenefits of inicroalgaemay also have theapproximate 34% reported byLaurence 977! resultedfrom direct ingestion of the algae, which forwinter flounder larvae raised at 8oCand pro- hasbeen observed in a numberof marinefish lar- videdwith 3000wild zooplankters/l.Our lower vae,including northern anchovy Moffatt 1981!, observedpercentages may have resulted from our turbot Howell 1979; Last 1979!, halibut Reitanet fluctuating,and slightly warmer,incubation tern- al. 1993!cod van der Meeren1991a! and wild- peratures. caughtwinter flounder Pearcy 1962b!. Both the We found no significantdifference mechanismofmicoalgal ingestion, which may in- p>0,05!in the final mean lengths of larvae,or in volveeither drinking or filter-feeding, and the nu- percentsurvival, between the two five prey treat- tritiveevalue of the ingested microalgae areopen to inents.Among the components of any larval fish speculation Van der Meeren 1991a!. Studies with diet,it iswell documented thatf attyacids, particu- larvalcod vander Meeren 1991. a!, turbot Howell larlyn-3 highly unsaturated fatty acids HUFA!, 1979!and halibut Reitan et al. 1993!suggest that areimportant to the nutritionof marinefish larvae assimilationofthe microalgae bythe larval gut is Watanabeetal. 1983b;Van Ballaer et al. 1985; low. Despitethis, Tytler et al. 997! foundthat Kovenet al. 1990!,including winter flounder IGein- turbotlarvae had chlorophyll containing apical vacu- MacPheeetal, 1980!. Because wild zooplankton olesin thegut enterocytes 3 days after hatching, aretypically rich in these essential fatty acids com- andthey suggested thatalthough assimilation effi- paredto culturedlive foodorganisms Watanabe ciencywas low, the larvae may obtainsmall etal. 1980,1983a; van Ballaer et al, 1985;Leger amountsof essentialfatty acids, amino acids, and et al. 1986;Naess et al. 1995!,experiments in carotenoidsfrom themicroalgal cell pigments. which marinefish larvaehave been fed culturrxf Microalgaemay also enter the larval gut indirectly preyand wild zooplankton have generally shown throughingesting microalgae-fed rotifers Reitan tha growth and survival are higher in thosefed etal. 1993!.It hasalso been suggested that thc wild zooplankton Skjolddal et al. 1990!. It has additionof rnicroalgaeleads to theestablishment alsobeen shown that some larvae e.g. turbot! ofan early larval intestinal microflora Skjermo and selectwild zooplanktonover the rotifer Brachionus Vadstein1993; Bergh et al. 1994!. Thisin turn, pkcatilisif givena choice van der Meeren 1991c!. mayenable the digestion of algalcells Rimmer Ourfinding that there was no significant differ- andWiebe 1987!, may provide amino acids, fatty encein lengthsor survivalbetween larvae fed cul- acidsand vitamins Kashiwada and Teshima 1966; turedrotifers and those fed wild zooplankton sug- Fongand Mann 1980; Ringe et al. 1992!,and may geststhat the two foodtypes were similar in their inhibitbacterial pathogens Olsson et al. 1992!. nutritionalvalue. Reitan et al. 993! haveshown Apartfrom stimulating first feedingand/ thatrotifers fed Isochrysis galbana T. Iso!have or providingeither direct or indirectnutrition, relativelyhigh levels of lipidsand 22:6 n-3 highly microalgaemay also act to control b~ growth unsauratedfatty acid HUFA!compared to those intanks by releasing natural bacteriostatic agents. in clear water, and that turbot larvae fed these Austinet al. 992! for example,found that thc microalgae-enrichedrotifers have higher growth exudatesfrom one species of algae Terraselmis andsurvival than those fed rotifers grown in clear suecica!inhibited certain bacterial fish pathogens. water.Presumably this was due to the micioalgae Themicroalgae may also stabilize water quality by providinga sourceof micronutrientsand HUFA to absorbingwaste products and producing oxygen thelarvae, both of whichare essential for growth Houde1975, 1978!. Becauseboth bacteriostatic and survival Fukushoet al. 1984; Brown et al. agentsand waterquality wou]d effect survival, it 1997!, Moreover,Reitan et al. 993! found that 74 UJXR TecbtucalRepor bio. Z6 totallipids and fatty acids remained relatively high ments 7%/wk! comparedto thosein clear water in uneatenrotifers living in greenwater systems treatments %/wk! for thisearly period. Results becausethey were consuming the microalgae. of the study alsosuggest that thereis little differ- Thusculture tanks receiving microalgae promoted encebetween wild zooplanktonand culturedroti- a continuoussupply of highlynutritious rotifers for fers asa firstfeeding diet for winter flounderlar- the fish larvae. BecauseIsochrysrs galhana is vae. We note, however, that our rotifers were a]- knownto haverelatively high ]eve]sof essential mostcertainly enriched, particu]arly in essential fatty acids Brown et al. 1997!, and becausea fatty acids,by thcinicroalgae with whichthey were number of authors have shown that the levels of cultured,and that their tendency, although not sta- n 3 HUFA can be increased in cultured food or- tisticallysignificant, for survivalto be higher in ganisms,including rotifers, by feedingthem uni- green water treatmentsthan in clear water treat- ce]lularmarine a]gae rich in n-3 HUFA Kitajima ments. These resultsindicate that rnicroalgae et al, 1979; Scott and Middleton 1979; Koven et shouldbe used when culturing winter flounder lar- al, 1990;Reitan et al. ]993!, we believeour roti- vae. Thismay be particularly important during the ferswere enriched to levelscoinparablc to thewild first weekfollowing yolk-sac absorption, as indi- zooplanktonwe used, thereby accounting for simi- catedby the greatestdisparity in the instantaneous lar perfonnanceof the larvaefed thesetwo diets, growthrate of larvaein greenwater treatments Comparisonsof larval lengths from the experiment ]7%/wk! comparedto thosein c]ear watertreat- supportthis theory of microa]galenrichment Table ments %/wk! for this early period. Resultsof 3!. We found,for exainple,that larvae fed both thestudy alsosuggest that thereis little difference culturedrotifers and wild zooplanktonin the pres- betweenwild zooplanktonand cultured rotifers as ence of microalgaewere larger than those fed a firstfeeding diet for winterflounder larvae. We culturedrotifers and wild zooplanktonin the ab- note, however, that our rotifers were a]most cer- senceof microa]gae,at everyweekly time inter- tainlyenriched, particular! y inessential fatty acids, val. Whilethe microalgaemay have been having by the rnicroalgaewith which they werccultured, a numberof effects see above!. it is possib]ethat andthat this presumed nutritional quality was prob- it mayhave been improving the nutritional value of ably maintainedover time by the addition of boththe rotifers and wild zooplanktonsuch that microalgaeto thelarval fish cultures.It is likely theywere nutritionally equivalent. We a]so found that rotifersgrown in the absenceof microa]gae thatthere was no significant difference in theincan wouldnot promote comparable growth. lengthsoflarvae fed rotifers and wild zooplankton inthe absence ofmicroalgae. This result also sug- ACKNOW LEDGMENTS geststhat the two diets were nutritiona]]y equiva- lent.In thiscase, however, the equivalence was We wish to thank Deborah Bidwell, Eliza- probablydue to the rotifershaving been fed beth Fairchild, Andrea Tornlinson and Carrie Hill microalgaeasthey were being cultured. for assistingwith all aspectsof the research,and In thisfive weekexperiment, we found Noel Car]son, Manager of the UNH Coastal Ma- thatlarvae in thegreen water treatments grew to rine Laboratory,for a]1his efforts. UNH Center largermean lengths than larvae in the c]ear water forMarine Biology/Jackson Estuarine Laboratory treatments,regardless of food type, We also found Contribution Series ¹33g. thatthere was a tendency,although not statisti- callysignificant, forsurvival tobe higher in green LITERATURE CITED water treaunents than in clear water treatments. Theseresults indicate that microa]gae should be Alves,D., D.A. Bengtsonand J.L. Speckcr.1997. usedwhen cu]turing winter flounder larvae. This Investigationsinto the causesof larvalmor- maybe particularly important during the first week tality in cultured summer flounder fo]]owingyolk-sac absorption, asindicated by the Paralichthysdenhzrur L!, Symposiumon ]p atestdisparity in the instantaneousinstanta- MarineFinfish and ShellfishAquacu]ture, n nis growthrate of larvaein greenwater treat- Marine StockEnhancement, and Open Kiss sad Hosea 15 OceanEngineering. Univ, of NewHamp- and survival of ood larvae in an enclosure. shire,Sept. 1997. abstract!. Experimentsand a mathematicalmodel. Austin, B., E. Baudet and M. Stobie. 1992. Inhibi- RappP,-V. Reun. Cons. int. Exptor.Mer, tionof bacterialpathogens by Tetraselrnis 178: 45-57, suecica, J. Fish, Diseases, 15: 55-61. Fong,W. and K.H. Mann. I 980.Role of gut flora Bergh,8., K.E. Naasand T, Harboe. 1994. 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Arnold.1972. Sepa- coastalcod populations. Doctoral Disserta- ratingand incubating winter flounder eggs. tion.Norwegian College of Fishery Science, Prog.Fish Cult., 34:113. Univ.of Trornso, Trornso, Norway. 141 p, Sruigielski,A.S, 1975.Hormone induced ovulation Pedersen,T., J.E Eliassen,H,C. Eilertsen,K,S. of thewinter flounder, Pseudopleuronecres Tande,and R.E. Olsen.1989. Feeding, arnericanus Fish. Bull. U,S., 73: 431-438. growth,lipid composition,and survivalof Tytler,P., J. Ireland and L. Murray.1997. A study larval cod Gadusrnorhua L,! in relationto of theassimilation of fluorescentpigments environmentalconditions in an enclosureat of microalgaelsochrysis galbana by thc 70o in northernNorway, Rapp.- V. Reun. earlystages of turbotand herring. J. Fish Cons.perm. int. Explor. Mer, 191: 409-420. Biol., 50: 999-1009. Reitan,K.I., J.R. Rainuzzo,G. Oie andY, Olsen. vanBallaer, E F. Amat,F. Hontoria,P Leger, 1993.Nutritional effects of algal addition in andP. Sorgeloos, 1985. Preliminary results first-feedingof turbot Scophthalrnas rnaxi- and nutritional evaluation of w-3-HUFA musL.! larvae.Aquaculture, 118: 257-275, enrichedAnemia naupliifor larvae of the Ricker,W. E. 1979, Growthrates and models. In: seabass, Dicerrrrarchus labrax. Aquacul- W. S, Hoar, D J. Randalland J. R. Brett, ture, 49: 223-229. Editors!.Fish physiology, Vol. VHI. Bioen- 7s UJNR TecbatcarRcport No. 26 vander Meeren, T. 1991a.Algae as first foodfor Scopthalamusnuuimus L. rearedon dif- codlarvae, Gadus morhua L.: filter feed- ferentfood organisms with special regard ingor ingestionby accident? J.Fish BioL, tolong-chain polyunsaturated fattyacids, 39: 225-237. Aqua.Engin 3: 177-190, vander Meeren, T. 199lb. Productionof marine Siestad,V P.G. Kvenseth, and A. Folkvord.1985. fishfry in Norway. J,World Aqua, Soc 22: Massproduction of Atlantic cod juveniles 37-40, Gaditsmorhira! in a Norwegiansaltwater vander Meeren, T. 1991cSelective feeding and pond.Trans. Amer. Fish. Soc., 114;590- predicnve food consumption inturbot larvae 595. Scoprhalmasmaximus L.! rearedon the rotiferBrachronus pticatiiis and natural zooplankton.Aquaculture, 93: 35-55. vander Meeren, T. andT, Naess. 1993. How does cod Gadus moritua! cope with variability in feedingconditions during early larval stages?Marine Biology, 116; 637-647. vander Meeren, T., J. Klungsoyr, S.Wilhelmsen, andP, G. Kvenseth.1993. Fatty acid corn- positionof unfedcod larvae Gadus morhua L,!and cod larvae feeding onnatural zoop- lanlttoninlarge enclosures, J.World Aqua. Soc., 24: 167-185, Vasquez-Yeomans, L., E, Carrillo-Barrios-Gomez andE. Sosa-Cordero.1990. The effect of thenanoflagellate Tetraselmis suecica on the growthand survival of grunion,Leuresres ienuis,larvae. Environ. Biol. Fish., 29: 193- 200. Watanabe,T.,F. Oowa, C. Kitijirna, and S. Fujita. 1980Relationship between dietary value of brineshrimp Arremia salina and their con- tentof n-3highly unsaturated fatty acids. BukkJapan. Soc. Sci. Fish 46: 35-41. Watanabe,T., C. Kitajima,and S. Fujita.1983a. Nutritionalvalues of live organisms used in Japanfor masspmpogation of fish:a re- view.Aquaculture, 34: 115-143. Watanabe,T., T. Tamiya,A. Oka,M. Hirata,C. Kitajima,and S. Fujita. 1983b. Improvement of dietaryvalue of live foodsfor fishlarvae byfeeding them on highly unsaturated fatty acidsand fat soluable vitamins Bull. Jap. Soc.Sci. Fish., 49: 471-479. Waters,E.B. 1996. Sustainableflounder culture and fisheries. Univ. of Ncnth Carolina Sea GrantCollege Program, Raleigh, N.C, Pub. No.UNC-SG-96-14, 12 pp. Witt,U., G. Quantz, D. Kuhlmann,and G. Kattner. 1984.Survival and growth of turbotlarvae Kurukawa and Suznki 79 DEVELOPMKNTAI PROCESS OF DIGESTIVE ORGANS AND THEIR FUNCTIONS IN JAPANESE FLOUNDER PARA LICH THYS OLIVACEUS Tadahidc Kurokawa and Tohru Suzuki NationalResearch Institute of Aquacultureof FisheriesAgency Nansei,Mie 516-0193,Japan e-mail:kurokawa@nria,affrc.go.jp ABSTRACT Toelucidate the developmental process of the digestive function of the pancrea.i and the intestine during the larvalstage of Japaneseflounder. distribution of aminopeptidaseand bypsinogen was traced by imrnunohis- tocbemicslmethods, Aminopeptidase wasdetected from the brush border of theposterior intestine athatching. At 2 dayspost hatching dpb!, the rectum had diTferentiated morphologically, and both the brush border of the intestineand rectum showing a strongreaction to anti-aminopeptidase antibody anti-rAmp!, but the reaction to theantibody in therectum was reduced at 3 dph. Thus,the intesdnal epithelial cells of flounderlarvae had alreadystarted tosynthesize digestive enzymes onto the brush border at hatclung, and the functional differemia- tionof therectum from the intestine occurs at thefirst feedingat 3 dph.Trypsinogen was detected in thc pancreasbeginning at 2 dph,The trypsinogen secretion into the pancreatic duct was found at 3 dph.Thus, the pancreasofflounder larva acquires exocrine function bythc time of the first feeding at3 dpb.The pancreas was a sinallcoropact organ at 3 dph,and it itartedto elongatealong the veins of theintesune at20 dpb Ttms,the pancreasof the fl ounder completes theformation from the compact-type organof larva to the diffuse-type organ ofthe adult at metamorphosis, atthe time of development of the gastric glands in thestomach wall. Therefore, thedigestive organs of theJapanese flounder, other than tbe stomach pancreas, intestine aml rectum!, have acquiredthe digestive ability by the time of first feeding and their digestivc system becomes ful!y developed after meuunorphosi s. INTRODUCTION trusslarvae, aminopeptidase activity was found on the intestinalbrush border Cousinet al. 1987!.In Japaneseflounder Parafiehthys oui vaceus thelarval stage of flounder, therefore, ingested food is an importantspecies in both aquacultureand is passeddirectly into the intestine, where it is di- commercialfisheries in Japan,The recentincrease gestedby thepancreatic enzytrn.s and the enzymes in theintensive production ofjuveniles for seeding of theintestinal brush border and undigested pro- hasnecessitated a more detailed understanding of teinsare absorbed by rectalcells. theearly developmentof flounderlarvae. Becausemarine fish larvae cannot prop- Sincethe gastric glands of Japanesefloun- erlyutilize artificial diets due to their undeveloped derhave not fully developed tnttil metamorphosis digestiveorgans Graff and Sorenson 1970, Braid Miwa et al. 1992!,the pancreas is thesole exo- and Shell 1981,Baragi and Lovell 1986,Beccaria crineorgan responsible forsecreting digesti ve en- et al. 1991!,rotifers and brine-shrimp nauplii are zymesduring the larval stage. In sunnnerflounder essentialfor rearinglarvae. In order to establish Paralfchfhysdenrarus larvae, the epithefiafcells an efficientrearing system for flounderlarvae in- of the intestineand the rectumabsorb lipid and cludingthe development of completeartificiaI di- protein,respectively Bisbal and Bengtson 1995!. ets for early larvae, it is important to understand This absorptionof proteinby rectalcells indicates their digestiveability at the larval stage. In this interred.'ffulardigestion via pinocytosis Watanabe study,to elucidate the devefop~taf processof the 1981,Watanabe 1982, Georgopoulou et al. 1986, digestivefunction of the intestine,rectum, and pan- Govoniet aL 1986!.In turbotScophfhtahnus maxi- creasduring the larval stage of Japaneseflounder, Sn VJNR Technical Report Nu. 26 distribution of aminopcptidasc and trypsinogcn and to 30 dph, on brine-shrimp nauplii A rrerrtta sp. from absorption of rotifcr proteins derived from rotifers l 5 to 45 dph, and on an artificial diet Kyowa A-250, were traced by imrnunohistochemical methods. In Japan! from 20 dph. addition, the formation processof diffuse pancreas was followed. Immunohistoehemistry Larvae n=10! were fixed with 10% for- MATERIALS AND METHODS malin in 10 rnM Tris-buffered saline TBS! pH 7.5 for 24 h at 0, I, 2, 3, l 0, 20, 30, and 45 dph, I'ixed Larvae sampleswere dehydrated through a gradedethanol Larvae of the Japanese flounder series, embedded in paraffin, and cut into serial Paralichrhys olivaceu.r were kept in a tank sup- sections 6 pm thick. plied with running seawater 7+1 "C!. The larvae Sections were stained immunohistochemi- were fed on roti fcrs Brachi orttt.s pli cari lis frotn 3 cally using anti-ecl trypsinogenantibody anti-eTrg!, Figure I. Distribution of arninopeptidase in the digestive tract and absorption of proteins by rectal cells of carly Japanese flounder larvae. Sectionswere immunostainedwith anti-rAmp A-C! and anti-Rot Dl. A, posterior instestineat hatching,i3; anteriorintestine at 3 dph. C,D; rectum at 3 dph. in = intestine;no = notochord;re = rectum, Scalebars indicate 25 Jtm. Karokama and suzaki tu anti-red sea bream aminopeptidase anti-rAmp!, gans. The primordia of the pancreasand the liver and antibody againstwhole solubleproteins of ro- had differentiated from the gut at I dph {Fig. 2A!. tifers anti-Rot!, and weredeveloped by Histofine SlightimmunochemicaI staining from trypsinogen SAB-PO kit Nichirei, Japan!. was detected in the pancreasbeginning at 2 dph Fig. 28!. The irnmunoreactionto anti-cTrg in- RFSUI.TS AND DISCUSSION creasedfrom 2 to 3 dph, Trypsinogen secretion into the pancreaticduct was foundat 3 dph Fig. Development of the digestivefunction of gut 2C!. Thus. the pancreasof flounder larvae acquires Thc intestineof newly hatchedJapanese exocrine function by the time of first feedingat 3 flounder larvae was a narrowduct consisting of a dph. smoothsingle-layered epithelium with nomucosal Beccaria et al. 991! classified the dcvcl- folds. The brush border of the intestinalepithelial oprnentalprocess of pancreaticprimordia in fish cells wasstill indistinct,but slight immunochemi- as four types that were based on morphological cal staining for aminopeptidasewas observations. The pancreas of Japanese flounder detectedin thebrush borderof the posteriorintcs- showsa similar developmeritaltype to sea bass tinc Fig,lA!, At I dph, the brush border of thc Diceritrarchus labrax. anterior intestine also exhibited weak The pancreasof Japaneseflounder was a irnmunoreaction to anti-rArnp, The rectum had cotnpactorgan localizedaround the gallbladder at differentiatedmorphologically from the intestineat 3 dph Fig. 2D!. The intestinehad coiled and the 2 dph,and the brush borderof the intestineand the pancreas slightly elongated posteriorly at I 0 dph. rectum gavestrong signalsto anti-rArnp.The in- The coilingof the intestinewas more pronounced testinalepithelia had formed mucosalfolds at 3 dph, at 20 dph, The pancreaswas mainly localized The brushborder of the intestine showedstrong aroundthe gallbladderbut the posteriorpart of the signals to anti-rAmp, but the signals in the rectum pancreashad begun to elongatealong the vein on had beenreduced Fig. 1 Band C!. the intestine at this time Fig. 2E!. After the first feeding of rotifers at 3 dph, At 30 dph,the pyloric appendages had dif- strongimmunohistocheinical staining for proteins ferentiatedfrom the anteriorpart oi the intestine derivedfrotn rotifcrs was detected froin the epithe- andthe bile duct hadelongated. The pancreas was lial cellsof thelarval rectum Fig.1D!. This indi- distributed frotn the vicinity of the gallbladder to catesthat rectal cells of Japaneseflounder larvae the proximalpart of the pyloric appendages.In haveacquired the ability to absorbdietary proteins addition,the posterior part of thepancreas had elon- via pinocytosisat 3 dph, gatedfurther a.longthe vein on theintestine. Accordingly,the intestinalepithelial cells At 45 dph completionof metamorphosis!, havealready started to synthesizedigestive enzymes the gastric glandshad developedin the stomach onto the brushborder at hatching, andthe func- wall. The pancreaswas locahzed around the proxi- tional differentiation of the rectum from the intes- mal partof the pyloricappendages, along the bile tine occursby the time of first feeding at 3 dph in ductand along the veinsrunning tothe porta hepatis Japaneseflounder, from the stomach,pyloric appendages,spleen, and ln summer flounder Paraiichthysdentatus intestine Fig, 2F!. At thisstage, the pancreasbe- larvae, tnucosalfolds were formed in the intestine comessimilar in structureto the diffusepancreas at 4 dph andactive pinocytotic features were ob- of the adult flounder Kurokawa and Suzuki 1995}. servedin the rectalcells at this stage Bisbal and Thus, the Japaneseflounder pancreascompletes Bengtson1995!. Thus, the differentiation of the the transition from the compact-g~ organ of larva gut appears to be synchronized with the onset of to thediffuse-type organ of adultat metamorpho- exogenousfeeding in flounder. StS. The formation process of' the Developmentof exocrinepancreas diffuse-pancreasof sea bassD. tabrax was ob- At hatching,the larval gut of Japanese sewed using scanningelectron microscopy Diaz flounderwas a simple tubewithout accessory or- et al. 19S9!. The pancreas of sea bass also be- PB Figure 2, Distribution of trysinogenin the pancreasof carly Japaneseflounder larvaeand formation of diffuse pancreas. Sectionswere itnmunostainedwith anti-eTrg A-Cl. Thc morphologyof the pancreaswas reconstructedfrotn serial sectionsand is representedschematically D-F!. Shadedareas indicate pancreatictissue. A; l dpb. B; 2 dpb. C, D; 3 dph. E; 30 dph. F; 45 dph completion of metamorphosis!.bd = bile duct; es = esophagus;gb = gallbladder;hd = bepaLicduct; in = intestine;li = liver; pa = pancreas;pd = pancreaticduct; py = pyloric appendages;re = rectum; st = stomach.Scale bars indicate25 pm. KttrOtra+a and Stteuki Sa Daysafter hatching 0 1 Z 3 5 10 15 20 30 130 Stomach Pancreas tnstestine Rectum Startfeeding Metamorphosis Ftgtire3. Sehetntttieittustratiots showing ttte developmentof digestivefuncuons during larval andjuvenile stagesin Japanese flounder, comesa diffuseorgan by the juvenilestage. It turn, and pancreas!,have acquireddigestive func- appears,therefore, that the pancreasof teleosts tionsby the onset of exogenousfeedtng at 3 dph. whichpossess a diffuse pancreas commonly corn- Therefore, we concluded that thc acquisition of pletesdevelopment into a diffuseorgan by the ju- digestivefunctions in theintestine, rectum, and pan- venilestage. However, the biologicalsignificance creas are ofthe transformation from compacttodiffuse pan- presumablya requirementfor larvaeto startfeed- creas is unclear. lilg. Thedevelopmental process of thediges- Thepancreas comp letcs thc transformation tivefunction.s during the larval stage of Japanese froma compact-typeorgan of larvato a diffuse-type floundercould be surttrrtarizcd asshown inFigure organat metatnorphosis,It is known that the ga s- 3. Theintestine of theJapanese flounder have ex- ttic glandsdifferentiate and begin synthesis of pep- presseddigestive enzymes on thebrush border sinogenat metamorphosisin Japanesefioundcr metnbraneathatching, and the functional differen- Miwa et al. 1992!. Therefore,the digestive sys- tiationof rectum from iritestine occurs at3 dph, temof Japaneseflounder becomes fully developed Thepancreatic cells begin synthesis ofdigestive in theearly juvcnilc stage fl ounderfollowing tneta- enzymesat2 1phand secretion ofenzymes intothe morphosis.This tttay be one reason why artificial intestineat 3 d h. 3 dph.Thus, the digestive organs of dietscan be utilized by juveniles but not by larvae. flounderlarvae arvae, othero thari stomach intestine, rec- S4 UJVR TechaicalReport No. 26 LITERATURE CITED Miwa, S., K. Yarnano,and Y. Inui. 1992. Thyroid hormone stimulates gastric development Baragi,V. and R.T. Lovell. 1986, Digestiveen- in flounderlarvae during metamorphosis. zyme activities in striped bass from first J. Exp. Zool. 261: 424-430. feedingthrough larva development.Trans. Watanabe,Y. 1981. Ingestionof horseradishper- Atn, Fish, Soc. 115: 478-484. oxidase by the intestinal cells in larvae or Beccaria,C., J.P. Diaz, R. Connes, and B. Chatain, juveniles of someteleosts. Bull. Jpn.Soc. 1991. Organogcncsisof theexocrine pan- Sci. Fish. 47: 1299-1307. creas in the sea bass, Di certt rarchus labor Watanabe,Y. 1982. lntracellulardigestion of L., reared extensivelyand intensively. horseradishperoxidase by the intestinal Aquaculture 99: 339-354, cells of tclcost larvaeand juveniles. Bull. Bisbal,G.A. andD.A. Hengtson.1995. Develop- Jpn, Soc. Sci. Fish. 48: 37-42, tnentof the digestivetract in larval sum- rner flounder, J, Fish Biol. 47: 277-291. Braid, M.R. and E.W. Shell. 1981, Incidence of cannibalismamong striped bass fry in an intensive culturesystetn, Prog. Fish-Cult, 43: 210-212. Cousin, J.C.B., F. Baudin-Laurencin, and J. Gabaudan,1987. Ontogenyof enzymatic activities in fed and fasting turbot, Scophthalmusmaximus L, 3. Fish Biol. 30: 15-33. Diaz, J.-P, R. Connes, P. Divanach, and G. Barnabe.1989. Developpementdufoie et du pancrdas du loup, Dicenrrarchus lab A/E RATIO PROFILES OF THE ESSENTIAL AMINO ACID REQUIREMENTS AMONG VARIOUS FINFISH SPECIES Ichiro Oohara National ResearchInstitute of Aquaculture 422-1, Nakatsu, Nansei. Watarai, Mic 516-0193, JAPAN e-ma i!: ooharaC~ nria,affrc.go.j p Toshio Akiyarna National ResearchInstitute of Aquaculture, Inland Station Hiruta, Tamaki, Watarai, Mie 519-0423, JAPAN e-mail; akiyarnatCa'nria-tink,affrc.go.jp and Takes hi Yarnamoto Nationa! Research Institute of Aquaculture Hiruta, Tamaki, Watarai, Mie 5! 9-0423, JAPAN e-rnai!:takejpn Cd'nria-tmk.affrc.go.jp ABSTRACT Dietaryessential atnino acidrequirernents have been determined in several fishes, Recently, some researcherv haveapplied a newmethod based on theidea that thereshould be a coriela ionbetween whole body armno acid compositionand the dietary amino acid requirement. For instance, only one amino acid requircinent can he determinedby growthdata and the other nine can be estimated as being proportional tothe whole body amino acidcomposition. The authors tunk that this newmethod is problematic,because the essential amino acid requircmcntprofi!es based on fish growth assay appear io bedifferent among fish species than tho~e of body aminoacid composi ionsln orderto evaluate the dissiinilarity of essenualamino acid requiremen among s fish species,v e used he "A/E ratio" defined as fol low s: A/Eratio equals to 1 Each[ essential ammo acid byweight I I dividedby !All essentialamino acids by weight!1 .l 8 y usingthis index, fish growth stages and water empcraturc s do no affect the eva! nation coin pared with theabsolute values of theamino acid requuemenu. From the diagram obtainedbased on the A/E ra iopro ilesthrough the Fitch-Margohash method, close similariues of essenua! aminoacid requirements were found between carp and cat!a belonging to thefamily Cyprtnidae, and among chinooksalmon, chum salmon, and coho salmonin thefamily Sa!monidae.This sugges thes occurrenceof specificityin aminoacidrequirements among each fish family, and we suggest thatgrowth experunenis concerning eseen ia!ainino acid requirements should be conducted on atleas one fish speciesper family. INTRODUCTION We dietaryessential of 10 atninoacids such as arginine, histidine. isoleucine, leucine, From the viewpoint of practical fish lysine, mcthionine, phenylalanine,threortine. culture, proteinis the mostimportant constituent tryptophan,and valine has been estimated for fishes in fishfeed, not only asthe material for structural such as salmonids Ha!ver et al, 1957, Ha!ver and e!ernentsof animals, but also as the main energy Shanks ! 960, Shanks et al. 196'2, Akiyama et a!. source, The subject of dietaryprotein must be !985!, Europeancel Anguilia anguii a and dealtwith in regardto bothquality andquantity. Japaneseeel Artg siiirr japonica Arai et al. 1972!, Fishbody protein is composedof approximate!y commoncarp Cyprir sscarpio Noseet al, 1974!. 20 distinct amino acids. The amino acids which red sea bream Pagrrc.smajor Yone 1976!, and arenot synthesized entire!y or sufficiently for fish tilapia Tilapiazillii Mazidet. a!. ! 978!,based on needsmust be suppliedthrough fish feed. These growth response. In addition, the tracer are called the essential amino acids. experimentsusing "C have shown that plaice sit UJNRTechnicst Report No. 26 Pleuronecrespiaressa, sole Solea solea Cowey doubtif thenew method using the body amino acid et al. 1970!, and sea bass Dicetitrarchttslabrar coinpositionis scientificallyreliable, and aim to Wilson I 989!require the same I 0amino acids. It evaluatein detailthe dissimilarity of essentialamino is consideredat presentthat these 10 aminoacids acidrequirement profiles among different finlish areessential for all fish specie». species. One of the important factors in determiningthe efficiency of protein utilization for MATERIALS AND METHODS fishis thecoin position of essential ainino acids in dict. It is veryimportant to clarifywhether the a! Datafor essential amino acid requirements pattern»of aminoacid requirementarc identical or Thecoinplete quantitative iequiretnent for thespecificity exists ainong species, The present 10 essentialamino acids has already been studywas carried out focusing on the presence of dcterininedfor chinool salmon Oncorhyrtchus specificityin requirementof thee»sential ainino rshawyrscha Hal ver ct al. 1958,1959, Delong and acids usingalready published data. Halverl962, Chanceet al, l964, Halver1965, Sincethe studies on qualitativeand Klein and Halver 1970!, coho salmonO. kisurch quantitativeamino acid requireincntsin fish Arai and Ogai.a 1993!, chum salinon O. kera commencedin the United States in the1950s, data Aki yamaand Arai l 993!,channel catfish Icralurus forvarious fishes have been accumulated using punctartts Wilson and Poe 1985!, cominon carp differentmethods. The quantitati ve requirement Akiyamaet al. 1997!,catla Cada catla Ravi and wasdetermined based on growthresponses to Devaraj1991 !, Nile tilapia Oreochromis nilori cus dietarygraded levels of a consideredamino acid Santiagoand Lovell 1988!, milkfish Chanas especiallyearly in thestudies. chaiios Borlongan and Coloso I 993!, and Japanese Recently,however, soine researchers have eel Akiyamaet al. 1997!. As thesestudies have applieda newmethod based on the idea thatthere beenconducted by the sainc laboratory orresearch shouldbe a correlationbetween whole body amino groupsfor eachfish species,under almost identical acidpattern and amino acid requirement. Inamino experimentalconditions for the determinationof acidnutritiori for swine, the concept of an idea! all 10essential amino acid», i.e., similar basal diets, proteinbalance i s proposed,which is based on the feedinglevels, and environmental conditions water ideathat there should be a correlationbetween the qualityand temperature used!, test fish ages, sizes, bodyamino acid composition andthe dietary amino etc.,they givethe mostappropriate data for acidrequirement Agricultural Research Council comparingthe difference of requirements among 198I!. Basedon this theory,Wtlson and Poe fish species. 985! and Wilson I993! introduceda newmethod In traditionalmethod» based on growth forestimating theamino acid requirement intofish assays,fish must be fed graded levels of specified nutrition:i.e only the lysine requiretnent is amino acid in test diets containingeither only determinedfrom growth assays in feeding crystallineamino acids or a mixtureof casein, experiinentsand the other nine essential ainino acid gelat'm,crystafl ine amino acids, and other nitrogen requirementsareestimated as being proportional sources.The rearing experiments are repeated at tothe whole body amino acid coinposition pattern least l0 times for 10 essentialamino acids to includinglysine which is normally the first Iitniting completea seriesof studyin theone species, and atninoacid in most feedstuff. This methodwas the requirementvalues are estimated basedon the alsorecently applied to reddruin Moonand Ciatlin conventionalgrowth response curve. It is labor 1991!and juvenile Japanese flounder Forster and intensiveand expensive. But we shouldnote that Ogata,pers, cotmnun.!. only the data obtained from well-defined Consideringthat body ainino acid patterns experimentalconditions can be utilized for precise arenearly identical among fish species, as shown comparisonof theprofiles of essentialamino acid belowby a graphicalmethod, there would be little requirementsamong different finfish species. differencein thepatterns of requirementif they wereestimate-d by this new mcihod. The authors b! AIE ratio,as a toolfor comparingthe mode oahars et aL R'r of essential amino acid requirements the requircincnt of i-th essential amino acid for the The sumof essentialatnino acidsin dietary fish species"a " crude protein varies widely from 24% for coho The Di definedin equation } is a distance salinon to 399c.for catla andJapanese eel, and the index introducedby Prevosti et al. 975} and is averagevalue among nine species is approximately essentially the same measureas the Manhattan 349o. These values are inuch hiwer than those of distance Sneathand Sokal 1973} except for the feedstuffscotnmonly usedin fish feeds,which are standardization factor of 1/2. The D. defined in around 50% in fish tncal,soybean meal, and corn equation 1 is a distanceindex introducedby gluten meal. Roger~ in 1972 Nci 1987}, and is essentially thc Therefore, it is quite questionable to same measure as the Euclidean distance Sncath directly compareeach absolute value of csscntial and Sokal 1973!except for thc standardizingfactor aminoacid requirement atnong fish speciesdue to of 1/2. Bothof thedistances are quite frequently the differencesof proteinlevel in testdiet andthe usedfor evaluatingthe degreeof dissimilarity sumof essential ainino acids in dieuuy crudeprotein between two sets of continuous characters. For by fishspecies, To overcoinc this difticulty, Arai the species"a' and "b" with identical A/E ratios {]981! has introduced a conceptof the A/E ratio for all the 10 essential amino acids, both Di [a,b} into the field of fish nutrition studyas a usefultool and D, a,b} are equalto zero, Thc values of D, for evaluatingamino acid balance in dietary protein and D. increasewith increasing dissimilarity from the results of feedingexperiments using between the two A/E ratio profiles and the juvenile cohosalmon. The efficacyof this index maximum valuepossible is 1 for bothD, andD,. has beenreconfirmed by the feedingexperiments Thc respectiveD, and Dt values for the of cherry salmonOncor/tynchtts masott masott, and A/E ratios of amino acid requirement were arnagosaltnon 0, masottishikatvae Ogataet al. assembledinto a species-by-speciesdissimilarity 1983!. The A/F ratio is defined as [ each essential matrixof 8 x 8 dimensions.Essentially the same amino acid content/total essential amino acid procedurewas used to analyzethe A/E ratio profiles contentincluding cystine and tyrosine! x1 000]. This of the wholebody amino acid compositionsfor the index is not regardedas an absolute value of 12 fish species. quantitativercquireinent but gives attentionto the relativebalance among the 10esscniialainino acids. d! Visualizationof the dissmtHaritiesamong A/ Accordingly, the A/E ratios of 10 amino acid E ratio prufdes requirementswere calculated for the nine fish To visualize the dissirnilariry relation of species,to allow a standardizedcomparison. A/E ratio profiles among the fish species, dissinularitydiagrains were drawn basedon the c! Caleubttingthe dissimilarity indices from the dissimilarity matrices.The PHYLIP 3.5ccomputer A/E ratio pro5les package {Fclscnstcin 1993! was used for this For each pair of fish amongnine species purpose.Both theFitch-Margoliash incthod Fitch for which 10 A/E ratios of essential amino acid and Margoliashl967! and the neighbor-joining requirementsare known,the dissitnilarityor method Saitou and Nei 1987!were applied to each distanceindex was calculated as follows, matrix. RESULTS AND DISCUSSION As for the A/E ratio of the essential ainino r !* » a,b! acid requirements Table 1!, Cyprinidae. Nile 2 tilapia, andJapanese eel show ratherlower values in argininercquiremcnt, Jsoleucineand leucine are highly requiredby Japaneseeel and mil&ish. where"a" and "b" represented the two fish species Thrconine requirements in salmonidae and of beingcompared, and R indicatesthe A/E ratio of channel catfish are low, and tryptopban $8 1;JVRTeehaics} gepert >o. 26 SALHONIDAEQKIIIRIDAE CYPRIHIDAE CICHLIDAECHAN IDAE ANGUIll. IDAE Atsino Chum ChinookCoho Channel Cosmon Catl a Ni le Milk- Japanese acids l, salatcrnsaIeon salaon catfish carp tilapia fish eel Arginine 183 177 131 143 120 125 124 115 Histidine 46 53 37 51 59 64 51 53 53 I so leucine 67 65 49 86 61 92 107 102 Leucine 108 115 139 117 96 100 135 Lysine 140 147 156 171 159 182 l51 135 Met+Cys 86 118 111 78 87 87 95 87 Phe Tyr 177 150 184 166 182 161 164 139 Threonine 86 65 82 74 109 128 111 102 Tryptophan 21 15 20 17 22 25 30 28 Valine 86 94 77 98 101 92 83 102 Total lOGO 999 999 1001 1001 1001 1001 1000 Tablel. A/Eratios of dietaryamino ac~ds requirements for ninefish species. Aatno ChcrmChinook Coho Cherry RsinhoeAtlantic Channel~ Nile Yell~ lti1k Jap. acids 1 salmon salaon salem salaon trout sslaon catfish carp tilapia tait fish eel Arginine 1 15 119 115 119 123 126 132 124 137 125 124 133 His tidine 67 44 58 46 57 67 43 45 55 57 50 76 1so 1 sueinc 77 79 71 76 83 85 75 79 78 87 82 Leucinn 155 144 145 w6 147 146 136 143 142 158 145 Lysins 167 165 166 170 163 168 171 170 161 156 163 ket+Cys 80 82 92 86 71 53 75 75 75 86 68 77 Phe Tyr 147 148 146 168 149 149 147 157 136 150 147 137 Threonine 87 104 86 88 93 77 Tryptophan 29 10 27 16 18 18 15 20 16 23 21 13 Valine 88 112 83 93 98 97 102 94 103 91 Total 1000 1000 1000 998 1000 1 001 1000 1OD1 100D 999 1 000 999 Thble2. 4/E ratios of theessentaial amino acid composition in the wholebody tissueof i 2fish species. Oahara et al. 119 requircrnentsof Nile tilapta and Japaneseccl are rn ilk high when comparedwith thoseof the otherfishes. a! These findings suggest a certain degree of consistencyof requirement within fami!y or dissimilarity among farnilics from a viewpoint of relative balance. On the other hand, the A/E ratios pin of csscntial amino acid compositionsin the whole body tissueof fish species Table2! do not seemto cope display any remarkable variability in contrast to the A/E ratios of requirements. This point is o lie quantitatively examinedbelow. coho Table 3 showstwo dissitnilarity matrices obtainedfrotn the A/E ratio profiles of the essential aminoacid requirements: above the diagonal,the chinook Di indices; and below the diagonal, D, indices, Table4 showstwo dissimilaritymatrices obtained b eel tiiapia]chtnook from A/E ratioprofiles of wholebody amino acid I'92r composition,The D, valuesare in the rangefrom carp cha 0,0390 to 0,1320 in Table3, whereasin therange lowta i I of 0.0!95 to 0.0615 in Table 4. This indicates that Atla the dissimilarityindices are larger for the dietary c hum coho essentialamino acid requirementsthan for the body amino acid compositions.The sametendency is Figstre 1. Two dissimilarity diagrams, drawn to visualize the dissimilarity relation of A/E rano profiles among the also observed in thc D. indices. fish species, basedon the dissirniliarity matrices of Dl in To visualize thc dissitnilarities of A/E ratio Tables 3 and 4. a!, for the essentialannno acid require- profiles among the fish species, dissimilarity ments;and b!. for the whole body amino acid cornposi- diagramswere drawn based on thefour dissimilarity tions. The two diagramswere drawn with thc useof the Fitch-Margoliash 967! a gortthrn. species Chw Chinook Coho Channel ConnonCatla Nile ai!k- Japanese salaonnelson salaon catfish carp t i lapis f i ah eel Chw salaon G.0615 0, 0860 G.0755 o. 0795 0. 0925 G.0835 0. 1190 D. 10T5 Chinooksateen 0. 0352 G.0955 G.0770 0. 1120 0. 1090 D.1050 0. 1195 0. 1.140 salaon 0. 8505 G.0505 G.0915 D.0915 0. 1035 0. 0945 G.1320 G.1146 Channelcatfish 0. 0419 G,0455 0.0466 0. 0760 0. 0840 0. 07XI G.0975 0, D820 C canoncarp 0. 0527 D.0633 G.0508 0. 0401 0. 0390 0.0620 G.1125 D.D820 0 0567 G.0655 0. 0560 0. 0488 G.0229 D.0510 G.1045 0. 0930 Nile tilapia 0.0514 0,059G 0.0513 G.0396 G.0271 0. 0295 G.0905 D.Gaao Nitkfish 0, 0636 G.0681 0. GT24 D.0626 D.065 1 0. G621 D.0491 G.0555 Japaneseael G.0636 D.0661 G.0594 0. 0443 D.0489 0. D507 D.tl358 0. G322 Keble3. Dissimi!iaritymatrices oftbc A/F. ratio protdes ofdietary essential amino acid requireinents above tbe diagonaL D lindices; belowthe diagonal, Dz indices!. 1:JAR Terhlliral RePOrtNCC. 2CI CO O ICC I aIl: CCI ICC O a a a a a a C> a a O aCC! a a a ICC C! CCC a lh a a CII I O a CI a O a a a I~ I I CL a a a a aO aaCCC CC'. 00 O a a CCa aCl a C CC O IC92a CCCN R D~ I CIC Cv a a Cl a a O a CI Ch O a a a a~ C O IDCCI ~ I O O a CI a CC CI ICC IC lO CC a O a a O fh a a 0 JO WaCI O O CC!a 4 a a CC a a o CI a O P3 0CI a a a a a ~ I C0 a aO a 0 RCl Cll O 0 C! C C IXI I C OO a O I CV a a 5 Cl a CC a a Oa CC! a CC!O CC! Ol CIV I AI 8 CC a a a a a CC a a O CCI a 0 0SIC 0 CCCI CC Cl CI CI CI CC CI O L ~: 0 O CI IC 0RC CC oahsrs ct iu. 9i matrices in Tables3 and4. A typical pair of such lt may be true that the use of amino acid diagrams is shown in Figure i a! and b!, for the composition profile for the whole body of fish requireinentsand the compositions,respectively, especially wheredietary requirementdata are not which were drawn with the Fitch-Margoliash available,c: an bea usefultool in feedrnanagernent, 967! algorithm,from dissitnilarity matrices of D,. and it is certainly expected t.o be less time In this type of diagram,the sum of'the lengthof consuming and lessexpensive than thc traditional branchesalong the path connecting each pair of method with rcpcated feeding experiments. Thc species correspondsto the estimate of the determined levels of essential amino acid dissimilarity index best suited to the original requirement based on the concept of an "ideal" dissimilarity matrix data, protein, however, would be similar amongall fish It is obvious thatthe size of diagram a'! is species,because there are little differences in thc about twice the size of diagram b!, indicating body amino acid compositionamong species. Out higher variabilityof A/E ratio profiles of dietary findingsdeduced from growth data indicate that amino acid requirementsthan body amino acid specificities of amino acid requirementsamong compositions. Some researchers may argue that speciesor families exist as a concern in the balance thisdifference in diagramsize between a! and b! A/E ratio! of dietaryamino acids. We therefore is thereHection of largerexperimental error in the cannotdeny the necessityfor traditional methods processof determiningthe dietary requirements than in determiningessential atnino acid requirements in determiningthe body atnino acid compositions. by feedingexpcrimcnts. We suggestthat growth It shouldbe noted,however, that the speciesare experiments concerning essential amino acid notrandomly distributed in FigureI a!. Although requirements should be conductedon at least one thedistances along the path connecting species do fish speciesin a family. Further,accurate studies not seem to perfectlycorrespond to the genetic areneeded to ascertainspecificity of essentialamino distancesinferred fromthe phylogeneticrelations acidrequirements among different finfish species. suggestedby Greenwoodet al. 966!, thereare clustersof speciescorresponding to phylogenetic AC KNOW I.EDGMENTS classitications, Carp is neighboringwith catla, bothof whichbelong to the same family Cyprinidae. We thankDrs. T. Murai, K. Hosoya,and Further, churn salmon, chinook salmon, and coho T. Okazaki for their helpful suggestionson the salinonin the familySalrnonidae are located close manuscript,and Drs.D, A. Higgsand R, W. Hardy to one another.This cluster of salmonidspecies for providinguseful information. reflects the higher similarity of A/E ratios of requirements,in spite of the slightdifferences in LITERATURE CITED experimentalfactors such as the main nitrogen sourcesand the aminoacid composition of basal AgriculturalResearch Council. 1981. Proteinand diets, and rearing temperatures, Whereas, in aminoacid requirements, pp. 67-124, ln: diagrain b! from the A/E ratios of whole body AgriculturalResearch Council Working tissuecomposition, the fish speciesare randomly Party ed,!,The NutrientRequirements of locatedirrespective of classificationinto fainilies Pigs. Commonwealth Agricultural or phylogeneticcategories and the distance among Bureaux,Slough, England. fishes is much shorter. It indicates that the essential Akiyama, T. and S. Arai. 1993. Amino acid amino acid coinpositionsof whole body have requireinents of chum salinon fry and smallervariations than the dietaryrequirements supplementationof aminoacid to diet,pp. amongfish species, as alreadyreported by Wilson 35-48. In: M. R. Collie and J. P. McVey andCowey 985!, Exactlythe same topologies eds!, Proceedingsof the 20th U.S.-Japan wereobtained m thedissimilarity diagrams drawn Symposiumon Aquaculture Nutrition. usingthe neighbor-joining method Saitou and Nei Newport,OR. 1987! insteadof the Fitch-Margoliash 967! Akiyama,T., S. Arai,T. Murai.and T. Nose. 1985. method, Threon inc, hi st id inc and 1ysi ne fZ t;JtVR'reehaieat Report tea. Z6 andG. S,Myers. 1966.Genetic studies requirementsofchum sa!nMn fry, Nippon of teleosteanfishes, with a provisional SuisanGakkaishi 51: 635-639. Akiyama,T,.I. Oohara, andT. Yamamoto. ! 997. classiiicationof living forms. Bull. Amer. Comparisonof essential amino acid Mus.Nat. Hist, 131:339-456. requirements«ith A/E ratio among fish Ha!ver,J. E. 1965.Tryptophan requirements of species review paper!. Fish. Sci, 63; chinook,sockeye and silver salmon. Fed, Proc, 24: 169. 963-970, Arai,S. 1981.A purifiedtest diet for coho salmon, Halver, J. E., D, C. Delong, and E. T. Mertz, 1957, OncvrItynchuskisurch, fry, NipponSuisan Nutrition of salmonoid fishes V. C!assification of essentialamino acidsfor Gakkaishi 47: 547-550. Arai,S., T. Nose, and Y. Hashimoto, 1972. Amino chinook salmon. J. Nutr. 63: 95-105. acidsessential for the growthof eels, Ha!ver,J.E., D. C. Delong, and E. T, Mertz. 1958. AnguilloanguiIIa and A. japonica, Threonineand lysine requirementsof NipponSuisan Gakkaishi 38: 753-759. chinook salmon. Fed. Proc. 17; 478. Arai,S, and H. Ogata. 1993. Quantitative amino Halver,J. E., D. C, Delong,and E. T, Mertz. 1959. acidrequirements of fingerling coho Methionineand cystinc requirements of salmon,pp. 19-28. In: M. R.Collie and chinooksalmon. Fcd.Proc. 18: 2076. J.P. McVey eds!, Procmdings ofthe 20th HalverJ. E. and W, E. Shanks. 1960. Nutrition U.S.-JapanSymposium on Aquacu!turc of salrnonoidfishes VIII. Indispensable Nutrition,Newport, OR. aminoacids for sockeyesalmon, J. Nutr. Borlongan,I. G. andR. M. Coloso. 1993. 72: 340-346, Requirementsofjuvenile milkfish Chanos Klein, R. G. andJ. E, Ha!ver.1970. Nutritionof chanos Forsskal!for essentialamino salmonoidfishes: arginine and histidine acids, J. Nutr. 123: 125-132, requirementsofchinook salmon and coho Chance,R, E., E. T. Mertz,and J, E. Ha!ver. 1964. salinon, 3. Nutr. 100: 1105-1110. Nutrition of salmonoid fishes XI!, Mazid, M. A., V. Tanaka, T. Katayama, K. L. tsoleucine, !eucinc, valine and Simpson,and C. O. Chichester, 1978. pheny!a!aninerequirements of chinook Metabolisrnof amino acids in aquatic salmon and interrelations between anima!-III,Indispensable amino acids for isoleucincand leucine for growth.J. Nutr. Ti!apiaziIlii, NipponSuisan Gakkaishi 83: 177-185, 44: 739-742. Cowey,C. B., 3, W. Adron,and A. Blair. 1970. Moon, H. Y. andD. M. Gatlin !II. 1991. Total Studies on the nutrition of marine flatfish. sulfuramino acid requirement ofjuvenile Thcessential amino acid requirements of red drum, S ciaen ops oceliar us. plaiceand sole. J. Mar. Biol. Assoc.U.K. Aquaculture95: 97-106. 50: 87-95, Nei, M. 1987. MolecularEvolutionary Genetics. Delong,D. C. andJ. E. Ha!ver. 1962. Nutrition CohitnbiaUniv. Press,New York. 512 p. of salmonoid fishes X. Quantitative Nose,T., S. Arai, D. Lee,and Y. Hashi moto 1974. threoninc requirements of chinooksalmon A noteon aminoacids essential for growth at two watertemperatures. J. Nutr, 76: of youngcarp. NipponSuisan Gakkaishi 174-178. 40: 903-908. Felsenstein, J. ! 993. PHYLIP Phy!ogeny Ogata,H., S. Arai, andT. Nose. 1983. Growth inferencepackage! Version 3,5c, Dept. responsesof cherrysaimon OncorItynchus Genetics, S K-50, Univ, Washington, masouand amago sa!mon O. rItodurusfry Seattle. fedpurified casein diets supplemented with Fitch, W. M. and E, Margoliash. 1967. The aminoacids. Nippon Sui san Gakkaishi 49: construction of phylogenetic trees, 1381-1385. Science 155: 279-284. Prevosti, A., J. Ocana, and G. Alonso, 1975- Greenwood,P. H., D. E. Rosen,S. H, Weitzman, Distances between populations of Drosuphi Ia subobscuro based on chromosomearrangement frequencies. Theor, Appl. Gcnet.45: 231-241. Ravi,J, andK. V, Devaraj. 1991. Quantitative essentialamino acid requirementsfor growthof catla, Catiucalla Hamilton!. Aquaculture 96: 281-291. Saitou,N. and M. Nei, 1987.The neighbor-joining method:a newmethod for reconstructing phylogenetictrees. Mol. Biol. F.vol.45; 406-425. Santiago,C. B. and R. 'I'. Lovell. 1988. Amino acid requirementsfor growth of Nile tilapia. J. Nutr. 118: 1540-1546. Shanks, W. E., G. D, Gahiiner, and J. E. Halver. 1962. The indispensableamino acids for rainbowtrout. Prog. Fish-Cult. 24: 68-73, Sneath, H. P. A. and R. R. Sokal. 1973, Numerical Taxonomy.W, H. Freemanand Company, SanFrancisco, CA. 573 p. Wilson,R, P, 1989, Aminoacids and protein, pp. 111-151, in: J. E. Halver ed!, Fish Nutrition, 2d Fruition. Acadcrnic Press, Saii Diego, CA, Wilson, R. P, 1993. Amino acid nutrition of fish: a newmethod of estimatingrequirement values,pp. 49-54. In: M. R. Collieand J. P. McVey eds!, Proceedingsof the 2Gth U.S.-JapanSymposium on Aquaculture Nutrition,Newport, OR. Wilson, R. P. and C. B. Cowey. 1985. Amino acid compositionof wholebody tissue of rainbow trout and Atlantic salmon, Aquaculture 48: 373-376. Wilson,R, P, andW. E. Poe. 1985. Relationship of wholebody and egg essential amino acid patternsto aminoacid requirement patterns in channel catfish, 1ctalurus puncrares. Comp.Biocbem. Physiol, 8GB: 385-388, Yone, T. 1976, Nutritional studies of red sea bream.Rep. Fish. Res Lab. Kyushu Univ, 3: 87-101. i'n s himi os DEVELOPING A STOCK ENHANCEMENT PROGRAM BASED ON ARTIFICIAL SEEDLINGS:ACTIVITIES OF THE JAPAN SEA-FARM- ING ASSOCIATION JASFA! IN THE LAST DECADE Hiroshi Fushiini FukuyamaUniversity, Department of Marine Biotechitology Sanzo,Gakuen, Fukuyama, Hiroshima 721-0292, Japan e-inail:hfushirniCmma.fuma.fukuyama-u.ac.jp ABSTRACT The3apan Sea-Farming Association JASFA! was established in 1963 as the Seto inland Sca Farming Associa- tionand reorganized m]979 as JASFA. f ASFA has been engaged inthe task ofdeveloping techniques relating to thefarming fishery process. The term of farming fishery, injapanese Saibai-Gyogyou, means the ideal fishery systemwhich is composed of stock enhanccmen andfishery management. Farming fishery is based on the artificialseedlings technique which was constructed onsome componems.i.e ..broodsiock management. mducod spawning,incubation of fertihzed eggs, and rearing oi fryand juveniles. Stock enhattccmcnt of Ihe fanning fisherywas constructed onthc intermediate rearing innursery grounds toacclimatize artificiai juveniles totbe naturalenvironment inrc tea sing areas, seed release, management forreleased artificial seed inprc-tccrui t periods. and fishery management. INTRODUCTION JASFA!to developthe needed technology and lo overcome thc transitional period of financial Oshirna 984! reviewed ihe historical diffi c u hie. JASFAis mandatedby JFA to develop developinentofthe Japan Sea-Farming Association stockenhancement techniques based on artificial asfollows. In 1961,the Japan Fisheries Agency seedproduction. The systemand administrative JFA! establisheda plan to prornotccoastal rolesof farming fisheries are summarized in Figure fisheries by developingstock enhancement 1. Thenational government has been engaged in technologyutilizing potential and untapped thetechnological development of highly migratory productivityof the sea. The plan was put into action and migratory species. The prefectural in 1962,and the SetoInland Seawas selected as a governments are playing important roles in the model littoral zone of stock enhancetnentfor the developtnent and cornrnercial ization ol' migratory ranchingof juveniles, The SetoInland Sca Cubure andnearshore species. Public corporations and Fishery Center was establishedas the base of fisherycooperatives areorganizations in charge operation for the intended technological of operatingfarming fisheries for coastal species, developinent. Furthermore, the Seto Sea Fish exceptfor thetechnological devehipment of some FarmingAssociation was established in 1963, which speciessuch as the Japanese spiny lobster. The operated the center by commissionfrom the nationalgovernment takes responsibility for the government. This name was derived from the technologicaldevelopment of nearshorcspecies abbreviationof "Fish Farming Promotion such as the Japanese lobster because of the ActualizationCenter." This is the first time that difficulties and high risks involved which are "fish farming" wasused, Recently,the term beyondthe capabilities of prefectural governments "farmingfisheries" and "sea farming" have ~n Matuoka I 996!. usedto express fish farming. In this paper, farming Figure2 showsthe locations of 16national fisheriesconsists of stockenhancement based on sea-farmingcenters operated by JASFA. National artificial see.dlings.In 1978,the SetoInland Sea centersare located over a wide arearanging from Fish FarmingAssociation was reorganized and the Akkcsi Station,Hokkaido, in the subarcticzone renamedthe JapanSea-Farming Association closeto latitude43'N!, to the YaeyatrtaStation, 96 UJNR Technical Report No. 26 Progress of artificial seed production, release, and catch The technology of artificial seedproduction is makingsteady progress, In 1995,seed for stock enhancement was produced by 284 facilities for 80 species. The total production number was 3640 million individuals and thc total rclcasc number was 11billion individuals including natural seed Morita 1997!. The numbers of artificial seed production andrclcasc in 1995 were 3600million for 80 species and 3000 million for 69 species,respectively. The role of JASFA, as shown in Figure 1, is the technological development for highly migratory species, migratory species, and coastal water Figure 1. Schematicexplanation of roles in farming fisheries in Japan after Japan Fisherics Agency!. JASFA denotes Japan Sea-Farming Association, Okinawa, in the subtropical zone close to 24'N!, Thirty-nine prefectural govcrnrnents have been constructed and are operating 53 prefectural sea- farrningcenters Fig. 3!. Publiccorporations and fishery cooperatives have constructed sea-farming centers. Processof technologicaldevelopment in farming 6sheries The processof technological development in farming fisheries is schematically described in Figure 4, which shows the caseof Atlantic bluefin WES tuna Fushimi et al., in press.!. This figure focuses syus on artificial seed production. The technique of artificial seedproduction is composedof four parts, i.e., broodstock management, induced maturation and spawning, larval rearing, and live feed culture. Artificial seedproduced in sea-farmingcenters are transported to release areas, and then are reared in nursery grounds to acclimatize to the natural environment,or releasedimmediately if the sizeof juveniles is adequate for survival in the natural Figure 2. Locations of the JASFA Stations. environment. Fisherymanagement methodology 1. Akkcsi Stn. 2. Miyako Srn. 3. Norojima Stn. 4. ObamaStn, 5, Miyazu Srn. 6. 1Vlinami-IzuStn. 7. has to apply to artificial seedin pre-recruit and TarnanoSrn, 8, YashirnaStn. 9. HakarajimaStn. 10. post-recruit periods in order to maintain optimal Momoshima Srn, ll. Komame Srn. 12. Kaminra Srn. 13, yield from them. ShibuShiSrn. 14. GOtOSrn. 15. YaeyamaSrn, 16, Amami Stn. Fnsbimi 97 abalone Nordotis discus hannai, and disk abalone Nordotis discus discus; and one speciesof Echinodermata: northern green sca urchin Strongylocentrotus intermedius. Annual fluctuationsof the numberof seedproduction, release,and catch in some speciesare described as follows: Japaneseflounder Paralichthys olivaceus Figure 5 shows annual fluctuations of the nuinbcr of seed production, release, and catch of Japaneseflounder. The nuinbcrsof seedproduction andrelease are increasing steadily, and quantity of seedproduction and releases have surpassed that of red sea breamin 1995. Quantity of seed production in 1995 was 31 million individuals and releasewas 23 million individuals,respectively. Meanannual seed production and release numbers are19 million and 13 million individuals, respectively. Mean annual catch is 6800 tons, which fluctuated between5100 990! to 8200 986! tons, and catch hasbeen increasing since 1991, Red seabream Pagrusmajor Thetechnological development of farming fisheriesin Piscesis representedby redsea bream, andgood results in thetechnological development of this fish havebeen leading new trials for another Figure3. Locationsof prefectttralsea-farming centers, species.The numbersof seedproduction, release, andcatch of redsea bream are shown in Figure6. Meanannual seed production and release numbers species, Target speciesnumber and number of are26 million and 19 million indivictuals, respectively. seedproduction of JASFAin 1995were 36 species Mean annual catch is 14,000tons, and and120 million individuals, respectively, excluding annualcatch fluctuated between 13,000 tons 988! Molluscaand Echinodermata JASFA 1997a!. to 16,000tons 984!. Recently,it has become Public corporationsand fishery cooperatives apparentthat sport fisheries land similar quantities; engagedin interinediaterearing and releasing thus,regulation and symbiosis with sport fishing operations numbered 1387. are ncw problemsto solve Imai 1994,Imai et al. Over1 millionseed each are produced for 1994,Imai 1996,and Shinoda 1997!. 33 species,and over 10 million seedeach for ll species. They are three speciesof Pisces: Blacksea bream Acanthopagrtts schlegeli Japaneseflounder Paralichthys oiivaceus, red sea The numbersof seedproduction, release, bream Pagrus major, and black sea bream andcatch of blacksea bream are shown in Figure Acanthopagrus schiegeli; three species of 7. Mean annual seed production and release Crustacea:kuruma prawn Penaeus japonicus, numbersare 9 million and 6 million individuals, swiinmingcrab Portunustrituberculatus, and respectively. speckledshriinp Metapenaeus ensis; four species Mean annual catch is 3900 tons, and of Mollusca: scallopPatinopecten yesoensis, annualcatch fluctuated between 3600 tons 994! short-neckclam Tapesphilippinarum, Yeso to 4,300 tons 984!. This fish encountersthe same 98 I'JNR Technical Report Ntj. 26 Figure 4. Schematicexplanation of technologicaldevelopment in stock enhancementof Atlantic bluefin tuna after Fushimi et al., in press!. FIIShilali 99 uapeeeae f Sellnder 35.C00 30.000 X 900c ?0,000 C' OOC 'I5. 000 4 10, 000 5 000 0 1997 19SS 1999 1991 1991 1952 1993 1994 '995 Year Figure5. Annoa!fiuctatiOn Of seed production, retetlse snd CatCh Of Japattesc flIIultder Pardhchth» 42/Ilpl e243. Red Sea Sraae 30,000 26,005 2E.003 16,I2%'. a 24,%0 14.5'' 22,000 21,IOO 14,O20 IS,3IIO a 16,000 13 C14, OOO 4'I 'SI43 I 994 19SS ' 969 1%4 19% I 992 '.95 ' '1994 'I996 Year Figure6. Annualftuetat'Ion ofSeed produCtinn. release snd catch of red sea bream Pagrtls Illoyrrr probletnsas rcd seabream, i.e regulation and The numbersof seedproduction, release. symbiosiswith sportfishing. andcatch of thekuruina prawn are shown in Figure 8. Mean annualseed production and release Kurnrnaprawn Penaerssj apt2nictss numbers are 510 million and 305 rni11ion individuals. Thetechnological dcvclopinent in fattrung respectively. fisheries of kuruma prawn hasattained the role of Mean annual catch is 3000 tons, and pioneerin thisfield accompanied by redsea bream. annualcatch fluctuatedbetween '300 ton.s 9931 Thefirst guidebook publication of the kururna prawn to 3400 tons 984!. It seems that abundance of farming fi~hery was issuedby JASFA in 1986 thekuruma prawn has been recovering by farming Kurata et al. 1986!. fisheries. because thc mean annual catch had t tu t:J. R Technicalttepart bo. Zn 8 lart uua nreau I z ccn , u!QI , Ia 3130 2CQ 'JO C 38 OG o 5 Cut 5,Qno e 4.XIC 98 ~988 1983 I'tuC 199' 1992 1993 1939 ' eur Figure7. Floe uato[ on ced produc release ion. andcatch ofhlack sea area Aeanthnparrus sehiegei . auru au prawtI 85Q,QQQ c' t cap 3.200 C:, 850,000 TI 3,OX ~QQ. XQ ; 800 C ~ 35"QQQ I 'o 390,000 n e 35c xIQ 398u 985 1986 1982 1988 3989 1990 1991 3939 1993 1994 1995 Year Figure$. Annualfluctuadon of seed production, release and catch of t;urutnaprawn I'enaetesj apont'ccrs. declined to 1000 tons in the late 1960s, The case played an important role in this field, too. A ot Hamana Lake. a brackish lake in Shizuoka tnonograph and manual of seed production was prefecture,is well known F shirni 1983!. published by JASFA recently Hamasaki 1996, JA S FA 1 997b!. S98 immingCrab POrtunuS trituberCulatua The numbersof seedproduction, release, The technological development of andcatch of the s wimmingcTah are shown in Figure farming fisheries in the swimming crab has Mean attnual seed production and release 6 II5 hi 943I lui Saie~ Iba Crab 65.0:0 = 60.000 C55, 000 IGC 4' Z 45.GCG , 420 C ~ 60,000 D 35. 009 'C7o 30Oor 4 , 000 ~ 2a -0 C 20,OOJ 1964 I965 1966 1967 4936 '969 1990 IOOI 49ai 4993 I994 !995 446a. Figure IO, Annual t]ueruaripn Of Seedprpdueri4rn. release and parch O 5V irrrinrng CrabPI nurII4Srr'IIIIheri u urua numbers arc 52 niil lion and 28 million individuals. PM-0 to their culture water Nogami arid Maeda respecti vc1y. 1992,Nogarni et al., in press!. It is expectc4J t.hat Mean annual catch is 3900 tons, and this will bc usedin crustaceanseed production. annual catch has fluctuated between 30 Jfl tons 993! to 5300 tons 98f3!. Mean annual catch in Developnrent Of rearing larVae Of JtipatteSe the late 19605declined to nearly 100 Jtons. t.hus spiny lobSter PantolirusjaponicttS abundanceof the ssximming crab has recovered Si nce 1899, mans Japanese marine by famting fishcrics, too. biologists havetried to rear phyllosornu of P. g42ponicusf rhe firSt SuCCeSStn 3 inStar Was New frontier of farming fisheries attained in 1958 Nonaka et al. 1958!. After ihat, Exploitationof thefield of farmingfisheries therearing period wa.s graduall> improved, and la« has been continuing within JASFA. using the stagephyllosoma was attainedin 1981 lnoue accumulatedexperiences and knowledge of over 1981!. The first successfulrearing of juvenile> 30 yr. Technological developments in various was realized in 1989 Yamakawa et al- JASFA activitiesare makingsteady progress, some Kittaka and Kimuru 1989!. Success in r«r»g of which arc briefiy described bein~. larvaeof Pj aponicuswas not reproduced d« to diff'icultiesin rearing. JASFAestablished the Developinent of bioeOtttrOI fOr Seed Minami-Izu Station in 1988, to engage '" ' prodttctiottof the swimming crab develOpmentOf rearirta larvae Of P.j r2P~"" u ' Results of seed production of the Subsequently,theJASFA Minami-lzu Statroi3 "a- swimming crab is infiucnced by the flora of attainedthe completerearing of phyl!oscrrrra rnicroorganisrns, Bacterial strain PM-4. isolated lobster.It seemsthat development in hardw'~ v are of from a crustaceanculturing pond, improved thc therearing system for phy1losoma isthe rriar itn reasorI r~ growth of swimming crab larvae and repressed for this success 1 JASFA 1993h AS IGA growth of Vibrio anguiilarum in seawater. Minarni-IzuStation had produced 134 poe~ rulJ " 1and Methodology to apply this finding has been 48 juveniles in !994. and 284 puerul» a developed,and production of swimnungcrab larvae juvenileswere produced during 1990-1~9~. wasgreatly increasedby addin.gthe bacterialstrain tP2 UJVRTecboieai Report No. 26 Developmentofbroodstock xnanagement and rearinglarvae of Paciflc hlnefin tuna Thunnus techniquesforotholith using Alizarin-complexone rhynrtus ALC!,application ofstatistical survey techniques InJASFA, the development ofbroodstock forthc fisheries market, and foundation of a managementof Pacific bluefin tuna PBT! had cooperativesystem fax stock enhancement trials startedin1985 atthe Yaeyama Station inOkinawa, byfishery cooperatives, administration, and establishedin 1985. Broodstock ofPBT was research. rearedinnet cages, andwe observed veryrapid growthrate,but very low survival rate, due tohigh Developmentof seed production and release watertemperature inthe subtropical area, The ofcoonstripe shrimp Pandahcs hypsirtarus JASFAAinami Station further north was Coonstripeshrimp Pandalus hypsirrorus establishedin1995, and has been engaged inthe isone ofthe important targetspecies forthe deep developtnentofbroodstock management ofPBT. seapot fisheries, and the JASFA Obama Station, Thefirst spawning success of PBT establishedin 1983, has been engaged inthe bxoodstockofJASFA was attained in 1997, The technicaldevelopment ofthis shrimp. The JASFA firstsuccessful spawning wasobserved for he 9- ObamaStation has developed techniques for 10age group reared in40-m round-shaped net artificialseed production andsubsequent release cages10m deepon13 May 1997, and 1,500 JOO in200to 300m water depths, SurvivaJ rate of seed fertilizedeggs were collected ftumthis broodstock. productionanddensity of post-larvaehave been Spawningof7 age groups hadbeen induced by consistenflyattained at70% and 7000 individuals risingwater temperature inearly July 1997, and m',respectively, Experimental artificial seed 5,600,000fertilized eggs were collected injust 2 releasehasbeen carried out at Toyama Bay in days.Subsequently, eggquality ofthese fertilized 200to 300m depths, andresults ofthis experiment eggswas examined, Experimental rearing ofPBT pointtothe success ofstock abundance recovery larvaewas begun atthe Arnami Station in 1997 by artificialseed release, Yamazaki1997}. Manyspecies ofthe Pandulus group are importantcommercial fisherics, andtechnological Developmentof farming fisheries ofPacihc developmentsbythe JASFA Obama Station have attractedthe attention of peopleconcerned with herring resident type! Chcpeia paNusi thethe deep sea pot fishery. The technique of Pacificherring Cluitreia palkrri has sho~n seedproduction for sandfishArctacopous drasticstock abundance fluctuation, especially in japoiricusdeveloped bythe JASFA Notojima theHokkaido-Sakhalin stock.Local stock of Stationisunique. Larvae have been reared byusing Pacifichem ng resident type,RT herring! hasbeen naturalplankton, composed main!y of Copepodite, inhabitingoffeastern Hokkaido; theirspawning collectedby nightlighting.Trials of stock groundisdistributed m the Zosr era zone of brackish enhancementbased on seed release have been lakes,i.e., Notsuke Bay,Furen-ko, Akkesi Bay, continuedin Akita Prefecture, because of the andYudo-numa, andtheir migrating areais limited drasticdecline in abundance. tothe coastal area of easternHokkaido. The Accordingto this brief overviewof JASFAAkkesi Station, established in1981, has activitiesinfarming fisheries, it is evident that the beenengaged in the technical development of presenceof farmingfisheries is essentialfor fartningfisheries for RT herring since 1983 at exploitingandmai ntaining marine resources bythe Notsuke-ko.Population parameters of released Japanesecoastal f isberies, This review focuses RTherring were estimated recently, andthe stock onan overview of themain activities and some abundanceof RT herring isrecovering since newfrontiers. %e have faced many problems to artificialseed release was begun, with an estimated solveinorder toestablish theneeded technology recoveryrate at 6%, It isa successful example of of fanningfisheries, and continuing efforts are thetechnological development carried out by the required. JASFAAkkcsi Station on seed production, intermediaterearing, large scale marking ACKNOWLEDGMENTS Developtnentof larviculture of the Japanese spinylobSter, PariuliruS japonicus. Tech. I wish to expresssincere thanks to Dr. Rep.55, 128pp. [In Japanese], IzutniNakamura and Mrs, Reiko Nakamura, Kyoto JASFA Japan Sca-Farming Association !. 1997a. University,forediting scientific and English names Annualreport of Heisei7 nenndo.365 pp. of aquatic organisms, I thank the technical staff [In Japanese]. of JASFA,particularly Kinya Nogami, Tetsuo JASFA Japan Sea-Farming Association!, 1997b. Morita,Syukei Masurna, Shigenori Suzuki, Masato Principlesand practice in mass-larviculture Aritaki,Keita Hattori, Taizo Morioka, and Shintaro of the swimming crab, Porrunus Sekincfor helpful discussions. rriruberculatus,Saibai-gyogyou Gijutu Ser. 3, 181pp, [In Japanese]. LITERATURE CITED Kittaka,J. andK. Kimura. 1989. Cultureof the Japanesespiny lobster, Panulirusj aponicus Fushitni,H. 1983.Stock enhanceinent of Kuruma fromeggto juvenile stage. Jpn. J. Sci. Fish. prawn Penaeusjaponicus! in Hamana- 55 !; 963-970. ko lagoon!,pp. 236-253.. In: Y. Oshirna Kurata,H., F. Hiyama,and H. Fushuni,Editors, ed.!, TsukuruGyogyo, latest edition. [In 1986. Guidebook of thckuruina prawn Japanese], farmingfishery, Saibai-sosho1, JASFA, Fushimi,H., K. Kani,H, Nhhala,S. Nakamura,A. 306 pp. [In Japanese]. Abrehouch,K. Chebaki,and A. Berraho. Matuoka,T, 1996.Present state and prospects of In press. Attempt on resources Japan'ssea farming. Abstract. The enhancementof Atlantic bluefin tuna, International Symposiumon Marine presentstatus and futureperspective of Ranching,Ishikawa, Japan-Moroccancooperative project for Morita,T. 1997.Number of hatchery-rearedseeds aquacultureof Atlantic bluefin tuna. ICCAT andits release in theperiod April 1995- Special Publication on ICCAT 25th March 1996, Saibai82: 17-25. {In AnniversaryInternational Tuna Symposium. Japanese]. Hamasaki,K, 1996.Study on the reproduction Nogami, K. and M. Maeda. 1992 Bacteria as anddevelopment of the swinuningcrab, hiocontrolagents for rearing larvae of the Porruriustrirubercularus. JASFA Spec. crab Portunus rritubercuIarus. Can. J. Bull.8, 124pp. Fish.Aquat. Sci. 49: 2373-2376. Imai,T. 1994.Present status of fishingactivities Nogami,K. K. Hamasaki,M. Maeda,and K. onred sea bream with thepleasure boats in Hirayama. In press.Biocontrol method in KanagawaPrefecture. Saibai Giken 23 !: aquaculturefor the swimmingcrab larvae 85-93. [In Japanese]. Porrunusrrirubercularus, Special Bulletin Irnai, T, 1996, Estimatesof returnrates of red of the Facultyof Fisherics,Nagasaki seabream seeds released in Kanagawa University. Prefecture.Saibai Giken 25 !: 59-784.[In Nonaka,M., Y. Oshima,and R. Hirano. 1958. Japanese]. Rearingand ecdysis of phyllosomaof the Imai, T H. Takama, and I, Shibata 1994. Japanese spiny lobster, Panulirus Estirnatiesof the total amountof red sea j aponicur.Suisan-zosyoku 5 !: 13-15.[In breamcaught by ~ional partyboats in Japanese]. KanagawaPrefecture. Saibai Giken 23 !: Oshima,Y. 1984.Status of "fishfanning" and 77-83. {In Japanese]. relatedtechnological development in the Inoue,M. 1981.Study on rearing of phyllosoma cultivationofaquatic resources inJapan, In: of the Japanesespiny lobster,Panulirus !.C.Liao and R. Hirano eds !, Proceedings japonicus, Spec.Bull. KanagawaPref. of ROC-JAPAN Symposium on Fish.Exp. Stn. 1, 91 pp. [InJapanese]. Mariculture.Tungkang Mar. Lab. Conf, JASFA Japan Sea-Farining Association!, 1993. Proc. I: 1-11. t.g+R Tedaait:otReport Vo. 26 Shinoda,M. 1997.Sports fishing and comnercial fisheries of the red sea bream "Madai" in the sea off Kyoto Prefecture. Aquabiology19 !: 321-325. [In Japanese]. gatnakawa,T M. Nishimura,H. Matsuda,A Tsujigado,and N. Kamiya, 1989. Completelarval rearing of theJapanese spinylobster, Panulirusjaporu'cus. Jpn. J. Sci. Fish, 55 !: 745, yatnazaki,H, 1997. Spawningsuccess of the Pacificbluefin tuna in ArnanuStation, JASFA,Saibai 83: I. In Japanese]. Ohsaka aad Kashawtu t05 EFFECTS OF COVERING A TIDAL FLAT WITH SAND FOR STOCK ENHANCEMENT OF TONGUEFISH: A FKASH3ILITY STUDY AT ARIAKK SOUND IN KYUSHU, JAPAN Yukio Ohsaka and Yuichi Koshiishi Seikai National Fisheries Institute 49 Kokubu-machi,Nagasaki-shi, Nagasaki 850-0951, Japan e-tnail:ohsakaOsnf,affrc.go.jp ABSTRACT AriakeSound is characterized by a hightidal range of aboutti m atthe innermost part, and is knownto have highproductivity of commerciallyimpottant species. However, the production of certainspecies has shown decreasingtreads due to overfishiag and deterioration inenvironmenud conditions. Tonguefi ahare iraportant speciesfor gill netand trawl f i sheriesin dussound because of theirhigh cotnmercial values, but the annual catch ofCyn aglosras abbraviurtts hasbeen demeaning markedly during the last deem&. We asstnned thatcovering the muddytidal flat with sandas a raeaasof habitatrestoration would enhance the stock of thesefrsb. ln orderto studythe effects of sandcovering on growth and survival of toaguefish C.abbreviarus and C. joyttrrijavennes, wecarried out periodic satnplings bysmall beam trawl at the ianertnost part of thesound. A sand-coveredarea tnadein 199l, at aboutthe lowest low water level, to increasethe production of short-neckclams was selected ssthe survey area. The gear was towed along the lines set on the sand-covered area and a nearbymuddy area as a control.The periodic satapl ings revealed that occurrence of C. nbbreviarus inthe sand-covered area i ntneased withgrowth, but was not the case for C joyneri. Since the larger juveniles ofC, abbrevinius changed their prey animalsfrom copepods togarrnnarids andtnysids which were known tobe abundant in the sandy area, it was suggestedthat covering the mud with sand providedbenencial effects at leastfor the growth and sarvival of this species. INTRODUCTION species,together with C. rnbtssnrs,are important speciesfor gill netand trawl fisheriesin thesound Ariake Soundin Kyushuis characterized becauseof theirhigh commercial value, but the bya widetidal range of 6 mat the innermost part, annualcatch of thesefishes has been decreasing anda large 63 km' ! tidal flat that accountsfor duringthe lastdecade, 40% of the Japanesetidal flats Sugano1981!. The decreasing trends in catches of High productivity of this sound due to these tonguefishand othercotnmercially important topographicfeatures supports various kinds of speciescart be attributedto overfishingand fisheriesincluding laver culture whose annual output deterioration in environmental conditions. The is about40 billion yen ca.$330 million!, reductionof sandytidal flats is thoughtto beone Faunain the soundis uniqueand many ofthe serious environmental changes. An attempt speciesexist only here in Japan.Some of these to cover a muddy tidal flat with sand to restore the speciesare regarded as continental relics, including productionof theshort-neck clam has been camed the tonguefishCyrtoglosstrs abbreviartrs. The out, and somepositive achievemetits have been fishingof tonguefishis coriducted only in the sound demonstrated Ueda and Yamastta 1997!. The sand and a part of the Seto Inland Sea Ohsakaand coveringof the inuddyflat is predictedto make Koshiishi1997!. Anothertongueftsh, C.j oyrteri, conspicuouschanges in termsof theburrowing inhabits the coast in thesouthern part of Japan,but condition and food organism distribution for sometaxonomic studies are still ongoing since some tonguclishjuveniles that inhabit the tidal flat astheir motphologicaldifferences were found between the nurseryground. This research isto studythe effect fish in the soundand in otherwaters, Thesetwo of thismanipulation on theenhancement of these ltt6 UJNR Technical Report Vo. 26 fish. We hypothcsizcd that covering thc muddy control Fig. 1!. Samplings by the beam trawl nct flat with sand produces positive effects on the along each line were performed five times serially, growth and survival of tonguefish by meansof two times at flood tide, one time at high tide, and beneficial change in feeding conditions. two times at cbb tide when the water depths were about 2, 3,5, and 5 m, respectively. Since towing a MATERIALS AND METHODS beam trawl net by boat could not bc performed properly when the water depth decreasedbelow 2 A sand-covered area made from 1991 m, a small sct nct with a 10-m wing was also used through 1995 in an attempt to increase the to catch fishes, production of short-neck clams was selected as Some sediment sampleswere collected to the survey area. The muddy area of 300 tn by 900 analyze the particle size by wet sieving and the tn at about the lowest low water level was covered distribution of passible prey for tonguefish. The with a sand layer 40 cm thick Fig. 1!. Sampling digestive tract contentsof tongucfltshcollected in the was carried out on the days of the spring tide of previoussurvey were examinedto study prey animals. May, Junc, and August in 1994 and 1995. A beam trawl nct with a 2-m-mouth width and 2.l-rnm- RESULTS AND DISCUSSIONS mesh aperture was used as the satupling gear. The net was towed by a boatalong the two 200-m lines, Occurrence, distribution, and growth of one sct on thc sand-covered area made in 1991 tonguefish at the northeastern part of the and the other set on a nearby muddy area as a sound Our previous survey on the distribution of tonguefish from 1990 thorough 1993 revealed that four species of Cynoglossidac juveniles, i,e., Cyrtoglossus robustus, C, abbreviatus, C. joyrteri, and C. interruptus, occurred in water shallower than 25 rn at the northeastern part of the sound. Within the intertidal zone, C. abbreviatus and Cj oyneri were numericallydominant, so we focused our study on these two species. From the occurrence of juveniles less than 15 mm, we predicted that the periods for settlement of C. abbreviatus and C. joyneri were from March to May and from July ta October, respectively. Older 0-group -yr-old! C. abbreviatus seemed to migrate offshore or into deeper parts of thc sound, becausethe density of the juveniles in the shallow area decreased to nearly zero in winter Fig, 2a!. This seasonal migration was confirmed by the information obtained through a questionnaire on tonguefish occurrence sent out to fishermen Ohsaka and Koshiishi 1995!. Contrary to this, seasonal change in the density of C. joyneri was rather low in general. Though there was a certain depth migration, 0-group C.joytteri inhabited the area shallower than 10 m during their first year Figure 1. Mapof Ariake Sound.Screened area off Yanagawa Fig. 2b!. Mean body length of 1-yr-old C. City indicates the sand-covered area. The lines for hearn abbreviatus collected tn the early settling season trawl are indicated within the circle where S denotes the was about 150 mm and that of C,j oyneri was 130 line in the sand-covered area, and M denotes the line in the mm Koshiishi et al. 1994!. muddy control area, Ohsnks and Knshiishi 107 Figure2. Seasonalchange in inain distributian areaaf -group tanguefishin northeasternpart af Ariake Saund. tu Cy{!agiossi{s {!hbrev'rat{{a;b: Cynogiossnsg'ayneri,The dotted line indicatesS-!n isodepth. Five sainplingsurveys were carried out from May f991 through April 1992. Since the number of 0-group C, abbreviates collected in February was few, no illustration was presented. Distribution of tonguefish in the sand-covered area The ground levels of the sand-covered area and the control 1'muddy! area were about 50 cm and 10 cm above the lowest low water level, respectively. More than half of the sediment on the sand-covered area consisted of medium and coarse sand, and about 70% of that in the control area consisted of particles less than 63 rnm Fig, U "-'I! ! ! 0."o{ !U ! >p !{!{!{! .'i !aQ s !i! "s{! 3!. About 25 thousand fish of ca. 50 species were ]g. u i!~ s't ~ a ~/J!s' collected in our survey in 1994 and 1995 Table 1!, The fact that more thar! 90/o of these fish were juveniles confirmed the importance of the tidal flat as a nursery habitat for fish, as pointed out previously by Uchida ancl Tsukahara t'1955!. Among these species, several gobiidae species were numerically dominant. Cynoglossidaespecies were also collected in relatively large rtumbers. Though the lines on the sand-coveredarea and the muddy control area were set closely, only Figsrre 3. particle size composition of the sand-covered 50 m apart, the majority of each demersal fish area and nearby inuddy control area in 1994. tttt} I'JNR TccbnicatReport No. 2' Tablet. Fishspecies collected by smallbeaut trawl net and specieswas collected, throughout the survey setttet iu thesand-covered area and muddy control area in period, in either the sand-coveredarea or conttttl t 904and 1995. area Fig. 4!, indicating the strong effect of sediment condition on their distribution. As for Gobi'idae Clupeidae Sardine a zunasi species,almost all Favonigobiusgyrnnaucben Konosirus punctatus were collected in the sand-covered area while llisha elongata Acentrogobiuspflcturnii were collected in the Engraulididae Engraulis japonicus controlarea, The tendency of one-sidedcatch in Cor'lia nasus thesespecies was recognized regardless of their Congtidae Conger myriaster S alangidae Salan c ariattensis size,or age. Onthe otherhand, density ratios Plotosidac Plotosus lineatus betweenthe sand and control areas for juveniles Synodontidae sp. ofAcanthognbius hasta drastically increased with Belonidae sp. growth Table 2!, Thejuveniles collected in the Hemiramphidaesp. sand-coveredarea were about ]0% of thosein Syngnathidae SP. thecontrol area when less than 20 min in body Mugilidae SP. Athetinidae length,but became nearly 100% when they llvpoatherina valenciennei exceeded 60 mm. Leiognathidae SP. Sciaenidae Nihea albiflora As for Plcuronectiformes, almost all Argyroromusargentatus Japaneseflounder Paralichthys olivaceus and Trichiuridae Trichiurus lept urus stoneflounder Karei us bi coloratus were collected CentrolophidaePsenopsi s a nontahr inthe sand-covered area Fig. 4!. Contrarytothis, Sttotnateidae sp. only a fewC. joyneri juveniles were collected in GobiidaeAcent rogobi us pflaurnii thesand-covered area Fig. 5!. Thougha few Favonigobiusgymnauchen smalljuveniles were collected in thc sand-covered Silhouettea dotui areain August, the older 0-group fish were collected Tridentiger harbatus inthe control ana without exception, Thejuveniles Tridenti ger nudicervicus Tridentiger bifasciatus of C, ctbhreviatusshowed a sitnilardistribution Glossogobius olivaceus patternto A. hasta,The percentage ofjuveniles Chaenogobius uchidai collectedin the sand-coveredarea increased with Acantlrogobius flabimanus growth Fig. 5, Table 2!. Acanthogobius lrasta Figure6 showsthc body length fequency Amblychaeturichthvshexanenta of two tonguefishspecies caught by thetwo Apocryptodon punctatus Ctenotrypauchenrnicrocephalus samplinggears in 1994and 1995. In June,the Taeniaides ci rratus averagebody length of C. abbreviatuscollected Taenioi des rubi cundus by beamtrawl net in thesand-covered area was PlatycephahdaeCociello crocodilo larger than that in the controlarea, and the Platvcephatusindicus differenceswere significant in bothyears. This is CalhottymidaeRepornucenus richardsonil very interestingbecause no suchresults were Repornucenusvalenciennei obtainedfor Cj ovneri.In August,the number of ParalichthyidaeParalichthysolivaceus C. ttbbreviatuscollected by beamtrawl net Pseudorhornbusarsius decreasedmarkedly, and the nutnber collected by Pleuronectidae Pleuronichth> s cornutus Pleuronicltt'hyssp. setnet increased in turn. It is noteworthythat Kareius bicoloratus orethan half of the C. abbteviants collection by Soleidae Abrias zebra set net occurredwhen the water depth dec~ sp to less than 1 in of ebb tide. These results Cynogloss idae Cynoglossuslighti suggestedthat C, abbrevt'atusjuveniles expand Cynoglossusahhreviatus theirhabitat from the muddy tidal flat to the sandy Tetraodontidae Takifugu xanthopterus flat with growth. The increasein numberof fish Takifugu rubripes collectedby setnet in Augustmay indicate that Ahsaka anrt koshiishi itic Table 2 Mean density N/100mi'! of 0-group fish caught by beam trawl on the days of spring tide and respective mean body length 1994 1 995 MAY JUN AUG MAY JUN AUG Acanthogobitts hasta Sand-covered area Si 1. 6 1 2 03 102 136 1.1 Corttro I miiddy! area M,' 15 2 5 8 0 3 157 2 26 7 1 4 S/M 5! 10 21 106 7 51 77 Mean body length ren1 17. 5 45. 4 69. 2 13. 7 32, 5 64. 5 Cyrtogiossus abbrevtatus Sand-covered area S! 0. 3 01 01 02 08 02 Control muddy! area 'M! 8 9 43 03 20 17 06 S.'M 5! 4 2 15 8 46 36 Mean body length rrrtt! 22. 6 45. 0 113. 4 15. 7 31 8 110. 7 TndenhlterbrtasOarDS ~ 5sc TnrtsnbgerntrttteerveDS Tnttenatterbaestas DC 3D 3D DD 5D 4D 3D ZD 1D 1D O6 D D o ParahChetrStitvaCDDS k 33636nnuapu~ tVS 25 ID ~ MAY 1994 %JUN 1994 C3AUG 1994 ~ MAY 1995 V!JUN 1995 QAUG 1995 Figure4. Cumulativecatches nt muefish speciesby six senalsampltnes carried out in tbe sand-c<>vered are Si ttndnearbs muddi controlarea M!, Averagenuinber per urut area r f five to sixtov3 iugs m eachsampling series tvas cumulated. It I t.'JXRTechnical Report sv'p, 26 1994 1995 CynOgfoaSuSabb;etpat!JS Cynog!ossus abbre syatus I 01 10 c 0i1 20 1' P e c. ic CL' c sA ~ .utv M ~ = I@0th eI. " Mavs pept i1 ~ llto CVrtogtosst!s Joyvtcrt Cyriog!OSSuStOyrttttv so 40 eI JO 3 ! V J C.' '0 !II tl eI ac 10 AMGs Jt!N Jlev MAV4 Capth us ' ilAVS t!cot!,a, MAvS FigureF. Change inrelattve density oft1mcuefich inthe sand-covered area S! and the muddy contnd area M! with t1dal level tnMay. June. and August. Total catch ofI!d otvtngs. lavein thc sand-covered areaand ftve in the muddy control area lowed at ditrcrentiidal leve!s,was deftnedas I IX!. migrationsynchronized with thetidal cycle was of particlesize in which 0-groupC. abbrevtatus notclear in Mayor June when the juveniles were couldburrow widened with growth. The juveniles still small, Comparedwith C. ahhrebiatu.r,thc of 70mm werc found to show high burrowing rates reverse pattern waS true for C. joyItteri. l ew in coarsesand as well asin fine sand.Figure 7 0-groupfish exceedingl00 mmin bodylength showsthe relative ratios of thedensities of 0-group were caught in the sand-coveredarea, and the tongueftshin sandyand muddy areas which was j uvenilescollected by setnet were the smaller ones. calculatedfrom the number of fishcaught by beam Ourlaboratory experiment shop ed that the tJawlnet in ourprevious survey in 1991and l 992. C.abbreviattar juveniles of about40 rnmin body Sedimentwas classified into two categories,i.c lengthcould burrowin fine sand bul could not sandand mud, along the lines of towing. Theratio whenthc bottomwas coarse sand fOhsaka et al, of 0-groupfish caught in sandysediment tended to l 997!.This experitnent also showed that the range increase with growth in both C abhreviattasand Ohbaka aad Knahii~hi Cynoplosso abbrev, af os Csrjog 'ossos . oyr.er r I MAY 1995 trlAY 1994 r 2 Set net i r .ad+ sand t 0 Beam trav satjd. 5 Beattt '.raw,'t mud! ! IIIllltu G acj JUN 1995 JUN 1994 I I II Ig u, II, J, I . > 7 / G 2G AUG 1995 AUG 1995 G 2G 0 IG IG c Sr S 72-, bp a, b $Ot, I1, . 92 2 la. I>e I tr ! r q ' l . b Body I erjgth rttttt, f tshGau9h t hy tjeartttraa1 rretand ~1 tret ra day and the muddy COntrotarea. 112 t.'2XR lechttieai Rcport h'a. 26 C'ynogfossusjoyr>en f:ynogtossus abhreviafus f. t.0 BL=64. 7tnm OCT BL= 339. 6rnm OCT BL=35. 4-mm AUG BL=95. Brnm ttlud ALJG BL =30. bmm 20 4! 60 80 f06 D ?0 40 60 80 300 ReIative ratio of densities Figiire7, Reilauve raiiuuf the deesiues stf0-group tuttgueftsh caught byhearn trav I net tit our previous surveys ttt1991 attd 1992 Datafrigate 25-.'I5 sample ttgs aluuglives distnhuted >stitorttieas terripart of Anake Sound >tteach rntiuth were used. C jt>vrteri. Howeverthe tendencywas much abbrevt'atus,C.jovrieri of overSO nitwit preyed clearerin C. abbret'it2ru.sthan joyrteri. These priinaril> nn polychaetes. resultscoincided with thepattern of distributionof We t.ried to comparethc amount of food tonguef~shjuveniles in the sand-ccvered area. organisinsdistributed in the sand-coveredarea and control area. Usingseveral satnpl.ers such as the Preyanimals of the two tonguefishand their core sampler,grab, and slednet. four series of distribution samplingv ere carrted Out frOm May thrOugh July Prey aitirnals of both C, abbreriarus and in 1995 and l996. Unfortunately, no clear C. gciyrtericonsisted of small crustaceanssuch as distribution pattern was found in gammands, copepods,gammarids, and polychaetes Fig 8j. mysids,and cuinaceas. However, a largernumber Bothspecie» preyed mainly on copepods when their ofharpacticoidcopcpods was always f'ound in thc body lengths were smaller than 50 mm. C. sand-covered area, and the reverse results werc abbreviaturepreyed primarily on harpacticoid found f'or polychaetes. copepodscompared to C.joyrieri whoseprey pritnarilyconsisted tif calanoidcopepods. The Effects of sajtd-coveringmanipttlation for importanceof copepodsas prey haddecreased in tOngVefiSb grOWth and SurVlval bothspecies when their bodylengths exceeded 50 Our sarnpline survey revealed that C. tntn. Gammarids and tnysids becamethe main abbret t'arusinhabited the sand-coveredarea, and prey of C. abbretiaras. Contrary to C. theypassed by in tidal migrationv hentheir body !hsaka and Knshiishi ii.t IZ Co E3Ga Zl 'U HIItiy U Ma NI Br R Po Cynogiossus abbfevi ates 100 181 h0 100 i50 20'a !0"o 60'a 80 r 100 + Cynogiossusj oyneri 100 164 50 100 0 0'i 60'.i 80'.-, 100':: Food composition in weight Figlrtv.8. Weightcompost ionof theprey ardraals by sizeof torso0-group touguensh. The compostuon was calculated b> potrtt method.Co: Copcpoda: Ga: Gammaridae.Cu. Cumacea;Vly. Mystdacea:Ma. Macrura,Br: Rrachyura:Po Polschaeta. lengthexceeded 40 mm. In orderto provesome small benthic crustaceandensities are higher in beneficialeffects of a sand-coveringmanipulation sandysedimenLs than muddy sediments Horikoshi for the growth and survival of 0-group C. and Kikuchi 1976, Kikuchi 1985 h We believe that abbret r'atus,the next two points must be the first point will be clarified il' more samplesare elucidated: ! thedensity of availableprey in the analyzed, sand-coveredarea is higherthan the nearby muddy To illustrate the second poini, C. flat area; and ! the 0-group fish in the abbretiarrrs juveniles collected by a 24-h serial sand-coveredarea actually preyed on food samplingin Junewere analyzedfor digestise tract organismsinhabiting the area. contents Table 3!, All juveniles caught in the As for gammaridand rnysid density, our daytime and at night were analyzedtogether. since data showed no consistent differcncc in densities there v'as no clear diurnal change in the whole between the sand-covered area and the control contents. Time interval of ihc sampling was area.But this inaybe partly because the sainpling changedfrom 1 to4 h accordingto tidal periodicity. sizewas not largeenough and the particlesize of There was no difference in the digestive tract the sandused for the short-neckclam project was fullness index between the fish collected in the toolarge for thesecrustaceans. General! y speaking, sand-covered area and the control area when fish 114 UJNR TecbtticalReport jvu. 2s Tabte3. Fullnessindex of digestivetract drycontent weight / drybody weight, ~/0! of C.abbreviafuscaught in the sand-coveredarea and the controlarea in June, 1995,The prey found as rntact appearance was classified as undigested. Bodylength Sand-coveredarea Control muddy! area rnrn! hlo. W/ho]eUndigested No. Whole Undigested 14.0 40.0 54 2 93 0.59 26 2.24 0.68 40. 1 78, 6 12 2 9l 0. G6 13 2. 28 0. 02 stnaller than 40 mm werc compared. However, Ohsaka, Y. and Y. Koshiishi. 1995. Results of a for thelarger juveniles, the amount of undigested questionnaireon fisheries and distribution of contentsof juveniles collectedin the sand-covered tonguefishcsin Ariake Soundsent out to area was clearly larger than that collectedin the fishermen, News Seikai Natl. Fish. Res, control area as shown in the fullness index of % Inst. 80!; 22-25. [In Japanese], dryweight of undigested content/dry body weight. Ohsaka, Y. and Y. Koshiishi, 1997, Becausethe time intervalwas relatively short,we Kourai-akashitabiratne Cvnog/os rtts supposedthat the amouritof undigestedprey in abbreviartts!,pp, 190-196. In: Biologyof thedigestive tract closely represented that of the t hreaten ed and/or possibly threatened preyingested in the areawhere the juveniles were aquaticanimals, Japan Fisheries Resources collected. Thus, the secondpoint is partially ConservationAssociation, Tokyo, [In explain&. JapaneseJ. Thoughour data is limited, we consider Ohsaka, Y Y, Koshiishi, and M. Sano, 1997. thatcovering the muddy flat with sandprovided Studieson fishingground development for beneficialeffects for the growth andsurvival of tonguefishesby sediment improvement, pp. C. abbreviates. 66-73, In: Reportsof theNational Fisheries ResearchInstitute on Coastal Fishing Ground LITERATURE CITED Improvementand Developmentfor 1995. FisheriesAgency, Tokyo. [In JapaneseJ. Horikoshi,M. andT. Kikuchi. 1976. Benthos,pp. Sugano,T. 1981, Natural Guideof Ariake Sea. 149-437. In: M. Iwashita, M. Hoshino, S. TokaiDaigaku Shuppan Kai, Tokyo. 194 p, Horibe, J, Masuzawa, and S. Motoda eds,!, [Io Japanese'. Algae andBenthos. Tokai Daigaku Shuppan Uchida, K. and H. Tsukahara. 1955. The Kai, Tokyo. [In Japanese]. fish-faunaof AriakeSound. Bull. Biogeogr, Kikuchi, T, 1985. Sedimentbottoin ecosystem Soc,Jpn, 16-19: 292-302. [In Japanese]. and benthos.Fish, Eng, 22 !: 25-33. [In Ueda, T. and T. Yamasita. 1997. The case of JapaneseJ. development of Japanese littleneck clain Koshiishi, Y Y, Ohsaka, Y. Sudo, and R. Ikemoto, Rudirapesphi/'rppinartun culture ground. 1994. Relationshipbetween depth migration Fish.Eng, 33 !.' 213-218,[In Japanese]. and environmental conditions of some O-groupflatfishes,pp 64-75. In: Reports of the National Fisheries Research Institute on CoastalFishing Ground Improvement and Development for 1992. Fisheries Agency, Tokyo. [In JapaneseJ. Mural and Koshiishi 115 PROSPECTS IN STOCK ENHANCEMENT OF JAPANESE FLOUNDER Takeshi Murai and Yuichi Koshiishi Seikai National Fisheries Research institute 49 Kokubu-rnachi,Nagasaki-shi, Nagasaki 850-0951, Japan c-tnail: [email protected],go.jp ABSTRACT Accordingtofisheries statistics, there has been no significant increase in cominercial catch of theJapancsc f!ounderPairrJicJtthy» olivaccus during the past 40 yrcven though releases of hatcheryreared Juveni!cs started in l 977and the numbers of juvenilesre!eaved increased linearly to 226 millionin totalby 1995.The extensive studiesconducted in manyinstitutions to improveperfonnance of the stock enhancement program indicate that: ! ! thcadaptability ofreared juveni!es tothe natural environmem ispoor. ! theirmortality. rale just after being rc!casedisextremely high due to cannibalismand predation froin various animals, and ! theirgrowth depends onthe availability of foodorganisms atthe release site which may be ! irnited and fluctuates annual!y Recently. however,positive achievements havebeen obtained in some areas where !arger-sized juvenile~ are released and strictmanagement ofthc mixed stocks of released and wild is observed. On the other hand, mass rc!eases ofthe hatchery-rearedfishes are alleged tocause a varietyofproblems includmg: I ! spreadofpathogens, ! limited numbersof broodfishdecrease genetic diversity, ! geneticconstitution and fitness of wild stocksare changed or diminished.and ! iinpactsof massre! eases on ecosystems are not well understood.In orderto makestock enhancementnot only economicallybut alsosctentifica!!y sound, conservation of the biodiversity caimot be ignored.As a countermeasuretothese issues, the Japanese government hasinitiated a newproject to clarify geneticeffects of stockenhancement onnatural populations and interactions between the released and native populations INTRODUCTION 1995as shown in Figure1 FisheriesAgency of Japan1997!, However,fisheries catch statistics Facedwith decliningmarine fish populations Ministry of Agriculture,Forestry and Fisheries worldwideand an expanding world population, marine 1997!show no significant change in cornrnercial fish enhancementhas been attracting global attentiott catchof this speciesduring the past 40 yr, Blankettshipand Leber 1.995!. In Japan,stock In the case of stock enhancementof chum enhancementprograms were initiated by the salmonOrtcorhynchrss keru, more than half a governmentofJapan as a national project in theearly centuryof hatcheryreleases inHokkaido produced 1960sin orderto restorethe stocksof corruncrcially noevidence of anincreased yield until the 1960s. importantmarine species whose populations were But as the annual number of releases increased decliningdue to overfishing,pollution, habitat from 0.5 billion in the 1970s to 1 billion in 1982, the degradation,or humaninfluences. Technology totalnumbers of catchstarted to increasegt adually developedin theseprojects has enabled us to produce andreached up to 440%over the historic record large numbersof marinefish arid shellfishlarvae of thepre-hatchery release period with the return beyondvulnerable juvenile stages, rateof about3% in 1990 Kaeriyama 1994!, Based Mass releases of the hatchery-reared on this achievement, some people believe the juveniles of Japanese flounder Para rchrhys numbersof flounder released are not sufficient to olivuceu.s widely distributedin coastalwaters of expecta sub.stantial increase incommercial catch, Japanfrom Hokkaidoto Kyushubegan in 1977. In orderto improvethe performance of Sincethen, numbersol' juvenilesreleased in all stock enhancementof flounder, extensive studies areasincreased linearly to 22.6 millionin total by have been conducted in many national and 110 t'J'.sR Technicaittepart 5e. 76 JL ice.,e ri 'qa',ed 'apl rear Figure 1. Armual Catch Region 7 East China Sea Figure 2. Coastalwaters around 3apan divided in X regions. Thelife historyof japaneseflounder ha» conditions or simulated natural conditions for a»hort been well elucidatedby exhaustivefield and peneriod before releaseimproves their fitnessto the experimentalstudies {Minami 199, 7, Noichi 1997, naturalenvironment {Yamashita 1997!, Tanaka1997!, Generally speaking, the juveniles lessthan 70 tnm in totallength {TL! prey mainly ENVIRONMENTAL CONDITION OF RELFASE SITE onthe availability offoocl organisms Noichi 1997!. Growth of thereleased juveniles depends Accordingto changesin feedingbehavior, they on the availability of food organismssuch as dispersefrom nursery ground ds in coastal waters rnysidson the nursery ground, although mortality b t ation maybe insignificant Yamashita et to offshore Koshiishiet al,, 1991!, !, In addition, phystologic and behavioralstudies indicate that al. 1994!.Koshiishi et al, unpublisheddata! avc the releasedfish showpoor swimming a i ity, shownthat abundance of rnysidssubstantially varies uliarfeeding behavior, and lack of predator annual!y and sea»onaily asshown in Figure 5. Also, ithas been reported by Koshiishi etal, 988! that avoidance,which result in p density ozf .my»i 'd. »a t therelease site isdrastica!Iy release Fu rut a 1996!.Although the ecological decreased wit hin day a after massre}eases of mechanismsarenot comp letel understoodit is juveniles.Thus, it is apparentthat thc carrying reportedthat rearing fis sh uunder less intensive 1!8 UJ2400TDCZlniral Report 'Pn.281 Rgggnntt~ 8OPZCh ~ drrani888rrle888d RaaI onI RngiOR9 I%0 l ZDOO I%0 E ZXO J IINQ ~ l O 0 0 cl 1000 1$lS I890 !010 ID15 8990 !985 1980 IMP 8, tf Raainn 9 RPRtnn0 g le 25M o ICOO IJOD l ZDOD 150 1u 0 0 1905 0 1870 1815 I QO l%5 'lac IN5 1010 I 815 ISKI 1985 1099 1095 Year FigPDre3. Annual catCheS ofJananeSe flOunder andItttnther ofjuveniles relca5ed IuregiOnS I -4 ~bra~0 ~tdt ~ Jtrcrt 185rclCrt5:d RDI!tnt' 5 Rraints9 1JOC 15C5 1200 P POD 2 A! ODII 1500 IKC. ZOO ian 25 7 i'l66 19ZC 11I I IS3 '.985 I PDC 18 tDZO 1919 1980 189! 1855 Region1 Rnaton9 o ION «OO o JofJO E 8000 ZRÃ I DOC '1500 JCP 0 0 KR I Jsi 'lP'0 IP!5 IR4' III% le% I NG 0 1956 IP,D zp5 8000 19% Year Vtgure4.Anttuul CDCCheS OfJapane Se flotrtrder Rnduulnher ofjuveuiles releaSed inregions 5-B. Mural anctknshti~ht 1 t9 capacityof thc nurseryground is limited and fluctuatesannually, The availabilityof food 1509 OrganiSmSiSa CruCial factor lo sustaingoOd grOwth 'K of juvenileflounde since they stay in particular 1909 nurserygrounds until becomingpiscivorou9. This sx early marine-life behaviorol'!apanese flounder is different from chum salmon. 0 1991 A major causeof thc high mortality after therelease of flounderis knownto be predation by crustaceansand fishes including wild flounde Yarnashitaet al. 1993!, so that the presenceof sandy ground providinga hiding place is also a critical element.To lninimize predationproblems. various measuresare being practiced sllch as releasingsmall-sized juveniles 0-50 mm in TL! beforewild flounderappear on thenursery ground, ' 599 orraising juvenilesto largersizes up to l00 rnm in CC: TL! whi ch are less vulnerable to predat i on hc Yarnashitaet al. l 993!. CC 1991 FISHF.RY MANAGKlVfKNT High survival and growth can be expected if large-sizedjuveniles are releasedaccording to thecarrying capacity of a targetnursery area having suitablehabitat at a timewhen food organisms are Figurc 5. Annualand seasonal changes in thedensity of abundant. Even though thesefactors are taken rnysids into consideration and the best methods are ~hnttua I t:atCh ~ Juverti I eS released 1600 4500 1400 O 35M v 1200 CD 3000 c= co 1000 I 25M 800 2000 600 1500 400 1000 2 C: 200 0 1990 1995 1975 1990 Year Figlsre6.Annual CatC O f! apas ese . Hounder and number ofiuvenuec released inAornori Prefecture. Aririual catch ~Suver!iles released 000 800 700 700 ru w 800 Cg c oi ED 0 '~ +J 4ao 400 0 W ai /j " '00 300 oi 5$ ID E ai 200 100 100 0 1970 1975 1980 1985 1990 1995 Year t tgiire7.Annual catchof !apanesc flounderand nuniher ofjuveni!es releasedin Fukusbiina Prefecture. cmploycdinre]casing juveniles, the results differ amongregions. One ol' the reasons forthe lack of thcprc fee rural government implemented a guideline successinmany regions seems tobe thc bycatch forfisheries management of this species with a of leesvaluable stnall-sized fishin commercial consensusof the fishermenafter laborious fishing, dia]oguesthrough the Fisheries Cooperative Asmentioned earlier, positive results for Associations.Theguideline contains various releasingjuvenile flounder have been obtained in regulationstoprotect thebroodstoc]c andjuveniles certainregions like the Seto Inland Sea, ofboth native and released flounder, and Additiiinally,positive rc!ationships between the designationofthe important nursery grounds asa arinualcatches andnumbers ofanimals being sanctuary.Inaccordance withimplementation of releasedinthc other spec icssuch asred sca bream thisguide]ine, 2Amillion juveniles about 50rnm in andcrustaceans havebccn achieved inthis region TLhave been produced usingfunds contributed IOgawa1995!. TheSeto fnland Scaisa relatively bythe fishermen %of income from flounder closedarcs which limits migration ofreleased fishing!andhave been released annually since animalsoutof this region. Also, self-imposed 1990.The minimum sizetobe harvested wasraised regulationsbyfishcnncn arcwelt abided tonot graduallyfrom25 cm in 1990 to 35 crn in 1995. h arvcstanimals lessthan certain sizesand tore- Asa resuft ofthese efforts, theannual catch of rcfcascthem if captured. flounderhasbeen linearly increasing andit Sofar, the most successful achievement surpassedtheINo-ton level almost 5-fold in~ h,asbeen attained inAornori Prefecture, The in7 yr! in 1996 Aomorr Prefecture 1997!. annualcatch ofJapanese flounder inAomori Theseresults indicate that fisheries Pret'ccturcusedtohc the highest inJapan about managementisa keyfactor for successful stock %of thc total catch inJapan! anditis designated enhancementactivity.Also, thc fact that positive «sthe prefecture's fish,However, theannual catch resultshavebeen obtained inregions 4 andg, ofover X! tons in 1976 dropped to224 t wheretheannual catches before thestart of I9ft9 FiFig.6!. In response tothe drastico dechne,tons in stockingwererelative]y sma]lor in Aomori Prefecturewherethestock wasdepleted, indicate the importanceof theinitial stocksize for a other areas as well if a.cost-benefit analysis is successfulstocking program, which in turnmay conducted. Thus, encouragingprospects do exist indicatethat the carrying capacity for juvenile in a stock cnhancernentprogram for Japanese floundcris limited and varies ftom region n tooregion. re ' flounde, although fish stocking may not be a Thus,unlike the chum salmon project, large panaceato the problem of declining populations. increasesin numbersof juvenileflounder for As fishcrics res.ources management has releasein inanyregions may not be feasible. developedand expanded, the usc of andneed for However,a substantialincrease in sizemay be culturedfishes have increased Schramm and Piper effectivin certain areas if the production of larger 1995!. However, mass releasesof cultured fish juveniles becomescost effective, havebeen alleged to causea variety of problems suchas: spreadof pathogens;liinited numbersof FVTVRK DIRECTION the broodiish decreasesgenetic diversity; genetic In FukushiinaPrefecture, about 0,2-0.4 constitutionand fitnessof wi!d stock arechanged million juveniles having 70-100 mm in TL have or diminished; and so forth Edward and Nickurn beenreleased annually since 1987 but so far have 1993!, Also, conservationof thc speciesand producedno evidence of anincreased yield Fig. ecologicaldiversities became an internationalissue 7!.However, the detailed market survey along with after the Convention on Biological Diversity came field study indicate that the recapture rate of into effect. Under these circumstances, tnany releasedflounder by yearclass was in a rangeof symposiaor workshopsrelated to theseissues have 16-31% with averagevalue of 24'%for 4 yr 987 been held worldwide. through1990! whichis almost8-fold higherthan For instance, the US-Japan Natural the return rate of chum salmon. The reasons for Resources UJNR! AquaculturePanel held the attaining a high recapturerate in Fukushima Symposiumon Interaction betweenCultured Prefectureare reported to be that the survivalof Speciesand Naturally OccurringSpecies in the releasedjuveniles is highdue to useof large-sized Environment in Alaska in 1993, and the International fish; mysids areabundant around the releasesites; Symposiuinand Workshop on the Usesand Effects fishing effort for flounderis intensive; and the of CulturedFishes in Aquatic Ecosysternswere fishermen refrain from harvesting flounder less held in New Mexico in 1994. In order to make a than 30 cm in TL 'Fujitaet al. 1993!. They also stockenhancement program not only economically inadea cost-benefitanalysis for the entire operation but also scientifically sound, inore academic in Fukushima Prefecture, and found that the information on the impactof massreleases of flounderstocking tesulted in annualprofits of $410- hatchery-rearedfish is needed.The Agriculture, 670 thousandassuming wholesale prices of Forestryand Fisheries Research Council of Japan flounderfor 1-yr-oldfish and for 2-yr-old fish were hasjust initiated a newproject "Effect of Fish Stock $21and $33/kg, respectively, and the benefit was Enhancementon Biodiversity," In this project,we 2-3times higher than the entire costs of juvenile planto conduct studies on the genetic constitution productionandrelease. This resuh agrees very well and ecological effectsof stockenhancement on withthat of a marketsurvey conducted by theJapan the nativepopulations of Japaneseflounder, and to SeaFarming Association which indicates that stock develop technology for stock enhancement enhancementofflounder can pay off if therecapture minimizingadverse effects on biodiversity.Also, rateexceeds 20% Furusawa1994!, Fujita et al, a jointproject on flounderbetween Japan and the 993!suggested thatmuch higher profits can be USA is now ongoingand significantscientific obtainedif harvest restrictions of 0-yr-old flounder contributionsfrom theseprojects are expected. aremore strictly observed byfishermen because its A put andtake fishery whichis supposed wholesaleprice is only about $4/kg. to have,less impact on the geneticdiversities of Theseresults suggest that in certain natural populationscan be one of the future stockenhancement canbe economically feasible directions for stock enhanceinent programs, However,this practicewill permanentlydepend evenif it doesnot result in increasedyield. An economicallybeneficial effect inay be fo und '" ona stockingprogram like thechum salmon project tlat l:julia TcchntretReport 5n. ts CONCLUSIONS Giken22 I !;67-73. [In Japanese]. Furusawa,T. 1997.Key problems of sea-farming ~ Unlike the chum salmon project, large associatedwith its perspective,pp 117-126. increasesin numbersof juvenile flounder for In: T. Minami and M. Tanaka eds.!, Biology releasemay not be fcasiblcbccausc of the and Stock Enhancement of Japanese limitedcarrying capac ity. Flounder.Koseisha Koseikaku, Tokyo. [In ~ It' thc stock cnhanccrncntpriigram is carried J apanese1. outwhere the initial stock size is re 1ati vcl y small Furuta,S. 1996,Predation of juvcnilc Japanese or thc~tock is depleted,clear-cut cvidencc for flounder Paralichrhys oliuaceti.r! hy diurnal increasedyield ran bc obtainedeven at thc piscivorousfish; field observationand present level of rcleases. laboratoryexperiments, pp. 285-296. In.' Y. Watanabc, Y, Yamashita, and Y. Oozeki ~ As far as a high valued fish like flounder is concerned,if thcrecapture rate excccds 20%, eds,!, Survival Strategies in Early Life mass-releaseis cconoinicallyfeasible cvcn Stages ol Marine Resources, A.A. though no increasein thc catch is obtained. Balkerna, Rotterdam, Kaeriyarna, M, 1994. Trend of churn salmon ~ ln eithercase, stork ma.nagcmenthy fisherrncn productionin Japan, Kaiyo Monthly 26 8!: is a must, especially in thc restrictionof harvestingundervalued small fish, 471-473. !In Japanese]. Koshiishi,Y., T. Fujii, M, Noguchi,and Y, Hirota. In order ro make stock cnhanccment a 1988. Estimatedatnount of fccding by sustainableprogram, impacts of massreleases released 0-group flounder Paralichthys on thc ecosystemmust bc scientifically olivaceus! and other demersal fishes on e lucidated. rnysidsin shallowwaters off Igarashi-Harna, Niigata. Marine RanchingProgram LITERATURE CITED ProgressReport on FlounderProduction 3: 253-267, Seikai Natl. Res, Fish, Inst., Aomori Prcfecturc, 1997. Fisheries Statistics Nagasaki, [I n Japanese]. of Aomori Preferture. Aomori Prcfectural Koshiishi, Y H. Itano, and Y. Hirota. 1991. Government.Aomori. 197 p. [InJapanese[. Artificial stock-sizeiinprovement of the Blankenship,ll.L. and K.M. Lebcr. I995. A flounderParalichrhys Olivaceus: present responsible approachto marine stock statusof technologicalachievement. U.S. enhancement,pp. 167-175. In: H.L, Dept.Corntnerce, NOAA Tech, Rep. Schrammand R.G. Piper eds,!, Usesand Ef'fectsof CulturedFishes in Aquatic NMFS 102: 33-43, Ecosystcms.American I ishcries Society, Minami,T. 1997.Life history, pp. 9-24. In: T. Bethesda, MD, Minarniand M. Tanaka eds.} Biology and Fdward.G,B. and J.G. Nickum. 1993, Usc of StockEnhancement of Japanese Flounder. propagated fishes in Fish and Wildlife KoseishaKoseikaku, Tokyo. [In Japanese], Serviceprograms, UJNR Tech. Rep. No, Ministryof Agriculture,Forestry and Fisheries. 22: 41-44. ]997. AnnualStatistics of Fisheriesand FishericsAgency of Japan,1997. Statistics on AquacultureProduction. 309 p. [In Productionand Release of the Seedsfor Japanese]. StockFnhancement. The JapanSea Xoichi,T, 1997.Early life history,pp. 25-40. FarmingAssociation, Tokyo. 412 p. [In In:T. Minarni and M. Tanaka etIs.!, Biology J apane se ! . and StockEnhancement of Japanese Fujita,T T. Mizuno,and Y. Nemoto.1993. Flounder.Koseisha Koseikaku, Tokyo. [In Stockingeffectiveness ofJapanese flounder Japanese]. Paralichrhyso ivaceus fingerlings released Ogawa,T. 1995.Trends in number of the seeds inthe coast of FukushirnaPrefecture. Saibai releasedand annual catches. News Nansei Natl.Fish. Res. Inst. 55: 8-9. [In Japanese], sturli and Koshiishi 123 Schratnm,H.L. and R.G. Piper. 1995. Preface, pp.l. In: H.L. Schramtnand R.G. Piper eds.!, Uses and Effects of Cultured Fishes in Aquatic Ecosystems.American Fisheries Society, Bethesda, MD, Seikai, T. 1997, Mcchanismof metamorphosis, pp. 63-73. In: T, Minami and M. Tanaka eds.!, Biology and Stock Enhancement of JapaneseFlounder. KoseishaKoseikaku, Tokyo. [In JapaneseJ. Tanaka, M, 1997. Ecologicalsignificance of rnetarnorphosis,pp. 52-62. In: T. Minatni and M. Tanaka eds.!, Biology and Stock Enhancement of Japanese Flounder. KoseishaKoseikaku, Tokyo. [In Japanese], Yatnashita, Y. 1997. Ecology and releasing techniques,pp, l07-116. In: T. Minarniand M. Tanaka eds.!, Biology and Stock Enhancement of Japanese Flounder. KoseishaKoseikaku, Tokyo. [In Japanese]. Yamashita,Y., S. Nagahora,H. Yamada,and D. Kitagawa. 1994. Effects of release size on survivaland growthof Japaneseflounder Parul REPRODUCTIVE MECHANISMS IN MACROBR4CHIUM ROSFXBERGII AND PFNAFUS JAPONICUS: ENDOCRINOLOGICAL RESEARCH AND POTENTIAL APPLICATIONS IN AQUACULTURK Marcy N, Wilder Japanlntemational ResearchCenter for AgriculturalSciences Ministryof Agriculture, Forestryand Fisheries l -2 Ohwashi, Tsukuba, Ibaraki 305-8686, Japan tel. 01141-298-38-6630; fax 011-81-298-38-6316 c-mail: [email protected] ABSTRACT The giant freshwaterprawn Macrobrrrcriiurn roseobergii, cultured extensively throughout SouR Asia, andthe kuruma prawn Penorusjaporurus, targetedponmpally in Japan and Taiwan, are speciesof commercial itnpor- tance which have been widely studieil in terms of basic physiological function. ln Japan, P. japortiius is addiuonally asignificant target of artificial seedproducuon operations for restocking of coastatareas. ln order to ensure a sustainablemeans o artificial seed production for significant crustacean species,i is important to effectively control female molting and reproduction under artificial conditions. ln decapod Crustacea,thc physiological processesof molting and reproduction are linked and are under honuonal conuol, The role of ecdysteroidswhich serveas molting hormonesare well-established, but the physiological significanceof juvenile hormone-relatedsubstances is Justbeginning to becomeclear, Endocrtnoiogical researchin I rosenbergiiand P juponfciis Canbe putenttany applied tOaquaCulture OperatiOnS m the future, inCluding anifiCial SeedprOduCtiOn programs for P japnnicus. INTRODUCTION msenhergii. However,in all prawnspecies, il is importantto be able to cffectivcly control molting The establishment of sustainable prawn and reproductionunder artificial conditions in order culture dependson manyfactors and requires the to produce larval seed for further aquacultural integrationof variOus fields of expertiSC. Al growout. In Japan,Pj aponicusisadditiorially a present,inducing reproductionin captivityand significant target of artificial seed production controlling diseaseremain obstaclesto successful operationsfor restockingof coastal areas, culture, and solving these technological problems This paperaddresses the currentstate of will dependgreatly on basicresearch relating to endocrinologicalresearch in M. roserrbergifand the biologyand physiology of the animals being Pj aponictas, focusing onthe roles of ecdysteroids targeted. The giant freshwater prawn and juvenoidsin moltingand reproduction,and Macrobrachirarrtrosertbergii, cultured extensively discusses how basic research in this area can bc throughoutSouth Asia ChavezJusto 1991!, and potentially appliedto aquacultureoperations in the the kuruma prawn Penaerisjaponicus, targeted future. The status of artificial seed production principallyin Japanand Taiwan Liao and Chen programsfor P japorsicusand currentaquaculture 1994!,are species of commercialimportance which in Japanin this context are also discussed. have been widely studied in terms of basic physiologicalfunction. While widespreadviral RKPRODUCTIVK ENDOCRINOLOGY IN infection has becomea problem of increasing M. ROSENBERGII AND P. JAPON1CIjS magnitudein the culture of saltwaterPerraerts BackgrounrI species,disease outbreak has not beenof sigruficant In decapodCrustacea including prawns, concern in freshwater species such as M. shrimps, lobstersand crabs, the physiological tan UJNR Technical Report Ala. Zn processesof molting and reproduction are in any crustaceanspecies. Methyl farnesoate inextricably 1inked and under hormonalcontrol. MF!,the un-epox idated precursor of JH,has been Ecdysteroidssuch as 20-hydroxyecdysoneserve found in a limited number of' crustaceanspecies as "molting hormones" in Crustacea and are such as the American lobster Hornarus excretedfrom a tissueknown as the Y-organ, On americanus Tsukirnura and Rorst 1992! and the the otherhand, peptide substances such as rnolt- spidercrab Libinia ernerginara Laufer el al. inhibiting horrnonc MIH! and vitellogenesis- 1987!. In prevt'ousstudies of this author and co- inhibitinghormone V[H! originatingin thesinus workers, MF was detected in M. rasenbergii gland complex of the eyestalks exert negative Wilder ct al. 1995! but was not found in P. influence on molting and ovarian development. japonicus. MF has been shown to bc secreted Thereis evidencefor thc existenceof positive from the mandibular organs Sagi et al, ]99ti. stimulatory factors, including a putative While MF is considered to be the crustacean vitcllogcnesis-stimulating horrnonc VSH!, equivalentof JH, its role in crustaceanreproduction vitellogenis-stitnulatingovarian hormone VSOH!, remainsunclear. MF may possibly function asa andmolt-stimulating hormone MSH! whichmay VSH to stimulateyolk proteinproduction and uptake possiblybe secretedat the brain and thoracic assuggested in Figurc1. Thc structureof MF is ganglion Takayanagi et al. 1986,Meusy and Payen shownin Figure3 1988!; however, such factors have not been sufficiently isolated and identified. A general Eedysteroids and juvenoids in M. roserebergei schemefor the controlof moltingand reproduction and P jrrponicus in Crustaceais shownin Figure 1. Structuresof In bothM. rosenbergiiand P,japonicus, representativeecdysteroids in M. rosenbergiiare an ecdysteroidsurge occurs in thc hemolymph in shown in Figure 2. the late pre-molt stage and the predominant In insects,juvenile hormone JH!, a larval ccdysteroid species is observed to be 20- developmentalhormone, also appears in theadult hydroxyecdysonewith lesseramounts of highly fernaleto stimulateyolk proteinproduction and polarecdysteroids high polarity products HPP!!. uptake. To date.. JH itself has not been identifted In M. rosenbergii,peak titers are about 40 ng/ml Okumuraet al, I 992!, and in P j aponicus,these levelsreach nearly 200 ng/ml Okumuraet al. rl at factOrs Exter 1989!, 20-hydroxyecdysoneis generally considered to be the active form of the hormone in !t-organ Cen.rai .-e.;ous systen. mostcrustacean species, and it regulatesthe molting Sinus =tan= cycle. I In additionto involvetncntin molting, V I H ecdysteroidsare foundin newlylaid eggsand matureovaries of numerousinsect and crustacean Y-organ Ibl'Iar o a t species.In general,eggs ecdysteroids during the earlyembryonic stages are ovarian in origin and e:D serveas a stockfor purposes of earlydevelopment Vo0I-, until embryonicprothoracic glands or Y-organs vg syn:rtetl c Ovary differentiateand produce ecdysteroids de novo site Vg Spindleret al. 1987!, In M. rosenbergii, ecdysteroidsarepresent inmature ovaries 5 ngf Figtsrel, Generalscheme for the endocrinologicalcontrol of g! andnewly spawned eggs 6.9 ng/g! Wilder et moltingand reproduction in Crustacea,Abbreviations are al. 1990!.These ecdysteroids are accutnulated in indicatedin thefigure for molt-inhibitinghormone MIH!, ovariesduring the reproductive molt cycle mott viteliogenesis-inhibiting hormone VIH!, ecdysteroid cycleaccompanied bymaturation of the ovaries! ECD!,vitellogenes is-stimulating hormone Vt H!, methyl farnesoatetlVlF!,nnd vitettogenesis-stimu!anng ovarianhor- tolevels of 50ng per ovary. In contrast, during the mone VSOH!. commonmolt cycle mott cycle in whichovaries wilder lZ7 EcdysteroId structures > rluvIII~ < II IO Figttre 2. SUDC tureS Oi representative ecd ysteroid sin hf. roseahergiiIncl tIdi stg the acb ve form Of the hOrm one. M-hydrus yecdy sone, preCurSOreCdySOne, and rnetabOliteS20,2b-dihydrOXyeedysnne and 20-dihydrOxveedysonotcaCid. Reproductive {2E6E! Methyl farssesoate so Figure 3. Structure of crustaceanjuvenotd substance,me- thyi famesoate MFl. 4o cn ID remainimmature!, ecdysteroid content is 1.5 ng perov ary Wilderet al, 1991! Fig. 4!. Thisovarian ra 3D ecdysteroid accumulation which occurs in O cr synchronizationwith moltingmay signify a rolefor CL these ecdysteroidsin inducing germinal vesicle aD C breakdown GVBD! andsubsequent ovulation. CI This author has examined hf. rosertbergii 0 isI and P. japorricus for the presence in the as so hemolyrnph of juvenoid substancesincluding I O juvenilehormone Ill andmethyl farnesoate MF!. MF was present in females during both the repruductiveand conunon molt cyclesand in lnales D CIICI DII D tsI Dr a 8 in M. roserrbergir Wilderet al l995!, butwas not Molt stage detectablein P jrrporticus Wilder andAida 1995!. In M rosenbergii,MF fluctuatedduring the molt Figure4. OvarianecdysteroId accumulauoo m ovariesdw- cycle withoutcorlnection to ovariandevelopment, ing tbe repraduCtivemolt cycle andeedytterOid coIIlent beinghighest in theearly pre-molt stages Wilder duringthe common molt cyclein ftf. roserrbrrgrion ltasis et al, 1995!. Theseresults suggestthat MF may of a 25-gindividual. Ecdysteroids are abbreviated as ECD. UJNR recbniealReport .'so.zs be involvedin the moltingprocess, In Arremia, significantlyto thedevelopment of techniquesfor MF has been found to elevate Na/K-ATPase controllingmaturation and reproduction in captivity. activity in larval homogenates,additionally Theremainder of this paperwill introduce suggestinga role in osmoregulation Ahland Brown the current statusof Pj aponicusculture in Japan, 1991!. and artificial seed productionprograms for this Ina separateinvestigation, this authorand speciesand related applied research being carried co-workers testedthe effects of MF injection on out by the Japan Sea-Farming Association vitellogcninproduction in eyestalk-ablatedjuvenile JASFA!, The authoris engagedin a combined M. rosenhergii Wilder et al. 1994!. Although farmingsystems project in theMekong Delta region vitcllogenc»is in hf. rosenbergii will not be of Vietnam, focusing on M. rosenbergii seed discussedin detail here, there were no observed productionand aquaculture, but this will not bc increase»in vitellogeninproduction in responseto discu ssed in detail. MF treatment, These results indicate that MF alone could not promoteincreased vitcllogenin PFNAEUS JAPONICUS CULTURE IN production,but do not rulc out a role for MF in JAPAN crustaceanreproduction. Whether MF playsa role The following information is basedon a in inducingpatency of theovarian follicles, thereby 1995Fiscal Year Report of thc Norinchukin Bank, allowingthe uptake of vitellogeninin developing FisheriesDivision sce Fujiwara 1995!. In 1995, oocytes,as JH doesin insects Daveyand Huebner total marine culture productionin Japan was 1974, Davey et al. 1993!, needs to be further approximately 1.284 million tons with a market addressed. value of Y575,6billion, Major speciesof interest in Japaninclude ycllowtail,sca bream,flounder, Current perspectives in endocrinologicai and the kurumaprawn P. japorricus, Of thistotal research production, P. japonicrrs culture accounted for At present,it is we!1-established that 20- approximately2000 tons valued at Yl 3,514billion. hydroxyecdysoneand other ecdy steroids function During 1991-1994, productionof P,japorricus as moltinghormones in Crustacea,but it is still decreasedfrom nearly 2500 to 1500 tons due to unclearwhat roleecdysteroids play in conjunction severe viral outbreaks in western Japan, but withjuvenoid substances in stimulating reproductive evidence of recovery is beginning to be seen as processes, Much progress has been achieved causes of viral outbreak have come to be recentlyin theisolation and identification of eyestalk elucidated,The numberof operatorsin total has hormones,particularly of MIH, A Japanesegroup remainedfairly constantduring thisperiod, around has succeededin isolatinga putative MIH in P. 150-160 enterprises nationwide. Table 1 japoiricas Yanget al. 1996! and hasdemonstrated summarizes changes in enterprise number, it to have molt-inhibitingactivity by assessing productionvolume, and market volume from 1991 ecdysteroidsynthetic activity under Y-organ to 1995, culture, Thenature of VIH in bothM. roseirbergii P, japonicas culture began as early as and P.japonicus and MIH in hf. rosenbergii 1962with the development of artifrciaJpropagation remainsunclarified, Morc information concerning techniques,and production levels peaked in 198g the structures of these hormones and how titers at about3020 tons. Typically,culture operations fluctuateduring the molt cycle shouldgreatly arebegun in the spring,between March and May, improve knowledge of endocrinological and prawns are reared to market size of about 30- mechanismscontrolling molting and reproduction. 50 g by the endof the year or following spring. It is also of importanceto further elucidatethe Culture is thus carriedout on the basisof yearly physiologicalroles of MF, andto detemmewhether cycles, Commonfeeds includeminced sardine, putative brain and ovarian factors are involved in squid or clam, and artificial pellets Culture is hormonalprocesses. Finally, an understandingof focusedpredominantly in westernJapan, Kyushu, how such mechanisms operate in context of andOkinawa. In 1994,of a totalof 151 operators, environmentalfactors is expectedto contribute 52 were based in Kumarnoto Prefecture, 25 in year No, enterprises 161 156 163 151 Production volume tons! 2, 491 2, 187 1, 712 1, 519 a2, 000 hiarket volume billions of en 17, 176 7. 11-t 15. 76 . 514 ~ 'estimated Table1. Changesin enterprtse number, product ton volume and marketvolutne lnrLurunia prawn culture in Japanduring 1991-1995 P ecture No. ent r ' To La I ea tn' Avera e a ea m' Kutaamoto 52 1, 549, 000 30, 000 Kagoshittta 25 997, 000 40, 000 Okittasra 20 715. 000 36, 000 49, 000 Ehime 15 642, 000 9 00 Yama uchi l.3 5 000 Table2. Numberofenterprises engaging inkuruma prawn cuhure, and total and average areas under operation. Kagoshima,20in Okinawa, 15 in Ehitnc, and l 3 in stockingor onan experimentalbasis. These Yamaguchi.Table 2 sununarizesthese figures programswill beintroduced inthe next section. alongwith totaland average area per enterprise underculture. Three forms of culture arc typically A RTIFI CI A I. S FFD PRO DUCTION practiced:artificial pond culture, net culture, and PROGRAMS FOR P. JAPO/VICUS IN tankculture. Artificial pond culture and tank culture JAPAN aremost predominant with only a minorityof Current status Artificial seedproduction prograins for P. operatorsengaged in net culture. In 1992, widespreadviral outbreak japrrnicusare implemented by theJapan Sea- occurredasa resultof theintroduction ofinfected FarmingAssociation JASFA!, an auxiliary seedimported frotn China. Causes ofviral outbreak organizationof the JapanFisheries Agency. Programsrelating to kurutna prawn production are havesince been elucidated, and control measures, mamlyearned out at JASFA's Momoshima Station suchas the disinfecting of culture ponds, have in HiroshimaPrefecture, and Shibushi Station in helpedbring the situation under control. Operators KagoshitnaPrefecture. Actual operations are havehad to rely increasingly more on domestic principallycarried out at Shibushiwhile work sourcesof seed, In Okinawa, parent prawns relatingto thecultivation of female spawners is obtainedfrom Miyazaki, Oita, and Nagasaki beingdone at Motnoji ma. Prefecturesareused to secureseed. While there Figure5 showsstatistics forartificial seed is sometechnical assistance and cooperation productionandrelease during the years 1977-1995. carriedout between governtnental agencies and Productionlevels have generally ranged between privateoperators, thefortner is not permitted to 400and 600 inil lion seeds/yr, with figures for actual produceand sell seedto the privatesector; releasefluctuating around 300 million seeds/yr therefore,culturists need to rely on other privatc- JASFAstatistics, personal communication, M. sourcesforobtaining seed. Goverrtment-sponsored projectsrelating toartificial seed production are Kobayashi,Japan Sea-Farming Association, Kanda. implementedexplicitly forpurposes ofcoastal re- Tokyo!.At present, JASFA relies entirely on thc 130 1,:JVR Technical Report Kin. 26 X l j! 1 p // // 'i"~' 78 7'9 h ! Hl 8',~ h,", 8 1 jl;=, Ht' 87 Nit Hjl tl ! 9 ji'> H.'> Sil Yc tr f r !jtt It>77 ]HH~! Figure S. Statistics for artificial seedproduction and release of P japonicus in Japan from 1977 to 1995 courtesy of the Japan Sea- Farming Association!. use of natural spawnersfor obtaining seedswhich day. At Shibushi in 1995, purchasing operations are generally raised to a size of 12-18 mrn before were carried out 20 times between 25 April and 7 release,although "large-size" seedsfrotn 20-40 mm September,during which time 6283 parent prawns are occasionally produced. Seed production were obtained Miyajima 1995!, and actual seed operations are conducted principally from April to production operations were implemented on a total September, when water temperature is warm of 15 occasions. Of total prawns purchased, 6120 about 26'C! and parent spawners are readily individuals survived transportation to Shibushi available, In general, prawns with developed Station, and 2005 individuals actually spawned at ovaries are purchasedfrom commercial fishermen, an average spawning rate of 39,4%. This yielded and are brought to JASFA premises while chilled an initial total of 236 million seeds with a final slightly or kept on sawdust, and are then put into harvest figure of 92 million seeds. Table 3 shows stocking tanks. With tetnperature in the stocking actual figures for each spawning occasion, with tanks raised back to higher temperatures,spawning size of tank used, date, number of seeds obtained, usually occurs in the evening or by the following and density for initial stocking and harvest. % elder 131 Prod. Tant Bo. 4 Table 3. Artificial seed production at JASFA Shibushi Station in 1995: initial production, density, body length and tinal harvest values. Operations were similar in the previous two years not discuss these areas in detail, but one potential of 1993-1994 Sato 1993, Sato and Yoseta 1994!. means of marking is uropod-cutting, whereby the Transportof artificial seedsis usuallydone regenerateduropod differs in color and pattern from by truck, and seeds are supplied to various uncut ones, making individuals of artificial origin prefectural users. Some trips take up to 17-1g h. distinguishable Miyajirna et al. 1996!. Shipping density ranges from 5.6 - 55.7 million individuals/m' and is adjusted according to length RESEARCH RELATING TO SEED of the trip and size of the seed. Temperature is PRODUCTION IN P. JAPONICUS kept between 19.0-22.5'C in order to suppress Background metabolism. Mortality during shipping is virtually The Mornoshirna Station of JASFA has nil and seedsare observed to be in good condition developeda biopsymethod for determining the state uponarrival. of ovarian deve!Opmentin Pj aporticus Miyajima At present, it is not difficult to secure and Matsumoto 1996!. It was previously necessary parentfemales during rnid-spring to early autumn; to rely on assessing prawns for maturity by however, theavailability of spawnersobtained from observingthe visible developmentof the ovaries, natural sources makes it difficult to conduct and classifying them into A, B, C, and D ranks operationsearlier than April. In addition, while basedon relative size and visual appearanceof seed productionis generally successfulbased on thc ovaries. The A and B ranks in which ovaries placing these females in holding tanks prior to are enlarged were considered mature and the C spawning and collecting the seed, it is difficult to and D stages in which ovaries were still elongated control spawning time or to synchronize the were considered immature Miyajima and spawningof many individualswhich would make Matsurnoto 1996!. However, with these methods, operations more efficient, It is still therefore it was difficult to observe fine differences in necessaryto improve technology in order to provide ovarian maturity which would serve as an index a stable supply of seed for coastal restocking. for spawningpotential. The biopsymethods permit JASFA is engagingin basic and applied research detailed observationsof developing oocytes. In in order to addressthese problems. It alsoremains this method, a syringe is inserted into the ovaries difficult to assess the effectiveness of seed release via the soft area between the first abdominal programs. At present, JASFA is investigating segment and the carapace. Oocytes are then meansof marking seeddestined for releasein order collected and positioned onto a glass slide and to determinethe proportions of artificially-produced observedunder light microscopy at a magnification prawns in the natural habitat, This manuscript wiH of 100-200X. Oocytescan be differentiatedinto 13$ tlJNR TechnicalReport tsa. zs three stages which correlate with histological individuals in the third yolk globule stage were examination.In thethird yolk globulestage, cortical unilaterally eyestalkablated right-side!, ln these alveol i cannotbe seen,but other features are sinu! ar individuals, there was a 40% spawning rate with to thoseof theearly maturation stage. In this stage, about200,000 eggs per batchand 73.7% hatching. the cortical alveoli becomeapparent. Finally, in In individuals not ablated,spawning was 7.1%with the maturation stage,the cortical alveoli become no differencesbetween the ablatedgroup regarding elliptical, batch-size and hatching rate. These results Thesemethods are a usefultool in selecting demonstratedthat biopsy can be used to reliably spawnersfor seedproduction operations and in selectprawns which will spawn,and that unilateral implementingexperiments relating to inductionof ablation is effective in increasing spawning rates maturation. In JASFA during 1995 Miyajirna in individuals prior to reaching the maturation 1995!, a seriesof experitnentscarried out at the stages. MomoshimaStation relating to artificial inaturation In part ! of this study, the use of in context of selection of prawns, transport environmentalfactors to inducespawning in thethird conditions,and rearing conditionsare highlighted, yolk globulestage females was examined. Fifteen Thesestudies, irnpleinented in order to dcvclop I-yr-old femaleswith A rank ovariesin this stage technologyfor the control of maturation and were selectedand usedexperimentally for a period spawning,are briefly describedbelow, of 4-7 days. Three groups with differing light conditions,24h lights-on, 14 h lights-an,and 0 hlights- Developmentof technologyfor the control of on were maintained bctwecn 18.6-24.2'C. As a maturation and spawning result,one individual in the l4 h lights-ongroup only MtrsseLariomof spawrriIsg spawnedduring the experimentalperi iatt st e Ho. rastts go. s aaners S avnin ate No. da s batch size Hatchin rate 3rd Yolk globule 14 1 7. 1% l. 261, 000 32. 2% Earlr aatttration 22 22 214, 000+ 101,000 57. 1~36. 2% t t'o 3 +84 000 78. 9~ . 2% e 4. Spawningrates, number of eggsper batch and hatching rates for spawningstimulation experhnent in kurutna prawn at >ASFA. Wilder i33 wereelnployed. Prawns were reared for a period workingin theserespective areas should bc actively of about 2 monthsand then exatninedfor ovarian pursued. lnaturationand evidence Of mating depositionOf spermcase!, Mating rates were 50.0%, 83.7%, ACKNOWLEDGMENTS and93,8% in groups1, 2, and3, respectively. The authorexpresses thanks to Toru Maturationrates were 6.0%, 26.5%, and 0.0%. Thus,treatment 2 was mosteffective. Prawns Furusawa,Managing Director. Masato Kobayashi. and Kouichi Saotorncof the Japan Sea Farming, with lnature Ovarieswere further examinedby biopsyfor the presence of thecortical alveoli, but Association JASFA! for generouslyproviding JASFA in-houselnaterials and guidance. noindividuals exhibited this. Actual spawning rates were0.0% and 42.9%in groups1 and2, Thus, only group2 individualsspawned with hatchout LITERATURE CITED rates of 86,4%. This is the first time, however, Ahl, J.S.B.and J,J. Brown. 1991. The effectof that femaleswere inducedto matureand spawn outsideof their norlnalspawning seaSon without juvenilehormone III. methylfarnesoatc, and rnethopreneon Nw'K-ATPascactivity in using eyestalk ablation, by manipulating larvae of the brine shriinp,A rtemia. Comp. environmental parameters, Biochem.Physiol. 100A: 155-158. Chavez Justo,C. 1990. Ecology,reproduction, PERSPECTIVES ON ARTIFICIAL SEED and culture of the giant freshwaterprawn. PRODUCTION AND CONCLUSIONS Macrobrachiumrosenbergii. pp. 163-191. ln; C. ChavezJusto ed!., Thc Aquaculture The abovestudies carried out by JASI'A havedemonstrated that it is possibleto control of Shrimp,Prawn and Crayfish in thcWorld. maturationand spawningin P.japorticus by Midori ShoboPubl., Tokyo. in Japanese! understandingthe effectsof the environmenton Davey,K.G. and E. Huebner,1974. The response theseprocesses, Atpresent, JASFA is cooperating of the follicle cellsof Rhodniusprolixus to with universitiesand other research organizations juvenilehormone and antigonadotropin in to increase knowledgeof mechanismsof vitro. Can. J, Zoot. 52: 1407-1412, maturationand to improve existing technology, ln Davey.K.G., V.L, Sevala, and D.R.B, Gordon. fish, knowledgeof the interactionof the 1993.The action of juvenile hormone and environment and endocrinology of significant antigonadotropinon the follicle cells of specieshas formed abasis for the development of Locustatnr'grataria. Invertebr.Reprod. usefultechnology. In manyspecies, it is known Dev. 24 39-36. thatfollowing ovarian maturation, the secretion of Fujiwara,T. 1995, Shuyoyoshokugyo saochi chosahokuku. Annual fiscal report of the steroid hormones which serve as maturation- inducing substance MIS! is triggered by Norinchukin Agricultural! Bank, 1-13-2 environlnental cues, Other basic knowledge has Yurakucho,Chiyoda-ku, Tokyo, l 00-0006, allowedthe developlnent of hormonal treatments Japan,106 p. In Japanese! to stimulatefinal maturationand spawning,such Laufer,H., D. Borst,F.C. Baker. C. Carrasco.M. asin theuse of humanchorionic gonadotropin. In Sinkus, C.C, Reuter, L.W Tsai, and D.A. Crustacea,while much progress has been achieved Schooley,1987. Identification of a juvenile inelucidating hormonat mechanisms, much remains hormone-likecompound in a crustacean. to be elucidated on how environmental factors Science235: 202-205. influence the secretionof hormoneswhich control Liao, I,C. andY-H. Chien. 1994. Culture of moltingand reproductive processes. Similar to fish, kurumaprawn Penaeusjapotticus Bate! whether an M IS existsis still unc!ear. In the future, in Asia. WorldAquacult. 25 I !; 18-33. it will be importantto link basicstudies to explain Meusy,J-J. and G.G. Payen, 1988. Female observationsand resultsof fieldwork and practical reprOductionin lnalacoStracan Crustacea. experiments,Cooperation between persons Zoot, Sci. 5:2l7-265. t:JNR Teehaieol Report 4o. Za Miyajima, Y. 1995. Artificial seedproduction in kyokai jigyo-nenpo. Annual report ttf the kurumaprawn at Mornoshima Station,pp. Japan Sea-FarmingAssociation JASI'A!, 43-51. In: Nippon saibaigyogyo kyokai 3-14-8 Uchikanda,Chiyoda-ku, Tokyo ] 01- jigyo-nenpo. Annual report of the Japan 0047, Japan. In Japanese! Sea-FarmingAssociation JASFA!, 3-14-8 Sato, H. and K. Yoseta. 1994. Promotion of Uchikanda,Chiyoda-ku, Tokyo 101-0047, kuruma prawn artificial seedproduction at Japan. In Japanese! Shibushi Station, pp. 324-326. In: Nippon Miyajima,Y. andMatsumoto, A. 1996.Maturity saibaigyogyo kyokaijigyo-nenpo. Annual classification using biopsyin pond-reared reportof the JapanSea-Farming Association broodstock of kururna prawn Penaeus JASFA!. In Japanese! japvnicus and efficient egg removal. Saihai Spindler,K,D., A. VanWorrnhoudt, D. Sellos,and Giken25: 37-40. In Japanese! M. Spindler-Barth. 1987.Ecdystcroid ]eve]s Miyajima, T., K. Toyota,Y, Harnanaka,and H. during embryogenesis in the shrimp, Komaki.1996. Efficiency of uropodcutting Palaentonserrarus CrustaceaDecapoda!: for markingyoung kuruma prawnPenaeus quantitative and qualitative changes.Gen. japvnicus. Saibai Giken 25: 41-46. Comp.Endocrinol. 66: 116-122. Mizuta,Y, 1995. Artificialseed production in Takayanagi,H., Y. Yamamoto,and N, Takeda, kururnaprawn, pp. 42-43. In: Nipponsaibai 1986. An ovary-stimulatingfactor in the gyogyokyokai jigyo-nenpo. Annual report shrimp, Pararya carnpressa.J. Exp. Zool. of the Japan Sea-Farming Association 240 203-209. JASFA!, 3-14-8 Uchikanda,Chiyoda-ku, Tsukimura,B. and D.W. Burst. 1992. Regulation Tokyo 101-0047,Japan. In Japanese! of methyl farnesoatein the hernolyinphand rishi, A. 1995. Promotionof kurumaprawn mandibular organ of the lobster,Hvrnarus artificialseed pmduction at ShibushiStation, antericanus.Gen. Comp.Endocrinol. 86: pp. 336-338. In: Nippon satbai gyogyo 297-303. kyokaijigyo-nenpo. Annual report of the Wilder, M.N. and K, Aida, 1995, Crustacean JapanSea-Farming Association JASFA!, ecdysteroidsand juvenoids: chemistry and 3-14-8Uchikanda, Chiyoda-ku, Tokyo 101- physiologicalroles in two speciesof prawn, 0047, Japan. In Japanese! Macrvbrachiurn rvsenbergii and Penaeus Okurnura,T., K, Nakamura,K, Aida,and I. Hanyu, japvnicus. Isr. J. Aquacult,Bamidgch, 47: 1989.Hernolymph ecdystemid ]evels during 129-136. themolt cycle in thekuruina prawnPenueus Wilder,M.N., T. Okurnura,K. Aida, and I. Hanyu. japvnicus. Nippon Suisan Gakkaishi 55: 1990. Ecdysteroid fluctuations during 2091-2098. embryogcnesisin the giant freshwater Okumura,T C.H Han, Y. Suzuki,and K. Atda. prawn,hfacrobrachiurn rvsenbergii, Gen, 1992.Changes in hernolymphvite]logenin Comp. Endocrinol. 80:93-100. and ecdysteroid levels during the Wilder, M.N., T. Okumura, and K. Aida. 1991. reproductiveand non-reprocluctivcmolt Accumulationof ovarianecdysteroids in cycles in the giant freshwater prawn, synchronizationwith gonadal development Macrvbrachiumirisenbergii. Zool. Sci. 9: in the giant fresh water prawn, 37-45. Macrvbrachiurnrvsenbergii. Zoo]. Sci. 8: Sagi, A..E. Homo]a, and H. Laufer. 1991. Methyl 919-927, farnesoatein the prawnMucrvbrachium Wilder, IVI.N., T. Okurnura, Y. Suzuki, N. Fusetani, rvsenbergii: synthesisby the tnandibular andK. Aida, 1994. Vite]iogeniitproduction organin vitroand titers in thc hemolyrnph. inducedby eyestalkablation in the giant Comp,8iochern. Physio].99B: 879-882. freshwater prawn Macrobrachiunt Sato,H. 1993. Promotionof kurumaprawn rosenbergii and trial methy] farnesoate artificialseed production at ShibushiStation, administratio~. Zoo], Sci. 11: 45-53 pp.361-362 In: Nipponsaibai gyogyo Wilder isa Wi!der,M,N S. Okada,N. Fusetani,and K, Aida, 1995. Hemolyrnphprofiles of juvenoid substancesin the giant freshwater prawn Macrobrachiurn rosenbergii in relation to reproductionand molting, Fish. Sci. 61: 175- 1 76, Yang,W-J., K, Aida,A. Tcrauchi,H. Sonobc,and H. Nagasawa,1996. Amino acid sequence ofa peptidewith molt-inhibiting activity from the kuruma prawn Penaeusjapnnicus, Peptides17; 197-202, Adachi et al. 137 PRIMARY PRODUCTIVITY OF SANDY SHORES K urn iko Adachi NationalResearch Institute of Fisheries Engineering Ebidai,Hasaki. Ibaraki 314-0421,Japan e-mail:kin C~'nrife.affrc,go.jp Katsunori Kimoto Seikai national FisheriesResearch Institute 49 Kokubu,Nagasaki 850-095 l. Japan c-mail:kimotoOsnf.affrc.go.jp JunyaHigano JapanInternational Research Center for Agriculture Science 1-2Ohwashi, Tsukuba, Ibaraki 305-0851,Japan e-mail;higa@'jircas.affrc,go.jp ABSTRACT Manykinds of aquaticorganisms «rcfound inhabiting the surf rxinc around exposed sandy shores and, in particular,plankton feeders such as the sandy beach clam is animportant species contributing to fishery resources.1'his fact shows that thc biontass of phytoplanktonin these areas is abundant.which comes from primaryproducuon. Thus, it inay be possible toestablish no-feed aquaculture andnursery culture ofbivaJves byusing abundant, natural phytoplankton asfeed in suchareas. However, because sandy shores are often utilizedfor varioushuman activities, the beach shape is sometimesartificially modified It istherefore necessary toclarify the mechanisms whichsupport primary production inexposed sandy shores in order to mainuun and improvebiological production m harmony witha varietyofcoastal uses. In this context, wc have investigated primaryproduction andcharacteristics of nutrients in the surf and outer turbulent zones of anexposed sandy shnrelocated in lbarakiprefecture, Japan. In genera!,it has been shown that the primary production rate in the otxanstrongly depends onlight intensity and water temperature. However. the results ofthe present investiganon onexposed sandy shores euggcst that the most important factor regulating primary production is nutrient concentration.Therefore, understanding the dynamics and mechanisms of nutrieni supply is consideredan importantstep in evsJuating primary production. Moreover, it has been shown that productivity ishigh in the surfzoiie as well as ol'fshore. It is thought that the physical characteristics ofthe surf zone, i.c., turbulence of watercaused by waves, run-up of seawater,infiltration ofrun-up water in sand.and exudation ofunderground water. are related to high primary production, Moreover,the supply mechanism ofnutrients which INTRODUCTION is an important factor to measurethe priinary It is thoughtthat biota are poor in thesurf productionis not well known. The main zone on exposed sandy shores because the characteristicsof exposedsandy shores are as turbulence of seawater is violent, and there are no follows. First, seawaler and sediments are always steadyadhesion bases. However, many kinds of turbulentbecause of wavesbreaking against thc aquaticorganisms inhabit such areas, and the sandy beach. Second, seawater infiltrates the sand beachclams are itnportantfishery resources on throughrun-up on the beach.Therefore, it is sandyshores. The fact that thereis an abundance thoughtthat the substance exchange between three of organismsof low trophiclevels, such as plankton spheres land, hydrosphere, and atinosphere! is feeder, areinhabited shows that thephytoplankton active.It is veryinteresting to know how nutrients biomassis enoughto supportthese organisms, aresupplied in suchenviroiunental conditions. However, primary productionresearch Sandyshores are utilized for various aroundthe surf zoneon exposedsandy shores has humanactivities, From thefishery point of view, been rare Brown and McLachlan, 1990!, sandyshores tnay be utilized as fishing grounds. ttJNR TechnicalReport No. 26 no feedaguaculture grounds and as bivalve nurseryculture grounds which take adavantage of Kashima-nadaisan exposed andshallow sandy abundantphytoplankton as feed. Moreover,in shore,and its total length along the shoreline from theOharai beach at the northern end to theHasaki reference to land developmentand coastal protection,beach shape isinodified artificial!y. It beachin the southern end is about 80 km, It is one is thereforenecessary toclarify the mechanisms of themajor sandy beaches in Japan. The beach is dividedinto two parts, the north and the south of substancecycling which supports primary sides,with the Kashima Port in themiddle, The productiononexposed sandy shores in orderto maintainand itnprove biological production in beachis flatand wide, but recently the coastal harmonywith a varietyof coastal uses. erosionhas occured in places,especially in the centeral part. Wehave been studying these mechani sms since1992. The temporal and spatial variation of Fieldresearch concerning variation of chlorophylla andnutricnts around the surfzone both phytoplanktonbiomass and nutrierlt concentrationin the surfzone were rescarched wascried outat the research pier near the Hasaki froin1992 to 1994,Primary production has been OceanographicalResearch Station HORS!, Port measuredinthe surf zone and of'fshore area every andHarbor Research Institute, Ministry of Transport,asshown in Figure 2. Thesandy beach seasonsince 1995. Moreover, research concerning thereis veryflat andwide. Thc nearshorcalso thebehavior of the underground water around the hasa gentlebottom slope and wide surf zone. beachbegan in 1996.Here, the research results Shorelinewater and bo hsea surface and bottom ofthe variation ofphytoplankton biomass, nutrients, waterat the offshore end of the pier, 380 m offshore andprimary production inthe surf zone and offshore area are introduced. fromthe shoteIine, 5 min depth, was sampled in orderto dcterinine chlorophyll a concentration and METHODS sizedistribution of phytoplankton.Because we wantedto obtaindetailed knowledge about the temporalvariation of phytoplankton concentration Theresearch area is Kashiina-nada, in thisresearch, the seawater was sampled locatedinthe southern part of Ibaraki Prefecture, approximately500times in 3 yr from1992 to 1994. Japan,onthe Pacific Ocean asshown inFigurc I, Nutrientconcentration wasdeterinined from 1993 Sea of Japan ic ocean Figttre1. Locationof Kashittsa-oadaattestthe Hasaki Oceanogtaphical Research Stattott. k4aetit et an }39 similarly measuredby the site method, The water temperature,salinity, and concentration of both chlorophyll a andnvtrients were also measured. In the offshore area, primary production wasmeasured eight timesaround noonort a day in july, August, andNovember 1995;February, May, july, and November l 996; and May 1997. In the surf zone, the research was carried out five times on the sameday or one of the samedays as offshore research, Chlorophy]la was analyzedas follosvs, After watersamples were filtcrcd througha I -Itin glass fiber filter and the pigments in the particle Figure2. Photographof the the HasakiOceanographical which had beencaught on the filter wereextracted ResearchStation. with acetone,concentration of the pigmentwas determined by thc spectrummethod Lorenzen 1967! Five kinds of nutrients nitrate, nitrite, on. The size distributionof phytoplanktonwas ammonium, phosphate, and silicate! were measured once a week from 1993 to 1994. determinedwith an autoanalyzer TRAACS-800, Chlorophylla distribution wasexamined Bran+RuebbeCo.! by absorption photometry. alongthc beach from Oharaibeach to Hasakibeach Stable isotope "C was determined with "C to characterizephytoplankton biomass in the surf analyzer Nippon-Bunkho Co.! located at the zone on Kashima-nada. This research was carried National Research Institute of Fisherics Science. outeight times from 1992 to 1994. Primary productionwas measuredby the sitetncthod pseudosite method in stormyweather! It t'ai at.Sta,3 .7 miles offshore from HORS, in 10-m r 2Xm ~ depths!and Sta.6 .7 milesoffshore from HORS, ' Kashrna Port in 40-mdepths! as shown in Figure3, First, vertical distribution of light quantum in the sea was measured. Next, sea surface water and seawater St 6 of each depthof quantum number at 50, 25, 10, t and1% in comparisonwith the surfacewater were RS .,St.3 sampled,and thesewater sampleswere divided into I-L transparentpolycarbonatc bottles. These i C bottleswere hung at originaldepths after carbon- 13 "C! reagentwas added and phytoplankton in thebottles was cultured for 3 or 4 h. Finally,the Rival Ton& I 35 45 I photosyntheticrate was estimatedby measuring I I thequantity of "C uptakeby phytoplankton while ! ! ! culturing.Additionally, water tetnperatttre, sahnity, I andthe concentrationof both chlorophyll a and nutrientswere measured at manypoints in thisarea induding rheresearch position. r 2am On the pierof HORS,the photosynthetic 14D' 40 140' 5Q rateof thesurface water at theshoreline part I m in depth!and both surfacewater and bottom water atthe offshoreend of thepier m indepth! were Figure 3. Location of the field researchon offshore area. 14tt U3'.ta TechnicalReport >o. 2a I r Ag ~ el 4 ~ ~ ~Ie g ' s' ~I l ~ W ~ 20~ ~ ~ O15 '. 10 35 ~ ~ p aI ~ ~, ~ e 8 30 pl iy+ o 25 50 40 ~tat30 20 ~g l ' ~ ~ - I 4 e i; pa~ ~ ~ P~ 4 7 10 ] 4 7 10 1 4 7 10 ] 992 1993 1994 Fttptre4. Thetetnporal vanatlon oftcmperatttre sahnt ty andchlorophyll a at the nhoreltne ofthe Hasaltt Oceanopaphtcal Research Station. Adachi et al. tent «Terrp, ~cal ~ t"hla ~I'U4-f' ~ N03-hl - ~ 25- 10 l! 4 20t- r L. 15 0z 1 0Z / i,", I i /'$ 5 600 ' - '~'* a 500 400 300 200' 100- 0 ,I 1993 1994 Figsrra5. Thevenation of tetnperantre,salinity, chlorophyll a. nitrate,phosphate and silicate at theshoreline of the Hasakt OceanographicalResearch Statton; discharge from River Tone; and rainfall at the Choshi Weather Station. Rainfall isshowed as sttm, and others are showed as mean value, Tetnporal and spatia! variation of the shore!ine frotn 1992 to 1994 was 9.5, 4.6, and phytoplankton biomass and nutrient 4.8 !tg/L,respectively, and the biomass from 1993 concentration in the surf zone to 1994 was low comparedwith that in 1992. Figure4 showsthe temporal variation of Bivalvejuveniles such as the Japanese surf clam ch!orophylla at the shoreline of HORSfrom 1992 Pseudocardium sachaltnensis appeared to !994, Monthly tnean values of the water abundant!y in this area in 1993where it hasgrown temperature,salinity, chlorophyll a, and nutrient we!l. Thereis a possibilitythat the ch!orophylla concentration at the shoreline of HORS are shown decreaseafter 1993 was causedby ingestion in theupper part of Figure5; totalrainfall at Choshi pressureby theseclams. andmean va!ue of dischargefrom the River Tone Chlorophylla concentrationshowed a Ministry of Construction!994! observedal. 76 tendencyto behigh at the shoreline compared with km abovethe river mouthare in the lowerpart of the offshoreend of the pier, Chlorophylla Figure 5. concentrationwas very high during one tnonth from Chlorophylla varied from about 1 to 20 the end of April to the endof May, and it was !tg/L. The atmua!mean value of chlorophyBa at thoughtthat this phenomenon was due to a spring t42 VJvR TcchnicalReport Na. zs 20 - 10 2 3 4 5 6 7 8 9 '0 1112 1 2 3 4 5 6 7 8 9 10 I I 1? 25 20 rye 15 10 O 1 2 3 4 5 6 7 8 9 10 II '2 1 2 3 4 5 6 7 8 9 10 ll 12 1993 MOnth 1994 Figure6. Thcvariation of chlorophylla concentrationwhich is classifiedaccording to thesize at theshoreline of the Hastdtt OceanographicalResearch Station and the bottom water of theoffshore end of thepter. phytoplanktonbloom. Chlorophyll a concentration However, a very high value was often seen in changeddrastically over a shortterm. Sucha diurnal variation, and it was thought that this changeoccurred because of a suddenchange in dependedon suddenchanges in weatheras well weatheror oceanographicphenomena such as as the chlorophyll a variation, The nutrient wave, current, wind, or dischargefrom the River concentrationdecreased during the springbloom Tone, becauseof uptakeby phytoplankton.That was Thoughfloating diatoms were dominant in exhaustedin the surnrner and recovered gradually the suspendedmatter, many fecal pelletsand in the autumn. At HORS, nutrient concentration detritus were seen. at the shorelinewas a little higher than at the The variation of the nutrient concentration offshoreend of the pier. at theshoreline is describedas fo]lows,Usually, It is believedthat the main supplysources concentrationof nitrate,silicate, and phosphate was of nutrients are the offshore bottom water, the lessthan 10, 20 and0,4 IsM, respectively. inland waters such as the river water, and the beach Adacbi et ab 143 Qharai m yA ~ mga I Io I I Noda I ! s I Ohtake I ~ ~ I I, Kohya a ~ 4 e I Or i tstl fj ~ .' 4 ~ a ~ I ~ 1' I a a= aa ' 4a ! Hasaki ~ ~ k ~ 5 a ~ 0 l9 l' *" 'li 't " 3s 'l 'I ' i i 6 ' ' r ' i r i';t Temp. 'C! Sal. psu! chl.a >9!'l.! NO3-N ~M! Si02-S; ~14! 3/26/92 7/3/92 8/26/92 3/6/93 B/24/93 3,'3l/94 6/29/94 7/22/94 Figure 7 Thc variation of temperature,salinity, chlorophyll u, nitrate and silicate at some shoreline points along the Kashima-narfa. undergroundwater, in addition to the regenerated f tne, while he beach in the center part has a steep nutrients in theecosystem. A correlationwas seen incline where sand is coarse. The River tlaka betweensalinity and both the concentration of flows into the north end of the Kashima-nada and nitrate and silicate; therefore, it was thought that the River Tone flows into its south end. There is a theinfluence of theinland waterwas a strongsupply tendencyfor river water to go southwardafter source of nutrients for the surf zone. flowing into the sea. Therefore, in the southern Figure 6 showsthe annual variation of' part with few influences of the river water, salinity chlorophyll a concentration that is classified was high and nutrient concentrationwas low. according to phytoplankton size at both the However,the level of chlorophylla was high at shoreline and the bottom at the offshore end of HORS. This suggeststhat primary productionis the pier. Phytoplanktonsize was large during being influencedby the shape of the sand, in the winter and spring months, especially the addition to the influence of river water, springbloom, and dominantsize was 20-60 pm. It was shown that the size was smaller in Estimatesof primary production summer. At the offshore end of the pier, the Figure8 showsthe optical quantum vertical mean size of phytoplanktonwas larger than at distributionat the observation points Sta 3 and the shoreline and it was thought that large Sta.6! in theoffshore area of Kashima-nadaLight suspended particles were disposed to sink transmittance short in winter and spring and long althoughturbulence of the water was violent. in summerand autunm, During the entire research Figure7 showsthe chlorophyll a distribution period,light reached the seabedand the value was of the shoreline water along Kashima-nada, from 2.5 to 12% comparedwith the surfaceat Chlorophyll a concentration was high in both the Sta. 3. Therefore, it can be said that all layers northand south, but low in the central part. The were productive,euphotic zones. At Sta. 6, the beach at both ends is flat and wide where sand is compensationdepth, depths at 1%of light intensity ttgga TecbtucalReport Xa, 2$ QuartturttNumber lt6! 0.1 0 1995 .1995 1995 4 .1996 E yl996 Jul. 1996 6i Nov.1996 .-- M ay1997 0.1 0.5 10 E + 20 a 30 40 Ftgure8. Thevertical distribution ufthe optical quantum number atSta. 3 andSta. 6. atthe sea surface, varied fiom 15 to 35 m, widelyfrom 0 to1G m in depth,and the seawater ChloroPhylla, measured when primary wasbrown, in May 1997, productionwas measured, showed the same Figure9 showsan example of thevertical tendencyasthe previous research onthe surf zone. distributionof thenutrient concentration atSta. 3 lnsummary, concentration washigh from early andSta, 6. Thenutrient concentration in the offshore sprirtgtoaround Mayand low, around l p.g/L, from wasvertically the same in autumnand winter. On e rainyseason, June and July, to August theother hand, it wasexhausted during July and in boththe surf zone and the offshore. A high Augustin theupper part of thepycnocline because concentrationlayer,from 1 Gto 40 lt~, existed thesupply of nutrients was cut off with stratification Adachi et ai. 145 1996 11.23 NG3-N, NH4-N StO2-Si< p iM! NO3-N, NH4-N 'Sl02-St y M,l 0 20 0 IO 0 0 w: e 10l tI~ NG3 c. 20 ~02 Q C3 e - --NH4 30' St.ti s" Jjl ~04 IQ 02 04 06 0,8 0.2 0.4 G.6 0,8 - Si02 N02-N. PO4 - P i p M! NG2-N, 1 G4-P p M~ <995 8 8 NG3"N NH4-N, Si02-St p M! NO3-N, NH4-N, Si02 St p M,'t 0 2 4 6 8 10 0 2 4 6 8 10 7 Y tO~- ! Q 30-fl St,6 0 1 6.2 0.3 0.4 0 5 0 01 62 03 04 05 NQZ-N, PO4-P] p M! NG2-N, PO4 P p M! Figure9. Thevertical distribution of thenutrients at Sta.3 andSta. 6 inAugust ! 995and November 1996. after largenutrient consumption during the spring result can be called equal or a higher value bloom, comparedwith the value of the offshore station. Figure 10 shows an exampleof vertical However, there is no winter measureinent yet. distributionof theprimary productionrate. At Sta. Assimilation number pg-C/ttrg-chl.a/h} 6, the production rate per day was high at the was comparedas an index of the photosynthetic surface with a tendency to rapidly decreasein the activity, Assimilation numberranged from 0.4 to depthof stratificationand to graduallydecrease in 5.0 in the offshore surface, and from 0.7 to 3.1 in the depthof verticalmixing. Sta.3 showedroughly the surf zone. Because vertical distribution of the same verticality. Moreover, the production chlorophylla wasroughly the sameexcept in May, maximumlayer wasseen where the chlorophyll a vertical distribution of the assimilation number maximumlayer existedin the spring, In order to showedthe sametendency as vertical distribution make a clear temporal and spatial difference in of production.A clearcorrelation was not obtained primaryproduction, the valueson eachsurface of between assimilation number and water the observation station are shown in Figure 11. In temperature. the summer,the pnmary production rates were very In estimatingprimary production vertically small in the offshoresurface, ranging from 7 to 12 for the whole water column, though the production p.g-C/L/day.In theother season,the valuesranged per unit areaat Sta.6 wasnaturally large compared from 15to 100p,g-C/Uday. Values from 11to 245 with the onshore area where the depth was ling-C/L/daywere obtained at the shoreline.This shallowerthan the compensationdepth, there was 146 UJNR Technical Report 'No. 26 Quantum %%u!,Producti on . ate ~g-C/L/day! Quantum %%d!, Producti on rate ~g-C/L /day! 0 20 40 60 80 100 0 20 40 60 80 100 10 4 ~ 20 IR 8 30 100 2 4 6 8 10 400 2 4 6 8 10 Chl.a erg/L! Chl.a p.g/L! Quantum Fo!, Producti or. rate ~g/L/day! Quantum %%u!,Producti on rate wg-C/L /day! 0 20 40 60 80 100 0 20 40 60 80 100 10 4 ~ 20 to ~ 30 100 2 4 6 8 10 400 2 4 6 8 10 Chl.a ag/L! Chl, a ~g/I. ! Quantum /!, Producti on rate ~ g-C/L/day! Quan92.um X!,Production rate w g-C/L/day! 0 0 20 40 60 80 100 0 20 40 60 80 ' 00 0 ~ 10 48 6 20 ~ 30 0 2 4 6 8 10 0 2 4 6 8 Chl.a ~g/L! Chl, a ~g/1 ! Quantum !,Production rate 10 4 20 cx 6 pR 8 ~ 30 IO 2 4 6 8 10 400 2 4 6 & 10 Chl.a >g/L! Chl.a ~g/L! ~uantum o Chl.a c production rate Figure 10. The vertical distribution of the primary productionrate per day at Sta, 3 and Sta. 6 in 1996. Adachi et ah 147 10 8 6 0.6 C! 0.2 2 a t 1995 ]996 1997 >995 1996 1997 10 5 8 4 4 m 4 2 C3 0Lo Z Q 1995 1996 1997 1995 1996 1997 ~ 5000 ~100 E EOOO 500 5O D g 100 50 o 10 C3 0 l996 1997 1995 1996 1997 ~ Shoreline > End of pier + St. 3 < St. 6 Figure11. Thevalues of nutxients,chlorophyll a, assimilationnumber and primary production at seasurface and production per eachunit areaof water column on the surf zonealong the HasakiOceanographical Research Station and offshore area. 148 UJNRTecaatcai Report Yio. 26 no markeddifference as thedepth changed, It CoastalOceanography Research Committee, was supposedthat the reasonfor this was the ThcOceanographical Society of Japan, tendencyfor a largechlorophyll a concentrationin the surf zonecompared to the offshorearea. Editors.1985. Coastal Oceanography of JapaneseIslands. Tokai Univ. Press, Tokyo. Resultsfrom Sta. 6 were about the sameas 1106p. reportedas themean value in the wholearea of theSeto Inland Sea of Japan .38g-C/rn'/day! Harrison,W. G. andT. Platt. 1980. Variationsin assimilation number of coastal marine CoastalOceanography Research Committee 1985!. phytoplankton:effects of environmentalco- variates.J. PlanktonRes. 2 249-259. Primaryproduction on the sandy shore in Lorenzen,C.J. 1967. Detertnination ofchlorophyll Kashima-nadawas roughlythe sameas that on semi-shelteredareas observed in other research. andpheo-pigments: spectrophototnetric Moreover,it was shown to bcvery high in very equation.Limnol. Oceanogr. 12. 343-346. RiverBureau, Ministry of Construction,Editors. shallowareas such as near the surfzone, But chlorophylla decreases insummer and so follows 1992-1994.Annual Report of Discharge. primaryproduction. In general,the assimilation Vol.43-45. Japan River Association, Tokyo. [In JapaneseJ, numberin theocean strongly depends on water temperatureand light intensityunder water Harrisonand Platt 1980!.However, our research resultson exposedsandy shores showed that the assimilationnutnber was not so high in thesummer althoughconditions ofboth light and temperature were ideal. This suggeststhat nutrient concentrationis also an itnportant factor in primary production. Abundantbivalves inhabit the surf zone. Becausebivalve irigestionrises with water temperature,estimates of primary production may below in sununer because phytoplankton isbeing consumed.We believe that nutrients supp!ied are largerthan the quantityestimated, and are used promptly.It isimportant to understandthe behavior andsupply mechanism of nutrientsin orderto evaluatethe biological productivity onexposed sandyshores more closely. ACKNOWLEDGMENT We thankthc staffsof thePort andHarbor ResearchInstitute, the R/V Taka-maru,and the Marine ProductionDivision of the National ResearchInstitute ofFisheries Science for the field studiesinKashima-nada andcarbon analysis, LITERATURE CITED Brown,A.C. and A McLachlan.1990. Ecology of SandyShores. Elsevier, Amsterdam, Netherlands.328 p. stakasoneru al. t49 NUTRIENT CONCENTRATIONS IN CROUNDWATER THROUGH SANDY BEACHES TakumaNakasone, Kumiko Adachi, Tomoyuki Takeuchi NationalResearch Institute of FisheriesEngineering Ebidai,Hasaki, Iharaki, 314-0421Japan e-mail:takumaCnrife.affrc.go jp; kinOnrife.affrc.gojp; [email protected] J unya Higano JapanInternational Research Center for AgriculturalSciences 1-2 Ohwashi,Tsukuba, Ibaraki, 305-8686Japan e-maik higa@jircas,affrc.go.jp and HiroshiYagi TokyoInstitute of Technology 2-12-1Oookayama, Meguro, Tokyo, 152-8552 Japan e-mail:[email protected],ac.jp ABSTRACT Theecological functions of sandybeaches are mruntaimng biological productivity and purifying coastal water quality.Quanti tati ve estimation of thesefunctions is accessary for conservauon of sandybeaches and maintenance ofbiological producti vity. We studied the f'unction of maintainingbiological productivity. Generally, groundwater whichflows throughsandy beaches to coastalwaters is consideredto be thesource of outrientsthat supports the biologicalproductivity of coastalwaters. Samples of groundwaterwere collected in anexposed sandy beach at Xasaki,Ibaraki Prefecture. J npan,and the concentrations of nitrate,nitrite, ammonium, phosphate, silicate. and salinitycontained in the groundwaterwere analyzed. Eight sample pipes at different locationsand at various depthsfrom theshoreline to the backbeachwere sct. Thesampling period was from July 1996to Januaryl 997 Thequantity of nutrientsinto coastal water was estimated, considering the moving volume of watercaused by thechange of groundwaterlevel followingtidal changeFrom this experiruent, it vas foundthat the nutrieuts of freshwaterin thebackbeach flowed intocoastal water mixing with seawateraud decreasing the concentrauon. INTRODUCTION productivity of coastalwaters. Generally, biological productivity of the nearshoreocean is very high. In Japan, thereused to be 10,000 km of This high productivityis supportedby the high sandy beaches out of 30,000 km of coastline. concentration of nutrients in coastal waters, The Presently, natural sandy beaches have been main source of nutrients is considered to bc reducedto only 4000 krn long due to various freshwaterfrom rivers,upwelled waters from the constructionsfor disasterprevention, ports, and deepsca, and groundwater through sandy beaches. ainenityfacilities, On the other hand,many Now,aquacultuie without artificial feeding has been researchershave reportedthe importanceof the proposedto relieve the organicload to offshore, ecologicalfunctions of sandybeaches Brown and andthe knowledge on thedynainics of nutricntsin McLachlan 1990, Morimoto 1993, Adachi et al. groundwaterthrough sandy beacheswill help 1994!.The ecological functions of sandybeaches develop the technologyof aquaculture without aremaintaining biological productivity andpurifying artificialfeedings. coastalwater quality. Quantitativeestimation of In thispaper, we investigatedthe function thesefunctions is necessaryfor conservationof of maintainingbiological productivity. We ~sured sandybeaches and maintenanceof biological the nutrient concentratiOnSiil grOundWater thrOugh iso ~yR technicalReport No. 26 aki anographical earch Station HORS! ibaraki Prefecture FigureI. Samplingsite. sandybeaches to understandthe role of nutrients in groundwater. MATERIALS AND METHODS SAMPLING SITE Weperformed sampling inthe sandy beach around the research pier at the Hasaki OceanographicalResearch Station HORS! of the Portand Harbor Research Institute, Ministry of Transport,Japan Fig. 1!. Thelength of the researchpier is 427 m tooffshore. This coastal areais an exposed sandy beach facing the Pacific Ocean.The mouth of Tone-gawa gawa means riverin Japan! which has the largest river basin in Figure2. HasakiOceanographic Research Station HORS!. Japanis located16 kin south. The fish and the c]amswhich live in this nearshore oceanare very va]uablcresources for fisheries, offshore,and froin the bottomlayer at 380 m 80-B!. Groundwatersatnples were collected SAMPLINGDESIGN througheach pipe using a pumpfrom July 1996 to W«ankeight sample pipes at different January]997. From 31 July to 1 August1996, we loca«onsandat various depths from the shore]ine sampledcontinua]ly every 2 or 3 h. «dtcb«kbeach. Sampling locations were0 m Watersamples were collected in 300-inl Pl!,25 m P2!and 65 m p3!from the base of bottles.Subsamples fordetermining concentration p'e t «backbeach Fig. 2!. Considering of dissolvednutrients were filtered through the»fluenceofthe drain to the ocean which is 0.45-mmmembrane filters to rcmovesuspended solidsand were kept frozen until analysis toavoid "of thepier, we estabIished P4at the bio]ogica]change of the nutrients.We usedan ofP2 Fig,2!. At each point, we sank autoanalyzer for analysis of nutrients, The '"reep~pes atvarious depths Fig, 3'I. retnainingwater samples were used to detertninc w«oil ectedseawater sainp]es from the shore]in initywith an inductively coup]ed sa]inotnete. At andsurface area at 200 m water HORS, the basiccharacteristics data of thecoasta] m!and 380 m waterdepth of 5 m! enviromnenthas been collected periodically. Wc e of thepier 00-0, 380-0!to the used this datafor analysis. Nakasoae et, al. 151 E 2 Figure 3. Sampling pipes. The term D.L denotesdatum ieveh 7131/96 9:00 350 30 ~ 300 zs> Kw 250 20 g ~ 200 z CO 150 0 j 100 ~ 50 5 Z Z 0 O~ Zl O f t as e ~ Pl I ~l N> P! I I ~ Al P! 92 t-92 AlI 92 0 O O ~ e- CV c> tel O tu t1. 0 Q. Ct. Q. K L. G. Q. Q. Q. Yp eo hJ O lo Figure 4. Nutrient concentrationsin surnrner. 12/25I96 3 6:00 200 12 180 g 160 10K ~140 80 ~ 120 Z 100 6 80 8 0 60 4 Q x 20 R 0 0 0 E O I I I cu os I I ~ pa ~ f p! ~ ~fu I p! I O o cu cv co tts O tu Q o! p! N 0 CL 0 Q G. CL CL Q CL Q 0 Figure S. Nutrient concentrationsm winter. l52 ugtstt TecitnieatRePnrt No, 26 7/31/96 9:00 12/25/96 16:00 P1-1 24.5 21.8 P1-2 21.1 31.7 P2-1 6.3 26,1 P2-2 4,3 25.8 P2-3 3.7 23.4 P3-1 0.5 P3-2 0.8 P3-3 0.5 0.5 P4-1 2.6 6.5 P4-2 5.8 P4-3 shoreline 34.2 2004 33.1 3804 33.2 34.3 380-8 33.2 Table1. Salinityconcentration ppt!. RESULTS AND DISCUSSIONS and from the drain. At P4, nitrate concentration varied The groundwater level at P2 varied with between100 and 120 pM; silicatevaried between theinfluence of tide but at P3 it wasuniform without 130and 170pM; andphosphate varied between anytidal influence. The salinity at P3 was usually 0.5and 2 ItlVI,Usually, the nutrient concentrations lessthan 1 ppt,and is consideredto be near y atthe location of theupland side were higher than freshwater.In the sumtner season, salinity at P2 thatof thesea side, This suggests that the nutrients was2-5 and at P1 was 20-30 ppt, respectively. In were suppliedby freshwaterin thebackbeach and thewinter season, due to theincrease of seawater flowedinto coastal water mixing with seawater. surgesinshore following decrease of ground There was no distinct feature for vertical elevation,salinity increased toapproximately 25 di stribution of nutrients. ppt. Wefound that the salinity at shallow points The nutrient concentrationsat shoreline washigher than at deep poi nts TableI !.Nutrient becainehigh when the discharge of groundwater concentrationsof coastal seawater except increased,noticeable by observingtide and phosphatewerelow in sutntner and high in winter. groundwaterlevel fluctuations Fig. 6!. The However,this tendency was not distinctly groundwaterlevel wasconsistently higher than recognizedin the case of groundwater.Nitrate seawaterIeveL This suggeststhe contributionof concentrationof groundwater varied between 150 groundwater to the nutrients of coastal water. and250 ItM, andsilicate concentration varied However,this phenomenon wasobserved only in between80and 150 ItM, which is very high thc summer season. cotnparedwith the nutrients ofseawater Figs. 4, Mixing of groundwaterwith seawater 5!. Inmost cases, the concentration ofammonium createsgradients of nutrientsand salinity. When in groundwaterwasless than 2 ItM,and the salinity is consideredas a conservative tracer of concentrationof nitrite was less than I pM. mixing,then it is possibleto quantifythe uptake However,the concentration atP3 and P4 increased andrelease of biologically and chemicaHy-reactive occasionally.Thisphenomenon isthought tooccur compounds within the mixing zone. If the bythe inflow of freshwaterfrotn the backbeach correlation betweensalinity and the nutrient %atasone ec ai. l53 2.5 4.5 E X o 3.5 0 3 K I 2.5 0 y 2 0 X Q CO o~ 0 0,5 -U X o X CD ~o ~o go ~o CB CV Igl EO ~ NH4 ~ NO2 NO3 ~ PO4 ~ Si02 ~--Groundwater Level at P2 + - Tide Figure 6. Time varianon of nutrient concentration ai shoreline. 350 concentrationis linear,nutrients tnay beconsidered $00 g 200 conservative. Therefore, we used the relation ~ 200 betweensalinity andnutrients Fig, 7!. This figure t5' 100 2 100 is calledthe 'mixing diagram.' Nitrate and nitrite 50 concentrationsbecame very high in thc case of 0 sotne concentrationsof salinity. The nitrate 0 10 20 40 Salinity concentrationbecatne especially high when thc salinity was 3-5 ppt which mainly occurred at P2, an and the nitrite concentrationbecame high when 50 the salinity was approximately 20 ppt which g 40 occurred mainly at Pl, This phenomenon i» 30 consideredto bcnitrification by nitrifying bacteria. 0 20 z Reportedly,the activity of bacteriawhich oxidizcs 10 nitrite is maximutn when the salinity concentration 0 is 3-5 ppt Kurihara1988!. Ourresults correspond 0 10 20 30 Salinity to that report. Furthertnore,from our results we can infer the presenceof dissolvedoxygen in the groundwater,Frotn Figure7 c!, therewas a linear 200 relationbetween salinity and silicate. Therefore, silicate seemedto be supplied by freshwaterin the O 100 backbeachand flowed into the coastal water tnixing CO 50 with seawater. The fluctuation of thc concentration of 0 0 20 30 nitrate at P2 seemed to correlate with nitritc at Pl, Salinity ammoniumat P2, and nitrite at P2 Fig. 8!, We consider that it was the facilitation of nitrification Ftgare7. Mixingdiagram, due to the in~ of amrnoniurn, l54 UJVR TechnicalReport Co.26 350 14 300 12 N 0 250 10 Zg O 200 8 0-~ X 0 o ~ Z $50 xO l X Z CVi lL Z CVi 100 4 ~ Q I CV 0 50 0 G 7/31/96 9:00 7/31/96 16:00 7/31/96 23f00 8/1/96 6:00 8/1/96 13:06 Figure8. Titnevariation of mtrate,nitrite anti ammonium. 3SO 300 Q 250 ~ ZOO V I O4 4. 150 0 50 0 IO In 8oo o o o 8 O lO O 8 O cd ol 8 CD ID O 8888 CO O el Dl CO O N CO N e nl eo ~ cd CI~ N IO 9 v Cd ID Ol CD IO CO CD IO CO CD Id CD I Ol IS IO CD IO IO ID Ol Ol Ol Ol Ol CD ~ n ol ol ol ol CD Ol a I f a N N ol cel Di el ib Q Q el cu c|l co i' a|l g g Figure 9. %e variatinn Of nitrate conCentratiOn. n'akasnaeet. a!, iSS 35 ~ 30 v 25 20 f5 ~ fo 8 8 888888 8 M s92 o % o 8 8 tk cl 8 8 888 O ~ ii v92a ~ e e ct cv cn ii o iv ut re i ri bi w e I I Igsilti -aatjla I I os a Figure t0. The variation of nitrite concentrate. Nutrient Quantity rnol.! Ammonium NHe! 4.3 Nitrite NO2! 10 Nitrate NOs! Phosphate POa! Silicate Si02! 260 'table 2. Estimationof quantityof nutrients for l km coastalinefor a tidal cycle!. The concentrations of freshwater nutrients the caseof phosphateand silicate, the measured which were dilutedby seawaterwere calculated concentrationcorresponds to the calculatedone, by estimating therate of mixingof freshwaterand hut there were a few cases which did not seawater. Hy these calculated values, we can correspond Figs. 11, 12!. Noncorrespondence estimate the quantity of nutrientswhich werc was consideredto be the releasefollowing the biologicallyor chemicallyreleased or uptaken.Thc decotnposition of organic compounds by mixing rate was calculated from salinity microorganismsor adsorptionto sandparticles or concentration which was considered to be not suspendedsolids Sewell 1982, Johannesand Hearn reactivebiologically or chemically.The tneasupxl 1985!. concentrationof nitrate was higher than the We estitnatedthe quantity of nuuients calculated concentration at P2 and the measured which flow into the coastal sca. We calculated concentration of nitrite was higher than the the dischargeof groundwaterinto thc coastalsca calculatedone at Pl Figs. 9, 10!. This indicates by the Sakatnoto method Sakarnoto 1991!. that nitrificationoccurred in the sandybeach. In Sakamoto proposed that the discharge of t;JNR TechnicalReport No, 26 3.5 2 2.5 CV CV 0- i.5 o L oo Se 883 o ICI o o o g o o ~ 0 ~ O 0 8 ~o 8@a 0 o e i lo bi o e o e ii$i gag V ih e! ~le a a I t% lIl l r Figure11 Thevariatron of phosphateconcentration. 3.5 X Z.5 Al 2 1.5 0 o o o o o o a e o o g e e 8 3 8 e o g o o g In l4 O c- ln ai o ei 'lo e e r ii e o i4 OI e e eai i cn Pi w e eCew e I ii> eN IN es 4ft Figure 12. The variationot silicateconcentration. 158 p3ga Techntca}Repor Changcs s 0 ofgrain distribution ofbed material in me su rfz«zone field observation atHasak i phicaIResearch Facility. Rep. P«Harbor Res.Inst. 29 Z!:38-61, [In JapanesewithEnglish summary], Kurthara,Y., Editor. 1988, Fcology Ecotechnologyin Fstuarine-Coastal Area. Univ.Tokai Press. 335 p. [InJapanese]. Morimoto,K. 1993,Nutrients budget and water ctrculationat intertidalzone. Bull Coacta] Oceanogr.30 !; 208-223-[In Japanese withEnglish summary]. Sakamoto,I. I 991.Use of respirationinthe sandy beachor on the t>da]Hat. ] Perm able~ndy beach.Mar. Potlut.Bull. 23; 123-13P, Sewell, P. L. 1982. Urban groundwateras a possiblenutrient source for an estuarine benthicalgal bloom, Fstuarine Coastal Shelf Sci. 15: 569-576. V ukay a ma l 59 CYSTEDlE METABOLISM IN RAINBOW TROUT Masahito Yokoyama NationalResearch Institute of FisheriesScience 2-12-4,Fukuura, Kanazawa-ku. Yokohama 236-8648, Japan e-mail;[email protected],jp ABSTRACT A seriesofexperiments werecarried out to characterize sulfuramino acid metabolism inlish, in particular taurineformation fromsulfur amino acids. Rainbow trout was used asthe experimental fish.Results ofboth feedinganda tracerexpenmeot indicated thatcysteine isthe main starting substance fortaurine biosynthesis, and thisis metabolized through cysteittesulfinatc asan intermediate tohy potaurine, taurine, and sulfate inrainbow trout.Cysteine dioxygcnase CDO!which catalyzes theoxygenation ofcysteine toform cysteioesu.lfinatc was consideredtoplay a regulativeroleof the cysteine ntetabolism inrainbow trout. On the other hand. alarge dose ofcysteine showed a serious toxicity torainbow trout. Considering therapid rise of the hepatic CDO activity afterthe cysteine intake, thisenzyme presumably playsa role indetoxication byconverting cysteine intonon- toxic cysteinesulfinate. was first discovered in bovine bile, and it is a INTRODUCTION generallyaccepted idea that taurine is one of the Becauseof the liinited globalsupply of final inetabolitesof sulfur amino acidsin mainmal s fishinealand its highprice, many fish nutritionists Griffith1987}. This sulfur-containing substance havesearched for suitablealternal.ive sources of is consideredto havesuch physiological futtctiotts proteinfor fish feed Watanabe1994}. Soybean in tnammals as membrane protection. mealmay be utilized first, which affords a relatively detoxification,andantioxidation Wright et al. 1986!. largesupply of cheaper protein, However, soybean Taurine also has attracted many fisheries proteints lowin sulfurairiino acids, especially biochemistswith regard to its physiological function, methionine,which is knownas one of theessential particularlyitsparticipation inthe osmotic regulation aminoacicLs for fish Noseand Murai 1990}, Many andits origin, because ofits high concentration in studies,therefore, have been performed on sulfur fish tissues Sakaguchiand Murata 1988!. aminoacid requirements for severalspecies of However,there has been little study on the culturedfish Ketola1982, Moon and Gati in 1991}. metabolismof sulfur amino acid in fish,especial! y In previousinvestigations on sulfur amino acid onthat of cysteine;only a fewstudies suggested requirementsforfish, little attention has been paid that taurinein fish body originatedfrom dietary to sulfur amino acid metabolisinincluding its sulfur aminoacids Walton et al, 1982,Cowey ct regulatorymechanism. al, 1992}.The inetabolic pathway of sulfuramino Thereare numerousreports of analysisof acidsin fish is still far from being understood. freeamino acids and amino acid-related substances Investigationonsulfur amino acid metabolism in in fishand shellfish, some of which areknown as fish, of course, must be important in thc aminoacid metabolitesin mammals Sakaguchi improvemetttof the fish feed, 1994}.It hasbeen frequently tnentioned that some In thispaper, the authordescribes the of these amino acids and ainino acid-related resultsof someexperiments that were carried out substancesvary greatlyiii contentwith species, to elucidatesulfur amino acid metabolism, organ,age of fish.seasons, and physiological especiallycysteine ~lism, infish fiom the point conditionof f ish. In particular,taurine is presentat of view of sulfuramino acid nutrition and that of a considerablyhigh concenlration in fish. Taurine taurineformation aswell. Rainbow trout was used tsar u3~RTedtatcar Report No. aa Table1 Compositionofexperimental diets '8! Vokoyamaand Nakazoe 1992!. Diet No. 3 tngredient Casein 50 50 50 50 L.Methionine 503 t,-Cystlne 1 Taurine 1 «-starch 15 15 15 15 15 Dextrin 13 12 12 10 I l.ipid' 10 Cellulose 1052 5 10 52 105 105 5 5 Mineral mixture 5 Vitamin mixture 2 2 2 siu}furamino acid content /4!t Methionine 1.2 2.2 1.2 1.2 1.2 Cystine 0.2 0.2 1.2 3.2 0.2 Taurine 1.0 ~ Soybeanoil: Pollockliver oil = 3: 2 t Thevalues were calculated from amino acid composition of casein and supplemented amino acids. as thc experimentalfish. All of the rearing 20 et0 experimentswere conducted at 15'C. 0 t5 2 METABOLIC FATE OF CYSTEINE 10 To confirmthe metabolic fate of cystcine a in rainbow trout, both tracer and feeding I-I experirnemsfocusing on the metabolicpathway from cysteineto taurinein rainbow trout were conducted. Diat t Diiet 2 Diet 3 Diet 4 Diet 5 Accumulation and excretion of taurine in fish Figure t. Taurine content of whole body of rainbow trout fed dietssupplemented with methionine, fed five different diets Yokoyama aod t'ai~ 19921. See Table 1 for the composition of five diets given in cystine, and taurine the figure. The efFectsof the amount of sulfur amino acids administered on the tissue levels of the coiiesportdingarltino acids and their rnetabolites, and However,the levels of cysteineand cystine showed onthe arrount of raurincexcreted were examined as only a slightchange in ail dietarygroups, The taurine a preliminarystudy to look into the pathway from content in the liver increased to some extent in all ~~ve sul amino acids to taurine. Rainbow dietarygroups. In the wholefish body,there was ~t weighingabout 10 g wercfed with casein-based little difference in taurine content between the dietssupplemented withmethionine, cystine, and controlgroup diet 1! and the 1% rnethionine group taurtnefor 1> days. The composition ofexperimental diet 2! Fig, l!. On the otherhand, the taurine dietsis given in Tablel. level in the fishfed eitherthe 1% cystinediet diet ln fishfed the methionine-supplement& 3! or the 3% cystinediet diet4! wassignificantly diet diet 2!, methionine andcystathiorun«ontents higher thanthat of the controlfish Consequently, in theliver wereobserved to be about25 and thefish fedcystine- or taurine-supplementeddiets timeshigher respectively, thanthose inthe liver of accumulatedtaurine at a highlevel in theirbodies. thefish in the other four dietary groups Table 2!, Administration of sulfur amino acids and taurine Yak oy a ma 161 Table 2 The free amino acid and glutathione levels in the liver pmoUg tissue! of rainbow trout fed diets differin in sulfur amino acid contents Yoko arneand Nakazoe1992!. Diet No. Amino acid 3 4 5 Methionine 0,05+0.01 1.21+0,86 0,05+0.01 0,0&0,D I 0.05M.D I Cystathioninc 0,15&.07 0.43+0.06 0.21+0,11 0,17+0,05 0.17+0,05 Cysteme 0.21+0,03 0.22+0.03 0.2&0.03 0.4 0.02 0.2&0.04 Cystine tr. tr. tr. G.DI+0.00 tr. Taurine 23.40+2.52 26.84+4.87 29.31+8.59 25.4&2.34 31,45+3.04 Ghttathi one 3.0%0.36 3.01&.34 2,96s:0.48 2,8 I+0.40 2,6~.51 Aspltic acid 1.10+0.30 0.5&0.04 0.8 0.55 0.65xG.I 8 0,86j.0.24 Threonine 0.48&.12 0.33tO.I 0 0.45&.10 0,55*0.4S 0.53s-0.07 Serine 0.38+0.11 0.07+0.05 0.3&0.07 0.3&0.13 0.38+0.10 Glutamic acid 3.78*1.14 2.73*0.42 3.90%2,52 3.77&L89 3,07s-.0,42 Glutamine 0.63+0.54 0.43M.32 G.56s:0. 54 0,71'.53 0,58s=0.17 Proline 2.51k 1.00 1,62+0.67 3.21+1.57 1.94M.55 2.49*0.85 Giycine I.QH:0.15 I.D&0.16 1.63&0.34 1.70s:0.17 1.7 0,30 Alanine 5.72+0.83 4.7&s0. 65 5.86+1.15 5.53+1.01 5.55+0.87 Valine 1.17M.23 1.G3+0. 16 1.31+0.38 1.34M.36 1.18&.15 1soieucine 0.50sO.13 0.43~0.06 0.57&.16 0.5320,09 0,5Ze0,05 Leucine 0.9trt0.25 0.78M.11 1.07sO.30 0,9&0,12 0.97+0.10 Tyrosme O.I ILtO.05 0.13M.Dt 0.1&0.02 0,14+0.04 0.12s:D.D2 Phenylalanine 0.1&F0.02 0.4&F0.04 0. 16s:0.0I D.ItitO.D3 0, 14&.DI Tryptophan 0,05M.DG 0.05M.OI 0,05aO.O'I 0.04+0.01 0.04M.D2 Lysine 0,26-sO.G7 0,18+0,04 0.36+0,25 0,53+0.47 0.19s:0.04 Histidine 1.37+0.14 1.37M.08 I.4&0.12 1.2&0.20 1.34+0.09 Arginine 0.0&0.02 0.01*0.00 0.02*0.02 0.0&0.10 O.DIM.DD 3.15+0.39 2.74+0,17 2,81M.40 3.07+0.62 3.0 IW.43 The vatuesare means of five individualtneaswements + standard deviation. tr: traceamount. showed no apparent effects an the levels of the for the cystathionine biasynthesis, although no non-sulfur amino acids in the liver and muscle analysis was made in this study for hamacysteine, except that of serine. which is another substitute for cystathianine According to Finkelstein and Martin formation. Thesefacts also imply that cystathionine 986!, the methionine level in the rat liver was as biosynthesis proceeds relatively rapidly, while Iow as 0.08 pmoVg, even when the rat was fed a cysteine biosynthesis from cystathianine proceeds diet containing 3% methionine for 7 days. In the very slowly. Therefore, both cystathionine and its present study, however, the methionine content in precursor methionine might be accuniulated in the the liver of fish fed the 1% rnethionine diet was tissues of rainbow trout when the fish is fed excess estimated to be as high as 1.2 pmoVg, Similar high dietary methionine. In any event, it is evident that metkianine levels in the serum Nose 1974! and the excess methionine ingested was not used the liver Walton et al. 1982! of rainbow trout were efficiently for cysteine biosynthesisin the liver, and reported for the fish fed a diet suppletnented with did not lead ta apparent accumulation of cysteine excessmethion inc. The relatively high contentof and taurine. cystathionine in the liver observed in this study Hosokawa et al. 988! observed for rats suggeststhat this ainino acid was synthesized from that taurine excretion into urine increased the excess rnethionine via hornocysteine. Since remarkably when a high level protein diet was serineis oneof the raw materialsfor cystathionine administered. As can be seen in Table 3, the biosynthesis Finkelstein and Martin 1986!, the amount of ordinary amino acids excreted from the decreasein free serinecontent of the liver might rainbow trout for 24 h after final feedingdid not be attributable to the consumption of free serine differ greatly between the control fish diet I ! and i62 UJNR TeeheicatReport No. Za Table3 Aminoacid excretion ofthe rainbow trout fed diets supplemented withsulfur amino acids pmoUg body weight/day! Yokoyama and Nakazoe 1992!. Diet No. Aminoacid 2 3 4 5 Tnunne 0.23 0.35 1.31 1.31 2.37 Asparticacid 0.08 0.09 0.10 0.07 0,1 I Thleonnle 0.22 0.20 0.21 0.15 0.3 5 Senne 0.42 0.42 0.33 0.20 0.54 Glutamicacid 0.38 040 0.43 0.41 0.51 Glycine 0.25 0.20 0,43 0.27 0.42 Alanine 0.45 0.47 0.53 0.38 0.63 Vatioe 0.49 0.46 0.53 0.38 0.63 Cystine ND 0.04 0,18 0.01 M ethionine 0.25 0.76 0,18 0.12 0,31 holcucine 0.38 0.39 0.26 0.25 0,53 Leucine t.l2 1.33 0.74 0.54 1.15 Tyrosate 0.69 0.85 0.32 0.20 0.56 Phenylatanine 0,52 0.71 0.6 0.15 0.44 I.ysioe 0.95 0.97 0.48 0.35 0.93 Hintidine 0.17 0.16 0.10 0.08 0.23 Arginine 0.56 0.63 0.32 0.22 0. 50 Anunonia 26.50 27.10 22.70 23.80 24.00 the fish administeredmethionine diet 2!, cystine Metabolites derived front L-[ 'S]cysteirte diet3 and4! ortaurine diet5'!, with the exception injected into the peritoneal cavity of slightly increasedmethioninc excretion from the When cystinewas given to the fish, the methionine-administered fish, Fish fed the 1% content of taurine tnarkedly increased;when methionine diet excreted almost the same levels of methionine was given, no such increment was taurine as fish fed the control diet, whcrcas, fish observed. This meatusthat cysteineis the main fcd the cystine- and taurine-suppletnenteddiets starting material for the taurinebiosynthesis in excreted a large amount of taurine. Taurine rainbowtrout, Therefore,the fate of cysteinewas excretionof thefish fed cystine-supplementeddiets tracedby injecting radiochemically-labeled cyste inc was four times higher than that of the fish fed into the peritoneal cavity of rainbow trout, rnethionine-supplementeddiets. Clearly, the higher Metabolitesderived from the radioactive cysteine the taurinecontent of the whole body,the larger werc examinedfrom whole fish body and the the excretion. This suggests that the taurine excreta.Each of thefish weighing about 10 g was excretion becomes active when thc net kept in an Erlenmeyerflask containing 1 Lof water accumulationof taurinein the fish bodyexceeds a for an of appropriateamount of L-PS]cysteine. certain level. The wholebody of the frozenfish samplewas cut AlthoughTateishi et al. 977! reported intosmall pieces and homogenized with 4 volumes for the rat thathepatic g latathione plays a roleas a of distilledwater. The homogenate was separated cysteinereservoir, there was no obviouschange in into a protein fraction and a soluble fraction. Thc the glutathionecontent of cithcrliver or musclein soluble fraction was further fractionated into anyexperimental group, This agreeswell with the severaltnetabolitc fractionsby the methodof results reported by Walton et al. 982!. At least Yamaguchi and Veda 976! with minor for rainbowtrout, glutathione does not seem to serve modification. The water in which rainbow trout as a cysteine reservoir when fish are fed excess was reared was fractionated in a similar manner amounts of sulfur amino acids such as methionine as above. or cystine. Irt vivo composition of radioactive substancesderived froin L-[" S]cysteincinjection voko rorno i as Table 4 Distributionof "S substancesin the whole body of rainbowtrout which wereinjected with two differentdose levels of L-[ S!cysteine Yokoyarna et al. 1997! Dose of "S Substances corno Vgbody weight! L-[ S]cysteine /rmoVgbody weight! Total Taunne Hypotaurine Sulfate Protein Others 1.50 0450 0.108 0.041 0.068 0.157 0,076 0.15 0,067 0.011 0.004 0.008 0.035 0.009 Table5 Excretionof "S subsiaocesintothe water where ihe rainbow trout was kept for 24 h alterIhe i: ["S!cysteineinjection Yokoyama et al. 1997!. Dose of S SubstancesQrnol/g body weight/day! L-["S]cysteine QmoVgbody weight! Total Taurine Hypotaurine Sulfate Others 1.50 0,107 0.251 0212 0.426 0.15 0.079 0,014 0.020 0.022 0,023 and that in the rearing water were tabulated in rnetaholitessuch as taurine, hypotaurine, and sulfate Tables4 and 5, respectively.When the fish was was found to be excretedinto the rearing water injectedwith 1.5 lirnol L-[isS Jcysteine/g fish body within the 24-h experimentalrearing. Howcvcr. weight, one-thirdof the total radioactivitywas in the caseof 1.5 lrmoVgbody weight injection, the retained, whereas one-half remained in the fish amount of radioactive substances corresponding injected with 0.15 p.rnol/g. In both cases, the to about one-third of the total activity could noi be remainderof radioactivitywas recovered from the identified Table 5!. A part of the injected rearingwater. From the data in Table 4, taurine, L-[-"S]cysteineis likely excreteddirectly into the hypotaurine,and sulfate can be saidto bethe major water,because the proportionof the unidentifirxl metabolitcspresent in the sohrblefraction of fish activitywas much greater in the largedose than in bodyregardless of thedose size, though a relative!y thcsmall dose Fromthe resuhs given in Tables4 largeamount of radioactivityhad been incorporated and 5, the total amounts of taurine, hypotaurine, intothe protein fraction. Even though it is notclear andsulfate formedfrom injected L-cysteinewere that the radioactivecysteine is incorporatedinto calculated as 14, 19, and 19% of a dose of 1.5 theprotein inolecule eirher as a disulfide linkageor lrmoVgbody, respectively, Regardless of thedose as a constituent part, this fact indicates that thc size,more than 50% of L-cysteinewas mctabol ized incorporationof cysteineinto the protein molecule within24 h afterinjection. Evidently, a largeportion occurredvery rapidly. of the cysteineadministered was metabolized A considerably large amount of the rapidlyto taurine vio hypotaurine,and the pathway 164 U5NR TedssiiealReport No. 26 is accompaniedby sulfateformation, although the detailsof pathwayhad not necessarily been clarified ~ 8 00 by this experiment. Oxidation of cysteineto cysteinesulfinate O is believed to be the major step of cysteine 6 catabolismin mammals,particularly whencysteine availabilityis high Wheldrakeand Pastcrnak 1967, tv 4 Yamaguchi et al. 1973, Stipanuk 1979!. P-sulfinylpyruvate, a productof cysteinesulfinate transaminationreaction, decomptxsesspontaneously intopyruvate and sulfite; sulfite is further oxidized by sulfite oxidaseto sulfate Griffith 1987!. The 0C4 0 formationof taurine,hypotaurine, and sulfate from L-cysteine suggeststhat rainbow trout has the L-cysteine-metabolicpathway similarto that of Days mammals where cysteinesulfinate plays a key role Figure 2. Changesin hypotaurine contents in several tissues as the intermediate, of rainbow trout during feedingon cystine-supplemented The mechani sm of conversion of diet Yokoyama and Nakazoe t998!. Results are ex- hypotaurineto taurinehas not been elucidated yet. pressedas the mean s standarddeviation of five indi- Bothenzyinatic Oja andKontro 1981, Kontro and viduals. Oja 1985! andnon-enzyinatic Fellrnan and Roth 1985! reactions, however, have been considered to 4 days. The hypotaurinecontent in the muscle to be involved in the taurine formation. On the tissue was almost zero at the very beginning, other hand, as for rainbow trout, much of the increasinggradually during feeding. The max iinum hypotaurinewas observed to be excretedinto the level in thcmuscle, however, was only one-eighth rearing water, i,e a considerableamount of of that in the liver, hypotaurineis excretedwithout being converted Changesin taurineand cysteinesulfinate into taurine. contentsin severaltissues are shownin Figures3 and 4, respectively A large amountof taurine Changes in tissue level of the major eysteine existedin the kidney and the liver at a level of etabolltes by the continuatiott of oral about25 limoVg tissue;the content was very low adtnlntstration of excesscystlne in the muscle and plasma. The taurinecontent An attempt was made to ascertainthat appeared to be constantthroughout the feeding taurine is originated from dietary cysteine by period. The levels of cysteinesulfinatetended to exaininingthe effectof the oral administrationof increase in the liver and kidney by the cystine cystine on the tissue contents of the major administration. However, these values were metabolites.A feedingexperiment was conducted extrelnclylow comparedwith those of taurineand usingthe 50% caseindiet supplementedwith 1% hypotaurine;even at themaximum on the4th day, cystine, Contentsof hypotaurine,taurine, and cysteinesulfmatewas only 0.015 tunoUg. ln the cysteinesulftnatein the tissuesof rainbowtrout muscletissue and plasma,there was no changein were periodicallymeasured throughout the 8-day the cysteinesulfinatecontent. feeding.The change in hypotaurinecontent m four The cysteinesulfinatecontent remained differenttissues is given in Figure2. The hepatic low throughoutthe experimental period, although hypotaurinelevel at the startof theex~nt was it was consideredto be affectedby the excess about1 pmoUgtissue, Excess ditxary administration cystine administration,This fact suggeststhat in of cystine brought about the hypotaurine rainbowtrout the cysteine metabolism is controlled accumulationboth in the liver and thekidney within mainlyat thecysteine oxidation step rather than at the first 2 days. The accumulationin thesetissues the step of cysteinesulfinate breakdown. reacheda maximumlevel about4 tunol/g!after 2 Therefore,the cysteineoxidation enzyme must Vakoyarna t65 have fundamental significance in cysteine 40 metabolism in the fish, Hypotaurineis regarded as an antioxidant by scavenginghighly reactivehydroxyl radicals, O 30 and to play an important role in preventing the attack by oxidants tn viva Aruoma et al. 19881. 20 Since taurine is the oxidative product of hypolaurine, theoxidation of hypotaurineto taurinein fish should prove to be interesting regarding physiological 10 protectionagainst the attack of radicals. The physiological role of hypotaurinc should be investigatedin the future in conjunction with the biological function of taurine. Days CYSTEIWE DIOXYCrENASE CDO! ACTIVITY AS A DOMINANT FACTOR IN Figure 3. Changesin taurinecontents in severaltissues of CYSTEINE METABOLISM rainbowtrout during feeding on cystine-supplemented diet Yokoyama and 1Vakazoe1998!. Results areexpressed as the incan a standard deisation of five individuals. Cystcinesulfinateis a key intermediate both in the catabolic pathway to pyruvate and sulfate, andin themetabolic pathway to taurinein inammals Griffith 1987!. CDO [EC 1.13.11.20! catalyzingthe oxygenationof L-cystcine to L- 0. 04 cysteinesulfinate plays an important role in mamtnaliancystei ne metabolisrn Yarnaguchi and 4 ~0. 03 Hosokawa 1987, Kohashi et al. 1978!. It hasbecome apparent that cysteine was btt0.02 metabolizedinto cysteinesulfinate as thevery first step in rainbow trout, as in the caseof mammals. O.Cl Mostprobab!y, this enzyme participates incysteine metabolism in rainbow trout as well. Since no information about CDO in fish has yet been V 0 0 2 4 6 8 availablc,response of CDO activityin the liver to Days the level of sulfur amino acid administrated was investigatedto confirm the participationof thc enzymein this oxidationreaction. Figure 4. Changes in cystetnesu!ftnatecontents in sevend tissuesof rainbowtrout during feeding on cystine-supple- mented diet Yokoyama and Nakazoe 1998k Resuhs are Enhancement of CDO activity by dietary expressedas the incan u standarddeviation of five indi- supplementationof sulfur asniuoacids viduals. Since CDO was considered to function in the initial stepof cysteinemetabolisin, the tissue distributionof CDO activity and the effectsof a large excessof sulfuramino acids in thediet on CDO activityin the tissuewere examined. The fish were fed 1%sulfur amino acid-supplemented 40% caseindiets for 10 days, The activities in the liver of both the fish fed either methionine-or cysteine-supplemented diets were significantlyhigher than thoseof the tilts UJNR TechnicalReport Nrr. 2rs control group, This enhancementin the liver As a preliminarytest, L-cysteine was suggeststhat a highsulfur amino acid intake induced injectedintraperitoneally torainbow trout in doses the hepaticCDO. On thc other hand, no such of 2.5,5.0, and 10.0 !into!/g of fish bodyweight, remarkablerise in CDO activity was observedin Eighteenhours after injection, the hepatic CDO othertissues Fig. 5!, Thisfinding implies that a activitywas measured, The hepal.ic CDO activity cysteinecatabolic pathway to taurine via of thefish given by injection in a doseof 2.5!tntol! cystcinesulfinatcexists in the liver,and thesystem g ofbody weight rose as much as two times that works in concert with dietary sulfur amino acid of thecontrol fish injecled only with salinesolution levels as ascertainedin mammals Kohashi et al. resultsnot shown!.However, the fish given doses 197 S!. above5.0 1trnol/gof bodyweight died with hcavy hemorrhagewithin 30 min after injection. Enhancement of hepatic CDO activity by Consideringthe toxicity of excessdosage of L- intraperltonealinjection of sulfur amino acids cysteinefor rainbow trout, 2.5 !tmol/gof body lt was observed that CDO occurs in the weightwas employed as thedose level for the liverof rainbowtrout, andits activity was enhanced subsequentexperiments onthe response time. As by dietarysupplementation of an excessamount shownin Figure6, theactivity of hepaticCDO of of both methionineand cystine. These findings rainbow trout increasedrapidly within the first 4 h suggestthat the sulfur amino acid metabolism is after the injection. The activity reached a control!edby thisenzyme in rainbowtrout. ln order tnaximurn level at 4 h, andthe activity was about to ascertainif the CDO activity is controlledby 2.5times that at the beginning, lt increasedrapidly, the sulfur amino acid level in the fish, and thc ltassedthrough the maximum, and fell off gradually enzymeis specificto cysteine, the effect of the within thesubscqucnt 1g h. Next,the experimental dosesize of cysteineand somedifferent kinds of conditionwas re-designedto examinethe dose- sulfur-containingcompounds on hepatic CDO response,The activity was measured4 h after activity was examine by intraperitoneal injection. injection of different doses. The results are shown in Figure7. Theactivity of hepaticCDO increased in proportionto the increasingdtise in the very limiteddose range, i,e., below 1.5 pinoVg of body l.a "~ps 4 2,0 A Q en ~ p.s ~ c 0 0 aa t.s sn~H ps lp ~ A ~ ls p.z D t p p Liver Brain !teart K.idncy Xluaeie all p 5 0 Hgttre 5. Cysteine dioxygenaseactivity in the dssueof rain- a bow trout fed experimentaldiets having different sulfur U 0 aminoacid contents Yokoyama and N skate t 989!.The P 4 8 I? I6 ig 24 activity of liver is expressedas mean + SD of sevenfish. Othersare the measurementsof thepooled samp!e of seven hours fish. One unit Ul of enzyme activity was defined as the amountof enzyme prOducing One rrmnle Of cysteinesulfmate Figttre6.Effects ofL-cysteine injection onhepatic cysteine in! hat37 C. dioxygenascacuvity in rainbowtrout Yokoyama and Nakazoe.1996!, Curve was fitted to representthe meso values~ SDfor thesix sample fish. Ynkoyama 167 2.0 .6 LJ so 1.5 0Ga i.oU 6 E [.0 V D.5 '4 CI 0 0,5 0 Q o.o U ea a- 0 03 1.0 I.s 2.0 X.S Cyseeioedose psnal/g body«eig6r ! tl rA Figure 7. Effects of L-cysteine dose level on the hepatic cysteine dioxygenase activity in rainbow trout Yokoyama and Nakazoe 1996!, Values are means of measurements Figure 8, Effects of injection of L-cysteine and its deriva- made on six fish per treatment, tives on hepatic cysteine dioxygenase activity in rainbow trout Yokoyama and Nakazoe 1996!. Mean values not sharing a common letter are significantly different p<0,05!, weight, and there was no additional increase in the activity above a doseof 1,5 pmoVg of body weight. To elucidate how the hepatic CDO is induced and how its activity is controlled by sulfur cysteine by CDO, nor taurine, the final substance amino acidsper se, different forms of sulfur amino of sulfur amino acid metabolic pathway, induced acids were injected into the peritoneal cavity of the activity. L-homocysteine which has the rainbow trout. The specificity for the induction of structure similar to that of L-cysteine and is an hepatic CDO was examined. Based on the above intermediate involved in methioninc metabolism to mentioned results of the dose-response cysteine transsulfuration pathway! also showed experiments,the dose and the induction period were negative effects. fixed as 1.5 pmol/g of body weight and 4 h, The activity of hepatic CDO increased respectively. L-cysteine and its analogues which linearly with the increasing doseof L-cysteine, and have a similar chemical structure to L-cysteine the response was rapid and significantly specific were selected as the substances. The relationship to L-cysteine. These facts strongly indicate that between inductive activity and Inolecular structure the hepatic CDO activity, i.e., cysteine metabolism, was determined. Results are shown in Figure 8. Inight be controlled by the tissue concentration of Among thesesubstances, L-cysteine and S-methyl- sulfur amino acid, precisely of L-cysteine, in L-cysteine showed the strongest induction of the rainbow trout as in the case of rats Kohashi et al, enzyme activity, and other cysteine analoguessuch 1978!. Excess intake of cysteine upon both oral as D-cysteine, S-carboxymethyl-L-cysteine, administration and intraperitoneal injection brought cysteamine, N-acetyl-L-cysteine, and L-cysteic about the induction of hepatic CDO activity, and acid did not induce the activity. Further studies cysteine might be metabolized to cysteinesulfinate with other intermediates of sulfur amino acid as the intermediate product. This also suggests metabolism and related compounds were that the cysteine level might be kept at a low level perfortned. Results are shown in Figure 9. L- in rainbow trout body. Therefore, there Inust be a methionine did not affect the activity at all, and regulative mechanism in the cysteine metabolism neither L-cysteinesulfinic acid produced from L- of rainbow trout similar to that of mammals. I6ti UJNR Technical Report Nn. 26 60 O 40 ~~ZO U ae 3 0 0 20 40 Dietary protein %! Figure 10. Effect of dietaryprotein levelson percentweight Figure 9. Effectsof injection of sulfur amino acidsinvolved gain of rainbowtrout Yokoyamaet al. I994! ~: eggwhite in sulfur amino acid metabolism on hepatic cysteine albumin diets, 0; casein diets. dioxygenaseactivity in rainbow trout Yokoyama and Nakazoe1996!. Mean vaiues not sharinga connnonletter aresignificantly different P<0,051. ~ I,2 H I,O Influence of dietary protein levels on hepatic CDO activity 0,8 A feedingexperiment of rainbowtrout was conductedby use of either the diets containing w 0,6 somedifferent levels of egg white albumin, or casein as a sole dietary protein source, becausethese 0.4 proteins differ in amino acid composition. The 0 ~ 02 relation between the hepatic CDO activity in V rainbow trout and the dietary protein level was determined. Either egg white albumin denatured 0.0 0 20 40 60 with hot ethanoi under reflux for 6 h, or vitarnin- free caseinwas employed as a soledietary protein Dietary protein %! source. Figure ll. Effect of dietary protein levels on hepatic cys- Rainbow trout weighing about 17 g were teinedioxygenase activity of rainbow trout Yokoyamaet divided into 12 experimental groups of 18 al. 1994!. Values are means ~ SD of five fish. ~: egg white individuals.Body weightgain of rainbowtrout fed albuinin diets, 0: casern diets. the experitnentaldiets for 10 days is shown in Figure 10. The maximumgrowth rate obtainedby feeding casein diets was somewhat lower than the of enzymeactivity defined as the amountof enzyme Inaximum growth rate observed in fish fed the producingone ljmole of cysteinesulfinatein 1 hat albumin diets. 37 C. Also, the CDO activity in the dietary groups As shown in Figure 11, the activity of CDO of casein increased with the increase of the dietary in the liver of rainbow trout fed egg white albumin protein level up to 26%, while further increasein dietsincreased exponentially from 0.2 U/mg protein the casein level failed in enhancing the activity. to 0.9 U/mg protein as dietary protein level Hosokawa et al. 988! observed that the increasedup to 51%. The unit U denotesone unit hepatic CDO activity in rats, which were fed a Yoknvama 169 Figure 12. Optical microscopic images of the gill tissue of rainbow uout administrated with and without L-cysteinc by injecuon Yoltoyama and Saitaguchi 1996!. A, Fish injected with 10.0/rmol L-cysteine/g body weight; B, Control fish which were injected with only 0.9% saline solution. Bars indicate 100 pm. Reproduction permitted from the lapanese Society of Fisheries Sciences!. casein diet or soybean protein diet, was boosted toxicity of cysteineinjected into peritonealcavity by supplementing methionine which is the first limiting amino acid of thoseproteins, but the activity As mentioned before, cysteine was lowered by supplementing lysine, the first administration by injection caused heavy toxicity limiting amino acid to wheat gluten diet. In this to rainbow trout, although this amino acid is one of experiment, the activity of CDO in the liver the physiologically important amino acids. There increased along with the increasing level of egg have been some papersthat pointed out the toxicity white albumin. However, the activity in the fish of cysteine to animals Anderson arid Meister 1987, fed the casein diets remained low. Total sulfur Griffith 1987, Olney et al. 1990!; however, scarce amino acid content in egg albumin was almost twice information is available on the toxicity of cysteine as high as that in casein. The marked increase in for fish. The functional significance of the pathway activity of hepatic CDO observed in rainbow trout of cysteine catabolisin in rainbow trout is fed the egg albumin diets probably reflects the rise investigated in connection with its toxicity. in dietary levels of sulfur amino acids, and the rise A dose of L-cysteine ,5 to 10,0 pmol/g might depend on the cyst e!ine level rather than body weight! was injected to ten individuals each the protein level per se. That is to say, the hepatic to estimate LDse by probit analysis, A large dose CDO may be involved in the regulation of sulfur of cysteine led to mortality of rainbow trout with amino acid metabolism, reflecting the sulfur amino serious hemorrhage. LD,a values within 2, 3, and acid balance in dietary protein in fish. Therefore, 4 h were 7.5, 4.8, and 4.5 limol/g body weight, there is a possibility that the hepatic CDO activity respectively. A histological observation was made is a useful index for evaluating the appropriate with several tissues of rainbow trout which died of sulfur amino acid content in feed for rainbow trout. an injection. No appreciablehistological changewas observed in the tissues examined except for the THE FUNCTIONAL SIGNIFICANCE OF gill tissues. Figure 12-A is the photograph of the THE CATABOLIC PATHWAY OF most typical change observed in the gill tissue CYSTEINE WITH RESPECT TO Figure 12-B is the photograph of the gill of control DETOXICATION OF CYSTEINE: Acute fish!. The epithelia of secondary lamellae of the I'rtt UJNR Technitst Report No. 26 Table6 Effect ofthe injection ofsulf'ur amino acids onthe degree ofbleeding inthe gills Yokoyatna and Sakaguchi.l 996!. Anuno acid OD+,Ix10 L~sle inc 3,5 0. I &!.0 5.0 0.8W.2 7.1 0.7'.3 10,0 2,2W.5 o-Cysteine 5.0 0.6&,2 10.0 1,~.6 iV-Acetyl-L-cysteine 10,0 0.~.0 S-Methyl-L-cysteinc 10.0 0.3W. I L-Cysteinesulfinicacid 10,0 0.~.0 t.Wysteicacid 10.0 O,Ot0'.0 Hypotaurine 10,0 0.~,0 Taurine 10.0 0.Otal 0 Glutathi one 4.0 0.2MO L-Methionine 4.0 0, M.O t pmoldose per kg body weight. f Thedegree was expressed asthe optical density ofrearing water at 413 nm, Valuesare means of eight individual measurements * standattl error. gills swelledmarkedly. Bloodstains were also not,Other sulfur amino acids, L-cystcinesulfinic observedall overthe gill, Thecontrol fish showed acid, hypotaurine,and taurine involved in the nosign of suchhistological change. This anomalous cysteine catabolic route also showed no heavyhemorrhage might be accountedfor by a hemorrhagiceffect. Large doses of L-methionine functionaldisorder of cell membrane:the declined andg Jutathionewhich is a tripeptidehaving a free osmoreguhtionfunction of the epithelial membrane SH group couldnot be adrninistcredto fishdue to bringsabout the swelling of thecell, resulting in theirpoor solubility in water.However, glutathione thedestruction of thccapillary vessel, showeda slight toxiceffect even in a smalldose. Next,the acute toxicity of severalcysteine NeitherL-methionine nor L-cysteic acid showed analogues was examined, For the sake of any effect. Thesefindings indicatethat the convenience,thetoxicity was determined by the sulfhydrylgroup in themolecule might be involved extentof bleeding,because this methodis simpler in toxicityto rainbowtrout. andmare reproducible than the ~ usingmortahty. The fishinjected werc kept in I L of waterwith CON CL US ION aeration,Thirty minutesafter theinjection, the degreeof bleedingwas measured asthe optional Resultsof bothfeeding experiments and a densityof thewater at 413nm where hemoglobin tracerexperiment indicated that cysteine seemed showsits absorption maxima. The readings were to be the actual starting substancefor taurine correctedfor the body weight of 30g. Resultsare biosynthesisin rainbowtrout. Cysteinewas shownin Table6. Thedegree of bleeding OD at metabolized through cysteinesulfinate as an 413nm! was almost proportional tothe L-cysteine intermediateto hypotaurine,taurine and sulfate. dosein therange of 3.5to 100 pmoVgof body Cysteine dioxygenasewhich catalyzes the weight.Bleeding was observed in thegills within oxygenationof cysteineto form cysteinesulfinate 30 trunafter injection even though the dosewas wasconsidered toplay a regulativerole in cysteine only 3.5 pmal.This dosageis lessthan LD ~ for4- metabolisrn. Thus, it was suggestedthat the h lethaltime. D-cysteine showed similar toxic effect enzyme activity in the liver can be used for to thatof L-cystcine.N-acetyl-L-cysteine showed evaluationof sulfuramino acid availability in diets hemorrhagiceffect but S-methyl-L-cysteinedid for thefish. On theother hand, cysteine had serious vekevama t71 toxicityfor rainbowtrout, Consideringhoth the Kohashi,N., K. Yamaguchi,Y. Hosokawa,Y, Korr, rapidresponse of hepaticcysteine dioxygenase O. Fujii, and I. Lieda. ]978. Dietary activity to cysteine and low toxicity of controlof cysteinedioxygenase in rat liver. cysteinesu]finate,this enzymeprcsurnab]y plays a J, Biochem. 84: 159-168, rolein detoxicationby converting cy stcineinto norl- Kontro, P. and S.S. Oja. 1985. Hypotaurinc toxic cysteinesu]finate. oxidationby mouseliver tissue,pp. 83-9 l. In: S.S.Oja, L. Ahtee,P. Kontro, and M.K. LITERATURE CITED Paasonen eds.!, Taurine: Biological Actions and Clinical Perspectives. Alan Anderson, M.E. and A. Meister, 1987. R. Liss, Inc., Nev York. Intracellular delivery of cysteine,pp, 313- Moon, H.Y, and D.M, Gatlin III. 1991. Total sulfur 324. In: W.B. Jakoby and O.W. Griffith amino acid requirement of juvenile red eds.!,Methods in Enzymology,Vol. ] 43, drum, .Sciaenopsocellorus. Aquaculture Academic Press, New York, 95: 97-106, Aruoma, O.l., B. Hal]iwc]l, B.M. Hoey, and J. Nose T. 1974. Effects of amino acids Butler, 1988. The antioxidant actiorr of supplemented to petroleum yeast on taurine,hypotaurine and their metabolic growth of rainbow trout fingeriings-I1. precursors. Biochem, J, 256: 251-255. Methionineand cystine. Bull. Freshw.Fish. Cowey, C.B., C Y. Cho, J.G. Sivak, J.A. Res, Lab. 24: 101-109. Weerheim, and D.D. Stuart, 1992. Oja, S.S. and P. Kontro. 1981. Oxidationof Methiouinc intake in rainbow trout hypotaurinein vitro by mouseliver and Oncorhynchrr.r I@kiss!, relation to braintissues. Biochim. Biophys, Acta 677: cataract formation and the metabolisrn of 350-357. methionine. J. Nutr. 122: 1154-1163. Olney J.W., C, Zorurnski, M.T. Price, and J. Fellman, J.H.and E.S. Roth. 1985, Thc biological Labruyere. 1990. L-Cysteine. a oxidation of hypotaurine to taurine: bicarbonate-sensi tive en dogeno us Hypotaurincas a antioxidant,pp. 71-82. excitotoxin. Science 248: 596-599, Irr: S.S.Oja, L. Ahtee,P. Kontro, andM, K. SakaguchiM. 1994. Nitrogenousextractive Paasonen eds.!, Taurine: B iological components, pp. 287-292. ]n: The Actionsand Clinical Perspectives, Alan JapaneseSociety of Fisheries Science R. Liss, inc., New York. eds.!, Recent Advance in Fisheries Finkelstein,J.D. and J.J.Martin. 1986. Methionine Science. Kouseisha-Kouseikaku,Tokyo. metabolismin mammals: adaptationto Pn Japanese]. rnethionine excess. J. Biol. Chem. 26]: Sakaguchi,M. andM. Murata 1988. Taurine,pp. 1582-1587 56-65, In: M, Sakaguchi ed.!, Extractive Griffith, O.W. ]987, Mammalian sulfur airuno Components of Fish and Shellfish. acid metabolism:an overview,pp, 366- Ko use ish a-Kou seik aku, Tokyo.,fin 376, Inr W.B, Jakobyand O.W. Griffith Japanese]. eds,!, Methods in Enzymology, Vol. 143. Stipanuk,M. 1979. Effectof excessdietary Academic Press, New York. methionine on the catabolism of cystcine Hosokawa,Y., S. Niizeki, H. Tojo, I Sato,and K. in rats, J. Nutr. 109; 2126-2139, Yamaguchi, ]988, Hepatic cysteine Tateishi,N., T. Higashi,A. Naruse,K. Nakashima, dioxygcnaseactivity and sulfur amino acid H. Shiozaki, and H. Sakamoto. 1977. Rat metabolismin rat: possibleindicators in liver glutathione:]xrssib]e rale as a reservoir the evaluation of protein quality. J. Nutr. of cysteine.J. Nutr, 107:51-60. 118: 456-46], Walton,M.J., C.B. Cowey,and J,W, Adron. 1982, Ketola, H.G. 1982. Amino acid nutrition of tish: Methioninc metabolism in rainbow trout requirementsand supplementation of diets, fed diets of differing rnethionine and Comp.Biochern. Physio], 73B; 17-24. 172 UJNR TeehoieolReport No. 16 cystine content, J. Nutr. 122: 1525-1535. oral administration of L-c ystine on Watanabe,T Editor, 1994. Useof alternative hypotaurinelevel in rainbow trout, Fish. proteinsource in aquai;ulture.Kouseisha- Sci, 64: 144-147. Kouseikaku,Tokyo, 118 p. [InJapanese]. Yokoyama,M. andM. Sakaguchi.1996. Acute Wheldrake, J,F. and C.A, Pasternak. 1967. toxiceffect of L-cystcineinjected into the. Oxidationof cyst e!ineto sulfate in peritonealcavity of the rainbow trout. cultured neoplasticmast cells, Biochem. Fish. Sci. 62: 660-661. J, 102:45p-46p. Yokoyama,M.,M, Kaneni wa, and M. Sakaguchi, WrightC,E., H.H, Tal lan, Y, Y. Lin, and G E.Gaull. 1997. Metabolitesof L-[-'S]cysteine 1986,Taurine: niological update. Annu. injectedinto the peritonealcavityof Rev.Biochem. 55: 427-453. rainbowtrout. Fish. Sci. 63: 799-801. Yamaguchi,K.and Y. Hosokawa. 1987, Cysteine Yokoyarna,M., M. Udagawa,and J. Nakazoc. dioxygcnase,pp. 395-403. In: W.B. 1994.Influence of dietaryprotein levels Jakobyand O.W. Griffith eds.!, Methods on hepaticcysteine dioxygenase activity inEnzymology, Vol. 143, Academic Press, in rainbowtrout. Fish,Sci. 60: 229-233, New York. Yamaguchi,K. andI, Ueda. 1976. Methodsfor quantitativeanalysis of cysteineand its sulfinicand sulfonic derivatives, pp. 451- 453. In: The JapaneseBiochemical Society eds.!, Vol. 11. TokyoKagaku Dojin,Tokyo. [In JapaneseJ. Yamaguchi,K S. Sakakibara, J.Asamizu, and I, Ueda,1973. Inductionand activation of cysteine oxidase of rat liver II. The measurernenof cysteinemetabolism in vivoand the activation of in vivoactivity of cysteineoxidase. Biochim. Biophys. Acta 297: 48-59. Yokoyatna,M, andJ. Nakazoe.1989. Induction ofcysteine dioxygenase activity inrainbow troutliver bydietay sulfur amino acids, pp. 367-372. In: M. Takedaand T. Watanabe eds,!, Proceedings of the Third InternationalSymposium onFeeding and Nutritionin Fish. eda M, and Watanabe T.!. JapanTranslation Center, Tokyo. Yokoyarna, M. And J. Nakazoe, 1992. Accumulationand excretion of taurine in rainbowtrou t feddiets supplemented with methionine,cystine, and Taurine. Comp. Biochem.Physiol. 102A; 565-568. Yokoyama, M, and J. Nakazoe. 1996. Intraperitonealinjection of sulfur amino acids enhancethe hepatic cysteine dioxygenaseactivity in rainbowtrout Oncorhynchrtsrnykiss!. Fish Physiol. Biochem. 15: 143-148. Yokoyama,1'. andJ. Nakazoe,1998. Effectof Kiaa et ai. 173 THE EFFECT OF STOCKING DENSITY ON THE GROWTH OF JUVENILE SUMMER FLOUNDER PARALICHTHYS DFXTATU5 Nicholas King, W. Huntting Howell~, and E. Fairchild Universityof New Hampshire Dcpartment of Zoology Durham, NH 03824 e-tnai]: whh 8'christa. unh.edu * Author to whom correspondence should he addressed ABSTRACT The effect of stocking density on the growth of rwo size classesof]uveoile summer flounder was studied in experiments which lastedfor 40 and 58 days. In eachexperiment, density treatmentsof 100. l50, and 200% fish coverage of tank bottom surface areawere tested. Fish were fed to satiation and randomly sampledtor length, weight, and ventral surface area. Resuhs from this study tndicated tha small approx. l g, approx. 50 mm! juvenile summer flounderwere unaffected by stocking density of at least 200% over 40 days. Larger tapprox. 10 g, approx. lOOnunl juveriile summer flounder were affectedby the nominal stocking densities, with fish initially stocked at 100% coveragegrowing to slightly larger sires during the 58-day experiment. INTRODUCTION feed conversion,aggressive behavior, and oxygen depletion Refstie and Kittelsen 1976,Refstie 1977, With an increasingdetnand for high quality Vijayan and Leathcrland1988, Holm et al. 1990, Ratfiish in dorncsticand overseasseafood tnarkets, Kindschi andKoby 1994!, Similarly, for flatfish the summerflounder Paralichrhys dertratushas species like Japanese flounder Paralichrhys become a new and promising candidate for olivaceous, turbot Scophrhalrrtusmaximus, and worldwide fish farming. Its potentialhas been Atlantichalibut Hippoglossus hippoglossus, there studied in the United Statesfor severalyears, and appear to be some effects of higher stocking commercial cultivation has been initiated in several densities on growth rate and feed efficiency locationsalong the eastcoast, Early researchhas Martinez-Tapiaand Fernandez-Pato1991, Jcon focusedon larvaldevelopment and production et al. 1993, Bjornsson1994, Chang et a.l. 1995!. Bisbaland Bengtson 1991a,b, Malloy and Targett To date, few studieshave demonstratedoptimal 1991,Bisbal and Bengtson 1993, Keefe and Able stockingdensity for juvenileflatfish under 50 g in 1993, Bisbal and Bengtson1995a,b.c!, but. less weight, and none have examined juvenile summer research has been directed towards issues flounder stockingdensity. This research was associatedwith ju venilegrow-out. The ability to undertakento estimatethe optimal stockingdensity raisethe fingerling s ata relativelyhigh density, thus of early juvenile summer flounder in an maximizingwater usage and fish production,is of experitncntal recirculating system. Two size particularimportance to commercial operation. groups, with initial weights of 0.7 and 7.8 g, were However,parameters which affect growth and examined in two separate experiments. survival,such as feedingefficiency, disease, and water quality, should be considered when METHODS determiningan optimalstocking density for a particularsystem. Studies examining high rearing The recirculating system densitiesof severalsalmonid species attribute The experimental recirculating system growthinhibition toreduced feed consumption, poor consisted of l4 round, 190-L, fitberglass tanks t 1NR TeehoicotReport No, 26 associatedwith a 26-L biological filter. Water Becausethe rrlationship had a highcoefficient of flowed gravity! fromthe biologicalfilter, which determination Ra = 0.957!,it waspossible to usc contained"Bio-Fill" media and nitrifying bacteria, eachfish's total length mm! to estimateits ventral throughan ultravioletlight sterilizing unit to a surface area cm'!. distribution manifold above the tanks. Overflow from thetanks went into a centralcollection channel Experiment l - group 1 juveniles that wasfilled with a coarsepolyester fiber mat Newly weaned juvenile» were stocked for the removal of large particulate waste. This intowhite, plastic, 20-L aquaria, each with a bottom partially clarified water fell into a suinp tank, and surfacearea of 506 cm-'. Each aquaria was set wasthen pumped through two cartridgefilters 5 into the largertanks of t.hcrecirculating system pm! back to the biofiltcr. Water flow to the tank and supplied with seawater lg C!. Mean fish was rcgulatcd using valves, and each tank was length, weight, and surfacearea was 43 mrn,0.7 gently aerated. The systein was inoculait:d with g, and6,35 cin', respectively Table 1!. The three nitrifying bacteriaand run for 6 weeksprior to the densitytreatments of 10' .1 kg/rn'!, 150% ,7 introduction of any fish. After the systemwas kg/m"-!,and 200% .2 kg/m'-!coverage of tank established,salinity, tcinperature, ammonia,and bottomwere established by stocking80, 120,«nd nitritewerc ineasured daily. while dissolved oxygen 160individuals into each of the threercplicates andalkalinity were measured periodically. per treatment,respectively Table l !. As mortality occurredthrough the course of the experiment,fish Relating total length to ventral surface area were replacedto maintain nominalstocking Fishdensity was measuredas percent densities. All fish were fed to satiation twice a coverageof the tank bottom, Fish ventral surface day using Moore-Clark%formulated feed. The area wasestimated by tracing live, anesthetized experimentwas terminated after 40 days,and 25 speci mens from several different size classesonto individual~froin eachreplicate were weighed and a 1 cinx 1cm paper grid, and counting the number measured.Final stocking density percent cover! of cm"grids within eachoutline Totallength was wasdetermined for eachreplicate by multiplying alsomeasured for each speciinen, The curvilinear the numberof fish by the incan ventral surface relationshipbetween fish length and ventral surface area of the fish. This total fish surface area value areawas determined using regression analysis. was then expressedas a percentageof the surface Day0 Day 40 Nominal % cover 100 150 200 too 150 200 Mean length rnm! 43 43 43 84 e/-16.7! 84 e/-15.9! 82 ei-19.4! Meanweight g! 0.7 0.7 0.7 6.2 +/- 3.45! 6.3 +/- 3,43! 6.3 +/- 4.58! Meansurface area crn2! 635 635 635 15,38 15.38 14,94 Observedmean % cover 100 150 200 243 1.1 1.7 2.2 9.8 14.7 19.8 Table 1. Sumnmy oi'data for experiment 1 e/-> is standarddeviauoo. Kiaa et a!. 175 Day0 tv'ominn!% tsMr 150 3X! Meanlen8th ntm! 0 8 8! ! 28 a-252! 122 - 153! ! 24 +'-!.! 5! 155 '.153! 9 t/.4'! 8 i 458! 7.8 7.8 7.8 24 1 12! 20 -.067! 211+-! .42! 465 +/-!56! 35.!I+ !65! 4!.1 4 '1! Meanstr!at@atra ai ! 2! '! 2 2! ' 3' 34.1 35 5 IVhe!Xtunnanvivn! !C! !Ki l I 96 -4.6! 78 i/-8.4! 96 -75! 77 +-88! 3.7 55 75 11n 13.! 153 2!8 2@5 Tabk 2, Sonttnaryof data for experiment 2 +/-! is standarddeviation. area of the bottomof the experimentalaquaria. replaced only during the first week of the Biomassper unit area kg/m'! wasdetermined for experiment. The flounder were fed Moore-Clark N eachreplicate by multiplyingthe mean fish weight formulatedfeed twice daily to satiation.Random by the numberof fish. Mean total biomasspcr samples of fish were measuredand weighedon treatmentwas expressedas a proportionof the day 27 andat the conclusionof theexperiment on surfacearea m-'! of the bottotnof the experimental day 58. Stockingdensities percent cover! at days aquaria. Data were analyzed using one-way 27 and 58 were determined for each replicate by analysis of variance ANOVA!. multiplyingthe number of fishby the meMt ventral surface area of the fish, This total fish surface Experiment 2 - group 2 juveniles area value was then expressed as a percentage 3uvenileswere stockedinto gray,plastic, of the surface area of the bottom of the 20-L aquaria,each with a bottomsurface area of cxpcrirnentalaquaria. Biomass per unit area kg/ 962 crn'. Eachaquaria was set into thc larger m'-! was determined for each replicate by tanksof the recirculatingsystem and suppliedwith multiplyingthe mean fish weight by the number of seawater 8 C!, Initialmean fish length, weight, fish. Totaltreatment biomass was expressed» a and surfacearea were 89 nun, 7.8 g, and 21.2 proportionof the surfacearea rn'-!of the bottom crn', respectively Table 2!. The threedensity of theexperimental aquaria. Data were anal! zed treatmentsof 100% .7 kg/m-'!,150% 5 kg/ usingANGVA, followed by Tukey'smultipl~ m'!, and200% .3 kg/rn'!coverage of tankbottom comparisontests when significant differences wei e were establishedby stocking 45, 68, and 90 found. individualsinto eachof thethree replicates per treatment, respectively Table 2!. Mortalities probablydue to handlingand transfer stress! were t-gtVrt TechnicalReport No. 26 120 100 8 S0 20 75 100 125 150 '1Js 200 Total Length rtrrrt! Figurer. Relationship between totallength and ventral surface areain juvenile summer flonttder. Repession equation isbased on 59 individuals. The term ' denotesmultiply by.! RKSULTS bottomwas 243, 361, and466%. Estimatesof biotnassper unit area at the end of theexperiment Relationshipbetween fish lengthaud surface were9,8, 14.7, and 19.8 kg/m' for stocking densities al'ea of 100, 150,and 200%,respectively, These Curvilinearregression analysis was used represent7 to 8 fold increasesover the courseof torelate total fish length to theventral surface area theexperiment. Even at these high final densities, of thef ish Fig, 1!. Theequation which describes nosignificant differences Po0.05! io totallength thisrelationship, forsummer flounder ranging froin or wetweight were found between juveniles in approximately50-200 mm total length, is; any of the threetreatments. Surfacearea cm'! = 2,720x lpottos'~~' Experiment2 - group 2 juveniles Meanlengths, weights, surface areas, and Experiment1 - groupI juvernles densitiesare reported in Table2. Following A totalof ]9 fish werereplaced among mortalityreplacement in the first week,final incan replicatesof the 100%treatment, and 17 fish survivalswere 100,96, and78% for treatmentsof amongreplicates in each of the 150 and 200% 100, 150,and 200% coverage, respectively. At treatmentsAdditionally, a replicate was lost in the end of this 58-day experiment,incan fish the200% coverage treatment due to accidental densities had reached 355, 399, and 493% bottom stoppageof waterand subsequent depletion of coverage.Final estitnates of biomassper unit area oxygenMean lengths, weights, surface areas, and were21.8, 23.7, and 29.5 kg/m' for the100, 150, densitiesarereported inTable 1. Meanlengths and200% treaunents,respectively. At day 27, andwe'ghts increased byapproximately 40mm fish initiallystocked at 100%coverage were g 'neach treatment. Fish density increased significantly P<0,05! larger, both in lengthand y about140% ineach treatment asjuveniles grew weight,than those initially stocked at 150and 200% bothin lengthand weight over the 40-day coverage Table 3!, Therewas no significant experimentalPeriod Final coverage of thetank difference P>0.05! in eitherlength or weight Day 27 Length Weight Length Weight l 00 vs. 15IH 100 vs. 200% ns ns 150 vs 200% ns ns '1!tb]e3. Resultsfrom orre way analysisof variance AisiOVA! comparing mean lengths and weights from treatmentsin experiment2. ns = noi significant PA!.05!;* = p<0.05!. whichbegan with largerfish initialsize of about8 between the 150 and 200% treatments at this time g and 90 mm! and ran for a longertime, indicate Table 3!. At theend of the experiment day 58!, that stockingdensity does affect the growth of these fish initially stockedat ]00% coveragewere still ]arger individuals. In this instance,fish stockedat significantlylonger and heavierthan those in the the lowestdensity l00% coverage!grew faster 150%coverage treatrncnt P ,05!, but notlonger than those in both of the other treatments from the or heavier than those in the 200% coverage startof the experimentuntil day 27, Duringthis treatment P .05!. At day 58, there was no time, the mean length of fish in this treatment differencein the lengthsand weights of fish in the increasedby about44%, whichwas slightly higher 150 and 200% coveragetreatments P>0.05! than increases seen in the 150% 7% increase! Table 3!. and 200% 9% increase!treatments. Similar ly, meanweight of fish in the 100%density treatment DISCUSSION increasedby about208%, which wasdramatical]y higherthan the increases seen in the 150% 56% Resultsfrom experiment 1 in this study increase! and 200% 68% increase! treatments, indicate that small, recently weaned summer It hasbeen suggested that high stocking densities flounders initial size of about1 gand 40 mm! can can]cad to poor water quality high ammonia, ]ow be stockedat densitiesof at least 200%, and raised oxygen!which in turn canlead lo reducedgrowth for at least40 dayswithout negative]y affecting performance Brett 1979, Pickering and pottinger growth.In fact,wc recordeddensities greater than ] 987,Kebus et a], 1992,Kindschi and Koby 1994. 400% coveragear the end of thisexperiment Table Wagneret al. ]995!.It is extreme.lyunlikely that I! andsaw no indicationthat growth was being poorwater quality was a factorin thisstudy. F" impaired.Resu]ts from thisexperiment prevent us becauseall treatmentswere associatedwith the fromsuggesting an upper limit onstockmg densitic.s samerecirculating water and bio]ogical ft]ter, water for tish of this size,but it is certainly greaterthan qualitywas probab]y identical in al] treattnents. 200% coverage.We areunaware of any other Second,we foundthat nonionized arnrnonia never densitystudies that have been done with flatfishof ex.ceeded0,05 ppm and dissolvedoxygen never this size. fell belowsaturation. Lastly, we observed noloss Results from experiment 2, however, of appetitein anyof rhetreatinents that cou]d l7$ t:JAR TerhairtttReport No. gati indicatepoor waterquality and/or stress. Thus, brhavior increaseswith numerical density, which our recirculatingsystein and biological filter werc in turn increasesstress and reducesgrowth rate, it capableof maintainingammonia below, and is possiblethat a reduction in agonism over time dissolvedoxygen above. normally stressfullevels. explains the compensatorygrowth we observed. In cffcct, the systemin which we conductedthe In thc 200%density treatment,the mean number cxpcrirnent eliminated two of the variables high of fish per replicate decreasedfrom 90 to 69, thus ammonia,low oxygen!that arc often associated possiblyreducing aggressive, agonistic mteractions. with high stocking densities. Aside from water Alternatively,the fish at thehigher density 00%! quality issues, food consumption and feeding simply could have becomemore "accustomed' to behaviormay also bc affected by stockingdensity this densityas time progressed,thereby reducing Holm ct al, 1990,M artinez-Tapia and Fcrnandez- stressand resulting in compensatorygrowth. A Pato1991!. In suchinstances, "crowding" high thirdpossible explanation is that suminerflounder numberof fishpcr unit area!can cause an increase responddifferently to stockingdensity as they in agonistic leeding behavior, which in turn increasein size. If, f'or example,larger fish are increases stress and decreasesgrowth. It is morctolerant ofhigh density at larger sizes,then it possiblethat these factors contributed to thcresults wouldexplain why fish at the higher density 00%! we found. Summer floundcr arc known to be exhibitedcompensatory growth during the second aggressivefeeders Bigelow and Schroeder 1953!, part of this experiment. andwc occasionally observed aggressive feeding Fishheld at the intermediatedensity behavior c,g, chasing, tail biting! in this experiment. 50%! weresignificantly smaller than thoseat If suchbehavior increased with numerical density, 100% on days27 and Sg, but not different from it tspossible that fish in thc lowest stocking density thoseat the200% density on eitherday. These l00%!, whichcontained only 45 fish in each results partially support our hypothesis that triplicate,benefited from low numerical abundance, numericalabundance and associated agonistic Thefact that we saw no density efTect inexperiment feedingbehavior may be affecting the growth of I, whichwas dane with smaller fish, suggests that juvenilesummer flounders. Because of mortaIities thismechanism, if applicable, may not operate until in replicates of the 200% treatment, mean thefish are somewhat older and larger. Density- numericalabundance in the 150% treatment nW5! dependentbehavioral changes are wd1 docu inc n ted was nearly identical to that of the 200% treatment Fendersonand Carpenter 1971, Refstieand n=69!. Thesesimilarities would explain why Kittelsen1976!, and Wagner et al. 996! found growthwas nearly identicalin the 150 and200'7t: that agonisticbehavior in rainbowtrout fry treatments,and why fish in bothof thesehigher increasedwith age. Thesestudies support our density treatmentswere smaller than thosem the contentionthatbehavioral mechanisms, which may 100% density treatmentwhich had a lowermean changewith fish size,were responsible forthe numericalabundance n=45!. Thefact thatfish in differencesweobserve. At the end of experiment the200% treatment deinonstrated compensatory 2 {day58!, fish in the 100% density treatment were growth,while those i nthe 150% treatment did not, stilllarger than fish in the 15G% treatment, butnot isdifficult to explain. It ispossible that ihe loss of thefish in the 200% treatment. The parity of fish fish in the 200% treatmenttriggered the inthe 100 and 200% density treatments atthe end compensatorygrowth we observed, and that this ofthe experiment, butnot at day 21, is an indication did not occurin the 150%treatment because thata sizeconvergence occurred between days numericalabundance was relatively stable 21 and58. Thus.it appearsthai fish stocked at throughoutthe experiment.This is very densitiesof 100 and 200% grow at different rates speculative,and additional researchwould be for a periodof time the first half of this needed to addressthis issue. experiment!,butfish held at thehigher density Ourresults are similar to those.of the few 00%!were able to "ratch up" compensatorystockingdensity studies which have been done with growth!as time went on thesecond half of this otherflatfish species. Jeon et al. 993!, who experiment!.If, as speculated above, agonistic worked with young JapaneseHounder Paralichthys olivaceous, evaluated stocking that recently weaned summer flounder ca.n be densities of 33, 50, 100, 200, and 300% bottom stocked at densities of at least 200%, but that coverage,and found that the highest feeding rate stocking density should be reducedto 100% for and growth occurredat 200%coverage Similarly, larger juveniles, at least for several weeks. and Chang et al. 995!, who worked with larger 0- that densities could then be allowed to increase as 7S mm! Japanese flounder, in a semi-closed, the fish grow in size. Furtherobservations and recirculating seawater system, reported final research, which develop with the commercial densitiesas high as 260% 6.3 kg/m'-'!.Although summerflounder industry, will undoubtedlyrefine our experimental fish were not grown to harvest our understandingof optimum stocking density. size at our nonunal stockingdensities, results with both turbot Scophthalrnasmaximus and Atlantic ACKNOWLEDGMENTS halibut Hippoglossushippoglossus suggestthat larger sizedHatfish can begrown at relatively high We thankChris Duffy, GeorgeNardi, and densities. Martinez-Tapia and Fernandez-Pato Andrea Tomlinson for their dedicate assistance. 991! found no ill effectsat a stockingdensity of The researchwas supported by a grant from the 68 kg/m' for turbot, and suggestedthat specific Saltonstall-Kennedyprogram. UNH Centerfor growth andfood conversion were greater at h.igher Marine Biology/JacksonEstuarine Laboratory densities. Bjornsson 994! conducted research Contribution Series ¹335. with relatively largehalibut initial size 1.8-3.2kg! at stockingdensities of 50, 100and 160% coverage. LIT ERATU RE CITED Although he observeda maximum coverageof 215% 95 kg/m'!, he indicatedthat growthrate was reducedin the highest coverage 60%! Bigelow,H,B, andW.C, Schroeder.1953, Fishes treatment,and that optimal stockingdensity was of the Gulf of Maine. Fish, Bull, U.S. 74: somewherebetween one andtwo layersof fishon 1-577. Bisbal, G.A. and D.A. Bengtson, 1991a, the tank bottom. This study,as well as thosewith halibut, Characterization of starved versus fed Japaneseflounder, andturbot indicate that Hatfish summerflounder, Parali chthys dentatus, speciesare able to grow effectivelyat stocking larvaeand juveniles, pp. 216-218. In; P, densitiesof 100-200% oneto two layersthick on Lavens,P. Sorgeloos,E. Jaspersand F. thetank bottom!. Indeed, in this studywe observed Ollevie eds,!, Larvi '91, European that fish, when not feeding, would crowd and AquacultureSociety, Spec.Publ, No. 15, overlapone anothereven when empty spacewas Gent, Belgium. availablc,and that thisoccurred at all stocking Bisbal,G.A. and D.A. Bengtson.1991b, Effect densities. Similar "layering" behaviorhas been of dietary n-3! HUFA enrichment on observedin halibut Bjomsson1994!. Further, survival and growthof summer fiounder, bioinassdensities can be relativelyhigh. In this Paralichthysdenratus, larvae, pp. 56-57. study,with relatively small fish, biomassdensities In: P. Lavens, P. Sorgeloos,E. Jaspers reachedonly 29,5 kg/m', but work with Japanese andI. OHevie eds.!, Larvi '91, European flounder Chang et al. 1995!,turbot Martinez-Tapia AquacultureSociety, Spec.Publ, No. 15, andFernandez-Pato 1991!, and halibut Bjornsson Gent,Belgium, 1994! suggesi.that biomass densities of 36.3,68.0, Bisbal,G.A. and D.A. Bengtson.1993. Reversed and 9S kg/m2, respectively.were possible. The asynunetryin laboratory-rearedsuinmer combinationof layering,and tolerance of high Hounder, Prog. Fish-Cult. 55: 106-108, biomassdensities, suggest that flounderscan be Bisbal, G.A, and D,A. Bengtson, 1995a. raisedat highdensities. This could be anenormous Descriptionof the starving conditionin advantageto the grow-outfarmer, provided that summerflounder Paralichthysdenratus ! therecirculating system is capable of supporting earlylife stages:morphometrics, histology, thesehigh biomasses, Results of thisstudy suggest andbiochemistry. Fish. Bull, 93: 217-230, tsa UJlttRTecbaical Report >n, 26 Bisbal, G.A. and D.A. Ben gtson, 1995b, Performanceand oxygen consutnptionof Developmentof thedigestive tract in larval Snake River cutthroat trout reared at four sutnmerflounder, Parali chthysdentatus, densitieswith supplementaloxygen. Prog J. Fish Biol. 47:277-291. Fish-Cult. 56: 13-18. Bisbal,G.A. andD.A. Bengtson 1995c. Effects Malloy, K,D. and T.E. Targett, 1991, Feeding, of delayedfeeding on survival and growth growthand survival of juvenile flounder of summer flounder, Paralichrhys Parali chthys dentatus: experimental dentarus,larvae. Mar. Ecol. Prog. Ser. analysisof the effects of temperatureand 121: 301-306. salinity. Mar. Ecoh Prog. Ser. 72: 213- Bjomsson,B. 1994, Effectsof stocking density 223. on thc growth rate of halibut Martinez-Tapia, C. and C.A. Fernandez-Pato IIippoglossus hippoglossusL.! reared 1991. Influenccof stockdensity on turbot in large circular tanks for three years. Scophthalamusmaximus! growth, Int Aquaculture 123: 259-270. Counc. Explor.Sea C.M. 1991/F:20. Brett, J.R. 1979. Environmental factors and Pickering, A.D. and T.G. Pottinger. 1987, Poor growth,pp, 599-675. In: W.S.Hoar, D.J. water quality suppressesthe cortisol Randall and J.R. Brett eds.!, Fish responseof salmonidfish to handling and Physiology,Vol. VIII. Bioenergeticsand confinement, J, Fish Biol. 30: 363-374. Growth. Academic Press, New York, Refstie, T. 1977. Effect of density on growth and Chang, Y.J S,H. Kitn, and H,S, Yang, 1995. survival of rainbowtrout. Aquaculture 11: Cultureof the oliveflounder Paralichthys 329-334, olivaceotts! in a semi-closedrecirculating Refstie, T. and A. Kittelsen. 1976. Effect of seawater system, J, Korean Fish Soc. density on growth and survival of 28 ! . 457-468, artificially reared Atlantic salmon, Fenderson,O,C, and M.R. Carpenter. 1971. Aquaculture8; 319-326. Effectsof crowdingon the behaviourof Vijayan, M.M. andJ,F. Leatherland. 1988, Effect juvenile hatcheryand wild landlocked of density on the growth and stress- Atlanticsalmon Salmosalar L.!. Aniin. response in brook charr, Salvelintts Behav. 19: 439-447. fonrinalis. Aquaculture 75: 159-170. Holm, J,C,, T. Refstie, and S. Bo. 1990. The Wagner, E.J., S.A. Miller, and T. Bosakowski. effectof fishdensity and feeding regimes 1995. Atnrnoniaexcretion by rainbow onindividual growth rate and mortality in trout.over a 24-hourperiod at two densitics rainbow trout Oncorhynchusmykiss!. duringoxygen injection. Prog. Fish-Cult. Aquaculture 89: 225-232, 57: 199-205. Jeon,I.G., K,S, Min, J.M. ~, K.S, Kim, and M.H. Wagner,E J.,S.S. Intelmann, and M.D. Routledge. Son. 1993. Optimalstocking density for 1996. The effectsof fry rearingdensity olive flounder,Paralichihys olivaceous, onhatchery performance, fin condition,and rearing in tanks. Bull. Natl. Fish. Res. agonistic behavior of rainbow trout Dev. AgencyKorea 48: 57-70. Oncorhynchusmykiss fry. J. World Kebus,M.J., M. T, Collins,M.S. Brownfield,C.H. Aquacult Soc.27: 264-274. Amundson,T.B. Kayes,and J.A. Mallison. 1992. Effectsof rearing densityon the stressresponse and growth of rainbow trout. J. Aquat. Anim Health4: 1-6. Keefe, M. and K,W. Able. 1993, Patterns of metamorphosisin summer flounder, Paralirhthysdenrarus, J. Fish Biol. 42: 7] 3-728. Kindschi, G.A. and R.F. Koby Jr. 1994. Kaaazawa 181 IMPORTANCE OF DIETARY LIPIDS IN FLATFISH Akio Kanazawa KagoshimaUniversity, Facultyof Fisheries 4-50-20 Shirnoarata,Kagoshima 890-005ft, Japan ABSTRACT Constderab!eanenuon has been focused on the n-3 po!yunsaturatcd fatty acid PUFA! ret!uiretnentsof marine fish larvae. To clarify the physiologica! role of dietary eicosapentaenoic acid EPA! and docosahexaenoicacid DHA! in the body of fish larvae. accumu!ation of dietary EPA and DHA in the eye including retina, brain, and !iver of Japaneseflounder was analyzed. Dietary DHA was rapidly incorporated into phospholipids of retina, hrain, and liver suggesting that DHA may play an iinportant role foi' fish !arvae. On the albinism in the ocular side of flatfish, which resuhs from the deficiency of pigmems, and widely occurs during the processof seedproduction, l found that albirusm resu!tswhen ! 0-day-o!d larvae were fed w!th nutritionally deficient experimenta! microparticu!ate diets. ! suggest that. the rhodopsin formation of the eye retina was hindered when fat-so!uble vitamin vitainin A! and n-3 high!y unsaturated fatty acid DHA! were deficient.in foods, resu!ting io the interruption of h!ac!t pigment mc! anin! formation. On the effect of dietary phospho!ipids on the stress to!erance of Japaneseflounder investigated using feedingtrials, I studiedthc toleranceof Japaneseflounder to variousstress factors such as changes in water tempeiature and salinity, and exposure to !owdissolved oxygen, and noted that dietary soybean !ecithin and kri!! phospholipid were effective m increasing the tolerance of flatfish to the various suess condinons. INTRODUCTION ACCUMULATION OF DIETARY EPA AND DHA IN BRAIN AND RETINA OF Dietary lipids are importantsources of JAPANESE FLOUNDER LARVAE energy and essential fatty acids for all animals, The n-3 fatty acid such as eicosapentaenoicacid Introduction EPA, 20:5n-3! and docosahexaenoic acid Considerable attention has been focused DHA, 22 n-3! are highly unsaturatedfatty onthe n-3 HUFA requirementsof marinefish larvae acids HUFA! that are commonly found in Kanazawa 1985!. Studieson speciessuch as marine organisins. The useful roles and Scnprhalmusmaximus Witt et al, l 984!, Spanrs beneficial effects of these fatty acids have been aurata Kovenct al. 1989!, Cnryphaertahippurus recognizedfor marineanimals and human health. Ostrowskiand Divakaran 1990!, and Oplegrmrhus It has be.cn demonstrated that EPA is fasciatus Kanarawa 1993a! larvae have shown biosynthesizedby phytoplanktonand it then is that DHA is strongly retainedand is essentialfor assimilatedby zooplanktonof which a part of these marine fish. When turbot Scnptharmus EPA is bioconverted into DHA. Both n-3 HUFA maximus larvae were fed on a pcllcted diet are depositeda.nd accumulated tn marinefish. containing13-fold mote DHA than Arremia, DHA Accumulationof dietaryEPA and DHA in brain is rapidlyincorporated into the brain phospholipid, and retina of Japaneseflounder, nutritional particularlyin thephosphatidylcholine Mourente mechanisms involved in the occurrence of and Tocher 1992!. abnormal pigmentation in h atch ery -reared Japaneseflounder, andeffect of phosholipidson Matenstls and Methods stress tolerance of Japaneseflounder were To clarifythe physiological role of dietary studiedto illustrate the importanceof dietary EPA and DHA in the body of fish larvae. lipids in flatfish. accumulationof dietaryEPA and DHA in thebrain, ia2 UJ'NR TeeitnicalReport No, 24 eyeincluding retina, and liver of Japanese flounder Results Paralichfhysolivaceous was analyzed. A feeding After 30 days,EPA and DHA contentsin experimentwas carried out usingseini-purified brain, retina, and liver of flounder larvae fed with microparticuiatediets containing 2% of eitherEPA EPAor DHA diets werecompared with those fed or DHA, The proteinsources in the diet were a HUFA-free diet. The brain and liver accumulated casein,white fish meal and squid meal, and gluten morcEPA and DHA in the polar lipid thanin the wasused as the binder. The diet wasa drypellet neutral lipid Figs. 1, 2!. In the retina, EPA was typeand thc basal diet composition isgiven in Table accumulated in both neutral and polar lipids; 1.The ingredients were added in the following order however,DHA washigher in polar thanin neutral and mixedwell in every addition: {1! protein lipid fraction Fig. 3j. DietaryDHA wasrapidly sources,water-soluble vitamins, minerals, activated incorporatedinto phospholipids of brain,retina, and gluten, etc.; ! fat-soluble vitamins, soybean liver suggestingthat DHA mayplay an important lecithin,EPA or DHA,oleic acid; ! waterat 30 role for thelarvae of this species. ml/100g dict, The well-mixed dough was pelletized threetimes by a meatmincer with 2.5 mm die. Thc NUTRITIONAL MECHANISMS pellets were then oven-dried at 40'C for 8 h, INVOLVED IN THE OCCURRENCE OF steamed for 90 sec, broken down into 1.9-mrn ABNORMAL PIGMENTATION IN particlesizes, and stored at -20'C. Pelletswere HATCHERY-REARED JAPANESE txioledto toom temperature before feeding, Twenty FLOUNDER P. olivacerrslarvae, 30 daysafter hatching total length35.32 ~ 2.18mm; weight 0 35 + 0.05 g!, Introdu ebon were stocked in a 100-L tank, Seawater was Thedepigmentation {albinism! in theocular allowedto flow at 2.4 Dmin with temperature sideof flatfish,which resuhed from thedeficiency rangingfrom 15to 18'C. Fishwere fed three times ofpigments, haswidely occurred during the process a day. of seedproduction. Although many researchers g/1I g diet Ingredient Free DHA 2% EPA 2% DHA' 2.3 0.0 EPA' O.G 0.0 73 Oleic acid' 7.0 4,7 4.7 Soybeanlecithin 6.0 6.0 6.0 Basalingredients' 97,0 97,0 97,0 'Ethylestcrs: purity 87% 'Basalingredients g/100 8,diet!: casein, 20.0; wite fish meal, 18.0; squid meal, 20.0; dextrin,6 3; vitamin tnixture,' 5,3; rnincrd inixture,'~ 5,0; activated gluten, 8.0; lysine HCl, 2.2; tryptophan.0,7; attiactants,~~'1.5. 'Vitaminmixture mg/100 g diet!: p-aminobenzoic acid 144.48, biotin 2,18, inositol 1450.69,nicotinir. acid 290.11, Ca-pantotbmate 101.57, pyridoxine HC1 17.28, riboflavin 72.51,ttuanun HC1 21.76, metutdione 1728, vitamin A palmitate 71.00, «-tocopherol 145.09, eyanctcobalaminc0.03.calciferol 3.65, APM 25.31, folic acid 5.44, choline chloride 2965.31. "Mineralmixture mg/100 g dict!:U.S.P. JGI No. 2: NaCI183.8, MgSG, 7H,O 685.0, NaH,PO,2H,O 436.0, KHp%, 1199.0, Ca HJ%,!,H 0 679.0,Fe citrate 148.5, Ca lactate 1635.0;trace elements J.E. Halver!: A1Cl,611,0 0.9, 7 'tttbtet. Cotnpositionof rett diet containiag eicosapeutaettoic acid EPA!or docnaatteaaenoic acidiDHA! for Japaneseflotnwter. Ken ezvwa 183 HUFA free diet HUFAfree diet 2% DHAdiel 2% OHA diet 2ssEPA diet 2'll, CPA diet 1DG 200 320 tv0! 100 200 300 yg! EPAcontent in liver EPA content in tsretn HUFA free diet HUFAtree diet 5 PL 2'!aOHA diet 2% DHAdiet 2% EPA diet 2% EPA diet 100 2 DO 300 leel 0 100 200 300 ug! rHIA conlentie liver DHA content In brain Ftgttre l. Eicosapentaenoicacid EPA! aud docosahexaenoic Figure 2. EPA and DHA contents !tg!mg! in polar IPL! and acid DHA! contents p.g/tng! in polar PL! and neutral neutra! ltpids HL! in the hvcr of Japanesef!ounder fed on lipids NL! in the brain of Japaneseflounder fcd on EPA EPA or DHA diet i or 30 days. Data are the meanof three or DHA diet for 30 days. Data are the mean of three rephcales. repi icates. Fish used JapaneSeflOUnder Age 4 days after hatching Total length 4,2 inm Number of fish 890/tank Rearingand feedingmethods Feeding period 65 days Tank 100 L Water temperature 15.0-20.0 C Flow rate 0.2-1.0 L/nun Feeding frequency 10times/day Type of diet Micxoparticulate diet Table X. Fish used and rearing tnethods for tbc abnormal pigmentation study i;JNR TechnicalRepert Na. 2tt have investigated the incchanisms of depigmentation,little is known in this field. The authorfound that depigmentationresulted when HUFA free diet l0-day-oldlarvae were fed with nutritionally deficientexperimental microparticulate diets. It 2'5 D HA diet wassuggested that the rhodopsin formation of eye retina washindered when vitamin A, DHA, and 2' KPA diet phospholipidwere deficient in foods,resulting in theinterruption of black pigment melanin!formation 100 200 300 app trgl Kanazawa 1993b!, EPA content in retina Materials and Methods Newly hatchedJapanese flounder P. alivacetrs larvae were fed with rotifers for 4 days. HUFA free diet Thereafter,800 fish were divided into experimental groups,and fed withinicroparticulatc diets reared 2% DHA diet under conditions listed in Table 2. The microparticulatediets werc mainly composed of 2', Er Ad et vitamin-freecasein, dextrin, lipids, mineral mixture, andvitamin mixture. As the binder, k-Carrageenan 100 200 300 a 00 JJg! wasused. The experimentaltreatments were: diet OHA content In retina l, completediet; diet 2. n-3HUFA- deficient diet; diet3, fat-soluble vitainin deficient diet; diet 4, live food rotiferand Arrerrtia!. The appearance of albinism in the ocular side of P. olivaceuswas determined65 days after feeding with thc various test diets. Ftgttre3. EPAand DHA contents itg/mg! m polar PL! and neonal lipids NL! in theretina of Japaneseflounder fed on EPAor DHA diet for 30 days.Data are the mean of three Results and Discussion replicates. Albinism completelyabnormal and partially abnormal!in flatfish was 23.1% in the groupfed withthe complete diet, but 83.4% in the Diet Abnormal + partialabnormal %! Complete 23,1" n-3 HUFA-deficient 83.4' Fat-soluble vitamin deficient 43.2~ Livefood rotiferand Artemia! 533 a Valueswith the same superscripts aresignificantly different at5'Y» level ~.05! Eachtreatment was conducted intriplicate groups. 'l|tbie3. Depigmentationof Japanese flonn der fed with nit tritionally deficient diets Ranaza» a IN n-3 HUFA-deficient diet and 43.2% in the fat- The author assumed that in flatfltsh, thc soluble vitamin deficient diets Table 3!. It has rhodopsinformation of theretina is interruptedwhen been suggestedthat DHA in lhe n-3 HUFA and vitalnin A, DHA, and phospholipid are deficient in vitamin A in the fat-soluble vitamins were essential initial foods after hatching of the eggs. For this in the reduction of albinisln in hatchery-reared reason, visual transmission from the retina is not Japaneseflouttder Kanazawa1995!, Rhodopsin transferred to the central nervous system, so that in the rod cells conducting vision in the dark is the melanophore-stimulatinghormone from the composed of opsin protein!, retinal vitamin A endocrine organdoes not secrete,resu!ting in the aldehyde!,and phospho!ipid phosphatidylcholine! interruption of the black pigment formation. i ncluding DHA. Therefore, when microparticul ate diets or rotifers enriched with vitamin A. DHA. and phospholipid soybeanlecithin! are fed on flatt tsh 10 daysafter Fishused J spanese fi ounder hatching,the preventionof albinism is possible. Totallength 35. 13i 0. 36 mnt Body weight 042& 0 I2 g ]4umherof fish 20 fish/tank EFFECT OF PHOSPHOLIPIDS ON STRESS TOLERANCE OF JAPANESE FLOUNDER Rearingand feeding methods Introduction Fling periods 40 days Tank 100k Marine fish larvae were found to have a Flowrate 500-600mtimin requirementfor phospholipidson growth and Watertemperature 16.5+ 0.3'C survival Kanazawa ct al.1985, Kanazawa 1993c, F cedingfrequ«ney 4 times/day Kanazawa 1997!. The present research was Feedinglevel 5%%dof body weight conductedto determine the effectof phospholipids Typeof diet Dry pellet Sizeof diet Diameter1.6 mrn on stresstolerance such as the changesin water temperature and salinity, and exposure to !ow dissolvedoxygen DO!. Table 4. Fishused and rearing methods for ihe stressstudy Pbosphohpid Phospholipid Phospholipid 1'Yi Pro as krill assoyhean phosphohpid! lecithin Survival '%%d! at tetnperature Risefiorn 16.5to ~U.OC andkept at 22 O'C far 30 mtn Risefrom 22.0 to 27.0 C aad kept at 27 0 C for 30 Risefmm 27.0 to 33,0 C andkept at 33.0'C for 30 20 ]tisefrom 33,0 to 34,0'C and kept at 34.0 C for 30 llaw salinity 5 to 0 ppt! '1'une min! when5 P%%dof fish 83.tJ 278.3 11 l.6 grouplaid down Low dissolvedoxygen to 0.80 tnl/L! Time min! when5%< of fishgroup 7. 7 15.6 ]2,1 laiddown Valuesare the meanof threereplieates. Table 5. To]eranceof ]apaneseflounder to stressdue to increasedtemperature, reduced salinity, and dissolved oxygen tlJNR Techttit:aiReport tstt. 2ts Materials and Methods Kanazawa,A. 1993c.Essential phospholipids of Japaneseflounder P. ofivaceuswere fed fish and crustaceans, pp.S19-530. In: diets containing soybeanlecithin % as S.J,Kaushikand P. Luquet cds.!, Fish phosphatidylcholine!andkrill phospholipid % as Nutrition in Practice, INRA Edition, Paris phosphatidylcholine!under the conditions listed in LesColloques, No,61. Table4, Thediet was mainly composed of vitamin- Kanazawa,A. 1995. Nutrition of larval fish, free casein, defatted squid meal and white fish pp.S0-59. In: C,E, Lim and D,J, Sessa meal,dextrin, lipids, mineralmixture, and vitamin eds.!,Nutrition and Utilization Technology mixture.Activated gluten was usedas the binder in Aquaculture,AOCS Press, Chatnpaign, see Table 1!, IL. Kanazawa, A. 1997. Effects of docosahexaenoic Results acid and phospholipidson stresstolerance After the feedingexperitnent, Japanese of fish, Aquaculture 155: 129-134. flounderwerc tested as to theirresponse to stress Kanazawa,A., S. Teshirna, and M. Sakamoto. dueto low dissolvedoxygen, low salinity,and 1985. Effects of dietary bonito-egg increasedwater temperature Table 5!. Japanese phospholipidsand some phospholipids on flounderin increasedwater temperature at growthand survival of the larval ayu 33,0'C! showedthat those fed with the soybean Plecoglossusalti verbis. Z. Angew, lecithinand krill phospholipiddiet had higher Ichthyol.4: 165-170, tolerancethan thc phospholipid-freediet. When Koven,W. M G. lvl, Kissil, andA. Tandler.1989. Japaneseflounder were exposed to low dissolved Lipidand n-3 requirement of Sparus aurara oxygenand low salinity,dietary soybean lecithin larvae during starvationand feeding. andkrill phospholipid were effective inincreasing Aquaculture79: 185-191. thetolerance of fish. Phospholipidswere not only Mourente,G. and D. R. Tocher.1992. Effects ot indispensablenutrients for thegrowth of fish,but weaning onto a pelleted diet on wereeffective in increasingtheir tolerance to the docosahexaenoicacid 2:6n-3! levels in various stressful conditions. brainof developingturbot Scophthalmus maximusL !. Aquaculture105: 363-377, Ostrowski, A. C, and S. Di vakaran. 1990. Survival LITKRATURZ CITED andbioconversion of n-3 fatty acids during earlydevelopment of dolphin Coryphaena Kanazwwa,A, 1985. Essentialfatty acid and hippurus!larvae fed oil-enrichedrotifers. lipidrequirement of fish,pp.281-298. In; Aquacu1ture 89: 273-285, C.B. Cowey, A,M. Mackie and J.G. Bell Witt, U., C, Quantz,D. Kuhlman,and G. Kattner. eds.!, Nutrition and Feedingin Fish 1984. Survivaland growthof turbotlarvae Academic Press, London. Scophrhalmus maximus L. reared on Kanazawa,A, 1993a.Itnportance of dietary differentfood organisms with specialregard docosahexaenoicacid on growth and to organismspolyunsaturated fatty acids, survival of fish larvae,pp 87-95.In: C.S. Aquacult. Eng. 3: 177-190. Lee, M.S. Su, and I.C. Liao eds.!, Proceedingsof Finfish Hatcheryin Asia '91, TungkangMarine Laboratory,TFRI, Taiwan. Kanazawa,A. 1993b. Nutritionalmechanisms involved in the occurrence of abnormal pigmentationin hatchery-rearedflatfish Heterosorna.J. World Aquacult. Soc, 24: 23-27, Oaniets and 8orski t87 EFFECTS OF LOW SALINITY ON GROWTH AND SURVIVAL OF SOUTHERN FLOUNDER PAR4LICHTHYS LFTHOSTIGMA! LARVAE AND JUVENILES Harry V, Daniels North Carolina StateUniversity, Dcpartmcntof Zoology 207 Research Station Road Plytnouth, NC 27962 and Russell J. Borski North Carolina State University, Department of Zoology Box 7617 Raleigh, NC 27695 ABSTRACT The Southernflounder IParafichrhvs ferhosrigritrt!is a euryhalineflatfish with a naturalrange that extendsfrom North Carolina to Mexico. Since adult flounder are commonly found in freshwater ~ound~and rivers, it appears that there is potential for culture in fresh water. A seriesuf studies was conductedto determine the eflects of! ow salituiy on growth and survival of flounder from metamorphosisthrough the advancedjuvenile stage.Survival of larval flounder was significantly lower P<0.05! when exposedto salinities below 20 ptn during metamorphosis. but postmeiarnorphicflounde werc noi adverselyaffected by saliniuesas low as0 ppt. Two separatestudies wereconducted to determinethe growthrate and survival of Southernflounder stocked in low salimtywater and fed pelleted feed. ln itic first study,juvenile flounder with an averageweight of approximately 7.0 g weregrown in salinities of 0, 5, and l0 ppi water for 84 days. 1n the secondstudy, advancedjuvemle fl ounderwith an average weight of approximately 32,0g were grown in salinities of 0, 5, IO, and 20 ppt for 58 days. The speciflc growth rate < SGR! of juvenile flounder ranged from ] .0to 1.09%/dayand was noi sifpiiftcantly diiTerent amongtseaunents. SGR of advancedjuvenile flounder ranged from 1.6 to I.71%/day and was not significantly different between beatnients Po0.05!. Protein efficiencv ratio, feed conversion efficiency FCE! and daily feed consumpuon DFC}values were not significantly different Po0.05!between veaunems, These results indicate that Southern floundercan be grown in salinitiesas low as0 pptwithm daysafler completing metamorphosis without affecting growth or survival. INTRODUCTION its high market value and apparent tolerance of low salinities. PostinetarnorphicSouthern flounder The salinity of culture water is a critical are commonlycaught in freshwalcrsounds arid parameterthat directly affects fish growth. Fish rivers Reaganand Wingo 1985!, Premctamorphic that expendenergy in osmotegulationto compensate Southern flounder have been found in salinities as for extremelyhigh or low saline conditionshave low as 17 ppt Burke et al. 1991!, and juvenile lessenergy available for growth Gran et al. 1994!. Southernflounder appear to spend most of their Defining the salinityrange neededfor optimum time in water at less than 20 ppt salinity fStokcs growth is important for achieving maximum 1977!, Flounderonly tnigratcout to the ocean to performance of cultured fish, spawnonce they reach sexual maturity. Culturing The Southern flounder {Paralichfhys Aounder in low salinity water offers several Ierhosrigma!is a euryhalineflatfish with a natural advantagesfor US mariculture;I l thefacilities for rangethat extends from North Carolinato northern flounderculture can be locatedaway from high- Mexico, There is considerable interest in the costcoastal land, 2! competitionfor limited coastal potential culture of Southernflounder becauseof spaceand waterresources is reducedthereby ttjlstt TeebnieatReport Na. 2a lesseningmulti-user conflicts, and 3! di sease twicedaily at a totalof 4%body weight, Fish causingparasites such as Atrty/oditttttrt sp. and wercweighed weekly to the nearest0.1 g then toxic algae such as Pfisreria piscicida can be harvestedafter 84 days. avoidedin water with less than 3 pptsalinity. Advancedjuvenile flounder weighing Hovyever,little informationis availableon the approximately30 g eachwere stocked into nine growthperformance of Southern flounder in low 10-Lplastic tanks containing water with 30 ppt sa]initywater. This informationwould be useful salinityat a densityof threefish per tank, Salinities forevaluating the potential for raisingflounder in werereduced to 0, 5, 10,and 20ppt over a I-week inland areas. periodby replacing seawater with fresh water. Each treatmenthad three rcplicates in separateclosed MATERIALS AND METHODS recirculatingsystems. Water temperature was maintainedat25 C withheat pumps Aquanetics Fourstudies were conducted on different modelAHP-D!, Fish were fed twice daily with a ]ife stagesof Southernf]ounder to determinethe commercialpelleted feed 5% protein;Corey Feed effectsof ]owsalinity on growth and surviva]. Mill,New Brunswick, Canada! ata dailyfeed rate Hatchery-rearedlarvae were obtained from strip-spawnedbroodstock Berlinsky et «]. 1996! of3% ofbody weight, One hour after each feeding andcultured according to methods described by uneatenpellets werecounted and removedto Danielsetal. 996! . Larvae day 25! werc stocked estimatefeed consumption, Fishwere weighed weeklyto thenearest 0,1 g andharvested after 58 into20-L glass aquaria with water containing 30 days. pptsalinity at a density of I fish/L,The salinity of thewater was gradual]y reduced over a5-day period RESULTSAND DISCUSSION withfresh water from a well 00 mg/Ltotal hardnessand350 mg/L total a]ka]ini ty!until target Tolerancetolow sa] inity increased assoon salinitylevels of 0, 10,and 20 ppt werereached. asfish comp/ eted metamorphosis, Fishexposed to Threereplicates were used per treatment, Fish wei e salinities of0 to10 ppt during metamorphosis had harvested,measured to the nearest 0,5 mrnand countedon day 60 posthatch. significantlylower survival than those in the20 and30ppt treattnents TableI!. All thefish in the Recentlymetamorphosed fl ounder day 60 posthatch!were stocked ata densityof I/L into 0 ppttreatment diedwithin 24 h ofreaching this 20-Laquaria containing waterwith 30 ppt salinity. salinity.But postrnetamorpic flounder atday 60 Thesalinity levels were abrupt]y reduced within a posthatchwere able to withstand anabrupt drop in sixhour period to0 pptby rep]acing saline water withfresh water. Aquaria were harvested after 5 daysand fish were counted todetermine survival. Sabnity ppt! Survival %! Standard Southernflounder juveniles weighing length mm! approximately5 g were caught by trawl in the Pam]icoSound and weaned onto pelleted feed over a three-weekperiod. Fish werc then stocked into nine,I 0L, plastic tanks ata densityofeight fish 10 29b l 2.5a pertank containing waterwith 30 ppt salinity. Sa]initiesweregradually lowered to0, 5, and 10 20 S9c I 1.] a pptover a two-weekperiod by rep]acing saline waterw'ith fresh water. Three repiicates werc used 30 S2c 1].2a pertreatment Tanks at eachsalinity were in a rVteansrauowed try different letterS between groupr or sepmu- 'c]used ttecircu]ating system. Temperature treateinentsare significantlydifrerent tpcO.OS!. wasmaintained at 20 C. Fish werefed a «mmercia]extruded pe]]eted feed 2% protein; Table1. Meanpercent survival and final length of southern HounderParalichrhys iethrrsbgrrur exposed to difl'cram couthinStates. Fartnville, NorthCarolina, USA! sattntuesduring metamorphosis. Daniels and Borsiti 189 in salinities as low as ppt, hencethe survival of' !naia1 Salinaylppt! Survival %! some fish at ! 0ppt is to bc expected. Lasswcll ct al, 977! reported survival rates of 0' for 10 postrnetamorphicflounder exposed to 0 pptsalinity after only a thrcc hour acclimation period, so the high survival rates observedin this study are not 20 surprising. The results of thc two growth trials ai Iow salinities showed similar trends Tables 3 and 4!. 30 80 Growth and feed conversion efficiency were not significantly different Po0,05! between any of the Table 2. Mean percentsurvival of postmctamnrphtcsouthern flounder Paralichrltys lerhostigmaexposed to 0 pptsalinity salinitiesalthough total weightgain for juvenile and after a 6-h acclimation period. advancedjuveniles was slightly higher in the 10 ppt treattnent. Survival was not affected by the long-term exposureto low salinitiesin eitherof the salinity from 30ppt to0 ppt within a six-hourperiod studies, The mortality of advanced juvenile fish in without a significant ~.05! reductionin survival the 5 ppt treatment was caused by a mechanical when compared to fish in the other treatments fai!ure that resulted in a loss of water circu!ation Table 2!. Fish in a!! treatmentsshowed few signs to someof the testcontainers, With the exception of stressafter the rapid acclimation; most fish were of the loss of this one container of fish, there were feedingand swimming actively withina few hours no mortalities duringeither of thesestudies. of reaching0 ppt salinity. Burke et al. 991! These results indicate that salinities as low as reported that prernetamorphicSouthern flounder 0 ppt are aseffecti ve ashigher salinities on growth were found in North Carolina sounds and estuaries and survival of postmetamorphic Southern Salinity ppt! Variable Initialwt g! 7.7 7.5 Finalwt g! 17,0 17,0 20,0 Gain g! 10.0 ! 0.2 12.5 S~ %! 96 Specificgrowth rate %/day! 1.2 Pxyteineflicency ratio' 2.2 2.0 Feed conversionef!ici ncy %! 67 91 Dai!yfeed consumption % 1.3 1.4 bw/day! ' pmtein efrtebm:yratint wejpe gaitldbnuy pmteit intake! " .FeedconversiMt efficietx:y weight ~ intake x 1001 Table 3. productionvariable for juvenile southern flounder Paralichrhyslerhosrigma grown at different sahnitiesdunng an g-'t-day period. 190 UJ'NRToeboteot Report No. 2O Salinity ppt! Variable 0 5 10 20 Initial wt g! 33.4 32.2 32.8 30.6 Finalwt g! 71.6 70,4 78.5 67.2 Gain g! 38. 2 38,2 45,7 36,6 S urvival %! 1.6 1.6 1.7 1 .6 Specdicgrowth rate '7o/day! 100 66 89 100 Proteinef&ietb-y ratiO' 1.3 1.2 1,5 1.3 Feedconversion e5ciency %!' 61 58 70 63 Dailyfeed consumption % bwlday! 2.0 2.2 1.9 2.0 ' Proteinetliciency ratasn weight gain/di:tary protest intake! ' -Feedconverskrn e cency weight gain/feed intake x l00! Table4,Production variablefor advancedjuvenile sotrthermllounder Paraiichrhys ierhosrigmrs grownatdifferent salinitieo duringa 58-day period. flounder. We found considerabledifferences in LITERATURE CITED growthof fltshwithin eachtreatment which may reflect genetic variability inherentto wild Arnold,C. R W. H. Bailey,T. D. Williams,A. populations.Further research is neededusing a Johnson and J. L. Lasswell. 1977. greater numberof fish per treatmentto detertnine Laboratoryspawning and larval rearing of if theslight differences in feedconsumption and red drum and Southern flounder. conversionefficiency at 0 ppt can be detected. Proceedingsof the Annual Conferenceof Comparisonof the growth/salinity relationship Southeastern Association of Fish and among wild-caught and domesticatedfish should WiMlife Agencies31:437-440. also bc explored. Taken together,these Berlinsky,D, L., W, King V, T, I. J. Smith,R. D. investigations clearly indicate that Southern HamiltonII, J. HoHoway,Jr. And C, V, flounderlarvae must be culturedin seawaterbut Sullivan. 1996. Induced ovulation of postmetamorphicflounder can be maintained in Southern flounder Parafrchthys freshwater without hindering their rate of growth lethosrigma using gonadotropin releasing or feedcan version efficiency. hormOneanalogue itnplant. Journalof the World AquacultureSociety 27 ! 143- ACKNOWLEDGMENTS 152. Burke, J. S., J. M. Miller and E. E Hoss. 199]. Thiswork wasfunded in partby UNC Sea immigratiort and settlementpattern of Grantproject no. 5-4811 L Paralichrhysderrrarus and P, lethosrigrrtp in an estuarine nursery ground, North Carolina, U.S.A. Netherlands Journal of Sea Research 27:393-405, Daniels, H, V., D. L. Berlinsky,R, G, Hodson and C. V. Sullivan,1996. Effects of stocking density,salinity, and light intensityon growth and survival of Southern flounder Paralichthys lethristigma larvae. Journal of the World Aquaculture Society 27 !: 15 3-159. Grau, E. G., N, H. Richinon, and R. J, Borski. l 994. Ostnoreccption and simple endocrine reflex of the prolactin cell of Oreochremi s mns.sambicus.Proceedings XIII Intl, Cong. Comp. Endocrinal. In "Perspectives in cornparati ve endocrinology".Eds, K. G, Davey,R. E Peter,and S. S. Tobe, NRCC, Ottawa.pp 251-256. Lasswell, J. L., G. Garzaand W. H. Bailey. 1977. Status of marine fish introductions into freshwater of Texas. Texas Parks and WildlifeDeparttnent, PWD ReportNo 3000-35. Reagan,R. E., Jr.And W. M. Wingo.1985. Species profiles: life historiesand environmental requirements of coastal fishes and invertebrates Gulf'of Mexico! - southern floundcr. U,S, Fish and Wildlife Service BiologicalRcport 82 1.30!. U.S, Army Corpsof EngineersTR EL-82-4,9, Stokes, G. M. 1977. Life history studies of Southern flounder Paralichthys lethnstigma! and gulf flounder P. albigutra! in the AransasBay area of Texas.Tx. ParksWildl, Dept, Tech Ser. 25. Zar,J. H. 1984. Biostatisticalanalysis, 2~ edition Prentice-Hall,Inc Englewood Cliffs, New Jersey,USA. Rust and ttarrows t93 AN IMAGE ANALYSIS APPROACH TO DETERMINE MICROPARTICULATK FEED ACCEPTABILITY BY LARVAL FISH Michael B, Rust National Marine Fisheries Service, NOAA Northwest Fisheries Science Center 2725 Montlake Blvd. E Seattle, WA 98122 c-mail: mike,rustCdinoaa.gov and Fredric T. Barrows U. S. Fish and Wildlife Service Fish TechnologyCenter 4050 Bridger Canyon Rd, Bozeinan, MT 59715 c-mail: rbarrowsC~motttana.campus.rnci.net ABSTRACT Methodswere developed to determinedietary preferences acceptability! by first-feedinglarval fish duringa single feeding event These methods involved: t! detertruning the feeding incidence and ! ineasuring the cross-sectional optical areaof the bolus using irnagc analysis. Both methods followed a short,defined feeding period. Both methods were usedto determinespray-dried feedstuff preferencesfor larval rebraftsh ftruchydanio rerio andmicroparticulate diet formulationpreferences for larvalgoldfish Cararsius aurasus. Feeding incidence was 100%for all diets with both species;however, diets differed significantly p<0.05!in meanholus sir~, indicating that larvae of these two speciesvary feeding rates with diet type even when diets are similar in formulation and manufacturing process. 1NTRODUCTION reared or captured from the wild. The most cornrnon live feed cultured for larval fishes is the A key impediment to intensive rearing of brine shrimp Arremia Lavens et al. 1986!. many altricial larvae of tnarine and freshwater fish Although Arremia i» adequate for some fishes is the lack of high quality microparticulate diets Lavens et al. 1986!,it is nutritionaBydeficient for that are acceptable,digestible, and which meetthe many other species Dendrinos and Thorpe 1987!, nutritional needs of the larvae. As aquaculture Culture of zooplankton for feeding fish larvae is and fishery enhancement efforts grow, thi» labor intensive, expensive, and prone to sudden impedimentwill becomemore acute, especially in "crashes." The uncertainty and expense of live the marine fish culture industry. dietsprovide motivation to developformulated diets There are a variety of feeding strategies that are nutritionally cotnplete, highly digestible, currently usedfor first-feeding larval andjuvenile palatable, inexpensive,and easyto feed. fish, These strategies include use of live diets, Mictoparticulatediets that are uniform both formulated diets, or a combination of live and in sizeand nutritional quality can ~cally i~ formulated diets, intensive systemsoften rely on the rearingsu~» of speciessuch as lake whitefish, cultured or wild caught live feeds for rearing fish Coregortntrsclttpeaformis Zitzow and Millard larvae in tanks. Available live foods for intensive 1988!,carp, Cyprinus carpio Lubzcnset aL 1984!, culture are litnited to those which can be easily smalltnouth bass,Micropferus dolomietst' Ehrlich et al, 1989!,and tnuskellunge, Essox masquinangy Brachvdanio rerio and goldfish Carassius Zitzow 19&6!.Survival in productionhatcheries of aiiratus,methods are applicableto other species larval walleye Sti ostedion vitreum fed only with transparentlarvae. The methodis illustrated microparticu]atediets averaged approximate]y 60% with two experiments: the first to determine after 30day» Bariowsand Ellis 1996!, Stripedbass, feedstuff preferencesfor larval zebrafish,and the !Hotionesaxatilis, has not been successfully reared secondto definethc optiinalkri]1 meal: fish meal onany formulated diet. In spiteof successwith some ratio for larval goldfish. speciesfed exclusive]y micropaiticu late diets, survival during the early larval periodgenerally has not been MATERIALS AND MFTHODS asgood as when larvaeare fed on live diets, Ourunderstanding of larvalfish feeding is Near first-feeding, 6-day post-hatch .5 limited. Currentlyavailable techniques that have intn! zebrafish or 9-day post-hatch 9.0 mm! beenused with larger fish to determinefeeding goldfish larvae,both producedin our laboratory, responsesare not appropriatefor usewith larvae. were selectedat randoinfrotn holdingtanks and Elucidationof the deve]opmenta]sequence of stocked five larvae/tank into clear plastic tanks feedingresponse and the development of methods containing ]00 inl of 5 pm filtered cu]ture water. to assess the nutritional needs of ]arval fish are A]1 tankswerc then placedin a water hath heldat importantto the scientificcoininunity, feed 28'C. Larvaewhich hadbeen feedingwere left companies,hatcheries, fishery managers,and withoutfood overnight to allowany residual feed aquacu]turists.The differences in successamong to pass through the gut prior to the start of each species fed solely microparticulate diets, and trial, betweenlive andmicroparticulated diets for a given Three tankswere randoinly assignedto species,may be related to differencesin diet eachdietary trcatrnent. The zebrafishtrial useda acceptability,digestibility, orcomposition. Before commercialdict FFKB-250,Kyowa Hakko Kogyo inicroparticulatediet digestibilityor nutrient Co., Ltd,Tokyo, Japan! known to producegood compositionstudies can proceed, the diet inust first growthand survivalas a positivecontrol; unfed be ingestedby a highpercentage of the larvae in larvaeserved as a negativecontrol. For the goldfish thetank. There is a needfor a methodto distinguish trial,live Artemia were used as the positive control amongdietary treatmentsthat is quick and not and unfed larvae servedas the negative control. compromisedby cannibalismand low survival rates Zebrafish test diets were spray-dried commonto larvalfeeding trials. approximately100 12average size! chickenmeat, Effectiveinicroparticulate diets need to: egg, or liver American DehydratedFoods, Inc ! efficicnt]yretain small,soluble nutrients after Springfield,MO!. The experiinentaldiets for theparticles are suspended in water, ! possess goldfishvaried in thekri1 1 mealand fish meal content physicaland chemical characteristics that result in Table I!. Kril] meal varied in 10% increments theiringestion byfish larvae; ! bcreadily digested frotn ] 4%to 54%, while herring meal variedfrom and assimilatedby larvae;and ! consistof an 6% to 46% of the diet. optimalnutrient composition for maxiinurn larval Thediets fed to thegoldfish were produced survival,development, and growth. Before nutrient usingthe micro-extrusion/marumerization ME1Vf! digestioncan occur, inicrocapsules must first be inethod Barrowset al. ]993!. Maruinerizationis ingestedby the larval fish. a pmcessof shapingand smoothingan extrudate This research describes a method to achievedby using a cylindrical machinein which differentiateainong microparticu]ate diets based the bottomof the cylinderrotates at veryhigh uponthe amount of dietingested degree of fullness! speeds.The rotationalforces within the machine by larval fishover a shorttime frame. This method result in a smoothing and densificationof the will helpto developmicroparticulate diets that surfaceof the extrudate. This processinvolves satisfy the secondaspect of an effective two piecesof equipinentfor the productionof microparticu]atedietlisted above. Although the particles. An LCI, Inc. system Charlotte,NC! method was developed wi th zebrafish included a radial discharge EXDC F!S-60! Rust ttntt 8arrom't 195 extruderand a QJ-400marumerizer. All ingredients Feeding incidcnccwas determined f'or were combined and inixed in a 1Vlarion ribbon mixer eachdietary treatmentby counting the numberof prior to addition of the fish oil, Thirty-two percent fish in eachimage with and without feed visible in water was added to the mix before extrusion the gut and expressingthe ratio as a percentage through a 500-pm screen. The mash was extruded feeding. No further analysisof feeding incidence at an augerspeed of 19 rprn to form wet noodles. data was undertaken as all larvae fed diets had Thesenoodles were then placedin the trtarurnerizer visiblefeed in theirguts after 1.5 h of feeding, which consists of a cylindrical chainber with a rotatingplate on the bottoin. The pl atewas grooved RESULTS to imp«rt energy from the rnarumerizer to the feed. This energy breaks the noodles, reshapes, and For both species, feeding incidence was densifies the particles, The marumerizer is 100%for all treatmentsreceiving diets, while feed equippedwith a variablespeed motor to allow for consuinption differed significantly amongdiets. ln a rangefrom 300 to 1210rpm. Thc noodlesof all the zebrafishtrial Fig, 1!, spray-driedegg .141 dietswere first processedat 1060 rpm for 10 sec ~ 0.016 mm-, mean+ standarderror! and chicken followedby about90 secat 500 rprn. The shaped .138 a 0.014 mm'! were consumed at. particles were then placed in an ambient significantly higher rates pc0.05! than the liver temperature about ]7'C! forced-airdryer until ,104 ~ 0.012mm-'! or positivecontrol dieLs .098 moisture levels were less than ] 0%, Moisture was ~ 0.007 trun'!. Significantdifference p<0.028! determinedusing a 30-minutecycle of 125'C on were found between all groupsand the negative an Oh«usMB 200 moistureanalyzer, The feed control group .002 ~ 0.007 mm'!, was then ~ifted to the proper sizes and stored in In the goldfish trial Fig, 2!, acceptability nitrogen-f]ushed,vacuum-packed plastic bags and of live A rremia ,81 a 0.01 mm-! was significantly storedat roomtemperature until used. greater pc0.05! than all other diets. The diets Each tank was then fed 0,1 g of the containing46% fish meal ,59*0,05 mm'! were appropriate feed. After 1.5 h, the larvae were significantly more acceptablethan diets contaiiung anesthetized with MS-222 Massee et al. 1995! ]essthan 26% fish meal. The dietcontaining 36% and videotaped using a dissecting microscope with fish meal .49 ~ 0,04 mm'! wasnot significantly a video recording systetn. Larvae were oriented different than any other fish meal-containing diet. ontheir sides so that the bolus wasvisible through the transparentlarvae as a cross-section, A stage DISCUSSION micrometerwas positioned so that a readablesection of the micrometerwas visible in eachimage. Zebrafish larvae consumed al] three Each image on the tape was then printed spray-driedproducts at levels equalto or greater and the cross-sectional area of the bolus, and thanthe commercialdiet, indicating potetuial for pre-flexion standard! length was measured. spray-dried products as feedstuffs for larva] fish Cross-sectionalarea of the bolusprovides an index diets. Furthermals usingother species and other of the ainount of feed ingestedby each larva. feedstuffs are needed to determine if these materia]s Cross-sectionaloptical areas were determined are widely acceptable,Ry testingthe samediets using a planirneter The appropriate conversion with otherspecies, it will bepossible to determine factor for each measureinentwas determined by if zebrafish are a suitable surrogatefor other measuringthe image of a I-mm- 'area on the hard-to-obtainspecies. Once highly palatable micrometercoverslip which was in view in each feedstuffs are identified, then ii wi]1 be easier to of the printedimages. Data was reportedas the formulatediets that are high]yacceptable. cross-sectionaloptical area of materialin the gut Goldfishlarvae preferred diets high in fish mm-'!. Statisticalsignificance was determined mealand low in kril] meal, Theseresults appear to using analysisof varianceand meansseparated be in contrast to surviva] data obtained using the using Fisher's Protected Least Difference same diet formulations with larval walleye Significant PLSD! method Zar 1984!, Harrows ]994!. Larval walleye fed diets Rust uad Barraws 197 containing krill meal levels as low as 24% had programsand computer systemscan alii>be used. survival ratesequivalent to fish fed dietscontaining An additional benefit of using a 54% kril I, Reducing the krill contentof thc diet to computer-aidedmeasurement system is that the 14%, with 46% herring resulted in a decreasein cross-sectional area can be rotated in space to survival. Survival wasgreater for the fishfed the producean estimate of volume. Fora moreaccurate four high krill mealdiets than fish fed a commercial estimate of volume, a standard curve can be larval dict, Barrows 994! suggesteda beneficial producedby intubatinga knownvolume of colored effect of including at least 24% krill in larval liquid into the larval gut Rust et al,1993a,h! and walleye diets. It could not be determined in that determining the cross-sectionalarea of thc liquid 30-day feeding study if the effect was nutritionaJ dropletcontained within thegut. The resulting or dueto theacceptability of thediet. Combining regressionwill describethe relationship between fling incidenceand bolus measurement data with cross-sectionalarea and bolus volume for a given the survival data would have been beneficial and species at a given developmental stage. This may havebeen able to pinpoint the reasonfor the informationmay bc useful for determining differentia! survival. consumptionand developing bioenergetic models. Differences among dietary treatments No quantitative requirement for any could not bedetermined with feedingincidence data nutrienthas yet been determined for the larval stage for eitherspecies all exceptthe unfedtreatments of any speciesof fish. Diets areformulated based werc 100%!. While the feeding incidencemethod upon the composition of the fish larvae or the provides a coarseevaluation of diet acceptability, compositionof zooplankton, Unfortunately, this itdoes not work when feeding incidence isuniformly approachassumes that thc bioavailability of dietary high, such as was the case with zebrafish and nutrients are equal, an assumptionthat docs not goldfish, Measurementof the bolusprovided an hold for altricial larvae Rust et al. 1993c, Rust index which was more sensitive to smaller 1995!, Quantitative and qualitative nutrient differencesin dietacceptability, Conversely, under requirementsfor larval fish will be difficult to conditionswhere feeding incidence is low or determineuntil a microparticulatetest diet that is variable,measurement of thebolus maynot provide high!y acceptableto Jarvaifish is developed.The meaningful data. This is becausesamples consist firststep toNard determining requirements may be only of feeding larvae and are not a good to develop such a test diet using the methods representationof all thelarvae being fed. outlined here. Both methods are useful to determine diet Vision and chemoreceptionare the two acceptabilityover a veryshort time. Studies that most important sensory systems used by determine differences over a short time are not first-feeding larvae to Jocate and ingest food compromisedby high mortalityrates common to Blaxter 1988,Noakes and Godin 1988!. In order larval feeding trials. Sincediet compositionand for rnicroparticulatediets to be ingested,they must nutrient bioavailabilityare clirninated as potential be attractive and visible to the larvae and must be causesfor mortality or poorgrowth, acceptability presented under the proper environmental trials usingfeeding incidenceand/or bolus sizeas conditions.Fish larvae are primarily visual feeders, indicescan yield tneaningfuldata with speciesthat though tastebuds and olfaction are often also cannotcurrently be culturedintensively. functionalat this time in mostspecies Noakes and The methodfor determiningboJus size Godin 1988!. The microparticulatc dict lendsitself to computer-aidedimage analysis. We acceptabilitymethods developed provide meansto have successfully measured the bolus determine optimal environmental {light! and cross-sectionalarea in larvaeusing a Macintosh chcmicaJ taste, olfaction! propertiesfor successful 8100AV Apple Computers, Cupertino, CA! larval feeding. computerwith NIH-image software, NIH-image Once optimal feeding conditions are is a free software package developed by the definedand a highly acceptablenucroparticulate National Institutes of Health and is available a their test diet is availabJefor the larvae of a species, website http://rsb.info.nih.gov/nih-imagd!. Other then work can proceed more quickly nn lstt U JAR 'rerhaical Repun Na. 26 deterininationof requirements. It isnecessary to Artejnianauplii. J. WorldAquacult. Soc. first I! ensurethat the feeds wc are developing 20: 1-6, arebeing eaten by the larvae, and ! understand Lavens,P P. Sorgeloos,and A. Flores Tom. thedigestibility of those diets, before drawing 1986.Producion debiomasa, quistes y conclusionsasto larval nutrient requirements, naupliosde Arteniia en sistemas a flojo constanteen «1tadcnsidad, pp, 229-247, En: Mernoriasdel primer Cursosobre ACKNOWLEDGMENTS Arremia y su uso en camaronicultura. Institutotecho! 6gico del Mar,Guaymas, Theauthors thank Mark Sleeper for his Son, Mexico, timeand dedication spent on image analysis, We Lubzens, E., G. Sagie, G. Minkoff, E. Meragelman,and A. Schneller. 1984. alsothank Dr. Ronald Hardy for arranging for the Rotifers{Brachionus p/icari lis! improve spray-driedfcedstuffs and the opportunityto growth rate of carp Cyprinus carpio! conductthese studies in hislaboratory, Finally, larvae.Bamidgeh 36: 41-46. we thank Dr, ConradMahnken, Dr. Robert Massee,K. C.,M. B. Rust,R. W. Hardy,and R. Iwamoto,and Kate Guthrie for critical reviews of R. Stickney.1995. The effectivenessof tricaine, quinaldine sulfate and themanuscript. Useof trade names orpnxfucts in metomidate as anesthetics for larval fish. thisstudy does not imply endorsement by the Aquacu1ture 134: 351-359. NationalMarine Fisheries Service orthe U. S.Fish Noakes,D.L. and J-G, J. Godin. 1988. Ontogeny and Wildlife Service. ofbehavior and concurrent developmental changesin sensorysystems in teleost fishes,pp. 345-384. In: W. S, Hoar and LITERATURE CtTEO D.J. Randal 1 eds.!,Fish Physiology, Vol I, B, AcademicPress, San Diego. Barrows,F.T. 1994.Walleye research summary; Rust,M. B., 1995. Quantitative aspects ofnutrient 1993season. Annual Progress Report: assimilationin six speciesof fish larvae. Fisheriesand Federal Aid Project, Ph,D. dissertation,Univ. Washington, USFWS, Denver,CO. Seattle.147 p. Barrows,F, T. andW, A. Ellis. 1996. Dietand Rust,M, B., R, W. Hardy,and R, R. Stickney. nutrition, pp. 315-321. In: R. C, ]993a. How to force-feedfish larvae Summerfelt cd.!, WalleyeCulture video!.Washington Sea Grant Publ, No Manual. NCRAC Culture Series 101. WSG-AV-93-01, North CentralRegional Aquaculture Rust,M, B., R. W. Hardy,and R. R. Stickney. CenterPublications ONce, Iowa State 1993b.A newmethod for force-feeding Univ Ames. larvalfish. Aquaculture116: 341-352. Barrows,FT., R.E.Zitzow, and G.A. Kindschi. Rust,M. B R. W. Hardy,and R, R. Stickney 1993.Effects of surface water spray, diet 1993c.Assinulation of threebiochemical and phasefeeding on swim bladder formsof radio-labeledmethionine in four inflation,survival and cost of production speciesoffish larvae, p. 261. Abstract!. of intensivelyreared larval walleyes. ln: M, Carrillo,L. Dahle,J, Morales,P Prog.Fish-Cult. 55: 224-228. Sorgeloos,N. Svennevig, and J. Wyban Blaxter,J. H. S. 1988.Pattern and variety in coinpilers!, From Discovery to development,pp. 1-84. In: W. S, Hoar Commercialization, European andD. J.Randall eds.!, Fish Physiology, AquacultureSociety Publ. No. 19. Vol XI, A AcademicPress, San Diego, Zar, J. H. 1984. BiostatisticalAnalysis. Dendrinos,P. and J. P, Thorpe. 1987. Prentice-HallInternational, London, UK. Experimentsonthe artificial regulation 718 p. of theamino acid and fatty acidcontent~ Zitzow,R, E. 1986,Survival and growth of larval of foodorganisms to meetthe assessed Muskellunge Esox Itfasquinongy ! nutritional requirements of larval, initiallyfed formulated diets. Information post-larval,and juvenile Dover sole Leafl.No. 86-106, US FishWildl. Serv., So!casoIea!, Aquaculture 61: 121-154. ValleyCity, ND. Ehrlich,K. F.,M. C. Cantin,M. B. Rust,and B. Zitzow,R. E. andJ. L. Miliard.1988, Survival Grant. 1989. Growth and survivalof andgrowth of lake whitefish Coregonus larvaland postlarval smallmouth bass fed clupeafnrmis!larvae fed only formulated a cominercially prepared dry feed and/or dry diets. Aquaculture69: 105-113, snift ei al, 199 FISH CAGE PHYSICAL MODELING FOR SOFTWARE DEVELOPMENT AND DESlGN APPLICATIONS M. Robinson Swift M ichaelPalczynski Kenneth Kestler Derek Miche in Rarbaros Celikkol University of New Hampshire Durham, NH 03824 c-maih rnrswiftOchrista.unh.edu and Michael Gosz Illinois Institute of Technology Chicago,IL 606 1 6 ABSTRACT Fish cageresponse to waveswas investigated using a physicalmodel in the Umversityof Vew Hampshire wave tank. The tank was built with below-waterline windows placed for convenientobservation of moored cage models. Cage motion was measuredusing an opncal systemcomprised of a highresolution video camera,a frame grabber,and a computerwith expandedrandom accessmetnory RAlVl!, Targetson the cage, consisting of two black painted dots on a white background, were tracked usmg image processing software. The system was, therefore, noninvasive to the fluid environment and did not aher the cage inertial characteristics. Specific experiments werc donein support of a coinputermodeling effort which has resultedin a finite elementprogram for fish cage dynamics. Experimental data was obtained using a physicalmodel reduced in complexity in order to focus on basic parameters. Comparison of rank data with coinputcr predictions indicated that the computer simulation reproducedthe fundamental featuresof the observedcage motion. IN TROD U CT ION the wavernakingsystem was added in 1996 as describedby Washburn 996!. Thenext. step was Physicaland computer models are essential to incorporatea measurementsystem for physical tools for the design of offshorenet pen systems. modelmotion response, In the studydescribed herc, To avoidfailure, net pensand their inooringsmust this needwas realized using an optical systein.This bc engineeredto withstandboth severe storm events strategywas chosen because it offeredprecision and the cutnuiativeeffects of long-termwave and measurements without altering the fish cage currentloading. We developedmethods for testing dynarnlca. physicalmodels in the new University of New The physical modeling approach Hampshire UXH! wavetank. The experimental complementedthe UlieH finite elementcomputer methodologywas then usedto generatedata for programmingeffort which resultedin a net pen comparisonwith recentlydeveloped finite element dynamics program. As demonstrated by Gosz et computermodels of fish cageresponse to waves al. 996!, the programcan be used to predictcage and current, movementand structural loads for user-specified The new UNH wave tank wasdesigned wave and current environments To increase andbuilt with offshoreaquaculture applications in confidencein its predictions,however, it was mind. The tank itself and the buildinghousing this determinedthat an experimentalprogram should and other facHitieswere constructedin l 994, while be set up to generatespecialized, empirical cage motion datafor comparisonwith thefinite element asa functionof time for key pointson thecage, fnode]predictions, Theobjectives of this work may. therefore,bc summarized as: L'NH WAVE TANK ~ Deve]opment of testtank methodsfor fish cage experimentsin the UNH Thewave tank is 36.6 m long,3,66 tn wide, wave tank; and3.05 m deep.l tis usual!y filled with 2.44tn of e lrnplernentationof an opticalsystem water. A tow carriageis supportedand cable- for cagemotion measurement; drivenalong a singlemain rail onone side g!amel], ~ Obtainingspecialized data in support l 996kA lightweight,protected outrigger supports of nctpen coinputer modeling; thecarriage on the opposite s idewh ich is reserved ~ Comparisonwith predictionsfrotn thc for observers.A hydraulicallydriven, computer exi~tingUNH finiteelement program. controlled,f]ap-type wavetnaker is at one end Theseobjectives were addressed making Washburn, ]996!. Software allows thc user to Use of uniquefeatures of the tank. The optical run a regular wave ot' specified height and measurementsystem was positioned opposite bui]t- frequencyora randomsea of specifiedspectrum. in observationwindows located halfway along the Wavesare dissipated atthe opposite end using a lengthof the tank. Fishcage physical tnodels werc vertical"beach' consisting of vertical layers of thenconveniently tnoored for clear viewing. For geotechnica]cloth suspended from an angled the softwaredeveloptnent application, the cage fiberglassframe, physicalmodel was simp]ifted tofocus on inajor Midwaydown the observer side, a pit componentsand basicdynamic processes. The a]lows accessto two sidewindows in the wall- cotrtputer progratnwas applied directly to the onecovering the waterlirie and upper water co]umn physicalcage tnodel at its actualsize, with no and the secondplaced just abovethe f]oorof the potentia]lyerror-producing changes in scale.The tank, A mid-widthfloor window can also be used eva]nationwas done by comparing displacement from a tunnelbeneath the tank which is entered Test Object Computer Figaret, Schematicof opticalmeasttremcnt systt.'m. Swift et aL 201 ! Cameracaptures image. Image is transferred to computer and stored. Positioninformation generated. Softwareprocesses images. Figure 2. Informationflow in the ofnicaI measurementsys em. from the baseof thc ptt. Thesewindows are very a white backgroundare paintedon thc test object, convenient for viewing the net pen system Horizontal,vertical, and angular changes planar experimentsand allow optical monitoring of motion motion! can be inferred from the movement of the variables. spots. The UNH system has been successfulin resolvingthe gray-scalecontrast between black dot Optical measurementsystem andlight background,so the useof potentiallyerror- An optical rneasurernentsystem for producing light sources on the test object is determiningtest objectmotions was developed to unnecessary.The cameracaptures a sequenceof take advantage of the observation window images and transfers each fraroe, via the frame opportunities. By using a noninvasive optical grabber, to the cotnputer for temporary storage. system,no dynamicaltering sensors are attached Later, speciallywritten software is usedto search to the test object. Figure 1 shows how images of eachframe for the gray-scaledifferenc indicating the test object are capturedby a cameraand fed the presenceof the targetdots, Dot position as a into a computer for analysis. The essential function of frame number converted to time! is componentsof the present UNH systcrn see then used to calculate test object linear and angular Michelin and Stott, 1996! include: displacernent components as a function of time. A high resolution,black and white Pulnix videocamera which canoperate Model testing at 30 frames/second; While the optical positionmeasurcmcnt ~ A framegrabber to transf'er the images systemis adaptableto any fish cage model, the to a computer; presentstudy made use of a specialmodel to obtain ~ A personalcomputer with expand& data for comparison with finite element computer random accessmemory RAM!, predictions.This skeleton model consisted only of ~ Software to analyze the stored a rectangularparallel-piped structuralframe, a sequenceof images, bridle,and a singlemooring linc seeFig, 3!. Thus, The stepsinvolved in motion rneasurernent the comparison between empirical data and begin, asindicated in Figure2, with establishinga computer predictions represented a focused target on the testobject, Two small black dotson evaluation of basic fluid mechanic processes 202 t'JTAIRTechnical Report 1Vo.2t> STILLvAT ER LEVEL BABA V*VETA»K Vi»BPV 099Am O ASS BLACKDDI, e ss RIA, i CE>>TEAE»m BcvEL BDT>>SIRES> I >RD699 IT>> >EiT B~ BI* >RSITRB>CLINE Figure3. Ske!etoitfish Cage physical mt>de1. &gare4 Expertmeitta!setup Oppoute upper Wittdow invulvingthe main net peri COinpoitents. Upon sinusoidalwaves. Frequencies used ranged from satisfactory validation, further complexities inthe 0.5to 1.2Hertz, and wave slopes were on the order formof netting and small appendages will be added of I/15, Afterthe leading edge of thewave train in future work, passedthe model and thc model appeared to be Beforewave tank testing, preliminary oscillatingwith the waves, position measurements experimentsonthe physical model were carried out wercrecorded over three wave cycles. The optical inthe UNH recirculating flurne,This 12.19 rnlong, systemsoftware was thenused to calculate time 1.22m wide, and 1.22 m high facility provided a seriesof horizontal and vertical position of the two steadycurrent environment enabling thestatic drag targetpoints shown in Figure4. Usingrelative characteristicsofthe tnodel to be measured. After heightdifference and the distance between target applyingthe finite element model to this case and points,time series for the cage pitch anglewerc obtainingasatisfactory comparison, thephysical also calculated. modelwas deployed fordynamic testing inthe wave tank. Thefinite element computer program was runfor identical conditions. The finite element cage Themodel was moored, using the setup model, shownin Figure 5, used the exact showninFigure 4,so that the f'u1ly submerged cage dimensionsand weights as the skeleton physical wasdirectly infront of the upper sidewall window. model. Theexcitation consisted of the samecases Thehigh resolution camera was positioned toview of regularwave forcing. lt shouldbe notedthat thecage through thewindow over its full range of motion. thecomputer program input corresponded directly to the actualmodel dimensions. Thus, therewas Themodel was allowed tocome to vertical no needfor eitherFroude or ReynoMsnumber equilibriumandwas then excited byregular, scale-up of results. Swift et aL 203 RF.SULTS The wave tank,physical modelresponse to regular wavesis summarizedin Table l. For eachseparate test, wave period T, waveheight H, and average amplitudes for pitch angle and horizontal X! and vertical Y! displacementof thc two targetpoints seeFig, 4! areprovided. Average amplitude is one-half the peakto trough difference averaged over the three waves measured. Representativetime seriesof horizontal and vertical displacementof the target poinb are plotted in Figures 6 and 7, Thc regular wave responseis gcncr@lysinusoidal with the horizontal motionof thetarget pointsnearly equal and greater than the vertical motion, A drift can be seen which is due to a persistenttransient initiated when the regular wave train encounteredthe upright cage/ mooringsystem. Closeexamination of the time series revealsthat thc angular motion is opposite to thatof an inverted pendulum, At the extremes of the horizontal displacement,the sideof the cage Figure 5 Fmite eletnentcage model. T = wave period; H = wave he'ght; angle - pitch angle with respect to the horizontal; X., X2 and Yl, Yz are horizontal and vertical displacement cotnponents of the two target points 3, 2 showrt in Figttre 4. Table L Measured cageresponse to regularwaves. t;JNR TechnicalReport Vo. 26 6 S.00 L cs O 1st 10.00 CO lO 2,0 3.0 60 6.0 60 Time seconds! Figure* Measttredcageresponse toa regu/arwave. Pertod = 1.67 seconds audwave height = 0.28neuter. 2,0 1,0 cs 8 0.0 CL Cl -2.0 -3,0 0.0 0.5 1D 1.5 2.0 6.0 Time seconds! easuredcage response to a regularwave. Period = 1.25seconds and wave height = 0.16tneters. gwifit ct al, X5 12 n 05 1 1.5 2 2.5 3 35 4 4S 5 mme seconds! Figure L Finite clement model predications of cage response. Period = 1.67 secondsand wave height = 0.2R meters. 2.5 1.5 E s3 Oh C I o I Q Ctt CL ELS CO Cl D nk 1 1.5 2 25 3 3S 4 45 5 Tame secands! Figure 9. Finite element inode1predictions of cage response Period = 1.25 secondsand wave height = 0,16 meters. ttJNR 'fggbatcatRePort Wo.26 towardsthc mooring dips down. This is apparently Universityof NewHampshire. Critical reviews dueto waveaction on the mooring/bridle system providedby Professors Igor Tsukrov and Kenneth havingnegligible inertia! kicking out thecage Baldwinwere very helpful and are appreciated. bottom, Finite element inodel predictions LITERATURE CITED correspondingtothe Figures 6 and7 experiment areshown in Figures8 and 9, respectively.The Darnetl,L, 1996.A towingcarriage for the sametype of horizontaland vertical sinusoidal Universityof NewHampshire towing and motion is seenand transient behavior is evident. wavemakingbasin. M.S. thesis,Univ, New Thcsame bottom kick- out type angular inotion is Hampshire,Durham, NH. 178p. also evident, though vertical motion is inure Gosz,M., K, Kestler,R. Swift,and B. Celikkol. symmetric in the computer model output. Overall, 1996. Finiie-elementinodeling of thefinite element inodel is seen to replicate the basic aquaculturenet-pens, pp. 523-541. In: processesand motion responserecorded in the OpenOcean Aquaculture, Proceedings of empirical data. Direct comparisonof the an InternationalConference, Portland, quantitativeresults, however, shows that the finite Maine.New Hampshire/Maine SeaGrant e/ementmodel somewhat underpredicts themotion CollegeProgram Report No. UNHMP-CP- amplitudes. SG-96-9. It shouldbe noted that coefficients in the Michelin,D,and S. Stott. 1996. Optical positioning Morisonequation fluid forcing inodelwere intrumcntationand evaluation.Ocean calculatedusing accepted theory sec Gosz et al ProjectsCourse final report,Univ Ncw 1996!and were not tuned for this physical model Hampshire,Durham, NH. 85p. application.Symmetry seen in the predicttxI vertical Washburn,S. 1996. A wavegenerator and motionbut not as evident in thephysical model responsemay be due to using a linear wave theory waveabsorber system for theUniversity of New Hampshirewave/tow tank. M.S. inthe program. Thewave loading ando ther coding thesis,Univ. New Hampshire, Durham, issuesare currently under review inthe ongoing NH. 224p. modelimprovement effort. CONCLUSIONS TheUNH wave tank is ideally configured fortesting offshore fish cage physical models. Convenientlyplaced observation windows allow precise,noninvasive measurement ofcage motion usinga passiveoptical technique, Theskeleton model approach reduces complexityallowing evahuation of how well computerprograms simulate basic processes governingfishcage dynamics inwaves. The Gosz etal, l 996!finite element program wasfound to replicatethefundamental characteristics ofthe physicalmodel motion, butwork isongoing to obtainmore exact numerical agreement. ACKNOWLEDGE@ NTS Theauthors aregrateful forthe support providedbythe National Sea Grant ONce to the Taka st 207 CREATON OF OFFSHORE AQUACULTURE GROUND BY FLOATING BREAKWATER N, Takagi National ResearchInstitute of FisheriesEngineering Ebidai, Hasaki, Kasitna, Ibaraki 314-0421, JAPAN e-mail:[email protected],jp ABSTRACT In Japan,cage culture hasexpandedmainlyincalmwa erazeassuch as the Seto inland Sea The presentculture condition can he described asoverly intensive, and this has causedthe detenorationof water quality. hindering the expansion of fish culture. ln order to expand lish culture, n is necessary to enhanceotfshore culture. An imponaot task is to develop a nursery system which can withstand the rough wave conditions of the ocean around Japan.Here, we introduce some offshore typesof floadng breakwaternow in useand a floating hreakwater equippedwith aquacuhurenet cageswhich is underdevelopment. in orderto realizeoffshore culture INTRODUCTION seawater exchange, mixing, and diffusion, so The coastal fisheries productionis on a water quality is stable level. Coastal fisheries resources have been tnaintained; enlargedby thecoastal fishing grounddevelopment, ! In the deep sea >20m!, such as construction of artificial reefs and fishery the floatingbreakwater is nurserygrounds for propagation.Only aquaculture tnore economical than the has shown an upward tendency in area and gravity type; pmductionquantity, {4! The floating breakwater Aquaculture was developed in the inland isconvenient for planning seasand calm baysbecause the wave conditions and tnaintenance. in the open sea were severe, thus detrimental to Reliability for safety of the floating aquaculturefacilities. The floating breakwaterwas breakwater has been established with actual results. developedin order to enlarge the aquaculture In addition, aquaculture grounds have expanded grounds,and at many locations in Japanhas been fromthe bay andinland seaareas to the opensea constructed to create more suitable grounds for because of environtnental change and aquaculture. overcrowding.However, it is clear that thenortnal It is necessary to maintain calm seas at typeof floating breakwatercosts too much in order theaquaculture grounds for safetyand workability, to achievethe requiredperfortnance, and in the andfor an optimalenvironment for breeding fish. case of aquaculturegrounds in the open sea, is Therefore,the floatingbreakwater has been usmi very difficultto construct.A newtype of floating to createaquaculture grounds because it has the breakwaterwhich canabsorb big and long waves followingcharacteristics: effectively is warranted,Moreover, its the caseof I! Transmitted waves can aquaculturegrounds in the opensea far from the becontrolled by the scale fishing port or fishing village, the floating and wave absorption breakwater must have additional functions, such principle of the floating as thecultivation of bmodstock andnursery culture. breakwater; I wouldlike to introducetwo examplesof ! The floating breakwater the floaung breakwaterconstructed in the open does not obstruct the sea,and to describethe direction of its research in U3hiRTechnical Rcport Yo. 26 Figure1. Projectsite and layout platt. the future. 12l Wave condition Designwave Designwave for Floatingbreakwater constructed in Takahama function structuralstabili v District,Fukui Prefecture srgruficantwave height 1.3 m 1.4 m 3.9 rn Thisfloating breakwater was planned as significantwave period 7.5 sec 6.0 see 11 7 scc Wavelength 87.tt m 56.2 rn 171,4 rn partof thecreation ofncw aquaculture grounds in Wavedirection 'sfNW 7C N TakahamaDistrict, Fukui Prefecture, located in the Transmissioncoeffiment 0.6 0.5 rtudd!epart of Japan along the Sea of Japan, This wasthe first oneconstructed along the Sea of Theprinciple of floatingbreakwater Japan,which protects theaquaculture ground I 3 Thefloating breakwater shown inFigure ha!from big waves, Figures 1 and2 showits 3 absorbswaves by imeractionbetween thc air layoutand the aquaculture ground, and Figure 3 flowand the internalwater movement, It has showsits structure. chamberson both sides and air ducts connected to eachchamber, Itsscale isas foliows: l unit length DesignCondition 68.0m; width 14.5 m; height 8.7 m; total length Ya ural rondinon 228 rn units!. Averagewater depth :28m Tidal range . I m Tidal current . 0.5 rrv'scc Characteristicsof floating breakwater Wind ve!ocny 28.0 rttrsec Wavefunction design requires a relatively sediment . Silt nosedwith fine sand longperiod, so this floating breakwateris categorizedasthe open sca type, It ismoored by Ta k agi 209 Figure 2, An airplane view of the floating breakwater. six cross lines connected with anchors because Air ducts wave direction is not normal to it. Moreover, a construction craft cannot be used because there is not one large enough in this region, and to move it from anotherplace to the project site would be very expensive. The anchor is divided into three parts. Figurc 4 showsthe three.-partanchor. This anchor was used for the first time, and is applied to the open seabecause of its size. In this case, its applicability to the oblique waves and problems in constructing in the open seawere made clear. The floating breakwater has performed very well 3 yr after installation, though high waves are frequent in the winter. Fishing ground constructed in Aba District, IVagasaki Prefecture Figure 3, Structure of the floating bteakwa er. This fishing ground was planned as part of the creation for a multipurpose calm area, which enhancesdeveloping coastal fishing grounds. The 210 UJNR Technical Report Ve. 26 ~ I Figure 4. Parts of the new type of attchor plan included the gravity-type breakwater and thc ! Wave condition new floating type that applies the results of the Design wave Design wave for function structural stability Takahama case described earlier. The project site 'g! is located in the northern part of Kyushu Island Significant wave height 2.0 m 3.9 m along the east China Sea, Figure 5 shows the Significant wave period 6.7 sec l 3,3 sec Wave length 70.0 m 276.0 m project site and the layout of facilities, and Figure Wave direction SSW SSW 6 shows thc new type of floating breakwater. Transmission coefficient 0.5 Design Condition The prin ciple of fl oating breakw ater l l Natural condition Water depth : 17-28 m Fi gure 5 shows the cross section of the new type Tidal range 38m of floatin g breakw ater. The sha peof itsabsorbing ';::,.''~>jji Tidal current : 0.761 m/sec chambers metrl cal bec au se threeabsorption '';::,'!~~ Wind velocity : 40.0 m/sec is asym Sediment . clay principles are consi dere dto wo rk effectively:first is:,"kg~>! Takagi 211 Gf8Ylbp t,pp8 bK88k%'RIM' PM Figure 5. Project site and layout plan. 212 1;3VR Technical Report Vo, 26 terr!p~~ B"! Figure 6, The floating breakwatn absorption by scattering waves due to the motion Characteristics of floating breakwater of the floating body; second is by interaction The bottom at the project site is very soft between the air motion and the internal water and large waves are often generated, so it is surface motion in the chambers; and third is by difficult to moor the floating breakwater. This dissipation due to the water currents in the floating breakwater was very unique, which chambers. The floating body could be madesmaller worked effectively in thc open sca with its mooring than the normal type by changing the length of the system designed for ground stability, Its internal current in the chambers to achieve thc construction began in 1993 and was completed in required performance. Because its performance 1995. It now works effectively although typhoons was also estimated by numerical simulation and often attack this area. Its performance was also the hydraulic model test, this floating breakwater checked by the field survey. was applied to the Aba aquaculture ground for the first time in Japan. Its scale is as follows: 1 unit Development of new type of floating length 57.0 m; width 11.0 m; height 8.3 rn; total breakwater length 200 m units!. Up to now, various types of floating breakwater have been proposed and applied in the creation of aquaculture grounds. Moreover, a new Tatragi Z13 Cross section of floating breakwater l,o 6 o OS & 0.00 2 6 8 l0 The ratioof wave Length to the widtlr of flaatingbreakwater Figure 7. Cross section of the floating breakwater for the open sea and its performance. type,one applicable to theopen sea, was developed Orifice andconstructed. Recently, for utilizing thefloating breakwater in rnultiples, the one with culture cages hasbeen proposed. By constructingthis new type ent wave which has culture cages and the facilities for management, offshore aquaculture will becoine safer and more efficient. Current state of development Figure 8 shows the cross section of the multipurpose breakwater. The front part absorbs waves by controlling air and water currents through oriflices attached to the ceiling of each chamber. Figure L Cross section of the floating breakwater attached The back part is a culture cage. The main problem with culture cages. is reducingthe wave motion which damagesfish in thecage. The width andheight of the absorption part must be considered for the safety of aquaculture and stability of the cage. Its UJNtt Teehntealrteport No. 26 sorbingsection Ftttttre9. A newtype of tnn/tipnrposebreakwater. performanceandthe water environment inthe cage Takagi,N., Y.Ohtnur,M, Ozaki,Y. Isozaki,A. basedon theimage shown in Figure9 arenow Arami,A. Kadono,and A. Nagano.1995. beingstudied. Experirrtentalstudy on applicationof new typefloating breakwater for open sea area. EPILOGUE AND ACKNOWLEDGMENTS Bull. Natl.Res. Inst. Fish. Eng. No,16, pp. 29-57. These studieswill serve as the basic Kagiyama,H. 1995. Development of ne w floating technologyfor creatingoffshore aquaculture breakwater.Sci. Tech, J. Jpn. Int. Mar. Sci groundsand the offshore fisheries base. So we Technol.Fed. 8 !: 31-40. continuetostudy steadily. Finally, I express special Ohkusu,M., M. Kashiwagi, K. Ikegarni, M. Ozaki, thanksto thepeople who offered important andY. Isozaki. 1991. Study on perfonnance photographs«nddata. In addition, development of offloating breakwater utilizing the relative thenew type of floatingbreakwater was carried motionof insidewater. J, Soc.Nav. Archit. outin cooperationwith the National Research Jpn.169: 215-222, InstituteofFisheries Engineering, Mihubishi Hea ndustryCo.,Ltd.. Ishikawajima-Harirna Heavy IndustryCo., Ltd., and Hitachi Engineering ShipbuildingCo., Ltd. LITERATURE CITED Takagi,N.and S, Akeda. 1992,. Hydraul icmodel teston formation of pisciculture ground at Takahama,Fukui Pref. Tech. Rep, Natl. Res.Inst, Fish. Eng., Aquacult Fish, Port Eng.No.14, pp. 37-75, Dusnryet ai. ius AWATS: A NET-PEN AQUACULTURE WASTE TRANSPORT SHVIULA- TOR FOR MANAGEMENT PURPOSES RobertW. Dudley University of Maine Departmentof Civil Engineering Orono, ME 04469 e-mail:rwdudley!stvoyager,umeres.maine.edu Vijay G. Panchang National Sea Grant Office, NOAA Silver Spring, MD 20910 and Carter R. Newel! The Great Eastern Mussel Farms, inc, P,O, Box 141 Tenants Harbor, ME 04860 ABSTRACT An efficientmathematica! modeling package called Aquacu! ture WasteTransport Siinu!ator A WATS! providesfirst-order estimates of thephysical dispersion of finfish aquacuhurewastes forregulatory purpose~. Themode!ing strategy entails the utilization of a verttca!!yaveraged, iwo-dimensional !!ow model to produce t!ow-fie! d information;this information is input to a particletracking wastetransport model to simulatethe resultingtransport of wastes.Since earlier studies have shown that the transportmodeling resuhs are sensi- tive to the thresho!dshear stress at which setoedfish-pcn wastesare resuspended,fieldwork was conducted to improveihe parameterizationof erodibi!ity in the transportmodel. Applicationof AWATSto severalaquac- ulturesites in coasta!Maine se!ectedby the !vtaineDepartment of Environsnenta!Protection! shows thai it is a convenient tool in the regulatory process. INTRODUCTION hydrodynamicenvironment. A considerable effort is put forth by Due to high stockingdensities and feed regulatorsto tnonitorhydrodynatnic, water rates,net-pen aquaculture operations are regarded quality,and benthic conditions and to evaluate as potentialpolluters of the tnarineenvironment. environmentalimpacts of net-penaquaculture Net-penwastes, consisting primarily of fish feed operations.The efficiencyof thiswork may be andfecal pellets, can adversely impact the coastal significatitly enhancedthrough the usc of environmentthrough increased concentrations of mathematicalmodels that give more complete ammonia,decreased dissolved oxygen, and the infortnationregarding the physical conditions in formationof bacterial mats at particularly the domain. For example,Panchang et al. 997! prob!einaticsites. While rates of' deposition and have shownthat the useof blanketguidelines for accumulationof thesewastes in the vicinityof minimum currentspeed and water depth do not net-penopera.tions depends on many factors automaticallyensure favorable hydrodynamic includingstocking density, feeding rates and the conditionsfor a net-perioperation. The flow-fields amountof excessfeed waste, settling rates of seenin coastalMaine arecomplex and it is often wastetnaterial, fish metabolism,grazing, bacterial difficultto discernprevailing current direction and decomposition,etc.!, the degree of environmental overall flow-fields froin discrete, site-specific deterioration depends ultitnately on the measurementsover liinited time periods. Such datafail to ascertainthe spatialand tempaial notoffer tools readily available to regulators. variationsof thehydrodynainic environment such Wedescribe efforts to improveestimates as vorticity,wind, seasonal effects, ctc.! within forthe critical resuspension velocity of net-pen lease sites or the cumulative effects of several wastes,and to createa modelingpackage that operationswithin a coastalernbayrnent. The couldbe routinely used ta aid regulators with site complexand restrictive regulatory environment evaluationand decision-making. Specifically, is viewedas a limitingfactor in thc growthof the fieldmeasurements were made to estimatein situ aquaculture industry in the United States erodibility of net-pen waste materials. A Schneiderand Fridley 1993!, submarineannular flume called the Sea Carousel To resolvesome of theabove limitations, wasused; this devicewas designed by the Panchanget al. 997! developeda comprehensive GeologicalSurvey of Canadato studyseabed modelingstrategy involving an investigationof instabilitiesand the rncchanisrns involved Amos tidaland storm-induced currents, wave effects, et al. I992a!. The SeaCarouse! and thc field andnet-pen ~aste transportmechanisms such as measuremcnt programs are described in section settling,resuspension, and decay. This approach l. In theinterest of packagingthe modeling wasshown tobe successful inassessing theimpact technologyfor regulators, two reasonably well- of aquacultureoperations inCobscook Bay and knownflow models were evaluated foraccuracy ToothacherBay. First, a verticallyaveraged, two- and easeof use: a fiiuteelement model called diinensionalflow modelis constructedusing RMA2 and a finite-differencemodel called appropriatefield measurements,to simulatethe DUCHESS,R1VIA2, developed through funding currentsinduced by the tides and by storznwinds. fromthe US ArmyCorps of Engineersand Theresulting flow-fields were used as input to a coupledwith a sophisticatedgraphical interface, particletracking waste transport model. The is a public-domain, two-dimensional resultsshowed that at some sites, inferences drawn hydrodynamicmodel. DUCHESS,which was regardingthe wastedistribution using a developedat DelftUniversity, Netherlands, is combinationof modeling methods and field data widelyused for two-dimensionaltidal and storm couldbe quite different from those drawn using surgecomputations e.g., Booij 1989,Jin and isolatedfield measurements. Thepotential of the Kranenberg1993!. The transport model developed modelingmethods for siteselection and iri by Panchanget al. 997! wasenhanced and decidinga priori which sites needed a greater level packagedwith an interfaceused to extractflow of morntoringwas also demonstrated. solutionsand graphically display flow and Beforethe modeling techniques can be transportresults. This work led to a packagecalled adoptedinregulatory practice, however, the work A WATS AquacultureWaste Transport of Panchanget al, 997! suggeststhat two Simulator!,described insection 2. It wasapplied problemsneed further attention. First, a more to threesites selected by the Maine Department reliabledescription afthe resuspension ofsettled of EnvironmentalProtection for testingand wastesis needed.Since resuspension involves demonstrationpurposes as part of technology complexmechanisms that are not well- transferefforts. Applicationof AWATS to understood,it wasmodeled using a parameter modelinganaquaculture sitein Maine is presented U describinga thresholdor criticalcurrent in section 3. velocityatwhich settled waste material would be resuspended.Panchang etal. 997! found that I. Fieldworktoestimate erodibility thewaste dispersion and accumulation results In the initial developmentof the waste werevery sensitive to thethreshold of shearstress atwhich settled fish-pen wastes areresuspended, transportmodel, Panchang et al. 997! foundthat thuslimiting theusefulness ofthe models forsite the transportof net-penaquaculture waste was selection,Secondly, Panchang etaL 997! were sensitivetathe ability of the currents toresuspend materialonce it hadsettled on the bottom. With motivated more bya researchperspecti veand did settlingrates of 4-10crn/sec and typical depths Dudleyet aL 2l 7 beneathpens of 15-25m, net-pcn wastes will settle turbidity measuretnentscan be correlatedwith in thevicinity of the pensin a matterof minutes. shear velocity to provide values of critical ln constantlow-velocity environmentssuch as resuspensionvelocity e.g., Amos et a1. 1992b!. fjords, local settling can have adverse The Deep Cove site containsthree pen environlnental impaCtS;in high-velOCity systems Fig. 2! consisting of net-coveredcages environmentsthe material may be resuspended arrangedin rows of 10 cages, with two rows and lnore effectively dispersed, Lacking formingan independent floating pen system, each applicableinformation regarding the complex holding about5,000 fish. At this site, wc process of resuspension in aquaculture attelnptedto determinethe erosionthreshold and environments,Fanchang et al. ]997! usedcasual its variationin time and space. It wasestimated diver observationswhich suggestedthat net-pcn that the greatestamount of sedimentationwouJd wastes were eroded when the flow velocity be near the centerof the three pen systemsand exceededapproximately 30 cm/sec,In view of would decreaseoutwards. Since i waspossible theuncertainty, however, Panchang et al. 997! that the erosion threshold varied with the amount modeledmultiple transport scenarios by varying of materialalready acculnulated, the Sea Carousel the valuesof U, overa rangeand found that the was deployedat nine locations: three near the resultingwaste dispersion was very sensitive to centerof thesite, four locationsat differentpoints U,. Forexample, waste removal from the domain on the seditnentationgradient, and two control usedto examinea commerciallease site in Deep locations closer to land deemed to be unaffected Cove, CobscookBay, varied between83% and by thenet-pen operation, Data were collected at 0% whenU, was variedbetween 10 cm/sec and two differenttimes, one in April 1996and one in 40 cm/sec. The areaaffected by the wastesalso variedsubstantially. Erosion of sediments is a function of bottomstress which is often expressedas shear velocity. In this sense,U, is intended to be a measureof the threshoMstress at which net-pen wasteswouM be eroded and resuspended.To obtain more reliable informationregarding this mechanism, measurementswere made under the direction of Dr. Carl Amos of the Bedford Institute of Oceanography BIO! at the ConnorsBrothers Inc. comtnercial lease site at Deep Cove in CobscookBay near Eastport, Maine Fig. 1!. Figure 2 shows the locations of erodibility experimentsin relation to theDeep Cove net-pen systems,A devicecalled the SeaCarousel shown in Figure 3 was used to conduct the erosion experiments. The Sea Carousel is an annular flume designedby the Geological Survey of Canada to measure seabed erosion Upon loweringit to the benthosfrom the sideof a boat, a current was generated inside the flume and slowly increasedin magnitude in a stepwise fashion. At each step over the courseof the Figure1. NottheL~~Cohscook Bay illustrating the loca- erosionprogram, a video of the erosionprocess tionof theDeep Cove field sitr..Bathymetry in meters. was obtainedin conjunctionwith water samples and turbidity measurelnents. The resulting atg t;JblttT~ aepertNo' S Agurel, Deep Cove showing tbclocations ofApril and September 1996Sea Carousel and corrcnttncter deploytncnts inrelation toConnors Brothers inc,net-pen systems 5400, 5600.and 5700. Sea Carousel deployments arenumbered 1-9 September1996, since there is likelyto be seasonalvariation in theamounts of net-pen wastespresent due to higher feeding rates in the surnrner,and more frequentstorm-induced erosionalevents in thewinter!. Duringthe fteldwork, locations ofthe net- pensites, current gauges, and SeaCarousel deployrnentswere determined via theGlobal PositioningSystem GPS!. The SeaCaroUsel workprovided videos of theseabed erosion, samplesofsuspended sediments foreach velocity step Fig. 4!, seditnentcore samples, water velocities,andturbidity data, The erodibility data fromthe Deep Cove aquaculture site were analyzedbyDrs. Terri Sutherland andCarl AtrtQ$ ~ 5 fbeSea Caronrel ahnnttobe lnwe 2. Mathetnatical models Modelingthe physical transport of finfish aquaculturewaste requires detailed knowledge of thespatial and temporal variations in tideand wind-inducedcurrents in theparticular region of interest. Hydrodynanucmodels, driven and validatedwith field data,simulate these currents andprovide the necessary input information for transportmodels to cotnputethe resu!ting waste dispersion.Previous modeling work conducted for CobscookBay indicated that a two- Fltpsre4, Watersamples for suspendedparticulate maner analysiscollected dttrirtg a Sea Carouse! erosion pro- ditnensionalflow model based on the shallow gram coadncaedai the ConnorsEtrothers inc. atlttacut- water equationsthat. yields depth-averaged tnre site in Deep Cove. Frorrt left to right. each houie velocitycomponents isadequate forthis task. This corresponds to a water sampletatten 2 min after thc on- is fortunate,since three-ditnensional schemes setof eacb step-wise velocitymagnitude incrementgen- requireintensive computer resources, particularly erated inside the attttttlar flume. whenlarge areas such as the coastal domains of Maine are to be modeled. In addition,data collectednear aquaculture sites in Cobscook Bay indicatethar. the large tidal forcing leads to little verticalvariation in thehorizontal velocities in Rat! UJh!RToebnkal Report ivo. 26 a. Hydrodyttamiemodels Thc graphicaluser interface for RMA2 consistsof a softwarepackage called Surface- waterModeling Systein SMS! developedat the Brigham Young University Engineering Computer GraphicsLaboratory ECGL! in cooperationwith theArmy Corpsof Engineers Jones and Richards 1992, ECGL 1995!. This enables users to graphically construct finite elementmeshes required as input to RMA2 and 0.5 1.5 to displayhydrodynamic solutions frotn RMA2. Utsee! ntfa! The SMS softwareprovides the user with various toolsand pull-down menusto facilitatedigitizing scanned topography maps, constructing Ft!tttre5. Esumateof thc erosionthreshold for an Apri!!996 computational meshes, and displaying and SeaCwouse! dcp!oyment at station 4 in DeepCove, Maine. Ambientctmccntrations of suspcndcdponictt!ate matter con- animatingsolution data sets with colorcontouring centration SPM! we designatedby round symbo!s, whi!e arid vectors. tbceroded concentrations of SPM sse designated by triangu- A significantamount of modeling was lar symbo!s.Based on thc significantchange in SPMi!!os- pursuedusing RMA2 to assessits suitability for trated above. Uo«~ for this particular experiment is esti- tnatedto be 0. 33 m/sec. coastalmodeling associatedwith aquaculture rnanagernent.The evaluation of RMA2 included chosensince the performanceof DUCHESS had modeling of simple test cases as well as a alreadybeen well established viaprior modeling systematicinvestigation of meshconstruction and efforts Panchang et al. 1997!. refinement,boundary conditions, time step size, and floodingand dryingmechanisms for the coastalregion of CobscookBay, Maine. The 15.5 x 13.7km Cobscook Bay domain Fig. 6! had been Tts!s!et. ~ Su ~' o ~mUo«' values from Sea Carouse! data;deploytnents Apr!!and Scptem bcrl996 atConnors Brothers Inc.'aquacu!tare farm,Deep Cove, Maine. Dudley et ai, 221 y ngoluslymodeled and validated using notas satisfactory as those descri bed by Panchang UC ESS,Initial RMA2 flow model mns usin et al. 997! usingthe finite-differencemodel a 225-mresoJution mesh of theCobscook Bay DUCHESS.Ivloreover, DUCHESS could resolve domainresulted in problemswith flooding two tidalcycles for tltesame domain with a deing- If anynode comprising anelement constant75-rn resolutionand a 40-sectime step, the drying criteria,the entireelement to whichit in about40 min on the samePC. belongedbecatne "dry" and was removed from In sutritnary,though RMA2 hasbeca used computation.As a consequence,entire reaches in otherapplications. its implementation was of thc baywould be shutoff dueto dryingin extremely time-consuming andproblematic for th is shallow,narrow areas, resulting ina discontinuous particularapplication. It alsopresented added d.omain and inodel failure. coinplexityforregulators due to its sensitivity togrid Subsequentefforts, which invol ved size~,requiring greater efforts inthe construction and refiningthe computational mesh and adjustments refinement of finite element meshes. Mesh to variousmodel pararnetcrs such as timestep, constructionand refinement is a complex problem eddyviscosity, and bottom friction, met with only requiringevaluation of dotnaingeometry and moderatesuccess. Due largely to the sizeand bathymetry,1Vhile a.ll modelinginvolves a certain coinputationaldemands of the CobscookBay level of trial-and-en'>r beforesuccessful simulauons domain,the most successful model run usinga are obtained,it was felt thatworking with finite relativelycoarse mesh and 12-min time stepsran element models would be too cumbersoine from the in near-real time on our 200 MHz PC, The mesh, pointof view of routine management atits finest,had a resolutionof 75tn, the majority Most finite difference models, in of which was much coarser, with a maximutn of comparison, require only a single resolutiort 225 m seeFig. 6!. Theresulting simulations were throughout, and entail a straightforward gurntnaryinustrtttion for theTRA.'4S aquaculture net-pen waste tn developsat the University of Maine. 222 t;JitlL Techakd ReportIso. 26 relationshipbetween the time step and the grid quantity, Otheruser-specified parameters in the size Althoughthe flexibility of enhancingthe modelinclude: the simulation duration, begin and resolution in specific parts of the domain is end times for food and fecal matter introduction compromised,DUCHESS does allow options for eachday, the uneatenfood ratio as a percentof subsequentsimulations in "nested" domains. the dailyfood mass introduced, the daily fecal Flow modelinggroundwork for computing pelletproduction in g/kgof fish,percentage of aquaculturewaste transport was performed by orgariiccarbon contained in thewaste depending Panchanget al, 997! for CobscookBay and onthe feed used, and first-order decay coefficient ToothacherBay using DUCHESS. The model has estimates for food and fecal rnatter. alsobeen successfully applied to otherfisheries- The transportmodel computations relatedproblems Newell 1991!and has been involve breaking the daily feed and fecal foundto begenerally robust. introductionsdown into particlesand tracking One limitationof DUCHESSis that, their dispersionthroughout the model domain as unlikeRMA2, it lacksa convenientgraphical user theyare advected by the currentscomputed by interfaceto expeditethe modelingprocess by thehydrodynainic model Fig. 7!, Eachparticle aidingthe user in model construction andviewing represents a user-specified amount of mass andinterpreting model output. Forthis reason, representinga part of the total massintroduced we madeefforts to interfaceDUCHESS with SMS overthe course of the simulation.Each particle toavail users of thegraphical advantages of SMS. is trackeduntil it leavesthe transport domain at We developed a utility program called whichpoint it is consideredto havebeen flushed DUCHSMS which indirectly links the two away,and is not allowed to return. As the particles programs, The programfacilitates construction sink,they are advected bythc flow-field until they of themodel domain using SMS, and graphical reachthe bottom. For modelingpurposes, we viewingof the flow modeloutput. I enables chosesinking rates of 4 cm/secfor fecalparticles bathymetrydigitized with SMS to be exported in and 10 crn/secfor feedparticles variable upon a formrequired by DUCHESSas input and also feed type! Panchanget al. 1993!. Onceon the transformsDUCHESS output into a formreadable bottom,a checkis madeat eachtime step against bySMS. This allows easy graphical display and the specifiedU, to deterrnincwhether or not the animation of flow solutions obtained from particle is eroded froin the bottom and DUCHESS in SMS. resuspended in the water colutnn to be further transported. Partides can decreasein mass over b. Transport rriodel thecourse of a modelrun to first-order exponential A transport model called TRANS was decay. Values usedfor the decaycoefficient developedat theUruversity of Mainero simulate dependupon the environtnentand oxygen theadvection and dispersion of finfish aquaculture availability. Valuesin fjordshave been found to wastes;it is included inthe A WATSpackage, and vary between0.1 yr' to 0.5 yr' Aure and modelsthe mechanisms of settling, advection, and Stigebrandt1990, Hansen et al. 1991!. resuspensionto describe the physical transport of At the end of the simulation, TRANS fish-penwaste materials. To accomplishthis, outputswaste distributionsnapshots at a user- TRANSrequires spatial and temporal flow-field specifiedtime interval and a simulationsummary, information,bottom topographydata, and Particlesreinaining inside the modeldomain at propertiesdescribing the net-pen wastes such as theend of thesimulation contribute to organic resuspensionthreshold U,!, settlingrates, and carbonloading to the benthos. The loading thelocation and the frequency of theintroduction concentration,in g/m', is computedby dividing of wastesinto the water. Paraineters describing thetotal mass in eachtransport model grid by thc the aquaculturefarm are input by the user areaof the grid. TRANS will interpolatefor providingcoordinates of eachnet-pen in the transportmodel grid andtime stepsizes that are domaincoordinate system, as well asthe size of smallerthan those of theflow model.A typical eachpen, its stockingdensity, and daily feed transportscenario is runfor 15 daysto approach Dudlcsel sk 223 a steady-state loading pattern, The output This package con venientls applies the snapshotsrepresent the estimated concentration hydrodynamic and transport mode!s along with t>f net-penwastes asa measureof organic carbon associatedinformation regardmgthc net-pensand as it is distributed over time throughout the to obtainappropriate graphical displays. A WA IS domain. These snapshohsare output in a fornt inc!udesthe wastetransport program TRANS, thc readableby SMS for easygraphical display and graphical interface SMS, and thc flow model animation. TRANS reportsal l model parameters DUCHESS. In the event the user does noi have as wc!! a» the amount oi' material f!ushed out of DUCHESS, output from anotherflotx model may the domain, the residence time for material be used.! lt also includes the uti!ity program introduced on the first day of the simulation. and DUCHSMS which will extract t!ow and the rnaximiurn !oad rate and its location in th.e bathymetrydata for the subdomainof interest i.c .. model.domain in the surnrnaryfile. thc general vicinity of the net-pen, specified by thc userin the form of a rectangle[from the output c. The A WATS modelingpackage files of the flow rrtode! DUCHESS or a!ternati ve! We have constructed a package cal!ed and use this information io run TRANS. AWATS that may be suitable for regulatory use. It is helpfu! to describe the AWATS IICtuaaurlcse Nstp en Thedi spars on at' partible" is computed by TRANS eacharne ar fecal particle'rsp- rsasrds8 dtsctettzecl amountof messrep- F ecclP artitjes" ~ reaenttng8 verySiiles ee ~ o tstb an Otthe tatel p ~ messIntradutm cl. P erbdes"ski k tathe batterstd user-sped fied anting rates,ttv 0tstfh std sv* dl Z F cadPesttcfss unetdetlj TheSav4etd soiurton fran the hyCkcdynamia madelis inputto TRANS shetde scribes the verbc- allyeve.aged IXinent "s:.' «~c~.~. veKtctt es in the x endy directionsover tim e u tsarp> tr tsaiA! The'parboil ere ed veCted h tmsontsly in the P ardctes'me resuspe nded tmctsding to 8 cand- The eccumulali on of net-pen s tstdy diretttone ss kioml atgottthm.Ftx cacti bmestep tnthe sssuc- msfensf, espreSSed es the they ink to the batfotn: etiOn, T RAN S chettts vetetherOr nat e ~ iS messO Iatgsrco Carbon per d*=u a resbngcASe bosom,if so, TRANSperssnns 8 squaremeter, iS Cam puted by dy- V'fR checkon thecurrent velocity at the ~" loc- TRANSask ts ~by ation. If it esessdstheCriticelresuspen San vel- Tha til 8SS at ccky,C, the psrbcle"is rest spsnsiedarXI Stdveo- eootsttteabng net~ m atestd ted in the harizcrttddrectton; cen decreamavsrthe toutse S Onthe btttama af Ste mtttssb On SCCOC dng to I fno, the ~ cortinues to sink. uaermpected,trSI-Order I fyes, is theststremt e~ al daaayCaefric ere s If yss the partes isedvectedinx andy for cststime stepend settlesback to the bottom. !'sgttre7. Sontmaryillustration fOr the TRANS St!uaou!turettet-patt watte trans!xtnsimulator atttnrtt!tnt tfeveta~ttt at the Uttiversity of Maine. modeling package in terms of its data file conlponeilis: I ! DOMAIN.DOM, theoverall domain descriptor file containingthe size of theoverall domain, grid spacing,number of tiincsteps, and time step size in the flow solution file; 2! DOMAIN.TOP, the topography file, containing the depthsat differentgrid pointsof the overalt domainfor whichthe flow modelis run; 3! DOMAIN.FLW, the flow soIution file, containingthe x-directed and y-directed velocity fieldsat eachtime step; 4! DOMAIN,OZ,a one-zerofile usedby DUCHESSand A WATSto differentiatebetween "dry" ! landpoints and "wet" I! computational pointsin the overalldomain; S! SUBDQMAIN.BTH,containing depths of a subdomainin thc vicinity of thenet-pen the subdornanin which waste transport simulations areto bemade!; 6! SUBDOMAIN.XYZ,a bathymetryfile readableby SMS for use in constructingthe domaingeometry tographically view flow and transportsolutions; 7! SUBDOMAIN.UV,the flow solution file correspondingtothe subdomain to be used by TRANS; 8!SUBDOMAIN,DAT, a second flow solution Ftgareg. Operatiouaichartfor the A WATSpackage. filereadable bySMS that can bc used todisplay/ animatethe flow solution over the domain geometry; inducedvelocities using a flowmodel solution, 9!SUBDOMAIN.FRM, containing user-defined Thisinvolves the DOMAIN.* files. Sincethe net-pcnparameters: coordinates ofthe center of overallmodel is oftenlarger than the area of eachnet-pen, andthe volume, stocking density, interestnearthe net-pen, a subdomain definedby anddaily feed quantity ofeach individual pen; thefour corner points may be selected forfurther I 0!TRANSIN.DAT, whichcontains thetransport modeling.DUCHSMS uses the DOMAIN.* files modelgrid spacing and time step, simulation asinput to provide the necessary SUBDOMAIN, duration,dailystart and end times and frequency BTHand SUBDOMAIN. UVfiles, which are used offood/fecal manerintroduction, outputrequests, alongwith the additional data contained in the pcnlocation coordinate adjustments, critical SUBDOMAIN.FRM and TRANSIN,DAT files resuspcnsionvelocity U !,settling velocities for requiredtoconstruct a transport simulation. The food/fecal material, thefraction ofthe introduced creationofthe following output fiIes using foodand fecal maner that is organic carbon, the DUCHSMSfunctions requires theinput of the fractionofthc daily feed quantity thatiswasted, DOMAIN.DOMfileto coordinate theuse of the massoffecal pellet production perunit mass of otherinput files *.TOP,~.FLW, *.OZ!: fish,and fiirst-order exponential decaycoefficients SUBDOMAIN.BTH,thebathymetry file to be forfood and fecal rnatter. usedas part of thefarm description file; Thefirst step in the modeling procedure SUBDOMAJN,XYZ,a secondbathymetry file Fig.8! consists ofobtaining tidaland/or wind- readablebySMS for use in constructing the domaingeoinetry tographically viewflow and Dttdteyet ai. 2?5 transportsolutions; SUBDQMAIN,UV, the f!ow solutionfile correspondingto the subdomain to beu«d by TRANS;and SUBDOMAIN.DAT, a secondflow solution file readableby SMSthat canbe usedto display/animatethe flow solution overthe domain geometry. ExecutingTRANS produces two forms of output. First, the TRANSPORT,SUM file is a simulationsummary describing all user-defined parametersand also reports flushing efficiency of introducedparticles from the domain,residence time, aud the sedimentationrate and location of the point with greatestaccumulation in the subdomain. The other file TRANSPORT.PLT is a datafile that contains snapshots of the dispersion of net-penwastes over the simulation, suirab!e for plottingin SMS forviewing/animation. 3. Sitnulationof net-penwaste distributionin MachiasBay, Maine The AWATS modeling package was appliedto six aquaculturesites in Maine: threein CobscookBay, and one each in Blue Hill Bay, MachiasBay, and Cutler Harbor. Here, space perruitsthe descriptionof our simulations in Machias Bay, which is locatedin Washington Figure 9. Locatiou of Machias Bay itt WashingtonCourt ty, Maitre, The aquaculture operatiott at Cross islaitd itt County Fig, 9! in theGulf of Maine. The typical the mouth of Machias Bay is one of six sttesrttodetcd tida1range for MachiasBay is about4 m. The with the AWATS niodeiiag package. aquaculturesite of interest for this domain is operated by Atlantic Salmon of Maine, Inc. ASMI! locatedin NorthwestHarbor off Cross Departmentof Marine Resources DMR! in the Island. The island is situated in the mouth of vicinity of the aquaculturelease area: however, MachiasBay close to thc mainlandwhere it forms the flow patternsin otherareas of the mode! did the CrossIsland Narrows to its northeast Fig. 10!. not appearto be entirely realistic. For example, Figure10 showsthe 13 x 14 kmdomain while the modelproduced high currents in the geometryrepresenting the entire Machias Bay Cross Island Narrows as related by anecdotal area. Thc domain cotLst!ineand bathymetry were evidence!and varied over time, thc directionof digitizedto 75 m reso!utionin SMS usinga the current never reversed over the course of an computer-scannedimageof nauticalchart ! 3326 entire tida! cycle. Additionalcurrent data were of the National Oceanic and Atmospheric therefore collected in the Cross Island Narrows Adnunistration NOAA!. Thesebathymetry data usingan S4 currentmeter on 15 August !997, arestored in thetopography fileMACHIAS TOP. Thesedata allowed the adjustmentof tidal The flow mode! DUCHESS was usedwith this amp!itudesand phasesat eachopen boundary, bathymetrytosimulate tidal currents The model yieldinggreatly improved resu!ts not only near wasforced with specified tidal amplitudes at the the Cross Island Narrows, but for the overall Gulf of Maine/MachiasBay boundaryand the domainby providing a morecomplete picture of CrossIsland Narrows boundary. Initial effortsin the tidalforcing at the boundariesof the model. tuningthe model y ieldedreasonable simulations A snapshotofcurrent velocities just after high tide whichmatched current data provided by the Maine near CrossIs!and, take.nfrom the model resu!ts LJWR t'eehedcaiReport Xo,26 Figure10. MachiasBay domain, baihyine shownry ingray srale. Locaoon of theAS%i aquaculture siteis denoted byan asterisknear Cross island. The subdomain chosen for transport mot}cling isenclosed by a box.Grid squares are l km'-, depthsare given in nteterv. storedin MACHIAS.FLW,is shownin Figure11. configurations were locatedwith the aid of aerial For modeling nct-penwaste transport at photosfrom March1996 provided by T. Riggens the ASMI aquaculturesite, the subdotnainoutlined of the Maine DMR. Exact stocking and husbandry by thc box in I-igure ! ! waschosen; it is defined information for this site is confidential and so, for by specifying the coordinatesof the corners. modeling purposes, general aquaculture DUCHSMSwas used to extractthe hydrodynatnic husbandrydata obtained for ihe previousmodeling solution and depths for this areaof interest from study in Cobscook Bay courtesy of Connors the overall domain information contained in BrothersLitnited, Aquaculture Division! were MACHIAS.FLW and MACHIAS.TOP. Thc usedin conjunctionwith literature data Laird and resulting subdotnain information is contained in Veedham1988! to estimatepen stocking density, ASMI.BTH and ASMI.UV. Another file called daily feed quantitics pcr pcn, and fecal production ASMI.XYZ is also obtainedfrom DUCHSMS, per unit massof fish for the ASMI site, It is which is read into SMS in order to construct the important to note that this nominal aquaculture domaingeometry for plottingand animating flow- husbandry information was used to simulate the field solutionsand transport inodel output. dispersionand rates of sedimentationof waste In addition to the hydrodynamic solution effluent from this site in order to illustrate the file, TRAIslSrequires a farmdescription file applicationof A WATS. definingthe locations,voluines, stocking Running a 15-day transport s.cenariofor densities,and daily feed quantitiesfor eachpen, the CrossIsland site producedthe sutnmaryfile Eighty-sixASMI net-pensof various sizesand ASMITRANS.SUM and the organic carbon i!udfri et a1 7'i7 Figure12 Contourplot of l 5-daysimulated net-pcn aqua- Figure11. Tidal fiots-field solution ai theASM l Crossis- ulturewaste deposition ai the Cro~s island aqua 'ulture landaquaculture lease site immediately after high tide. site in Northwest Harhor Coniour interi a! s are lo ! g/ Vectorsde~ote magnitude and direction of currentve- locity.Gray-scale contours also represent velocity mag- m- organiccarbon. nitude. The circle indicatesthe approximatelocation of the leasearea. concentrationsfile ASMITRANS.PLTwhich is thc macrobenthos Drake and Arias 1997!.Findlay read into SMS andplotted. Figure L2 showsa andWatling 994! estimatedthat a constant6 snapshotillustrating the loading pattern of finfish cm/sec current can deliver enough dissolved aquaculturewaste deposition as g/tn'-organic oxygento sedimentsto supportthe theoretical carbonat the Crossisland aquaculturelease site maximumaerobic oxidation of nearly50 g/m-/ ai the end of the 15- day model run. For this dayof organiccarbon. The results demonstrate simulation, the U, value was set at 40 cm/sec. howAWATS can provide noi onlya pictureof Since no waste material introduced on day l of waste distribution, but information regarding the simulationwas transported beyond the Cross spatialand teinporal variations in currentvelocity Islanddomain bounds,average residence time was that can be used in conjunction with benthic notcomputed for the summary.The easternand oxygendemand data to determineif organic southeasternportions of the Leasearea received enrichmentin high-loadregions has thc potential thc.highest loading with one pointreceiving a to exceed the assimilative capacity of ihe inaximumorganic carbon loading rate |averaged environment. over 15days! of 38.9g/ma/per day. The mean and maximuinvelocities computed by the model for CONCLUSION thisparticular area were 6.3 crn/sec and 10.0 cm/ sec, respectively.Though not high enoughto In situ measurementsnear the Deep Cove exceed the U,, criterion for resuspension,the aquaculturesite suggested that bonom sediments currentsin this areacould supply sufficient oxygen nearnei-pen aquaculture sites are eroded ai I.'ur to the benthosfor adequaterates of decayof the velocitic.sgreater than about 40 cm/sccin the effluent as well as high rates of water exchange winier andabout 50 cm/sec in the suinrncr. These in the embaymentto prevent adverseimpacts on values are used in the development of the 228 L'JNRTechnical Re pert Nn. 26 modehng package A WATSwhich can be used for MacroinvertebrateCommunity of a Lagoon estimatingthe dispersalof net-penwastes in a Systemin the Bayof Cadiz Southwestern coastalen v ri onment with varyingcurrents. Spain!, Estuaries20 !: 677-688 Althoughnot described here, the package may be EngineeringComputer Graphics Laboratory. usedfor storm-drivencurrents and wave-induced 1995,SMS Surface Water Modeling System velocitiesaswell. Application tothe aquaculture ReferenceManual. Brigharn Young Univ., sitein Machias Bay and others in Maine suggest Provo, Utah. thatAWATS is a convenienttool that can be used Findlay,R. H. andL. Watling. 1994. Towarda to aidwith site evaluation and direction of field processlevel model to predict thc effectsof monitoringprograms for areasof coastalMaine. salmonnet-pen aquaculture on the benthos, pp.47-78. IIi: B.T.Hargrave [ed.], Modehng ACKNOWLEDGMENTS BenthicImpacts of OrganicEnrichment froin MarineAquaculture, Can. Tech. Rep, Fish. Thiswork was supported in part by the Aquat.Sci, 1949:xi+ 125p, NationalMarine Fisheries Service NMFS!/ Hansen,P. K K. Pittman,and A, Ervik. 1991. NOAAunder the Saltonstall-Kennedy Progratn Organic waste from marine fish farms- andby the University of MaineSchool of Marine effectson thesea bed, pp. 105-119.In; T. Sciences.We are grateful toDr. James Manning Makinen ed.!, Marine Aquacultureand of NMFS,John Sowles of theMaine Department Environment. Nord 1992: 22, Nordic of EnvironmentalProtection, Laurice Churchill Councilof Ministers,Copenhagen, Deninark, andTracey Riggens of theMaine Department of Jin, X. and C. Kranenberg.1993. Quasi-3d MarineResources, and to StephenDickson of the numerical modeling of shallow-water Maine GeologicalSurvey for their helpful circulation.J. Hydraulic Eng. 119: 458-472. assistanceand adviceover the courseof this Jones,N. L, and D. R. Richards. 1992. Mesh pfoJec L generation for estuarine flow models. J. WaterwayPort Coastal Ocean Eng. ASCE! 118 !: 599-614 LITERATURE CITED Laird,L. andT. Needham,Editors. 1988. Salmon and Trout Farming. Halsted Press, New Amos,C. L., J.Grant, G. R, Dabom,and K. Black. York, 271 p. 1992a. SeaCarousel a benthic,annular NcweH,C. R, 1991.Development of a modelto flume. EstuarineCoastal Shelf Sci.34: 557- seedmussel bottom leases to theircarrying 577. capacity. Phase2 report, ISI8809760, Amos,C. L., G, R. Dabom, H. A. Christian,A. National Science Foundation SBIR. Atkinson, and A, Robertson. 1992b. In situ Panchang,V.G., G.Cheng, and C. Newell, 1993, erosionmeasurements on fine-grained Applicationof rnathernaticalmodels in the sedinsmtsfrom the Bay of FundyMar Geol. environmentalregulation of aquaculture. 108: 175-196. Final reportto the National Marine Fisheries Aure,J. andA. Stigebrandt.1990. Quantitative Service4tNOAA. estimatesof theeutrophication effects of fish Panchang,V G.,G,Cheng,andC,Newell. 1997, fartning,on fjords. Aquaculture 90: 135-156. Modelinghydrodynamics and aquaculture Booij,N. 1989. UserManual for the program waste transport in coastal Maine. Estuaries DUCHESS, Delft Universitycoinputer 20 !: 14-41. programfor 2D horizontalestuary and sea Schneider,C, and R. Fridley 1993.Aquaculture surges,Department of Civil Engineering, andthe marine environment: the shaping of Delft Universityof Technology,Delft, The public policy Working Paper for the Netherlands, Workshop,Marine Biological Laboratory, Drake, P. and A, M. Arias. 1997. The Effect of WoodsHole, MA. 10p. Aquaculture Practices on the Benthic ENGINEERINGTECHNIQUES FOR ENHANCEMENT OF NEARSHOREROCKY HABITATS FOR SEAURCHIN AND ABALONE AQUACULTURE S, Kawarnata NationalResearch Institute of FisheriesEngineering Ebidai,Hasaki, Kashiina, Ibaraki 314-0421, Japan e-mail [email protected] ABSTRACT Commercialproduction ofabalone and sea urchins has been markedly reduced bylow availability ofalgal foodthey consume, There are two mech anisins responsible forthe liinited food: overgrazing bysca urchins, anda greatloss of driftalgae produced asthey becoine dissipated bywater inovement. The wave-induced watermotion msy inhibit sea urchin grazing, and as such tbe importance of the wave action to protect kelp abundancefrom the destructive grazing leads us to anengineering possibility of developingkelp beds by increasingwatervelocity. lnaddition, anew device was developed totrap drift algae. The device isa bait cage witha pendulum-likedoorand stoppers. The door is designed tobe opened inwards bywave-induced oscilia- toryflow but the stoppers prevent itsoutward opening. Laboratory scale-madel cxperhnents onthe trapping mechanism,effectiveness, andengineering design were carried out. Further field experiments demoisstrated thatthe device could trap drift kelp and sever lose thetn until they were consomcd byaggregated seaurchins. INTRODUCTION operationsof constructingartificial substrata, mainlywith concrete blocks and quarry rocks to The comtnercialproduction of abalone createkelp beds. However,the operations and seaurchins is frequentlylimited by the frequentlyfailed to establishkelp forests as availability of food, Two mechanismsare expected. Many of such failures seemed responsiblefor limited food in the habitats. First, attributableto sea urchin grazing. Thus, a number kelp,Lanunarian algae, are primary food resources ofattempts were made to protect kelp plants from necessaryfor growthof the animalsbut are animalgrazing with physical barriers, such as grid frequentlyovergrazed, especially by seaurchins. fences,plastic nettings and plastic-seaweed frills, In addition,most of thekelp production inay be as well as with chetnicalrepellents Kawamata sweptout of the shallowhabitats as drift algaeby 1994!.Nevertheless, notechnique is availablefor coastal water motion. These benthic herbivores the artificial developmentproject in seaurchin- areof greatitnpottance to thenearshore fisheries dorninatedbarren grounds. A recentstudy showed in Japan.Artificial structures built to establish that wave-induceddistu.rbance may restrict sea kelpbeds and to trapdrift algaehave been urchinfeeding, thereby maintaining kelp forests incorporatedin a long-termgovemrnent subsidy adjacenttosea urchin-dominated areas Kawarnata program.The programwas the Coastal Fishing in press!,This view would be of greatsa!ue to GroundImprovement and Development Project, engineeringpractices. called the Ensci Project, initiated in 1976 to On the other hand, the necessity of promotethe enhancement of artificial habitats in preservingdrift algaein nearshorerocky habitats Japanesecoastat waters {Stone et al. 1991!, hasbeen recognized by field researchersand It is well known that new substrata tnay fishermen involved in abalone and sea urchin lead to an increasedabundance of algae. This aquaculture,butlittle has been known about their empirical knowledgehas encouragedthe physicalbehavior in thefield. A seriesof engineeringstudies revealed the effectiveness of' supportingthe hypothesis that the absence of algal varioustypes of structuresof watermotion in the plantsfrom deeper or shelteredsites results from laboratoryand in thefield Kawamata1987, 1988, herbivorousgrazing but nat fram the shortage of 199 I, Kawamata et al. 1993!. The results were lightintensity or nutrients,First, experimental reflectedin thepublication of a guidebook issued removal of sca urchins led to re-establishmentof by the Japan Coastal Fisheries Promotion inacraalgae Iwate Prefectural Fisheries Association l993! for design of artificial ExperimentalStation 1988, Agatsuma et al. 1997!, structuresunder the EnseiPreject. The previous In addition,it is frequentlyfound that underwater structures were "stable," or consisted of fixed floating objects such as mooring rapes, which materials.It is predictedfrom theguide that any benthic herbivores can scarcely climb, are of the stablestructures could hardlytrap and overgrown by kelp, even immediately above preservedrift algaein manyof theshallow rocky barren beds. habitatsfor a longenough period. Ta copewith thedifficulty, recent studies e.g., Kawamata and EFFECT OF WATER MOTION ON SEA Suzuki 1995! developeda new trap with a URCHIN GRAZING pendulum-likedoor that is inovedby wave- A previousstudy Kawamatain press! inducedoscillatory flow. evaluated the restrictiveeffect of the wave-induced Thispaper describes the itnportance of the oscillating flow an feeding by the sea urchin wave-inducedwater motion to ecologicalbalan': Strorrgyloeentrotus rtudus. Thc sea urchin is betweenplants and herbivores, thereby showing commerciallyimportant but is frequentlya causal thepotential for establishmentof kelp bedsin agentin clearanceof macraalgaealong the coast engineering modifications. State-of-the-art of northern Japan, from Hokkaido to central technologyof trapping drift algae is also described Honshu.The methodof the studywas briefly as follows.A kelp Drrrirtariaspp.! food with given ARTIFICIAL DEVELOPMENT OF KELP dimensions was anchored to the bottom in an BEDS oscillatingflow tank, where starvedsea urchins It is welt knownthat feedingby sea werecontained. A feedingexperiment was then urchinsmay cause devastating effects an benthic conductedfor oneor two daysto examinethe marineplants Lawrence 1975, Lubchencoand feedingrate under a periodicoscillating flow. The Gaines 1981!. Unlike other benthicherbivores experimentsshowed that the restrictiveeffect of such as abalone, sea urchins have hard teeth and theoscillating flow on feedingrates somewhat therebyeasily feed on the stiff stipe and holdfast variedwith the animal size and food morphology, of macroalgaeSea urchins may aggregate and butindicated a mechanicalconstraint that strictly denudethe substratum of foliose algae, farming inhibitedurchin feeding at a moderatewater barrengrounds that are coveredsolely by velocity,approximating 30-40 crnisec. The sea encrustingcoralline algae. Seaurchin-dominated urchin's mouth is at the center of its attachment barrengrounds are widely observed a1ong the base,so that it mustmount a thallusby detaching coastsof the Japanesearchipelago. Such more than half the number of tube feet used to communitytypes show long-term persistence cling to the substratum. Sea urchins were becausesea urchins can survi ve and reproduce in dislodgedwhen they would try to eatat such faod-limitedenvironments, However, preferred moderatelyhigh velocities. These findings led to foliosealgae are frequently abundant in shallow a conclusionthat the urchin feeding on foliose waters next to the sea urchin-dominated barren algaeis nearlyinipossible beyond 40 cm/sec. zones.The wave-induced benthic oscillating flow Other sea urchins seem to show similar increaseswith decreasing depth more precisely, velocity limits for feeding. Kawamata up to a wave breakingpoint!, so that the water unpublisheddata! examined feeding rates of sea motionin theshallow depth constantly prevents urchinHerrricentrorus pulcherrimus in the seaurchins from feeding on algae,even during oscillatingflow in the samemethod as described calm sea periods,There is muchevidence above, The sea urchins of 45-rnrn test diamete~ approximatingthe maximuin size! showed higher in nature. However, recent studies Kuwahara et feedingrates at the highertemperature over the al. 1997, Kawamatain press!indicated that the peak velocity. However,the feedingrate under velocity limit for feedingmay give a reasonable bothtemperatures began to ceaseat approximately estimatefor the areawith kelp plantsexposed to 40 cm/sec. intensiveanimal grazing, The findingthat sea urchins cannot feed on kelp at the peakvelocity higher than 40 c m/sec ENGINEERING TECHNIqUES FOR might give quantitative estimates for ARTIFICIAL KELP BEDS understandingthe spatial distributionsof sea Several observations indicate an urchinsand kelp. In shallowsubtidal areas where engineeringpossibility of establishingkelp beds waves are constantly broken, kelp are usually by increasingthe water velocity: kelp overgro~ abundant,The wave-inducedpeak velocity u the uppermost parts of concrete blocks is estimatedfrom theequation Denny 1988!: immediatelybelow low water level while kelp are absentfrom the lower parts Terawaki et al. 1995!; rr = 0.3[g h+ H! f '~, ! kelp areabundant on the onshore side of permeable breakwaters but absent from the onshore side of whereg isthe gravitational acceleration = 9.8 rn/ less perineable ones. s'!, h thewater depth, and H thelocal wave height. Althoughit is easyto increasethe water When the bottom of the area is horizontal, the wave velocity with conventionalengineering structures, heightis solelyrelated to thedepth, approximating attentionshould be paid to otheraspects of the Denny 988! wave effect on algal populations,such as breakage and dislodgmentby waves. When an object is H= 078 h p!acedunder waves, the watervelocity is higher on the op of the object. The increased water The wave heightwithin the surf zonesomewhat velocityinay lead to the highermaximum water increasesas the bottomslope is steeper.Hence velocity at severewaves, thereby increasing the the wave-inducedbenthic peak velocity in the surf risk of kelp breakage and dislodgment. No zone is quantitauveinformation is availablefor estimating i4 ! 0.4 gh!'~ ! the breakageand dislodgment of kelp. In addition to this biologicalproblem, no practical methodis Eq,3 indicatesthat the velocity almost everywhere availablefor estimatingthe local water velocity in the surf zone exceedsthe limit for sea urchin on the surfaceof structuresunder waves. Despite feeding. Two othertypical examplesmight be theseproblems, the velocityliinit for seaurchin explainedby the spatial variation in wave-induced feedingwill beundoubtedly an important criterion watervelocity. First,the lowerlimit of kelp beds for decidinghow to designor allocateartificial tendsto bedeeper with increasingdegree of wave structuresfor kelp, exposure.Second, kelp occursolely on the uppermostpart of rockoutcrops and artificial DEVELOPMENT OF DRIFT-ALGAL TRAP structures,where absence of kelp fromthe lower BACKGROUND partis unlikely to beattributable to lightintensity Aimedat increasingfood availabilityby or drift sand Terawakiet al. 199S!. trappingdrift algae, varioustypes of artificial In general,the peakvelocity on the structures have been constructed in nearshore substratumproduced by surface waves is estimated rocky fishinggrounds of Japan.In general,these fromwave data through numerical computation structuresmay be dividedinto two types: block e.g., Kawamatain press!. Severalproblems or grid. The block typesettles drift algaeon the remainin accuratelypredicting kelp abundance upstreamand downstream sides by controllingthe in the field,including engineering problems on surroundingfluid motion while the grid type predictinglocal waterflow in thevicinity of obstructsdrift algae by nettings with litt!e variation rnicrohabitatand biological ones on algal growth of the flow. In practice,however, previous studies Z3Z Ujlt R TedttdetuReport Na. Za suggestedthatthe past attempts were too optinustic primarilyon the f]ow-shieldwhen thc wave- artdthat any of theconventional fixed structures inducedwater flow crosses the door inwards. INTRODUCTION Thenewly developed drift-algal trap consistsof a rectangu]arcage with a doorand stopper Fig.1!. Thedoor is a p]ateas a whole,or a flow-shieldandgrating atthe lower and upper Partsrespectively, which is suspendedbyhinges st attachedtothe cage. The stopper consists ofelastic bodiessuch asrubber and springs installed tostop hedoor frotn swinging outwards andlessening tlteshock ofcollision. Byp]acing thetrap on the e-abedasthe door confronts prevailing waves, the dooris openedinwards bydrag force exerted Ftgara1. Drift-algaltrap with a tnoveabledoor. r thcheight of the door, h, the heightof theflow- transportedwith progressivewaves near the trap shield, and u, the instantaneouswater velocity at from the front to the back of the door, and denote collision. The coefficient C is empirically the crosswise distance between the course of determinedas 1 Kawamataand Suzuki 1995!. oncomingdrift algaeand the axisof thetrap asy. A laboratoryscale model experiment Kawamata TRAPPING MECHANISMS andSuzuki 1995! showed that a trapwith the door A laboratory scale-model experiment properlydesigned has the followingability to trap Kawamataand Suzuki 1995! clarified the trapping drift algae.The probabilitythat drift algaecoming mechanismsand efficiencies. Under progressive in thecourse of y wifl be trappedcan behigh more waves, drift algae are transportedin the wave than approximately90%! when the courseis directionwith oscillatorymotion When a trap is within the door i.e., y < b/2! and thendecreases placedand oriented with thedoor facing the wave- as the course is more distant from the door, The corningdirection, drift algaeare trappedin three approaching course at which the trapping ways, First,drift algaestraightly approaching the probability begins to be zero may reachthe door doorarc trapped as described earlier. Second,drift width away from the side of' the door i.e., y = algaemoving in the courseoutside of thetrap are 1.5b!, The relationshipbetween the. trapping graduallydrawn towardthe side of thedoor, and probabilityand the ratioy/b seemedindependent then are moved back to the front of the door and of the absolute value of the door width. Hence, enterthe trap. Third, drift algaeapproaching the the trappingefficiency defined as the integration door from the front are once transportedto the of the trappingprobability overy divided by h may sideof thc door,and then are trappedin thesame reachalmost 2. This suggeststhat the trap may way as in the secondprocess. The fact thatdrift havethe equivalent of completelycapturing drift algae are carried obliquelyto the door and that algaepassing thmugh twice the width of the door. drift algaeonce passby the door and then are The trappingefficiency may remain at sucha high drawnback to the frontof the doorare explained level whenthe heightof theflow-shield is greater by thecrosswise flow producedas follows, When than a limit, which is never lower than 0.25 m in the direction of oscillatory flow turns from full scale.The higherthe door or theflow-shield!, outwards to inwards of the door, the door turns the greaterthe impulsiveforce on the stopper. back to the upright position and stops at the Thus,the optimal height of the flow-shieldis the stopper.However, the water iminediately outside limit, probablyapproximating 0.4 to 0.5 rn. of theflow-shield is entrainedby thefiuid passing over the flow-shield and then moves outwards, FIELD TESTS followedby the fluid fromits sides. The crosswise To verify the effectivenessof the trap. flow caniesdrift algaefmm thesides iminediately field experimentswere conductedwith a simple ahead of the door so that they are readily test device from August to December 1994 transportedinto the trap with the subsequent Kawatnataand Suzuki 1995! and werc redone inward flow. When the moment of the with a revisedtest model from August1995. The hydrodynanucforce on the dooris largecompared deviceswere placed at 9-m depthson a relatively with thatof the gravitationalforce on thedoor i.e, flat boulder area on the northeastern Pacific coast the door readily follows the flow!, drift algae of Honshu,Japan 8'22'N,141 26'W!. The site coining from the front of the door are mostly wasimmediately offshore of a steeplysloping bed trappedin the first process,Otherwise, they are that reachedthe shore. The shore was partly trappedin the third process,because the slowly protectedbut constantly washed by waves.Kelp tnovingdoor produces a highpressure region or K bicyelis were abundant immediately below low a separatedflow region!in frontof theflow-shield waterlevel while the deeperarea was barren with whenthe water begins io moveinwards of thedoor. a high densityof sea urchinStrorrgyfoceritrorus nudus. Abalone Haliotis discus hanriai occurred TRAPPING EFFICIENCIES mostly in the kelp bed but wit.h lower density. Let us consider that drift algae are Waveaction seemed to preventsea urchins from 234 UJMt TaettateaiReport No. 26 Figtsre2. Thefirst testdevice placed on theeaperitnenta! site Htttsra3. The titaniutndrift-algal trap testdevice. invadingthe shallow kelp bed, as describedby way. Final.ly, drift ke]p caught in the cage Kawamata in press!. accutnulatedfrom the innermostpart of' it but did The first devicewas a stainlesssteel cage notpile upapproximately 0.4. tn abovethe bottom, .4 x 2 x 0.6 m! with a door of 53 cm height, suggestingthat the height of the cage could be whoselower portionwas coveredwith a 5-mm- lowerwithout decreasing the effective capacity for thickfiber remforced plastic board to create a 286- drift algae. mm-highflow-shield. The massand the moment Improved in theserespects, the second of inertiaof the door were 11.1 kg and 1.54 kg/ devicewas made asa morepractical modeL The in', respecti vely. The trap wasfirmly anchoredto devicewas a 2.5 m !ongx 2,0 in wide x 43 cin- the bottomusing underwater drilling equipment high titaniumcage incorporated with a titanium and orientedwith the door facingthe shoreand doorand rubberstoppers Fig. 3!, The doorwas a perpendicularto the directionof oscillatoryflow 196 cin wide x 40 cm -high titanium plate Fig. 2!. Threeiron springs with 300-kgcapacity reinforcedwith thickerplates at the margin. Thc mountedon iron plates 5 x 30 x 1 5 cm! were mass and the moment of inertia of the door was separatelyembedded in front of the door as the 6,37kg and 0,361 kg/m', respectively. Iron weight stopper. The device successfullytrapped drift amountingto 1920kg wasplaced in the innermost Fisenita nearly to the full, In addition, partof thecage. Theweight necessary to stabilize investigationssuggested that the trap couldhold thetrap was estimated from Eq. 4 witha design driftalgae until they were consumed by aggregated water velocity of 1.2 mtsec,which was determined sea urchins. Despite such high effectiveness, fmmthe velocitymeasurement conducted in 1994. engineeringproblems for practicalapplication The improveddevice also succeeded in trapping surfacedduring the first test. First,stainless steel drift kelp up to thefull approximately80 kg wet may be corrodedin seawater,so that making a weight! Fig. 4!, and in holdingdrift algae until doorof stainlesssteel mightresult in a heavydoor theyare consumed by aggregatedsea urchins Fig. with a lowertrapping efficiency. Thus, it might 5!. Drift algae were almost absentaround the be better to use more cormsion-resistant metal with devicethroughout the year except in earlyautumn, a smaller specific inass density. Second, to whereasdrift algaefrequently reinained in thetrap facilitate the installation of traps,the stopper with intensivegrazing by sea urchins.The door shouldbe combinedwith the mainc-age and the wasamended because of slightdamage due to its trapshould be simplyfixed by weightsin a general insuflicientstiffness. With this amendment,the Kawasstata 235 Flgtsre4. The improvedtest device trapping drift kelp to Figure 5. Seaurehins aggregated at the trapped drif kelp. the full. devicehas been functioning over 2 yr, showing comparedwith a greatconsumptive capacity of proinising results, aggregatedsea urchins, it couldbe expected f'rom the hightrapping effectiveness that the device DISCUSSION tnightbe an effectivetechnique for nearshore rocky aquaculture. Observationssupported the hypothesis Only oneor twoabalorie were found in thatmost drift algaewere swept out of shallow the device,probably because of a low population habitatsbefore being consumed by animalsunder density. However,observation made in late natural conditions, Abalone and sea urchins August1997 recordednine adult shells in the occasionallycaptured small pieces of drifting kelp, device, but neverfed on entire detachedkelp plants. It Sincethe densities of drift algaeand their wasobserved that 1 arge amounts of driftkelp were herbivoresalso vary with the location,the trap accumulatedin a crevice near the trap in early shouldbe placed at the pathon a nearshore,deeper autumn.However, the drift kelp soondisappeared barrenground, through which plants detached fmm withoutaggregating animals. The driftkelp in the kelpbeds may frequently pass. Like this field test, creviceoccasionally oscillated itt a "huge"body, a small embayrnentwith a relatively flat and with the result that smallaniinals hardlygrasped depressedbottom near the opening is a potential them. ln contrast, the test devicesfrequently appropriatesite for application, caughtdrift algae and maintained them until they With high trappingefficiencies, the trap were consutncdby congregatedsea urchins. mayalso be used as an "automatic feeding system" The observedvariation in trappeddrift for underwatercage culture, e,g., by stocking algaesuggested that drift algaesporadically occur starvingadult sea urchins or leanadult abalone in at storms,especially during the first storm after a cagewith adequately small meshgrids placed summer,in whichkelp biomass reaches maximum. closelybehind the trap. Consideringsuch sporadicoccurrence of drift Finally,the durabilityrequired to resist algae,an effective drift-algal trap is a devicewhich therepeated collision should be exanuned by field can catcha largeamount of drift algaeoccurring tests,The present experiment will be continued at rough seas and can reserve them under to validateits practicalapplicability. subsequentlyrepeated severe waves. Although the capacity of the test devices was too small zs6 Usia TcchakA Rtyorl!va 26 CONCLUSION 60. In Japanese!. Kawamata,S. 1988. Developmentalstudies on In nearshorerocky fishing grounds with block-type drift alga traps with slit a highdensity of seaurchins, kelp bedsmay be openings.Bul!. Natl. Res.Inst, Fish. Eng. confined to areas where the wave-induced 9: 1-8. In Japanese!. oscillatoryflow cons4mtlyprevents sea urchin Kawatnata,S. 1991, Physicalconsiderations for grazing.The velocitylimit for feedingby sea the designof algal drift traps. U.S. Dept urchinsis approximately40 cmlsec, There is a Commerce,NOAA Tech.Rep. NMFS 102 possibilityof developingartificial kelp bed.s in sea 171-1 80. urchin-dominated areas by increasing the wave- Kawamata, S. 1994. Importanceof physical inducedwater velocity at calm sea periods. In disturbancefor artificia! developmentof suchareas, drift plantsmay be the primaryfood nearshorerocky fishing grounds,Fish. Eng. of seaurchins and abalone, but mostof thetnmay 31 !: 103-110. [In Japanese]. be swept out of hc shallow habitatsby water Kawamata,S. In press. Effect of wave-induced movement. The previous stable structurescould oscillatoryflow on grazingby a subtidalsea not trap drift algae in most shallow habitats urchin Srrongylocenrrorus nudus A. becauseof thehigh mobility of drift algaein water Agassiz!.J, Exp, Mar, Biol. Ecol. flow, The devicedeveloped by Kawamatain 1994 Kawamata,S, and H, Suzuki. 1995. Deve!opment couldcatch drift algaeeffectively and hold them of a drift-a!gal trap for nearshorerocky unti!consumed by animals, aquaculture,pp. 628-633 In: Proceedings of the International Conference on LITERATURE CITED Ecological System Enhancement Technology for Aquatic Environments Agatsuma,Y., K, Matsuyama, A. Nakata, T. S i xth In t. Con f. Aquat. Habitat Kawai, and N, Nishikawa. 1997. Marine Enhancement!.Japan International Marine algal successionan coralline flats after Scienceand Technology Federation, Tokyo, remova!of seaurchins in SuttsuBay on the Japan, Japan Sea Coast of Hokkaido, Japan. Kawamata,S., S, Hagino, and M. Yamamoto. NipponSuisan Gakkaishi 63: 672-680, [In 1993. Modome-shisetsu no kaiso-hosoku Japanese ]. koka ni kansuru kenlr.yu studies on the Denny,M.W. 1988, Biologyand the Mechanics trappingeffects of drift algaltraps!. Heisei of theWave-swept Envimnment. Princeton 3-Nendo Engan-Gyojo Seibi-Kaihatsu- Univ. Press,Princeton, NJ. 329 p. Jigyo ni Kansuru Suisancho-Kenkyusyo I watePrefectural Fisheries Experimental Station. Kenkyu Hokoku reportsof the research 1988. Ganshou-kaitei ni okeru moba-zosei- institutesof JapanFisheries Agency on the jouken ni kansurukenkyu study on the CoastalFishing Ground Irnprovernent and conditionof kelp-beddeve!opment on the Development Project!, pp.! 39-164. In rocky bottom!. Specific ResearchGrant Japanese!. Program Report, 1975-1977. Iwate Kuwahara, H., S. Akaike, H Hayashi, and Y, Prefectural Fisheries Experimental Station, Yammhita. 1997. Mechanismof group Kamaishi,Iwate. 15 p, [In Japanese]. seaweedformation in isoyakearea, Jpn. JapanCoastal Fisheries Promotion Association. Sac. Civil Eng. 44: 1181-1185. [In 1993. Structui'a!design guide for the Japanese]. CoastalFishing Ground Improvement and Lawrence, J.M. 1975. On the relationships Deve!oprnentProject Engan-GyojoSeibi- between marine p!ants and sea urchins, Kaihatsu-JigyoKozobutsu Sekkei-Shishin!. Oceanogr. Mar. Bio!. Ann. Rev. 13: 213- [In Japanese], 286. Kawamata,S, 1987. Drift alga trapsto feed Lubchenco, J. and S.D. Gaines. 1981. A unified benthica!ga! feeders. Fish. Eng. 24 !: 53- approach to marine plant-herbivore interactions. I. Populations and communitics,Annu. Rev. Ecol. Syst. 12: 405-437. Stone, R.B J.M. McGumn, L.M. Sprague, and W. Seaman, Jr. 199L Artificial habitatsof the world: synopsisand major trends, pp. 31-60. In: W. Seaman, Jr. and L.M. Spraque eds.!, Artificial Habitatsfor Marine and Freshwater Fisheries. AcadetnicPress, San Diego, CA, Terawaki, T., S. Arai, and Y. Kawasaki. ]995. Methods of subtnarine forest formation considering local limiting factors of distribution.Fish. Eng,32 !; 145-154. In Japanese!. Sraaintnu-Smith aud Messier 239 DESIGN CONCEPTS FOR INTEGRATION OF OPEN OCEAN AQUACULTURE AND OSPREY" TECHNOLOGY BnanBraginton-Smith The Conservation Consortium 4380 Main Street Yartnouthport,MA 02675 e- mail:consortI capecod.net and Richard H. Messier Universityof Mame Departmentof MechanicalEngineering Orono, ME 04469 e-mail:[email protected] ABSTRACT A uniqueopportunity exists to developa rnulu-benefit,cninmercial ocean enterprise off the coastof New England,with potentialsites reaching from Nantucket,Massachusetts, to Eastport, 5/lxi no. This enterprisecoutd generateelectric power,provide a basefor installationof openocean aquaculture facilities. help revitalize depletedfisheries, recycle discarded materials, and enhance economic de ve lo pment in severalcoastal industries, includingshipbuilding and tourism. The conceptis centetedon the Ocean Swell Powered Renewable Energy OSPREY!technology device developed by AppliedResearch dc Technology ART! of inverness,Scotland. The OSPREYis a hybridwind/wave energy generation system wherein wave energy is harnessedby meansof sn oscillatingwater column within a collectorchamber anchored to theocean floor by large, permanently insudled ballastuutks, fbe OSPREY formsthe centralelement in a multi-useocean structure.Successful establishment of aquaculturefacihties in thehigh energyenvironment off theNew Englandcoast will requiresolutions to a numberof engineeringchallenges including s~ integrity, security,operability, maintainability, and affordable design.The concept of designintegration of aquaculturenet pens into theOSPREY provides the opportunity to mee anumber of thesechallenges in a cost~ffective manner. Concepts for integration of submersibleand floating net penstructures and their operation into theOSPREY technology must be advanced,potentiat sites must be studied, and benefits to the aquaculture industry determined. INTRODUCTION prevalentduring the fall and winter seasons in these areas. Large, wind-driven waves and strong The purposefor this paper is to present currents are common in these latitudes, even anddiscuss ideas for anexisting technology which relativelyclose to the coastline.Permanently may providea viable technicaland commercial moorednet pen structuresmust be designedto partnerfor offshoreaquaculture facilities in the maintainstructural integrity and net integrityitl future. It is clear,from researchconducted by the conditionsapproaching sea stateseven perhaps present authors and many others, that the highersea state during hurricane season!, and establishmentof a viable offshoreaquaculture currentsup to 3 to 4 knots. industryin theNortheast United States will require Thc offshoreaquaculture facilities must meeting numerous technical and economic bedesigned to providefor logisticsupport from challenges. shoresidefacilities through the use of service An offshore location for aquaculture vessels. The service vessels must be able to come facilities will require structural design for alongside,dock, and offload personnel, supplies, survivability in the severe weather, which is andequipment, Furthermore, products will be ugNtt Teehaleelttepaet No. 26 Flgare1. Modniarfloating net pen, harvestedand delivered toshoreside facilities for marketcompetitiveness is expected to increase. processingby the sameservice vessels. The Thecost for purchase, tnaintenance, andoperation aquaculturefacility must provide for effective of offshorefacilities will be higher than such costs harvestingand net handling, for today'sinshore facilities. These trends are Oneof themajor concerns voiced by the clearlyin conflict.Future. offshore aquaculture operatorconununity has been associatedwith operatorswill be required toexplore new and novel securityof suchoffshore facilities. These techniquesfor reducing cost and increasing structureswill represent potentially large capital productivityinorder toremain competitive. investmentinnet pen structures and finfish stock. Severalnet pen architectures arecurrently Accessto thesefacilities will be limitedand/or in useor areunder consideration for offshore impossibleduring extended periods ofhad weather. facilities,Figures l through 4 illustrate floating, Thisis in contrasttothe existing operating modularnet pens, two types of individual floating conditionsinshore, where daily access and visual netpens, and a submersiblenet pen concept inspectionare possible during all weather advancedbythe Ocean Engineering Department conditions.Potential security concerns include at the Universityof NewHampshire. Each of accidentalcollisions bycommercial shipping and thesearchitectures provides relative advantages offshorefishing vessels, intrusion bymarine anddisadvantages. Each meets the projected mammals,human intrusion, and detectionof requirementswith varying degrees of success, structuraldegradation. Theeconomic challenges associated with NotionalTechnical Requiretneats establishmentofa viable offshore aquaculture In orderto visualizethe potential utdustryare substantiaI. The market conditions advantagestointegration ofaquaculture netpens forfinflsh products in the United States are withOSPREY technology,it isuseful to revie w extremelycompetitive today. As finfish specificnotional engineering technical requirements quacultureexpands in developing countries, foroffshore aquaculture. It is expected that these Sraginton-Sntith and Messier 24l Struetumlit!Supported TensionedCttge ME@i>!!!t!ttutl!!s [e!lc4N!ttteu!!!! L @lit!ke3 res!e Figure 2. Individual rigid floating net pen. Figure3. Individual flexible floating netpen. facilities mustmeet the following operationaland The structure must retain integrity during technical requirements: periods of severe weather. As described ~ The facility must have the capability for earlier,these periods would be primarilyduring independent operation without human the fall and winter seasons However, brief intervention for extended periods. These periodsof severeweather may occur during periods would be primarily due to weather spring and sutnmer seasonsas well. conditions. This operational need is distinct The facility tnustprovide a stableplatform for from today's practice of daily human personnel to perform operation and intervention which is, in most cases,continuous maintenance tasks during periods of during daylight hours, occupation. Walkways and enclosures are 242 t JAR Technical Repnri lSo. 26 aboard. ~ The facility must provide sheltered spaceswith suitable environment for electrical and electronic equipment, Such equipment may include sensorsand monitoring equipment for intrusion detection and alarming, net containlnent brcach, structural failure, weather COnditiOnS,autOlnated feeding Cquipment, navigational aids, telemetry electronics, actuators for surfacing and submerging of submersible net pens, materials handling equipmentsuch as cranes and hoists, electricity generation, and others. ~ The facility must provide security monitoring and alarming, Security includes human intrusion, predation, structural integrity, and net containment integrity. Thefacility mustprovide an on-boardelectrical power source for sensors, monitoring equipment, processors, navigational aids, telemetry electronics, materials handling, autornatcd feeding, and actuators. Through considerationof the operation and maintenance of a future offshore aquaculture ? TAOkmA??t?? facility, it becomes clear that the design of such a facility must be very carefully considered, with participation by experienced operators along with mechanical, electrical, oceanengineers, and marine Figure 4. Pull-up pen PUP!, submersible net pen, biologists. A successful design will require an interdisciplinary approach by a dedicated design team. needed. Personnel safety must be ensured. In considering a relatively mature Also, stability of the platform may be an issue technology for electrical power generation using for certain species of finfish envisioned for wave energy, known commercially as Ocean Swell offshore culture. Powered Renewable EnergY OSPREY !, it Provision for alongside operations by service became clear to the authors that an opportunity vessels is required. This consists of features for strong synergy between renewable power for tie-up, fending, loading and unloading of generation and aquaculture off the New England cargo and personnel, and conduct of net coast exists. Design of an aquaculture facility for handling operations. Successful performance integration with an OSPREY~ array offers the of these evolutions in open water will require potential to satisfy tnany if not all of the engineering careful engineering design and careful planning technical requirements for aquaculture described at time of execution. above. In addition, businessarrangements may be The facility must include sheltered storage feasible between the aquaculture operator and the space for feed, tools, spare parts, lines, nets, power generation commercial entity such as lease! and other miscellaneous equipment. which greatly reduce the cost of operation and The facility should provide habitability space maintenanceof the aquaculture facility, In the next for short overnight visits by personnel during section, we provide a brief overview of the periods of settled weather and high activity OSPREY" system and notional concepts for Bragiuton-Smith and Messier 243 integration of nct pcn designs. Thc rcadcr is estimated to cost $5 million US to build, including encouraged to consider the potcntia] advantages an NEG Micon I-Mw wind generator. Figures 5 for structural integrity, protection from wave action, through 7 depict OSPREY units in various settings. housing, storage, and electrical power which may Each unit will bc constructed in a local accrue from such intcgratcd design, shipyardand launched,towed or thrustcd to thc deployment site where it is scuttled and ballasted OSPREY" system description and concepts to thc bottom, then interconnected to the grid via for integration with aquaculture net pen submarine cable. Each unit will be designed for designs local wave resourceoptimization. When deployed, The concept of utilizing oceanwave action each unit will displace in excessof 8000 tons, This as an oscillating water column for energy formidable structure, when deployed in arrays of gcncration is an acceptedtechnology and is utilized five to ien units, will be able to enhance the wave in numerous applications around the globe. Most capture resource and the aquaculture resource of these installations are based upon coastal yield can be increased substantially, geology and therefore offer poor replicabi lity on a gcncral scale. The ability to fabricate such a device LIMPET Locally Installed Marine-Powered into a deployable configuration would have far- Energy Transformer! coastal harbor reaching impact on renewable ocean energy deployment unit utilization and sustainable-yield aquaculture Where practicable, structures could be development. This proven technology was realized deployedalong coastal harbors, improving harbor throughthe developmentof OSPREY by Applied defense against ocean wave action while Research&Technology of Inverness,Scotland. The generating clean renewable energy. We have OSPREY unit is fabricated of a closed rear identified our oceans as an important new frontier; bulkhead catamaran design. Each unit would we will need a platform to develop our coastal consist of 800 tons of steel or concrete and is marine technologies a.nd OSPREY is ideal, An Figure 5. Dimensional 3-D drawing of OSPREY. 244 UJNR Technical Report Nn. 26 Figure 6. Rocky coastal harbor unit, Figure 7. Ocean energy unit in storm conditions. arrayof ten OSPREYcould provide offshore inay increasethe cost competitiveness of finfish researchopportunities for a better understanding culture.Furthermore, the integration of aquaculture of our coastal ocean resources, The structure structures into the OSPREY" design cou!d couldprovide sheltered facilities for thesupport of probablybe accomplished wiih minimal impactto oceanranching as suggestedin the open ocean the OSPREY mission, cageculture section of thispaper. The structure The first stepmust bc to fully evaluatethe wouldpetfortn well as a structurefortneteorological potentialmarket detnand for commercialpower arrays, remotely operated vehicle ROV! generationwith the OSPREY~' system off the deploytnent,aquacul ture, and cage culture as well Northeast coast of the United States. h must bc asoffshore laboratory deployment. shown that OSPREY"~ can generatepower at significantlevels at a competitivecost with fossil Eaelt site will determine structure design or nuclear sources. I should be noted that with Thevariability of theocean wave resource theon-going deregulation of the powergeneration makes the specificdesign of the wave capture industry in the Northeast, several states are chainberand ancillary design criteria such as those consideringmandatory use of renewableenergy outlinedpreviously very site-specific.It is therefore sourcesat a percentageof thetotal energy usage. necessaryto understandthe resourcefrom a It must be shownthat OSPREY can compete regionalcoastal perspective with energetic resource with wind and hydropowersources. sitesgiven a priority developmentfocus. Coastal A systemsengineering study is required resources, demand characteristics, and to integratenet pen concepts with the OSPREY" detnographicswill detertninethe final econonuc structute, Conceptsfor aquacultureinechanical potentialof eachdeployment opportunity, and electrical systems, aquaculture primary The authors believe that the overall structurefor alternativeatchitectures, and concepts economicresource represented in thedeveloptnent for operationof boththe OSPREY systemand of regionalOSPREY centerswill be substantial, the associatedaquaculture facility must be Theaquaculture conununiry is in generalagreement developedand evaluated. An importantelement thatoffshore cage culture will becomean important of this systemsengineering effort must be cost componentof the futurefor sustainable-yield estimates for acquisition, construction, andoperation aquaculturedevelopmenL The OSPREY unit of the integratedfacility. presentsthe opportunityfor a myriadof inarine A final elementof thisearly work inust be technologyapplications as well. A detailedocean the examination of the range of commercial energyresource assessment should be conducted. arrangementswhich may be possible between the This study wilt determine the wave resource aquacultureoperators and energygeneration potentialand unit design criteria for a pre-designated operator.The objective of this systeins engineering area, ideally, from Florida, USA, through effort will be to establish the technical and Newfoundland, Canada. commercial feasibility of the integration of aquaculturewith OSPREY power generation for Recommendations for future work sites off the Northeast coast of the United States. Futureoffshore aquaculture facihties tnust be designedto meetchallenging engineering and technicalrequiretnents. Potential for meetingthese requirementsmay be substantiallyimproved tough integrationofthese facilities into the design of OSPREY" offshore power generation structures, The OSPREY~ technology is relativelymature and commerciallyavailable. Potential exists for attractive commercial arrangementsbetween the aquacultureoperators and the power generationbusiness entity which Tati ayan atti 247 WATER QUALITV GUIDELINES FDR AQUACULTURE: AN EXAMPLE IN JAPAN Kazuf'urniTakay anagi NationalResearch Institute of Aquaculture Nansei, Mie 516-0193, JAPAN e-mail:[email protected] ABSTRACT The basis for setting up water quality criteria WQC! for the protection of aquatic living resourcesset by the japanFisheries Resource Conservation Association FRCA! is thatgood water quality is needed to providefor thehealthy growth of fish andshellfish and to maintaintheir higheconomic values. Elevenparasneters are establishedto mainuunoptimal conditions. The p~s aredissolved oxygen DO!, chemical oxygen demand COD!,pH, suspendedparticulate matter SPM!, total phosphorus TP!, total nitrogen Thl!, amount of coliform bacteria, Escherichia cali, petroleurohydrocarbons, temperature, toxic chemicals,and sediments. INTRODUCTION economicallyhigh in valueso that fishermencan earnenough money for a living,These basic ideas In the 1960s, water pollution was an shouldalso be applicable to aquaculture. Human epidemicin Japanand manyfears concerning impactsare actually larger in aqusL~ltureasit is contamination of fish and shellfish were normallyoperated in semi-enclosedwaters where widespread.In responseto thosepublic concerns, waterquality tendsto get worseeven without in 1965,the Japan Fisheri es Resource Conservation anthropogenicperturbation. Association JFRCA! had established Water Quality Criteria WQC! for the Protectionof Items Related to Living Environment AquaticLiving Resources.The WQC havebeen Eleven items are listed in Table 1. These revisedseveral times, and a inajor revisionwas weremade by reviewingexisting data and also madein 1995 JFRCA 1995!reflecting the renewal consideringthe environmental quality legislation of theEnvironmental Quality Standards for Water EnvironmentalQuality Standards and its related Pollution EQSWP! set by the Environment legislation!issued by EAJ.Each item is discussed Agencyof Japan EAJ! TheseWQC in thetnarinc below. environmentare presentedand the strategyfor determiningWQC valuesis discussedin thisreport. Dissolvedoxygen DO! Normal environmental water contains Basisf' or Water Quality Criteria oxygenclose to its saturationdepending on The ideal situation in a good marine temperatureand sa!inity. But the concentration environment for fish and shellfish often is one mayfluctuate significantly ina shortperiod of time withoutany anthropogenicperturbation. But, that andit isdifficult to maintainat a steadylevel. Only is almostimpossible and it is not practicalto set a few studies have been reported for DO WQC basedon no huinanimpact. Therefore, we requireinentsfor saltwater fish. An excellent needto seeka way to reconcilehuman activities reviewby Davis 975! indicatesthat an oxygen with preservationof a goodmarme environment. saturation level less than 60% may induce Underthese circumstances, good water quality is physiologicalchanges insonM: marine species such the watercondition which producesnormal and aspile perch,dogfish, and dragonet. Saunders safe-to-eat fish. Harvested fish also need to be 963! evenreported that any reduction in ambient ti3NR TeebtticulReport No. 26 ~i t. WaterQuality Criteria fortire protection ofaquatic I; ;ttgresources; itetns related io living environtrtent by benthicorganisms suchas goby, shrimp, and crab 9FRCA!. canapparently swim away from Iow oxygen waters rnI/L!.By considering allthese facts, JFRCA judgedthe critical concentration level to induce a physiologicalchange insaltwater fish to be 3,0 tnl/ L = 4,3mg/L!. Considered as a safefactor, JFRCArecommends 6 mg/L as WQC. Chemicaloxygen dentand COD! ThcCOD is an amountof oxygen SPM moleculesequi valent to organic rnatter consumed byoxidizing chemical reagents. There are several methodsforthe determination of COD, Japanese 0.05 IndustrialStandards JIS! has listed three methods JapaneseIndustrial Standatd 1993!. Among them, thedichroinate reflexmethod isinost widely used forCOD CODst,!. But, this inethod isiriterfered bychloride ionand suffers reproducibility. Since thechloride ionis the major component ofseawater, thismethod isnot suited for seawater analysis. The recommendedprocedure isalkaline oxidation using potassiumpermanganate CODoH!. '&e CODis consideredan indicator of eutrophication.Ahigh load of nutrients andorganic rnatterfroin land disrupts themateriaI cyclings in coastalenvironinents andpruduces nutrient-rich conditionsandeutrophication Fig.1!. The target valueof CODottis notan anthropogenic perturbationlevel. ln themarine environment, reportedvalues of CODduring a pre-highly induslrializedperiod in Japanbetween 1931 and oxygenlevel produces a risc in ventilatory water 1940are less than I mg/L.However, some coastal flowin Atlantic cod. speciessuch as mullet, sea bass, and sardine are Forcommon Japanese cornrnercial fish knowntobe caught inhigher COD waters Satomi suchasjack mackerel, sandfish, yellowtail, black 1985!.Therefore, JFRCA recommends I mg/L seabream,redseabream, stingfish andpuffer, the asWQC for open waters and 2 mg/Lfor coastal incipientlethal level of DO ranges from 0.2 to 1,5 waters. rnI/L JFRCA l989! Chiba 983! reported, based onhis laboratory studies, thatthe 60% oxygen pH saturationlevelisnecessary forthe healthy gruwth Anevent of acidrain leads the public to ofsilver bteam. Fieldsurveys ofbenthic organisms anincreasing awareness ofa pHchange inaquatic inthe Seto Inland Seaby Imabayastu 983!show environments,especially inthe freshwater system. thatadecrease inDO causes a decrease inthe A suitablepH range for organisms is 6 5- 8.5.A speciesdiversity. Moreover, thenumber ofbenthic pHchange may induce a physiologicaldisturbance. %'mtsmsexponentially decreaseswith decreasing It mayalso promote a highertoxic effect of andbelow the DO concentration 2 mI/Ltheir chemicalsdissolved inwater to aquatic organisms. survivalbecomes very low. Similar studies in However,in themarine environtnent, the effect OmumuraBay by Mori et al. 973! indicate that ofacid rain is minimal due to the buffering capacity of seawater.JFRCA recommends natural levels Takay an a gi 249 Figure 1. Materialcycling in coastalenvironments modified after JFRCA!. of pH, 7.8-8,4 for seawater, as WQC. the baseline concentration and to set a WQC, especially in coastal waters. Coastal waters are Suspended particulate matter SPM! always influence by land runoff and offshore In general, aquatic organistns, especially currents. Nutrients are continuously supplied by bottom-dwelling fish, can adapt to high lands and diluted with open-ocean seawater. concentrationsof chemically non-reactive SPM. Furthermore,natural causes such as upwellingcan However, pelagic fish may show an escape bring nutrient-richwaters to the surface. movementfrom muddy waters. Furthermore,5 The TP and TN are closelyrelated to COD mg/L of SPM can be fatal to juvenile striped in seawater,and they are a sum of inorganic and knifejaw Oplegnathus fasciarus!, Natural organic forms of phosphorus and nitrogen, population of phytoplankton can also be considered respectively. Living organisms such as as SPM since SPM is defined as any substances phytoplankton are included in these fractions. By trapped on a filter. Therefore, JFRCA definition, theseliving organisins are also part of recommends the man-induced SPM such as from COD. Therefore,TN, TP,and COD arenecessary sewage and industrial effluents! of 2 mg/L for for good environtnental conditions for fisheries. seawater as WQC. There needsto be adequatelevels of TP and TN to supportgood fisheries. After all, phosphorus Total phosphorus TP! and total nitrogen TN! and nitrogen are the nutrients for primary producers These are also used as indicators for Fig, 2!. eutrophication, However, it is difficult to determine Sincethe input of thesenutrients and CODs 25ti t;JNR Teehnicttl Report No. 26 from landsis tightly regulatedby thewater pollution productionof rcd tide!. control legislation,TN, TP, and COD in seawater Satomi and Takayanagi 987! reviewed arc mainly thc results of organic production by theexisting data of TN,TP, and COD in unpolluted primaryproducers, especially in nonpollutedareas. coastal waters in Japan and recommendedthat However, in a semi-enclosed water body where 0.022 mg/L for TP and 0.32 mg/L for TN to meet water circulation is restricted, TP and TN may bc the EQSWPCOD value. Satomi 983! also accumulated in the water column and they may reported that higher populations of many triggerundesirable plankton bloom rcdtide!. An commerciallyavailable marine spccics are tound eventual collapse of the bloom can lead to the in the waterhaving TP 0,03 mg/L arid TN 0,3 consumption of DO and higher COD values. mg/L. On thc other hand,high populationsof Therefore, wc need to seek desirable TP and TN coastal fish such as mullet, sca bass, and sardine levels to sustainprimary producers,but not too arc found in thc waterhaving TP > 0.1 mg/L and much to produce high COD values such as a TN ! 1.0 mg/L Satomi 1985!. Figure 2, Nutrientlevels and fisheries. 'rakayanagi 251 Figure 3. Cycling of toxic substances in coastal environments modified after JFRCA!. By considering these reviews, JFRCA n-Hexane extracts divides water bodies into three categories, and Remnants of oil spills and tar balls are recommendsTP values of 0.03, 0.05, and 0.09 rng/ included as n-Hexane extracts. Toxic effects of L and TN values of 0.3, 0.6, and 1.0 mg/L for n-Hexaneextracts may not be clear,but fish can open waters, coastal waters and nearshore waters, accumulate odor-producing substances Motohiro respectively. 1973!. Bad smell certainly reduces the economic value. Recommended concentration is less than Coliform bacteria, Escherichia coli 0.001 mg/L, which is currently the detection limit. The main concern is for oyster aquaculture. The WQC needs to be detertnined based on the Sediment parameters safe cating of uncooked oysters, The value of 70/ Biogeochemical processesoccurring in the 100 ml is set by the food hygiene legislation for sediments can affect the water quality. Sediment oyster aquaculture. Therefore, JFRCA adapted may act as a secondary source of contamination this value for oyster aquaculture, and recommends and may worsen all thc water quality parameters l 000/100 ml. described above. Therefore, WQC is also considered for sediments. Sulfide, COD, and n- 252 UJNRTechnical Report No. 26 'Ishle2. Waterquality criteria for metais inseawater byJFRCA and EAJ. I JaptmFisheries Resources Conservation Association ~~Environment Agency of Jape Table 3. Water Quality Criteria for synthetic organic substancesin seawaterby JFRCA and EAJ. ~JapanFisheries ResourcesConservation Association *~Ettvironrnent Agency of Japan I-ITKRATURE CITED Chiba, K. 1983. The effectof dissolvedoxygen onthegrowthofyoung silver bream, Bull. Jpn.Soc. Sci. Fish.49: 601-610. Davis, J. D. 1975, Minimal dissolvedoxygen requirementsof aquaticlife withemphasis onCanadian species; a review.J. Fish. Res. Board Can. 32: 2295-2332. Japan Fisheries Resource Conservation Association.1989. Finalreportof 5-year carryingcapacity project, Japan Fisheries Resource Conservation Association, Tokyo.1003 p, [in Japanese! Japan Fisheries Resource Conservation Association,1995, Waterquality guidelinesfor livmg resources inaquatic ents. JapanFisheries Resource Baldwin nad Kraus 2S5 MARINE MAMMAL GEAR INTERACTIONS: PROBLEMS, ACOUSTIC MITIGATION STRATEGIES, OPEN OCEAN AQUACULTURE Kenneth C. Baldwin Universityof NewHampshire Ceriterfor OceanEngineering Durham, NH 03824 e-mail: kcb@christa,unh.edu and Scott D. Kraus New EnglandAquarium EdgertonResearch Laboratory Boston,MA 02110 e-mail: sdkrausCw neaq.org ABSTRACT Interactionsbetween maritte mammals and fishing gear have changed as fishing technology and activity have evolved.The interactions arewide rangi ng, and have included large seines for tuna with dolphins inthe eastern uopical Pacific,cod traps and humpbacks in Ne wfouudland, harbor porpoise and gil inets all around the northern hemisphere, andseals and saltuon aquaculture pens. Many of the mid gation strategies have been active acoustic devices targeted ata singlemarine mammal species. Moving aquaculture offshore tuthe open ocean piesenrs a situation where a varietyof interactionscould occur due to the many tnarine mammal species present. Resolving theinteractions will requitenew approaches tothe ruultiple species situation. Further. because offshore aquaculture maybe moving into significantiuarine rnarnrnal areas, special precautions mustbe taken toensure that any amflict mitigation does not lead to excludinga speciesfrom habitatcritical for its survival. INTRODUCTION shortsighteddesign that results in conflictswith marinemammals, and hencea difficult start. To meet the dcrnand for future grow- outspace for finfish,aquaculture activities will HISTORICAL OVERVIEW have to move offshore, especially in New As the demand for fish increased, the England. As offshoreaquaculture activity fishingindustry expanded to meetthis demand, begins,there is a needto considerall thepotential This required more gear in the water, situationsthat can develop with regard to marine Technologyevolved to optimize the ability of a mammals, and to address them from the singleboat to haulmore nets. Forexample, net beginning.This new level of aquacultureactivity twinewhich was traditionallya hempor cotton- will require placing large, fixed net-pen based material became monofilament nylon. structures at offshore sites, It is essential that Thisenhanced the fishing operation, but created these structures be designed to resist the problemsfor marine mammals. Nylon weather and seas, to accornrnodate the fish monofilamentis virtuallyacoustically transparent adequately,and to avoid negative interactions Vicedomine1991!, and its useled to marine with marine mammals. This ncw initiative in matnmalentang le ments,damaging fishing gear aquaculture should not be the victim of andkilling marinemammals, 256 UJNR TcchnicalReport 5a. 26 ln Newfoundland, Canada, this scenario evidencethat harborporpoise in British existedin the inshorecod fish traps, Collisions Columbia,Canada, have been displaced due with humpbacksalways occurred according to to thesound Olesiuk et al. 1995!. the older fishermen. The collisions resulted in In the examples stated above, there geardamage to the cottonand hemp lines. With were a variety of interactions with marine the adventof strongerlines, tnore collisions mammalsand fishing gear. The mitigation happenedwith more whale entanglements. Over strategieshave all beenacoustic, and have been theperiod from 1979-1990, Lien 994! reported a reactivefix to theproblcin. The mittgation that30% of themarine mammals entangled in strategy for one species interaction can fishinggear in Newfoundlandwere dead. The potentially lead to displacement or habitat responsetothis situationwas to begina program exclusionof anotherspecies, To effectively focusedon developing an acousticalarm to warn address the potential for marine marnrnal marinemammals about the presenceof the interactionsand acoustic deterrcnts in open fishinggear. A 4-kHz acousticalarm resulted oceanaquaculture, it is necessaryto formulate and was effective in reducingthe marine a basicmodel of theacoustic propagation and mammal-fishinggear conflict. A similarscenario exists with the gillnet how marinetnamrnals fit into this environment and interact acoustically. This acoustic fisheryin the northernhemisphere with the interactionscenario will provide a basis for entanglernentof harbor porpoise, The old twine netshad tninirnal problems with entanglements, understandingmitigation strategies. but the netswhen soaked were difficult to haul dueto theweight. The movement to synthetic ACOUSTIC PROPAGATION MODEL monofilamentnets improved the hauling The objective to developinga efficiency,but renderedthe netsvirtually propagationmodel is to havean understanding acousticallytransparent. En tanglements of of how a soundthat is generatedat a source harbor porpoise became relatively travelsa path and is received. Models can be commonplace.The mitigation strategy was to simpleand qualitative inwhich case they identify use an acoustic pinger to warn the harbor the salientfeatures such as source,path, porpoiseabout the existence ofthe gilinets. This receiver.Or theycan be a veryquantitative acousticpinger has a fundamentalfrequency of sonarmodel with all the complexitiesone 10kHz wi th harmonics upto the 100-kHz range desires,The goal here is to presenta simple witha sourcelevel of approximately 135dB re modelthat explains qualitati vel y thepropagation 1 liPa.The initial testing of thispinger, in environmentfor marinemammals and fishing experimentswith a validstatistical design, gear. indicatesthat it isvery effective atdeterring The basicpropagation model has a harborporpoises from gillnets Kraus et al., source,path, and a receiver.This generic 1997!. scenarioisdepicted in Figure1, Thesource in Presently,the primary conflict between theaquaculture case could be thesound froin a marinemammals and aquaculture gear is with deterrentdevice or soundsassociated with the thesalmon net pens. Seals are predators to aquacultureactivity. The former is meantto keepmarine manunals away, but the latter could thefish in the net pens and the industry has be a curiosityenhancer. developedthe practice of usingacoustic The pathis the oceanbetween the source harassmentdevices AHD! to scare the seals andreceiver. This path is most likely short when awayfrom the pens. A commonlyusedAHD consideringthe size of theoceans, but still it broadcastsa IO-kHz signal at210 dB re 1 @Pa canbe cotnplicated due to localpropaga«on witha 10%duty cycle every 4 secThis is an effects.Topography, temperature profiles, and effectivedeterrent forthe seals, but the sound ambientnoise are three factors that can readily propagatesa long distance, and there is some effectthe path. Figure 2 isa plotof thevarious cotnponentsof ambientnoise, The spectral Baldwin and Krnns 257 SOURCE: PATH: RECEIVER: GENERATES PROPAGATION DETECTION, SOUND EFFECTS HEARING Figure1. Source Pa h ReceiverInodcl for definingbasic acoustic parameters. 'I90 IUTCIINITTCOT*kO LQCOLCFFCCTC CKKTPCIJKKC~ PPOCPPLPKIOK ~ ~ ' ~ IOLO4C~ POCCIPITKTIO1 'lrI r KKKICC ! 00 190 'l ctsv 5 r4 ~ I.IWT ~ OFPPCTPlLWK KOIKC LI OWO-KCPCCOCCFPPKKLCPOP KPPKP 11lKE O'O'LOP-FKEOPCOTT VCOPKPPLLOP-OKTCP "'.".".'-;;:::;,1 '"' = I ~IPO OKPKOPCCCC PCCPTPKECOITKTKW 55 a0 ~ PCKPF'TOKFFIK OOIKC TOO >LnlrrkOKOKL TKKFF4 141K - KOKLLIWOCTC ~ ~ OKINL TKKFFCllOICC OKCP OKIKK TPKOOKL1451 l,r IPirl I CIIC'11IPET TC11 OF 1OIKC I ~ Oll l 11TNOOJKC'I ~ 111 CPPLOKPWK a CKTOKHECT KWK $0 ~ I rls~, 'l LU 141 FO11C IOCIIIFOCTI IU EL1 .''O'F l X 40 ~ 0 0 IU ~ ',L~ IL ~ 1 IU 5 90 In Ol IU IU h. ~ . IPIICVKILIUOHOCOC9 CK CI TOEPOI.COT PPC~ KIMC X D 4 0 ~OKCKOIO TKKPFIK~ ~ Nl 1 OLE'~ COO 5 1 1K KIIKFKCKOPPCK~CC~PCK POC544C CFTCCT ~ IKCIKWO OPCKCKOIWOI 90 IO 10 CO 10' IO FiCKOUNCY-CP0 FigTcre2, Ambient noise spectruln Richardsonet al. 1989! content is presentedas sound pressurelevel or This basic model canprovide insight into sound pressure spectrum level. The latter how a soundis propagatedand its soundpressure presentationis more readily acceptedin practice level, how loud it is, at range from the source. as the units !tP!2/Hz infer frequency There are two basic concepts ta understand here: dependence. The basic trends show that there there is a zone of audibility and a zone of is a higher level of ambient noise at lower influence Richardson et al. 19S9!. frequencies with a steady decreasein level as The zone of audibility is determinedby frequency increases. Shipping and industrial the ambient noise level, As sound propagates activity typically have a frequency range from out from a source, its level is diminished due to 10 Hz to 10 kHz. Biological sounds cover a spreading losses geotnetric! and attenuation wider range of the spectrum. true energyloss!. Thesetwo loss mechanisms 258 UJNR Technical Report No. 26 ZONE OF AUD IBILI'I'Y are defined as transmission loss in sonar modeling. On a plot of sound pressure level vs, range, and a constant ambient noise level vs. + 130 0 range, the intersection of these two lines defines 5 110 thc zone of potential audibility. When the received sound pressure level is greater than ~ 30 the constant ambient noise level, then the sound o X ro can be heard. This occurs at shorter ranges, At longer ranges, the ambient noise is greater 50 10 20 30 OO 30 than the receivedsound pressure level; the sound nalcncofrom lndcotrlol slto km! is effectively inaudible. This concept is shown in Figure 3A. ZONE OF INFLUENCE The zone of influence is a modification to thc above as it incorporates a response 2 threshold. The response threshold is a sound 0 pressure level above the atnbient noise that is R 110 required for a marine rnamrnal to determine that a specific sound is present. The effect of this response threshold is to reduce the range at which thc two plots intersect. Hence, the zone of potential influence is a shorter range from 10 20 30 so 30 the source than the zone of potential audibility. Dioroncotram Indootrlot aire tkml This is shown in Figure 3B. This basic model is useful in defining the Figure 3. Graphical definition~ of Zone of Audibility A! 2nd Zone of Influence B! Richardson et al. l 989! 100 00 00 30102 100 100 100 100 FXWyatkaey ES! Figure 4. Underwateraudiograms of severalodontocetes; A! white whale White et al. 1978;Awbry et al, 1988!;killer whale Hall and Johnson1972!; harbor porpoise anderson1970a!; B! bottlenosedolphin Johnson1968a; Ljunblad et al, 1982c!;Amozon river dolphin or boutu Jacobs and Hall 1972!; false killer whale Thomas et al. 1978!, Richardson et al, 1989! Baldwin and Kraus 259 above concepts. The limitation is thc usc of a characteristics. Upon close examination of single number, constant level, for thc ambient marine mammal acoustics, it is apparent that noise and the response threshold, As shown they too have spectral characteristics to their earlier, ambient noise has definite spectral acoustic behavior, 4 0 4 4 Fla0aatlcp Ha! Figure5. Underwateraudiograms of severalodontocetes: A! white whale White et al. 1978;Awbrey et al. 1988!;killer whale Hall andJohnson 1972!; harborporpoise Andersen1970a!; {B! bottlenosedolphin Johnson1968a; Ljungblad et al. 1982c!;Amazon river dolphin or boutu {Jacobsand Hall 1972!;false killer whale {Thomaset al. 1978!. Richardonet al. 1989! 100 100 100 100 OO 00 10 10 10 10 10 r~assstJf Figure 6. Underwater audiograms of several pinnipeds: California sea lion Schusterman et al. 1972!,' average of two fur seals Moore and Schusterman 1987!; harbor seal {Mnttht 19681!; average of two ringed seals Terhune and Ronald 1975a!; harp seal Tehrune and Ronald 1972!. Richardson et al. 1989! 260 UJNR Technics! Report No. 26 MARINE MAMMAL ACOUSTICS odontocctesare typically more sensttivebetween Marine mamtnals are the receiver in the 10 and 108 kHz, while pinnipeds are more previous model. They have spectral sensitive between 5 and 40 kHz. Also, the actual characteristics associated with their receiving threshold for the odontocetes is in the 40-60 db mechanisms, and also they have spectral content area while thc threshold for thc pinnipeds is in associated with various vocalization used for the 60-80 db area, cotnmunication, navigating, and foraging. The The other aspect of marine mammal effect of ambient noise and noise generated from acoustics is their vocalizations. This activity has aquacultureactivities can potentially affect the both spectral and source level characteristics. intent of thc marine mammal vocalizations. From Table 1, it is scen that a variety of type of Audiograms are the method of defining sounds are produced. These sounds have the spectral characteristics of hearing. Most different dominant frequencies, bandwidths, and audiograms have a band of frequencies over source levels. The functions of the sounds are which the hearing is most sensitive. This band short and long distance communication, varieS fOr different marine marnlnals, as well as echolocation, and reproductive displays. the threshold or hearing sensitivity. The Introducingother sounds into the water audiogramsin Figures 4, 5, and 6 show typical column, on a continual basis, that can interfere results for a variety of marine marnrnals,both with these behaviors can have adverse effects odontocetes and pinnipeds. There is a difference on the species. These "introduced sounds" can in the basic structure of these audiogratns. The impact the ability of an animal to hear the Tree»cotyEsege Seal»set ScarceLO«et T79»of el torotisetuea Pre«»»acies 0 re 1 «9» «ocsiieatioa tba ! ibas! ~ t t a! la!et«»ceo «bi»ties 0 2620 2-5.9 Sjsrea»4 Saich 1906»,h 7»ts»4to»os 0.4-12 t< Sjsree»4 Saith 1900»,~ ~ aiel«ocatisstioes 0.5-16 4 ~-3 Sjaree»4 Saith 1900e,~ echelocetioacliche 40-130 «sriohle le at el. !905, 1907 Elllec «halo «bietlas !e5-18 0-12 Stereos»t sl 1979;tet4 a»4 risher 1983; 0«ts»4~ 0.5 25 1% 4»hr»7et el. 02; ror4 o»4 richer 1983; Scbeoilloai ttetktas1966 ~ cholocatioeclicks 0.1 35 11-25 tts»4ee4 E«oao 191! Sortheceright- cticks 40»7 100 SichO»4 ltrrt r976 «hateiot ohio «hi»ties 7 t& L»ether«o»4sa4 0»thor 1979 et»co t 8. 3 Leather«o»4o»4 0»!hor 1979 9»ctiic«ktts- «hi»elec 2-30s i ll E«eae1973; Cola»et! s»4 C»14»etl 1971 ~ i4»44»tphia Neo!»catt»oclicks 0.2 150 4040 Sr»os1973 0»lt'~ 9»rgatse cliche 0.0i- , 125-135 120-140 E«eaa1973; E«eeo o»4 4»hr»7 1904 cliche 100-1IO 130 131149 ttOtle»4 4»4»ross 1973 cliche 2 100 S»sae!a»4 Saiegsic 1960»; Scbeeil1 et al. 1909 0»»St»each»4 cticks Sorctseoc E«s»o l 967 ~ olghla «hi»ties i 7 See»els»4 tet»4aic 19ddh Short-Siss»4 »hi»ties 0. 5-20c 100 Fishe»4 ter! 1'076:C»14»ett e»4 e»14»ell 1940 eliot «hei» ~ cholocetioa cliche 0.1-100 100 0~ i973 09»ta«hate 0.!-30 160100 Sech«aa»4 Schecill 1906tLereasm 1974; Qttkteo 1900» 0»sh»4tthale I@i»ties 3-10 «tat et ~ 1.1970» cticks 0.5 26» Oieaec et. 1970» Table1. Characteristicsof underwater sounds produced by Alaskanodontocetes whales, Richardson et aL, 1989! behavioral sounds,thus potentially driving a Lien, J, 1994, Entraprnentsof large cetaceans speciesfrom a site.This is obviouslythe goal in passive inshore fishing gear in of acoustic deterrent devices. The key to Newfoundland and Labrador l979- effectivedesign of thesedevices is to targeta 1990!. Report of the International single species that is problematic to the WhalingCommission, Special Issue 15. aquacultureoperation and minimize the effect Olesiuk,P. F., L. M. Nichol, P, J. Sowden,and on other species. J. K. B. Ford. 1995. Effects of sounds generatedby «nacoustic deterrent on the DISCUSSION AND SVMMARY abundanceof harborporpoise Phocoena phocoeira!in Retreat Passage,British Historically,most fishing interactions Columbia.Department of Fisheriesand havebeen single species and a reactivefix has Oceans, paci fic Biological St ati on, beenfound. The key to an effectiveacoustic Nanaimo, B, C, fix is to assume that the initialization of onc Richardson,W. John,J. P. Hickie, R. A. Davis, speciesinteraction is notexclusive for another. D. H. Thomson,and C. R. Greene, 1989. Alternatively,what detersone speciescould Effectsof offshorepetroleum operations easilybe the dinner-beB for another,A deterrent on cold water inarine rnaminals; a for one speciescould effectivelyimpact the literature review. API Publ. No. 4485, vocalizationresponses for another.Long tenn American Petroleum Institute, studies on inarine mammal exclusion due to Washingt.on DC. deterrentsounds have not beendone yet. When Vicedomine, J, 1991. Investigation of the consideringanacoustic deterrent for aquaculture acoustic interaction between harbor sites,specific local information about the ambient porpoise Phocoena phocnena! and Gulf noise,propagation path and marinemarnrnals of Maine cotnmercialgillnet fishing. MS needsto be assimilatedfor an objective,focused thesis, Ocean Engineering Program, approachfor solvingthe problem. Universityof New Hampshire,Durham, To facilitate objective approachesto NH. marine mammal-aquaculture acoustic interactions, the following points need consideration. The database for auditory responseof marine rnaintnalsneeds to be enhanced. Threshold levels where sounds producedetriinental effects need to be clearly defined, Developing a better understanding betweenspecies vocalizations, environmental noise, and deterrent sounds is critical to effectiveuse of soundas a warning/deterrent. These efforts will lead to a rational context for designingspecies specific acoustic alarms for use in offshore aquaculture, LITERATURE CITED Kraus, S., A.D. ReaL, A. Solow, K. Baldwin, T. Spradlin, E. Anderson, and J. Williamson. 1997, Acoustic alarins reduceporpoise mortality. Nature 388: 525. Goldstetn 263 NORTH AMERICAN LOBSTER CULTURE HOMARUS AMERIC41VUS!, HATCHERY METHODS, AND TECHNIQUES: A TOOL FOR MARINE STOCK ENHANCEMENT? Jason S. Goldstein New EnglandAquarium EdgertonResearch Laboratory l Centra! Wharf, Boston, MA 02] 10 e-mail:jsg olden eaq.org ABSTRACT The North American lobster Homarmsaasericonstr Milne-Edwards! is fishedintensivelythroughout tts l 1,000- bn rangeresulting in only a fractionof theseanimals survi ving longenough ta reproduce.Success in marine stock enhancetnentprograms suggestthat enhancementmay be possiblefor lobstersas well. Personnelat the lobster rearing facility of the New England Aquarium are studying the methodologiesused in the culture af larval and juvemle iobsters,as well asexplore the tnajor considerationsinvolved for inibating andcarrying out a producuve andeffective lobster stock enhancementprogram. INTRODUCTION value. Lobster stock enhancement is not a new concept.Efforts to rear andculture lobstershave The Americanlobster is found naturally existed since the early 1880s Addison and alongthe eastcoast of NorthAmerica, from North Bannister 1994!. Lobsterhatcheries originated in Carsylinato Labrador,being tnostabundant from Franceand Norwayover 130 yr ago,hatching and Nova Scotia to New York Herrick 1895!. The raisingthe European lobster Horrtartas garrvnartas major populationcenters, and thereforeinshore Herrick 1909!. The first successfulattempt at fisheries, are located within the Gulf of Maine and hatchingH, amert'cartuslarvae was in 1885 at the in the New Brunswick and Nova Scotian coastal newly establishedlaboratory of the U.S. Fish waters,where over 90% of the inshorelandings Camrriission in Woods Hole, Massachusetts arc made Cobb and Phillips 1980!. During the Rathbun1886!. Theworld's f irst lobsterculture last decade,areal expansion of the lobsterfishery facility was completedat St. Andrews,New and the continued intense inshore fishery have Bruns-wick,Canada, in 1974, The work done at focused attentionon the relationshipbetween St. Andrews closedthe lobster cycle from egg to animals in inshore and offshore areas. If consistent broodstock, Becausethe private sector was not recruitment in coastal areas dependson cgg preparedfor the direct transfer ofthis technology, productionfrom offshore,heavy exploitation of potentialgovernmental and academic research offshorepopulations could impact aII fisheries.The programsstalled, The popular perception of lobster New England Fishery Management Council aquacultureis that it is analogousto farming,in NEFMC! currently considersthe American whichlobsters produce eggs that are hatched, lobsterresource overexploited. There is currently reared,and then sent to market.The other potential no commercial culture for the North Atnerican applicationhowever, isin marine stock or resource lobster;however, these animals have long been enhancement,whereby young of yearlobsters are rearedin pilot scaleprojects, and havereceived a culturedand releasedto enhancenatural stocks, considerable amount of attention froin Tobe ableto moveforward in this area of research aquaculturists.The reason for theattention is clear. however,it is imporumt to disseminate thedetails lobsteris a verypopular seafood with a highmarket ofculturing such animals for these types of studies, 264 UJNtt Techrdcai Report No, 26 and relate what others have done to contrtbutc to inspectedfor lesions,infections, abnormalities, or lobster stock enhancement. anything cise which looks or appearssuspicious. ln mostcases, this procedureis often doneoff-site MATERIALS AND METHODS prior to bringing animalsinto the facility so asnot to introduce diseased or abnormal animals. A year-round culturing facility for thc Animals then undergo a seriesof dips, swabs, and American lobster is located in the Harold E. brushes. They are sprayed lightly with distilled EdgcrtonResearch Laboratory of theNew England water and large portions of thc shell and joint areas Aquarium. The culturingfacility incorporatestwo are swabbed and/or brushed with a 10ok solution separatesystems: a cold seawater system 8- of betadine. A smail sample of eggs are removed lOoC! for holding gravid female lobsters, and a and inspectedmicroscopically for any severecases warm seawatersystem goC! for the production of fungal or bacterialgrowth andare also analyzed andgrowout of larvaeand juvenile lobsters, for their current embryonic developmental stage by applying the Perkins eye index formula Perkins Broodstock system: 1972!. In some cases,new lobsters are weighed, Female lobsters obtained through the measured, and tagged as well. These lobsters Marine Research Station Vineyard Haven, remainin quarantinefor up to 3 wk at which point Massachusetts!, New England Aquarium staff they can be integratedinto the rest of the system. divers, or other similar sources are put through a strict quarantineprocess before they areintegrated I.arval system: into theprimary lobster recirculating system, When The larval rearingsystem consists of eight obtained, wild-caught females are first given a circular tanks or "kreisels" with cross-section physical checkup in which they are visually conical bases Figs. 1 and2! eachof 40-L capacity. i pprrepprapp ~ «J a sowcae pernppi-r Io8t4per M+~ ~~ appppppr~perppp precppavsfsT pt' pnapp ra*a Figure 1. The kteisel or circulating rearing tank, Modified frotn Schuuret al t976, With permission.! 'ntdstein 265 Figure 2. Planktonic kreisel. These 40-L tanks are ideal for Figure 3. Juvenile holding tank tray. Probably one of the larval culture. Cannibalism can be avoided by keeping the most unique attributes to lobster lab culture is the use of water moving and intensively feeding. individual rearing compartments. Elevated and seated in shallow recirculating seawater trays, to flush out organics and keep water circulating, compartmentalizing animals allows for careful identification and cataloging of lobsters, as well as a tool for easy sorting and grading, Water is pumped into the baseof the tanks through a manifold with a series of offset holes producing a cyclonic upwelling water circulation. This is necessary to keep larvae dispersed and hence Life Support: The Key to a Successful reduce lossesby cannibalism. Kreisels are typically Hatchery stocked with 2000 larvae each. Larvae are fed To efficiently culture and produce animals frozen brine shrimp Arterrti a salina, frozen mysids, for potential resource enhancement, a sound and and live amphipods. Larvae are typically held for well-designed system should be used. 4 wk at 18-2PC during which they molt three times. Recirculating aquaculture systems RAS! offer Survival to the post-larval stage is variable, with complete control over environmental growing 10%-15% typically surviving. conditionssuch as temperature, salinity, and water quality, while eliminating conventional concerns Juvenile system: about weatherand clitnatic conditions. Additionally, Once larvae have molted to the post-larval stock managementand inventory control are greatly stage stage IV!, when they normally would be simplified. A properly designedRAS can be placed ready to assume a benthic existence in the wild, almost anywhere and used to produce a wide they are removed from the larval system and placed variety of aquatic animals. The following three in individual holding trays or "condo trays" Fig. components are excellent additions to any lobster 3!. These shallow seawater trays, perforated with hatchery setup. holes, allow them to be flushed of uneaten food and wastes and also allow for identification and Chiller units: cataloguing of lobsters. Water exchange in the Continuous seed larval! supply requires cubicles is achieved by continuous circulation of manipulation of either or both the spawning or water along the trays. These animals are then hatching cycles Waddy and Aiken 1992!. weaned off of the brine shrimp diet and introduced Temperature is the dominant regulator for the to a gelatin-bound diet called "Supergel" Figs, 4 continuance of year-round larval lobsters. To assist and 5!, in this challenge, having one or more chiller units 266 UJNR Technical Report Vo. 26 Figure 4. Aside from temperature, nutrition is the most Figure 5. This I -month-old,9-min H aniericanusis ready important parametergoverning the survival and growth lor transfer into the seawater condo tray system. of culturedlobsters. Adult brine shrimpis the staplefood used here as well as enriched brine shrimp nauplii. Juvenile lobsters receive a gelatin-bound diet of brine shriinp, krill, kelp, spirulina, soy lecithin, bone meal, and crabrneai all pictured here. fourth stagestill exposesthem to heavypredation. One manageinent practice used in Europe is to rearjuvenile lobstersfor up to 1 yror morebefore stocking them in appropriate benthic substrates allows incubation periods which result in staggered which offer the proper habitat release component. hatch-out periods. This application of temperature One excellent case study on lobster stock greatly increases the chances to regulate and enhancementinvolves a comprehensive6-yr study sustain a year-round population of larval lobsters. in CardiganBay in theNorthwestern Wales district, UK Cook 1995!, The stock enhancement Ultroviolet disinfection: experiment carried out at Aberystwyth has Ultraviolet radiation in the 200-300 nm demonstrated that releases of hatchery-reared range is extremely effective in killing most juveniles onto carefully selectedsubstrate can result microorganisms. in a good rate of recaptureby the commercial fishery, with peakreturns occurring between4-6 Protein skimnrer foam fractionator!: yr after release. Differences are apparent between Foam fractionation is a cost-effective and the inshore and offshore release sites. The efficient meansof removing fine suspendedsolids Aberystwyth experiment failed to establish the 0 Jtm in size! and dissolved organic rnatter, optimum release size for juvenile lobsters. Foam fractionation is accomplishedby bubbling air Carapace lengths as low as 14 mm produced very throughwater to trap fine solidsand organics which good returns, but the recapturerate of small lobsters then generates a foam Weeks et al. 1991!. 9 mm carapacelength! was extremely poor Cook 1995!. This is an important aspect of lobster DISCUS SION restocking because post-larval lobsters would be very inexpensive to produce in large numbers by A lobster does not achieve its benthic mass rearing. It still seemsinevitable that to rear existenceuntil somepoint in the fourth stageof juveniles through several molts with acceptablylow development Botero and Atema, 1987,Govind and mortality, individual compartments will be Pearce 1989!. Releasing these animals during necessary to avoid cannibalism. The consequent GoMstein xs7 satcreasein spacerequired, and particularly in and North Wales-Sea Fisheries Commits husbandry and heatingcosts, ineans that the uni Report,Uruv. Lancaster, 32 p. productioncost of lobstersrises steeply the longer Factor,J,R., Editor!. 1995, Biologyof theLobster chey are kept. Advantagesto later releasemeans Hontarus americanas, Academic Press, a. greatlyincreased survival; the disadvantage is at New York. 528 p. m muchgreater cost. Thc long periodof intensive Govind,C.K. andJ. Pearcc,1989, Critical period cultivation priorto releaseraises. the potential stock for determining claw asymmetry in enhancement cost considerably Factor 1995!. developinglobsters. J. Exp.Zook 249: 31- Wny future lobsterstock enhancement program 35. should consider the f'ollowing factors: ! more Herrick, F.H. 1895, The American lobster; a detailed andextensive surveys of potentialareas study of its habitsand development.Bull. ~f suitablesubstrate within thc particulartest site U.S. Fish Comin. 15: 1-252, mr area, ! an assessment of the natural Herrick, F.H. 1909. Natural history of the ~ruitment patternsand populationstructure of American lobster, Bull. U.S. Bur, Fish, 29: lobsters withinthese areas with a viewto assessing 148-408+20plates. their suitability for enhancement, ! a inorc Perkins, H.C. 1972. Developmentalrates at ~gorousinvestigation of the optimumrelease size various temperatures of embryos of the f' or lobsters, ! developmentof improvedhatchery northern lobster Homarus americanus techniques in orderto reducethe unit costof the Milne-Edwards!. Fish. Bull. 70: 95-99. j xivenile lobsters,and ! a detailedappraisal of the Rathbun, R. 1886. Notes on lobsterculture. BulL c=conamics of lobster stock enhanceinent. U.S. Fish Conun. 6; l7-32, Schuur, A., W,S. Fisher, J,C. Van 01st, J.M. ACKNOWLEDGMENTS Carlberg,J.T. Hughes,R.A. Shleser,and R.F. Ford. 1976. Hatcherymethods for I ain grateful to the New England theprnduction of juvenile lobsters Homarus aquarium andthe EdgertonResearch Laboratory americanus, Sea Grant Pubk No. 48, Univ. f or providingthe support and expertise of sotnany California, Inst. Mar, Resour.,La Jolla tatiented staff members,in particular Marianne Waddy,S,L andD.E. Aiken. 1992.Environmental harrington andAlbert Barker, Also my gratitutc interventionin the reproductive processof ao W. Cook of the University of Lancaster, UK. the American lobster, Homarus Iffy thanks also go to continuousand past funding aneri carms lnvertebr Reprod,Dev. 22: as.geneies, the National Science Foundation. Grass 1-3, 245-252. & oundation, and the National Institutes of Health, Weeks, N.C., M.B. Tiininons, and S Chen. 1991. Feasibilityof usingfoam fractionation for thc X ITKRATURE CITED removaJof dissolvedand suspended solids forfish culture. Aquaculture 7!: 101-106. addison, J.T. and R.C.A. Bannister. 1994. Re- stockingand enhancement of clawedlobster stocks: a review. Crustaceana 67 !: l31- 155. X3otero, L. and J. Atema. 1987. Behavior and substrateselection during larval settling in the lobster, Homarus americanus. J. Crust. Biol. 2: 59-69. cobb, J.S. andB,F. Phillips,Editors, 1980. The Biologyand Management of Lobsters,Vol, L Acadenuc Press,New York. 463 p. ~k, W. 1995. A lobster stock enhancement experimentin CanliganBay, NorthWestern Klkuebi 2si9 gLUE MPSSELS IN THE DIET OF JUVENILE JA.IsANESE FLOUNDER Kotaio Kikuchi CentralResearch Institute of Electric PowerIndustry Abiko ResearchLaboratory Abiko,Chiba 270-1194, Japan [email protected],or.jp ABSTRACT Blue musselshfyrilus golloprovincialis were usedin the diet of juvenile Japaneseflounder. The control diet mainly consisted af white fish meal, potato starch. and pollack liver oil. and part of the control diet was exchangedwith freeze-driedmeat, fresh meat,or freshwhale blue musselin experimental INTRODUCTION examinedto developtechniques for practicaluse of the blue mussel in the diet of fish. Blue mussels area nuisanceorganism for electric power plantslocated along the coastof MATERIALS AND METHODS Japan. Excessive inusselgrowth along water intakepipes constricts and iinpedesthe inflow of EXPERIMENTAL DIET coolingwater. Generally,they arecollected once The formulationand composition of the or twice a year and buried in the landfill of the experimentaldiets are shownin Table 1, The plant after incinerationof organicmatter. The controldiet basically consisted of 8].7% whitefish removal and disposalrequire considerable cost; tneal,10. 8% potatostarch, 3.0% inineralmixture, moreover,landfill space rapidly fills. and4.5% vitamin mixture. Thebasic ingredients Several studies on the utilization of of the controldiet were replacedwith freshblue collected mussels have been conducted to work musselmeat at a rate of 17 and 33% weightt out the disposalproblems; however, no effective weight! in diets2 and3, respectively.Five percent ways but as a source of fertilizer have been of the basicingredients in thecontrol diet were developed,We previouslyreported that. the freeze- replacedby freeze-driedmussel meat in diet 4, and dried meat of blue tnussels My fi Jtts 20 and 30% with fresh whole mussel in diets 5 and galloprtsviitcialr's,predominant in power plants of 6, respectively.Live bluemussel s obtainedfioiu a Japan,can effectively replace fish meal asa main fish market were used, ln diets 2, 3, 5, and 6, ingredientin thediet of juvenileJapanese flounder fresh blue mussel meat and whole blue mussels ParalicJtfhysolivaceus, In addition,blue mussel weretninced and gmund into a liquid mostly meatappears to stimulatethe feeding behavior of water!,and usedas an ingredientin the diet. the flounder Kikuchi and Sakaguchi]997!. In Freeze-dried mussel meat was prepared as this study,freeze-dried blue musselmeat, fresh describedpreviously Kikuchi and Sakaguchi 1997!. titusselmeat, and fresh wholemussel werc used, Bluemussels in diets 2 through6 replaced fish meal andeffects on the growth of juvenileflounder were suchthat the ratio of white fish meal proteinto 279 UJARTechaiesl Repart No. 26 I O,' O I Ch %' f hl Q O 8 rt 0 lA M W & cA IO E0 W f ~ IA sA C! W cA Al I I I O 4l ~ CV 0 f O 0! IA Eh , 'o CP IB v a/! O 0 0S 0 4l 'U L7 0 Ql Cl Si S4 Q ~ lee~ I e~ 5 I S ClI S S l5 lb' H 0 N Jt S Il', 0 U S C g S Egl S E 0 0 0S ~e a ~ S S C E S S gkc S A IJ ~ l 0 u ml IO a I5 0 0 4 0 g W S S 0 a S u 4, S M 4 C 0 E Q'S O S 0 Cl S C 0C t7 W 4 S I S E el '0 Ig u C 4 Q Ol 0 M 0 4 C C4 44 cl d7 JL'~ 0 0 0 K D U O 0 R H ~ g4 4 Ol Kibac bi bluetnussel protein was 98.7:1.3, 97.5:2.5,97.5;25, experiment,thefish were tran sferred into floating 99,3:0.7,and 98.5:1.5, respecti vely. All feedstuffs, netcages 5 x 35 x 25 cm - W x L x H! within he exceptthe pollack liver oil, were ground,minced, aquarium,20 fish/cage,with threereplications per andformed into spheres of about 2 and4 mmin dietarytreatment. Fish werefed to satiationtwice diatneterusing a twin extruderwith additions of daily for 6 days/wkwith each experimentaldiet. tap water. The formulateddiets were driedin an The bodyweight of eachfish wasmeasured at the air dryer at 2PC, and an equalamount of pollack beginningand at the endof the study afterthe fish liver oil was added to each diet. Diets were dried were starvedfor 36 h, At the endof experiment, again and storedat -35"C until used. analysesof proximatecomposition of the whole As shown in Table 1, the crude lipid body and of hematologicaland hematochemical contents of' all diets were similar to each other, parameters were conducted by the methods however,the crudeprotein of diet 6 was lower describedpreviously Kikuchi et al, 1994a.b!. than that of the others. Differencesin proximatecomposition of the whole body, and the hematological and EXPERIMENTAL PROCEDURE heinatocheinicalparameters among treatments In 3uly 1996,juvenile flounder of about1 were tested for significance using the Mann- g inbody weight were transported frotn the Chiba Whitneytest Campbell1983! Dataon final body PrefecturalFisheries Experimental Station to our weight,weight gain, feed efficiency, and protein laboratoryin Chiba Prefecture. Fish werereared efficiencyratio were analyzed for significanceby in 2000-L tanks at 2 FC with a commercial diet Duncan'smultiple range test {Duncan1955!. usedfor Japaneseflounder Higashimaru Foods Inc.!,until thestart of the feedingexperiment. The RESULTS feedingexperiments were conducted for 8 wk beginningin August 1996 in two 2000-L tanks The growthand feed performancedata equippedwith a closedrecirculating seawater areshown in Table2. All fish soonaccepted the system. The tankswerc placed in a roomunder experimentaldiets and fed actively for theduration naturallight conditions and the water temperature of theexperiment. Survival rates were high in all waskept at 20i 1'C. At thestart of thefeeding dietarygroups and mostof themortality was from '2 Diet Average body weight g! Weight gain Fr, pm' ' DF" Survival initial Final ! 4.6 28.2 ' 513 144 3.2 2 1 1 2 9 7%bc 4.6 525' 149' 3. 3 97 4.5 3o.8~ 569 l47 3.2 2.1 97 4.6 31. 3' 573 148 3.3 2.1 98 4.5 27. 44 498 137 3. ! 2 2 98 4.6 28. 8 520 133' aVeragevalue Of tripliCate fOr eachdietary gZ~. Feed efficiency %, weight gain/feed proteinefficiency ratio weightgain/dietary protein intake!. Daily feed consveption batty weight.!. s Valuesin the samecolustn having satae superscript are not significantlydifferent !»0. 05 't!sbte2. Growth data of Japanese !ovnder fedsir erpenrnental dietsfor 8wtt» ' UJNRTechnical Report No. 26 Rmayh~ Hanal~t lax!bleed cell protein tg/10Qnt! <~> MO'/heal!tg/It!Qat ! Gltxxxau Rcsphate Ctlciua t3iio~ tng/100at!tng/10Ctnl ! tag/IQthtt ! tng/IOQal! 27.1'.1 s.m.P 3- Jd0. 1 4.049.2 24,3t5. tf 24.66.8 7.6'.7' 11.583.5 136t7.7 5.2tt!.P 3.m.5' 4.5tI. I' 5!Qb?BS 21.7i4.3' 7.720.6 11.5u!.6' 1342. ~ 27.4' 4 5.7'.1 3.Is0. 1' 3-5t0.1 6249235 22.Qt6.6' 23.Qt2.4' 6.8t0.2 11.420.4 138t5.4 5.200.7 3.040.9 3.5'!.l 34~7 6.8t0.6 11.3t0.3' ~.8' 25.ts4.d 4.8 .4' 3.Qt0. I' 73&fZPf Zt.at1.6' 3I.Qt2.8' 5.550.8 13.2'.5' 3. Iio. 5' 3.5t0.8 74~ ' Harmand ae axatttt ekiviaticna tbrfive ~ 2e.2t6.9' 6. Bto.2 11.4' I 1$85.tf a Sact2e 5xaxx:eecf Table 2. Thbleexperimen3.Hematological tat diets for 8characteittucs wk*' ajid contents nfsome ptasma cunuituents ofJapanese flounder fedsix fishthat jumped outof the net cages. Final body DISCUSSION weightand weight gain of fishfed diets 3 and4 weresignificantly higher than those in thecontrol p <0.05!. Theseparameters in the other Similartrends asshown inthe previous experimentalgroups were comparable to the report Kikuchi and Sakaguchi 1997! superior control.Feed efficiency was similar for diets 1 to growth,comparable feedefficiency, andprotein 4,and significantly higher than that of diets 5 and6 efficiencyratio to the control were observed m p <0.05!.Some differences were observed in dietarygroups which contained blue mussel meat proteinefficiency ratio.' however, a significantly diets2 to4! inthis study. Therefore, it isclear lowervalue vs, the control was obtained onlyin thatthe inclusion ofblue inussel meat in the diet diet5 p<0.05!, Final body weights aadweight promotesfeeding and growth ofthe flounder, even gainof fishincreased asblue mussel inthe diet if it isa verysmall amount %on a drybasis!. increased diets 1 to3!. Furthermore,thepositive effect on growth was notdifferent whether the mussel was fresh or Thehematological characteristics andthe plasmaconstituents areshown in Table3. All freeze-driedaccording tothe results ofthis study dietarygroups were identical interms ofred blood diets3 and4, Table2!. cellcounts. Although therewere some fluctuations Manystudies have been coiiducted on in hemoglobinandhematocrit values, no stimulatingthefeexling behavior offish with various experitnentaldietarygroups werc significaritly kindsof organistns, Soine amino acids such as differentfry!mthose ofthe control. Plasma protein glycine,alanine, andvaline showed activity tofish, indiets 3 and4,and triglyceride indict 4 werc especiallywhen two or threeof themform significantlylowerthan those inthe control; complexes e.g The Japanese Society ofFisheries however,therewere not any differences inthe Science1981!, Ina and Higashi 978! andlna otherparameters. 986!reported that amino acid fractions ofblue mussels hfyiilas echdis stimulated feeding activity Thewhole body composition of the ulturedfishis shown inTable 4.No significant of redsea bream Chrysophrys major, There is differenceswereobserved amongdietary groups nouseful information onfeeding attractants for n thebasis pf crude protein, crudelipid, and crude Japaneseflounder; however, it is consideredthat ashcontents. Theinoisture content offish fed aminoacids in themussel may contribute to the diet6 wassigficant]y higher thanthat offish fed promotionoffeeding activity of the flounder. leis1 and3 pc0.05!. Thefinal body weight and weight gain offish fed diets containiug whole blue mussel were similarto those in thecontrol, although the feed Klkucbi Diet Moisture Crude rotein Crude li id Crude aah 74.410.2 11. 4i 1. 4 4.2a0.6' 3 410.2' 74.520.9 18.221.9 4.310.5 3.510.1 74.4t0.5 17.3a2.6' 4.e*0.6 3.510.1' 74.3%0.8 16.5%1.1 4.7%0.3 3-4~0.1' 75.1fl.l 17.211.5 4. &0.3 3.410.1' 75. 2%0. 4 17.712.1 4. 5i0. 7 3.390.1 * See the footnote of Table 3. Table 4. Proxintatecomposition of the whole body of Japaneseflounder fed sixexpetimental diets for 8 wk %! + efficiency was lower due to higher crude ash show that blue mussel meat can be considered as content in the diet. This means that even whole a replaceinentfor fish meal in the diet for inany musselcan be usedas an ingredientin the diet for kindsof fish. However,considering the high market theflounder if theinclusion level is low upto 30% priceof bluemussels, it is hardto saythat mussels as fresh whole blue mussel!, because fish feed canbe usedas an economically attractive ingredient actively andcompensate for the high ashcontent for fish meal in the diet. Moreover, becausemany of the diet as shown in increasing daily feed of thernussels obtained from power plants are shell consumption Table 2!. Whole blue musselsM, and sometiines contain sludges and other edtdiswere usedby Berge and Austreng 989! containinants,acquiring a largeamount of meatis assubstitute for fish argentineArgenrintts silas! not considered to bc cost effective. Therefore, in a moistpellet for rainbowtrout Saba gaiIdneri. froma practicalpoint of view,it wouldbe better to They showedthat there was a tendencytoward utilizeits effecton feeding stimulation as an additive poorergmwth and feed efficiency with increasing in thediet thanas a inainingredient, at leastfor level of the blue mussel in the diet; however, juvenileJapanese flounder. statisticaldifference were not observedamong dietarygroups as shownin thisstudy. They also ACKNOWLEDGMENTS statedthat the musselmeat may be a goodprotein The authorwishes to thank Dr H- sourceand has a favorablefatty acidcomposition. Daniels,Department of Zoology, North Carolina On the otherhand, Grave et al. 979! StateUniversity, for his valuable ad»ce a reportedthat rainbow trout fed frozen blue inussel readingof themanuscript. ~ a M. edsdismeat showed much higher growth, feed Mr T Furuta,Central Research In»tute efficiency,and proteinefficiency than thosefed powerindustry, andMr, T Sato,Tokyo Uni"e~i y coinmercialtrout pellets, when the daily ration of Fisheries,for theirtechnical tts»sta"~. levels were in the samerange. Kitamuraet al 981! reportedthat the growthof red seabream LITERATURE CITED Pagrusmajor fed inoistpellets containing a powder of freeze-dried blue mussel M. Berge,G,M. and E, Austreng. 1989- Blu galloprovinc'ialismeat as a main ingredientwas iil feedfor rainbowtrout. Aquacul&m almostequal to that of fishfed commercial pelleted 81: 79-90, dietswhen the moistpellets were supplementcxi Campbell,R.C 1983.Mann-Whitn y "" p ' with Vitamin B l. 55-g9.Jn: Statistics forBiologists, 2nd These studiesand our previouswork ed. BaifukanTokyo [ JaP z74 UJNRTechmcsl Report No. zs Duncan,D.B, 1955, Multiplerange and F tests. Biometrics 11: 1-42, grave, H., A. Schultz, andR. Van Thielen. 1979, The influenceof blue mussel, Mytilas edulis,and krill, Euphausio superba,on growth and proximatecomposition of rainbowtrout, Salmo gai rdnerr, pp. 575- 586, ln: J.E.HalverandK. Tiews cds,!, Proceedingsof theWorld Symposium on Finfish Nutrition and Fishfeed Technology,Vol. 1. Heeneinann,Berlin. Ina, K, 1986. Artificialdiet with plantproteinfor fish. Suisanno Kenkyu 5: 85-91. [In Japanesej Ina, K. andK. Higashi. 1978. Surveysof feeding stimulants of the sea bream Ch rysophrys maj or!l. Ni ppon Nogeikagaku Kaishi 52: 7-12. [In Japanese] Kikuchi,K. andI. Sakaguchi.1997.Blue mussel as an ingredientin the diet of juvenile Japaneseflounder. Fish. Sci. 63: 837- 838. Kikuchi, K., T, Furuta, and H. Honda, 1994a, Utilization of feathermeal as a protein sourcein the diet of juvenileJapanese flounder Fish. Sci. 60: 203-206. Kikuchi, K., T. Furuta, and H, Honda. 1994b, Utilizationof soybeanmeal as a protein sourcein the diet of juvenileJapanese flounder, Paralichrhys oli vaceus. Suisanzoshoku 42: 601-604. Kitamura. H., H. Mizutani, and Y. Dotsu. 1981. Rearingexperiment on youngof same fishesand decapodid crustaceans fed on the meat of thecommon mussel,Mytilus edrdisgallopmvincialis. Mar Foul 3: 23-27. [In Japanesel The JapaneseSociety of FisheriesScience. 1981. Chemical sense of fish and feeding stimulants.Koseisha Koseikaku, Tokyo. 128 p. ]In Japanese!. 8e e seeoa 275 SUMMARYOF THE PANEL DISCUSSION ONCULTURE HELD AT THE CONCLUSIONOFTHE UJNR AQUACULTURE PANEL'S SCIENTIFIC SYMPOSIUMIN DUIQKAM,NEW HAMPSFIIRE, USA, 18 SEPTEMBER 1997 David A, Bengtson Universityof RhodeIsland, Rapporteur Theconveners ofthe 1997 UJNR Aquaculture Scientific Symposium included inthe final day's program an afternoonsessionduring which Japanese andU.S. scientists coulddiscuss theirmost iinpoitant research needsandtiy to identify areasand strategies forcollaboration, Thediscussion onculture wasfacilitated by Drs.D.Bengtson, K.Fukusho, andM Wilder andabout 30scientists participated otherpanels onstock enhancementandoffshore aquaculture wereheld concurrently inseparate roozns!. Theprimary research ~eeds are presented herein outline form: I. A definitionof"egg quality." Weall talk about theimportance ofqu.ality ofgametes tothe growthandsurvival ofoffspring andwe are interested inthe parental contribution togamete quality,but we do not have a standarddefirution of"quality," II. Naturalspawning. Theneed here isfor information transferfrom Japanese scientists toU.S. scientists. III. Broodstocknutrition. Collaborative research would aidthe understanding ofthe contribution of nutritionofthe parents tothe quality oftheir gametes, however wedefine that quality. IV, Cryopreservationofeggs. This issomething thatindustry wants, butthe panel identified itas high-riskresearch because oflack of previous success inthis area. V. Selectivebreeding. Especially inthe areas ofdisease resistance andreducing problems of inbreeding. VI. Larvalrearing. Themain areas offocus should beunderstanding themicrobial ecology of rearingtanks and the replacement oflive foods with formulated diets. A. Investigationofthe potential forspread ofdiseases fromaquaculture facilities to naturalpopulations. B, Workisspecifically needed ondiseases ofcultured abalone andflounder andon mechanismsforbetter import controls oa foreign seed. C. Researchand developinent of new diagnostic technology. D. Researchand development of new vaccine technology. E, Researchon disease prevention and trcatxnent. VIII. Recircul ation systems, A, Engineeringof moreefficient systems. B. Biologyand physiology of theorganisms in the systems. C. Nutritionof theorganisms in thesystem, especially to reduce the ammonia and solidwaste outputs. D. Economics related to A-C above. IX. Nutritionalrequirements of "new" species. We still lack detailed knowledge of the nutritional requirementsof manyspecies that are in coinmercialculture. X. Developmentof culture techniques for new species, especially fry production technology. The listidentified bythe panel included: grouper, Seriola, tuna, true cod Pacific!, Sebastes, tautog, seabass, haddock, and northern species of flatfish sole, Pacific halibut, Atlantic halibut!, Whilethe identification ofresearch needs was relatively easy for this group, the identification ofstrategies andfunding for collaboration wasnot. The UJNR meetings arean excellent way for scientists from the two couniriestomeet each other, but there should be more funding available on both sides for attendance atUJNR meetings.This panel discussion wasa goodway for the scientists tothink about, and agree on, research needs andit wouldmake sense for the UJNR Panel on Aquaculture tocontinue asa focalpoint for exchanges. Flounderresearch isa goodexainple ofa joint research project. Interested scientists canuse existing programsforexchange e.g STA and JSPS post-doctoral fellowships, NSF and USDA prograins, and Fulbrightexchanges!, butthe group felt that the existing programs wereinsuSicient forthe kind of long-term collaborationthat woukd be necessary to solve many ol' the problems identified above. New ideas and programs,probably involving additional goveminent funding, will be required if significant collaborative effortsare to take place in addressing theabove research priorities. Acknowledgments Theeditors would like tothank Dr, AnnBttcklin for allof herefforts in organizingand funding this symposium;Mary Masterson, Linda MacPherson, Meriel Bunker, Jane Pittroff and Susie K. Hines for theirassistance in many ways; Rollie 8arnaby for organizingthe tour and social events; Deborah Bidwell,Elizabeth Fairchild, and Nicholas King for their cheerful help during the meetings; and all thepeople whose attendance made this symposium somemorable. Credits Theseproceedings were produced bythe University ofNew Hampshire Sea Grant College Program, whichis supportedby theNational Sea Grant Office, National Oceanic and Atmospheric Adnunistration,U.S, Departtnentof Commerce, SeaGrant is a partnershipof university, government, and industry focusing on inarine research, educationand advisory service. Nationally, Sea Grant began in 1966with the passage of the Sea GrantProgram and College Act. Thisnational network rnects the changing environmental and economicneeds of peoplein ourcoastal, ocean, and Great Lakes regions. U.S, Departmentof Commerce William M. Daley, Secretary NationalOceanic and Atmospheric Administration NOAA! D. JamesBaker, Under Secretary and Adxninistrator Oceanicand Attnospheric Research, NOAA ElbertW. Friday,Jr. AssistantAdministrator NationalSea Grant College Program, NOAA Ronald Baird, Director S7n I;JNR Technic@Rcport No. 26 ATTENDEES Robert Bayer University of Maine 22 Coburn Hall Orono, ME 04469, USA Kumiko Adachi NationalResearch Institute of FisheriesEngineering Brian Beat Ebidai, Hasaki University of Maine at Machias Ibaraki 314-042l, Japan Machias, ME, USA ToshioAki yama Kenneth L. Beat NationalResearch Institute of Agriculture NMFS 422-1, Nakatsu, Watarai I Blackburn Drive Mie 516-0l, Japan Gloucester, MA 01930, USA Jennifer Allen GregoryBeckrnan NOAA/NMFS Great Bay Aquafarms,Inc. NarragansettLaboratory I 53 Gosling Rd 28 Tarzwell Dr. Pottsrnouth, NH 03801, USA Narragansett,RI 02882, USA David Bengtson David Alves Departtnentof FisheriesAquatic Veterinary Departmentof Fisheries,Animal and Veterinary Science Science Universityof RhodeIsland Universityof RhodeIsland Kingston, RI 02881, USA FisheriesCenter, East Farm Kingston,RI 0288I, USA David Berlinsky Building9, l4 EastFarm Masanori Azeta Universityof RhodeIsland Marinoforum21, ShibataBuilding Kingston, RI 02881, USA Uchi-Kanda 2-6-3 Chiyoda,Tokyo I 01,Japan Deborah Bi dwell Departmentof Zoology Kenneth C. Baldwin Universityof New Hampshire OceanEngineering SpauldingHall JereChase Building Durham,NH 03824, USA Universityof New Hampshire Durham, NH 03824, USA Nathan G. Birnbaum 4700 River Road, Unit 148 Roland8arnaby Riverdale, MD 20737-1231, USA Marine AdvisoryService Taylor Hall Russell Borski Universityof New Hampshire Bott 7617 Durham, NH 03824, USA Departmentof Zoology North CarohneState Umverstty Fredric T. Barrows Raleigh, NC 27695, USA U.S. Fish and Wildhfe Service FishTechnology Center BradfordD. Bourque 4050 BrigerCanyon Road I 8l Snake Meadow Rd Bozeman,MT 59715,USA Moosup, CT 06354, USA BraddBaskerviBe-Bridges Universityof Maine 5763 RogersHall Orono, ME 04469, USA Aneadees 279 TerenceBradley Harry V. Daniels Departmentof Fisheries Aquatic Veterinary Science North Carolina State University Universityof RhodeIsland 207 Research Station Road Building tt I 4 East Farm Plymouth,NC 27962,USA Kingston,RI 02881,USA Ian Davison Brian Braginton-Smith 5715 Coburn Hall The Conservation Consortium Universityof Maine Orono,ME 04469-571S,USA 4380 Main Street Yarmouthport,MA 02675,USA Amy R. Day LawrenceJ, Buckley 113 North Road Universityof RhodeIsland Brentwood,NH 03833, USA GraduateSchool of Oceanography SouthFerry Road Darrel] W Donahue Narragansett,RI 02882,USA 5710 Bio-Resource Oruno,ME 04469-5710, USA Lorne Brousseau 15 Industrial Boulevard RobertDudley TurnersFalls, MA 01376, USA 201 Libby Hall Universityof Maine JosephA, Brown Orono,ME 04469, USA Ocean Sciences Center MemorialUniversity of Newfoundland Chris Duffy S Johns,Newfoundland ALC 5S7 Great Bay Aquafarms,Inc. CANADA 153 Gosling Rd Portsmouth. NH 03801, USA Ann Buck1 in UNH SeaGrant Pmgram StephenD. Eddy Universityof New Hampshire GreatBay Aquafarms, Inc. Morse Hall 1S3Gosling Rd Durham,NH 03824, USA Portsmouth, NH 03801, USA Joseph Buttner StephenEllis Departmentof Biology VeterinaryM ed Office Salem State College RR3, Box 6888 Salem, MA 01970, USA Vassalboro, ME 04989, USA AnthonyCalabrese John Ewart NMFS Milford Lab Universityof Delaware 212 Rogers Avenue Collegeof MarineStudies Milford, CT 06460, USA Lewes, DE 19958, USA GeorgeChamberlain Elizabeth Fairchild Ralston Purina International Departinentof Zoology CheckerboardSquare I 1-T Universityof NewHampshire St. Louis, MO 63164, USA SpauMingHall Durham, NH 03824, USA R. ChristopherChambers 74 MagruderRoad K. Fukusho NOAA/NMFS Fish ReproductionDivision Highlands,NJ 07732, USA National ResearchInstitute of Aquaculture Nansei, Nakasuhama Mie 516-01, Japan tJJtaaTedmiad Reyort tso. 26 HtrofutntFututta CharlesE. Helseley NationalResearch Institute of Aquaculture 1000 PopeRoad, MSB 220 Nansei Honolulu, HI 96822, USA Met 516-», I Inm Junya Higano AtsushiFurukawa JapanInt. ResearchCenter Makabadai4-26-927 Asahiku 1-2 Ohwashi, Tsukuba Yokohama Kanagawa241 J~ Ibaraki305-8686, Japan HiroshiFushimi Kenji Hotta FukuyamaUniversity 7-24-1 Naraslurodai Departmentof MarineBiotechnology Funabashi-shi Santo. Gakuen, Fukuyatna Chiba27X, Japan Hiroshima 721-0292,Japan Hunt Howell JasonGoldstein Departmentof Zoology New EnglandAquarium Universityof New Hampshire Central Wharf SpauldingHall Boston, MA 02110 USA Durham, NH 03824, USA Cliff Goudey Nakahiro Iwata MassachusettsInstitute of Technology Central ResearchInstitute of Electric Power BuildingE 38-370 Industry Cambridge,MA 02139, USA 1646 Abiko, Abiko-Shi Chiha 270-11,Japan Neil Greenberg Universityof Maine Carro11 Jones 128 Hitcher Hall VeterinaryDiagnostic Lab Orono, ME 04469, USA Universityof New Hampshire Kendall Hall NaregGngorian Durham, NH 03824, USA 54 West Dane Street Beverly,MA 01915,USA Steve Jones Jackson Estuarine Lab DanielG ruenberg Universityof NewHampshire 9-5.26-606 Akasaka Durham, NH 03824, USA Mirato-ku Tokyo107, Japan Uday V. Joshi 4628 River Road HeatherHamlin Bethesda, MD 20816, USA 202Rogers Hal} Umversityof Maine Akio Kanazawa Omno,ME 04469,USA KagoshimaUniversity 4-50-20 Shimoarata WilliamR Heard Kagoshima890-0056, Japan NattonalMarine Fisheries Service A e BayLabora 113056Gheirer Hwy Shin'ichiro Kawai Juneau.AK 99801,USA Departmentof Human Sciences KobeCogege, Okadayama 4-1 Nishinomiya Hyogo662, Japan Aneadces 281 ShigcruKawamata Thomas Lauttenbach NationalRes. Inst. of Fisheries Engineering 54 West Dane Street Ebidai, Hasaki, Kashima Beverly,MA 01915,USA Ibaraki 314-0421, Japan Dale Leavitt Jane Keller WHOI Seagrant U.S. Departmentof Commerce MS ¹2 904 South Morris Street Woods Hole, MA 02543, USA Oxford, MD 2 54, USA Ken liber Kotaro Kikuchi Mote Marine Laboratory Central Research Institute of Electric Power 1600 Ken ThompsonParkway Industry Sarasota, FL 34236, USA 1646 Abiko, Abiko Chiba 270-1194, Japan Scott Lindell Aqua Future,Inc. NicholasKing 15 Industrial Road TurnerFalls, MA 01376, USA Departmentof Zoology Universityof New Hatnpshire SpauldingHall Jeff Lotz Durham, NH 03824, USA Gulf Coast Research Lab P.O. Box 7000 Grace Klein-MacPhee OceanSprings, MS 37566, USA URI GraduateSchool of Oceanography SouthFerry Road James P. McVey Narragansett,RI 02882, USA National Sea Gram Program 1315 EastWest Highway Linda Kling SSMC 3, Room 11838 Universityof Maine SilverSpring, MD 20910,USA 202 RogersHall Orono, ME 04469, USA Richard H. Messier 14 Boardman Hall David R. Kluesener Universityof Maine P,O. Box 2042 Orono, ME 04469, USA Concord, NH 03301, USA Takeshi Murai Yuichi Koshiishi Seikai National Fisheries Institute Seikai National Fisheries Institute 49 Kokubu-machi,Nagaski-shi 49 Kokubu-machi, Nag aski-shi Nagasaki 850-0951,Japan Nagasaki,850-0951, Japan Takuma Nakastme Tadahide Kurokawa National ResearchInstitute of FisheriesEngineering NationalResearch Institute of Aquaculture Ebidai, Haski Nansei Ibarnki314-0421, Japan Mei 516-0193, Japan GeorgeNardi JosephLannutti Great Bay AquafarmsInc Office of Research West 153 GoslingRoad 515 Keen Building Portsmouth, NH 03801, USA Florida StateUniversity Tallahassee, FL 32306-3016, USA Debra Oda Bruce Scamman Section of Fishes 69 Portsmouth Avenue 900 ExpositionBoulevard Stratham,NH 03885,USA Los Angeles, CA 90007, USA Tadahisa Seikai NobuyukiOhkubo Fi sheries ResearchStation Hokkaido National Fish. Res, Institute KyotoUniversity 116 Katsurakoi, Kushiro Maizuru Hokkaido085-0802, J apan Kyoto625-0086, Japan Yukio Ohsaka Tonic Siinmons Seikai Nauonal Fisheries Institute 33 Oak Street 49 Kokubu-machi,Nagasaki-Shi Old Town, ME 04468, USA Nagasaki850-0951, Japan Theodore I.J. Smith Ichiro Oohara SouthCarolina Department of NaturalResources NationalResearch Institute of Aquaculture P.O. Box 12559 422-1 Nakatsu,Nansei, Watarai Charleston,SC 29422,USA Mie 516-0193, Japan RobertStickney Paul Kilho Park 1716 Brtarcrest 904 South Morris Street Suite 702 Oxford,MD 21654-9724,USA Bryan.TX, USA DeanM. Perry James J. Sullivan NMFSLaboratory 9500 Gilman Drive ¹023 212 RogersAvenue La Jolla,CA 92093-0232,USA Milford,CT 06460, USA Rob Swift Gary D. Pruder OceanEngineering The Oceanic Institute Universityof NewHampshire 41-202Kalanianaole Highway JereChase Building Waimanalo,Hl 96795, USA Durham,NH 03824, USA William L. Rickards JamesP. Szyper VirginiaSea Grant College Program PacificIinter. Ctr. High Tech. Res. 170Rugby Road 2800 Woodlawn Dr.ive Charlottesville,VA 22903,USA Honolulu,HI 96822, USA JohnRiley NorimasaTakagi 5710Bio-Resource Engineering NationalResearch Institute of FisheriesEngineering Universityof Maine Ebidai,Hasaki, Kashiina Orono.ME 04469-5710,USA 1baraU314-0421, Japan JamesM. Rounds KazufumiTakayanagi Section of Fishes NauonalResearch Insntute of Aquaculture 900 ExpositionBoulevard Nansei LosAngeles, CA 90007,USA Mie 5I6-0193, Japan Michael Rust Masura Tanaka NationalMarine Fisheries Service Divisionof AppliedBiosciences NorthwestFisheries Science Ceoter GraduateSchool of Agrtcuhure 2725Montlake Bou.levard KyotoUniversity Seattle,WA 981l2, USA Kitashirakawa,Kyoto 606-01, Japan Timothy E, Targett Wayne Zei'Lenga Universityof Deleware 1 l 6 State Slreet Lewes, DE 19958, USA Montpelier,VT 05602, USA Andrew Tate David A. Ziemann 6 LighthouseWay The Oceanic Institute Lewes, DE 19958, USA 41-202 Kalanianaole Highway Waimanalo, HI 96795, USA Toln TsurutanI PacificIinter. Ctr. High Tech, Res. 2800 Woodlawn Drive Honolulu, HI 96822-1843, USA Yukio Uekita 190-25-305Gokasyoura N amseityo Mei, Japan NagahisaUki JapanSea National Instituteof FisheriesScience Suido-cho.Niigata 951, Japan Charles W. Walker Departmentof Zoology University of New Hampshire Durham, NH 03824, USA StephanieWarrington 15 Industrial Blvd Turner Falls, MA 01376, USA Caro! Watts 1315 EastWest Highway SSMC3, 2nd Hoor Silver Spring,MD 20910, USA Don Webster Universityof Maryland P O. Box 169 Queenstown,MD 21658, USA StephenWeglarz 54 West Dane Street Beverly, MA 01915, USA Marcy WiMer Ministry of Agriculture,Forestry and Fisheries 1-2 Ohwashi, Tsukuba Ibaraki 305-8686, Japan MashahitoYokoyama National Res, Inst, of Fisheries Science 2-12-4 Fukuura, Kanazawa-ku Yokohama236-8648, Japan