T recce 121

SHRIMP MATURATION AND SPAWNING

Granvi 1 D. Treece TexasARM University, SeaGrant CollegeProgram 1716 Briarcrest, Suite 702 Bryan, Texas 77802 USA Tel: 09! 845 7527; Fax; 09! 845-7525;e-inail: gtreece.unix.tamu.edu

ABSTRACT

The lack of a reliablesupply of disease-resistantpostlarvae PL! continuesto contributeto the uncertainty, inefficiency,and economic loss facing shrimp farmers worldwide. Many of theworld's estimated 375 913 shrimp farms reiyheavily on wild stocks for broodand seed to stockponds! and many of the5777 hatcheries Rosenberry 1999! rely onready-to-spawn adult females from the oceans as a sourceof nauplii.As theshrimp aquaculture industry has matured, thenumber of farms relying on hatcheriesfor seedhas increased and hatcheries are adopting technology tocontrol the reproductiveprocess and to producegeneration after generauon of shrimpwithout totally relying on the wild populanons. Thistechnology offers independence from the unpredictable fluctuations in wild populations,accessibility to thesuperior non-indigenousspecies, improvement in performancethrough artificial selection, and somecontrol overthe diseases found in feral stocksthrough development of disease-resistantstrains. Thetechnology for controlof shrimpreproduction has not changed much since the most important breakthroughs in this areaoccurred more than 20 yr ago.The UnitedStates Department of Agricultute USDA! ShrimpFarming Consortiumhas made progress toward domesticating thewestern white shrimp Pen«ieus vannairiei, the newly proposed genusby Perezand Kensley998! is Liropenaet«s!,and has worked toward developing High Health, Geneticaliy Improved HHGI! for the industry. More progress is neededto stayahead of theshrimp virus problems plaguing theindustry. Problems with maleshrimp quality have beenovercome to someextent by using artificialinsemination techniques but, again, more research is needed, The technologyfor controlling shrimp reproductionis under constantrefinement by commercialand academic groups.Initial breakthroughsoccurred more than 65 yr agowhen a Japaneseresearcher published the first written accountof shrimpculture Hudinaga 1935!, The seine researcher spawned the kuruina shrimp and described the techniques in detail Hudinaga 1942!. Panouse 943! describedshrimp eyestalk ablation, but it wasnot usedin commercial shrimp inaturationuntil theearly I 970s.A few advancedfarms were familiar with the techniques, but there was reluctance to shareinformation. For many years, the industry generally preferred wild-caught PL overhatchery-reared PL,but with the adventof shrimpviruses like theWhite Spot Syndrome Virus WSSV!and evidence of measuredgrowth from capuvestocks, the industry is graduallybecoming more dependent on captive stocks, Disease-resistant, hatchery-reared animalsare becoming more popular for pond stocking, Practicing the HHGI concept involves strict biosecuiity measures at thehatchery and farin tocontrol disease which generally involve limiting access and maintaining strict quarantine procedures.Pond growout comparisonshave been made with PL from different sources,and it is documentedthat domesticationandthe HHGI concept with added biosecurity measures practiced! have benefits over wild-caught stocks. TheUSDA funded US MarineShrimp Farming Program, The Oceanic Institute, and a numberof otherorganizations havemade progress in domesticatingand selecliiig faster-growing, disease-resistant families of shrimpand have brought themforth in their breedingprograms. Several private coinpanies are now utilizingthe offspringfrom this workto producefuture generationsof shrimpfor the aquacultureindustry Dr, JamesWyban, Hawaii, personalcommunication, http://www.hihealthshrimp.corn!. Several research groups have selected animals through numerous generations that do not requireablalion and the resulting animals selected spawn without ablation Dr. RobertShleser, Hawaii, personal communication!,Through selective breeding, virus-resistant strains of P. srylirostriscalled the Super Shrimp were developedin Venezuelawithout eyestalk ablation Chris Howell, Venezuela,personal communication! and this has assistedMexico to reestablishitself as oneof the topproducing shrimp mariculture countries in LatinAmerica.

INTRODUCTION j aponicusfemales and reared the resultinglarvae Maturation - ResearchHistory to subadults Hudinaga 1942!, The capture of wild Dr. MotosakuFujinaga Hudinaga1935! females with mature ovaries for immediate made someof the most important contributions spawningin captivity, known as "sourcing", was to the development of shrimp culture when he first the only method known and practiced for inducing accomplished captive spawning of mature P. penaeidfemales to spawn in captivity until the 122 UJNR Technical Report No. 28

early1970s. As discussedearlier, shrimp eyestalk Lawrence992!, In the 1990s,shrimp viruses ablation was not used in commercial shritnp forced the industry to rely less on feral maturation until the early 1970s. populations,adopt biosecurity measures, and look Thesourcing of gravidfemale shrimp is more closely at perfecting domesticationof still widelypracticed today in manycountries with .The shrimp aquacultureindustry has an abundantsupply of wild brood in nearby followedsimilar steps taken earlier by thepoultry waters.In the past,Japan had a totaloutput of and swine industries in an attempt to control 600 to 700 million PL shrimp annually using diseases. sourcedfemale P.japonicus. About 80% were used to restock coastal fisheries and the rest were Maturation aud SpawningResearch used in commercial culture Liao and Chao 1983!. Highlightsin the United States Sourcing,or obtainingready-to-spawn females Johnsonand Fielding 956! reportedthe from the wild, has been used worldwide for first successfulmaturation and spawning with experimentaland commercial culture of numerous fertilizedeggs! of P.setiferus in theUS, butthis otherspecies. This is particularlytrue in Southeast was in ponds.In 1959,the National Marine Asiawhere a singleP. rnonodon female can sell FisheriesService NMFS! hadbegun to adoptand for US$500-$2,000or more. Sourcing,however, use a modification of the Japanese culture limits culturists to the use of indigenousspecies techniqueto assistwith closingthe cycleof thatmay or maynot be the best, or evena suitable, importantspecies for theshrimp fishery in Texas. culture speciesand is dependenton seasonal Cummings961! describedmaturation and availability, migratory movements,weather, spawningin thepink shrimpP. duorarurn,Dr. naturalrhythms, and diseases in feralpopulations. Fujinagavisited the NMFS Laboratoryin Effortsto inducepenaeid reproduction in captivity Galveston,Texas, in 1963 with the intention of continuedso that a consistent,reliable sourceof scoutingthe area for a shrimpmariculture facility. PL seedstock could be obtained to support Instead,a facility waslater built in the stateof commercialculture operations and establish the Florida. Soineof the other researchpublications basis for genetic selectionto develop ideal from the NMFS lab were Brown and Patlen domesticstocks with stronggrowth and survival 974!, Brownet al. 979, 1980!,and Duronslet characteristics resistance to diseases!.Annie et al. 975!, TheGalveston Laboratory, serving Laubier-Bonichon and L. Laubier at the Centre asan important demonstration and training center Oceanologiquede Bretagnein Brest, France, for maturation-hatcherybiologists worldwide, developedthe "Laiibier method" of shrimp continued to refine maturation, hatching and maturation which involves maturatio~ of P, larval-rearingmethods throughout the 1970s. The japonicususing temperature and photoperiod methodsutilized by theNMFS researchers are still manipulation,without ablation or theremoval of widely knownas the intensivemethod or "the oneeye Laubier-Bonichonand Laubier 1976; GalvestonLaboratory Technique" Klima 1978; Laubier-Bonichon 1978!, The Laubier method McVey 1983! sometimesreferred to as the workedfor P j aponicuson a smallscale but was clearwatermethod. The methodsused today are not dependablefor commercialuse. The French basicallymodifications of this intensive method, made other important advancesin shrimp andmethods developed in Asiaand other parts of maturation Aquacop 1977a, 1977b, 1979, 1984!. the world, NMFS continuedresearch and training Additionally, the SoutheastAsian Fisheries at the Galvestonlaboratory in the early 1980s, DevelopmentCenter SEAFDEC! in the andlater under Texas AsrM University Lawrence Philippinesmade very important contributions to et al. 1980!the research continued along similar our presentknowledge of shrimp maturation lines. Similar research occurred at Texas AkM Primavera1978, 1979; Primavera et al. 1980!. University TAMU! maincampus Chamberlain Good literature reviews of maturation and and Gervais 1984! and the TAMU Texas reproductionin penaeidshrimp were done by AgriculturalExperiment Station labs in Corpus Primavera985!, Harrison990!, andBray and Christi andPort Aransas, Texas, Other institutions T recce 123

like The OceanicInstitute in Hawaii Oyama et vannarnei spawners from the Ecuadorian coast al. 1988! worked with shrimp maturation and might contradict the above Roeland Wouters, spawning. Since the late 1980s, research and CENAIM/ESPOL, Ecuador, The following developmentby the US Marine Shrimp Farming equationwas calculatedfrom 612 spawns some Program has contributed to the successof the US of themwere repeat spawns! from spawnerswith shrimp aquacultureindustry. Commercialtrials weightsranging from 27 to 80 g: y = 3665 x + 22 with the domestication of P. vannamei have 660 with R-squared= 0.1892 [eggsper spawn= resultedin diseaseresistant strains using the HHGI 665 x spawner weight in g! + 22 660j. The concept and animals are provided to the US largestspawn in this groupwas 621 000 eggsfrom industry that havebeen tested commercially and a 45-g female.Pond shrimp n=51! give similar selected from numerous families. results to wild spawners. A significant positive Hybridization of penaeid shrimp was correlationbetween fecundity and spawner weight attemptedwith P. setiferus + P. stylirostris and P.05; P.001! has been shown in most other species at TAMU in the 1980s, but their batchesof wild P. vannamei spawners.When offspringwere sterile Lawrenceet al, 1984;Bray fi]ling in datain the previouslylisted equation,it et al. 1990!.Reproduction of penaeidspecies was can be noticedthat this equationis okay for wild detailed by Bray and Lawrence 992! but litt1e broodstock,while domesticatedanimals produce to no work has been done since then on 30% less than predicted. The latter observation is hybridization. only basedon 25 samples,which is not enoughto draw solid conclusions, but similar results have Fecundity beenreported by numerous hatcheries.This could Martosubroto 974! showed that there indicate that lower fecundity of domesticated is a directcorrelation between size of the shrimp broodstock is not only due to lower spawner and the number of eggs per spawn. Other weight, but that other factors,such as inadequate referencesshowing higher egg numbers with feed, could be involved as well. Somemanagers largerammals are Emmerson 980!, Ottogalli et feed their animals excellent feeds artificial al. 988!, Hansford and Marsden 995!, and broodstock diets and fresh diets! and often Beardet al, 977!. Evidenceindicates multiple overcomethis problem in ponds.Most hatchery spawningof unablatedP. setiferus five spawns operations report that it generally takes three to per lifetime! and at least two spawnsper season four generationsto obtain pond broodstock of from P, setiferus,P. duorarurn,P. japonicus and equal or better quality than wild broodstock. Metapenaeus affinis. Multiple spawning of Operationswhich dependupon wild broodstock unablated P. japonicus and pond-raised P. shouldstart serious breeding programs as soon as vannamei has been shown in captivity and in one possible and over time the benefits will become case, an unablated female spawned 19 times in 7 apparent.Fecundity is just one of a long list of mo. Unablated P. nierguiensis have been noted to traits to select for in a breeding program. spawnan averageof 2.6 mo in captivitycompared Spawning without ablation, rapid growth, and with an average2.8 mo for Pjaponicus. resistance to disease might be others, There are conflicting data, but wild P. Other data availableon fecundity Peter vannamei generally produce average spawns of Larkins, personal communication! are: between55 000 and 150000 eggs,whereas pond- domesticatedstocks from Colombia Wt 37 g! raised females of the same species and size eggs/fernale 105,000 n=25!; wild stocks from produce22 000-100000 eggs. Larger species such Panama pond raised from wild nauplii kept in as P. monodoncan produce 700 000 to over 1 maturation tanks with Wt 29.5 g! eggs/female million eggs/spawn,For example,a 290-g0.2- 116,000 n=25!; and Ecuadorian wild broodstock oz! femaleP. monodon might spawn700 000 eggs, kept in maturation tanks with Wt 60 g! eggs/ whereasa 454-g-LB! femalemight spawn1,4 female 230 000 n=130!. to 1.8 million eggs each spawn personal Other data Dr. Henry C. Clifford, experiencein Indonesia!.Some data for wild P. personalcommunication! indicate that the pond- 124 UJNR Technical Report No, 28 reared, domesticated,P. vannamei broodstock beenreported by Bray et al. 985! andLeung- raised and maintainedunder normal maturation Trujillo andLawrence 987!. conditions, naturally mated, unablatedand ablated,averaging around 45 g females!typically FemaleReproductive System producein therange of 120,000-160,000viable Thefemale reproductive system consists nauplii/spawn.It has been more than 15 yr since of pairedovaries, paired oviducts and a single Clifford measuredthe percent fertilization per thelycum;the first two are internal and the last is centof fertileeggs in eachspawn!, but he recalled an externalorgan and was thoroughly discussed it generallyvaried from 60-90% in viablespawns. by Motoh 981!. Naturalspawning percent mating! generally were on the order of 7-12% of the female population Descriptionof CurrentTechnology in perd whenthe maturation system was "healthy" !. ShrimpMaturation and Spawning The author's experiencehas been that the Almost all hatcheriesrequire availability percentageof fertileeggs of ablatedfemale P. of oceanic-qualitywater on a 24-hbasis. Salinity vannameiis in the 90% + range shortly after and temperatureare the most importantwater ablationand tapers off with time.After 3 mo,the parametersimpacting production of shrimpin the animalshad to be replaced. hatchery,and mustbe maintainedin a narrow In contrast,Preston et al. 999! showed range,between 27 and 36 ppt salinityand 28 C that wild kurumashrimp broodstock produced 82 F! plus or minus two degreesfor most about the samenumber of eggs as equal-sized penaeids.These and other important factors in the domesticatedkuruma shrimp broodstock, but the maturationand spawning of penaeidshrimp are survival of larvae from the domesticatedstock was discussedin detailby Treeceand Fox 993!. half that of larvae from wild broodstock. They found that it would take 12 domesticatedbrood Parametersfor TropicalShrimp Maturation to produceenough PL to stocka 1-hapond, and ALlowableranges/24 hr. whereas,it would only take six wildbrood to stock Sahnity the samepond. However, the costsof postlarvae 27-36 ppt +/- 0.5 productionusing wild broodstock isAus$851 per Temperature pondcompared to Aus$390using domesticated 28 C +/- 2 80.5-84.2 F! broodstockto stock a 1-hapond with postlarvae, pH Thehigh cost of sourcingwild brood contributed 7.8 +/- 0.2 to the difference.See Magarelli 981! for further Light information, primarily with referenceto P. 14L, 10D styiirostrisproduction and the importanceof D.O. nutrition. 5 ppm

Male ReproductiveSystem Other parametersto consider in the Themale genital system was thoroughly maturationof penaeidsare nitrogen levels in the discussedby Motoh 981!. Referenceson the water especiallyammonia and nitrites! which maleshrimp spermatophore and spermatozoaare shouldbe very low to non-existent.Average sea Jeri 998!; Bauer986!; Heitzmanand Diter water has: 0.02-0.04 mg/L ppm! NH,-N = 993!; Bauerand Cash991!; Pascualet al. ammonium ion total ammonianitrogen!, 0.01- 998!. Thespermatozoa are non-motile and have 0,02mg/L ppm!NO,-N nitrite!, and 0.1-0.2 mg/ beendescribed as resembling a golf ball on a tee. L ppm!NO,-N nitrate!. Chen and Chin 988! Leung-Trujillo990! foundthat the number of found that 0.1 mg/L ppm! nitrite or abovecan spermatozoais directly related to thesize of the affect reproduction. male.She found that a 35-gmale might carry 70 Nutrition of broodstock is another million spermper compoundspermatophore. important aspect of shrimp maturation. Methods for assessingmale spermquality have Middleditchet al. 980! showedthat P.vannamei Treeee 125

grown in captivity reachedsexual maturity when 1998!" edited by ShaunMoss, Shrimp Program fed diets similar in fatty acid profiles to that of Manager with The Oceanic Institute US Marine marinebloodworms. Bloodworms have a highn- Shrimp Farming Program or see 3 andn-6 PUFAratio and this variableis thought www oceanicins itute,or . The proceedings by some to be the key factor necessaryfor a providea goodoverview of theUS MarineShrimp maturationdiet. Lytle aud Lytle 989! lookedat Farming Program biosecurity strategy and an the fatty acid composition and variations in interestingglimpse at someprivate sectorshrimp individual bloodworms, Dechan and Chen 975! farms in the United States. describedthe processrequired for the cultureof a similar Lugworin species, which could be Biosecurity Measures and Suggested modified for bloodworms. Results indicate that Criteria For Countries Importing Live squid,oysters and a diet supplementmade from Arteniia called Marilla appear to best match the ShrimpThefollowing aresuggested for fatty acidprofile of bloodworms.A largehatchery maintainingbiosecurity within a countryor area in Panamareported that Marilla, when addedto and suggestedcriteria for importing live shrimp. the maturationdiet, savedapproximately US$27 The criteria should have a sunsetof 1 yr and 000/yr by increasingthe frequency of females should be reviewed and inodified, if necessary, spawning, the total number of viable eggs yearly. spawned and the survival rate of the nauplii All imports should be restrictedto closed cycle produced. Magarelli 981! also found sex- hatcherieswith at least 2 yr experience specificnutritional requirements for crudeprotein with this larval production method. and fat in cultured P. styiirostris broodstock. He Hatcheriesshould have at leasta 2yr performance foundthat femaleshrimp required a higherprotein and health record of producing shrimp level, a lower fat level, a higher protein/calorie broodstock, nauplii, and postlarvae! ratio and a much higher protein/fat ratio than preferably with past imports to the coun- males, Again, a combination diet is most often try, which could verify in a practicalway used so that all of the essential requirements for the health statusof the larval production both males and females are met. centers. Hatcheries should be clear of It has also been demonstrated that marine Taura Syndrome Virus TSV! and WSSV polychaetescan be replaced successfullywith for the past 6 mo. Arteniia biomass for shrimp maturation and Selectedhatcheries should be askedas early as reproduction and that the culture of Artemia possible to perform a general health cer- biomass can be done under intensive or extensive tification using polymerase chain reaction conditions Naessens et al. 1997!. Roeland PCR! assay for WSSV, and in situ hy- Wouters of CENAIM/ESPOL in Ecuador and bridization for TSV in a diagnostic lab, researchersat the Laboratory of Aquaculture and for their broodstocktanks or ponds and Artemia ReferenceCenter at the University of maturationtanks in production.A repu- Gent, Belgium, have replaced polychaetes with table diagnostic lab should do such certi- Arternia, and CENAIM reports to have fication once a month before the season successfully replaced both polychaetes and begins, and during stocking season Artemia with an experimental artificial diet. postlarvaeproduced should be spot-tested for WSSV and TSV using 6-day-old PL Shrimp Biosecurity to 30-day-old PL samples. Hatcheries Biosecuritymeasures are a inustnow that shouldpractice the amplificationmethod seriousdiseases such as the White SpotSyndrome on brood if brood die, then body parts Virus WSSV! and others have plagued the are fed to other brood to amplify the ef- industrysince the early 1990s.The seriousreader fects!, should obtain the -Proceedings of the U.S. Marine If needed selected hatcheries should be visited at ShrimpFarming B iosecurityWorkshop February least two mo prior to shipments into the 126 UJNR ~col Report No. 28 country,by designatedrepresentatives of specific Otsu 1963; Boinirski et al. 1981!. the industry and regulatory authority to Researchon otheraspects of hormonalcontrol collaborate biosecurity proceduresand over maturationhas been done by: Bliss 966!; general health status. Kamemoto et al. 966!; Adiyodi and Adiyodi Samplingmethods in thebroodstock farins, matu- 970!; Fingerman970!; Silverthorn975!; ration and hatcherytanks, for viral diag- VanHerp et al. 977!; Bollenbacheretal. 978!; nostics,should be standardizedand con- Changand O'Conner 978!; Highnam978!; ductedby a reputablediagnostic labora- Kleinholz978!; Andrewand Saleuddin 979!; tory. Kulakovskii and Baturin 979!; Emmerson The country of Australia has published 980!; Bellon-Humbertet al. 981!; Faureet on the Interneta reportdetailing the country's al. 981!; Adiyodi and Subramoniam983!; animalquarantine policy, a descriptionof the Quackenbush and Herrnkind 983!; importrisk analysisprocess for shrimp,and Quackenbushand Keeley986!; Lauferet al. categorizationof shrimpdisease agents ~h:// 986, 1987!and Bradfield et al, 989!. www. is. ov.au/d an ' / 8- 86b.doc, According to Caillouet 972!, Aquacop 975!, andDuronslet et al. 975!, ovain female Mating of Open-Thelycum Shrimp shrimp are typically reabsorbed without The inatingof open-thelycumshrimp was subsequentspawning. These problems were discussedby Aquacop977b!, Primavera979! alleviatedby the ablationof only one eyestalk andDe Saint-Brisson985!. Many otherauthors unilateraleyestalk ablation!, which provided havededuced the presence of sexpheromones in moderatehormonal stimulus without reabsorption decapodcrustaceans see Dunham 1978!. of ovaor excessivemortality Arnsteinand Beard 1975;Wear and Santiago 1977!. Consequently, Mating of Closed-The!ycum Shrimp unilateral eyestalkablation rapidly emerged Matingof closed-thelycumpenaeids was worMwideas a simpleprocedure for inducing describedby Hudinaga942!, Primavera979, reproductionof numerousspecies of penaeid 1985!,Yano 987! amongothers. Spawning lasts shrimpreared in captivity.Some researchers have even used ablation to improve growth rate of from 2 to 7 min. shrimp Hameedand Dwivedi 1977!. Eyestalkablation has beenperformed Maturation Research On Hormonal usinga varietyof methodsdescribed by Duronslet Control et aL 975! andPrimavera 978,1985! and is Researchers in the 1980s were able to summarizedby Fox andTreece 000!. Shrimp isolate and characterize hormonal systems that are ablated as they prepare to enter their involved in maturation/reproductionof the spiny reproductivepeak are more conditioned to yield lobster Quackenbush and Herrnkind 1983!. a reproductive as opposed to molting!response Researchers later undertook similar work with than those entering a reproductively dormant penaeidshrimp Chanet al. 1988;Bradfield et al. period Bliss 1966!. Within a moltcycle, ablation 1989!.The pink shrimpP. duorarum was the first performedduring premolt leads to molting; penaeidto beresearched and researchers atTexas ablationimmediately after molting causes death; ARM Universitylooked into the eliminationof andablation during intermolt leads to maturation someof the husbandryproblems associated with Adiyodiand Adiyodi 1970!. maturation egg fertility, decreased spawning rate The fecundity and viability of spawns over time!, but progresshas been slow. from ablated females have sometimes been Panouse943! wasthe first to recognize inferiorto spawnsfroin femalesmatured in the that removalof the X organ/sinusgland complex wild Adiyodi andAdiyodi 1970;Beard and by eyestalkablation often results in prematureor Wickins 1980; Emmerson1980; Lumar 1981!. nonseasonalgonadal hypertrophy. This effecthas Other important works dealing with crustacean been attributed to removal of gonad inhibiting hormonal control, ablation and crustacean hormone GIH!, whichis neithersex- nor species- Treeee 127

reproduction can be found in the following as identification of cDNA markers and references; Otsu 963!; Fingerman 970!; microsatellite markersfor growth performance Silverthorn 975!; Laubier-Bonichon and and viral diseaseresistance, may lead to a higher Laubier 976!; Santiago977!; Van Herp et al. rate of geneticimprovement in shrimp Acaciaet 977!; Dunham 978!; Kleinholz 978!; al. 1997!. Andrew and Saleuddin979!; Aquacop979!; Kulakovskii and Baturin 979!; Primavera et al. Other Research on Maturation and 980!; Bellon-Humbert et al. 981!; Bomirski Spawning et al. 981!; Faure et al. 981!; Adiyodi and A few researchers and few commercial Subramoniam983!; and Bray and Lawrence hatcheries in Venezuela, Ecuador, Australia and 992!. Mexico have been able to select families in a breedingprogram and obtain egg development, Ablation of Male PenaeidShrimp matingand spawning of captivepenaeids without Male ablation causes precocious ablation, Temperature and photoperiod maturation of P. monodon and P. merguiensis manipulationalone have not produced sustainable Alikunhi et al. 1975!; however, it has also been commercial operations without ablation. As a rule, shownto increasegonad size and double mating hatcheries have not been able to base a frequencyof smaller 5-30 g! P. vannameiin long-lasting, profitable, highly-productive comparison to similar-size, unablated control commercial operation without ablation, The shrimp Chamberlain and Lawrence 1981!. nonablation approach is the desired route for Eyestalkablation of maleshrimp has rarely been hatcheriesin the future, but presentlyonly makes considered useful and the author does not up a very small percentage. Some of the research recommend its use under practical culture toward this end was discussedby Benzie 997! conditions. and Browdy 998!, Most of the researchdeals Further information on ablation and with endocrinology, particularly the research shrimp reproductive physiology can be found in aiming at isolatingand identifying substancesthat Treece and Yates [1990] 1993!, Treece and Fox promote maturation. 993! and Fox and Treece 000!. A shrimp maturation unit or tank is describedin detail by Treeceand Fox 993! and Broodstock Diseases the procedures for operation are covered in that Consult Brock and Main 995! if publication. working with P. vannanrei and Alday de Research on broodstock nutrition often Graindorgeand Flegel 999! if working with P. focuses on developing diets that enhance nronodon. An Internet web site where shrimp maturation Harrison 1990, 1997!. From the diseasesare discussed is http://www.aqis.gov.au, literature, personalexperience, and discussions and a 50+ page document can be downloaded at with maturationmanagers, it appearsto be much http: //www.aqis.gov.au/docs/anpolicy/98- easierto maturedomesticated shrimp than wild 086b.doc. shrimp. Progress has been made toward High health broodstock are available from domesticatingshrimp or pond-raisedstock anda commercialhatcheries. One example is at Internet growing number of hatcheries are doing this web site h://www.hih hrirn .com. without eyestalkablation. Mendoza et al. 997! According to Itami et al. 998!, discussed the influence of squid extracts on establishmentof disease-resistantshrimp strains triggering of maturation. is one possiblesolution to the diseaseproblems UnitedStates: Texas A&M Universityhas now threatening the world shrimp industry. just completeda maturationfacility studydealing Becauseartificial breedingtechniques of kuruma with closed filter types and concludedthat the shrimphave not beensufficiently developed,this cohmn filter developedby JohnOgle at the Gulf will be an important area for future research. Coast Marine ResearchLaboratory in Ocean Recentdevelopments in molecularbiology such Springs,Mississippi, improved shrimp maturation 128 UJNR Technical Report No. 28 and increased the number of females spawning Australia: Dr. Michael Hall at the eachnight compared to thebead filters used and Australian Institute of Marine Science AIMS! has on the open market Bray, personal beenworking on replacement of eyestalkablation communication!. Researchers at Texas A&M University Mexico: Eduardo Figueras, Hatchery havebeen working on isolating and characterizing Director,lndustrias Pecis, Merida, YucaM, http:/ the hormonesin P. vannarneiusing monoclonal antibodies, and have synthesizedand injected beenconducting research on the domestication brood animals Dr. Larry Keeley personal of P. vannamei broodstock and found that cominunication!. The commercial hatchery in maturationimproves with eachgeneration. Some Texasis not interestedin the injectedform because organismswere kept unablated and their offspring shrimpdo nothandle the stress of injectionwell. normally spawnedwithout ablation. He reports A fewyears before, Dr. ScottQuackenbush one that some hatcheries in Mexico are seeing of Dr, Keeley's colleagues! isolated and excellent results with unablated females, after characterized the hormones in the pink shrimp P. their third domesticatedgeneration. At present, duorarutn after leaving TAMU to join a Florida their hatchery had a secondgeneration of University, broodstock and almost 309oof their females were Ecuador: CENAIM in Ecuador is spawningwithout ablation. The interesting thing presently conducting a study entitled aboutthis is that the averagenumber of females "Improvementof reproductionand egg quality of spawninghas improved from 5% daily to 12% penaeidshrimp by induction of ovarymaturation and the spawnsare aboutthe samesize as the by neuropeptides and by diet" http: // ablatedcontrols. They have also found that an www.cenaim.espol.edu.ec!.Thisproject contains unablatedfemale can produce high quality nauplii twoparts: ! endocrinology,coordinated by Julie throughalmost 200 d in comparedto 120d for Nieto julianietoolatinmail.corn!who is also the ablated females. doingsome research at the CatholicUniversity of Leuven Belgium!,and ! nutrition.In the LITERATI JRE CITED endocrinologystudy, the identification and purificationof 30novel pure peptides from shrimp Acacia,A.-W,A., A.K. Dhar,W.H. Carr and J.M. centralnervous system is the majoroutcome of Lotz. 1997. Developmentof molecular the projectthus far. However,it is importantto markers in Penaeus vannatnei for resis- stressthat these peptides were not purified based tanceto Taurasyndrome virus TSV! in- on a maturation assay due to the difficulty fection. Book of Abstracts, WorldAquac- encountered in establishing a reliable assay, ulture'97, Seattle, WA. p. 8. Nevertheless,these novel peptidesare present in Adiyodi,R.G. and T. Subrainoniam,1983. PLrthro- thenervous system during the maturation process poda-crustacea.In: K.G. and R.G. andtherefore may be directly or indirectly related Adiyodi eds.!,Reproductive Biology of to suchphysiological processes. Purified peptides Invertebrates.Vol. I; Oogenesis,Ovipo- need to be characterized, determining their sition, and Oosorption. John Wiley & physiologicalpathway of actionand their kinetics SonsLtd., Chichester,NY. pp. 443-495. during the maturation process. The major Adiyodi,K.G. and R.G, Adiyodi. 1970, Endocrine limitation encounteredduring this projectwas the control of reproductionin decapodcrus- establishmentof a homologousbioassay which tacea. Biol. Rev. 45: 121-165. proveda roleof the peptidein inaturation.The AMayde Graindorge,V, andT.W. Flegel. 1999. bioassay is the key to the successin the Interactive CD entitled "Diagnosis of purificationof peptides.In-vivo experiments had shrimpdiseases, with emphasison the nopositive results since P. vannantei are sensitive blacktiger shrimp P.monodon!." ISBN to manipulation.Working with in-vitro assayswas 974-662-093-2, FAO, Rome, Italy, and the secondoption usedand is presentlyunderway. T recce 129

Multimedia Asia Co. Ltd., Bangkok, pod crustaceans. Journal of Crustacean Thailand. Biology. 6!: 313-325. Alikunhi, K.H., A. Poernomo, S. Adisufresno, M. Bauer,R.T. andC,E, Cash.1991. Spermatophore Budiono and S. Busman. 1975. Prelimi- structure and anatomy of the ejaculatory nary observations on induction of matu- duct in Penaeus setiferus, P. duorarum, rity and spawning in Penaeus monodon andP, aztecus crustacea: decapoda!: ho- Fabricius! andPenaeus merquiensis de mologies and functional significance. Man! by eyestalk extirpation. Bull. Transactions of the American Micro- Shrimp Cult. Res.Cent, Jepara.1!: 1- scopical Society, 110!: 144-162. 11. Beard, T.W., J.F. Wickins and D.R. Arnstein. 1977. Andrew, R.D. and A.S.M. Saleuddin. 1979, Two- The breeding and growth of Penaeus dimensional electrophoresisof neuro- merguiensis de Man in laboratory recir- secretory polypeptides in crustaceaneye- culating systems.Aquaculture. 10: 275- stalk. J. Comp. Physiol. B, 134!: 303- 289. 314. Beard,T.W. and J.F.Wickins. 1980.Breeding of Aquacop. 1975. Maturation and spawning in cap- Penaeus monodon Fabricius! in labora- tivity of penaeid shrimp: Penaeus tory recirculationsystems. Aquaculture. merguiensis de Man!, P. japonicus 20: 79-89. Bate!, P. aztecus Ives!, Metapenaeus Bellon-Humbert, C., F. Van Herp, G.E.C.M. ensis de Haan! and P. semisulcatus de Strolenberg and J.M. Denuce. 1981. Haan!. Proc. World Maricul. Soc. 6: 123- Histlogical and physiological aspectsof 132. the medulla externa X organ, a neuro Aquacop. 1977a.Reproduction in captivity and secretory cell group in the eyestalk of growth of P, monodon Fabricius! in Palaemon serratus Crustacea, Polynesia. Proc. World Maricul. Soc. 8: Decapoda!. Natantia. Biol. Bull. Woods 927-945. Hole!. 160: 11-30. Aquacop. 1977b, Observations on the maturation Benzie, J,A.H. 1997. A review of the effect of and reproduction of penaeid shrimp in genetics and environment on the matura- captivity in a tropicalmedium. Aquacul- tion and larval quality of the giant tiger ture Workshop. ICES. May 1977. Brest, prawn P.monodon. Aquaculture. 155: 69- France. Work done in Taravao, Tahiti; 85. CNEXO-COP, P.O.B. 7004!. Bliss, D.E. 1966.Relation between reproduction Aquacop. 1979. Penaeid reared broodstock: clos- and growth in decapod crustaceans.Am. ing the cycle of P, monodon, P. stylirostris Zool. 6: 231-233. and P. vannamei. Proc. World Maricul. Bollenbacher, W.E., S.L. Smith and L.I. Gilbert. Soc. 10: 445-452. 1978.The biosynthesisof a andb ecdys- Aquacop. 1984. Observations surla naturation et one: in vivo, in vitro, and cell free ap- la reproduction en captivite des crevettes proaches,pp. 491-494. In: P.J.Gallare and Peneidesen milieu tropical, pp. 132-152. Boer, H.H. eds.!, Comparative Endocri- In: Aquaculture en Milieu Tropical, nology. Elsevier/North-Holland Biomedi- 1FREMER, Brest, France. cal Press, Amsterdam. Arnstein, D.R. and T.W. Beard. 1975. Induced Bomirski, A., M. Arendarczyk, E. Kawinska and maturation of prawn Penaeus orientalis L.H. Kleinholz. 1981. Partial character- Kishinouyi! in the laboratoryby means ization of crustaceangonad-inhibiting of eyestalk removal. Aquaculture. 5: 411- hormone.Int. J. Invert. Reprod.3: 213- 412. 219. Bauer, R. 1986. Phylogenetic trends in sperm Bradfield, J.Y., R,L. Berlin, S.M. Rankin and L.L. transfer and storage complexity in deca- Keeley. 1989. Cloned cDNA and anti- body for an ovarian cortical granule 130 U JNRTechnical Report No. 28

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van Maaren et al. 135

TEMPERATURE TOLERANCE AND OXYGEN CONSUMPTION RATES FOR JUVENILE SOUTHERN PARALICHTH YS LETHOSTIGMA ACCLIMATED TO FIVE DIFFERENT TEMPERATURES

Chris C. van Maaren, North CarolinaState University, Departmentof Zoology, Plymouth,NC 27962USA Jun Kita, CentralLaboratory, Marine Ecology Research Institute, Onjuku-machi, Chiba, Japan and Harry V, Daniels~ North CarolinaState University, Department af Zoology 207 ResearchStation Rd., Plymouth,NC 27962USA Tel: 52! 793-4428xt. 150;Fax: 52! 793-5142;e-mail:Harry DanielsINCSU,edu

ABSTRACT

Criticalthermal maximum CTM!, lethal thermal tolerance LT!, and oxygen consumption rates p,g Ogg fish/min! of juvenilesouthern flounder average weight 5.5 g! weredetermmed for acclimationtemperatures AT! of 13,17, 21, 25,and 29 C. A totalof 75 fish wereused to measureCTM and LT in salinitiesof 0, 12,and 34 ppt, Three replicates of twofish eachwere used to measureoxygen consumption rates in 34ppt, Salinity had a significanteffect on both CTM andLT P<0.05!.The mean CTM for 0 pptwas 0.46 C lowerthan the mean CTM for 12ppt and0.84 C lowerthan the meanfor 34ppt. The mean LT for 0 ppt was0.40 C lowerthan the mean LT for 12ppt and0.61 C lowerthan the mean for 34ppt. The LT was20.39 C higherthan acclimation temperature at 13C butonly 9.85 C higherat 29 C, Thehighest LTwas 38.85 C forfish acclimated to29 C. The oxygen consumption rate increased from 1.26 to 4.53 lig Oggfish/min astemperature increased from 13to 29C. The highest Q, values,for oxygenconsumption, occurred between 21 C and 25C. Between 13 C aud17 C theQ» was 2.37, between 17 C and21 C theQ~p was 2.50, between 21 C slid25 C theQpp was2.68, and between 25 C and29 C theQwas 1.29.Based on the relationship between LT andpreferred temperature, andthe observed decline in Q[pfor oxygenconsumption, we calculatedthat the final preferredtemperature FP! for juvenile southernflounder is between25 C and29 C in salinitiesf'rom 0 to 34 ppt.

INTRODUCTION 1996! and larviculture methods Daniels et al. 1996; Denson and Smith 1997; Jenkins and Smith The Paralichfhys 1997; Smith et al. 1999!. However, little lethostigma has potential as a species for information is available on the environmental aquaculture Waters 1999!. Little is known about requirementsfor grow-out. the effect of acclimation temperature on thermal The natural geographic range of the tolerance and oxygen consumption, and southern flounder in the wild extends from the knowledgeof the thermaltolerance for juvenile Albemarle Sound of North Carolina to Jupiter southernflounder will aid in grow-outfacility site Inlet, Florida, USA, on the Atlantic Coast and selection.Likewise, oxygen consumption data from CaloosahatcheeEstuary Florida, USA, to will help in the development of oxygen northern Mexico in the Gulf of Mexico Ross management strategies. 1980!. Adult southern flounder can be found in Southern flounder grow weil in fresh and coastalwater systemsin salinities ranging from salt water Lasswell et al. 1977; Daniels et al. full-strengthsea water to fresh water.During the 1996!. This euryhaline ability has generated months of December through January they interestin their potentialfor aquaculture.Several migrateto the openocean to spawn Smith et al. studies have been done to develop captive 1975!. The pelagic eggs hatch at sea and the spawning techniques Arnold et al. 1977; developinglarvae typically reenterthe estuaries Henderson-Arzapaloet al. 1988;Berlinsky et al. 136 UJNR TechmcalReport No. 28 justas they are completing metamorphosis Burke MATERIALS AND METHODS et al. 1991!. Preferred temperature is commonly Hatchery-raised juvenile southern determinedthrough the use of eithera vertically flounder from the Tidewater Research Station or horizontallyarranged temperature gradient tank TRS! hatcheryin Plymouth,North Carolina, Tsuchidaand Fukataki 1991!. These test tanks, USA,weighing 5,5 g+ 1,9g, wereshipped to the however,are inadequatefor flounder as this MarineEcology Research Institute in Onjuku- specieshas a strongnatural desire to stayhidden machi,Chiba, Japan on 9 June1999. Two wk prior bynot moving rather than to seekout a preferred to theexperiments, juveniles were acclimated to temperaturewithin a tank JunKita personal thefive testteinperatures 3, 17,21, 25 and29 communication, Marine Ecology Research C! andfed frozen kriH and moist pellets. Institute, Chiba, Japan!. Recent studies by Tsuchida995! and Kita et al. 996! have Thermal Tolerance reportedan indirect method of determiningthe Fish at eachtemperature were given 3 d preferredtemperature of fish. Tsuchida995! to acclimate to salinities of 0, 12, and 34 ppt. reporteda linearrelationship between final Fifteen thermal tolerance tests were run, one for preferredtemperature and lethal tolerance. The eachtemperature and salinity combination. A total final preferredteinperatures of 14 marinefish of 75 fish were transferredto the experimental specieswere plotted against their lethal tanks1 h prior to the initiationof the tests.The temperaturesyielding a linearrelationship with experimental tanks were closed systems an r' of 0 981 LT = 0.741FP+ 17.549!.This controlledby a ceramicheater and cooling device relationshipcan be usedto calculatethe final Matsushita Electric NU-301 AHD! and a preferredtemperature of other species, given the programmablethermostat ShimadenFP21!. relationshipbetween lethal temperatureand From the acclimationtemperatures, the testwater acclimationtemperature LT = aAT + b!. This was heatedin incrementsof 5 C/h. The critical calculationis possiblebecause the final preferred thermalmaximum CTM!, wherethe fish losttheir temperatureis definedas the temperature where equilibrium,and lethal temperature LT!, where preferredtemperature equals acclimation opercularmovement ceased, were recorded as teinperature.Thus, by replacingacclimation described by Tsuchida 995!. After the temperaturewith final preferredtemperature, the experiments,water samples were taken to confirm formulas for the two equationscan be combined salinities via an inductively coupledsalinometer leavingthe final preferredtemperature to be 01 MK-III Yo-KAL Environmental solved aFP+ b = 0.741FP+ 17.549!. Electronics!. Thefinal preferred temperature coincides with optimumtemperature for growth Brett1971; OxygenConsumption Kelloggand Gift 1983!.The point where the Q Five oxygenconsumption tests were run, for oxygenconsumption starts to decreasewith one for each acclimation temperatureat 34 ppt increasing acclimation temperature also salinity.A totalof 10fish were transferred to the correspondsto the optimal temperature for growth respirometrychamber Fig, 1! approximately20 Kita et al. 1996!.Thus, the final preferred h prior to the start of the experiment.The temperaturemay be determined indirectly, based respirometrychamber was sunk in a waterbath onthe relationship between oxygen consumption maintained at the desired acclimation temperature and acclimation temperature. + 0.2 C! by a waterheater and chiller AQUA The purposesof this study were 1! to C101A-5!. The flow into the respirometry determinehow acclimation temperatureaffects chamber was cut off and the dissolved oxygen thermaltolerance and oxygenconsumption rates concentrationwas measured every 20 secwith an and2! to indirectlydetermine the final preferred oxygen probe TOA DO-25A with OE-211 temperaturefor juvenilesouthern flounder. oxygen electrode TOA Electronics Ltd.!. Measurements continued until the oxygen van Mastreu et aL 137

probepaint asrrat oxvsppINobe Temp 2 = the higher of the two temperatures used to determine oxygen consumption

RES ULTS

Temperature Tolerance The salinity varied as follows: 0.20 + 0,15, 12.10+ 3.43 and 34.24+ 0.09ppt. Salinity had a significant P.05! effect on both CTM Figure 1. RespirometryChamber used to measureoxygen and LT. The mean CTM for 0 ppt was 0.46 C cousumptiou of southern flouuder lethostigma. lower than the meanCTM for 12 ppt and 0.84 C lower than the meanfor 34 ppt. The meanLT for concentration decreased from 100% to 80% 0 ppt was 0.40 C lower than the mean LT for 12 saturation over a period ranging from 2-4 h. ppt and 0.61 C lower than the mean for 34 ppt. Oxygenconsumption rate pg 0,/g fish/min!was The difference between the CTM at 0 ppt and the calculated according to Kita et al. 996!. From CTM at 34 ppt was greatest at 13 C. A similar theoxygen consumption rates, Qwas calculated trend was found for LT Table 1!. The mean LT as: was20.39 C higherthan acclimation temperature at 13C butonly 9.85C higherat 29 C Fig. 2!. A Q 10= Rate 1/Rate 2!""i e' ~si» where, siinilar trend was found for CTM Fig. 3!. The Rate 1 =the oxygenconsumption rate at highest mean LT was 38.85 C for fish acclimated temperature 1 to 29 C. Rate2 = the oxygenconsumption rate at Although salinity had a statistically temperature 2 significant effect on LT, the magmtudeof this Temp 1 = the lower of the two effect is on averageless than 0.5 C. Thus,a single temperatures used to determine oxygen function acrossall salinities was developedto consumption calculate the final preferred tetnperature LT = 0.336AT+ 29.54!. Using the function for this line,

Tablel. Effectof salinityou critical thermal maxiiuuiu CTM! and lethal temperature LT! atfive diFereut acclimation temperatures for juvenilesouthern flounde Para li chthyslethosti gma. Values are meatts i+ sd!for five fish,1VJeatis followed by a difFerent letter are statisdcallydifferent P<0.05!.

Critical thermal maximum Temperature C! Salinity ppt! 13 17 21 25 29

32.56 + 0.36a 34.78+ 0.08a 36.36+ 0.26a 37.18+ 0.13a 38.04g 0.1&a

12 32.92 + 0.08a 35.36 + 0.09b 36.72 + 0.18b 37.66 + 0.21 b 38.50+ 0.25b

34 33.74 + 0.75b 36.00+ 0.12c 37.08+ 0.&c 37.78 + 0.05b 38.58 + 0.00b Lethal temperature Temperature C! Salinity ppt! 13 17 21 25 29 0 32.88 + 0.57a 34.9& + 0.13a 36.80+ 0.14a 37.98 + 0.05a 38.70 + 0.14a 12 33.14 + 0.05a 35.84 + 0.26b 37.16 + 0.08b 38.12 + 0.05b 38,94 g 0.06b 34 34.08 + 0,80b 36.10 + 0.00c 37.22 + 0.14b 38.18 + 0.08b 38.96 + 0.06b 138 VER TechrriealReport No. 28

4.5 !s 4 33 35 3T 0 3'5 Kc < 35 Eg 3- 34 32 43c 15

O 30 0.5 10 15 20 25 0 Ace!immi!entempemerre C! 13 tr 21 25 20 ACCamauttntarnartttura C! Figure 2. Lethal temperaturevs, acclimation temperature forjuvenile southern flounder Para fichrhys lerhasiigma Figure 5. Juvenile southern flounder Paralichthys at three different salinities. lethostigmaoxygen consumption rate fitg/ghnin! at five different temperatures.

to 4.53p,g 0,/g fish/min Fig. 5!. The highest Q tp values occurred between 21 C and 25 C. Between E j 32 13 C and17 C theQwas 2.37,between 17 C S 35 m 35 and21 C theQwas 2.50,between 21 C and25 C theQwas 2.68,and between 25 C and29 C 1 'N theQ pl was1 29. . Using the point where a dropin 32 theQbecomes apparent Kita et al. 1996!, these resultssuggest that thefinal preferredtemperature m 15 25 Acdimrttcn~ C! of juvenilesouthern flounder is between25 and Figure 3. Critical thermal maximumvs. acclimation 29 C. temperaturefor juvenile southern flounder Parafi ch hys le br!srigrrraat threedifferent salinities. MSCUSSION

thecalculated final preferred temperature is 29.61 Althoughsalinity has a significanteffect C. This correspondsto the point wherethe line on the thermal tolerance of juvenile southern for lethal tolerance vs. acclimation temperature flounder,the magnitude of this effectis so small for southern flounder crosses the line for lethal .5 C! thatit is of little practicalsignificance to temperaturevs. final preferred temperature for 14 culturists. Furthermore, salinity has no effect on marinespecies Tsuchida 1995; Fig. 4!. juvenile southernflounder growth and survival Danielsand Borski 1998!.Therefore, southern OxygenConsumption flounder can be considered to thrive in a wide Theoxygen consumption rate increased rangeof salinity.This providesthe following withincreasing temperature and ranged from 1.26 advantagesto culturists;1! flexibility in water sourceselection and 2! the ability to usevarying salinities to combat pathogens. 13 Te IT Comparedto the14 species examined by 33 Tsuchida 995! the southern flounder has a K» epecie3 I-5 u relativelyhigh thermaltolerance, similar to the 33 ~ TT seabass Larealabrax japonica andthe black sea ~ ArI bream Acar!topagnus schlegeli. This high 92 ~ 12 I~ 1~ 13 2 ~ 22 21 23 23 toleranceis necessaryfor outsideculture in eastern Acclimationtemperature C! eeli4line! Fine!preferre4 temperature C! etted line} North Carolina as water temperaturescan reach Figure4. Lethaltemperature vs. accliinationtemperature the low to middle 30 C rangeduring the summer for southern flounder Paralic trays !eritosrigma and months. lethal temperaturevs. final preferred temperatureas describedby Tsuchida995!, van Maaren et aI. 139

Oxygenconsumption rates were found to LITERATURE CITED be temperaturedependent for juvenile southern flounder. However, oxygen consumption rate Arnold, C.R., W,H. Bailey, T.D. Williams, A. varies with fish weight. Burke 998! has Johnson and J. L. Lasswell. 1977. Labo- determined the effect of weight on southern ratory spawningand larval rearingof red flounderoxygen consumption. By combiningthe drumand southern flounder. Proceedings relationship between temperature,weight and of the Annual Conference of Southeast- oxygen consumption, culturists will be able to ern Association of Fish and Wildlife predict the oxygen needs of their stock and thus Agencies. 31: 437-440. manage oxygen input accordingly. Berlinsky, D.L., W. King, T.I.J. Smith, R.D, The preferred temperaturefor juvenile Hamilton II, J. Holloway Jr. and C.V. southernflounder, weighing 5.5 g + 1.9 g, as Sullivan. 1996. Induced ovulation of calculated by the combination of the thermal southern flounder Paralichthys tolerancetests and the oxygen consumptionrate 1ethostigma using gonadatropin releasing tests, falls within the range of 25-29 C, Davis hormone analogue implants. Journal of 998! found that the optimal temperaturefor the World Aquacu1tureSociety. 27: 143- growth of juvenile southern flounder was 25 C. 152, This correlation, between final preferred Brett, J.R. 1971. Energetic responses of temperatureand optimum temperature,supports to temperature.A study of some thermal previousstudies that show that fish prefer to live relations in the physiology and freshwa- in the temperaturewhere growth is optimized ter ecology of sockeye salmon Brett 1971; Kellogg and Gift 1983!, Oncorhynchus nerka!. Am. Zool. 11: 99- This study wascompleted with southern 113. flounderraised from eggscollected from a single Burke, J.S., J.M. Miller and D.E, Hoss. 1991. female. Additional replications of this work are Immigration and settlement pattern of needed to account for possible variations in the Paralichthysdentatusand P. lethostigma thermaltolerance and oxygen consumptionrates in an estuarinenursery ground, North that may exist within the North Carolina Carolina, U.S.A. Netherlands Journal of popu1ation and between the different strains that Sea Research. 27: 393-405. occur over the entire natural range. Burke, B.J. 1998. The dynamics of a linked for- aging and bioenergetics model for south- ACKNOWLEDGMENTS ern flounder Paralichthys lethostigrna. M.S. Thesis, Department of Zoology, The authors thank Seiji Machidori and North CarolinaState University, Raleigh. Katsutoshi Kido, Marine Ecology Research 43p. Institute, Onjuku-machi, Japanfor agreeingto Daniels, H.V., D.L.Berlinsky, R.G. Hodson and host Chris Van Maaren as a participant in the C,V, Sullivan. 1996.Effects of stocking Summer Institute Program. We also thank density, salinity, and light intensity on ProfessorEmeritus Isao Hanyu, University of growth and survival of southern floun- Tokyo and Jim Rice, North Carolina State derParalichthys lethostigtna larvae. Jour- University,for valuableadvice on the manuscript. nal of the WorldAquaculture Society. 27; This research was supported in part by the 153-159. National ScienceFoundation Summer Programs Daniels, H.V. and Borski, R.J. 1998. Effects of in Japan and Korea. low salinity on growth and survival of southern flounder Paralichthys lethostigrna larvae and juveniles. Pro- ceedingsof the twenty-sixth V.S.-Japan aquaculture symposium. 187-191. 140 U3NR TechnicalReport 1Vo.28

Davis, D.D. 1998. The role of temperatureon flounderParalichthys lethostigma. Jour- somaticand otolithgrowth on juvenile nal of the World AquacultureSociety. southern flounder Paralichthys 30!: 236-244. lethostigma!in North Carolina estuaries. Smith,W.G., J.D. Sibunkaand A. Wells. 1975. M.S. Thesis, Departmentof Zoology, Seasonaldistributions of larval North Carolina State University, Plueronectiformes!on the continental Raleigh. 50 p. shelfbetween Cape , Massachusetts, Denson,M.R. andT.I.J. Smith. 1997. Diet and andCape Lookout, North Carolina, 1965- lightintensity effects on survival, growth 1966.NOAA Tech.Rep. NMFS SSRF- andpigmentation of southernflounder 691: 1-68, Paralichthyslethostignta. Journal of the Tsuchida,S. andH. Fukataki.1991. Structure and WorldAquaculture Society. 28: 366-373. performanceof a laboratoryapparatus Henderson-Arzapalo,A., R.L. Coluraand A,F. newlydesigned toform a thermallystrati- Maciorowski.1988. Temperatureand fied sea water column.Report of the photoperiodinduced maturation ofsouth- MarineEcology Research Institute No. ern flounder.Texas Parks and Wildlife 91201. DepartmentManagement Data Series No. Tsuchida,S, 1995.The relationshipbetween up- 154. per temperaturetolerance and final Jenkins,W.E. andT.I.J. Smith. 1997. Production preferendumof Japanese marine fish. J. of southern flounder Paralichthys Therm. Biol. 20/2!; 35-4. lethostig dnajuveniles in anoutdoor nurs- erypond. Journal of theWorld Aquacul- tureSociety. 202. 211-214. Lasswell,J.LG. Garzaand W.H. Bailey.1977. Statusof marine fish introductionsinto freshwaterTexas. Texas Parks and Wild- lifeDepartment. PWD Report No. 3000- 35. Kellog,R.L. andJ.J. Gift. 1983.Relationships betweenoptimum temperatures for growthand preferred temperatures forthe youngof fourfish species. Trans. Am. Fish, Soc. 112: 424-430. Kita,J., S. Tsuchida and T. Setoguma.1996. Tern- peraturepreference and tolerance, and oxygenconsumption ofthe marbled rock- fish, Sebastiscttsmarntorattts. Marine Biology.125: 467-471. Ross,S.W. 1980. G. Jordon and Gilbert, SouthernFlounder, p. 829.In: D. S.Lee, C. R. Gilbert,C. H. Hocutt, R.E, Jenkins, D. E. McAllister and J. R. Stauffer eds.!, Atlas of North American freshwaterfishes, North Caro- linaBiological Survey Publication 1980- 12, Raleigh,North Carolina, USA, Smith,T.I.J., M.R. Denson,L.D. Heyward,W.E. Jenkins and L.M. Carter. 1999. Salinity effectson early life stagesof Southern Watanabe et al. 141

RECENT PROGRESS IN CONTROLLED REPRODUCTION OF SOUTHERN FLOUNDER PARALICHTHYS LETHOSTIGMA

Wade O. Watanabe*, Patrick M. Carroll, The University of North Carolinaat Wilmington, Centerfor Marine Science 7205Wrightsville AveWilmington, NC 28403 USA *Tel: 910! 256-3721ext 245; Fax: 910! 256-8856;e-mail:[email protected] and Harry V. Daniels North Carolina StateUniversity, Departmentof Zoology 207 ResearchStation Road,Plymouth, NC 27962USA

ABSTRACT

Hormone-inducedspawning of southernflounder Paralichthys Jethosrigmohas producedsubstanlial numbers of viableeggs, but widevariations in percentfertilization and percent hatching have been reported. Recently, sustained natural spawning of southern flounder broodstock, without hormone induction, has been achieved at the Center for MarineScience, University of NorthCarolina at Wilmington UNCW!, USA, Adults avg.wt. = 1.12kg; n = 25! were stockedin two4.8-m' controlled-environment tanksin October1998 and held under natural phototheimal conditions until January1999, when an artiflcial winter photoperiod of 10L: 14D wasmaintained through April 1999.Natural spawningwas observed in earlyDecember 1998 and increased in frequency to a peakin March 1999, before declining in lateApril. Water temperature ranged from 13,9to 24.5C duringThe spawning period. Natural spawns over 142d produceda totalof 18,3x 10'eggs, with an average percent fertilization of 28.0% range = 0-100%!,yielding 4.94 x 10' viableeggs. The percentage of eggsthat remained buoyant in full-strengthsea water averaged 41.3% -99%!, whilethe percenthatching of buoyanteggs averaged 37.3% -100%! andsurvival of yolksaclarvae to thefirst-feeding stage averaged30,2% -100%!. A preliminarycomparison suggests that natural spawning may produce higher egg quality than hormone-inducedspawning in terms of fertilization andhatching success.These results suggestthat natural and hormone-inducedspawning of photothcrmally condiuoned fish will help produce the large numbers of eggsrequired to supportcommercial producuon, Additional research is neededto addressthe problem of variableegg quality.

INTRODUCTION freshand brackishwater ponds as weil asin coastal areas Berlinsky et al.1996; Daniels et al.1996; The southern flounder Paralichthys Jenkins and Smith 1999; Smith et al.1999a!. At Iethasfigrrta is a high-value recreational and present,reliable methodsfor controlledbreeding commercially harvested found in andproduction of high quality eggsare needed to estuarine and shelf waters of the Atlantic and Gulf acceleratehatchery research and development of coasts from North Carolina, USA, to Mexico commercial grow-out systems. Gilbert 1986!. With the implementation of In Japan, commercial farmers rely fishery quotas for the in the mainly on photothermalconditioning and riatural early 1990s, landings of southern flounder have spawning of Japanese flounder P. 0livaceus increased. Today, the southern flounder is the broodstockto supply the large numbersof high number one flatfish species landed in North quality eggs needed to support cominercial Carolina Copeland et al. 1999!. hatchery operations Ijima et aL 1986; Min 1988; Interest in the southern flounder as an Tsujigado et al. 1989!. In the United States,~ aquaculture candidate in the southeasternUnited spawns of the southern flounder have been rare, Statesis relatedto the wide rangeof teinperature and researchers have therefore focused on and salinity toleranceof this species;50-d-old hormone-induced spawning. Intramuscular juvenilescan tolerate salinities as low asfive ppt, implantation of a cholesterol-cellulose pellet while older juveniles can tolerate fresh water containing gonadotropin releasing hormone- Smith et al. 1999a!. This suggeststhat this analogue GnRH-a! has produced repetitive speciescould potentially be cultivatedin inland spawningand substantial numbers of viable eggs l42 UJMt TeehnteatReport No. 28 in Southern flounder, but wide variations in = 0.76m; depth= 0.76m; vol. = 0.24m'! before percentfertilization and percent hatching have enteringa reservoirtank diam.= 1.54m; depth beenreported Berlinsky et al. 1996!. = 1 m; vol. = 1.86 m'!, froin which water was Natural spawning without hormone pumpedto thebiofilter system. Water flow to each inductionof captivesouthern flounder braodstock tankwas approximately 38 Vmin andwater was was recentlyachieved in our laboratory,The exchangedat a rateof approximately10%/d, objectivesof this paperare to describethe Immersionheaters placed in the reservoirtank environmental and culture conditions and the controlled water temperature. reproductiveperformance associated with natural In October 1998,one broodfish tank tank spawning.A preliminarycomparison of natural 1! wasstocked with 13fish ,73/m'! consisting spawningand hormone-induced spawning is also of six laboratory-rearedand sevenwild-caught made. adultswith an averageweight of 0.948kg .59 kg/m'!.At stocking, the sex ratio was 8 female:3 NIATERIALS AND METHODS males: 2 unknown. In November 1998, the second broodfish tank tank 2! was stockedwith twelve fish .52/m'!, consistingof only wild-caught Natural Spawns The southern flounder broodfish used in adults,with an averageweight of 1.28kg .23 thisstudy originated from two sources. One group kg/m'!.The sex ratio was unknown at the time of laboratory-reared!originated &om fish collected stocking. asjuveniles at 100-105 mm SL in thesummer of Gonadalinaturity of individual brooders 1993 near Beaufort, North Carolina, USA. A was assessedperiodically by biopsy of secondgroup wild-caught!originated as adults anaesthetized.3 g/L 2-phenoxyethanol!fish, capturedby commercialfishermen during using a polyethylenecannula .57 mm o.d. x September1998 in PamlicoSound, North 1.14-mm i.d.! Shehadehet al. 1973!. Ovarian Carolina,USA. Wild-caughtfish wereheld in 20- sampleswere fixed in a solutionof 10%formalin in seawater. General stage of oocytedevelopment m diameteroutdoor concrete tanks supplied with flow-throughbrackish water 2-20 g/Lsalinity! i.e,pre-vitellogenic, cortical vesicle, vitellogenic for 6 wkbefore transport to theCenter for Marine andatretic! was determined from the microscopic Science, University of North Carolina at appearance,and maleswere identified by the Wilmington UNCW!, where this study took place presenceof milt whenpressure was applied to the betweenDecember 1998 and April 1999. Fish gonadal area. wereindividually tagged and held for 3 wk in Fish were fed to satiation once daily flow-through sea water 4 ppt! tanks under approximately0900!, a diet that consisted primarilyof Atlantic silversides Menidia menidia ambient conditions before stocking into a supplementedwith squid, krill, andcommercially- controlled-environment broodfish system. The controlled-environment broodfish prepared diets containing 45% INVE systemconsisted of two circularfiberglass tanks Aquaculture,Grantsville, Utah, USA! and 55% diam,= 2.46m; depth= 1 m; vol.= 4.76m'!. protein Corey Feed MiHs LtdNew Brunswick, Canada!and 16%fat. The feedingrate of the Situated out of doors, the broodfish tanks were insulatedand provided with a conicalfiberglass broodstockaveraged about 1% BW/d. To obtain gonadal maturation and cover fitted with a timer-controlled, fluorescent fixture, containingtwo 20-W daylight bulbs, spawningin January,presumed to be the natural Averagelight intensityat the watersurface was reproductiveperiod of southernflounder in North Carolinawaters Berlinsky et aL 1996!,fish were 234 lux. Both tanks were supportedby a water- exposedto ambient light and temperature recirculatingsystem, consisting of a high-rate conditionsuntil 9 January1999, when timers were used to maintain a constantwinter photoperiod sandfilter, fluidized bed biofilter, foam fractionater,and ultraviolet sterilizer.Water from of 10 L: 14 D, and a water temperature that did eachtank drainedthrough an egg collector diam. not fall below approximately14.5 C Fig. 1!, Watamtbe et al. 143

30 19 29 26 17 24 19 8 22 19 20 14 el 13 o I 19 12 Q 14

10

Ol ill gI 4I g ill Ot pop z 8 lO 0 l CV N Cl IN O lit g e l' lO Figttre 1. Photoperiodand temperatureconditions during natural spawning of Southern flounder broodstock in 4.76 m' tanks.

Egg collectors were checked daily for Water Quality spawnedeggs. Once daily, eggs were siphoned Temperature, salinity, and dissolved from the collector, transferred to a separatory oxygen were monitored daily, while pH, tota1 funnel in seawater 2-37 ppt!, andbuoyant eggs ammonia-nitrogen, nitrite and nitrate were " floaters"! were separatedfrom sinking eggs monitored weekly. Average daily values and "sinkers"!. The numbersof eggsin eachfraction ranges!were asfollows: salinity, 35.2 2-37! g/ were estimated using volumetric methods. L; dissolvedoxygen, 7.64 .01-9,01! mg/L; pH Floaters were transferred to 15-L airlift 8.12.8-8.4!; total ammonia-nitrogen, 0.029 - "in-tank" incubatorsplaced inside the reservoir 0.08! mg/L, nitrite-nitrogen, 0.022 ,003-0.051! tank or situatedin an indoor laboratory.In-tank rng/L; nitrate-nitrogen, 2,75 .6-9.4! mg/L. incubatorswere stocked at a densityof 1,000eggs/ Temperature,salinity, dissolvedoxygen, and pH L, while indoor incubators were stocked at in the incubators were monitored once at the end densitiesof 300-600 eggs/L. Indoor incubators of the incubation period. Average values and were provided with 1-pm-mesh filtered seawater ranges!were as follows. salinity, 35.5 4-38! g/ sterilized by ultraviolet light! at ]6-19 C and L; dissolvedoxygen, 8.50 .59-9.27! mg/L; pH, suppliedwith diftusedaeration. Using volumetric 8.36 8.2-8.5!; temperature, 17,4 6-18.6! C. methods, survival of embryos were monitored through hatching d 2 to d 3 after fertilization! Horxnone-inducedSpawns and at the first-feeding stage d 6 to d 7 after Hormone-induced spawning trials were hatching!, when 100% of the Iarvae possessed conducted at the Tidewater Research Station functional fully pigmented! eyes, mouth, and North Carolina State University! in Plymouth, alimentary tract. North Carolina, USA. Adult Southern flounder Percent fertilization was determined as avg. wt. 1.2 kg! originated from the same source the percentageof viable embryos,while percent as the wild-caught brooders used for natural hatching was determinedas the percentageof spawning trials at UNCW, Fish were stocked into viablelarvae hatched from fertilizedeggs. Percent tanks diam. = 3.0 m; depth = 1.0 m; vol. = 7.4 fertilization andpercent hatching were expressed ms! supplied with recirculating sea water. as percentagesof total eggsand of buoyanteggs. Broodfish wereexposed to artificial photoperiod Survival to the first-feedingstage was expressed and temperature conditions simulating ambient, as a percentageof total and of buoyanteggs. reaching9 h L:15 h D and 16 C by 15 December. Beginning in January 1999, females with 144 UJNR Technical Report No. 28 maximum oocyte diameters of 500 p.m were beentreated with hormones,indicating that natural selected for hormone-induced spawning. To spawninghad occurred. Spawning increased in inducespawning, females were implanted with a frequencyto a peakin March 1999before 95% cholesterol and 5% cellulose pellet decliningin mid-April.Fertilized eggs were first Sherwoodet al. 1988!containing [D-Ala' Des- observed in tank 1 on 12 January 1999. Gly"] LHRH ethylamide GnRH-a, Sigma On 5 February1999, fully hydratedova ChemicalCo., St. Louis,Missouri, USA! at a dose werefirst observedin theegg collector from tank of 100ling/kg Berlinsky et al. 1996!. 2, consistingentirely of wild-caught adults. In some hormone-induced spawning Naturalspawning in tank2 increasedin frequency trials, femaleswere allowed to spawnvolitionally to a peakin late Matchand early April before in the tanks "tank spawns"!. In others trials, decliningin lateApril andfertilized eggs were ovulatedfemales were strip-spawned by applying first observed on 2 March 1999. gentlepressure tothe abdomen, Eggs from a single Duringa 142-dspawning period from 3 femalewere collected in a glassbeaker and mixed December1998 to 23April, eggswere collected with thesperm from two males Berlinskyet al. on 70 daysin tank 1 andon 53 daysin tank 2 1996!,then left undisturbedin at least100 ml of TableI !.Numbers of eggscollected/d from each seawater for 1 h.The floating eggswere separated tank rangedfrom 5,490to 601,250,averaging from the sinking eggsin a separatoryfunnel. 159,596in tank 1 and133,104 in tank2. For the Embryoswere incubatedin a 70-L round durationof the 142-d spawningperiod, a totalof fiberglasstank containing 34 ppt filtered sea water 11,331,319eggs were collected from tank 1 and at 16C andat a maximumdensity of 500 eggs/L. 7,054,489were collectedfrotn tank 2, for a total Percentfertilization and percent hatching were of 18,385,808eggs from both tanks. determined as described above. Eggbuoyancy, Fertilization, and Hatching Percent fertihzation of naturally spawned Natural Spawns eggsvaried day to dayfrom 0 to97.2%, averaging On 3-5 December 1998, fully hydrated 30.6% in tank 1 and 24.9% in tank 2, with an ova were first observedin the egg collectorfrom overallaverage of 28.0%for bothtanks Table tank I, consistingof bothlaboratory-reared and 1!. A total of 3,470,516 fertilized eggs were wild-caughtadults. None of theseindividuals had producedin tank I, while 1,472,577 were

TableL Summarizeddataon natural spawning of southern flounder broodstock in two 4.76-rn3 tanks December1998 to 23 April2000!. Each tank was stocked with 12-13 adults. Data are presented foreach tank and for both tanks combined.

Hatching Tank No. Total eggs No. of Floaters Fertilization Fertilization No. of rate % of No. of days spawned floaters %! range! rate % overall! rate % of fertilizedeggs floaters! eggs No. spawned No. per range! floaters! range! observed per day! day! range! range! 1 70 11,331,3195,737,902 46.6 30.6 50.2 3,470,516 ,490- - - o- - o- 601,250! 412,750! 97.5! 99.0! 100! 177,300! 2 53 7,054,4892,199,867 34.2 24.9 50.6 1,472,577 2,750- - - - - - 419,000! 244,000! 96,5! 95.2! 100! 233,996! 1 +2 123 18,385,8087,937,769 41.3 28.0 50.4 4,943,093 Estimatedas percent hatching % overall!= floaters %! x percenthatching % of floaters!. Estimatedas survival to first-feeding % overall!= floaters %! x survivalto first-feeding % of floaters!. Watannhe et al. 145

produced in tank 2, for a total of 4,943,092 for that thesesix individualsparticipated in spawning, both tankscombined. On daysthat spawningwas an averageof 3,064,301eggs was releasedper observed,an averageof 34,811 fertilized eggs femaleduring the study,Two individuals in tank were collected from each tank. 1 and one individual in tank 2 from which ovarian During incubationof eggsin full-strength tissue was sampled were also observed to emit a seawater 2-36 g/L!, the percentageof eggsthat milky fluid from the urogenital openingon the remainedbuoyant i.e.,"floaters"! varied among dorsal ocular! sidewhen pressure was applied to spawnsfrom 0 to99.2%, with anaverage of 41.3% the abdominalregion. Microscopic examination for both tanks Table 1!. For both tanks 1 and 2, of this fluid revealed highly motile 1-2 p.m percent hatching averaged 37.3% of floaters particles,presumably spermatozoa, attd indicating 5.4% overall!, while survival of yolk-saclarvae hermaphroditic individuals. to the first-feeding stage averaged 30.2% of floaters 2.5% overall!. Hormone-induced Spawns In 1999, a total of 31 hormone-induced Thermal Regime spawningtrials wereconducted, producing a total From 5 January to 23 April 1999, the of 1,101,000 eggs of which 62% were floaters period during which the majority of spawns Table 2!. Of the floaters, 19% were fertilized occurred, water temperatures varied over a wide 2% overall!. The percent hatching of floaters rangeof 13.9C to 24.5 C Fig. 1!. Fertilizedeggs was 16.5% 9.9% overall!. were also obtainedunder this temperaturerange, Of the 31 hormone-inducedspawning althoughavailability of fertilized eggsincreased trials. 14 tank spawnsyielded 345,000eggs, of to a peak during March and early April, while which 55% were floaters, but no fertilization was water temperature averaged arouttd 17.5 C. The obtainedunder this method Table2!. Seventeen number of eggs spawned decreased as water strip-spawning trials yielded 756,000 eggs, of temperatures increased steadily in April. which 68% were floaters, with 40% of these Spawningappeared to be stimulatedby a change f1oatersbeing fertilized 7% overall!. Percent in weatherconditions; a warmingor coolingtrend hatching of the floaters averaged 30% 0,4% was followed by an increasein egg release. overall!.

Growth and Sex Ratios of Broodstock DISCUSSION A gonadal examination of al] broodstock madeon 11January 1999 revealed the following Natural Spawning sex ratios: 8 females; 3 males: 2 unknown in tank Through sustained,natural spawningof 1 and 5 females: 2 males: 5 unknown in tank 2. A captive southern flounder broodstock, gonadal examination of a11broodstock made on commercially significant quantities of viable 27 April 1999 revealed four individuals in tank 1 embryos were produced in this study. Previous andtwo individualsin tank2 withatretic, hydrated attemptsto obtainnatural spawning of this species eggs, indicating probable spawners.Assuming met with limited success.Using photoperiods and

Table2, Suinmarizeddata on hormone-inducedspawns of southernflounder Paralichthyslethostigma! at theTidewater Research Station North Carolina State University! in 1999,including eggs collected from both tank and strip spawns, Average percent hatching range! was determinedwithin 24 h of first observedhatch for floating eggsonly.

Method of Batches Total eggs Floaters Fertilization Fertilization No, of HatchingHatching spawllillg spawned %! rate % rate % of fertihzed rate % rate % overall! floaters! eggs of floater! overall!

Tank 14 345,000 190,000 55 0 0 0 0 0 Strip 17 756,000 514,000 68 27 40 206,000 30 20.4 Total 31 1,101,000 704,000 62 12 19 l 34,000 16.5 9.9 146 UJNR Tecbnieal Report No. 28 temperatureswhich simulated natural seasonal for the duration of the study. In addition to the changes,Arnold et al. 977! obtainednatural phatothermalconditions, which maintained fish spawningfrom 3 of6 femalesfor 13consecutive in a reproductivestate fram December through daysin 30-m'tanks producing 120,000 eggs with April, factorssuch as diet, tank size and color, a percentfertilization of 30-50%and a percent andlow light intensitiesapparently minimized hatching of 6-35% of the fertilized eggs. stressand wereconducive to natural spawning. Controlledphotoperiod and temperature were also Based on our observation that egg release effective in stimulating releaseof 200,000eggs increased following a change in weather from 5 femalesover 2 spawningseasons, but no conditions,pulsatile temperature conditions may fertilization was obtained Henderson-Arzapala havebeen an importantstimulus for sustained et al. 1988!. naturalspawning in thisstudy. Smith et aL999b! The resultsof this studydemonstrate that reportedthat a dropin watertemperature from 17 wild-caughtsouthern flounder adults, conditioned ta 14C inhibitedspawning in southernflounder, throughphotothermal manipulation for only8 wk, whichresumed when temperature was returned can be spawnedsuccessfully without hormone to 17C. In reddrum, spawning is stimulatedby inductionduring their first seasonin captivity.This gentlyraising or loweringthe water temperature is important to avoid a prolongedperiod of Roberts 1990!. acclimationta captivity.In wild-caughtturbot Sustainedrelease GnRH-a pellet implants Scoprhatmusmaximus, efficient spawning are highlyeffective in inducingovulation of occurredonly after 2 yrof habituationto captivity successive batches of eggs in the southern Devauchelleet al. 1988!.In southernflounder, flounder Berlinksy et al. 1996!, allowing onlyfemales ! 2 kgspawned naturally m captivity repetitivestrip spawning. These workers produced Arnold et al. 1977!and successfulhorrnone- substantial nuinbers .6 x 10'! of eggs from induced tank spawning was attained with multiplestrip spawns of 12females over a 2-wk braodstockheld in captivityfor atleast 1.5 yr and period, but percentfertilization varied receiving photothermal conditioning for a considerably-95%! betweenfemales and minimumof 12 wk prior ta spawning Smith et betweenspawns fram individual fish. As an al. 1999b!. alternative to strip-spawning,photothermal The naturalspawning period for southern conditioningcoupled with GnRH-a implants have flounder is believed ta be December and January also resulted in successful tank spawning of Henderson-Atzapalo1988;Berlinksy et al. 1996; southernflounder Smith et al. 1999b!. From a Smith et al. 1999b!,In this study,viable embryos broodstockconsisting of 3 females,these workers wereproduced from Januarythrough late April collectedan average of 277,844eggs during a 99- 1999,indicating that the extendedphotoperiod dayperiod for a totalof 17,782,000of which regimewas effective in extendingthe spawning 32.8%were fertilized range= 0-82%!,although seasonat least2 mabeyond the natural spawning survivalthrough hatching was not reported.A periodfor this species. Using a combinationof combinationof photothermalconditioning and photothermalconditioning and GnRH-a impiants, GnRH-aimplants apparently reduced stress and southern flounder braadstock spawned resultedin higheregg production and an extended volitionallyin tanksover an extended period of spawningperiod Smith et al.,1999b!. 99 d from Januaryto late April Smith et al. Results af this study demonstratethat 1999b!. photothermalconditioning of southernflounder It is likely that natural spawningof canproduce natural spawning, without the use of Southernflounder in this study waspromoted by hormones,resulting in relativelyhigh spawning a number of factors. The fish were received from successin termsof egg productionand quality fishermenin good health and water quality andthe duration of thespawning period. Natural parametersin broodtanks were maintained within spawningof southernflounder broodstock optimalranges throughout the study. The fish fed produceda totalof 18.4million eggs, from 5 well and showed little or no evidence of disease females,with an overallpercent fertilization of Watanabe et aL 147

28% and an overall percenthatching of 9%. In Berlinsky, D.L., W,V, King, T.I.J. Smith, R.D. comparison,31 hormone-inducedspawning trials Hamilton II, J. Holloway, Jr. and C.V. produced 1,101,000 eggs, with lower overall Sullivan. 1996. Induced ovulation of percentfertilization andpercent hatching of 12% Southern flounder Paralichthys and 9.9%, respectively, While spawning success Iethostigma using gonadotropin releasing varied widely under both natural and hormone- hormone analogue implants. Journal of induced spawning trials, the data suggestthat the World AquacultureSociety, 27: 143- natural spawns generally resulted in higher egg 152. quality. Bromage, N. 1995. Broodstock managementand Higher egg quality under natural seedquality generalconsiderations, pp, spawning may be related to minimal handling of 1-24. In. N. R. Bromage and R. J. Rob- the broodfish, since stresscan reducespawning erts eds.!, Broodstock management and success Kjesbu 1989!, Inappropriate hormone egg and larval quality Blackwell Science dosages can also affect egg quality Lam 1994!, Ltd., Oxford, U.K. andstrip spawningmay cause varying egg quality Copeland, B.JJ. Miller and E.B. Waters. 1999, due to over ripening or disturbances in the The potential for flounder and red drum ovulation process Bromage1995!. stock enhancement in North Carolina, A major disadvantage of natural North Carolina Sea Grant, North Caro- spawningis the inability to time the spawnsto lina State University, Raleigh, N.C. suit the needs of the fish culturist. Both natural Daniels, H.V, D.L. Berlinsky, R,G, Hodson and andGnRH-a induced spawns will help supplythe C.V. Sullivan, 1996.Effects of stocking large numbers of eggs necessary to support density, salinity and light intensity on commercial hatcheries. Additional research is growth and survival of Southern floun- needed on hormonal, nutritional and der Paralichthys lethostigma larvae. Jour- environmentaleffects on egg quality. nal World Aquaculture Society. 27: 153- 159. ACKNOWLEDGMENTS Devauchelle, NJ.C. Alexandre, N. Le Corre and Y. Letty. 1988. Spawning of The authors thank Dr. John Burke for Scapthalmus maximus in captivity. donating the "laboratory-reared" Southern Aquaculture. 69: 159-184. flounderbroodstock, and Christopher Woolridge, Gilbert, C. R. 1986. Species profiles: Life histo- Andrew Rhyne and Todd Guerdatfor technical ries and environmentalrequirements of assistance and Dr. Ron Hodson and Mr. Robert coastal fishes and invertebrates south Wicklund for helpful advice. This research was Florida! Southern, Gulf and summer supportedby the U.S.Department of Agriculture, .Biological Report82 1.54!, Cooperative State Research Education and U.S. Fish and Wildlife Service, Washing- Extension Service Grant Na. 98-38854-6009! ton, DC, USA. andby the North CarolinaSea Grant Grant Nos. Henderson-Arzapalo, A., R,L, Colura and A.F. R-MG9804 and NA86R003!. Maciorowski. 1988. Temperature and photoperiod induced maturation of South- LITERATURE CITED ern flounder. ManagementData Series No. 154, Texas Parks and Wildlife De- Arnold, C.R., T.D. Williams, A. Johnson, W.H. partment. Austin, TX, USA, 20 pp. Bailey and J.L. Lasswell, 1977. Labora- Ijima, T., T, Abe, R, Hirakawa and Y. Torishima, tory spawning and larval rearing of red 1986. Early spawning of Japanesefloun- drum and Southern flounder, Proceedings der Paralichthys oli vaceuswith elongated Southeastern Association Fish Wildlife day treatment.Saibai-giken. 15: 57-62. Agencies. 31: 437-440. 148 UJNR Tecbnieal Report No. 28

Jenkins,W. andT.I J. Smith. 1999.Pond nursery Tsujigado,A., T. Yamakawa,H. Matsudaand N. production of Southern flounder Kamiya.1989. Advanced spawning of Paralichthyslethostigma and weaningto the flounder, Paralichthys olivaceus,in commercialdiets, Aquaculture. 176: 173- an indoor tank with combined manipula- 180, tion of water temperature and Kjsebu,O.S. 1989. The spawning activity of cod, photopeirod.Int. J.Aq. Fish.Technol. 1: Gadus morhua L. Journal of Fish Biol- 351-356. ogy. 34: 95-206. Lam, T. 1994.Hormone and egg/larval quality. JournalWorld AquacultureSociety. 25: 2-12. Min, B.S. 1988. Maturation and spawning of flounder Paralichthys olivaceus under captiveconditions. Aquaculture. 1: 25-39. Roberts,D.E. Jr. 1990.Photoperiod/temperature control in the commercialproduction of red drum Sciaenops ocellatus eggs, pp. 35-43. In: G.W. Chamberlain, R.J. Miget andM.G. Haby, eds.!.Red Drum Aquac- ulture, Texas A&M University Sea Grant CollegeProgram No. TAIVIU-SG-90-603. Shehadeh, Z.H., C.-M. Kuo and K..K, Milisen, 1973. Validation of an in viva method for monitoringovarian development in the greymullet Mugil cephalusL.! Journal of Fish Biology. 5: 479-487. Sherwood,N.M., L.W. Crim, J. Carolsfeld and S.M. Walters, 1988. Sustained hormone release: I. Characteristics of in vitro re- leaseof gonadotropin-releasinghormone analogue GnRH-a! from pellets.Aquac- ulture. 74: 75-86. Smith,T.I.J., M.R. Denson,L.D. Heyward,Sr. and W.E. Jenkins. 1999a. Salinity effects on early life stagesof Southernflounder Paralichthyslethosti g ma.Journal World AquacultureSociety. 30: 236-244. Smith, T.I, D.C. McVey, W.E, Jenkins,M.R. Denson,L,D. Heyward,C.V. Sullivan and D.L. Berlmsky.1999b. Broodstock man- agementand spawning of Southernfloun- der, Parali chthyslethostigrna. Aquacul- ture 176: 87-99.