Lipid metabolism-related gene expression pattern of Atlantic bluefin tuna (Thunnus thynnus L.) larvae fed on live prey

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1 Fish Physiol Biohem (27) 43: DOI.7/s Lipid metolism-relted gene expression pttern of Atlnti luefin tun (Thunnus thynnus L.) lrve fed on live prey Móni B. Betnor & Aurelio Orteg & Fernndo de l Gándr & Dougls. Toher & Griel Mourente eeived: 9 My 26 /Aepted: 8 Otoer 26 /Pulished online: 4 Novemer 26 # The Author(s) 26. This rtile is pulished with open ess t Springerlink.om Astrt The present study is the first to evlute lipid metolism in first-feeding Atlnti luefin tun (ABT; Thunnus thynnus L.) lrve fed different live prey inluding enrihed rotifers Brhionus plitilis nd Arti sp. opepod nuplii from 2 dys fter hth. Understnding the moleulr sis of lipid metolism nd regultion in ABT will provide insights to optimize diet formultions for this high-vlue speies new to quulture. To this end, we investigted the effet of dietry lipid on whole lrve lipid lss nd ftty id ompositions nd the expression of key genes involved in lipid metolism in first feeding ABT lrve fed different live prey. Additionlly, the expression of lipid metolism genes in tissues of dult roodstok ABT ws evluted. Growth nd survivl dt indited tht opepods were the est live prey for first feeding ABT nd tht differenes in growth performne nd lipid metolism oserved etween lrve from different yer lsses ould e onsequene of roodstok nutrition. In ddition, expression ptterns of lipid metoli genes oserved in ABT lrve in the trils ould reflet differenes in lipid lss nd ftty id ompositions of the live prey. The lipid nutritionl requirements, inluding essentil ftty id requirements of lrvl ABT during the erly feeding stges, re unknown, nd the present study represents first step in ddressing these highly relevnt issues. However, further studies re required to determine nutritionl requirements nd understnd lipid metolism during development of ABT lrve nd to pply the knowledge to the ommeril ulture of this ioni speies. Eletroni supplementry mteril The online version of this rtile (doi:.7/s ) ontins supplementry mteril, whih is ville to uthorized users. M. B. Betnor (*): D.. Toher Institute of Aquulture, University of Stirling, Stirling, Sotlnd FK9 4LA, UK e-mil: m..etnor@stir..uk A. Orteg : F. de l Gándr Plnt Experimentl de ultivos Mrinos, Instituto Espñol de Oenogrfí (IEO), 386 Puerto de Mzrrón (Muri), Mdrid, Spin G. Mourente Deprtmento de Biologí, Fultd de ienis del Mr y Amientles, Universidd de ádiz, 5 Puerto el, ádiz, Spin Keywords Bluefin tun. Lrve. otifer. opepods. Lipid ontent. Lipid lsses. Ftty id omposition. DNA. Gene expression Arevitions ABT Atlnti luefin tun o Ayl oa oxidse AA Arhidoni id (2:4n-6) Free holesterol pt rnitine plmitoyl trnsferse I dh Dys fter hth DHA Dooshexenoi id (22:6n-3) EFA Essentil ftty id EPA Eiospentenoi id (2:5n-3) elovl5 Ftty yl elongse 5

2 494 Fish Physiol Biohem (27) 43: fp2 Ftty id inding protein 2 (intestinl) ftty id fp4 Ftty id inding protein 4 (dipoyte) fp7 Ftty id inding protein 7 (rin-type) fds2d6 Delt-6 ftty yl desturse FAME Fttyidmethylester fs Ftty id synthse hmgl 3-Hydroxy-3-methylglutryl-oA lyse HPTL High performne thin-lyer hromtogrphy L- Long-hin polyunsturted ftty id PUFA lpl Lipoprotein lipse lxr Liver X reeptor MUFA Monounsturted ftty id PBT Pifi luefin tun P Phosphtidylholine PE Phosphtidylethnolmine PI Phosphtidylinositol pprα Peroxisome prolifertor-tivted reeptor lph pprγ Peroxisome prolifertor-tivted reeptor gmm PS Phosphtidylserine qp Quntittive rel-time P rxr etinoid X reeptor SE Steryl ester srep Sterol regultory element-inding protein srep2 Sterol regultory element-inding protein 2 TAG Triylglyerol TF Trnsription ftor Introdution Improvement in the prodution of Atlnti luefin tun (ABT; Thunnus thynnus L) lrve nd juveniles is essentil to estlish full-yle ulture tehnology for this speies. The supply of vile eggs nd optimizing the nutritionl vlue of live prey (e.g., rotifers, Artemi, opepods, fish yolk-s lrve) nd juvenile diets re prmount to hieve this gol. To dte, stndrd live feeds nd rtifiil diet feeding protools for lrve nd juvenile ABT re giving poor survivl nd growth nd stress resistne. Moreover, size vrition, low swimldder infltion rtes, skeletl nomlies, nd tnk wll ollisions re ommon, not only in ABT ulture ut lso in other luefin tuns speies (Ysunori 22). In fish, lipids nd their onstituent ftty ids (FAs) ply essentil roles in mintining optimum growth, survivl, feed effiieny, helth, neurl nd visul development, nd response to stressors in ddition to generlly eing the min energy soure (Srgent et l. 989, 22; Toher, 23, 2). Among the lipids nd their onstituents, phospholipids s well s omeg 6 nd 3 (n-6 nd n-3, respetively) long-hin polyunsturted ftty ids (L- PUFA) re prtiulrly importnt due to their ritil roles in the physiologil proesses ove. Indeed, the limited glol supply of the n-3 L-PUFA, eiospentenoi id (2:5n-3; EPA) nd dooshexenoi id (22:6n-3; DHA) (Ton nd Metin 28; Toher 25), is mjor issue for the ulture of top predtor speies suh s ABT, whih mkes it ritil to understnd the mehnisms y whih fish llote energy from lipids for metolism, development, growth, nd reprodution. Approprite uptke nd umultion of lipids improve growth nd survivl of ll fish, ut, in prtiulr, lipids re reltively more importnt nd key in highly tive migrtory fish speies suh s tuns (Mourente nd Toher 23, 29) given tht the fish otin energy for the migrtions from flesh lipid reserves (ly 988). A key hrteristi of tissue FA ompositions in lrge tun speies is tht they disply high DHA ontents (>2 %) nd high DHA/EPA rtios (Mourente et l. 22; Mourente nd Toher 23, 29; Orteg nd Mourente 2). Besides, in ABT nd other regionlly endothermi tive-migrtory speies, the DHA ontent nd DHA/EPA rtio of musle re muh higher thn in non-migrtory teleost speies (Nkmur et l. 27; Osko et l. 29). This my lso suggest tht tun speies my hve dietry requirement for high DHA nd high DHA/EPA rtio. However, there is seletive utiliztion of monoenoi nd sturted FAs reltive to PUFA s energy soures in tun, nd so the high DHA/EPA rtio ould lso indite seletive tolism of EPA reltive to DHA (Mourente nd Toher 23, 29; Sholefield et l. 25). Furthermore, the pity for endogenous synthesis of EPA nd DHA is limited in ABT, nd so the lipid iohemistry underpinning the high tissue DHA nd DHA/EPA rtio is unler (Gregory et l. 2; Moris et l. 2; Sholefield et l. 25). eently, we investigted lipid nd FA metolism during erly development of yolk-s ABT lrve inluding the loning nd funtionl hrteriztion of

3 Fish Physiol Biohem (27) 43: omplementry DNAs (DNAs) for key enzymes involved in L-PUFA synthesis, ftty yl desturse 6 (fds2d6), nd elongse of very long-hin ftty ids 5 (elovl5) (Moris et l. 2). In unfed lrve, the level of DHA ws mintined until 4 dys fter hth (dh), ut the proportion of EPA delined, nd so the DHA/EPA rtio inresed during yolk-s utiliztion. As desried ove, this ould e due to the reltive retention of DHA during period of high FA oxidtion nd utiliztion, ut it ws noteworthy tht there ws lso inresed expression of fds2d6 nd elovl5 with lrvl development potentilly leding to endogenous synthesis of DHA (Moris et l. 2). This suggested tht inresed tivity of these enzymes ould e ruil for norml development of ABT lrve, possily relted to the provision of suffiient DHA for the formtion of memrnes, prtiulrly in neurl tissues (Mourente 23). Hene, studies tht emphsize FA metolism nd L- PUFA synthesis/deposition in different tissues nd the trnsriptionl ontrol mehnisms tht regulte these proesses re key to understnding lipid nutrition in this speies. The regultion of lipid homeostsis in fish is omplex lne etween lipid uptke, trnsport, storge, energy utiliztion, nd iosynthesis with eh proess eing ontrolled independently nd lso in onjuntion with other proesses (Lever et l. 28; Toher 23). eent studies investigting glol gene expression using trnsriptomi nd proteomi pprohes hve shown tht dietry lipid ontent nd omposition hve signifint effets on gene expression in slmonids (Kolditz et l. 28; Pnsert et l. 28; Higgs et l. 29; Mrtinez-uio et l. 23), fltfish (ho et l. 29, 22; unh et l. 23; Peng et l. 24; Yun et l. 25), nd other mrine speies (Tsi et l. 28; Dong et l. 25;Lietl.25, 26), s well s Pifi luefin tun (PBT) (Agw et l. 22). Thus, studying the impt of dietry lipid on lipid nd FA metolism, inluding effets on whole lrve lipid nd FA ompositions nd the expression of genes of mjor lipid metoli pthwys inluding lipogenesis, lipid deposition, FA β-oxidtion, nd L-PUFA synthesis in ABT, is highly relevnt (Lever et l. 28). Furthermore, key to this understnding is knowledge of the lipid-regulted trnsription ftors (TFs) nd nuler reeptors ontrolling nd regulting the expression of genes involved in FA/lipid metoli pthwys. In this sense, studies in mmmls estlished tht memers of the peroxisome prolifertor-tivted reeptor (PPA), liver X reeptor (LX), nd sterol regultory element inding protein (SEBP) TFs ontrol n integrted network of lipid nd FA metolism (Nkmur et l. 24). The ims of the present study were to investigte the effet of dietry lipid on lipid nd ftty id ompositions s well s on the expression of key genes involved in lipid metolism in ABT lrve fed different live prey. Speifi ojetives were first to lone DNAs of ABT genes involved in mjor lipid metolism pthwys nd their ontrol nd regultion, inluding ftty id nd L-PUFA iosynthesis, lipid deposition, nd β-oxidtion, for the evlution of gene expression. The seond im is to determine the expression of these genes in first feeding ABT lrve 4 dh, nd third, to determine the expression of these genes nd the mjor lipid pthwys in tissues of dult ABT. Our overrhing hypothesis is tht understnding the moleulr sis of lipid metolism nd regultion will provide insight to optimize diet formultions nd the effetive use of sustinle dietry lipid soures in ABT quulture. Mterils nd methods Atlnti luefin tun lrve rering onditions The ABT lrve used in this study were otined from two onseutive lrvl rering trils performed in July 23 nd July 24, respetively. The ABTeggs were otined from roodstok omposed of 35 fish with n estimted men ody weight of kg. The roodstok were mintined in ptivity for severl yers in floting ge loted t El Gorguel By, off rtgen ost, South Est Spin. ptive ABT roodstok fish spwned nturlly nd spontneously (during the nturl spwning seson in June July). A.5-m polyvinyl sheet ws pled round the inside of the ge to void eggs drifting wy from the ge y mens of urrents or wves nd floting eggs olleted inside the ge y mens of net of 5-μm mesh sreen size. olleted eggs were trnsported in 5-L plsti tnk supplied with pure oxygen to the IEO Plnt Experimentl de ultivos Mrinos (Puerto de Mzrrón, Muri, Spin) quulture filities nd pled in -L tnks with gentle ertion nd flow-through sterilized sewter. After h, ertion nd wter flow were stopped to seprte uoynt (vile) from non-uoynt (nonvile) eggs. After wshing nd ounting, the eggs were inuted in 5-L ylindril tnks t density of

4 496 Fish Physiol Biohem (27) 43: eggs L. Inution ws rried out t 25 26, 37 slinity, nd ontinuous photoperiod, with light intensity of lx. An upwelling flow-through with gentle ertion ws employed in order to mintin oxygen levels ner to sturtion. Lrve hthed pproximtely 32 h fter fertiliztion, with hthing rte of lmost 9 %, nd were fed with rotifers or opepod nuplii 2 dh. A mixture of the mirolge Isohrysis sp. (T-Iso, Thitin strin of Isohrysis)nd hlorell (V2 DHAenrihed, Pifi Trding o., Jpn) ws dded to tnks t density of 2 3, ells ml s green wter. During the trils, photoperiod ws mintined t 4/ h light/drk (light intensity out lx), temperture rnged etween 22.9 nd 24.8, nd dily wter renewl ws 5 7 %. Inoming sewter ws filtered through -μm sieve nd UV sterilized. An upwelling urrent ws reted to void lrve sinking (minly t night) nd mintin oxygen level.. Lrvl tril 23. Two different feeding tretments were tested from the eginning of exogenous feeding 2 to 4 dh: (i) L-type rotifers (Brhionus plitilis), ultured with DHA-enrihed hlorell (hlorell V2, Pifi Trding o.) nd enrihed with turine (5 ppm, dded to rotifer ulture tnks during 8 h efore hrvesting) nd Skretting OI- Green over 3 h t dose of.3 g per million rotifers, nd (ii) nuplii of the opepod Arti grni fed on Isohrysis T-Iso nd opepodites fed on mixture of Isohrysis T-Iso nd Tetrselmis sp. To mintin onstnt live prey onentrtion ( rotifer ml or five opepod nuplii/opepodite ml ) within eh experimentl tnk, three wter smples ( ml) from eh tnk were smpled nd ounted efore supplying new feed. 2. Lrvl tril 24. Three feeding regimes were tested 2 to 4 dh: (i) enrihed L-type rotifers (B. plitilis) supplemented with turine nd Skretting OI-Green s ove, (ii) nuplii of the opepod Arti tons s ove, nd (iii) o-feeding (5:5) of enrihed rotifers nd Arti nuplii. Arti tons eggs were inuted t 3 slinity nd nd, fter hthing, were fed on hodomons lti nd Isohrysis ff. gln T-Iso for 2 4 dys. Density of the live prey ws s follows: rotifers were mintined t rotifers per milliliter, A. tons t five nuplii per milliliter, nd o-feeding of rotifers nd Arti t five individuls per milliliter s 5 % mixture of oth orgnisms. Smple olletion Lrve (2 for eh rering ondition) were nesthetized (.2 % phenoxyethnol) nd totl lengths mesured. eplites of preweighed smples (pproximtely 5 mg wet weight) were mintined t for 24 h nd dry weights determined fter ooling in vuo for h. Triplite smples of rotifers nd opepods (Arti) were wshed nd filtered, exess wter drined nd lotted with filter pper, immeditely frozen in liquid N 2 nd stored t 8 prior to lipid nlysis. Two susets of triplite smples of 4 dh ABT lrve fed the different live prey used in 23 nd 24 feeding trils were olleted. One suset of smples ws pled in ml of NAlter (Amion, Mdrid, Spin) for NA extrtion, nd seond suset ws frozen in liquid N 2 nd stored t 8 for lipid nlysis. Eight roodstok tun (four mles nd four femles), ulled for reprodutive stge ssessment, were used for olleting tissue smples for tissue expression of lipid metolism genes. After srifie, triplite sets of smples of rin, gills, hert, kidney, spleen, liver, intestine, white musle, red musle, dipose tissue ovry, nd testis were olleted. Eh replite (out mg) ws pled in ml of NAlter (Amion), stored t 4 overnight efore trnsferring to 2, nd susequently stored prior to NA extrtion. All proedures were rried out ording to the urrent Spnish ndeuropenunionlegisltiononthehndlingof experimentl nimls. Lipid ontent, lipid lss omposition, nd ftty id nlysis Totl lipid of live feeds (enrihed rotifers nd opepods) nd ABT lrve fed the different regimes ws extrted from triplite pooled smples ording to the method of Folh et l. (957). Approximtely g of ABT lrve ws pled in 2 ml of ie-old hloroform/methnol (2:, y vol.) nd homogenized with n Ultr-Turrx tissue disrupter (Fisher Sientifi, Loughorough, UK). The non-lipid nd lipid lyers were seprted y ddition of 5 ml of.88 % (w/v) Kl nd llowed to seprte on ie for h. The upper non-lipid lyer ws spirted nd the lower lipid lyer evported to dryness under oxygen-free nitrogen. The lipid ontent ws determined grvimetrilly fter drying overnight in vuum desitor.

5 Fish Physiol Biohem (27) 43: Lipid lss omposition ws determined y highperformne thin-lyer hromtogrphy (HPTL) using -m pltes (VW, Lutterworth, Englnd). Approximtely μg of totl lipid ws pplied s single spot nd the pltes developed in methyl ette/isopropnol/hloroform/methnol/.25 % queous Kl (25:25:25::9, y vol.) to two thirds up the plte. After drying for 2 min, the plte ws fully developed with isohexne/diethyl ether/eti id (85:5:, y vol.). The lipid lsses were visulized y hrring t 6 for 5 min fterspryingwith3%(w/v) queous upri ette ontining 8 % (v/v) phosphori id nd quntified y densitometry using AMAG-3 TL Snner (version Firmwre.4.6) (Henderson nd Toher 992). Snned imges were reorded utomtilly nd nlyzed y omputer using winats Plnr hromtogrphy Mnger (version.2.). Ftty id methyl esters (FAMEs) were prepred from totl lipid y id-tlyzed trnsesterifition t 5 for 6 h ording to the method of hristie (993). The FAME were seprted nd quntified y gs-liquid hromtogrphy (rlo Er Veg 86, Miln, Itly) using 3 m.32-mm i.d. pillry olumn (P-Wx 52B, hrompk, London, UK) nd on-olumn injetion t 5. Hydrogen ws used s rrier gs, nd temperture progrmming ws from 5 to 5 t 4 min ndthento 23 t 2. min. Individul methyl esters were identified y omprison with known stndrds nd y referene to pulished dt (Akmn 98; Toher nd Hrvie 988). Dt were olleted nd proessed using hromrd for Windows (version.9). Tissue NA extrtion nd DNA synthesis Approximtely mg of pooled lrve (n =3per tretment) nd dult ABT tissues from eight individuls (see ove) were pled in NAlter (Sigm-Aldrih, Dorset, UK) nd frozen t 2 for totl NA extrtion. Smples were homogenized in ml of TI egent (Sigm-Aldrih) NA extrtion uffer using ed tissue disruptor (BioSpe, Brtlesville, OK, USA). Totl NA ws isolted following mnufturer s instrutions nd quntity nd qulity determined y spetrophotometry using NnoDrop ND- (Lteh Int., Est Sussex, UK) nd eletrophoresis using 2 ng of totl NA in % grose gel. DNA ws synthesized using 2 μg of totl NA nd rndom primers in 2-μL retions nd the High pity everse Trnsription Kit without Nse inhiitor ording to the mnufturer s protool (Applied Biosystems, Wrrington, UK). Sequening of genes of interest Severl genes relted to lipid nd ftty id metolism were evluted in the present study. Quntittive reltime P (qp) ws rried out on DNAs enoding the trnsription ftors pprα, pprγ, lxr, rxr, srep, nd srep2; ftty id iosynthesis genes fds2d6 nd elovl5; nd ftty id metolism genes fs, pt, o, fp2, fp4, fp7, lpl, nd hmgl (Supplementry Tle ). Sequenes orresponding to the open reding frme (OF) of srep, srep2, pprα, lxr, nd pti from severl fish speies were ligned, nd primers were designed on ommon onserved regions. GenBnk ession numers of the sequenes used in these lignments were JF5269. (Signus nliultus), XM_ (Esox luius), XM_ (Stegstes prtitus), nd XM_ (Pundmili nyererei) forsrep; NM_9589. (Slmo slr), XM_8343. (S. prtitus), XM_ (Oreohromis nilotius), XM_ (Hplohromis urtoni), XM_ (Mylndi zer), nd XM_ (Neolmprologus rihrdi) for srep2; NM_6333. (Dnio rerio), FJ (tenophryngodon idell), FJ (yprinus rpio), NM_2356. (S. slr), NM_972. (Onorhynhus mykiss), AB (Pgrus mjor), JN97. (Onorhynhus nerk), FJ (Lteolrx jponius), JX (Sophthlmus mximus), JQ (Synehogoius ommturus), EU (Pimephles promels), nd HM (Meglorm mlyephl) for pprα; NM_4542. (S. slr), NM_ (O. mykiss), AB (Prlihthys oliveus), FJ (. idell), FJ (. rpio), nd NM_7545. (D. rerio) forlxr; nd HM (Epinephelus oioides), JQ (S. ommturus), NM_ (O. mykiss), nd JQ (Sestius mrmortus) for pti. Frgments were otined y reverse-trnsription P (MyTq HS Mix, Bioline, London, UK) from DNA (High pity

6 498 Fish Physiol Biohem (27) 43: everse Trnsription Kit; Applied Biosystems, Wrrington, UK) otined from 2 μg of totl NA pooled from dult ABT tissues nd the primers designed for the onserved regions for eh gene. P produts were ligted into plsmid p2. (TA loning Kit, Invitrogen, Pisley, UK) nd sequened (Snger ABI373xl, GAT Bioteh, Konstnz, Germny) nd primers for qp designed. Sequenes for fds2d6 nd elovl5 were lredy ville for ABT (Moris et l. 2). Primers for fp2, 4, nd7; rxr; ndhmgl were designed on existing expressed sequene tgs (ESTs) derived from ABT liver, ovries, nd testis (EG99964, E9273, EG999669, E9299, nd EH668469, respetively; hini et l. 28). Pprγ primers were designed on the pprγ sequene of Thunnus orientlis (AB57433.). Primers for o, fs, nd lpl were designed on sequenes inluded in n ABT oligonuleotide DNA mirorry (ArryExpress dtse ession numer E-MTAB-342; Trumić et l. 25). qp nlysis Expression of genes of interest ws determined y qp of ll the NA smples. Elongtion ftor-α (elfα) ndβ-tin were used s referene genes to study nutritionl regultion. The DNA ws diluted 2-fold with Milli-Q wter. The effiieny of the primers for eh gene ws previously evluted y seril dilutions of DNA pooled from the smples to gurntee it ws >85 % for ll primer pirs. qp ws performed using Biometr TOptil Thermoyler (Anlytik Jen, Goettingen, Germny) in 96-well pltes in duplite 2-μL retion volumes ontining μl of Luminris olor HiGreen qp Mster Mix (Thermo Sientifi, Hemel Hempsted,UK),μL of the primer orresponding to the nlyzed gene ( pmol), 3 μl ofmoleulriology grde wter, nd 5 μl of DNA (/2 diluted). In ddition, mplifitions were rried out with systemti negtive ontrol (NT, no templte ontrol) ontining no DNA. Stndrd mplifition prmeters ontined UDG pretretment t 5 for 2 min nd n initil denturtion step t 95 for min, followed y 35 yles: 5 s t 95, 3 s t the nneling temprture, nd 3 s t 72. The reltive expression of eh gene mong the tissues ws lulted s rirry units fter normliztion ginst the expression level of the housekeeping gene elfα. One ritrry unit ws equl to the expression level of the gene expressed t the lowest level per eh set of genes. Sttistil nlysis esults for iometry, lipid lss, nd ftty id ompositions re presented s mens ± SD (n =2 for iometry nd n = 3 for survivl, lipid lss, nd ftty id ompositions). The dt were heked for homogeneity of the vrines y the Brtlett test nd, where neessry, rsine-trnsformed efore further sttistil nlysis. Differenes etween men vlues were nlyzed y t test nd one-wy nlysis of vrine (ANOVA), followed when pertinent y multiple omprison test (Tukey). Differenes were reported s sttistilly signifint when P <.5(Zr999). Gene expression results were nlyzed using the reltive expression softwre tool (EST 29), whih employs pirwise fixed rellotion rndomiztion test (, rndomiztions) with effiieny orretion (Pfffl et l. 22) to determine the sttistil signifine of expression rtios (gene expression fold hnges) etween two tretments. In ddition, supervised hierrhil lustering ws pplied employing the reltive gene expression rtio for eh gene sed on the P effiieny nd yle threshold (t) of smple ompred to the ontrol, ording to Pfffl s mthemtil model (Pfffl 2). esults ABT lrve iometry nd survivl in feeding trils Totl length, individul dry mss, nd survivl of 4-dh ABT lrve re shown in Tle. In the 23 tril, totl length of ABT lrve fed on enrihed rotifers ws signifintly greter thn tht of lrve fed on opepods, lthough totl dry mss ws not signifintly different. However, survivl ws nerly 2-fold higher in the ABT lrve fed opepods. In 24, totl length nd totl dry mss were highest for ABT lrve fed opepods nd lowest in lrve fed rotifers, with intermedite vlues in lrve o-fed with rotifers nd opepods. However, survivl ws highest in o-fed lrve followed y lrve fed opepods nd rotifers, respetively.

7 Fish Physiol Biohem (27) 43: Tle ering performne of 4-dy fter hth Atlnti Bluefin tun (Thunnus thynnus) lrve fed enrihed rotifers Brhionus plitilis, Arti sp. opepod nuplii, nd o-feeding rotifer + opepod in 23 nd 24 feeding trils Tril ABT + rotifer ABT + opepod ABT + rotifer ABT + opepod ABT + rotifer + opepod Totl length (mm) 7.75 ± ±.5* 7.2 ± ± ±.22 Dry mss (mg).66 ±.3.6 ±.3.35 ±.3.77 ±..5 ±.6 Survivl (%) 3.8 ± ±.93* 2.88 ± ±.7.24 ± 3.5 esults re mens ± SD (n = 2 for totl length nd dry mss nd n = 3 for survivl). Different supersript letters denote signifint differene (P <.5) for one-wy ANOVA nd Tukey multiple omprison tests in 24 tril ABT Atlnti luefin tun *Signifint differene (p <.5) for t tests nlysis in 23 tril Totl lipid ontent nd lipid lss omposition of the live feeds nd the ABT lrve Totl lipid ontent of the enrihed rotifers ws 3 6 % of dry mss nd signifintly higher thn the totl lipid ontent of the opepods (6 9.5 % of dry mss) in the two trils (Tles 2 nd 3). The lipid ontent of the ABT lrve fed rotifers ws..2 % for oth yers wheres the lrve fed the opepods or o-fed the rotifers nd the opepods hd totl lipid ontent tht ws.8 % of live mss, lthough these differenes were not signifint (Tles 2 nd 3). In 23, the lipids of the enrihed rotifers were predominntly polr lipids ( 56 %), minly phosphtidylholine (P, 5 %) nd phosphtidylethnolmine (PE, 3 %), with totl neutrl lipids ounting for 44 %, primrily triylglyerol (TAG, 25 %) (Tle 2). ompred to the rotifers, the opepods in 23 hd lower proportions of PE, phosphtidylserine (PS), phosphtidylinositol (PI), Tle 2 Tril in 23 totl lipid (% dry mss for prey nd % live mss for ABT lrve) nd lipid lss omposition (% of totl lipid) of () rotifer Brhionus plitilis enrihed with Skretting OI-Green, () nuplii of the opedod Arti grnii fed Isorysis T-Iso, nd 4-dy fter hth Atlnti luefin tun (ABT) lrve fed rotifers () nd opepods (d) () otifer () opepod () ABT + rotifer (d) ABT + opepod Totl lipid (% dry mss) 3.4 ± ±.3*. ±..8 ±.2 Lipid lss Phosphtidylholine 4.7 ± ± ± ±.4 Phosphtidylethnolmine 3.3 ± ±.4*.4 ±.6. ±.3 Phosphtidylserine 4.9 ±.2.6 ±.9* 7.3 ± ±.7* Phosphtidylinositol 7.7 ±.5.3 ±.* 3.7 ±. 4.6 ±.6 Phosphtidi id/rdiolipin nd 3.5 ±.8 3. ±. 2. ±.2* Sphingomyelin.5 ±. 5.2 ±.* 2.3 ± ±.3 Lyso-phosphtidylholine 2.4 ±. 3.5 ±.2*.8 ±.3.6 ±. Totl polr lipids 55.9 ± ± ± ±.7 holesterol 4. ± ±.7*.6 ± ±.9* Triylglyerol 25.5 ± ± ±.9.6 ±.8 Steryl/wx ester 5.4 ±.2. ±.* 3. ±.9 5. ±.5 Free ftty id 8.9 ± ± ±. 5. ±.3 Totl neutrl lipids 44. ± ± ± ±.7 Triylglyerol:holesterol 6.2 ±.7.8 ±.2 *.3 ±..8 ±.2 esults re mens ± SD (n =3) *Signifint differene (P <.5) for t test nlysis

8 5 Fish Physiol Biohem (27) 43: Tle 3 Tril in 24 totl lipid (% dry mss for prey nd % live mss for ABT lrve) nd lipid lss omposition (% of totl lipid) of rotifer Brhionus plitilis enrihed with Skretting OI-Green (), nuplii of the opedod Arti tons fed on hodomons slin nd Isohrysis T-Iso (), nd 4-dy fter hth Atlnti luefin tun (Thunnus thynnus) lrve fed rotifers (), opepod (d), nd rotifer + opepod (e) () otifer () opepod () ABT + rotifer (d) ABT + opepod (e) ABT + rotifer + opepod TL (% dry mss) 5.9 ±.6 9. ±.6*.2 ±..8 ±..8 ±.3 Lipid lss Phosphtidylholine 2. ± ±.5* 22.2 ± ± ±. Phosphtidylethnolmine 3.2 ± ±.3* 4.2 ± ± ±. Phosphtidylserine 4.9 ± ± ± ± ±.6 Phosphtidylinositol 8.8 ±.6 5. ±.3* 5.7 ± ±.2 4. ±.4 Phosphtidi id/rdiolipin.6 ± ±.*.5 ±..6 ±.2.7 ±.2 Sphingomyelin.5 ±. 5.6 ±.4* 2.6 ±.2 2. ±.2 2. ±.2 Lyso-phosphtidylholine.8 ± ±.2.6 ±..6 ±..9 ±. Totl polr Lipids 52.8 ± ± ± ± ±.9 holesterol.2 ±.2.9 ±.3. ±.6. ±.4. ±.3 Triylglyerol 23.6 ± ± ±.7 5. ± ± 2.8 Steryl/wx ester 5.6 ±.3.7 ±.* 3.6 ±.2 6. ± ±.8 Free ftty id 6.7 ±..9 ±.4* 5.5 ± ±.2 5. ±.8 Totl neutrl Lipids 47.4 ± ± ± ± ±.9 TAG/ 2. ±.3.9 ±..4 ±..4 ±..6 ±.3 esults re men ± SD (n = 3). A different supersript letter denotes signifintly different (p <.5) for one-wy ANOVA nd Tukey multiple omprison tests in 24 tril TAG/ triylglyerol/olesterol rtio. ABT Atlnti luefin tun *indites signifintly different (p <.5) for t-tests nlysis mong the live preys nd steryl ester (SE) nd higher proportions of sphingomyelin (SM), phosphtidi id/rdiolipin, nd holesterol. Irrespetive of the live feed used, the lipid lss omposition of the ABT lrve showed reltive high levels of polr lipids (63 66 %) with high proportions of P (22 25 %) followed y PE ( %), nd the feed hd reltively little effet on the lrve lipid lss, lthough lrve fed opepods showed higher proportions of PS nd holesterol thn lrve fed rotifers (Tle 2). In 24, the lipid lss omposition of the enrihed rotifers ws very similr to tht in 23, ut tht of the opepods ws slightly different (Tle 3). However, the differenes etween the rotifers nd the opepods were lrgely similr to those in 23 with opepods showing lower proportions of PE nd PI nd higher proportions of P, SM, phosphtidi id/rdiolipin, nd free fttyidompredtothe rotifers (Tle 3). As in the 23 tril, the live feeds hd little impt on the lipid lss omposition of the ABT lrve. Lrve fed rotifers hd higher proportions of polr lipids ( 67 %) ompred to lrve fed opepods or o-fed rotifers nd opepods ( 62 %), due to inresed perentges of ll phospholipid lsses, espeilly PE nd PS, nd lower proportions of TAG nd SE (Tle 3). o-fed lrve showed very similr lipid lss profile to those fed opepods. Totl lipid ftty id ompositions of the live feeds nd the ABT lrve Totl lipids of rotifers in 23 were hrterized y 25 % sturted ftty ids (primrily 6: followed y 8:), lmost 3 % monounsturted ftty ids (MUFA), primrily 8:n-9, nd round 6 % PUFA, primrily 8:2n-6 ( 2 %) with 8:3n-3 ( 7 %)(Tle4). opepods showed similr levels of sturted ftty ids leit with higher proportions of 4: nd lower proportions of 6:. Additionlly, similr proportions of MUFA (higher 6:n-7 nd lower 2:n-9) nd lower 8:2n-6 nd 8:3n-3 ut higher 8:4n-3 nd n-3:n-6 PUFA rtio were oserved in opepods ompred to rotifers

9 Fish Physiol Biohem (27) 43: Tle 4 Totl lipid ftty id omposition (weight %) of rotifers B. plitilis enrihed with Skretting OI-Green (), nuplii of the opepod Arti grnii fed on hodomons slin (), nd 4-dy Live preys fter hth Atlnti luefin tun (Thunnus thynnus L.) lrve fed with rotifers () nd opepods (d) in the 23 tril ABT lrve () () () (d) Ftty id 4:.9 ±. 8.9 ±.4*.8 ±..8 ±.3* 6: 9.6 ± ±.7* 8.4 ± ±.8 8: 3.4 ±. 2.9 ±.* 9.3 ± ±.4 Totl sturted 25. ± ±.6* 3. ± ±.3 6:n-7.8 ±. 5. ±.*.2 ± ±.* 8:n ±. 5.2 ±.* 6.3 ±.9 6. ±.2 8:n-7.9 ±..3 ±.2.6 ± ±. 2:n-9 2. ±.3.3 ±.*.3 ±.2.4 ±.* Totl monoenes 2.7 ± ± ± ±.3 6 PUFA 6.5 ±.3.7 ±.* 2.6 ±..9 ±.2* 8:2n ± ±.2* 3. ± ±.3* 2:4n-6.6 ±..2 ±.*.3 ±..5 ±. 22:5n-6.5 ±. 2.5 ±.*.6 ±. 2.2 ±.2* Totl n-6pufa 27.9 ± ±.* 8. ±.7.5 ±.3* 8:3n ±. 3.9 ±.* 3. ±..7 ±.2* 8:4n-3.2 ±. 3.4 ±.*.4 ±..9 ±.2* 2:4n-3. ±.. ±.*.9 ±..6 ±.* 2:5n ±. 8.9 ±.* 5.4 ±. 5.9 ±.4 22:5n ±.2.3 ±.* 4.3 ±.4. ±.* 22:6n-3 2. ± ±.9* 7.5 ± ±.* Totl n-3pufa d 32. ± ±.9* 33.8 ± ±.7* Totl PUFA 6. ± ± 3.9* 5.9 ± ± 2. n-3/n-6. ±. 2.9 ±.*.9 ±. 3.9 ±.* DHA/EPA 2.7 ±. 2.8 ±. 3.2 ±.2 5. ±.* esults re mens ± SD (n = 3). An SD of. implies n SD of <.5 DHA dooshexenoi id, EPA eiospentenoi id, PUFA polyunsturted ftty id *Signifintly different (P <.5) Totls inlude 5:, 2:, 22:, nd 24: Totls inlude 6:n-9, 8:n-, 2:n-7, 22: isomers, nd 24: Totls inlude 8:3n-6, 2:2n-6, 22:4n-6, nd 22:5n-6 d Totls inlude 2:3n-3 nd 22:3n-3 (Tle 4). Besides, rotifers nd opepods provided similr DHA/EPA rtios (2.7 nd 2.8, respetively) ut perentges of DHA nd EPA were twie s high in opepods (25 nd 9 %, respetively) ompred to rotifers (2 nd 4.5 %, respetively). Higher proportions of 22:5n-3 were found in rotifers ompred to opepods. Totl lipid of ABT lrve ws out 3 32 % sturted ftty ids nd 3 5 % MUFA irrespetive of live feed used with few importnt differenes (Tle 3). However, the PUFA ompositions of the live feeds were refleted in ABT lrve ompositions nd thus lrve fed opepods showed lower 8:2n-6, 8:3n-3, nd 22:5n-3 nd higher DHA nd n-3:n-6 PUFA thn lrve fed rotifers. Interestingly, the proportions of EPA were lower nd DHA nd the DHA/ EPA rtio higher in lrve fed opepods thn in the opepods themselves (Tle 4).

10 52 Fish Physiol Biohem (27) 43: otifers nd opepods in 24 showed similr proportions of sturted ftty ids (27 28 %) nd MUFA ( %) s in 23 with the sme differenes etween the live feeds in the proportions of 4:, 6:, 6:n-7, nd 2:n-9 (Tle 5). Similrly, opepods showed lower 8:2n-6 ut higher 8:3n-3, 8:4n-3, nd n-3/n-6 PUFA rtio thn rotifers. In 24, oth rotifers nd opepods provided higher DHA/EPA rtios thn in 23 with the rtio eing signifintly higher in opepods (6.9) ompred to rotifers (3.8). As in 23, the perentge of DHA ws higher in opepods ut oth EPA nd 22:5n-3 were higher in rotifers (Tle 5). As in 23, the PUFA omposition of ABT lrve in 24 tended to reflet the dietry ompositions Tle 5 Ftty id omposition (weight %) of totl lipid of rotifers B. plitilis enrihed with Skretting OI-Green (), nuplii of the opepod Arti tons fed hodomons slin nd Live prey Isohrysis T-Iso (), nd 4-dy fter hth Atlnti luefin tun (Thunnus thynnus L.) lrve fed with rotifers (), opepods (d), nd o-feed rotifer + opepod (e) in the 24 tril ABT lrve () () () (d) (e) Ftty id 4:.7 ±.. ±.2*.7 ±. 2.2 ±.2.6 ±.3 6: 22.7 ±.9.9 ±.2* 6.9 ± ±.5 7. ±.4 8: 2.9 ±. 2. ±.* 8.6 ± ±. 8.3 ±.4 Totl sturted 28. ± ± ± ± ±.3 6:n-7.7 ±. 3.9 ±.*.7 ± ±..4 ±. 8:n ± ± ± ±.2 5. ±.2 8:n-7.9 ±..2 ±..9 ±.2 2. ±..7 ±. 2:n-9.9 ±..3 ±.*.2 ±..3 ±..9 ±. Totl monoenes.6±.2.8±.4 2.9± ±.3 4. ±.3 6 PUFA 3.6 ±.3. ±.* 3. ±. 2.2 ±. 2.8 ±. 8:2n ±. 8. ±.2* 2.7 ± ±..3 ±.9 2:4n-6.7 ±..6 ±. 2.6 ±.3.6 ±..4 ±. 22:5n-6.9 ± ±.*.4 ±. 2.6 ±.2.8 ±.3 Totl n-6pufa 2.4 ±.3 4. ±.* 9.4 ± ±.2 2. ±. 8:3n ± ±.3* 2.3 ±. 2.5 ± ±. 8:4n-3.3 ±. 8.2 ±.6*.2 ±. 2.2 ±.2. ±.2 2:4n-3.7 ±.. ±.*.7 ±..9 ±.2.8 ±.2 2:5n ±. 3.8 ±.* 6.6 ± ±. 4.8 ±. 22:5n ±.3.3 ±.* 4.6 ±.2.6 ±. 2.2 ±.3 22:6n ± ±.9* 7.6 ± ± ±.5 Totl n-3pufa d 34.6 ± ±.3* 34.5 ± ±.3 4. ±.3 Totl PUFA 55. ± ± ±.6 5. ± ±.3 n-3/n-6.7 ± ±.*.7 ±.3 3. ±. 2. ±.2 DHA/EPA 3.8 ± ±.6* 2.7 ±.2 5. ±.2 5. ±.2 esults re mens ± SD (n = 3). An SD of. implies n SD of <.5. Vlues ering different supersript letters (ABT lrve) re signifintly different (P <.5) DHA dooshexenoi id, EPA eiospentenoi id, PUFA polyunsturted ftty id *Vlues within live prey re signifintly different (P <.5) Totls inlude 5:, 2:, 22:, nd 24: Totls inlude 6:n-9, 8:n-, 2:n-7, 22: isomers, nd 24: Totls inlude 8:3n-6, 2:2n-6, nd 22:4n-6 d Totls inlude 2:3n-3 nd 22:3n-3

11 Fish Physiol Biohem (27) 43: nd therefore lrve fed opepods showed lower 8:2n-6, EPA, nd 22:5n-3 nd higher DHA, DHA/ EPA, nd n-3/n-6 PUFA rtios thn lrve fed rotifers (Tle 5). Lrve o-fed rotifers nd opepods tended to give intermedite vlues s oserved, for instne, with 8:2n-6, 22:5n-3, DHA, nd n-3/n-6 rtio (Tle 5). Expression of lipid metolism genes ftty id iosynthesis nd trnsription ftor genes in ABT lrve Differing results were oserved regrding expression of fds2d6 etween the two experiments. In this regrd, in the 23 experiment, the lrve fed opepods showed higher expression of fds2d6 ompred to the lrve fed rotifers (Fig. ). In ontrst, expression of fds2d6 ws the lowest in opepod-fed lrve in 24 (Fig. ). No differenes were oserved regrding elovl5 expression, lthough there ws tendeny for higher expression in opepod-fed lrve (Figs., ). In the 23 experiment, no differenes in expression were oserved in trnsription ftors etween lrve fed opepods or rotifers (Fig. 2), wheres some genes presented differing expression mong lrve fed the three dietry tretments in 24 (Fig. 3). In this regrd, srep expression ws the highest in o-fed lrve, with the lrve fed rotifers displying the lowest expression of this gene (Fig. 3). Differenes etween lrve fed the three dietry tretments in 24 were lso oserved for pprγ with lowest expression displyed y o-fed lrve nd highest expression oserved in lrve fed rotifers (Fig. 3). Although no signifint differenes were found in lxr or rxr mna levels, the sme expression ptterns were oserved in lrve from the two trils, with low expression of these TFs in lrve fed rotifers ompred to lrve fed opepod, with o-fed lrve showing intermedite expression levels in the 24 tril (Figs. 2 nd 3). The expression profiles of lipid homeostsis genes re shown in Figs. 4 nd 5. In generl, expression of fps showed reltively stle ptterns Fig. Nutritionl regultion of fds2d6 nd elovl5 gene trnsription in whole lrve of Atlnti luefin tun fed different dietry tretments in the 23 () or 24 () trils. Feeds were either enrihed rotifers (), nuplii of opepods (), or ofeeding of oth live preys (). Vlues re normlized expression rtios, orresponding to n verge of six individuls (n =6) with stndrd errors (SEM). Different supersript letters denote differenes etween the dietry tretments. fds2d6 delt- 6 ftty yl desturse; elovl5 ftty yl elongse 5 A B fds2d6 fds2d elovl5 elovl

12 54 Fish Physiol Biohem (27) 43: lxr srep srep rxr pprα pprγ Fig. 2 Nutritionl regultion of trnsription ftor genes in whole lrve of Atlnti luefin tun fed different dietry tretments in the 23 tril. Feeds were either enrihed rotifers () or nuplii of opepods (). Vlues re normlized expression rtios, orresponding to n verge of six individuls (n = 6)with stndrd errors (SEM). Different supersript letters denote differenes etween the dietry tretments. lxr liver X reeptor; srep sterol regultory element-inding protein ; srep2 sterol regultory element-inding protein 2; rxr retinoid X reeptor; pprα peroxisome prolifertor-tivted reeptor lph; pprγ peroxisome prolifertor-tivted reeptor gmm in oth yers, with higher expression in lrve fed rotifers thn in lrve fed opepods, or o-fed lrve. However, signifint differenes were only oserved in fp4 expression for 23 lrve nd in fp4 nd 2 for 24 lrve. The expression of fs lso showed differenes etween tretments in oth yers, with highest expression in lrve fed opepods, nd there ws no differene etween lrve fed solely on rotifer or o-fed rotifers nd opepods in 24. In ontrst to the 23 experiment, o displyed differenes in expression mong dietry tretments in lrve in the 24 tril. In this se, lrve fed rotifers showed the highest expression with no differenes etween opepod-fed or o-fed lrve. Lrve fed rotifers showed the lowest expression of lpl in 23 with similr ut non-signifnt tendeny oserved in 24. No differenes mong tretments in either yer were found in the expression of fp7, pt, orhmgl. Tissue distriution of lipid nd ftty id metolism nd trnsription ftor genes in dult ABT The tissue expression profiles showed tht oth L-PUFA iosyntheti pthwy genes (elovl5 nd fds2d6) were expressed in ll tissues exmined, with the highest expression levels in rin followed y liver nd testis (Fig. 6). The elovl5 trnsript ws more undnt thn fds2d6 in ll other tissues, with prtiulrly low expression of fds2d6 in red musle nd ovries (Fig. 6). egrding trnsription ftors, pprα nd pprγ showed prllel expression, with dipose tissue exhiiting the highest reltive opy numer

13 Fish Physiol Biohem (27) 43: lxr srep srep rxr pprα pprγ Fig. 3 Nutritionl regultion of trnsription ftor genes in whole lrve of Atlnti luefin tun fed different dietry tretments in the 24 tril. Feeds were either enrihed rotifers (), nuplii of opepods (), or o-feeding of oth live preys (). Vlues re normlized expression rtios, orresponding to n verge of six individuls (n = 6) with stndrd errors (SEM). Different supersript letters denote differenes etween the dietry tretments. lxr liver X reeptor; srep sterol regultory elementinding protein ; srep2 sterol regultory element-inding protein 2; rxr retinoid X reeptor; pprα peroxisome prolifertortivted reeptor lph; pprγ peroxisome prolifertor-tivted reeptor gmm (Fig. 7) of ll the evluted tissues, followed y intestine > testis > liver. The expression of lxr ws surprisingly low in liver, with highest expression in testis, rin, nd kidney. Similrly, rxr ws poorly expressed in liver with higher levels of expression in oth white nd red musle, followed y spleen nd rin (Fig. 7). The rnk order of expression of srep ws rin > testis > ovry > intestines > kidney > gill > liver > white musle > spleen > hert nd red musle (Fig. 7). For srep2, the highest expression ws lso oserved in rin followed y testis nd dipose tissue with lowest expression in hert nd white musle (Fig. 7). The expression of fp2 (intestinl isoform) ws highest in intestine, followed y rin nd hert, with lower levels of expression in liver, red musle, dipose tissue, nd kidney (Fig. 7d). In ontrst, the dipoyte isoform fp4 showed highest expression in ovries with liver showing the lowest vlues, with red musle, hert, nd dipose tissue showing intermedite vlues (Fig. 7d). The rin isoform fp7 showed highest expression levels in rin nd testis, followed y red nd white musle, hert, nd kidney, with lowest vlues in liver (Fig. 7d). The highest expression of pti ws in rin nd the lowest expression in liver (Fig. 7e). Similrly, the reltive opy numer for fs ws highest in rin followed y gonds, gill, nd liver, with white musle showing the lowest expression (Fig. 7e). The expression of o ws highest in dipose tissue nd intestine, followed y liver, kidney, nd rin (Fig. 7f). The expression of hmgl ws highest in

14 56 Fish Physiol Biohem (27) 43: fp4.6 fp7.6 fp2.6 fs pt.6.4 o.6.4 lpl.6.4 hmgl Fig. 4 Nutritionl regultion of lipid metolism gene trnsription in whole lrve of Atlnti luefin tun fed different dietry tretments in the 23 tril. Feeds were either enrihed rotifers () or nuplii of opepods (). Vlues re normlized expression rtios, orresponding to n verge of six individuls (n = 6) with stndrd errors (SEM). Different supersript letters denote differenes etween the dietry tretments. fp4 ftty id inding protein 4 (dipoyte); fp7 ftty id inding protein 7 (rintype); fp2 ftty id inding protein 2 (intestinl); fs ftty id synthse; pt rnitine plmitoyl trnsferse I; o yl oa oxidse; lpl lipoprotein lipse; hmgl 3-hydroxy-3-methylglutryl-oA lyse ovry followed y dipose tissue, rin, nd testis, with lowest expression in liver (Fig. 7f). Finlly, the expression of lpl ws highest in testis nd lowest in ovry, with liver nd white musle, dipose tissue, hert, gills nd red musle, kidney, nd intestine showing intermedite levels of expression (Fig. 7f). Disussion The present study reports on the expression of ftty id iosynthesis, lipid metolism, nd trnsription ftor genes in ABT lrve during first feeding with different live prey, speifilly enrihed rotifers versus opepod nuplii nd opepodites. Due to the short spwning seson of ABT in nturl onditions (only June nd July) nd the sre vilility of fertilized vile eggs, trils on first feeding ABT lrve were performed over two periods orresponding to 23 nd 24 spwning sesons. Bluefin tun lrve predte opepods in the wild (Uotni et l. 99), nd, in generl, mrine fish lrve fed on opepods grow fster thn lrve fed enrihed rotifers or Artemi, s hs een shown with

15 Fish Physiol Biohem (27) 43: fp4 fp7 fp2 fs pt o lpl hmgl Fig. 5 Nutritionl regultion of lipid metolism gene trnsription in whole lrve of Atlnti luefin tun fed different dietry tretments in the 24 tril. Feeds were either enrihed rotifers (), nuplii of opepods (), or o-feeding of oth live preys (). Vlues re normlized expression rtios, orresponding to n verge of six individuls (n = 6) with stndrd errors (SEM). Different supersript letters denote differenes etween the dietry tretments. fp4 ftty id inding protein 4 (dipoyte); fp7 ftty id inding protein 7 (rin-type); fp2 ftty id inding protein 2 (intestinl); fs ftty id synthse; pt rnitine plmitoyl trnsferse I; o yl oa oxidse; lpl lipoprotein lipse; hmgl 3-hydroxy-3-methylglutryl-oA lyse Log normlized E Fig. 6 Tissue distriution of fds2d6 (lk) nd elovl5 (white) trnsripts. The trnsript expression level ws determined y qp in 2 tissues. Vlues orrespond to the log-normlized (efα) reltive expression (E) of the trget genes in eh tissue. For omprison, the expression level of fds2d6 in ovry, whih B G H K S L I W A O T 6fd elovl5 ws the lowest, ws defined s. The results represent the verge of six individuls (n = 6) with stndrd errors (SEM). B rin; G gills; H hert; K kidney; S spleen; L liver; I intestine; red musle; W white musle; A dipose tissue; O ovry; T testis

16 58 Fish Physiol Biohem (27) 43: A E ppr pprg B G H K S L I W A O T D Log normlized E B G H K S L I W A O T fp2 fp4 fp7 B E lxr rxr B G H K S L I W A O T E Log normlized E B G H K S L I W A O T pti fs E d d d d d d d d srep srep2 d B G H K S L I W A O T Fig. 7 Tissue distriution of pprα nd γ (), lxr nd rxr (), srep nd 2 (), fp genes (d), pt nd fs (e) nd o, nd hmgl nd lpl (f). The trnsript expression level ws determinedyqpin2tissues.vluesorrespondtothenormlized (efα) reltiveexpression(e)ofthetrgetgenesin eh tissue. For omprison, the expression level of pprα in gills (), rxr in ovry (), srep2 in white musle (), fp2 in F Log normlized E B G H K S L I W A O T o hmgl ovry (d), pti in liver (e), nd lpl in spleen (f) were lowest nd defined s. The results represent the verge of six individuls (n = 6) with stndrd errors (SEM). B rin; G gills; H hert; K kidney; S spleen; L liver; I intestine; red musle; W white musle; A dipose tissue; O ovry; T testis. Different letters indite signifint differenes mong tissues for eh of the genes (P <.5) lpl od (Gdus morhu; Hmre 26), grouper (E. oioides; Toledo et l. 999), or turot (Sophtlmus mximus; Wittetl.984). In ddition, in previous study, ABT lrve fed on A. tons opepods showed high growth nd survivl rtes (Yufer et l. 24). Our hypothesis ws tht opepodsmyeetterlivepreythnartemi due to their high DHA nd DHA/EPA rtio. Furthermore, lthough n-3 L-PUFA levels n e similr in enrihed Artemi nd opepods, they re found within the neutrl lipids in the former (Srgent et l. 999), wheres they re predominntly found in polr lipids (e.g., phospholipids) in opepods nd my e more esily ssimilted y the lrve (Shields et l. 999). In the present study, ABT lrvl survivl ws similr in oth yers, with opepod-fed lrve (nd o-fed lrve) showing higher survivl thn lrve fed rotifers, proly inditing tht opepods were more suitle live prey for lrve of this fstgrowing fish speies. onversely, growth differed in the 2 yers, with lrve fed rotifers showing higher growth rtes thn those fed opepods in 23, ut the opposite in 24. otifers supplied more lipid per dry mss thn opepods in oth yers, so this on its own does not orrelte with

17 Fish Physiol Biohem (27) 43: survivl or growth. Similrly, differenes in lipid lss omposition etween rotifers nd opepods were essentilly the sme in oth yers ut, in ontrst, ftty id ompositions of the live feeds vried. Thus, in oth 23 nd 24 trils, opepods supplied onsiderly more DHA on reltive sis thn rotifers. The DHA/EPA rtio ws similr etween opepod- nd rotifer-fed lrve in 23, eing higher in opepods thn in rotifers in 24. This my suggest tht the solute DHA level my e importnt for the survivl of ABT lrve ut tht the DHA/EPA rtio my e reltively more importnt for lrvl growth. However, neither DHA level nor the DHA/EPA rtio on their own n explin the vrition etween the 2 yers. It hs een shown tht roodstok nutrition, prtiulrly dietry lipid, n gretly ffet lrvl survivl nd growth (Srgent 995; Wiegnd 996; Izquierdo et l. 2). Although ABT roodstok were not evluted in the present trils, it is likely tht fertilized eggs elonging to different spwning groups led to differing results in lrvl performne etween the 2 yers. Additionlly, roodstok dietry differenes etween the 2 yers ould lso hve influened lrvl performne. The present tril imed to provide further insight into the effets of nutrition on ABT lrvl performne y investigting lipid metolism y determining expression ptterns of some key genes, rther thn fousing on ssoitions etween dietry ompositions nd resultnt lrvl ompositions. To the uthors knowledge, this is the first report to evlute expression of lipid metolism genes in ABT lrve under different feeding regimes t 4 dh. One ovious nd potentilly key pthwy to investigte is L-PUFA iosynthesis. Modultion of fds2d6 ws oserved in oth yers, ut with differing diretion of expression, whih my e relted to differenes in ftty id profiles of the live prey s differenes in the levels of n-3 L- PUFAwereoservedetweenthe2yers.Inontrst, expression of elovl5 in the present study showed no signifint regultion. Generlly, upregultion of fds2d6 expression hs een oserved in fish fed low dietry levels of n-3 L-PUFA, for instne when high levels of vegetle oil re inluded in feeds (Moris et l. 22; Betnor et l. 25). This is wht ws oserved in ABT lrve fed enrihed rotifers nd opepods in 23, ut not in 24. However, the tivities of fds2d6 nd elovl5 in mrine fish hve een the sujet of disussion nd speultion s most mrine speies hve only low pity for L-PUFA synthesis from 8 PUFA preursors suh s 8:3n-3, proly s onsequene of them otining high levels of n-3 L-PUFA in their nturl diet (Toher 2). However, n lterntive role for the fds2d6 nd elovl5 enzymes in mrine fish hs een suggested sed on requirement to mintin memrne DHA levels, espeilly in neurl tissues, t times of high demnd suh s emryoni nd lrvl development (Toher et l. 26; Zheng et l. 29; Mohd-Yusof et l. 2). onsistent with this, the present study hs shown tht oth fds2d6 nd elovl5 were expressed t highest levels in dult tun rin, showing the typil mrine speies expression pttern suh s previously shown in Atlnti od (G. morhu) nd oi (hyentron ndum) (Toher et l. 26; Zheng et l. 29; Xue et l. 24), given tht freshwter speies tend to hve higher levels of expression in liver thn in rin (Ooh et l. 26). The lterntive role of these genes in mrine fish in mintining (neurl) tissue DHA levels (y desturtion nd elongtion of EPA) my t lest prtly explin the differenes in expression oserved. Thus, in 23, the high level of dietry EPA in opepods resulted in higher expression of fds2d6 in ABT lrve. In 24, the level of EPA ws lower, nd DHA higher, in opepods thn in rotifers (nd opepods in 23), nd this high DHA/EPA rtio in opepods ppered to inhiit expression of fds2d6 in ABT lrve fed them. Therefore, the higher DHA levels found in opepod-fed ABT lrve in 23 thn in opepods themselves my e onsequene of oth desturtion of EPA nd/or seletive retention of this ftty id. A previous study evluting the expression of oth fds2d6 nd elovl5 in unfed ABT lrve showed up-regultion of these genes during erly development (Moris et l. 2). onsistent with the inresed expression, the DHA/EPA rtio in the ABT lrve lso inresed during development, ut retention of DHA in polr lipids nd seletive tolism of EPA lso offer lterntive mehnisms. Irrespetive of the primry mehnism for the inresed DHA/EPA rtio in the study with unfed lrve, the inresing expression of fds2d6 nd