fluorochrome marking of out-planted green sea urchins ...tetracycline (sigma-aldrich tetracycline...

Fluorochrome Marking of Out-planted Green Sea Urchins, Strongylocentrotus droebachiensis, for Sea Ranching and Restocking

Programs in the Gulf of Maine, USA

PameliaFraungruber＊1,StephenD.Eddy＊1,NicholasP.Brown＊1,andLarryHarris＊2

Abstract:Markingcalcifiedstructureswithfluorochromesisdoneinavarietyofvertebrateandinvertebratespecies to tag individuals forgrowth,population,andecologicalstudies.Here,wedescribetheuseof thefluorochrometetracycline to identifyhatcheryrearedgreenseaurchinsreleasedon-bottomonto twoaquaculture leasesknownasJobandSloop, located in theGulfofMaine,USA.Thiswasdonetoexaminetheviabilityofsearanchingandstockenhancementbylookingatrecoveryratesandgrowthofreseededjuvenilesoverthecourseoftwoyears. 21,000hatchery rearedgreen seaurchin juveniles (Strongylocentrotus droebachiensis)weremarkedwiththefluorochrometetracyclinewhentheywereat10- 20mmtestdiameter,andreleasedonto400m2studyareas locatedateach lease. Juveniles fromthesamehatcherycohortweresimultaneouslyrearedinaland-basedrecirculatingaquaculturesystemsothatsearanchingcouldbecomparedwithtankfarming.ThereleaseareasweresurveyedbySCUBAdiversat3-5monthintervals forovertwoyears.Urchinswerecollected fromthefield,measured,anddissectedtoremovethejawstructures,whichwerethenexaminedwithfluorescencemicroscopy.Tetracyclinefluorescencewasdetected forupto27monthspost-release inrecapturedurchins.Numbersofrecapturedmarkedurchinsfluctuatedovertime,causing largevariability inpopulationsurvivalestimatesforeachsiteateachsampleinterval.Sizemeasurementsofrecapturedurchinsshowedadecline inaveragetestdiameterat theJobsite,butat theSloopsiteaveragetestdiameterincreasedduringthetwoyearstudy.Greenseaurchins fromthesamehatcherycohortrearedina land-basedtanksystemhadsignificantlybettergrowththanthoserecaptured fromeitherleasesite.Environmental factors, rather thangenetic factors (hatcherysource),were likely thecauseofthesizedifferencesobservedbetweenhatcheryseedrecapturedfromtheleasesitesandthosereared intankculture.Site factorsmayhaveresulted insizedependantmortalityand/orout-migrationof largerurchins.Oneof the limitationsof themark/recaptureapproachwithseaurchinsisthatdivesurveysneedtoexpandovertimetoaccountforurchinmovementawayfromthereleasearea.Giventhehighcostofsuchefforts, thismaynotbepracticalorcosteffective.Becausethemarkedjawstructureswereinternallylocated,itwasnotpossibletoidentifymarkedseaurchins in the field, and the animalshad tobesacrificed for laboratory analysis.Recentadvances in fluorochromemarkingandvisualizationcouldallow field identificationofmarkedurchins.ThiswouldenhancetheabilityofresourcemanagerstoevaluaterestockingprogramsintheGulfofMaine,aswellastoassignprovenanceorownershipofsearanchedurchins.

Key Words:greenseaurchins,fluorochromes,sea-ranching

2015年 1 月30日受理（ReceivedonJanuary30, 2015）＊1 UniversityofMaine,CenterforCooperativeAquacultureResearch,Franklin,Maine,USA＊2 UniversityofNewHampshire,Durham,NH,[email protected] Correspondingauthor:StephenD.EddyE-mail:[email protected]

水研センター研報，第40号，135－143，平成27年Bull.Fish.Res.Agen.No. 40，135－143，2015

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Pamelia Fraungruber, Stephen D. Eddy, Nicholas P. Brown, and Larry Harris

　The green sea urchin Strongylocentrotus droebachiensishasbeenaneconomically importantfisheriesspecies in theGulfofMaine,USA (GOM)since the 1980’s.Catch levels peaked in 1993 at19,050metric tons,andthefisheryvaluepeaked in1995 at $35,604,275.However, these large annualharvests couldn’t be sustained, and ecologicalchangeshavecontributedtoasteepdeclineinwildstocks (Stenecket al., 2004).Since2008theannualcatch inMainehasaveragedabout1,300mt,withan average value of about $5.5million.Althoughthefisherymaynotreturnto1990 levels,resourcemanagersandfishermenbelievethatwildstockscanandshouldberebuilttoallowforincreasedharvestlevels.Severalmanagementapproacheshavebeentaken tohelprebuildnatural stocks,mostlybasedonfishingrestrictions.Stockenhancementthroughreleaseofhatcheryseedhasalsobeendiscussed,butuncertainty regarding itsecological andeconomicviability has discouraged public funding of anysustainedstockenhancementprogramsintheGOM.　Evaluating the effectiveness and benefits ofsea urchin reseeding programs is an essentialbut complex task.The economic returnwill bea function of seedproduction costs, growth andsurvivalofout-plantedseed,andmarketpricesatthe timeof recapture. In Japan, seaurchin stockenhancement,knownas reseeding,hasbeendoneatlargescale(>50millionseedannually)forover20years (Agatsumaet al., 2004;Sakaiet al., 2004). InHokkaidoitcost4-10JPY(4-8UScents)toproduceoneseedof5mmtestdiameter,andnearly3xthatfor20mmseed (Sakai et al., 2004).TheeconomicbenefitsofreseedingtotheJapanesefisheryremainuncertain; in some cases catches have declinedor remained staticdespitewidespread reseeding(e.g.Strongylocentrotus intermedius inHokkaido) ,whereasinothercasesreseedingiscorrelatedwithimprovedcatch levels (e.g.S. nudusatEsannearHakodateCity) (Agatsuma 2014, in publication).Ultimately, thecostsofseedproductionversustheeconomicreturntothefisherymustbeconsideredin the context of cultural values and ecologicalconsequences.　Althoughincreasedcatchlevelsmightimplythatrestockinghasbeensuccessful, itisnotevidenceofacauseandeffectrelationship.Ecologicalchanges,

increasedrecruitment, or intensified fishingeffortcanalsoleadtoimprovedstocksorincreasedcatchlevels.Measuresofsurvival,growth,andreturntothe fisheryareneeded to assess the cost/benefitof stock enhancement.This can only be done ifreleasedstockcanbedifferentiatedfromwildstock,but there arenodiscernible externaldifferencesbetweenhatcheryandwildurchins (Agatsumaet al., 2004).However, seaurchins canbe internallymarkedwithfluorochromes,eitherthroughinjectionorbathimmersion.KobayashiandTaki(1969)werethefirsttousetetracyclinetomarktheseaurchinS.intermediusforgrowthstudies.Sincethattime,avarietyofstudieshaveusedfluorescentmarkerstoidentifyseaurchinsinthelaborinthefield(EllersandJohnson, 2009).Thispaperdescribestheuseoffluorochromemarkingto identifyhatcheryseedofS. droebachiensisreleasedontooceanbottomleasesintheGOMinordertoevaluatesearanching.Searanching is similar to reseeding, but in this casethe juvenileswere released onto privately heldaquaculture leases.TheprojectwascarriedoutbytheCenter forCooperativeAquacultureResearch(CCAR),workingwith industrypartnerFriendshipInternational (FI), a seaurchin trading companybasedinMaine.Wewereinterestedinascertainingwhetherthisprivatizedmodeofreseedingcouldbeaviablemodelforthefishery.Todothisweneededtodetermine if releasedseedwouldremainwithinlease site boundaries, andwhether growth andrecoveryrateswouldbesufficienttorealizeareturntotheleasesiteoperatorortothefishery.

Materials and Methods

Hatchery:TheCCARisamulti-speciesaquacultureresearchanddevelopment facilityoperatedbytheUniversityofMaine(http://www.ccar.um.maine.edu/index.html).HatcheryproductionfortheprojectwascarriedoutattheCCARinthespringof2009 (Feb.-June).Green sea urchinswere induced to spawn(N=39 females and30males) toprovidegametesthatwere fertilized for larval rearing.Laboratoryspawning, fertilizationand larval rearingmethodsforS. droebachiensisaresimilar to thosedescribedformanyotherseaurchinspecies (McBride, 2005).Larvaewerereared inconicalbottom230Lclear

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Mark Recapture of Green Sea Urchins in the Gulf of Maine

fiberglass vats continuously suppliedwith freshseawateratabout12 ℃.Dunaliella tertiolecta and Rhodomonas salinawere theprimaryalgal feeds.Thelarvaewerecompetentforsettlementat24-30dayspost-fertilization.Followingsettlement,juvenileswererearedforanadditionalperiodof8-10monthsinalandbasednurserysystem.Duringthenurseryperiod theywereheld inplastichydroponicplantbaskets inshallow fiberglassraceways,and fedad libitumwithfreshlyharvestedSaccharina latissima.Fluorochrome marking and visualization : Thejuvenilesweremarked (tagged)with tetracyclineaboutfourmonthsbeforereleaseontotheleasesites,usingmethods adapted fromEllers and Johnson(2009).Juvenileurchinsweregradedintoperforatedbaskets,and immersed for24hours in tanks filledwith0.2µmfilteredseawaterand37.5mgperL－1tetracycline (Sigma-AldrichTetracyclineT3258).Urchinswere fed to satiationbefore andduringtaggingtoensureactivegrowthanduptakeof thefluorochrome into thecalcareousexoskeleton.Twoweeks followingmarkingtwenty-fiveurchinswereexaminedusingafluorescencemicroscope.Thejawsof each individualwere removedandplaced inasodiumhypochloritesolutiontodissolveallorganicmaterial, leavingonly thecalcareous jawsbehind.These structureswere then examinedthrough aGIBfilterusingaZeissAxioImagerZ1fluorescencemicroscope.Oxytetracyclinegoesthroughexcitationat 390nm and emission at 560nm.Tags appearasabright lineof fluorescencespanning the jawhorizontally, and for themost partwere easilyidentifiable(Fig. 1). 100%ofthoseexamineddirectlyaftertagginghadclearlyvisibletags.Sea ranching:The tagged juvenileswerereleasedattwoaquacultureleaseslocatedinPenobscotBay,Maine.Site1 (Sloop)was locatedoffofNorthaven,Maine near Sloop Island (44°12.2’N 68°50.1’W)andSite2 (Job)wasoffofCamden,MainenearJobIsland (44°13.5’N68°50’W).Each site comprisedtwoacres(0.81ha)ofseabottom,withameanwaterdepth of about 2-5m.The leasesweremarkedwithbuoys to indicate thatharvestingurchinsbydraggingnetsacrossthebottomwasprohibited. InFebruaryof 2010, 10,500 juvenileswere releasedat each site onto a small study area locatedapproximatelywithinthemiddleofeach lease.The

juvenilesweretransferredinplasticbagsbydiversonto the bottom and distributed along transectlineslaidoutto15minallfourcompassdirections,encompassingatotalareaof400m2.Between1,000and1,500 juvenileswere releasedat 5mand10mmarkersalong the transects toensureanevendistribution.The juvenileswerenotenclosedandthereforewere free tomove.No feeding or anyotherhusbandryactivitywasconductedduringthetwoyearsfollowingtherelease.Site surveys: The siteswere characterized in aprevious study (Kirchhoff et al., 2008), but priorto out-planting an initial transect divewasdoneto estimate the extent of existing sea urchins,predators, and bottom cover. At each releasearea a baseline was laid out in a North-Southorientationandfivetransect lineswere laidoutonaperpendicular(East-West)bearingextendingto10m.Samplequadratsconsistingofa1m2PVCframewereplacedatthe10mmarkerineachdirection,atthecenterofthetransect,andjustoverthebaseline(0montransect),foratotalof15quadratspersite.Duringthepre-releasesurveythebottomsubstratewas characterizedand thenumbers ofpredators(crabs,seastarts,etc.)andnaturallyoccurring(pre-existing) urchinswere counted.The extent andcompositionofalgalfeedwasalsoobservedforeachsite.Theout-plantedareaswerethendivesurveyedonsixmoreoccasionsat3-5month intervalsoverthe course of 27months.Allurchinswithin each

Fig. 1.A tetracyclinemarkedgreen sea urchinStrongylocentrotus droebachiensis demipyramidviewed using a WIB filter on a fluorescencecompoundmicroscope.

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samplequadratwereenumeratedandthosebetween4-30mmTDwere collected in numberedmeshtubestobetakentothelaboratoryformeasurementandidentification(absence/presenceoffluorochromemarker).Urchinssmallerormuch larger than theoriginal release sizewere not collected in earlysurveys, butduring later surveys largerurchinswerecollectedtoaccountforanygrowth.Tank culture:During the twoyear sea ranchingstudy 9,500 green sea urchins from the samehatchery cohort as the lease site urchinswerereared ina tanksystemat theCCAR, tocomparegrowth and survival of lease site urchinswithjuvenilesrearedonland.ThejuvenileswerestockedintoracewaysassembledtoformaslantedVinteriorprofile(V-trough),withaperforatedbottomplatetoremovewastes.TheV-troughswereplumbedintoarecirculatingseawateraquaculturesystemequippedwithaparabolicfilterforsolidsremoval,movingbedbiofilter, foam fractionator, oxygen injection, 3hpchiller,andUVsterilizer.Rearingtemperatureswereheldbetween6-16 ℃yearroundandthejuvenileswerefedhighqualityformulateddiets (NofimadietfromNorway). Itwasanticipated thatseaurchinsreared under these conditionswould have goodgrowthand survival, toprovideabenchmarkbywhichtheleasesiteurchinscouldbecompared.Specimen analysis : Specimen bags containingurchins from the samplequadratswerebroughtback to the lab,drainedand frozenuntil analysis.Thesewere later (within 2-6weeks) thawed inseawater,andall individualswereblotteddryandweighedtothenearest0.1g.Testdiameter(TD)wasmeasuredtothenearest0.1mmwithdigitalcalipers(modelCD-6PMXMitutoyoCorporation,Kawasaki,Japan).Eachseaurchincollectedfromtheleasesiteswasanalyzed for thepresenceof oxytetracyclinemarking,asdescribedabove.Insomecasesmultipleorsinglebandsofauto-fluorescencewereseenthatappearedatypicalorambiguous(e.g.diffuse).Sourcesof ambiguityand thereforeerror in identificationincludedsizeofthejaws,intensityofthelightusedtomakethetagsfluoresce,andambient light fromthesurroundingroom.Iftheresultswereuncertain,thenthejawswereeitherreevaluatedormarkedas“untagged”.Urchins thatwereclearly taggedwereconsideredasrecaptured(hatcheryorigin).

　Urchins reared in the land-based tank systemweresampledat intervalscoincidingwiththeleasesite surveys.Thirtyurchins fromeach tankwererandomlyremovedandmeasuredforweighttothenearest0.1g,andTDtothenearest0.1mmusingdigitalcalipers.Data analysis:Theaveragenumberoftotalurchins(tagged and untagged) per square meter wascalculated for each study area and survey dateas the total number ofurchins collectedper sitedividedbythenumberofsamplequadrats (usually15).Thenumberofreleasedseedremainingateachsiteandsurveydatewasestimatedastheaveragenumberofrecaptured (tagged)urchinspersamplequadrat (m2)x400m2 (thesizeof thereleaseareaasawhole).Themean,minimumandmaximumtestdiameterof recapturedurchinswascalculated foreachsiteandsurveydate.Chisquaredtestswereusedtodeterminewhetherornot thenumbersoftagged anduntaggedurchinswere significantlydifferent fromeachother.Thestandarddeviationofthemeantestdiameterwasdeterminedtoseeiftheaveragesizeofrecapturedurchinssignificantlydiffered(±1SD)betweenthetwositesandfromthetankrearedurchins.Datawereplotted todisplaytrends innumbers,averageTD,andmaximumTDofrecapturedurchinsateachsiteovertime,andtheTDofleasesiteurchinswascomparedwiththatoftankrearedurchins.

Results

Site characteristics:The twositeswere less thansixnauticalmiles apart andof comparabledepth(2-6mmeanwater),but theydiffered in termsofexposure,current,bottomsubstrate,andpopulationdensityofnaturallyoccurring(pre-existing)urchins.The Job Island sitehad relativelyuniformdepth,butwassubjecttoperiodsofextremeslacktideandperiodsofstrongcurrent.ThebottomsubstrateatJobwas80%rockcobblewithseveralsmallbouldersthroughout,whichwerepopulatedwithmacroalgae,butrelatively littledriftalgaewasfound.Predatorswerenot found inabundance,withonlyone largeJonah crab (Cancer borealis) observed, and theinitialpopulationdensityofpre-existingurchinsattheJobsitewas2.25animals/m2.AttheSloopsite,

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thebottom substratewas 80% shell hash,whichprovidedabundantrefuges forsmallandmedium-sizedurchins.Thestudyareawasonaslopedledge,sothedepthvariedacrosstheareacomparedwithJob,whichwasmoreflat.AfewsmallboulderswerefoundontheSloopsitewithmacroalgaegrowingonthem,anddriftalgae,mostlykelp,wereabundant.TheSloop Island sitehadanabundance of largeurchinsandseastarspresenton itatout-planting.Theinitialpopulationdensityofpre-existingurchinsattheSloopsitewas4.5animals/m2.Recapture rates: Seaurchinswere foundonbothstudyareas at every survey for over twoyears.The totalnumber (taggedanduntagged) foundateachsurveyrangedfrom4- 674attheJobsiteandfrom194- 397at theSloop site.Taggedurchins(hatcheryorigin)wererecapturedatbothsitesandateverydivesurveyuptothelast, 27monthspost-release.Recaptureratesdeclined in the firstyearbut thensignificantlyspiked in thesummerof thefollowingyearatbothsites,beforeagaindeclininginsubsequentsurveys(Fig. 2).AttheJobsite10%to100%of theurchinscollectedduringeachdivesurveyweredetermined tobeofhatcheryorigin,andatSloop35%to71%ofcollectedurchinswereofhatcheryorigin.It’simportanttonotethatontheoneoccasionwhen100%of the animals collectedat Jobwere tagged, theentire samplepopulationconsisted of just four animals, all very small (<7mmTD).Atthefinalsurveyatotalof107urchinswere collected from Job and about 30%of thesewere tagged.At the finalSloopsurveytheurchinpopulation showed a significant decline from

previous levels,andtherewasevidence (disturbedgrounds,geartracks,andbrokentests)thatthesitehadbeenrecentlyfishedbyadraggerboat.Population estimates : Population estimates ofhatchery origin urchins remaining within the400m2 release areas at each survey varied indirectproportionwiththerecapturerates (Fig. 3).Originally, 10,500urchinswere released at eachstudy area. Extrapolation from dive surveysindicatedthatthenumberofhatcheryoriginurchinsremaining within the Job release area at eachsurveyrangedfrom45to36,894;with3,306projectedasstillremainingat27monthspost-release.AttheSloopsite,populationestimatesofhatcheryurchinsremaining at each survey ranged from 3,680 to18,165;with7,360projectedasstillremainingatthefinalsurvey, 27monthspost-release(Fig. 3).Average and maximum size : The average testdiameter(TD)ofhatcheryoriginurchinsrecapturedattheJobsitedeclinedto5.1mmoverthecourseofthestudy,whichwastheminimalreleasesize,butTDincreasedattheSloopsite (Fig. 4).AtJob, theaverageTDdeclinedfrom10.6mmatreleaseto5.1mm27monthspost-release,whereasatSloop theaverageTD increased from11.3mmatrelease to18.3mmat27months.The largestmarkedurchinrecaptured fromanyof thesurveysatJobduringthecourseof the studywas19.7mm (Aug2010),and at Sloop itwas 49.3mmTD (Sept 2011, 19monthspost-release) (Fig. 5).The Job sitehadadisproportionatenumberofsmallurchinsremainingon itateverysurveythroughoutthecourseof the

Fig. 2. Total numbers of hatchery origin S. droebachiensisrecapturedfromtworeleasesites inPenobscotBay,Maineateachsurvey.

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study.Of the totalnumberofurchins (sumof sixsurveys)recapturedfromJob, 84%were≤6mm.AttheSloopIslandsite,only1%ofthetotalrecapturedurchinswere≤6mm.　Growth rates diverged between land and seabasedhatcheryurchinswithinthefirstyearof thestudy.Seaurchinsreared inthe land-basedculturesystemweremuch larger onaverageat theendof the twoyearstudythanthoserecaptured fromeitheroceanleasesite(Fig. 4).After27monthsthelargesturchinsampled in the tankculturesystemwas53.4mm,and≈ 1/3ofthetankrearedurchinswere≥40mm.

Discussion

　Fewprevious studies inNorthAmerica havemonitoredsurvivalandgrowthoftaggedseaurchinsreleasedintothefield.Dumontet al. (2004)releasedthreesizegroupsofgreenseaurchinstaggedwithtetracyclineontoasmallstudyarea.Similar to thepresent study, they found that recapture ratesweresizeand timespecific: 69% for<10mmand2% for>15mmurchinsafterninedays, and25%and0%respectivelyafterfortydays.InastudybyRogers-Bennett et al. (1994), redurchin juveniles(Strongylocentrotus franciscanus)were taggedand

Fig. 4. Average test diameter of hatchery origin S. droebachiensisrecapturedattworeleasesitesinPenobscotBay,Maineateachdivesurvey,andintankcultureattheCCAR.Errorbars= ±1standarddeviationfromthemean.

Fig. 5.MaximumtestdiameterofhatcheryoriginS. droebachiensisrecapturedat tworeleasesites inPenobscotBay,Maineateachdivesurvey.

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released onto study areas that varied in depth.Recovery rates after 12monthswere 21% fromshallowhabitatsand11%fromdeephabitats.　The present study provides evidence thathatchery reared green sea urchins can besuccessfully out-planted for reseeding or searanching in theGulf ofMaine.Success isdefinedas theabilityofseedtosurviveandgrowto legalharvestsize (52mm)within5yearsofrelease.Wesawthatreleased juvenilessurvivedandremainedfor an extendedperiod (27months)within eachreleasearea.However, recaptured juvenilesweredisproportionatelysmalleratonesite (Job) thanatthe other (Sloop).This suggests that site factorsmodified the size distribution of surviving orremainingout-plants indifferentwaysat the twosites.TheSloopIslandsitemayhavehadahabitatmorefavorableforseaurchins,withmoreandlargerrefuges,andgreaterfeedabundance.　Followingsettlement, juvenilegreenseaurchinstakerefugeunderrocks,increvices,orunderdebrisas an adaptation to escape predation (CameronandSchroeter, 1980;Dumont et al., 2004).Heretheygrazeondiatoms,corallinealgaeanddetritus(RaymondandScheibling, 1987).Whileboth sitesin thepresent study supportedurchins, the shellhashattheSloopIslandsitewasfullofcracks,holesand largerspaces,providingrefuges forabroadersizerangeof juveniles.Therockcobbleat theJobIslandsitewas, for themostpart, flatagainst thesediment,with fewerandsmallerhidingplaces forjuveniles.Therockcobblehadsmallintersticesthatwerewellsuited for juvenilesatorbelow5-6mm,buttoosmallforlargerurchins.Mostoftheurchinsrecovered fromtheJobsitesurveys,whetherwildortagged,were< 15mmTD.　TheJobsitegenerallyhad lowerrecaptureratesof taggedseed than theSloopsite, indicating thatitwas lesshospitable forout-planted seaurchins.The notable exception occurred in the summerof the secondyear,whena large and significantnumberofsmallurchinswerecapturedattheJune2011Jobsitesurvey,andsubsequentlyidentifiedashatcheryoriginduetopresenceofthefluorochromemark.Thisspike inrecapturenumberscouldhavebeenduetomisidentification (e.g.detectingautoorpseudo-fluorescenceandattributingittothetag),or

itcouldhavebeenasamplingartifact.Presumably,misidentificationwouldhave occurred equally atSloopat thissurveydate,and itdidnot.AlthoughSloophadhigherrecapturenumbers (perquadratandtotal)atthissurveydatethanatothersurveys,theywerenotsignificantlydifferent fromtheotherSloopsurveys.Also,wewereconcernedaboutthisissueandanyspecimenswithatypicalfluorescencepatternswereconsideredasunmarked.For thesereasons,we believe that the spike in recapturenumbers observed at Job during the June 2011surveywasasampleartifact.Everysamplequadrathadtobethoroughlyandequallysearched,oftenbyoverturningrocksandshellstofindhiddenurchins.This effort had to be consistent between sitesand surveydates,which inpracticewasdifficultto accomplish.Undervarying field conditions ofbottomsubstrate, current, turbidity,ambient light,and temperature, it’s likely that the success ratefor findingurchinswouldvarybetweensitesanddates. In addition, randommovementpatterns ofurchinsontoandoff of the studyareasprobablyoccurred,becauseurchinsmoveinresponsetofoodavailabilityand thepresence/absenceofpredators(Dumontet al., 2007).Atabout15mmTDseaurchinjuvenilesarelessvulnerabletopredation,andashiftfromcryptic toactive foragingoccurs (Dumontet al., 2004).Migrationofurchins larger than10mmTDawayfromthereleaseareainsearchoffeedorrefugemightexplainthedisproportionatenumbersofsmalltaggedurchinsseenattheJobsite.　Active foraging enhances the availability andqualityofmacroalgae, increasing thegrowthrate.When there is abundant food sea urchins willaggregate inhighdensities, and theycan remainstationaryforseveralmonthsor longer (Dumontet al., 2007). Inthepresentstudy,bothsitesprovidedfeedintheformofencrustingalgaeandparticulatemacro-algae.However, the Sloop sitewasmoreexposedandhadgreatercurrents (Kirchhoffet al., 2008), andurchinsat this site thushadaccess tolargepiecesofdrift algae,mostlykelp, thatwerecarriedonto thesiteby thecurrent.Thisgreaterfeed availabilitymight explainwhy recapturedurchinshad a larger average andmaximumTDatSloopthanatJob.The lackofasubstantial foodsource at the Job site might have encouraged

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moreof the largerurchins to leave thesite,whilealso causing slow growth of the small urchinsthat remained,due to low food intake.Green seaurchingrowthratescanbehighlyvariable in thenaturalenvironment,primarily inresponse to feedavailability and type (Nestler andHarris, 1994;BradyandScheibling, 2006).Growthcanbeveryslowandratesof ≤ 0.25mmperyearhavebeendocumentedforurchinsfoundintidepools(Russell, 1998).Weobserved thatgreen seaurchins fromthesamehatcherycohortreared in the land-basedculturesystemhadsignificantlybettergrowththanthoserecapturedateitherleasesite.Thisisfurtherevidence thatgrowthpotential at the lease siteswas limitedmoreby environmental factors thanbygeneticsorbythefactthattheurchinswereofhatcheryorigin.　Inthepresentstudywewereabletodifferentiatehatchery origin fromwild urchins for up to 27months in the field. Johnsonet al. (2013) reportedthat tetracycline fluorescence could bedetectedforat least twoyears ingreenseaurchinsheld inthe lab,when tetracyclinewas administeredviainjection.Thefactthatfluorochromescanpersistforsuchextendedperiodsmakesthismarking/taggingmethodinvaluableforlongtermlabandfieldstudiesof seaurchins, andwasessential to carryingouttheresearchdescribedabove.Recentadvances intheapplicationandvisualizationof fluorochromesoffer furtheradvantages,whichcouldbringdowncostsand improve theeffectivenessof seaurchinmark/recapturestudies.EllersandJohnson (2009)describemethods tocreatemultiplemarkson thedemipyramids (e.g. at intervals orwithmultiplefluorochromes),whichwouldallowfordifferentiationofmultiple year classes released into the field.The same authors also describe visualization offluorochromes on external structures such asthe skeletalplates (test) andspines,whichallowstagged individuals tobe identifiedwithoutsacrifice(Johnsonet al., 2013).Ultimately,developmentofafieldportabledevice forvisualizing fluorochromesseems feasible, to allow reliable identification ofstocks in situwhileminimizingadverse impactonthepopulation(Johnsonet al., 2013).Thesemethodsprovidepowerful tools forevaluatingtheresultsoffuture restockingand sea ranchingprograms for

greenseaurchinsintheGulfofMaine.

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AgatsumaY., 2014:Reseeding of sea urchins inJapan. In:BrownN.P., andEddyS.D. (eds)Aquaculture of Echinoderms, In press JohnWileyandSons,NewYork,NY

BradyS.M.andScheiblingR.E., 2006:Changes ingrowthandreproductionofgreenseaurchins,Strongylocentrotus droebachiensis, duringrepopulationof theshallowsubtidalzoneaftermassmortality. J. Exp. Mar. Bio. Ecol., 335, 277-291.

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fluorochrome marking of out-planted green sea urchins ...tetracycline (sigma-aldrich tetracycline...

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