Live prey enrichment and artificial microdiets for larviculture of Atlantic red porgy Pagrus pagrus

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ContentslistsavailableatScienceDirect

Aquaculture

Reports

j ou rn a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / a q r e p

Live

prey

enrichment

and

artificial

microdiets

for

larviculture

of

Atlantic

red

porgy

Pagrus

pagrus

Wade

O.

Watanabe

a,∗

,

Md.

Shah

Alam

a

,

Andrew

D.

Ostrowski

a,1

,

Frank

A.

Montgomery

a

,

Jennifer

E.

Gabel

a

,

James

A.

Morris

Jr.

b

,

Pamela

J.

Seaton

c

aCenterforMarineScience,UniversityofNorthCarolinaWilmington,USA

bCenterforCoastalFisheriesandHabitatResearch,NationalOceanService(NOS),NOAA,USA cDepartmentofChemistryandBiochemistry,UniversityofNorthCarolinaWilmington,USA

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received31January2015

Receivedinrevisedform4January2016 Accepted9January2016

Availableonline22January2016

Keywords:

Redporgy Livepreyenrichment Rotifers

Artemia

Microdiets Microbounddiets Essentialfattyacids

a

b

s

t

r

a

c

t

Inthefirstexperimenttheeffectsofrotiferenrichmentandfeedingfrequencyonlarvalperformance ofredporgyPagruspagruswerestudied.Larvae(2dayspost-hatching=2dph)werefeds-typerotifers (∼20rotifers/mL)enrichedwithoneofthefourdifferenttreatmentmedia:RotiferDiet(microalgae Nan-nochloropsisoculataandTetraselmischuii),DHAProteinSelco,Algamac3000(Schizochytriumsp.)and Algamac+ARA(arachidonicacid).Larvaewerefeddailyatfullrationortwicedailyathalfration.Larval growthandsurvival(mean=22.8%)weresatisfactorythrough16dphunderalltreatments;however, resistancetohyposalinechallenge(SurvivalActivityIndex=SAI)waspositivelycorrelated(P<0.01)with DHAconcentrationofrotifers,andSAIappearedhighestintheAlgamac+ARAtreatment.Inthesecond experimenttheeffectsofArtemiaenrichmentonlarvalperformancewerecomparedfrom18dphthrough pre-metamorphosis(33dph).LarvaewerefedArtemia(0.5–3.0/mL)enrichedwithtwodifferentmedia Algamac3000andDCDHASelco,orunenrichedArtemia(control).BothmediaimprovedDHAlevelsin Artemiaandgrowthandsurvival(36.7–54.6%)oflarvae,whilelarvaefedunenrichedArtemiashowed poorgrowthandsurvival(5.2%).InthethirdexperimentaUniversityofNorthCarolinaWilmington microbounddiet(MBD)andtwocommercialmicrodiets(GemmaMicroandOtohime)wereevaluated. TheMBDcontaineddifferentproteinsources(i.e.,menhaden,squidandkrillmeal,soyprotein concen-trate)andattractants.Beginning16dph,livefeedsandmicrodietswereco-fedtothreetreatmentgroups oflarvae:(1)Gemma,(2)MBD,and(3)Otohime.LarvalperformanceontheUNCW-MBDwascomparable tothecommercialmicrodiets,withnosignificantdifferencesinlarvalsurvival,DHA,ortotaln-3PUFA contentthrough32dph.ResultsdelineatemoreeffectiverearingprotocolsforlarvicultureofAtlanticred porgyjuveniles.

©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Abbreviations: MD,microdiets;MBD,microbounddiets;EFAs,essentialfatty acids;PVC,polyvinylchloride;LRTs,larvalrearingtanks;2dph,2days post-hatching;EPA,eicosapentaenoicacid;DHA,docosahexaenoicacid;ARA,arachidonic acid;DPA,docosapentaenoicacid;ALA,alphalinolenicacid;MUFA, monounsatu-ratedfattyacid;PUFA,polyunsaturatedfattyacid;SFA,saturatedfattyacid;TFA, totalfattyacids;ANOVA,analysisofvariance;FBW,finalbodyweight;SAI,survival activityindex;FAMEs,fattyacidmethylesters.

∗ Correspondingauthorat:UniversityofNorthCarolinaWilmington,Center forMarineScience,601 SouthCollege Rd.,Wilmington,NC 28403-5927,USA. Fax:+19109622868.

E-mailaddresses:watanabew@uncw.edu,wwatanabe@ec.rr.com,

watanabe.wo@gmail.com(W.O.Watanabe).

1 Presentaddress:CenterforCoastalFisheriesandHabitatResearch,National

MarineFisheriesService,NOAA,USA.

1. Introduction

TheredporgyPagruspagrus,alsoknownasseabream,silver snapper,orpinksnapper,isavaluablemarinefinfishinthe fam-ilySparidae,inhabitingtheMediterraneanSea,theeasternAtlantic (fromtheBritishIslestoSenegal),andthewesternAtlantic(from NorthCarolinatoMexicoandfromVenezuelatoArgentina)(Morris etal.,2008).Animportantcomponentofthesnapper–grouper com-plexinthecoastalAtlanticofftheSEUS(particularlyNCandSC), redporgypopulationshavedeclinedseverely(SAFMC,2006,2010; VaughanandPrager,2002), andstringentrequirementsfor cap-tureofredporgyhavebeenestablished(SAFMC,2006,2010).Due todecliningnaturalpopulations,highmarketvalue,andsuitability forintensivecultureintanksandcages,redporgyareconsidered apromisingspeciesforfarmingintheMediterranean(Koliosetal., http://dx.doi.org/10.1016/j.aqrep.2016.01.003

2352-5134/©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4. 0/).

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1997;Hernandez-Cruzetal.,1999;Rooetal.,1999;Mihelakakis etal.,2001a,b;Papandroulakisetal.,2004;Morrisetal.,2008;Roo etal.,2010).

Studies to date indicate that, aside from high early mor-tality related to swim bladder inflation (Mihelakakis, 2001b; Papandroulakisetal.,2004),redporgyareadurablespeciesunder hatcheryconditions(Rooetal.,1999;Mihelakakisetal.,2001b; Papandroulakisetal.,2004;Bukeetal.,2005;Morrisetal.,2008; Rooetal.,2010).Significantprogresshasbeenmadein understand-ingthespawningandlarvalculturerequirementsofAtlanticred porgy(Morrisetal.,2008;Ostrowskietal.,2011)andproducing post-metamorphicjuvenilesinthelaboratory(Morrisetal.,2008). However,greaterknowledgeofpracticallarvalrearingtechnology isneededforreliablemasspropagationandtoremovean impor-tantconstrainttocommercialfarmingofthisspecies(Cavalinand Weirich,2009;Andradeetal.,2012).

Livefeedscommonlyusedinmarinefinfishhatcheries, partic-ularlyrotifersBrachionusspp.andbrineshrimpArtemiaspp.,are inadequateandvariableinnutritionalvalue.Therefore,itis criti-caltoenrichthesepreyitemswithessentialnutrients(e.g.,fatty acids)beforefeedingthemtothefishlarvae(Izquierdoetal.,1989; Hernandez-Cruzetal.,1999;Sargentetal.,1999;Sorgeloosetal., 2001).Previousstudieshave showngrowthperformance of lar-valredporgyisaffectedbylevelsofhighlyunsaturatedfattyacids (HUFAs) docosahexaenoic acid(DHA, 22-6:n-3) and eicosapen-taenoicacid(EPA,20:5n-3)providedinliveprey(Hernandez-Cruz etal.,1999; Rooet al.,2009).Morerecent studieshave shown thatlevelsofthen-6HUFAarachidonicacid(ARA,20:4n-6)inlive prey canalso affect growth or stress resistancein marine fin-fishlarvae(Kovenetal.,2001b;Rezeketal.,2010;Carrieretal., 2011;Watanabeetal.,2014).Numerouscommercialmediahave beenformulatedtoboostHUFAprofilesandotheressential nutri-entsinliveprey.Theseincludepreservedmicroalgaeconcentrates, yeastandlipidpowdersandsemi-moistpastes,andfreeze-dried heterotrophically-grownmicroalgae.Theseproductsyield differ-entnutritionalbiochemicalprofilesinlivepreywhich markedly affectgrowthandstressresistanceofthemarinefishlarvaefed enriched prey(Faulkand Holt,2005; Faulket al., 2005; Garcia etal.,2008;CavalinandWeirich,2009).Experimentaldata compar-ingtheefficaciesofpopularlivepreyenrichmentmediaforlarval cultureofmostmarinefinfishisfragmentaryandisof consider-ablepracticalimportance.Nostudieshavecomparedcommercial enrichmentmediaonlarvalgrowthperformanceandstress resis-tanceinredporgy.

Availableinformationsuggeststhatlarvalgrowthand produc-tioncostscanbeaffectedbyfeedingfrequencyofliveprey,which affectspreyquality,digestionandassimilationbylarvae,aswell asfeedwasteandwaterquality.Theoptimumfeedingfrequency andratevarydependingonspecies,fishsizeandrearingsystem (RabeandBrown,2000;Choetal.,2003;Russo,2013;Woolleyand Partridge,2015).

Duetohighlaborrequirementsandcostsforraisingliveprey, minimizingoreliminatingtheuseoflivefeedsbysubstituting arti-ficialformulatedmicrodiets(MDs)willalsobeimportantformore cost-effective production protocolsin marinefinfish hatcheries (Yufera etal.,1999; Baskerville-Bridgesand Kling,2000;Koven etal.,2001a;CahuandZambonino-Infante,2001;Teshimaetal., 2004;Kolkovsky,2009;SalhiandBessonart,2012;Andradeetal., 2012;Alametal.,2013).Zein-boundmicrodietswereused suc-cessfully in thelarval rearing of red seabream Pagrusmajor a congeneroftheredporgy(Kanazawaetal.,1989;Teshimaetal., 2000;2004).Zeinisahighmolecularweight,non-toxic,edible pro-teinwithamphotericandthermoplasticproperties.Zeinissoluble in40–90%alcohol,makingpreparationofzeinmicroparticlesless toxiccomparedwithpreparationmethodsthatinvolvetheuseof toxicsolvents.Ethanolcanalsobeeasilyremovedbyevaporation

orspray-dryingtoform zein-boundcomplexparticles. Further-more,aminoacidsandmanyotherlowmolecularwater-soluble materialsaresignificantlylesssolubleinethanolthanpurewater, reducingtheleakagelossofcorematerialsduringparticle prepa-ration(Langdonetal.,2007).Experimentalstudiesontheuseof artificialmicrodietsforweaninglarvaefromliveprey,including theefficaciesofzein-boundandcommerciallyavailableproducts, areneededtoadvancereliablelarge-scalehatcheryproductionof redporgyfingerlings.

Theoverallobjectiveofthisstudywastocontributeto optimiz-ingfeedingprotocolsduringhatcheryrearingofredporgy.Specific objectiveswereto(1)optimizeenrichmentmethodsforlivefeeds bycomparingpracticalenrichmentmediaandfeedingfrequency oflivepreyonlarvalperformance,and(2)tominimizereliance onlivepreythroughartificialmicrodiets(MDs)forco-feedingand earlyweaning.

2. Methods

2.1. Experiment1:comparisonofrotiferenrichmentmedia 2.1.1. Experimentalanimals

Redporgybroodstock(N=20,mean weight=373±2g,mean totallength=306±4.7mm)werecollectedbyhookandlineoff thecoastofNorthCarolinaandmaintainedinarecirculating31-m3 roundtankattheCenterforCoastalFisheriesandHabitatResearch, NationalOceanicandAtmosphericAdministration(Beaufort,NC, USA).Broodstockwereheldunderconditionssimulatingseasonal offshorebottom temperaturesoff North Carolina from27–33m (ParkerandDixon,1998)andunderambientphotoperiod. Brood-stockspawnedvolitionallyincaptivity(Morrisetal.,2008)during theirnaturalspawningseason(January–April),andeggswere col-lectedbyfilteringsurfacewaterfromthespawningtankthrough a505-␮mmeshnitexnet.EggsweretransferredtotheUniversity ofNorthCarolinaWilmingtonCenterforMarineScience (UNCW-CMS)AquacultureFacility(WrightsvilleBeach,NC,USA)inplastic bagscontainingoxygenatedseawater.Eggswereplacedina con-icalplexiglasstank(15-Lvolume)in32g/Lseawaterwhereviable (buoyant)eggswereseparatedfromnonviable(non-buoyant)eggs beforetheexperiment. Seawaterfor larval cultureexperiments waspumpedfromtheAtlanticIntracoastalWaterwayadjacentto WrightsvilleBeach,passedthrougha1-␮mfilterandUV-treated beforebeingaddedtorearingtanks.

2.1.2. Experimentalsystem

Redporgylarvaewererearedin15-Lcylindrical,black polyethy-lenetanksheldinfourtemperature-controlledwaterbaths(213-L) inacontrolled-environmentallaboratory.Lightintensityatthe sur-faceofeachtankwas500lx(492.3±3.9)(OstrowskiandWatanabe, UNCW,unpublisheddata)suppliedbytwo40-Wfull-spectrum flu-orescentlightbulbssuspendedovereachtable.Photoperiodwas 16L:8Dstartingat0700handendingat2300h.Extraneouslight wasexcludedbyblackcurtainssurroundingthewaterbath.

To begin the experiment, yolksac larvae (0days post-hatching=0dph)wereinitiallystockedintoeachtankatadensity of60larvae/Lat34salinityand19◦C.Temperaturewasadjusted 1◦Cperdayafterhatchinguntilanexperimentaltemperatureof 21◦Cwasreachedby2dph(Ostrowskietal.,2011).Airwas sup-pliedtoeachtankthroughanairdiffuserpositionedatthebottom centerofeachtank.Airflowwas90mL/mintoavoidinjuryfrom excessiveturbulence(ManginoandWatanabe,2006).

2.1.3. Experimentaldesign

Usingastandardizedlarvalfeedingregimedevelopedforred porgyatUNCW(Morrisetal.,2008;Ostrowskietal.,2011),different rotiferenrichmentmediaweretestedconcurrently.First-feeding

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stagelarvae werefed, beginning 2dph and continuingthrough 18dph,s-typerotifersBrachionusrotundiformis(5–20rotifers/mL) enrichedwithone offollowing fourdifferenttreatmentmedia: RotiferDietacombinationofthemicroalgaespecies Nannochlorop-sis oculata and Tetraselmis chuii (0.22% total fatty acids DHA, 40.72%EPA,4.34%ARA;ReedMariculture,Campbell,CA),DHA Pro-teinSelco(25mg/daydwDHA,10mg/daydwEPA,0ARA;INVE Aquaculture, Belgium), Algamac 3000 (heterotrophically-grown Schizochytrium sp.; 41.3% DHA, 14.72% DPA, 1.3%EPA, 0% ARA; BioMarineAquafauna, CA,USA)and Algamac+ARA (mixof75% Algamac3000and25%Algamac-arachidonicacid;2.4%DHA,0% DPA,0%EPA,37%ARA,BioMarineAquafauna,CA,USA).Rotifers werefedataconcentrationof5permLon2dph,increasingto7, 10,12,15,and20permLby4,7,9,12and14dph,respectively.

Tostudythecombinedeffectsrotiferenrichmentandfeeding frequencyonlarvalperformance,larvaeunderthefourdifferent enrichmenttreatmentswereeitherfedoncedaily(0830h)atfull ration (20rotifers/mL),or twicedaily(0830 and 1600h) athalf ration(10rotifers/mL).Fourreplicateunitswereassignedtoeach treatmentcombinationofenrichmentmediaandfeedingfrequency ina4×2factorialdesign.RotiferswereculturedonRotiGrow(DHA 0%,EPA22.5%,ARA3.2%,HUFA36.0%TFA,ReedMaricultureInc., Campbell,CA)inacontinuousculturesystem(Bentleyetal.,2008) andwereharvesteddailyfromthesystemandenrichedfor12h accordingtoprotocolsrecommendedbythemanufacturerbefore beingfedtothelarvae.Afterthefirst(0830h)feeding,enriched rotiferswereheldat10◦Ctomaintainnutritionalprofilebeforethe second(1600h)feeding.Thefattyacidprofilesofrotifersenriched withthesedifferentmediaaresummarizedintheResultssection. 2.1.4. Experimentalconditions

Waterwasexchangeddaily(33%)bysiphoning5Lfromeach tankat0600handreplacingwithfilteredseawaterat22◦C.Water exchangewascompletedbetween0600and0830hbeforethefirst feeding.Fromd2tod16ph,anon-viable, condensedmicroalgae N.oculatapaste(ReedMariculture,Campbell,California,USA)was addedtoeachtankataconcentrationof750,000cells/mLas back-ground.Thewatersurfaceofeachtankwas“skimmed”dailyusing apapertoweltoremoveoilanddebris.

2.1.5. Waterquality

Temperature (22±0.1◦C), salinity (34±0.1), D.O. (7.21±0.08mg/L), pH (range=8.10–8.40) (YSI 560, Yellow Springs,Ohio,USA),andlightintensity(492±4lx)weremeasured twicedaily (0900 and 1600h) from alternating replicate tanks per treatment. Ammonia (0.2±0.08mg/L) was tested twice a week(HACHDR850,Loveland,Colorado,USA;0.02±0.005mg/L) and airflow to each tank was tested daily using a flow meter (GilmontInstrumentsInc.,Barrington,Illinois,USA;±1mL/min) andadjustedasnecessary.

2.1.6. Growthandsurvival

Larvalgrowthandsurvivalweremonitoredusingvolumetric methods (Henne and Watanabe, 2003; Moustakas et al., 2004; ManginoandWatanabe,2006).Larvaeweresampledbyplunging a2-cmPVCpipe,46cminlength,intothetankandthen transfer-ringthewaterandlarvaecollectedtoagraduatedcylinder.Prior tosampling,airflowwasincreasedtouniformlydistributethe lar-vaeintanks.Larvaewererandomlysampledfromeachreplicate tankon1,7,10,and16dphatapproximately0730h.Thenumber offishwerecountedanddividedbythewatervolumeremoved duringsamplingtodeterminesurvival.Afteranesthetizingthe lar-vaewith0.5mg/Lof2-phenoxyethanol,larvaewereplaced ina Petridish and notochordlengths (NL)weremeasuredusingan ocularmicrometeronadissectingmicroscope(MeijiTechnoCo., Ltd.,Japan).Dryweightwasdeterminedusinganelectrobalance

(±0.01mg)(Sartorius,Goettingen,Germany)byplacing10larvae onapre-weighedmicroscopeslideaftertheywerepouredontoa 23-␮mmeshscreen,whichwasblottedwithaspongefrom under-neathtoremoveexcesswater.Theslideswerethenplacedintoa laboratoryovenat60◦Cfor72handthenreweighedtodetermine dryweightsoflarvae.

2.1.7. Hyposalinestressresistance

Attheendoftheexperiment(16dph),ahyposalinestresstest wasperformedontheremaininglarvaefromeachtreatmentto measuretheirabilitytowithstandanabruptdecreaseinsalinity to24underconditionsof zerofeeding.Using volumetric meth-ods,survivalwasmonitoredevery12hfor48h.Asurvivalactivity index(SAI)totestlarvaequalitywascalculatedusingthefollowing equation: SAI=N1 k

i:1 (N−hi)×i

whereNisthetotalnumberofinitiallarvae,hiisthecumulative mortalitybytheithhour,andkisthenumberofhoursthathave elapseduntilalllarvaehavediedortheexperimentended(Matsuo etal.,2006).

2.1.8. Fattyacidcomposition

Fatty acid composition of the enriched rotifers from each treatment at the beginning and at the end of the experiment was determined by first extracting total lipids into chloro-form:methanol(Folchetal.,1957).Onemilliliterofa0.001gmL−1 solutionofC19:0fattyacidwasaddedtoeachsampleasan inter-nal standard. Lipidfatty acids were converted to their methyl esters(FAMEs)forGCanalysisbyrefluxingtheconcentratedlipid sample in1.0mLof0.5MNaOH/MeOH for30min,followedby additionof1.5mLofborontrifluoride-methanol(BF3)and reflux-ing for an additional 30min. The FAMEs were extracted into hexane,concentratedanddissolvedin1mLofchloroform.GC anal-ysis wasperformed ona HP-6890 GasChromatograph using a 25m×0.25␮mHP-5capillarycolumnwithFIDdetection (Depart-mentofChemistryandBiochemistry,UNCW).Heliumwasusedas thecarriergas.Thecolumntemperatureprofilewas:195◦C,hold for8min,rampto270at15◦Cmin−1andholdat270Cfor2min.

FAMEpeakswereintegratedusingtheHPChemstationsoftware packageandindividualFAMEswereidentifiedbycomparisonof retentiontimestostandards:GLC-84(NuChekPrepUSA)aswellas individualstandardsofsteariodonic,eicosapentaenoicand arachi-donicacidmethylesters(Sigma–Aldrich,MO,USA).FAMEsfrom allsampleswerequantifiedusingtheirpeakareascomparedtothe peakareaoftheC19:0internalstandards.

2.1.9. Dataanalyses

Quantitative values were expressed as treatment means±standard error.Prior toanalysis,percentage data were arcsine transformed. To meet the assumptions of ANOVA, all datawereanalyzedusingLevene’shomogeneityofvariancetest priortoatwo-wayANOVA.Ifthedataviolatedtheassumptions ofthetwo-way ANOVA,aKruskal–Wallistest wasrunto com-paretheindependenteffectsofenrichmentand feedfrequency. The effects of rotifer enrichment, feeding frequency and their interactionongrowth,survival,andstressresistance(SAI)were analyzed usinga two-way ANOVA.Whennointeractiveeffects were observed, rotifer enrichment treatments were combined across feeding frequencies and feeding frequency treatments werecombinedacrossenrichmenttreatments. ATukey–Kramer honestly significantdifference(HSD)testwasusedfor multiple comparisonsamongmeans(Kramer,1956).Allstatisticalanalyses

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wereperformedusingSASstatisticalsoftware(version9.1.3,SAS Institute, Cary, North Carolina, USA) and a P-value ≤0.05 was consideredsignificant.

2.2. Experiment2:comparisonofArtemiaenrichmentmediaand feedingfrequency

2.2.1. Experimentalsystem

ForExperiment1.2,larvaewereinitiallyrearedfromhatching through14dphin300-Llarvalrearingtanks(LRTs)supportedbya recirculatingsystemasdescribedbelow.Yolksacstagelarvaewere stockedatadensityof50larvae/Lat34salinityandat22◦C,and lar-vaewererearedonastandardfeedingregime(Morrisetal.,2008; Ostrowskietal.,2011)untilthestartoftheexperiment.During thepre-experimentalperiod,larvaewerefedrotifersculturedon RotiGrowandthenenrichedwithN-Rich(ReedMaricultureInc., Campbell,CA)beforefeedingtothelarvae.

Experimental units consisted of gray, 30-L cylindrical (diam.=38cm, depth=40cm) mini larval rearing tanks (mini LRTs)withcenterdrainingstandpipesandin-flowmanifolds.The miniLRTs were supported by a common recirculatingsystem, including a 0.11-m3 bubble bead filter (Aquaculture Systems Technology, New Orleans, LA); a 40-W UV sterilizer (Emperor Aquatics,Pottstown,PA);a1-HPheatpump,afoamfractionator (TopFathomTF300A,1/10HPFoamFractionatorPump) (Orange-burg,NY);

¾

HP pump(JacuzziStingray) (ChinoHills,CA);and a25-␮mcartridgefilter.Fullstrengthseawater(34salinity)was recirculatedthrougheachtankatanexchangerateof200%daily, andtemperaturewasheldat22◦Cusingaheatpump(Aqualogic, SanDiego,CA).Airwassuppliedtoeachtankbytwoairstoneson eithersideofthecenterstandpipeat90mL/min.Illuminationwas providedat500lxatthewatersurfacebytwo40-Wfullspectrum fluorescentlightbulbssuspendedoverthetanks.Photoperiodwas 18L:6Dstartingat0600handendingat2200h.

2.2.2. Experimentaldesign

Larvaewerestockedinto12miniLRTsatadensityof8larvae/L at34salinityand22◦C.Larvaewerefednewly-hatchedunenriched ArtemiafranciscanaInstar1nauplii(ArgentLaboratories,Redmond, WA,USA)from15to20dph.TostudytheeffectsofArtemia enrich-mentonlarvalperformance,larvaewerefed,beginning18dphand continuingthrough32dph,ArtemiaInstarIImetanaupliienriched withtwodifferenttreatmentmedia:Algamac3000andDCDHA Selco(15mg/gdw EPA,30mg/g dwDHA, 50mg/g dwsumn-3 HUFAs,INVEAquaculture,Belgium).Inaddition,larvaewerefed unenrichedArtemiaInstarIImetanaupliiasa controltreatment. Four replicate tanks were maintained per treatment. Enriched Artemiaconcentrationwas0.5/mLon18dphandthenincreased dailyto3.0/mLby23dphandcontinuingthroughthebeginningof themetamorphicperiodapproximately32dph.Artemiawerefed twicedaily(0830and1600h)tothetargetedlevel.Afterthefirst (0830h)feeding,enrichedArtemiawereheldat10◦Ctomaintain nutritionalprofilebeforethesecond(1600h)feeding.

2.2.3. Experimentalconditionsandlarvalgrowthandsurvival Experimentalconditionsandwaterqualityweremaintainedas describedforExperiment1above.Larvalgrowthwasdetermined usingvolumetricmethodson14,21,25and33dphasdescribedfor Experiment1above.Survivalwasdeterminedatterminalsampling on33dph.

2.2.4. Fattyacidcomposition(Experiment1.2)

FattyacidcompositionoftheenrichedArtemiaandlarvalwhole bodiesfromeachtreatmentatthebeginningandattheendofthe experimentweredeterminedasdescribedabove.Fattyacidprofiles aresummarizedintheresultssection.

2.2.5. Dataanalyses

DataanalysesfollowedthosedescribedforExperiment1above. However,inExperiment2,theeffectsofArtemiaenrichmenton growth,survivalandlarvalfattyacidcompositionwereanalyzed usingaone-wayANOVAandsignificantdifferencesamong treat-mentsweretestedbyTukey–KramerHSDtest.

2.3. Experiment3:minimizerelianceonlivepreythrough artificialmicrodiets(MDs)forco-feedingandearlyweaning. 2.3.1. Experimentalsystemandanimals

Experiment3wasconducted totesttheperformance ofred porgylarvaeondifferentmicrodiets.ForExperiment3,larvaewere initiallyrearedfromhatchingthrough15dphin300-LLRTs sup-portedbyarecirculatingsystemasdescribedaboveforExperiment 1.Embryos were stockedinto three300-L LRTs ata density of approximately39embryosperL(11,906pertank)at34salinity andat22◦C,andlarvaewererearedonastandardfeedingregime (Morrisetal.,2008;Ostrowskietal.,2011)untilthestartofthe experiment.Duringthepre-experimentalperiod,larvaewerefed rotifersculturedonRotiGrowandthenenrichedwithN-Rich(Reed MaricultureInc.,Campbell,CA)beforefeedingtothelarvae.

The experimental units consisted of gray, 30-L cylindrical (diam.=38cm,depth=40cm)minilarvalrearingtankswithcenter drainingstandpipesandin-flowmanifoldssupportedbya recircu-latingsystemasdescribedinExperiment1.2above.Fullstrength seawater(34salinity)wasrecirculatedthrough each tankatan exchangerateof200%dailyandwasheldat22◦C.Airwas sup-pliedtoeachtankbytwoairstonesoneithersideofthecenter standpipeat90mL/min.Illuminationwas500lxatthewater sur-face,andphotoperiodwas18L:6Dstartingat0800handendingat 0200h.

2.3.2. Experimentaldesign

On16dph,redporgylarvaewerestockedintotwenty-four 30-LminiLRTsataninitialdensityof6.2fish/L,butpossiblydueto transferstress,manyfishsuccumbedovernight,leavingastarting experimentaldensityof3.9fish/L.Tocomparetheperformanceof redporgylarvaeondifferentmicrodiets,fishwereco-fedArtemia and one of the following three microdiets: (1) Gemma Micro (Skretting,Nutreco,Canada),(2)aUNCW-formulatedzein-bound microdiet(MBD)and(3)Otohime(ReedMaricultureInc., Camp-bell,CA), and (Table1).Fish werereared accordingtoUNCW’s larvicultureprotocolforredporgy(Morrisetal.,2008;Ostrowski etal.,2011)(Table1).Fishwereco-fedlivefeedtwotimesperday (0830and1600h)andmicrodiets(1–6mg/fish/day)fourtimesper day(0830,1130,1430,and1630h)asreportedforothermarine fishlarvae(Teshimaetal.,2004).UNCWMBDandthecommercial microdiets(MDs)werefedusingasmallspoonandblowingthe feedintothewater.Microdietparticlesizeswereadjusted accord-ingtofishmouthsizesbasedonvisualobservation.

UNCWzein-microbound diet (MBD) was formulated onthe basis of previous studies on southern flounder Paralichthys lethostigma(Table2)(Alametal.,2013)andnutrientrequirement informationforothermarinefishlarvae(Teshimaetal.,2004).The UNCWMBDcontained58%crudeproteinand16%lipidandwas formulatedwithdifferenthighqualityproteinsources,including menhadenfishmeal,soybeanproteinconcentrate,squidmealand krillmeal.TheUNCWMBDalsocontained1%attractants(alanine, glycine,taurineandbetaine),0.5%methionine,0.5%lysine,5% vita-minand5%minerals.Menhadenfishoilandsoybeanlecithinwere addedaslipidsources(Table2).

Before preparing the MBD, all protein sources were sieved (mesh size125–250m). The MBD was prepared accordingto Teshimaetal.(1982)andKanazawaetal.(1989)andthenground andsievedintoparticlesrangingfrom125to550␮m.Thelipids

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Table1

Protocolusedtofeedlivefeedandcommercialmicrodiets(MDs)andtheUNCWmicrobounddiet(MBD)tolarvalredporgy.

1 5 10 15 20 25 31

Rotifers(5–20/mL)

Artemia(0.5–3/mL)

MDs/MBD(125–250␮m) MDs/MBD(250–355␮m) MDs/MBD(355–500␮m) Feedingoflivefeed 2times/day(0830and1600h)DanielsandWatanabe(2003),Morrisetal.(2008),Ostrowskietal.(2011)

FeedingrateofMFs/MBD 1–6mgfish/day(Teshimaetal.2004) FeedingfrequencyofMDs/MBD 4times/day(0830,1130,1430and1630h)

Table2

CompositionofUniversityofNorthCarolinaWilmingtonmicrobounddiet.

Ingredients g/100g Soyproteinconcentrate 8

Krillmeal 10 Squidmeal 35 Menhadenmeal 15 Zein 10 Menhadenfishoil 7 Soybeanlecithin 3 Vitamin 5 Mineral 5 Taurine 0.25 Alanine 0.25 Glycine 0.25 Betaine 0.25 Methionine 0.5 Lysine 0.5 Total 100 Protein% 58 Lipid% 16 Energykj/gdiet 19.4

sources(menhadenfishoilandsoybeanlecithin)weremixedwith 300mL of60% ethanol inan electricblender. Thezein andthe remainingingredientswereadded,mixedthoroughlyuntilallthe ethanolevaporated.Theresultingdoughwasfreezedriedfor48h ormoreasnecessary.Drydietsweresortedwithsieves(125,250, and355␮mmeshsizes)toobtainthedesiredsizesofdietsafter removalofethanolbyfreeze-dryingandcrumblingintoparticles. TheMBDwasstoredat−24◦Cuntilused.

2.3.3. Waterqualityparameters

Totalammonianitrogen(TAN)(mg/L)wasmonitoredweekly (HACHDR850,Loveland,CO,USA).Temperature,salinity,dissolved oxygenandpH(YSI55,YellowSprings,OH,USA)ofthewaterwere monitoredeverytwodays.Therangesofwaterqualityparameters weremaintainedatoptimallevels(Morrisetal.,2008)asfollows: temperature19.2–21.7◦C,pH8.2–8.4,salinity33.1–34.2,dissolved oxygen7.3–8.6mg/L,TAN0.1–0.3mg/L,andnitrite0.09–0.14mg/L. Lightintensity(470-580lx)atthewatersurfaceofeachtankwas measuredwithalightmeter(ExtechInstruments,Waltham,MA, USA).Airflow(40–60mL/min)toeachtankwasmonitoredusing aflowmeter(Cole-ParmerInstrument,VernorHills,IL,USA)and adjustedasneeded.Tanksurfaceswereskimmeddailywithpaper towelstoremoveoilfilms.

2.3.4. Growthperformance,survivaldetermination,andsample collection

Thefishweresampledinitiallyandonthefinalday(day16)of theexperimenttominimizehandlingstress.Survival(%frominitial stocking)wasmonitoredonday7,12,and16(finalsampling). Per-formanceoffishoneachtestmicrodietwasassessedbyfinalbody weight(FBW),totallength(TL),andsurvival(%survivalofinitial testfish).MeasurementsofTLwereconductedusingamicroscope with10fishrandomlycollectedfromeachtank.Forbodyweight,all

thelarvaewerecollected,rinsedwithfreshwaterandblotteddry andwetweights(mg/fish)weremeasuredbyweighingfishwith anelectronicmicrobalance.Thelarvaewerefreezedriedanddry weightsweremeasuredusingamicrobalanceandthenstoredat −80◦Cforproximateanalysis.

2.3.5. Larvalproximateandfattyacidcomposition

ProximateanalysisfollowedthemethodofAOAC(2000).Fatty acidcompositionofthelarvalwholebodies wasdeterminedas describedabove.

2.3.6. Dataanalyses

Data analyses followed those described for Experiment 1.1 above.Growthdatawereanalyzedbyone-wayANOVA(JMPver.7, SAS,USA).ATukey–KramerHSDtestwasusedformultiple com-parisonsamongmeans(Kramer,1956).

3. Results

3.1. Experiment1:comparisonsofrotiferenrichmentmediaand feedingfrequency

3.1.1. Growthandsurvival

Therewerenosignificant(P>0.05)mainorinteractiveeffects ofrotiferenrichmentmediaand feedingfrequencyonlarvalNL (Table3),dryweight(Table4)orsurvivalonanysamplingdayfor thedurationofthestudy.Initial(0dph)NLunderbothfeeding fre-quencieswas2.52mmamongtreatmentsandincreasedsteadilyto 5.14mmon18dph(Table3).Initial(0dph)dryweightunderboth feedingfrequencieswas0.02mgamongtreatmentsandincreased steadilyto0.35mgon18dph(Table4).Survivalunderboth feed-ingfrequencies,fellsharplyto42.3±5.2%by6dph,andthenmore graduallyto29.8± 3.3%on13dphandto22.8±3.5%on18dph. 3.1.2. Hyposalinestressresistance(SAI)

Therewerenosignificantmainorinteractiveeffectsofrotifer enrichmentmediaandfeedingfrequencyonlarvalSAIatany sam-pling period for the duration of thestudy (Fig.1). Under both feedingfrequencies, mean SAIvalues among enrichment treat-mentswere39.4,208.1,627.2and939.3after12,24,36and48h, respectively (Fig.1). From24hpost-transferonward, however, meanSAIvalueswereconsistentlyhighestforlarvaefedrotifers enrichedwithAlgamac-ARAfollowedbyAlgamac3000andthen proteinSelcotreatmentandwerelowestforRotiferDiet(Fig.1A). Underallenrichmenttreatments,meanSAIvalueswere consis-tently higher for larvae fed 2×/day than for larvae fed 1×/day (Fig.1B).After48hofthehyposalinechallenge,ahighly signifi-cantpositivecorrelation(r=0.99,P<0.01,N=4)betweenlarvalSAI androtiferDHAconcentrationwasevident.

3.1.3. Fattyacidcompositionofenrichedrotifers

Lipid and fatty acid composition of enriched rotifers are shown in Table 5. Values are presented for rotifers sampled before the morning (0800h) feeding and after the enriched

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Table3

Growth(notochordlength=NL)oflarvalredporgyunderdifferentrotiferenrichmentandfeedingfrequencytreatments.Datarepresentmeans(±SEM).Tocompare enrich-menttreatments,growthdatawerecombinedacrossbothfeedingfrequencies(N=8).Tocomparefeedingfrequencies,growthdatawerecombinedacrossallenrichment treatments(N=16).Nosignificant(P>0.05)treatmentdifferenceswereobserved.

Age(dph) Enrichment

RotiferDiet DHAProteinSelco Algamac Algamac+ARA 0 2.50±0.03 2.53±0.03 2.53±0.03 2.53±0.03 6 3.46±0.11 3.38±0.11 3.28±0.06 3.23±0.12 13 4.33±0.09 4.33±0.08 4.44±0.07 4.35±0.12 18 5.00±0.11 5.18±0.06 5.24±0.11 5.12±0.09 Age(dph) Feedingfrequency(times/day)

2×/day 1×/day 6 3.34±0.07 3.34±0.08 13 4.27±0.07 4.45±0.05 18 5.04±0.07 5.22±0.06

Table4

Growthindryweight(mg)oflarvalredporgyunderdifferentrotiferenrichmentandfeedingfrequencytreatments.Datarepresentmeans(±SEM).Tocompareenrichment treatments,growthdatawerecombinedacrossbothfeedingfrequencies(N=8).Tocomparefeedingfrequencies,growthdatawerecombinedacrossallenrichmenttreatments (N=16).Nosignificant(P>0.05)treatmentdifferenceswereobserved.

Age(dph) Enrichment

RotiferDiet DHAProteinSelco Algamac Algamac+ARA 0 0.02±0.01 0.02±0.01 0.02±0.01 0.02±0.01 6 0.03±0.00 0.03±0.01 0.03±0.00 0.04±0.01 13 0.13±0.01 0.10±0.02 0.15±0.01 0.13±0.01 18 0.35±0.03 0.37±0.02 0.33±0.02 0.36±0.03 Age(dph) Feedingfrequency(times/day)

2×/day 1×/day 0 0.02±0.01 0.02±0.01 6 0.03±0.00 0.03±0.00 13 0.14±0.01 0.12±0.01 18 0.35±0.02 0.36±0.02 Table5

Totallipidandfattyacidcomposition(%oftotalfattyacids)ofrotifersenrichedwithdifferentproducts(RotiferDiet,DCDHAProteinSelco,Algamac3000,Algamac3000+ARA). Valuesrepresentmeans(N=2)andarepresentedforrotiferssampledbeforethefirst(0800h)feedingandaftertheenrichedrotiferswereheldat10◦Cfor8hbeforethe

second(1600h)feeding.

Fattyacids Rotifertreatments

RotiferDietamRotiferDietpmDCDHASelcoamDCDHASelcopm Algamac3000amAlgamac3000pmAlgamac3000+ARAamAlgamac3000+ARApm 14:0 4.80 4.77 3.12 3.22 6.27 6.28 5.04 4.79 16:0 26.53 26.47 23.12 24.62 35.70 35.25 27.58 26.76 16:1n-7 16.59 16.29 10.67 11.22 12.14 10.96 13.97 13.60 18:0 5.65 5.24 6.24 6.29 6.42 5.81 4.94 5.39 18:1n-9 7.74 7.57 11.86 12.21 5.93 5.95 6.62 6.46 18:1n-11 5.65 5.52 6.79 6.84 4.32 4.21 5.29 5.24 18:2n-6 3.66 3.19 6.83 6.70 1.99 1.69 3.33 3.08 18:3n-3 0.96 0.93 1.47 1.51 0.73 0.74 0.82 0.80 18:4n-3 Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected 20:1 2.25 1.98 2.35 2.34 1.59 1.50 1.77 1.77 20:2n-6 0.27 Undetected 0.60 0.26 Undetected Undetected Undetected Undetected 20:4n-6 0.81 0.44 1.04 1.04 0.80 0.58 1.56 1.46 20:5n-3 4.64 3.79 5.48 4.60 2.79 2.66 5.52 4.91 22:5n-3 1.18 1.19 1.82 1.71 1.10 1.23 1.90 1.73 22:5n-6 Undetected Undetected Undetected Undetected Undetected Undetected Undetected Undetected 22:6n-3 Undetected Undetected 1.49 1.30 1.89 2.02 2.76 2.40

SFA 36.98 36.48 32.48 34.13 48.39 47.34 37.56 36.94

MUFA 32.23 31.36 31.67 32.61 23.98 22.62 27.65 27.07

n-3PUFA 6.78 5.91 10.26 9.12 6.51 6.65 11.00 9.84

n-6PUFA 4.74 3.63 8.47 8.00 2.79 2.27 4.89 4.54 Totallipidmg/g349.17 353.81 369.92 342.32 374.69 363.09 376.07 361.20 Fattyacidsintraceamounts(<0.5%)arenotreported.

Totallipidisreportedinmg/gdryweight.

rotiferswereheldat10◦Cfor8h(i.e.,“cold-banked”)beforethe afternoon(1600h)feeding.Sinceduplicatedeterminationswere madeforeach fattyacid,nostatisticalcomparisonsweremade. In general, afternoon values were similar to or slightly below the morning values. Total lipid was similar among treatments

(mean=361.2,range=342.3–376.1mg/gdrywt.)(Table5).Total SFAappearedtobehigherinrotifersfedAlgamac(47.34–48.39% TFA)thanintheothertreatments(36.48–37.56%TFA).TotalMUFA appearedtobehigherintheRotiferDietandDHAProteinSelco treatments(31.67–32.61%TFA)thanintheAlgamac+ARA

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treat-Hou

rs

0 12 24 36 48

SA

I

0 200 400 600 800 1000 1200 1400 1600 Roifter Diet Selco Algamac Algamac + ARA

Hou

rs

0 12 24 36 48

SA

I

0 200 400 600 800 1000 1200 1400 1600 2x 1x

B

A

Fig.1.SurvivalActivityIndex(SAI)oflarvalredporgyunderdifferentrotiferenrichment(A)andfeedingfrequency(times/day)treatments(B).Datarepresentmeans(±SEM). InA,datawerecombinedacrossbothfeedingfrequencies(N=8).InB,datawerecombinedacrossallenrichmenttreatments(N=16).NosignificantdifferencesinSAIamong treatmentswereobserved.

ment(27.07–27.65% TFA)andlowest intheAlgamac treatment (22.62–23.98%TFA).

Total n-3PUFA levels appeared to be higher in rotifers fed DHA Protein Selco (9.12–10.26% TFA) and Algamac 3000+ARA (9.84–11.00%TFA)thaninrotifersfedRotiferDiet(5.91–6.78%TFA) orAlgamac3000(6.51–6.65%TFA)(Table5).Amongthen-3PUFAs, concentrationof EPA 20:5n-3 appeared tobe lower in rotifers enrichedwithAlgamac3000(2.66–2.79%TFA)thanintheother treatments(3.79–5.52%TFA).DPAdocosapentaenoicacid22:5n-3

rangedfrom1.10to1.90%TFAamongtreatments.DHA 22:6n-3 wasundetectedinrotifersfedRotiferDiet,butrangedfrom1.30to 2.02%inrotifersfedDHAProteinSelcoandAlgamacandappeared tobehighestintheAlgamac+ARAtreatment(2.40–2.76%TFA).

Totaln-6PUFAlevelsofrotifersappearedtobelowestinthe Algamactreatment(2.27–2.79%TFA),intermediateintheRotifer Diet(3.63–4.74%TFA)andAlgamac+ARA(4.54–4.89%TFA) treat-mentsandhighestintheDHAProteinSelcotreatment(8.00-8.47% TFA) (Table 5). Among the n-6 PUFAs, rotifers enriched with

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Algamac3000+ARAappearedtohavehigherARAconcentrations (1.46–1.56%TFA)thantheothertreatments(0.44–1.04%TFA).

3.2. Experiment2:comparisonsofArtemiaenrichmentmedia 3.2.1. Growthandsurvival

From14to21dph,growth(NLand dryweight) oflarvae in thecontroltreatmentfedunenrichedArtemiawas indistinguish-ablefromgrowth oflarvae fed Artemiaenriched withAlgamac 3000andDC DHA Selco(Fig.2).However,a cleardeparturein growth was observed from 25dph, when larvae fed enriched Artemiashowedmarkedlyimprovedgrowth.LarvalNLon14dph was4.83mmamong treatments,withnosignificantdifferences (Fig.2A).MeanNLremainedsimilaramongtreatmentsthrough 21dph(6.64mm)and25dph(7.64mm)(Fig.2A).By33dph, how-ever,therewerehighlysignificant(P<0.001)differencesamong treatments,withtheAlgamac3000(12.15mm)andDCDHASelco (11.68mm)treatmentsproducinghigherlarvalNLthanthe con-troltreatment(7.85mm),butnotsignificantlydifferentbetween enrichmenttreatments(Fig.2).Larvaldryweighton14dphwas 0.2mginalltreatments,withnosignificantdifferences(Fig.2B). Dry weight remainedsimilar in all treatments through 21dph (1.18mg)(Fig.2B).By25dph,however,therewerehighly signif-icant(P<0.01)differences amongtreatments,withtheDC DHA Selco(3.08mg)andAlgamac3000(2.80mg)treatments produc-inghigher dry weightthan the controltreatment(2.0mg), but withnodifferencesbetweenenrichmenttreatments(Fig.2B).On 33dph,theAlgamac3000(11.3mg)andtheDCDHASelco(9.17mg) treatments were significantly largerthan the control (2.18mg) treatment,butwithnodifferencesbetweenenrichmenttreatments (Fig.2B).Ond33dph,survival(mean+SEM,N=4)wasmarkedly higherintheDCDHASelcotreatment(54.61+6.43%)thaninthe Algamac3000treatment(36.7+4.03%)andwaslowestinthe con-troltreatment(5.23+2.58%).

3.2.2. FattyacidcompositionofenrichedArtemia

LipidandfattyacidcompositionofenrichedArtemiaareshown inTable6.ValuesarepresentedforArtemiasampledbeforethe morning(0800h)feedingandaftertheenrichedArtemiawereheld at10◦Cfor8h(i.e.,“coldbanked”)beforetheafternoon(1600h) feeding. No significant (P>0.05) differences between morning andafternoon concentrations wereobservedfor anyfatty acid. Nosignificanttreatmentdifferenceswereobservedintotallipid (290mg/gdrywt.)orinMUFAs(41.9%TFA)(Table6).TotalSFA waslowestintheDCDHASelco(27.5%TFA)andtheunenriched (control)(29.8%)highest(P<0.05)intheAlgamac3000(33.08% TFA)treatments.

Nosignificantdifferenceswereobservedintotaln-3PUFA lev-elsamongArtemiaenrichedwithdifferenttreatments(21.3%TFA) (Table6).Amongthen-3PUFAs,concentrationofstearidonicacid (STD)18:4n-3 was higher (P<0.05) in the control (3.79% TFA) andAlgamacenrichedArtemia(3.22%TFA)comparedtoArtemia enrichedwithDCDHASelco(2.14%TFA).Therewerenosignificant differencesinEPA20:5n-3(2.53%TFA)amongtreatments. Con-centrationofDPAdocosapentaenoicacid22:5n-3averaged(1.45% TFA)inDCDHASelcoandAlgamac-enrichedArtemia,butwasnot detectedin thecontrolArtemia. DHA 22:6n-3levelsin Artemia fedDCDHA Selcoand Algamac(2.40%TFA),although consider-ablylowerthanthoseprovidedintheirdiets(Algamac=41%DHA, DCDHASelco=30%DHA),weresignificantly(P<0.05)higherthan thosefoundinunenrichedcontrolArtemia(0.66%TFA)(Table6).No significanttreatmentdifferenceswereobservedintotaln-6PUFA levelsofArtemia(6.62%TFA)(Table6).Amongthen-6PUFAs, con-centrationofARAwashigher(P<0.05)inArtemiaenrichedwith

Algamac3000(2.17%TFA)thanwithDCDHASelco(0.32%TFA)and waslowestintheunenrichedcontrols(0.29%TFA).

3.2.3. Larvalfattyacidcomposition

Wholebodylipidandfattyacidcompositionofredporgylarvae on13dph(initial)andon25dph(final)areshowninTable7.On 13dph,meantotallipidlevelamongtreatmentswas306mg/gdry wt.Amongthen-3PUFAs,DHAaveraged11.8%TFA,EPA8.01%,and ALA12.9%.Amongthen-6PUFAs,ARAaveraged4.45%TFA,andLA linoleic(18:2n-6)5.42%.

On25dph,nosignificantdifferencesamongtreatmentsintotal lipid(170mg/gdrywt.)wereobserved.On25dph,concentration ofSFAwashigher(P<0.05)inlarvaefedtheunenrichedcontrol Artemia(30.6%TFA)thaninlarvaefedpreyenrichedwith Alga-mac3000(27.8%TFA)andwaslowestinlarvaefedpreyenriched withDCDHA Selco(25.7%TFA).TotalSFAwashigher(P<0.05) in13dphlarvae(30.3%TFA)comparedto25dphlarvaefedprey enrichedwithDCDHASelco(25.7%TFA).On25dph,nosignificant differencesamongtreatmentswereobservedintotalMUFA(33.7% TFA).

On25dph,nosignificantdifferencesamongtreatmentswere observedintotaln-3PUFAlevels(25.5%TFA)(Table7).Amongthe n-3PUFAs,EPAwashigher(P<0.05)inlarvaefedArtemiaenriched withDCDHASelco(8.02%TFA)thaninlarvaefedArtemiaenriched withAlgamac(7.24%TFA),orfedunenrichedArtemia(6.92%TFA). On25dph,EPAwashigher(P<0.05)intheDCDHASelco treat-ment(8.02%TFA)thanintheAlgamac(7.24%)orcontrol(6.92%TFA) treatments.On25dph,DHAlevelswerehigher(P<0.05)inlarvae fedArtemiaenrichedwithDCDHASelco(10.3%TFA)thanin lar-vaefedArtemiaenrichedwithAlgamac(7.09%TFA)andwaslowest (P<0.05)inlarvaefedunenrichedArtemia(3.55%TFA)(Table7).

Thetotaln-6PUFAconcentrationwashigher(P<0.05)in lar-vaefedArtemiaenrichedwithAlgamac(13.6%TFA)thaninlarvae fedArtemiaenrichedwithDCDHASelco(11.0%TFA)orunenriched Artemia(10.2%TFA)(Table7).Amongthen-6PUFA,concentration ofARAwashigher(P<0.05)inlarvaefedArtemiaenrichedwith Algamac(5.42%TFA)than inlarvaefed DCDHA Selco-enriched (4.10%TFA),orunenrichedArtemia(4.63%TFA)(Table7).ARA con-centrationin larvae enrichedwithAlgamac washigherthan in thosefoundon13dph(4.45%TFA).

Wholebodylipidandfattyacidcompositionofredporgy lar-vaeon33dphareshowninTable8.Nosignificantdifferenceswere observedintotallipid(148mg/gdrywt.),SFA(27.0%TFA),orMUFA (30.6%TFA)amongtreatments.On 33dph, nosignificant differ-encesamongtreatmentswereobservedintotaln-3PUFAlevels (28.6%TFA).Amongthen-3PUFAs,EPAwashigherincontrol lar-vaefedunenrichedArtemia(7.63%TFA)thaninlarvaefedArtemia enrichedwithDC DHASelco(6.23%TFA)orwithAlgamac3000 (5.79%TFA).ConcentrationofDHAwashigher(P<0.05)inlarvae fedArtemiaenrichedwithDCDHASelco(10.6%TFA)thaninlarvae fedunenrichedArtemia(6.40%TFA).

On33dph,totaln-6PUFAconcentrationwashighest(P<0.05) inlarvaefedunenrichedArtemia(13.7%TFA)comparedtolarvae fedArtemiaenrichedwithAlgamac(12.5%TFA),orwithDCDHA Selco(10.5%TFA)(Table8).Amongthen-6PUFAs,concentration of18:2n-6linoleicacid(LA)washighest(P<0.05)inlarvaefed theunenrichedArtemia(7.03TFA)comparedtolarvaefedArtemia enriched withAlgamac (4.73% TFA). Concentration of ARA was higher(P<0.05)inlarvaefedtheunenrichedArtemia(6.66%TFA) than in larvaefed Artemiaenriched with DC DHA Selco(3.42% TFA)andAlgamac(4.52%TFA).Concentrationof22:5n-6 docos-apentaenoicacid(DPA)washigher(P<0.05)inlarvaefedArtemia enriched with Algamac (3.28% TFA) than in larvae fed Artemia enrichedwithDCDHASelco(0.85%TFA),orinunenrichedArtemia (undetected).

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A

Days Post Hatch

10 15 20 25 30 35

)

m

m(

ht

g

n

e

L

dr

o

h

c

ot

o

N

2 4 6 8 10 12 14 16 DC DHA Selco Algamac 3000 Control

**

B

Days Post Hatch

10 15 20 25 30 35

)

g

m(

t

h

gi

e

W

yr

D

0 2 4 6 8 10 12 14 16 18 DC DHA Selco Algamac 3000 Control

*

***

a

a

b

a

a

a

b

a

a

b

Fig.2.Growthinnotochordlength(mm)(A)andindryweight(mg)(B)oflarvalredporgyunderdifferentArtemiaenrichmenttreatments(Experiment1.2).Datarepresent means±SEM(N=4).Asterisksindicatesignificanttreatmentdifferences(*P<0.05,**P<0.01,***P<0.001,ANOVA).Meansnotsharingaletterincommonaresignificantly (P<0.05,Tukey–Kramertest)different.

3.3. Experiment3:artificialmicrodiets(MDs)forco-feedingand earlyweaning

Themeaninitialtotallengthoftheredporgylarvaewas5.4mm. Otohimeproducedafinalmeanlengthof14.4±0.4mm,followed byGemmaat13.7±0.5mmandUNCWat13.5±0.4mm(Table9). Meaninitialwetweightofredporgylarvaewas1.54mg.Otohime produced a mean wet weight per fish of 53.65±5.28mg, fol-lowedbyGemmaat47.06±5.06mgandUNCWat46.11±4.17mg (Table9).Nosignificantdifferenceswerefoundinfinaltotallength orwet weightamong treatmentsafter the16-dayfeeding trial

(Table 9). No significant differences in survival were observed amonglarvaefedthedifferentmicrodietsondays7,12 and16 ofthefeedingtrial(Table9).Finalsurvival(N=3)after16dayswas 39±9,45±8and43±11%forfishfedGemma,UNCWandOtohime MDs,respectively.

After the feeding trial, crude protein (range=63.0±0.58 to 64.6±0.67% dry basis, N=3) and total lipid contents (range=15.0±1.2to16.0±2.0%drybasis)inredporgylarvalwhole bodies fed different microdiets were not significantly different amongtreatments.Fattyacidcompositionoftheredporgylarvae fedthedifferentmicrodietsarepresentedinTable10.Linoleicacid

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Table6

Totallipidandfattyacidcomposition(%oftotalfattyacids)(mean±SEM,N=3)ofArtemiaenrichedwithtwodifferentenrichmentproducts,Algamac3000andDCDHA Selco,andanunenriched(control)group.ValuesarepresentedforArtemiasampledbeforethefirst(0800h)feedingandaftertheenrichedrotiferswereheldat10◦Cfor8h beforethesecond(1600h)feeding.

Fattyacids Artemiatreatments

Controlam Controlpm DCDHASelcoam DCDHASelcopm Algamac3000am Algamac3000pm 14:0 2.63±0.19 2.63±0.06 2.94±0.15 2.90±0.17 3.35±0.45 3.37±0.07 16:0 15.2±0.83a 15.2±0.56a 16.3±0.41ab 16.2±0.50ab 19.4±1.56bc 20.3±0.35c 16:1n-7 3.71±0.60a 4.57±0.23a 7.29±0.13b 6.87±0.09b 3.84±0.12a 4.18±0.21a 17:0 2.61±0.28 2.68±0.05 2.47±0.10 2.41±0.14 2.51±0.22 2.47±0.04 18:0 11.2±0.92b 10.6±0.63b 7.88±0.16a 7.95±0.29a 10.1±0.32ab 10.2±0.36ab 18:1n-9 23.5±1.37 22.9±0.82 22.6±0.38 25.7±2.79 19.9±1.12 19.6±0.22 18:1n-11 11.9±0.40c 10.8±0.63bc 8.16±0.14a 8.25±0.11a 9.71±0.60ab 9.43±0.14ab 18:2n-6 4.80±0.20 4.82±0.22 4.45±0.43 4.50±0.39 4.02±0.22 3.90±0.22 18:3n-3 13.3±2.55 13.9±1.53 15.7±0.09 12.2±4.25 11.3±0.94 11.6±0.39 18:4n-3 3.60±0.23c 3.98±0.33c 1.86±0.01a 2.42±0.09ab 3.13±0.10c 3.30±0.12c 20:1 3.08±0.22 1.96±0.98 1.17±1.17 Undetected 2.66±0.19 1.76±0.88 20:4n-6 0.58±0.58ab Undetected Undetected 0.64±0.64ab 2.26±0.12b 2.07±0.08b 20:5n-3 2.78±0.13 2.69±0.05 1.69±0.86 2.53±0.23 2.62±0.39 2.89±0.10 22:5n-3 Undetected Undetected 2.11±0.61 1.45±0.73 1.44±1.44 0.81±0.81 22:6n-3 1.32±1.32 Undetected 2.36±0.16 2.60±0.24 2.84±0.27 1.78±0.89

SFA 30.2±1.56ab 29.3±0.53ab 27.4±1.29a 27.6±0.30a 32.1±1.20ab 33.8±0.52b

MUFA 44.8±1.79 42.9±1.06 42.1±1.08 43.7±2.93 39.4±1.63 38.4±0.67

n-3PUFA 19.6±2.67 21.9±0.61 23.8±0.84 21.2±3.68 20.9±0.44 20.4±0.20

n-6PUFA 5.38±0.76 5.76±0.79 6.70±1.46 7.46±0.81 6.95±0.65 7.45±0.72 Totallipidmg/g 291±14.5 294±23.8 329±16.2 267±11.9 260±27.0 299±17.0 Fattyacidsintraceamounts(<0.5%)arenotreported.

Meansnotsharingacommonletteraresignificantly(P<0.05)different. Totallipidisreportedinmg/gdryweight.

Table7

Totallipidandfattyacidcomposition(%oftotalfattyacids)(mean±SEM,N=3)of13dphredporgylarvaeatthebeginningoftheexperimentandon25dphafterfeeding

Artemiaenrichedwithtwodifferentenrichmentproducts,Algamac3000andDCDHASelco,andanunenriched(control)group. Fattyacids 13dph 25dph

Control DCDHASelco Algamac3000 16:0 17.4±0.34b 14.2±0.35a 14.0±0.39a 14.3±0.16a 16:1n-7 6.24±0.30b 3.54±0.15a 4.37±0.21a 3.53±0.25a 17:0 3.12±0.29 2.65±0.10 2.47±0.22 2.74±0.33 18:0 9.83±0.19a 10.8±0.44b 9.30±0.21a 10.3±0.09b 18:1n-9 Undetected 20.0±2.69 19.4±1.49 16.2±1.90 18:1n-11 6.39±0.09 9.12±0.55b 7.71±0.16a 8.23±0.12ab 18:2n-6 5.42±0.39 5.53±0.11 6.90±0.03 5.09±0.18 18:3n-3 12.9±0.51 6.04±3.20 5.24±1.19 7.64±1.66 18:4n-3 Undetected 3.02±0.14 2.35±0.21 2.81±0.30 20:0 Undetected 2.98±0.13b Undetected 0.50±0.50a 20:1 3.52±0.11 3.90±0.12 2.18±0.78 1.63±0.94 20:3 Undetected Undetected 0.77±0.71 2.50±0.83 20:4n-6 4.45±0.21a 4.63±0.07a 4.10±0.67a 5.42±0.15b 20:5n-3 8.01±0.07bc 6.92±0.27a 8.02±0.14c 7.24±0.13ab

22:5n-3 5.40±0.62 Undetected Undetected Undetected 22:5n-6 Undetected Undetected Undetected 3.07±0.27 22:6n-3 11.8±0.03d 3.55±0.22a 10.3±0.23c 7.09±0.20b

SFA 30.3±0.65bc 30.6±0.65c 25.7±0.48a 27.8±0.45b

MUFA 21.6±1.38a 37.3±3.48b 33.6±0.82b 30.4±2.34b

n-3PUFA 38.1±1.03b 21.9±1.96a 28.9±1.31a 25.7±1.50a

n-6PUFA 9.86±0.59a 10.2±0.16a 11.0±0.19a 13.6±0.59b Totallipidmg/g 306±57.8b 172±2.21a 165±7.67a 173±12.3a

Fattyacidsintraceamounts(<0.5%)arenotreported.

Meansnotsharingacommonletteraresignificantly(P<0.05)different. Totallipidisreportedinmg/gdryweight.

(18-2n-6)wassignificantlyhigher(P<0.05)inlarvaefedGemma MicrothaninOtohime,buttherewerenosignificantdifferences betweenGemmaMicroandUNCWMBD.However,EPA (20:5n-3)contentinlarvaefedOtohimewassignificantlyhigherthanin larvaefedGemma.DHA(22:6n-3),andtotaln-3PUFAcontentin fishlarvaewerenotsignificantlydifferentamongthetreatments. However,totaln-6PUFAsweresignificantlyhigherinlarvaefedthe GemmamicrothanthelarvaefedOtohime(Table10).TotalPUFAs werenotsignificantly differentbetweenthelarvae fedGemma MicroandUNCWMBD.

4. Discussion

4.1. Experiment1:comparisonsofrotiferenrichmentmediaand feedingfrequency

Onlymodestlevelsofrotiferenrichmentwereachievedinall treatments,withpost-enrichmentessentialpolyunsaturatedfatty acid (PUFA) levels in rotifers well below those found in their respectivemedia.Despitegenerallylowenrichmentlevelsacross treatments,therelativePUFAlevelsinenrichedrotiferssuggested

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Table8

Totallipidandfattyacidcomposition(%oftotalfattyacids)(mean±SEM,N=3) of33dphredporgylarvaefedArtemiaenrichedwithtwodifferentenrichment products,Algamac3000andDCDHASelcoandanunenriched(control)group.

Fattyacids 33dph

Control DCDHASelco Algamac3000 16:0 13.3±0.12 13.5±0.63 14.3±0.04 16:1n-7 5.59±1.19b 3.90±0.38ab 2.83±0.13a 17:0 1.88±1.88 2.03±0.07 2.09±0.05 18:0 10.1±0.44 8.64±0.76 10.3±0.10 18:1n-9 12.6±0.01a 19.8±1.54b 15.8±1.14ab 18:1n-11 7.90±0.74 7.07±0.36 7.75±0.14 18:2n-6 7.03±0.64b 6.18±0.60ab 4.73±0.13a 18:3n-3 9.49±0.24 8.60±0.93 11.1±0.77 18:4n-3 1.85±1.85 2.12±0.04 2.33±0.03 20:0 1.84±1.84 0.99±0.58 2.23±0.05 20:1 5.40±1.19b 1.44±0.83a Undetected 20:3 Undetected 1.49±0.86 3.08±0.03 20:4n-6 6.66±0.65b 3.42±0.38a 4.52±0.11a 20:5n-3 7.63±0.55b 6.23±0.21a 5.79±0.11a 22:5n-3 1.75±1.75 1.82±0.61 1.36±0.46 22:5n-6 Undetected 0.85±0.85a 3.28±0.08b 22:6n-3 6.40±1.46a 10.6±0.79b 8.59±0.23ab

SFA 27.2±3.51 25.1±1.94 28.8±0.15

MUFA 31.5±3.12 33.5±2.54 26.8±1.12

n-3PUFA 27.1±1.36 29.4±0.73 29.2±0.81

n-6PUFA 13.7±1.29b 10.5±0.67a 12.5±0.27ab Totallipidmg/g 152±23.8 126±13.9 167±25.4 Fattyacidsintraceamounts(<0.5%)arenotreported.

Meansnotsharingacommonletteraresignificantly(P<0.05)different. Totallipidisreportedinmg/gdryweight.

Table9

Survival(%),totallength(mm)andwetbodyweight(mg)ofredporgylarvae fedUNCWMBDandcommercialmicrodietsfor16days.Valuesaremeans±SEM (N=3).Nosignificantdifferencesamongdiettreatmentswereobservedforthese parameters.

Parameter Feeding period (day)

Microdiet

Gemma UNCW Otohime Totallength(mm) 16 13.7±0.5 13.5±0.4 14.4±0.4 Bodyweight(mg) 16 47.06±5.06 46.11±4.17 53.65±5.28 Survival(%) 7 55±5 60±2 56±8 12 49±6 52±6 46±6 16 39±9 45±8 43±11 Table10

Fattyacidcompositionofredporgylarvaefeddifferentmicrodiets(%oftotalfatty acids).Valuesaremeans±SEM(N=3).Meansinarownotsharingaletterin com-monaresignificantly(P<0.05)different.

Fattyacids Gemma UNCW Otohime 16:1n-7 2.18±0.03 2.63±0.19 2.86±0.25 16:0 12.7±0.29 12.9±0.06 13.6±0.27 18:4n-3 1.26±0.11 1.23±0.17 1.30±0.08 18:2n-6 8.01±0.49b 6.60±0.33ab 5.79±0.40a 18:3n-3 7.33±0.97 7.35±0.31 7.83±0.78 18:1n-9 14.3±0.91 13.8±0.83 11.3±0.60 18:1n-11 4.38±0.04 4.49±0.17 4.43±0.18 18:0 7.47±0.08 7.54±0.20 6.98±0.08 20:4n-6 2.43±0.08b 2.38±0.10ab 2.07±0.06a 20:5n-3 7.00±0.09a 7.43±0.21ab 7.98±0.27b 20:0 0.75±0.02 0.78±0.05 0.70±0.04 20:1 2.19±0.06 2.09±0.08 2.46±0.12 22:6n-3 11.7±0.37 10.7±1.02 12.0±0.43 22:5n-3 1.70±0.12 1.89±0.13 1.84±0.04 SUMofn-3PUFA 31.4±0.76 31.0±1.33 33.0±0.80 SUMofn-6PUFA 10.4±0.50b 8.98±0.23ab 7.85±0.44a

thatconcentrationsofPUFAsweremodestlyimprovedby bioen-capsulation.Lower thanexpected amountsof EPA in allrotifer treatmentscouldinpartbearesultoftherotifersconvertingsome EPA(20:5n-3)toDPA(22:5n-3),whichrangedfrom1.10to1.90% amongtreatments(Table3).ARAlevelswerehighestin rotifers enrichedwithAlgamac+ARAandcomparativelylowinrotifersfed AlgamacandDHAProteinSelco,bothofwhichweredevoidofARA. ThesmallamountofARAfoundinrotifersenrichedwithAlgamac andDHAProteinSelco(bothARAdeficient)mayalsoreflectthefact thatrotiferswereraisedonRotiGrowwhichcontained3.2%ARA. AnumberofreportsdescribeadirectrelationshipbetweenPUFA levelsinrotifersandintheiremulsiondiets(Watanabeetal.,1983; Rainuzzoetal.,1989;Kovenetal.,2001b;Rezeketal.,2010;Carrier etal.,2011).Thesurprisinglylowenrichmentlevelsobservedinthe presentstudyarepresumablyrelatedtofactorsaffectinguptakeof emulsionmediabyrotifers,suchasexcessivepre-enrichment feed-ing levels,insufficient enrichmenttime, temperature, dissolved oxygen,pH,orotherconditionsthatmayhaveinfluenced bioen-capsulationofnutrients.

Consistent with very minor differences in lipid and fatty acid composition of rotifers enriched with the different prod-ucts(RotiferDiet,DCDHAProteinSelco,Algamac3000,Algamac 3000+ARA)inthisstudy,therewerenosignificantmainor inter-activeeffectsofrotiferenrichmentmediaandfeedingfrequencyon larvalgrowth,orsurvivalonanysamplingdayforthedurationof thestudy.Final(18dph)survivalamongalltreatmentswas22.8%, consideredmoderateforcultureofotherlarvalmarinefinfishsuch assouthernflounderandblackseabassraisedundersimilar con-ditions(DanielsandWatanabe,2003;Watanabe,2011).Thelackof obviousdetrimentaleffectsofrotifersfedRotiferDiet(which con-tainedundetectablelevelsofDHA)indicatethatredporgylarvae couldbesuccessfullyrearedonrotiferswithrelativelylowlevels ofPUFAandwithDHA:EPAratios(0.27–0.75:1)lowerthanthe recommendedminimumof 2:1DHA:EPA formarinefinfish lar-vae(TocherandSargent1984;Sargentetal.,1999).Thisfurther suggestedthatlarvaefed0%DHAmayhavederivedasignificant amountof DHA frommaternalyolk reserves,since marine fish eggsnormallycontainhighconcentrationofPUFAs,especiallyDHA (Tocheretal.,1985;Sargent1995;Bransdenetal.,2007). Alterna-tively,DHAmayhavebeensynthesizedinthelarvaefromprecursor fattyacids 18:3n-3,20:5n-3 or 22:5n-3.Conversion of 18:3n-3 and20:5n-3to22:6n-3(DHA),whilelimitedinmostmarine car-nivorousfishspecies, hasbeenreportedin turbotScophthalmus maximus(LinaresandHenderson1991;Mourenteetal.,1991), gilt-headseabreamSparusaurata(Mourenteetal.,1993),Senegalese soleSoleasenegalensis(Moraisetal.,2012)andthesouthern floun-der(Watanabeetal.,2014).Inthisstudy,themicroalgaN.oculata (31.4%EPA,0%DHAand3.94%ARA,ReedMaricultureInc., Camp-bell,CA)wasaddedtothelarvalrearingtanksasa background and mayhave alsosupplied20:5n-3and 20:4n-6tothelarvae eitherdirectly,orindirectlyviarotifers.Sincerotifersenrichedwith RotiferDiet(N.oculataand T.chuii),showednodetectableDHA (Table5),itisunlikelythatthefishlarvaeacquiredDHA(directly orindirectlyviarotifers) throughN.oculatausedinbackground microalgae.

Althoughgrowth andsurvival patterns andstress resistance levelsweresimilaramongallrotiferenrichmenttreatments, dis-tinctive cumulative survival patterns among treatments were observedunderhyposalinitychallengeandfooddeprivation,with consistentlygreatermeanSAIvaluesforlarvaefedrotifersenriched withAlgamac+ARAfollowedbyAlgamac,DHAProteinSelco,and then RotiferDiet andfor larvae fed2×/daythan for larvaefed 1×/day (Fig. 1). Due to highinter-replicate variability on each samplingdate, statisticallysignificantdifferences in SAIamong enrichmenttreatmentswerenotobserved.However,SAIwas pos-itivelycorrelatedwithDHAlevelsinrotifersenrichedwiththese

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treatmentsand indicatedthat subtledifferences instress resis-tanceweremanifestedevenwhennoobviousdifferencesinlarval growthandsurvivalwereevident(Akoetal.,1994).Highestmean SAIvalues werefoundin larvaefedARA-supplementedrotifers (i.e.,theAlgamac+ARAtreatment).ThisissimilartowhatKoven etal.(2001b)foundingiltheadseabream,whereARA supplemen-tationtorotifersdidnotaffect larvalgrowthorsurvival tothe endoftherotiferfeedingperiod(19dph),butresistanceofthese larvaetohandlingstresswasimproved.Inblackseabasslarvae, dietarysupplementationofARAwithintherangeof0–6%improved growthandsurvivalfromthefirstfeedingthroughmetamorphic stages(Rezeketal.,2010),andsupplementationofARAwithinthe rangeof6–12%promotedtheadaptivephysiologicalresponsesto hypersalinitystressandhypo-osmoregulatoryability(Carrieretal., 2011).Basedontheresultsofthepresentstudy,bioencapsulation ofrotiferswithacombinationofenrichmentproductsthat sup-plementedbothDHAandARAprovidedredporgylarvaewiththe higheststressresistanceandisrecommendedamong the treat-mentstested.Workisneededtoimproveuptakeofenrichment mediabyrotiferstofurtherelevatePUFAconcentrationoverthe modestlevelsattainedinthisstudy.

4.2. ComparisonsofArtemiaenrichmentmedia

Amongthen-3PUFAs,DHA22:6n-3levelsinArtemiafedDC DHASelcoand Algamacwerehigherthanthosefoundin unen-richedcontrolArtemia,reflectingrelativeDHAlevelsfoundinthese enrichment products (Algamac=41% DHA, DC DHA Selco=30% DHA,N.oculata=0%DHA).Despitethis,levelsofDHAenrichment attainedinArtemiaweremuchlowerthanthoseprovidedintheir diets.Artemiaenrichmentisdifficultduetotheirrapid develop-mentandcatabolismofDHA(Sorgeloosetal.,2001).Itispossible thathigherlevelsofDHAinArtemiacouldbeachievedifthe enrich-mentperiodisextendedfrom16h(asusedinthisstudy)to19–24h (McEvoyetal.,1995).EnrichmentsuccessinArtemiaalsodepends ongeneticcharacteristics.ChinesestrainsofArtemiaaccumulated higherlevelsofDHAthantheGreatSaltLakestrainortheSan Fran-ciscoBaystrainusedinthisstudy(Evjemoetal.,1997).Amongthe n-6PUFAs,concentrationofARAwashigherinArtemiaenriched withAlgamacthanwithDCDHASelcoandwaslowestinthe unen-riched controls.The presenceof ARA in Artemia fromall three treatments,although lackingin theenrichmentmedia,is likely attributabletoendogenousARA(0.8%TFA)inArtemia(Coutteau andMourente,1997).

Whereasnodifferencesinlarvalgrowthorsurvivalwere evi-dentthroughtherotiferfeedingphaseon18dph,distincttreatment effectsonlarvalperformanceemergedduringtheArtemiafeeding phase.Growthandsurvivalamonglarvaefedenrichedor unen-richedArtemiaremainedsimilarthrough21dph;however,from 25to33dph, larvaefedArtemiaenriched withAlgamacand DC DHASelcoweremarkedlylarger(Fig.2)andshowedmuchhigher survivalthancontrollarvaefedunenrichedArtemia.Superior per-formanceoflarvaeonArtemiaenrichedwithAlgamacandDCDHA SelcoisconsistentwiththehigherlevelsofDHAprovidedbythese enrichmentproducts.Clearly,providingsufficientdietaryn-3PUFA (especiallyDHA)throughbioencapsulationofArtemiaiscrucialto normalgrowthandsurvivaloflarvalredporgy.Thisisinaccord withwhatwasreportedinredseabreamPagrusmajorwhere lar-vaefedrotifersenrichedwithhighlevelsofDHA(≥20%)grewfaster thanthosefedrotifersenrichedwith≤2%DHA(Watanabeetal., 1989).ImprovedgrowthwithhigherdietaryDHAhasalsobeen reportedinlarvalyellowtailSeriolaquinqueradiata(Furuitaetal., 1996;Copemanetal.,2002),yellowtailsnapperOcyuruschrysurus (FaulkandHolt,2005),cobiaRachycentroncanadum(Faulketal., 2005),andblackseabassCentropristisstriata(Rezeketal.,2010).

The lack of differences in growth and survival related to DHA-enrichmentofrotifersandthecleardifferencesobservedin responsetoDHA-enrichmentofArtemiaseemcontradictory. How-ever, significantdifferences in response to Artemia enrichment werenotevidentuntil25dphafter11daysoffeeding.Itis pos-sible,therefore,thatthe15-dayfeedingperiodofenrichedrotifers wasinsufficientfortreatmenteffectstobedemonstratedandthat differencesingrowthrateswereaccentuatedduringtheArtemia phasewhenlarvalfeedingandspecificgrowthratesweremuch higher.Andradeetal.,2012reportedthatinlarvalredporgy,live feeddemandincreasedsharplyatabout5.6mmTL(Andradeetal., 2012),abodylengthreachedaround18dphinthepresentstudy (Fig.2)whentherotiferfeedingExperiment1.1wascompleted.On 25dphandon33dph,levelsofessentialn-3PUFAsEPAandDHA weregenerallyhigherintissuesoflarvaefedArtemiaenrichedwith DCDHASelcoorAlgamacthaninlarvaefedunenrichedArtemia. ThisisconsistentwiththehigherdietarylevelsofDHAprovidedin thesetreatmentsandtheimportanceofDHAtolarvaldevelopment. 4.3. Experiment2:artificialmicrodietsforco-feedingandearly weaning

Total replacement of live food with microdiets has been accomplished in shrimp, but not in marine fish (Cahu and Zambonino-Infante,2001).Co-feedingliveandinertdietshasbeen foundtoproducehighergrowthandsurvivalthanfeedingeither livefeedsormicrodietsalone(Kolkovskietal.,1995;Wangetal., 2009).Inredporgylarvae,directweaningfromrotiferstodrydiet resultedinsignificantlylowergrowth,survival,pancreatic(trypsin andlipase),andintestinal(alkalinephosphatase)enzyme-specific activity,comparedtofeedingregimesusingArtemiaasan interme-diatestep(Andradeetal.,2012).‘Co-feeding’weaningprotocols appliedinthepresentexperimentsimultaneouslyusedinertand livedietstoallowafastandefficientchange-overperiodontodry microdietsfromlivefeed(Kovenetal.,2001a).ZeinboundMBDs wereselected asexperimentaldietsin this studybecause they wereusedsuccessfullyinthelarvalrearingofredseabreamand Japaneseflounder(Kanazawaetal.,1989;Teshimaetal.,2004). QualityMBDforlarvalandjuvenileaquaticanimalsareneededto meetthefollowingcriteria:inclusionofnutritionallywell-balanced nutrients,highwater-stability(minimalleachingofnutrients),high digestibility,andpropersuspensioninawatercolumn(Bengtson, 1993; Teshima et al., 2000; Izquierdo and Fernandez-Palacios, 2001).Inthisexperiment,larvaefedUNCWMBDcontainingboth fishmeal and squid meal asprotein sources showed compara-blegrowthperformancetothosefedthecommercialmicrodiets GemmaandOtohimeafter16daysoffeeding.Fishmealandsquid powderwere foundtohave a highnutritionalvalueasprotein sourcesforL.calcariferlarvaebyvirtueofasynergisticallyfavorable aminoacidprofileandmoderatetohighdigestibility(Nankervis andSouthgate,2006;SaenzdeRodriganezetal.,2011).

Inthisstudy,thelowsurvival(39–45%)ofredporgylarvae co-fedlivefeed andmicrodiets couldbedue toleaching lossesof essentialsolublenutrients.Rapidratesoflosshavebeenreported forfree amino acidssupplementingmicrodiets (Lopez-Alvarado and Kanazawa, 1994; Kvaleet al., 2006).Extensive leaching of nitrogenous compounds from microdiets based on intact fish protein sources demonstrated that leaching can also occur for water-solublemacromolecules.Atthesametime,someleachingof nutrientsmaybebeneficialduetotheirrolesasattractants(Koven etal.,2001a;Kolkovskietal.,2009).Aloweravailabilityofthe pro-teininformulateddietscomparedwithlivepreyisalsoconsidered tobeanimportantreasonforthelowperformanceofmarinefish larvaefedonlyformulateddiets(Kolkovskietal.,2009).However, larvaeinthisstudywerefedfourtimesadaytomitigatenutrient deficienciesduetoleachinginwater.Andradeetal.(2012)

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recom-mendedfeedingofinertdietstoredporgyfrom30dphonward, whenlarvaearephysiologicallyabletodigestmorecomplexfeed items.Inthepresentstudy,redporgylarvaewereco-fedlivefeed andmicrodietsfrom16dph,and limiteddigestivecapacitymay havereducedfeedutilizationandsurvivalinalltreatmentgroups. IntheUNCW-MBD,supplementationoffeedattractantslikely played animportantrole in acceptance ofdry dietsin fish lar-vaeduringtheweaning periodas wellasin enhancinggrowth asreported for otherspecies (Kolkovskiet al., 1997;Kolkovski 2000,2001).Thechemo-attractantpropertiesofsquidmealwere alsoconfirmedfortroutfingerlings(Lianetal.,2008)andgilthead seabream(Kolkovskiand Tandler,2000).The dietaryinclusion oftaurineproducedasignificantimprovementinthegrowthof juvenileJapaneseflounder(Kimetal.,2005).Carnivorousspecies usuallyshowapositivefeedingresponsetoalkalineandneutral substances,suchasglycine,proline,valine,taurine,and betaine (Takaokaetal.,1995).

Ingeneral,thefattyacidcompositionofthefishwholebody reflectedthecompositionoftheirdiets(SalhiandBessonart,2012). Thelipidsourcesofcommercialmicrodiets(OtohimeandGemma) areunknown.However,then-3PUFAlevelsinthelarvaefedthe UNCWformulatedMBDwerenotsignificantlydifferentfromthe twocommercialmicrodiets(OtohimeandGemma)after16daysof feeding.Thiswasconsistentwiththesimilargrowthandsurvival performancesobservedamongtreatments.ThehighDHAcontent ofredporgylarvaereflectstheimportanceofthisfattyacid,which isknowntobehigherinefficiencyasanessentialfattyacidthan EPA(WatanabeandKiron,1994).Growth,survivalandfattyacid compositionoflarvalwholebodiessuggestedthatredporgy lar-vaewereabletoutilizeUNCWformulatedzein-boundmicrodiets, andthattheresultswerecomparabletothecommerciallyavailable microdiets.

Basedontheresultsofthisstudy,amicrobounddietforred porgylarvaehasbeenformulatedusingacombinationofdifferent proteinsourcesandsupplementalattractantsthatproducedlarval performance(growth,survivalandwholebodyproximateandfatty acidprofiles)thatwerenotsignificantlydifferentfrompremium commercialmicrodietsOtohimeandGemmaMicro.Thesuccessin rearingthelarvalredporgy(16dph)withtheMBDcontainingfish meal,soyproteinconcentrate,squidmeal,krillmealand attrac-tantsasthemajornitrogensourcesindicatesthatredporgylarvae readilyassimilatethesedietaryproteinsourcesfornormalgrowth anddevelopment.Theseresultsprovideafirmbasisfor develop-ingandtestingimprovedformulationsthatcanpotentiallyyield highergrowth,survivalandearlierweaningfromlivepreyinred porgylarvae.

5. Conclusions

Redporgylarvalgrowth,survival andstress resistancewere improvedthroughbioencapsulationoflivepreyusing commer-ciallyavailableproductsandproperfeedingregimes.Duringthe earlyfeedingstageredporgylarvaeshowedhigherstressresistance whenfedrotifersenrichedwithastrategiccombinationof enrich-mentproductstoboostbothDHAandARA,andwhenlarvaewere fedtheirdailyrationallocatedovertwofeedingsratherthanone. Forolderlarvae,growthandsurvivalweremarkedlyhigherin lar-vaefedArtemiametanaupliienrichedwithDHA.Duringtransition fromlivepreytoartificialfeeds,aUNCW-formulatedmicrobound dietproducedlarvalperformancecomparabletopremium, com-mercialmicrodietsuptothemetamorphicstages.Resultsdefine protocolsformoreeffectivehatcheryproductionofredporgy juve-niles.

Acknowledgements

ThisstudywassupportedbygrantsfromtheNorth Carolina FisheryResource Grant Program (08-AM-02),the UnitedStates Department ofAgriculture,NationalInstituteof Foodand Agri-culture (USDA-NIFA Grant No. 2008-38854-18896) and Marine BiotechnologyinNorthCarolina(MARBIONC).We thankTroyC. Rezek,PatrickM.Carroll,WalkerD.Wright-Moore, andTylerF. Gibsonfortechnicalassistance.

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