Impact of essential fatty acid deficiency and temperature on tissues' fatty acid composition of European sea bass (Dicentrarchus labrax)

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1 Plese note tht this is n uthor-produced PDF of n rticle ccepted for publiction following peer review. The definitive publisher-uthenticted version is vilble on the publisher Web site Aquculture Vol. 255, Issues 1-4, 31 My 26, Pges Elsevier B.V. All rights reserved Archimer, rchive institutionnelle de l Ifremer Impct of essentil ftty cid deficiency nd temperture on tissues' ftty cid composition of Europen se bss (Dicentrrchus lbrx) A. Sklli, J.H. Robin b, *, N. Le Byon b, H. Le Delliou b nd J. Person-Le Ruyet b Deprtmento Biologí Animl y Ecologí, Fcultd de Ciencis Universidd de Grnd, Spin b UMR 167, Nutrition Aquculture Génomique des Poissons, Ifremer, Centre de Brest, BP 7, 2928 Plouzné, Frnce *: Corresponding uthor : Tel.: ; fx: ; emil: jrobin@ifremer.fr Abstrct: The effects of essentil ftty cid deficiency nd temperture on the ftty cid profiles of polr lipids () nd neutrl lipids () from vrious tissues (muscle, liver, gills, eyes nd brin) of Europen se bss juveniles were compred in two fctoril design. Fish (6 g) were held for 84 dys t 22 or 29 C (upper limit for growth) nd fed either t lower or higher level thn n 3 HUFA miniml requirement for growth (.4% nd 2.2% n 3 HUFA dry mtter for s nd HD, respectively). Essentil ftty cid deficiency hd mjor influence on ftty cids in frction of ll tissues, more moderte influence on of muscle, liver nd gills, while low nd very low influence ws observed on eyes nd brin ftty cid content, respectively. DHA nd EPA content in brin s well s DHA in eyes were not ffected by. DHA contents were similr in gill of 22-HD, 29- HD nd 22- but ws reduced in 29- fish. Most of brin ftty cids displyed significnt effect of temperture (t 29 C; 18 :, 18 : 1n 9 contents were higher, nd 2 : 5n 3, 22 : 6n 3, 2 : 4n 6 contents were lower thn t 22 C). Temperture hd more influence on thn on ftty cid content, except in liver. A lipogenic ctivity seemed to occur, both 16 : nd 18 : were high prticulrly in liver nd dependent on temperture (higher t 29 thn t 22 C). An enhnced 18 : 3n 6 content in fish fed on the deficient HUFA indicted desturtion ctivity, minly in liver nd gill, with higher rchidonic cid content in of gills thn other tissues. Muscle ftty cid profiles in nd were more similr to those of whole body thn other tissues, however totl lipid content then : rtio differed. This study shown tht beside known chrcteristics of ech tissue in term of ftty cid content, ech tissue hve lso chrcteristics in term of response to temperture nd ry deficiency influence. Among then, neurl tissue displyed the highest cpcity to regulte DHA content in, preserving functionlity despite HUFA deficiency. Keywords: Europen se bss; n 3 HUFA; Temperture; Tissue ftty cid composition 1

2 2 1 Introduction The nutritionl spects of essentil ftty cids (EFA) in fish hve been widely investigted (Srgent et l., 22; Tocher, 23). The n-3 highly unsturted ftty cid (n-3 HUFA) re required nutrients for mrine fish (Knzw et l., 1979), minly eicospentenoic cid (2:5n-3; EPA) nd docoshexenoic cid (22:6n-3; DHA). These ftty cids re mjor components of polr lipids in fish, insuring membrne functionlity (Srgent et l., 22). HUFAs re implicted in mny physiologicl processes, including fish dpttion to environmentl fctors s temperture (Hzel et l., 1992; Frks et l., 1994). However regultion of membrne fluidity my involve other components, s monounsturted/sturted ftty cids rtio (Wodtke nd Cossins, 1991) or cholesterol/phospholipid rtio (Robertson nd Hzel, 1996). In generl, the min lipid source used in fish feeds is fish oil, contributing with fish mel to bring high n-3 HUFA content in s. Replcement of fish oil by vegetble oil results in lower contents of EPA nd DHA, nd in higher contents of the 18C ftty cids (oleic, linoleic nd linolenic cids) in ll fish species (Alexis, 1997; Bell et l., 21; Izquierdo et l., 23; Regost et l., 23). The ftty cid composition of fish tissue lipids usully reflect those of the ry lipids (Jobling, 21). The vilble dt concerning the effects of temperture on tissues lipid composition in mrine fish re relted to low temperture (Ventrell et l., 1993; Sturnes et l., 1994), conversely the effects of high temperture re poorly documented. Although tissue ftty cid profiles cn be modified by ltering the sources of fts nd oils in formulting fish feeds, little is known on the combined effects of temperture nd ry n-3 HUFA in tissue ftty cid profiles, minly in mrine fish. The effects of two ry n-3 HUFA contents (.4 nd 2.2% DM) nd two stbilized tempertures (22 nd 29 C) hve been studied on feeding, growth, metbolism nd body composition of juveniles se bss (Person-Le-Ruyet et l., 24). These results showed tht specific growth rte ws significntly ffected both by nd temperture but there ws no significnt difference in finl fish proximte composition except decrese in moisture content with temperture. Whole body ftty cids were ffected by nd temperture with some interctions. This experiment ws designed in order to follow how fish my dpt to temperture when they were fed under or over HUFA requirement level, this dpttion should be tissue dependent. Fish receiving enough HUFAs my dpt their tissues polr lipids with less constrin thn fish fed deficient. A specific incorportion of n-3 HUFA in some tissues might lso induce reduced vilbility of HUFA for other tissues. The purpose of the present study ws to investigte these hypothesis nd to provide informtion on how these two fctors influence fish ftty cid composition of trget tissues: liver, muscle, gills, neurl tissues (brin nd eyes) fter 84 dys exposure under the four experimentl conditions tested. 2 Mterils nd methods 2.1. Experimentl design The fish of two temperture groups (22 nd 29 C) were fed to stition with two experimentl s. Diets were formulted to be isonitrogenous nd isolipidic, 5%

3 3 crude protein nd 16% crude lipid, nd to contin 2.2% or.4% n-3 HUFA (HD nd, respectively). The two s differed only by the oil dded, detils on s composition cn be found in Person-Le Ruyet et l. (24), ry ftty cids were lso presented compre to tissues ftty cids in Fig. 1 to 3. The experiment ws crried out using triplicte tnks of ech group (-22, -29, HD-22, HD-29). At the end of the experiment (dy 84), the fish were fsted for 24 h nd six fish per tnk were nesthetised in solution of ethylene-glycol-monophenyl-ether (.5%) before smpling. In ech fish, different tissues were tken s follow: gills rch wshed in.25m sucrose; whole liver; whole hed; bout 8 cm 2 of dorsl muscle tken under the first dorsl fin nd skin ws removed. They were immeditely frozen in nitrogen liquid nd stored t 8 C until nlysis. The gill filments, brin nd eyes were selected prior to nlyses Anlyticl methods The lipid nlyses of ll tissues (pooled smples per tnk) ws done ccording to Folch et l. (1957), chloroform being replced by dichloromethne. The seprtion of neutrl lipids nd phospholipids ws performed ccording to the procedure described by Juned nd Rocquelin (1985). A prt of ech smple ws trnsmethylted ccording to Morrison nd Smith (1964). Ftty cids methyl esters (FAME) were seprted by gs chromtogrphy s described previously in Person-Le-Ruyet et l. (24). FA composition ws expressed s percent of totl identified FA methyl esters Dt nlysis All results re expressed s men ± SE. Sttisticl nlyses were conducted using Sttistic for Windows, dt in % being rcsin squre-root trnsformed. For ech ftty cid, differences between the four experimentl conditions were tested by two-wy ANOVA, to identify the effects of tempertures nd s nd their possible interctions. When significnt interctions were observed, differences between mens were compred by Newmn-Keuls test, differences were considered significnt t P <.5. Similrities between ftty cid profiles between tretments within ech tissue, or between tissues within tretments were studied using the coefficient of distnce D (McIntire et l., 1969): n 1/2 D(h-j)=(Σ(Pih-Pij)²) i=1 where the D(h-j) is distnce between tretments h nd j, nd Pih nd Pij re percentge of the ftty cid i in tretments h nd j, for ech i ftty cid.

4 4 3. Results Neither nor temperture significntly ffected lipid content in the muscle, gills, eyes nd brin (Tble 1). A significnt interction of the two fctors studied ws observed for liver, liver of 29-HD fish hving lower liver ft content thn those of 22-HD nd 22-. A high vribility ws observed in muscle lipids in 22- nd 29-HD groups. The ftty cid (FA) composition of fish tissues is reported in Fig.1-3. Sum of the presented ftty cids ccount for 71 to 86% nd 87 to 93% of identified ftty cids for nd, respectively. Except in eyes nd brin (, ), plmitic cid (16:) contents were significntly influenced by ry contents (Fig.1). They were higher in fish tissues thn in s. Significnt temperture influences were observed in whole body, muscle, in liver ( higher vlues t 29 C thn t 22 C), nd brin (higher t 22 thn 29 C). No significnt influence of ws observed on steric cid (18:) contents. Temperture induced significnt differences in steric cid contents of muscle nd liver nd, whole body, eyes nd brin, with higher vlues t 29 C thn t 22 C. Conversely, in gills, 18: contents were lower t 29 thn t 22ºC. Compred to s, steric cid contents were very high in ll tissues, specilly in nd in brin. Oleic cid (18:1n-9) displyed vrious influences of nd temperture with severl interctions (in muscle, whole body nd liver ). In ll tissues, except in brin, significnt influence of ry 18:1n-9 ws observed. Temperture induced higher 18:1n- 9 content t 29 thn t 22 C in whole body, in muscle, gills nd, nd brin. Conversely, in liver 18:1n-9 contents were lower t 29 thn t 22 C with n interction (stronger temperture effect in thn in HD groups). The higher 18:2n-6 nd lower 2:4n-6 contents of compred to HD, ws reflected in ll tissues studied (Fig. 2). A noticeble exception in influence ws encountered for 18:3n-6; despite hving lower mount in thn in HD s, fish fed showed higher levels of 18:3n-6 thn fish fed HD, in both polr nd neutrl lipids of WB, muscle, liver nd gills (ns in eyes nd brin). A significnt effect of temperture on 18:3n-6 in gills nd muscle ws observed, with lower vlues t 29 thn t 22 C. Compred to s contents, high percentges of rchidonic cid (AA, 2:4n-6) were observed in polr lipids of ll tissues nlysed nd in brin. Gills displyed higher AA contents thn other tissues in polr lipids, x temperture interction occurred, with higher vlue t 29 thn t 22 C only in fish fed HD. Significnt effects of temperture were observed with higher AA content in muscle, nd lower vlues in brin nd whole body, t 29 thn t 22 C. Temperture lso influenced 18:2n-6 contents with higher vlues in gills nd whole body, nd lower vlues in muscle nd liver t 29 thn t 22 C. In comprison with ry levels, DHA contents were very high in ll tissues, while 18:3n-3 contents were low (Fig. 3). A significnt influence of s ws observed for n-3 FA in ll cses except brin nd 22:6n-3 in eyes. Eyes were very high in DHA, compre to other tissues including brin, EPA levels in brin nd eyes were lower thn the other tissues nlysed. In ll tissues, EPA nd DHA levels were clerly reduced in groups compred to HD groups. Liver hd lower levels of EPA nd DHA in thn other tissues. The highest levels of DHA in were encountered in brin even within fed fish. Among temperture significnt lower vlues t 29 thn t 22 C were

5 5 found for 18:3n-3 in WB, muscle, liver, eyes, nd in liver ; for EPA in eyes nd brin nd gills ; for DHA in WB, muscle, brin, nd gills. Conversely 18:3n- 3 ws higher in WB t 29 compred to 22 C. Some x temperture interctions were noticed. In whole body EPA nd DHA were significntly effected by temperture only in fish fed, but not in fish fed HD. Sme interction occurred for DHA in gills, in this cse 22 fish hd sme DHA content thn the HD groups. In eyes more noticeble influence of temperture on EPA ws observed in HD groups thn in groups. Nervonic cid (24:1n-9) ws lso found t substntil mount in brin (3.3 to 3.7% of totl FA) without significnt influence of the two studied fctors. The reltive influences of the two s nd tempertures on tissues ftty cid content were summrized using D, distnce coefficients (Tble 2) clculted with ll determined ftty cids. In fish, higher D vlues were observed between s thn between tempertures for ll tissues, except brin. The impct ws remrkbly low in brin (D=1.8 for ech temperture), nd moderte in eyes ( ). Temperture impct ws lower in eyes nd liver thn in other tissues. In, impct ws strong for ll tissues nlysed nd temperture impct ws low, except for liver. Compring these effects between tretments, FA tissues specificities were kept within ech tretment. Distnces were clculted between ech tissue within ech tretment, similr D vlues were obtined between 2 tissues whtever the tretments were. Tble 3 summrized these results presented s men vlues with vribility between tretments (S.E ) for sme comprison. This method indicte ftty cid ptterns were reltively similr in muscle, eyes nd gills nd close to whole body, while liver nd brin differed strongly. ftty cid ptterns were more specific of ech tissue, little less for muscle nd gills compred to whole body. 4. Discussion After 84 dy exposure of se bss juveniles to the four experimentl conditions tested (.4 or 2.2% n-3 HUFA in s x 22 or 29 C wter temperture), hs stronger effect on tissues FA profile thn temperture. Wheres temperture hd rther stronger effect on growth performnce thn s (Person-le Ruyet et l., 24). The effect of s ws expected s the ftty cid composition of fish tissues usully tend to reflect those of ry lipids (Bell et l., 21; Jobling, 21). As it generlly occurred, influence ws higher in neutrl lipids (more similr to ry ftty cid pttern) thn in polr lipids (Sklli nd Robin, 24). The reltive higher levels of HUFAs s DHA EPA nd AA in tissues compred to is in ccordnce with the preferentil incorportion of these ftty cids in phospholipids (Linres nd Henderson, 1991). In muscle, liver nd gills, the (low n-3 HUFA content) induced higher levels of oleic (18:1n-9), linoleic (18:2n- 6) nd linolenic (18:3n-3) cids in polr nd neutrl lipids thn the HD nd lower levels of n-3 HUFA. However, influence ws prticulrly low in brin. It ws lso reltively low on eyes, tht contined high DHA levels (not influenced by tretments). Then, even when fed below the n-3 HUFA requirement (Sklli nd Robin, 24), fish tend to mintin functionlity of neurl nd visul tissues, where DHA is of key importnce (Luritzen et l., 21). On the sme species Pglirni et l. (1986) obtined noticeble influence of vegetl oils on brin ftty cid content with mrked decrese of DHA in,

6 6 but with s more depleted in HUFA thn here. DHA is known to be present t high levels in brin, nd t very high levels in eyes of vrious fish species (Tocher nd Hrvie, 1988; Mourente, 23). In this experiment, high levels of sturtes were observed in tissues compred to s. Both plmitic (16:) nd steric (18:) cids were high especilly in liver. These ftty cids were lso present t high levels in polr lipids, s clssiclly ssocited with HUFAs in dicyl phospholipids of fish (Bell nd Dick, 1991). The high levels of tissue sturtes, my result from lipogenic ctivity (Dis et l., 1998). This finding is in greement with the higher feed intke nd feed conversion rtio of fish observed t 29 C thn t 22 C (Person-Le Ruyet et l., 24). In se bss juveniles, 29 C is temperture close to the upper tolernce limit, the temperture for mximum growth being 26 C (Person-Le Ruyet et l., 24b). Despite this pprent lipogenic ctivity reinforced t 29 C, lipid liver content tended to be lower t 29 thn t 22 C, ftty cids produced in liver being minly exported to other tissues; the min site of lipid storge in this species is periviscerl dipose tissue (Dis et l., 1998). The higher level of 18:3n-6 in fish fed compred to HD, strongly suggests it ws produced by desturtion of 18:2n-6, since 18:3n-6 content in ws very low nd 3 times lower thn in HD. Other observtions support this finding: i) the differences in 18:3n-6 between ry groups ws mrked in liver, tissue known to be involved in desturtion-elongtion ctivity; ii) in gill, high levels of 18:3n-6 were ssocited with high levels of 2:4n-6 (AA). Similr bio-conversion cpcity stimulted by HUFA ry deficiency hve been shown in freshwter nd mrine fish species (Buzzi et l., 1996; Seiliez et l., 21, 23). In gill the 18:3n-6 contents were lower t 29 thn t 22 C, corresponding to reduced desturtion with n increse in temperture (Tocher nd Srgent, 199). It my lso explin the interction observed between nd temperture on 2:4n-6 contents in gills, considering AA contents were both ffected by ry levels nd by desturtion process. The highest level of AA observed in gill, underlines the importnce of this FA for gill function (Henderson et l., 1985; nonymous, 1987; Bell et l., 1994 ). A desturtion-elongtion process, enlightened by 18:3n-6 contents, my lso contribute to mintin reltively high AA levels in phospholipid of fish fed despite the low AA content in this. However AA contents of ll tissues remined significntly lower in groups thn in HD groups. In this experiment, 18:3n-6 let to demonstrte desturtion ctivity more clerly thn FA of the n-3 series. Diets contined higher levels of intermedites nd finl products with lower precursors in n-3 thn in n-6 FA. Similr biotrnsformtions my lso occurred in the n-3 series but cnnot be evidenced. The effect of temperture on fish ftty cid composition ws less documented minly regrding mrine fish species. In Europen se bss juveniles, dpttion to low temperture did not induce mjor chnge in polr lipid ftty cid composition of liver nd hert (Trigri et l., 1992) or gill nd kidney (Ventrell et l., 1993). The present study let to observe severl significnt influences of temperture on polr lipids of vrious tissues s previously shown on whole body ftty cid composition (Person-Le Ruyet et l., 24). In slmonids, therml response corresponds to higher proportion of unsturted FA with low temperture (Hzel et l. 1992; Clbretti et l., 23). Similr effects of temperture were observed in the present study, with lower sturtes nd 18:1n-9 versus higher n-3 FA

7 7 contents t 22 thn t 29 C. Trditionlly, higher levels of n-3hufa (DHA + EPA) in fish t low temperture ws previously interpreted s wy to mintin membrne fluidity (homeoviscous regultion). This notion ws denied by Srgent et l. (22, pp ) who consider the intrinsic structure of 22:6n-3 is inherently resistnt to temperture chnge, its bundnce in fish dicyl phospholipids insuring membrne functionlity independently to environmentl vribles. Another determinnt of membrne fluidity cn be the rtio of monounsturted versus sturted ftty cids in dicyl phospholipids (Wodtke nd Cossins, 1991). However present results did not let to observe such regultion s both sturtes nd monoenes were higher t 29 thn t 22 C. According to Poon et l. (1981), cution should be exercised in inferring chnges in membrne fluidity bsed on lipid modultion of biologicl membrnes. Differences in ftty cid composition relted to temperture depended lso on the tissue exmined. As shown in by distnce coefficients, brin, gill nd muscle were more ffected by temperture thn liver nd eyes. The effect of temperture ws different in gill thn in brin nd muscle, concerning 18: nd n-3 FA. Mjor disprities between tissues with regrd to the influence of temperture on FA composition occurred in the n-6 series: t 29 C 18:2n-6 in ws lower in muscle but higher in gill compred to 22 C, nd 2:4n-6 in ws higher in muscle nd in gills (only for HD) but lower in brin. Differences in tissues composition should result from tissues specificities in ftty cid ffinity nd metbolism: high DHA versus low n-6 in eyes nd brin; high AA contents in gills compred to other tissues. In this experiment, the more generl temperture influence ws encountered in sturtes (16:, 18:) nd 18:1n-9, for ll tissues composition. These ftty cids re those produced by lipogenic ctivity, minly in liver. Then prt of observed differences could reflect n effect of temperture on fish energy metbolism, modifying vilble ftty cid pool. Inside this pool, ftty cids nd minly HUFAs were selectively retined for polr lipids. A more noticeble influence of temperture on thn on tissue content suggest some relevnce in terms of therml dpttion. As feed intke ws higher t 29 C thn t 22 C, higher n-3 HUFA quntities were ingested t 29 thn t 22 C. These quntities ingested filed to ttenute lck of n-3 HUFA in fed fish. In opposite, significnt effect of temperture occurring in vrious tissues corresponded to lower n-3 HUFA content t 29 thn t 22 C, with the deficient HUFA s well s with the sufficient. Muscle FA content ws more similr to whole body FA content thn other tissues. This seems logicl s muscle represent the mjor prt of fish. However the totl lipid content ws low in muscle compre to whole body. In this species most of re stored in periviscerl ft. Then muscle represent the mjor quntity of rther thn on whole body bsis. Within ech ry groups DHA nd EPA levels were reltively similr in of ll tissues nlysed, except liver, while more specificities between tissues were observed in content. Comprison of tissues content let to show different responses to temperture nd ry HUFA between tissues. These responses should depend on the ctive roles of HUFA for ech tissue functionlity nd on the cpcity of ech tissue to regulte its content in order to mintin this functionlity. Among the tissues nlysed, neurl tissue (brin nd eyes) hd the highest regultion cpcity inducing low influence on their content. References Alexis, M.N., Fish mel nd fish oil replcers in Mediterrnen mrine fish s. Ch. Options Mediterr. 22,

8 8 Anonymus, Potentil importnce of rchidonte in gill function of mrine fish. Nutrition Reviews 45 (2), Bell, J.G., Tocher, D.R., McDonld, F.M., Srgent, J.R., Effects of s rich in linoleic (18 :2n-6) cid nd linolenic (18 :3n-3) cids on the growth, lipid clss nd ftty cid compositions nd eicosnoid production in juvenile turbot (Scophthlmus mximus). Fish Physiol. Biochem. 13, Bell, J.G., McEvoy, J., Tocher, D.R., McGhee, F., Cmpbell, P.J., Srgent, J.R., 21. Replcement of fish oil with rpeseed oil in s of tlntic slmon (Slmo slr) ffects tissue lipid compositions nd heptocyte ftty cid metbolism. J. Nutr. 131, Bell, M.V., Dick, J.R., Moleculr species composition of the mjor dicyl glycerophospholipids from muscle liver, retin nd brin of cod (Gdus morhu). Lipids 26, Buzzi, M., Henderson, R.J., Srgent, J.R., The desturtion nd elongtion of linoleic cid nd eicospentenoic cid by heptocytes nd liver microsomes from rinbow trout (Oncorhynchus mykiss) fed s contining fish oil or olive oil. Biochim. Biophys. Act 1299, Clbretti, A., Cteni, F., Procid, G., Fvretto, L.G., 23. Influence of environmentl temperture on composition of lipids in edible flesh of rinbow trout (Oncorhynchus mykiss). J. Sci. Food Agric. 83, Dis, J., Alvrez, M.J., Diez, A., Corrze, G., Butist, J.M., Kushik, S.J., Regultion of heptic lipogenesis by ry protein/energy in juvenile Europen sebss (Dicentrrchus lbrx). Aquculture 161, Frks, T., Dey, I., Bud, C.S., Hlver, J.E., Role of phospholipid moleculr species in mintining lipid membrne structure in response to temperture. Biophys. Chem. 5, Folch, J., Lees, M., Stnley, G.H.S., A simple methods for the isoltion nd purifiction of totl lipids from niml tissues. J. Biol. Chem. 226, Hzel, J.R., McKinley, S.J., Willims, E.E., Therml dpttion in biologicl membrnes: Intercting effects of temperture nd ph. J. Comp. Physiol. B162, , Henderson, R.J., Bell, M.V., Srgent, J.R., The conversion of polyunsturted ftty cids to prostglndins by tissue homogentes of the turbot Scophthlmus mximus L. J. Exp. Mr. Biol. Ecol. 85, Izquierdo, M.S., Obch, A., Arntzmendi, L., Montero, D., Robin, L., Rosenlund, G., 23. Dietry lipid sources for sebrem nd sebss: growth performnce, tissue composition nd flesh qulity. Aqucult. Nutr. 9, Jobling, M., 21. Nutrient prtitioning nd the influence of feed composition on body composition. In: Houlihn, D., Boujrd, T., Jobling, M. (Eds.). Food Intke in Fish. Blckwell, Oxford, pp Juned, P., Rocquelin, G., Rpid nd convenient seprtion of phospholipids nd non-phosphorus lipids from rt hert using silic crtridges. Lipids 2, 4-41.

9 Knzw, A., Teshim, S.I., Ono, K., Reltionship between essentil ftty cid requirements of qutic nimls nd the cpcity for bioconversion of linolenic cid to highly unsturted ftty cids. Comp. Biochem. Physiol. 63B, Luritzen, L., Hnsen, H.S., Jorgensen, M.H., Michelsen, K.F., 21. The essentility of n- 3 ftty cids in reltion to development nd function of the brin nd retin. Prog. Lipid Res. 4, Linres, F., Henderson, R.J., Incorportion of 14C-lbelled polyunsturted ftty cids by juvenile turbot, Scophthlmus mximus (L.) in vivo. J. Fish Biol. 38, McIntire, C.D., Tinsley, I.J., Lowry, R.R., Ftty cids in lotic periphyton: nother mesure of community structure. J. Phycol. 5, Morrison, W., Smith, L., Preprtion of ftty cid methyl esters nd dimethylcetls from lipids with boron fluoride-methnol. J. Lipid Res. 5, Mourente, G., 23. Accumultion of DHA (docoshexneoic cid, 22:6n-3) in lrvl nd juvenile fish brin. In: Browmn, H.I., Skiftesvik, A.B. (Eds.), The Big Fish Bng, Proceedings of the 26th Ann. Lrvl Fish Conference. Institute of Mrine Reserch, Bergen Norwy, pp Pglirni, A., Pirini, M., Trigri, G., Ventrell, V., Effects of s contining different oils on brin ftty cid composition in se bss (Dicentrrchus lbrx). Comp. Biochem. Physiol. 83 B, Person-Le Ruyet, J., Sklli, A., Dulu, B., Le Byon, N., Le Delliou, H., Robin, J.H., 24. Does ry n-3 highly unsturted ftty cids level influence the Europen se bss (Dicentrrchus lbrx)cpcity to dpt to high temperture? Aquculture 242, Person-Le Ruyet, J., Mhé, K., Le Byon, N., Le Delliou, H., 24b. Effects of temperture on growth nd metbolism in Mediterrnen popultion of Europen se bss, Dicentrrchus lbrx. Aquculture 237, Poon, R., Richrds, J.M., Clrk, W.R., The reltionship between plsm membrne lipid composition nd physicl-chemicl properties. II Effect of phospholipid ftty cid modultion on plsm membrne physicl properties nd enzymtic ctivities. Biochem. Biophys. Act 649, Regost, C., Arzel, J., Robin, J., Rosenlund, G., Kushik, S.J., 23. Totl replcement of fish oil by soyben oil or linseed oil with return to fish oil in turbot (Psett mxim). 1 growth performnce, flesh ftty cid profile, nd lipid metbolism. Aquculture 217, Robertson, J.C., Hzel, J.R., Membrnes constrints to physiologicl function t different tempertures; does cholesterol stbilize membrnes t elevted tempertures? In: Woods, C.M., McDonld, D.G. (Eds.), Society for experimentl biology, seminr series 61: Globl wrming: implictions for freshwter nd mrine fish. Cmbridge University Press, pp Srgent, J.R., Tocher, D.R., Bell, J.G., 22. The lipids. In: Hlver, J.E., Hrdy, R.W. (Eds.). Fish Nutrition. Acdemic Press, New York, pp Seiliez, I., Pnsert, S., Kushik, S., Bergot, P., 21. Cloning, tissue distribution nd nutritionl regultion of Δ6-desturse-like enzyme in rinbow trout. Comp. Biochem. Physiol. 13 B, Seiliez, I., Pnsert, S., Corrze, G., Kushik, S., Bergot, P., 23. Cloning nd nutritionl regultion of Δ6-desturse-like enzyme in the mrine teleost gilthed sebrem (Sprus urt). Comp. Biochem. Physiol. 135 B,

10 Sklli, A., Robin, J.H., 24. Requirement of n-3 long chin polyunsturted ftty cids for Europen se bss (Dicentrrchus lbrx) juveniles: growth nd ftty cid composition. Aquculture 24, Sturnes, M., Rinuzzo, J.R., Sigholt, T., Joergensen, L., Acclimtion of Atlntic cod (Gdus morhu) to cold wter. Stress response, osmoregultion, gill lipid composition nd gill N-K-ATPse ctivity. Comp. Biochem. Physiol. 19A, Tocher, D.R., 23. Metbolism nd functions of lipids nd ftty cids in teleost fish. Rev. Fish. Sci. 11, Tocher, D.R., Hrvie, D.G., Ftty cid compositions of the mjor phosphoglycerides from fish neurl tissues; (n-3) nd (n-6) polyunsturted ftty cids in rinbow trout (Slmo grdneri) nd cod (Gdus moruhu) brins nd retins. Fish Physiol. Biochem. 5, Tocher, D.R., Srgent, J.R., 199. Effect of temperture on the incorportion into phospholipid clsses nd metbolism vi desturtion nd elongtion of n-3 nd n-6 polyunsturted ftty cids in fish cells in culture. Lipids 25, Trigri, G., Pirini, M., Ventrell, V., Pglirni, A., Trombetti, F., Borgtti, A.R., Lipid composition nd mitochondril respirtion in wrm- nd cold-cclimted se bss (Dicentrrchus lbrx L.). Lipids 27, Ventrell, V., Pglirni, A., Pirini, M., Trigri, G., Trombetti, F., Borgtti, A.R., Lipid composition nd microsoml ATPse ctivities in gills nd kidneys of wrm nd cold cclimted se bss (Dicentrrchus lbrx L.). Fish Physiol. Biochem. 12, Wodtke, E., Cossins, A.R., Rpid cold-induced chnges of membrne order nd delt-9-desturse ctivity in endoplsmic reticulum of crp liver: A time-course study of therml dpttion. Biochim. Biophys. Act 164,

11 11 Tble 1. Totl lipid content of the vrious tissues (% wet weight bsis ± SE), dt on whole body lipid content reported from Person-Le Ruyet et l. (24). Tretment 2 wy ANOVA 22-HD HD 29- Diet T D. Whole body 16.3 ± ± ± ±.6 ns ns ns T Muscle 3.±.2 3.9± ± ±.1 (ns) (ns) (ns) Liver 4.1 ± ± ±1.6b 35.4 ±2.2b ns * *** Gills 3.2±.2 3.5±.2 3.3±.2 3.3±.2 ns ns ns Eyes 1.1±.6 8.5±.6 9.5±.4 8.6±.6 ns ns ns Brin 9.4±.5 9.7±.2 9.7±.4 1.4±1. ns ns ns Vlues re mens ± stndrd error (n=3). 2 wy ANOVA sttisticl results: * P <.5; *** P <.1; ns: not significnt; (ns) unequl vrinces, rnk test nlysis. In cse of significnt interction mens hving different superscript letter in the sme rw re significntly different (Newmnn-Keuls test, P<.5) Tble 2. Comprison of ftty cid profiles by McIntyre distnce coefficient (D). Muscle Liver Gills Eyes Brin Polr lipids Among tempertures within : 22-HD/29-HD / Among s within temperture: 22-HD/ HD/ Neutrl lipids Among tempertures within : 22-HD/29-HD / Among s within temperture 22-HD/ HD/

12 12 Tble 3. Crossed comprison between tissues : Distnce (McIntyre distnce coefficient : D) between the ech tissues nd whole body (WB) ftty cid pttern, in neutrl lipids ( upper right prt) nd polr lipids (lower left prt) were clculted for ech tretment, men vlues (±ES between tretments) WB Muscle Liver Gills Eyes Brin WB 1.6 (.3) 14. (.9) 4.3 (.4) 2.1 (.3) 15.9 (.5) Muscle 7. (.4) 14.1 (1.2) 3.3 (.7) 1.7 (.1) 15.2 (1.1) Liver 13.2 (1.4) 9.9 (2.7) 15.2 (1.8) 15.1 (1.3) 22.8 (1.4) Gills 6.7 (.9) 1.2 (2.4) 14.2 (2.5) 3.5 (.7) 12.6 (1.4) Eyes 26. (1.7) 21.8 (3.4) 15.1 (1.5) 27.5 (2.4) 15.3 (1.2) Brin 12.4 (.4) 14.1 (1.6) 18.1 (2.9) 15.1 (1.2) 24.5 (2.1) Polr lipids N L

13 13 16: HD 1 22-HD HD 29- d** Fig : HD 18: 1 n HD 2 i* d c b d** d* * i*** c d b i* d b c 1 Fig.1- Chnges in composition of min sturted nd monounsturted ftty cids (16:, 18: nd 18:1n-9, respectively) of nd frctions in whole body (WB) nd different tissues studied. Mens re given with stndrd error (n = 3 replictes). Letters t nd d indicte significnt sttisticl differences between temperture nd s: *, P <.5; **, P <.1 nd ***, P <.1. = no significnt difference (p.5). Letter i indictes interction between the two fctors, brs with different superscript letters re significntly different (Newmnn-Keuls test, P<.5).

14 14 18: 2 n HD HD HD 29- * Fig. 2 18: 3 n-6 1,4 1,2 1,,8,6,4,2, HD d** d** d** 2: 4 n-6 7 i* c b d* 1 HD Fig.2- Chnges in composition of 18:2n-6, 18:3n-6 nd 2:4n-6 of nd frctions in WB nd different tissues studied. Mens re given with stndrd error (n = 3 replictes). Letters t nd d indicte significnt sttisticl differences between temperture nd s: *, P <.5; **, P <.1 nd ***, P <.1. = no significnt difference (p.5). Letter i indictes interction between the two fctors, brs with different superscript letters re significntly different (Newmnn-Keuls test, P<.5).

15 15 18: 3 n-3 22-HD HD 29- Fig HD * * d* 2: 5 n HD i* d c b i*** c c b 22: 6 n HD * d** d* * i* bbb i* c c b Fig. 3- Chnges in composition of 18:3n-3, 2:5n-3 nd 22:6n-3 of nd frctions in WB nd different tissues studied. Mens re given with stndrd error (n = 3 replictes). Letters t nd d indicte significnt sttisticl differences between temperture nd s: *, P <.5; **, P <.1 nd ***, P <.1. = no significnt difference (p.5). Letter i indictes interction between the two fctors, brs with different superscript letters re significntly different (Newmnn-Keuls test, P<.5).