Temperature adaptation in two bivalve species from different thermal habitats: energetics and remodelling of membrane lipids

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1 2999 The Journl of Experimentl Biology 2, Pulishe y The Compny of Biologists 2007 oi:.1242/je Temperture pttion in two ivlve speies from ifferent therml hitts: energetis n remoelling of memrne lipis Frie Pernet 1, *, Réjen Tremly 2, Lu Comeu 3 n Helg Guerley 4 1 Institut e Reherhe sur les Zones Côtières, 232B rue e l Église, Shippgn, Nouveu-Brunswik, E8S 1J2, Cn, 2 Institut es Sienes e l Mer, 3 llée es Ursulines, Rimouski, Quée, G5L 3A1, Cn, 3 Deprtment of Fisheries n Oens, Siene Brnh, Gulf Fisheries Centre, PO Box 5030, Monton, New Brunswik, E1C 9B6, Cn n 4 Déprtement e Biologie, Université Lvl, Quée, Quée, G1K 7P4, Cn *Author for orresponene (e-mil: fpernet@ums.) Aepte 18 June 2007 We ompre lipi ynmis n the physiologil responses of lue mussels Mytilus eulis, ol-pte speies, n oysters Crssostre virgini, wrmer-wter speies, uring simulte overwintering n pssge to spring onitions. To simulte overwintering, nimls were hel t 0 C, 4 C n 9 C for 3 months n then grully rought to n mintine t 20 C for 5 weeks to simulte spring summer onitions. Chnges in lipi lss n ftty i omposition were relte to lerne rte n oxygen onsumption. We foun mjor ifferenes etween speies in triglyerie (TAG) metolism uring overwintering. Mussels use igestive gln TAG stores for energy metolism or reproutive proesses uring the winter, wheres oysters i not umulte lrge TAG stores prior to overwintering. Mussel TAG ontine high levels of ompre to levels in oysters n in the iet. This my help to ountert the effet of low temperture y reuing the melting point of TAG n thus inresing the vilility of storge fts t low temperture. Mussels seeme etter le to moilise n thn other ftty is. We lso foun tht oth ivlves unerwent mjor remoelling of memrne phospholipis. The unsturtion Summry inex erese in the gills n igestive glns of oth speies uring the erly stges of wrming, priniplly ue to ereses in n. In igestive glns, the unsturtion inex i not inrese with eresing temperture eyon threshol ttine t 9 C wheres perfet negtive reltionship ws oserve in gills, s preite y homeovisous pttion. The presene of igestive enzymes n is in the igestive gln miroenvironment my le to speifi requirements for memrne stility. Tht oysters h lower metoli rtes thn mussels oinies with lower unsturtion inex of their lipis, s preite y Hulert s theory of memrnes s metoli pemkers. Both speies showe inrese levels in their tissues s temperture rose, suggesting n inresing vilility of this ftty i for eiosnoi iosynthesis uring stress responses. The ontrst etween the speies in TAG ynmis n the similrity of their phospholipi remoelling emphsises the essentil funtionl role of memrne phospholipi struture n the ontrsting use of TAG y oysters n mussels uring overwintering. Key wors: lipi, ftty i, triglyerie, phospholipi, homeovisous pttion, mollus, temperture pttion, limtion. Introution Temperture, ue to its impt upon ll levels of iologil orgnistion, is ruil eterminnt of the iogeogrphy n physiologil hrteristis of poikilotherms. Inee, temperture lters the veloity of hemil n enzymti retions, rtes of iffusion, memrne fluiity n protein struture (Hohhk n Somero, 2002). The therml sensitivity of memrne proesses is ue to the strong effet of temperture on the physil properties of memrne lipis, whih in turn hve mjor influene on ssoite proteins. A erese in temperture usully reues memrne fluiity, whih n le to memrne ysfuntion. Poikilotherms usully ountert this temperture effet y remoelling memrne lipis, proess known s homeovisous pttion (HVA), vi hnges in phospholipi hegroups, ftty i omposition n holesterol ontent tht ompenste for the effet of temperture on memrne struture (Sinensky, 1974; Hzel, 1995). Mny intertil orgnisms, whih ommonly withstn vritions in temperture of C on ily sis n enounter even wier therml rnges on sesonl sis, re le to regulte memrne fluiity in response to therml hnge. For exmple, the mussel Mytilus liforninus exhiits strong sesonl vritions in memrne fluiity tht re onsistent with HVA (Willims n Somero, 1996). Similrly, memrne fluiity in gill phospholipis of the se sllop Plopeten mgellnius is positively orrelte with n negtively orrelte

2 3000 F. Pernet n others with limtion temperture, presumly helping to mintin memrne funtion t low tempertures (Hll et l., 2002). Finlly, mjor remoelling of lipis onsistent with HVA ours in hr lms Merenri merenri expose to grul ooling from ~24 C to 0 C n limtistion t <0 C (Pernet et l., 2006). Blue mussels Mytilus eulis n estern oysters Crssostre virgini re two eurytherml suspension-feeing ivlves wiely istriute long the est ost of North Ameri. M. eulis rnges from Bffin Isln to North Crolin (Gosling, 1992; Fisk et l., 2003), wheres C. virgini is minly foun in the southern prt of North Ameri, from the Gulf of St Lwrene to the Gulf of Mexio (Gltsoff, 1964). In the Gulf of St Lwrene, C. virgini is restrite to wrm shllow ys n esturies wheres M. eulis is foun lmost everywhere. This reflets the therml preferenes of the two speies: they oth tolerte minimum temperture of 2 C ut mximl n optiml tempertures for M. eulis re muh lower (27 C n 20 C respetively) thn those of C. virgini (36 C n C, respetively) (Thompson n Newell, 1985; Shumwy, 1996). Therefore, low overwintering tempertures hve een suggeste s potentil explntion for spori overwintering mortlities of C. virgini in Atlnti Cn (Lvoie, 1995), wheres tempertures >20 C in this re oinie with summer mortlity in ertin popultions of M. eulis (Myrn n Gureult, 1995; Tremly et l., 1998). In the Gulf of St Lwrene, where mortlity of C. virgini osionlly ours, 0 C is typil overwintering temperture. No mortlity hs een reporte long the entrl Atlnti osts, where 4 C is typil overwintering temperture. The southern Atlnti ost from Chespeke By to South Crolin, whih hs overwintering tempertures of out 9 C, supports the highest nnul growth rte of C. virgini (Shumwy, 1996). Oysters re quiesent t 0 C, egin to fee t 4 C, n strt to grow t 9 C (Loosnoff, 1958). In this stuy, mussels n oysters from the Gulf of St Lwrene were overwintere t 0 C, 4 C n 9 C in the lortory for 3 months. The temperture ws grully rise to n hel t 20 C for 5 weeks to simulte spring summer onitions in the Gulf of St Lwrene. Animls were regulrly smple for lipi nlysis n physiologil mesurements. We fousse our stuy on hnges in lipi lss n ftty i omposition of igestive glns n gills in reltion to lerne rtes n oxygen onsumption. Digestive glns re the min site of extr- n intrellulr igestion; they typilly store lrge mounts of neutrl lipis. In ontrst, gills re involve in prtile proessing n gs exhnge, n gill lipis onsist minly of sterols n phospholipis. We preite tht (1) ivlves woul ountert therml effets on memrne fluiity y remoelling memrne lipis s stipulte y the HVA, (2) memrne lipis of M. eulis, speies pte to hrsh Cnin winters, woul e more unsturte thn those of C. virgini, speies tht is less tolernt to ol, n (3) interspeifi ifferenes in metoli rtes woul e relte to memrne unsturtion, s preite y Hulert s theory of memrnes s metoli pemkers (Hulert n Else, 1999; Hulert n Else, 2005). Unlike stuies tht only fous on the therml effets on memrne lipis, we lso exmine the ynmis of storge lipis in reltion to overwintering temperture. In mrine ivlves, lipis re primrily store s triglyerie (TAG) roplets in igestive glns (Giese, 1966), n the synthesis, storge n use of TAG usully show pronoune sesonl yles: TAG re sequestere uring perios of high foo vilility in lte summer n fll, n re susequently use for mintenne metolism uring perios of reue feeing in the winter n for the initition of gmetogenesis (De Zwn n Mthieu, 1992; Thompson et l., 1996). Beuse TAG re not linke to memrne funtion, they seem less likely thn memrne lipis to hnge with temperture. However, TAG n only e moilise if they re in flui stte (Flornt, 1998), ft tht poikilotherms living t low tempertures must ountert if lipi moilistion is to ontinue. Mterils n methos Animls Ault mussels Mytilus eulis (Linneus 1758) n oysters Crssostre virgini (Gmelin 1791) were otine from two jent quulture sites: Hvre e Shippgn (47 46 N; W, lese NB3D, mussel) n Bie e Misou (47 52 N; W, lese NB3K, oyster), Gulf of St Lwrene, NB, Cn. Wter temperture t the time of olletion ws 8 C n slinity ws 29. Animls were trnsporte on 15 Novemer 2004 to the Costl Zone Reserh Institute (CZRI, Shippgn, NB, Cn). Upon rrivl, 2 nimls of eh speies (men shell length=61.9±1.9 mm for mussels n 79.8±3.5 mm for oysters) were numere with ee tgs n limte to lortory onitions for 63 ys prior to strting the experiment. Animls were eqully istriute in two 300-l tnks with light ertion; the slinity ws 29, the nturl photoperio ws followe, n the temperture ws mintine t 9 C. The two speies were mintine together in these tnks over the entire experiment. Sewter ws irulte through 1/2 or 1/5 HP externl hillers (J&L Aqutis, Burny, BC, Cn) to mintin the require temperture; the wter temperture in eh tnk ws ontrolle seprtely. Animls were fe mixe suspension of Chetoeros muelleri (CHGRA) n Isohrysis gln (TISO). These two lgl speies showe equte hrteristis s foo for severl ivlve speies n omplementry profiles in essentil ftty is (Pernet et l., 2003). Animls were fe every 2 ys t 20 3 ells ml 1 (50:50 of eh lgl speies y ell numer). When sewter temperture ws rise on the 12 April (see next setion), the lgl onentrtion offere to mussels n oysters ws inrese from 20 to 50 3 ells ml 1. The ily sh-free ry mss (AFDM) of the lgl rtion vrie from % of the men AFDM of one niml uring the overwintering perio n inrese to % uring the spring simultion perio. Vrition in foo rtion within eh experimentl perio ws ue to the suessive removl of nimls use for lipi nlyses. Mirolgl stoks were otine from the Centre for Culture of Mrine Phytoplnkton (West Boothy Hror, ME, USA). Alge were thulture in 20-l roys (Pernet et l., 2003) n smple three times uring the experiment for ftty i etermintion (Tle 1). Temperture ws monitore every hour in eh tnk throughout the experiment with sumersile Vemo 8-it Minilog-TR t loggers (Sh By, NS, Cn).

3 Temperture pttion in two ivlve speies 3001 Tle 1. Ftty i omposition of mirolgl speies use in iet fe to mussels n oysters Chetoeros grilis Isohrysis gln Mixe iet (CHGRA:TISO, Vrile (CHGRA) (TISO) 50:50 y ell numer) Ftty i omposition (mol %) 7.3± ± ± ± ±0.6.7± ± ± ±0.2 SFA 19.4± ± ± ± ± ± ± ± ± ± ± ±0.1 MUFA 33.9± ± ±1.1 16:2n-6 0.9± ± ±0.4 16:2n-4 4.1± ± ± ±0.5.5± ±0.6 16:3n-4.4± ± ±0.8 18:3n-6 0.7± ± ± ± ± ± ±0.1.7± ± ± ± ± ± ±0.4.8±0.6 22:5n-6 0.2± ± ± ±0.2.4± ±0.3 PUFA 46.7± ± ±0.9 Unsturtion inex 221.8± ± ±4.6 Totl ftty is (nmol 6 ells) 5.6± ± ±1.0 Ftty i omposition is given s mol % of totl ftty i. The iohemil omposition of the mixe iet ws lulte from tht of its iniviul onstituents. All vlues represent the men ± s.., N=3 replites. Only ftty is ontriuting >1% in t lest one speies re reporte. Experimentl esign Mussels n oysters were rnomly ivie etween the three experimentl tretments in uplite tnks on 17 Jnury 2005: one group ws mintine t 9 C n two groups experiene grul temperture erese (~0.5 C/y), one to 4 C n one to 0 C (Fig. 1A). Thus, ll nimls rehe the esire overwintering temperture y 31 Jnury, fter whih these tempertures were mintine for 12 weeks. Mussels n oysters tht h overwintere t 9 C, 4 C n 0 C were then wrme y ~1 C/y strting on 19, 15 n 12 April, respetively, to simulte spring summer onitions. A 1 C/y inrese is representtive of fiel onitions in the spring (Brielj et l., in press). When nimls ttine 20 C on 4 My, they were hel t this temperture for 5 weeks, until 8 June. Digestive glns n gills were smple on 17 Jnury, efore pplying the overwintering tempertures; on 31 Jnury, fter ttining the overwintering temperture; on 14 Ferury, fter short-term winter limtion; on 12 April, whih reflets longterm winter limtion; on 4 My, fter ttining the summer temperture; n on 8 June, whih reflets long-term summer limtion (Fig. 1). Physiologil mesurements Six mussels n six oysters per tnk were use for physiologil mesurements. Physiologil rtes were Temperture ( C) T C Tnk Speies Tissue Dte 31-Jn Oyster DG 14-Fe Gills 12-Apr Mussel 04-My 08-Jun Jn Fe Mr Apr My Jun Jul Time Fig. 1. (A) Experimentl protool for low temperture n spring summer simultion experiment with mussel Mytilus eulis n oyster Crssostre virgini. Cirles inite tes of lerne rte mesurements n lipi smpling. Routine V O 2 ws mesure on 12 April, 4 My n 31 My n V O 2 min ws mesure on 8 June fter strving the nimls use for routine V O 2. (B) Shemti of the splitsplit plot experimentl esign. T C=9, 4 or 0. See Mterils n methos for further etils. B A

4 3002 F. Pernet n others iniviully mesure in 600 ml hmers; nimls were hel in their metoli hmers for 1 h efore mesurements egn. Six hmers were use simultneously, whih llowe us to mesure five nimls n one ontrol (empty shell) t time. Animls tht remine lose in the hmer were exlue from physiologil nlysis. On 12 April, mussels n oysters for physiologil mesurements were srifie for ry mss etermintion, whih ws mesure fter rying t 70 C for 72 h; ry msses were use to lulte mss-stnrise physiologil rtes for the overwintering perio. Physiologil rtes were mesure on other nimls uring the spring summer simultion, whih ourre etween 12 April n 8 June. Agin on 8 June, mussels n oysters use for physiologil mesurements were srifie for ry mss etermintion to normlise physiologil rtes for the spring summer simultion perio. Physiologil rtes were onverte to mss-speifi rtes for nimls of 1 g ry mss using pproprite weight exponent (=0.7) in the llometri equtions. The llometri eqution is Y = X, (1) where Y=physiologil rte, X=oy mss n n re fitte prmeters. Clerne rte Clerne rte (CR) is efine s the volume of wter lere of suspene prtiles per unit time n iomss (Wiows n Johnson, 1988). The CR ws etermine on 17 n 31 Jnury, 14 Ferury, 12 April, 4 My n 8 June for eh temperture tretment. Six nimls per tnk of eh speies were use for CR mesurement (12 nimls per overwintering temperture n speies). Animls were remove from their holing tnk n mintine iniviully in their experimentl hmers in whih the suspension ws mixe vi gentle ertion from the ottom of the hmer. Before eginning prtile onentrtion mesurements, nimls were left unisture for t lest 1 h to llow their vlves to open n feeing to egin. The CR ws etermine using stti system in whih the erese in prtile onentrtion ws monitore insie the hmers. At the eginning of the inution perio, iniviul nimls were provie TISO t n initil onentrtion of 3 ells ml 1. Foo prtiles were ounte every min for 60 min with n eletroni prtile ounter (Bekmn Coulter-ounter Z1 TM ) fitte with 0- m perture tue. The gretest ifferene etween two onseutive mesurements ws use to lulte CR (Gilek et l., 1992): CR = [ C mx S]V t 1 m 1, (2) where C mx =gretest ifferene in prtile onentrtion etween two onseutive mesurements, S=seimenttion onstnt (0.049 n for mussels n oysters, respetively) lulte s the men exponentil eline in prtile onentrtion in the hmers without niml, V=volume of suspension, t=time etween mesurements n m=ry tissue mss. Oxygen onsumption Routine oxygen onsumption (V O 2, or routine metoli rte) ws etermine t the en of the overwintering perio on 12 April in eh temperture tretment n uring the spring summer simultion on 4 My n 31 My. Minimum oxygen onsumption (V O 2 min, or stnr metoli rte) ws mesure t the en of the experiment on 8 June, fter strving the nimls for 8 ys. Oxygen onsumption for n iniviul niml ws etermine y seling the hmer n mesuring the reution in %O 2 with YSI (5331) polrogrphi nlyzer n eletroe (Yellow Springs, OH, USA). Sewter in the hmer ws mixe with mgneti stirrer. The output signl ws monitore ontinuously on hrt reorer until erese of t lest 20% O 2 ws rehe. Respirtion ws then expresse s ml O 2 h 1 g 1 tissue ry mss. Lipi nlysis Tissue smpling Mussel n oyster gills n igestive glns were smple on 17 n 31 Jnury, 14 Ferury, 12 April, 4 My n 8 June in eh temperture tretment for etermintion of lipi lss n ftty i ompositions. 2 3 mussels n oysters were rnomly smple in eh tnk for etermintion of shell length, tissue AFDM, n lipi omposition. Animls were issete n. 300 mg wet mss of tissue were store in lipi-free mer glss vils with Teflon TM -line ps uner nitrogen in 1 ml ihloromethne t 80 C for lter etermintion of lipi omposition. Alge were filtere on GF/C filters preomuste t 450 C n store in mer glss vils s previously esrie for tissues. Lipi lsses Lipis were extrte following the metho of Folh et l. (Folh et l., 1957), spotte onto S-III Chromros (Itron Lortories In., Tokyo, Jpn), n seprte into liphti hyrorons, sterol n wx esters, ketones, TAG, free ftty is, free ftty lohol, free sterols, iylglyerols, etone moile polr lipis n phospholipis (Prrish, 1999). Chromros were snne y flme ioniztion etetion system (Itrosn Mrk-VI, Itron Lortories In., Tokyo, Jpn) n hromtogrms were nlyze using integrtion softwre (Pek Simple version 3.2, SRI, Torrne, CA, USA). Neutrl n polr lipi seprtion Lipis were seprte into neutrl lipis (inluing triglyeries, free ftty is n sterols) n polr lipis (inluing minly phospholipis n minor mounts of glyolipis) using olumn hromtogrphy on sili gel hyrte with 6% wter s previously esrie (Pernet et l., 2006). Briefly, the 0 mg olumns were preonitione with 1 ml of methnol n 1 ml of hloroform. Smples (200 l) of lipi orresponing to ~1 mg of lipi were loe onto the soliphse extrtion olumn. Smples were gently rwn into the soli phse with slight vuum. Columns were wshe with 1 ml hloroform methnol (98:2 v/v) to elute neutrl lipis followe y 5 ml of methnol to elute polr lipis. The frtions elute were ollete in 7 ml tues positione in vuum mnifol pprtus. The vuum ws juste to generte flow rte of ~1 ml min 1. Ftty is Ftty i methyl esters (FAME) from neutrl n polr lipis

5 Temperture pttion in two ivlve speies 3003 were prepre using 12% BF 3 in CH 3 OH following the Amerin Oil Chemists Soiety metho (AOCS, 1989). FAME were run on Vrin CP3900 gs hromtogrph equippe with ZB-wx fuse-sili pillry olumn (20 m 0.18 mm i m film thikness; Supelo, Bellfonte, PA, USA). Helium ws use s the rrier gs (flow veloity: 1 ml min 1 ). FAME were injete t 250 C t 1: split rtio. The temperture rmp ws 140 C for 0.2 min, followe y n inrese of 40 C min 1 to 170 C, followe y n inrese of 4 C min 1 to 185 C, n finlly y n inrese of 2 C min 1 to 230 C. The etetor ws mintine t 260 C. FAME were ientifie y omprison of retention times with known stnrs (37 omponent FAME Mix, PUFA-3 n menhen oil; Supelo Bellefonte, PA, USA) n quntifie with nonenoi i (19:0) s n internl stnr. Chromtogrms were nlyze using the Glxie hromtogrphy t system (version , Vrin, Mississug, ON, Cn). Sttistil nlyses Anlyses of vrine (ANOVAs) were onute to etermine ifferenes in initil hrteristis of the neutrl n polr lipis etween the two ivlve speies, M. eulis n C. virgini, efore exposure to overwintering tempertures. Onewy ANOVAs were onute to etermine ifferenes etween the two speies in the reltive TAG onentrtion, the ftty i omposition n the unsturtion inex [verge numer of oule ons per yl hin s lulte in Logue et l. (Logue et l., 2000)] of the neutrl lipis in igestive glns. Minor mounts of TAG (<0.5%) were osionlly etete in gills n were not nlyze further. Two-wy ANOVAs were onute to etermine ifferenes in the initil hrteristis of the memrne lipis s funtion of ivlve speies n tissue (gills n igestive glns). Depenent vriles were the phospholipi to sterol rtio, the ftty i omposition n the unsturtion inex of the polr lipis. The unit of replition use in these nlyses ws the rering tnk in whih the nimls were mintine (N=2). Three-wy split-split plot ANOVAs were onute to etermine ifferenes in the physiologil rtes (CR n V O 2) n the neutrl lipis of igestive glns, i.e. the reltive TAG onentrtion, the unsturtion inex n the mjor polyunsturte ftty is (PUFA), nmely, n, s funtion of overwintering temperture, speies n te. The unit of replition ws the tnk in whih the overwintering temperture ws pplie (N=2 for eh temperture). The min plots were overwintering temperture levels (0, 4 n 9 C), suplots were speies levels (mussel n oyster), n su-suplots were smpling tes. Four-wy splitsplit plot ANOVAs were use to etermine ifferenes in the phospholipi to sterol rtio, the unsturtion inex n mjor PUFA of the polr lipi frtion, nmely,,, 22:2 n, s funtion of overwintering temperture, speies, tissue n te (Fig. 1B). Fetures of the four-wy split-split plot experimentl esign were similr to those of the three-wy plot exept tht su-suplots lso inlue tissue levels (gills n igestive glns). Here we use mixe liner moel, whih moels not only the mens of our t ut their vrines n ovrines. The nee for ovrine prmeters rose euse the experimentl units on whih the vriles were mesure were groupe into lusters n repete mesurements were tken on the sme experimentl unit. The repete option ws pplie to the intertion terms Dte n Tissue Dte for the three-wy n four-wy split-split plot experimentl esigns, respetively. These terms were omine with the ovrine struture of the mtrix to tke into ount sptil n temporl epenene (SAS Institute 2002). Where ifferenes were etete, lest-squre mens multiple omprison tests were use to etermine whih mens were signifintly ifferent. Resiuls were sreene for normlity using the expete norml proility plot n further teste using Shpiro Wilk. When neessry, t were log+1 or 1/squre root(x) trnsforme to hieve normlity of resiuls n homogeneity of vrines. Homogeneity of vrine ovrine mtries ws grphilly ssesse. Anlyses were rrie out using SAS (SAS Institute In., Cry, NC, USA). Results Physiologil mesurements Clerne rte (CR) vrie s funtion of temperture speies te (Fig. 2A; P=0.029, F=2.57,.f.=8). Clerne rte (l h 1 g 1 ry mss) Mussel Oyster A 0 C 4 C 9 C, B, 0.8,,e 4,e 0.6,e,e,e,e,e e,f f,g 2 f,g f,g 0.4 f,g f,g f,g f,g f,g f f,g f,g g g f,g e g f,g g e 0 Jn FeMr Apr My Jun FeMr Apr My Jun FeMr Apr My Jun Jul Jn FeMr Apr My Jun Jul Month Fig. 2. (A) Clerne rte (CR) n (B) oxygen onsumption rte of mussels n oysters for stnr niml 1 g in whole oy ry mss (men ± s.e.m, N=2 tnks). CR ws ffete y the omintion of temperture, speies n te wheres oxygen onsumption ws ffete only y the omintion of speies n te. Routine V O 2 ws mesure on 12 April, 4 My n 31 My, n V O 2 min ws mesure on 8 June fter strving the nimls use for routine V O 2. Oxygen onsumption (ml O 2 h 1 g 1 ry mss) Month V O2 V O2 min

6 3004 F. Pernet n others During the overwintering perio, mussels mintine t 4 C n 9 C showe higher CR thn those mintine t 0 C, where the CR ws <0.5 l h 1 g 1 ry mss; CR ws similrly low in overwintering oysters. During the spring summer simultion, the CR of mussels overwintere t 0 C inrese more rpily (y 16 ) thn the CR of mussels overwintere t higher tempertures (~2 ), resulting in CR vlues of ~5.1 l h 1 g 1 ry mss for the three overwintering groups t the en of the experiment (Fig. 2A). As in mussels, the CR of oysters inrese uring the spring summer simultion. However, the CR of mussels ws lwys higher thn tht of oysters, where it vrie etween 1.1 n 3.6 l h 1 g 1 ry mss s funtion of overwintering temperture. Oysters overwintere t 9 C showe greter inrese in CR thn those overwintere t lower tempertures. There ws no effet of overwintering temperture on the oxygen uptke of mussels n oysters (Temperture effet: P=0.904, F=0.,.f.=2; Temperture Speies effet: P=0.809, F=0.23,.f.=2; Temperture Dte effet: P=0.2, F=1.58,.f.=6; Temperture Speies Dte effet: P=0.808, F=0.49,.f.=6). At the en of the overwintering perio, mussels n oysters showe similr rtes of oxygen uptke (Fig. 2B, P=0.534), ut the inrese in V O 2 uring the spring summer simultion ws greter in mussels (4.8 ) thn in oysters, where the V O 2 inrese y only 3.2 (Speies Dte effet: P<0.002, F=11.2,.f.=3). After foo eprivtion, the oxygen uptke of mussels n oysters mintine t 20 C for 5 weeks erese y 2.2 n 1.8 respetively. The V O 2 min of mussels ws 1.6 higher thn tht of oysters t 20 C (Fig. 2B). Storge lipis The initil onentrtion of TAG in the igestive gln, expresse s mg g 1 AFDM, ws 3.7 higher in mussels thn in oysters (Tle 2). TAG levels in mussel igestive glns erese uring the entire stuy, wheres TAG remine lmost onstnt in oysters (Fig. 3, Tle 3). During the entire stuy, mussels use 48.9 mg g 1 AFDM of TAG, whih represente ~80% of the initil TAG level. The unsturtion inex of igestive gln TAG ws initilly 21% higher in mussels thn in oysters n in the iet (Tle 2, Fig. 3). The speies effet on the unsturtion inex ws minly ttriutle to PUFA n more prtiulrly to, whih ws higher in mussels thn in oysters n the foo, respetively. During the experiment, the unsturtion inex of the igestive gln TAG vrie s funtion of speies n te (Fig. 3, Tle 3). In mussels, the unsturtion inex remine elevte uring overwintering n erese only uring the spring summer simultion t 20 C. In oysters, the unsturtion inex inrese 12% uring overwintering n erese until ttining initil vlues when temperture ws rise to 20 C. A stepwise multiple regression moel using groups of ftty is s explntory vriles n the unsturtion inex s the response vrile showe tht the unsturtion inex ws positively orrelte with PUFA (y=5.2 PUFA 21.8; r 2 =0.909, N=60, P<0.001; Fig. 3). A seon regression moel using iniviul PUFA s explntory vriles showe tht vritions in the unsturtion inex were mostly ttriutle to for the two speies (y= ; r 2 =0.734, N=60, P<0.001; Fig. 3) n (y= ; Tle 2. Chrteristis of the neutrl lipis in igestive glns of mussels Mytilus eulis n oysters Crssostre virgini smple on 17 Jnury efore exposure to overwintering tempertures Vrile Mussel Oyster P Triglyerie (mg g 1 AFDM) 57.1± ±5.7 NS Ftty i (mol %) 5.7± ±0.3 ** 15:0 0.6± ±0.2 NS 14.3± ±1.1 * 17:0 0.8± ±0.1 * 2.0± ±0.7 NS SFA 23.5± ±1.7 * 11.5± ±0.6 ** 16:1n-5 0.4± ±0.1 NS 3.2± ±0.1 ** 3.0± ±0.2 NS 0.8± ±0.1 * 1.5± ±0.2 NS 1.0± ±0.1 ** MUFA 22.4± ±1.2 NS 16:2n-4 1.5± ±0.1 ** 16:2n-6 1.0± ±0.1 NS 2.5± ±0.2 NS 20:2NMI i,j 1.3± ±0.4 NS i,j 1.1± ±0.2 ** 2.6± ±0.6 NS 5.8± ±0.8 NS 1.6± ±0.2 NS 19.1± ±0.2 * 21:5n-3 1.6± ±0.2 * 22:5n-6 0.5± ±0.0 NS 22:5n-3 0.7± ±0.1 NS 11.1±0.0.1±0.8 NS PUFA 53.5± ±0.7 * Unsturtion inex 259.9± ±0.7 * All vlues represent the men ± s..; N=3 replite tnks, 2 nimls per tnk were smple. *Signifint ifferenes (P<0.05); **highly signifint ifferenes (P<0.01); NS, no signifint ifferene. Only ftty is ontriuting >1% in t lest one omintion of tretment re reporte. r 2 =0.735, N=60, P<0.001; Fig. 3). The ftty i inrese signifintly y 16% in the igestive gln TAG in the two speies uring overwintering efore eresing until the en of the stuy. The ftty is n in mussel igestive glns erese uring the entire stuy, wheres in oysters, erese only when the temperture ws inrese to 20 C n remine onstnt (Fig. 3, Tle 3). The reltive moilistion of 17 ftty is from mussel n oyster igestive glns ws pproximte s the rtio of their molr perentge (mol %) in initil TAG to tht in TAG t the en of the stuy perio (Fig. 4). A rtio greter thn, equl to, or lower thn unity inites tht the ftty i is moilise more, eqully, or less reily thn the totl TAG-ftty is,

7 Temperture pttion in two ivlve speies 3005 Triglyerie (mg g AFDM 1 ) A,,,, Mussel Oyster Diet (mol %) D Both speies PUFA (mol %) Unsturtion inex Jn B C, Fe Mr Apr Month,,, My, Jun Jul (mol %) (mol %) E F Jn,e,,, Fe Mr,,, Apr My Month e,f f, Jun Jul Fig. 3. Chrteristis of the neutrl lipis in mussel n oyster igestive glns s funtion of time. Dt presente here re (A) the onentrtion of triglyeries, (B) the unsturtion inex n (C F) the mol % of polyunsturte ftty is (PUFA; C), (D), (E) n (F). Vlues re mens ± s.e.m, N=2 6 tnks. The green line inites ietry vlues. The unsturtion inex is lulte s the sum of the molr perentge of eh unsturte ftty i multiplie y the numer of oule ons within tht ftty i. Dt from ifferent overwintering tempertures were poole s this effet ws not signifint. Different letters inite signifint ifferenes. Tle 3. Summry of the split-split plot three-wy ANOVAs on the effet of overwintering temperture, ivlve speies n smpling te on onentrtions of triglyerie, unsturtion inex, totl PUFA n mjor iniviul PUFA of neutrl lipis in igestive glns Triglyerie Unsturtion PUFA Soure of vrition.f. F P F P F P F P F P F P Min plot nlysis T C Error A 3 Suplot nlysis Sp T C Sp Error B 3 Su-suplot nlysis Dte < < < < T C Dte Sp. Dte < T C Sp. Dte Error C 24 Error A: Tnk (T C) Error B: Sp. Tnk (T C) Error C: Dte Tnk (T C),.f.=12; Sp. Dte Tnk (T C),.f.=12 Inepenent vriles were overwintering temperture (T C; 0 C, 4 C n 9 C), ivlve speies (Sp.; mussel n oyster) n Dte (31 Jnury, 14 Ferury, 12 April, 4 My n 8 June). Signifint proilities re in ol. Triglyerie t were log trnsforme n t were 1/squre root(x) trnsforme.

8 3006 F. Pernet n others A Overwintering Spring n summer Overll % TAG 17 Jn:12 Apr % TAG 12 Apr:8 Jun % TAG 17 Jn:8 Jun B C 9 C Mussel 4 C 0 C 9 C Oyster 4 C Seletive umultion (<1) / utiliztion (>1) SFA MUFA PUFA 4 0 C Fig. 4. See fing pge for legen.

9 Temperture pttion in two ivlve speies 3007 Tle 4. Chrteristis of the memrne lipis in igestive glns (DG) n gills of mussels Mytilus eulis n oysters Crssostre virgini smple on 17 Jnury efore exposure to overwintering tempertures Mussel Oyster Vrile DG Gills DG Gills Signifint effet Lipi lss PL/ST 8.7± ± ± ±0.7 Tissue ST (mg g 1 AFDM) 4.0± ± ± ±2.3 Tissue PL (mg g 1 AFDM) 34.7± ± ± ±14.5 NS Ftty i (mol %) 2.4± ± ± ±0.1 Sp. Tissue 15:0 0.8± ± ± ±0.0 Sp., Tissue 16.8± ± ± ±1.1 Sp. Tissue 17:0 1.1± ± ± ±0.1 Sp. 4.0± ± ± ±0.2 NS SFA 25.0± ± ± ±0.9 Sp. 4.0± ± ± ±0.2 Sp. Tissue 16:1n-5 0.8± ± ± ±0.1 Tissue 2.0± ± ± ±0.2 Sp. 2.2± ± ± ±0.1 Sp., Tissue 1.3± ± ± ±0.0 Sp. Tissue 2.1± ± ± ±0.2 Sp., Tissue 1.0± ± ± ±0.3 Sp. MUFA 14.3± ± ± ±0.6 Sp. Tissue 16:2n-6 2.2± ± ± ±0.0 Sp. Tissue 1.7± ± ± ±0.0 NS 20:2NMI i,j 3.4± ± ± ±0.4 Sp. Tissue i,j 4.7± ± ± ±0.1 Sp., Tissue 1.2± ± ± ±0.1 Tissue 2.2± ± ± ±0.3 Tissue 3.8± ± ± ±0.3 Sp., Tissue 18.8± ± ± ±0.2 Sp. Tissue 21:5n-3 1.1± ± ± ±0.1 NS 22:5n-6 0.9± ± ± ±0.1 NS 22:5n-3 1.3± ± ± ±0.1 Sp. Tissue 15.4± ± ± ±0.8 NS PUFA 59.5± ± ± ±0.3 Sp. Unsturtion inex 276.3± ± ± ±5.2 Sp., Tissue PL, phospholipis; ST, sterol. All vlues represent the men ± s.., N=2 replite tnks, 3 nimls per tnk were smple. Only ftty is ontriuting >1% in t lest one omintion of tretment re reporte. respetively. Overll, the most reily moilise ftty is in mussels were n, whih rnge from n , respetively. Interestingly, uring overwintering, seeme to e moilise less reily in mussels overwintere t 0 C n 4 C thn in mussels Fig. 4. Reltive moilistion of ftty is from the igestive glns of mussels n oysters overwintere t 0, 4 n 9 C uring the overwintering perio (A), the spring summer simultion (B) n the overll perio of stuy (C). The reltive moilistion of iniviul ftty is ws lulte s the rtio of initil to finl levels in the TAG for eh time intervl. Vlues re mens ± s.e.m, N=2. A rtio greter thn, equl to, or lower thn unity shows tht the ftty i is relese more, eqully, or less reily, respetively, thn the totl TAG-ftty is. Vlues for ftty is >2 mol % of the totl re rrnge in inresing orer of reltive moilistion from ottom to top. mintine t 9 C wheres the inverse ws true uring the spring summer simultion: seeme to e moilise more reily in mussels overwintere t 0 C thn in mussels mintine t higher tempertures. In oysters, the most reily moilise ftty i ws, whih rnge etween 1.34 n Due to the low ontriution of to the totl TAG-ftty is (<1%, Tle 2), seletive moilistion of only h mrginl effet on the ftty i omposition of TAG. Memrne lipis The phospholipi to sterol (ST) rtio vrie s funtion of time, tissue n speies (Fig. 5, Tle 4, Tle 5). Mussel n oyster tissues showe 1.2-fol inrese in their phospholipi to sterol rtio etween 12 April n 4 My, when temperture inrese from 0 C, 4 C or 9 C to 20 C. The phospholipi to

10 3008 F. Pernet n others sterol rtio of igestive glns ws 1.4 higher thn tht of gills (11.9 n 8.4, respetively; Fig. 5), minly ue to the higher ST ontent in gills (Tle 4). Interestingly, mussels were hrterise y phospholipi to sterol rtio 1.5 higher thn in oysters, irrespetive of time or tissue (Fig. 5). The unsturtion inex ws initilly 5.2% higher in mussels thn in oysters n 3.3% higher in igestive glns ompre to gills (Tle 4). The unsturtion inex vrie s funtion of tissue te. In gills, the unsturtion inex inrese slightly (y 3.6%) uring erly overwintering, remine elevte uring overwintering, n erese mrkely uring the spring Phospholipi / sterol Unsturtion inex PUFA (mol %) (mol %) (mol %) A B C D,,,, DG Gills,, DG Gills, Both speies, oth tissues Jn Fe Mr Apr My Jun Jul E, Both speies, oth tissues Mussel DG Oyster e Gills e 8 JnFe Mr Apr MyJun Fe Mr Apr My JunJul Month * e e DG Gills,,,, e f M DG M Gills O DG O Gills Mussel DG summer simultion t 20 C. In igestive glns, the unsturtion inex remine onstnt uring overwintering n erese mrkely uring the spring summer simultion. The unsturtion inex lso vrie s funtion of speies tissue (Fig. 5, Tle 5). As oserve for TAG, the unsturtion inex of polr lipis ws positively orrelte with PUFA (y=4.4 PUFA 6.4; r 2 =0.867, N=120, P<0.001; Fig. 5) n more prtiulrly with (y= ; r 2 =0.633, N=120, P<0.001; Fig. 5) n (y=4.6 22:6n ; r 2 =0.601, N=120, P<0.001; Fig. 5). The ftty i remine onstnt uring overwintering n erese y 35.5% uring the spring summer simultion, irrespetive of speies or tissue. The ftty i vrie s funtion of tissue te, speies te n speies tissue (Fig. 5, Tle 5). Although some minor ifferenes ourre in the ynmis of uring overwintering etween speies n tissues, erese uring the spring summer simultion in igestive glns n gills for Mussel Oyster oth mussels n oysters (Fig. 5). Overll, there ws no signifint effet of overwintering temperture on the ftty i omposition of memrne lipis. However, regression moels using temperture s n explntory vrile n the unsturtion inex of nimls limte t 0 C, 4 C, 9 C (April 12) n 20 C (June 8) s the response vrile showe tht the unsturtion inex in gills ws negtively orrelte with temperture (Fig. 6A). Interestingly, the unsturtion inex of mussels ws higher thn tht of oysters, irrespetive of limtion temperture. In mussel gills, showe wier rnge of vrition ( %) thn ( %) in response to limtion temperture, wheres in oyster gills, 20:5n- Oyster 3 n vrie to the sme extent (Fig. 6B). In ontrst to gills, the unsturtion inex in igestive glns ws not signifintly orrelte with limtion temperture: threshol vlue Gills M DG M Gills O DG O Gills Fig. 5. Chrteristis of the memrne lipis in mussel (M) n oyster (O) igestive glns (DG) n gills, s funtion of time (left) n speies n tissues (right). (A) The phospholipi to sterol rtio, (B) the unsturtion inex n (C E) the mol % of polyunsturte ftty is (PUFA; C), (D) n (E) in the polr lipis. Vlues re mens ± s.e.m, N=2 6 tnks. The unsturtion inex is lulte s the sum of the molr perentge of eh unsturte ftty i multiplie y the numer of oule ons within tht ftty i. Dt from ifferent overwintering tempertures were poole s this effet ws not signifint. Different letters inite signifint ifferenes.

11 Temperture pttion in two ivlve speies 3009 Tle 5. Summry of the split-split plot four-wy ANOVAs on the effet of overwintering temperture, ivlve speies, tissue n smpling te on the phospholipi to sterol rtio, unsturtion inex, totl PUFA n mjor iniviul PUFA of polr lipis PL/ST Unsturtion PUFA 22:2 NMI Soure of vrition.f. F P F P F P F P F P F P F P F P Min plot nlysis T C Error A 3 Suplot nlysis Sp < <0.001 T C Sp Error B 3 Su-suplot nlysis Tissue < < < < < < <0.001 T C Tissue Speies Tissue < <0.001 T C Speies Tissue Dte 4.0 < < < < < < T C Dte Speies Dte < < T C Speies Dte Tissue Dte T C Tissue Dte Speeis Tissue Dte T C Speies Tissue Dte Error C 54 Error A: Tnk (T C) Error B: Speies Tnk (T C) Error C: Tissue Tnk (T C),.f.=3; Speies Tissue Tnk (T C),.f.=3; Dte Tnk (T C),.f.= 12; Speies Dte Tnk (T C),.f.=12; Tissue Dte Tnk (T C),.f.=12; Speies Tissue Dte Tnk (T C),.f.=12 PL/ST, phospholipi to sterol rtio. Inepenent vriles were overwintering temperture (T C; 0 C, 4 C n 9 C), ivlve speies (mussel n oyster), tissue (gills n igestive glns) n smpling te (31 Jnury, 14 Ferury, 12 April, 4 My n 8 June). Signifint proilities re in ol. Dt of PL/ST, 20:2 n 22:2 NMI were log+1 trnsforme.

12 30 F. Pernet n others A Unsturtion inex B Mol % of totl ftty is Mussel DG Mussel gills, y= 2.1 temperture 288.6; r 2 =0.995, Oyster DG N=4, F=195.9,.f.=1, P=0.005 Oyster gills, y= 2.2 temperture 264.3; r 2 =0.980, N=4, F=47.8,.f.=1, P= Temperture ( C) Mussel gills PUFA 15 5 Temperture ( C) Oyster gills ws ttine t 9 C n i not inrese further with erese in temperture. The ftty i, n eiosnoi preursor of mny iologilly tive lipis, showe speies- n tissue-speifi ptterns (Tle 4). Inee, ws initilly lower in mussels (4.4%) thn in oysters (5.9%) n lower in igestive glns (4.3%) ompre to gills (6.0%, Tle 4). The ftty i vrie s funtion of overwintering temperture speies te (Fig. 7, Tle 5). During the overwintering perio, inrese in mussels wheres it remine stle in oysters. Therefore, t the en of the overwintering perio, mussels n oysters showe similr levels of. During the spring simultion perio, inrese mrkely in mussels, inrese moertely in oysters overwintere t 0 C n 9 C, n i not inrese signifintly in oysters overwintere t 4 C. Finlly, the non-methylene-interrupte ienoi ftty is (NMI), group of long-hin PUFA nturlly iosynthesise y molluss, showe speies- n tissue-speifi ptterns (Tle 4). Signifint levels of oth 20:2 n 22:2 NMI hrterise mussels, wheres 22:2 NMI ws the preominnt NMI ftty i in oysters. Aitionlly, 22:2 NMI ws lower in mussels thn in oysters n lower in igestive glns ompre to gills (Tle 4, Fig. 8). Levels of i not vry uring the experiment, wheres 22:2 NMI vrie s funtion of speies te n tissue te (Fig. 8, Tle 5). In oysters, 22: PUFA Fig. 6. (A) Regression moels using temperture s the explntory vrile n the unsturtion inex of nimls limte t 0 C, 4 C, 9 C (April 12) n 20 C (June 8) s the response vrile in igestive glns (DG) n gills of mussels n oysters. (B) Mol % of polyunsturte ftty is (PUFA),, n in the gills of mussels n oysters limte t 0 C, 4 C, 9 C (April 12) n 20 C (June 8) s funtion of temperture. (mol %) 12 Mussel, Oyster 0 C 4 C 9 C,, 8,e,,f,f,f,f,f,f,f e,h e,h 6 h,j f,i h,j h,j e,h e,h g,j g,j f,h f,h e,h i h,j i,j 4 Jn Fe Mr Apr My Jun Fe Mr Apr My Jun Fe Mr Apr My Jun Jul Month Fig. 7. Mol % of rhioni i () in the polr lipis s funtion of temperture speies te. Vlues re mens ± s.e.m, N=2 tnks). Dt from ifferent overwintering groups n tissues were poole s these effets were not signifint. Different letters inite signifint ifferenes. NMI inrese from 7.9 to 9.3% uring the limtion perio t 20 C wheres it remine low in mussels (Fig. 8). In gills, 22:2 NMI inrese from 7.3 to 8.4% uring the limtion perio t 20 C, wheres it ws mintine onstnt in igestive glns. Disussion Storge lipis: quisition n moilistion Mussels n oysters iffere in their lipi metolism uring overwintering. In M. eulis, speies pte to the hrsh winters of Atlnti Cn, high initil TAG onentrtions in the igestive gln eline mrkely uring the stuy, suggesting reline on TAG for energy metolism or reproutive proesses (De Zwn n Mthieu, 1992). In ontrst, C. virgini, whih generlly ours in wrmer hitts, showe low n onstnt TAG onentrtions uring overwintering. This mirrors iogeogrphil ptterns of reline on lipis for energy metolism mong mrine speies. For exmple, herivorous opepos from polr n temperte res use lipi stores for energy n reproutive nees uring the fll 22:2 NMI (mol %) A, Mussel Oyster e, e, e, DG Gills Fig. 8. Mol % of non-methylene interrupte ienoi ftty is (22:2 NMI) in the polr lipis s funtion of speies te (A; t from ifferent tissues poole) n tissue te (B; t from ifferent speies poole). Vlues re mesn ± s.e.m, N=2 6 tnks. Dt from ifferent overwintering tempertures were poole s this effet ws not signifint. Different letters inite signifint ifferenes., 4 Jn Fe Mr Apr My Jun Fe MrApr My Jun Jul Month B

13 Temperture pttion in two ivlve speies 3011 n winter, presumly in response to the yli nnul proutivity typil of northern foo wes, wheres zooplnkton from tropil iomes generlly o not umulte lrge lipi stores (Lee et l., 2006). Likewise, the pity for lipi storge inreses in ol-pte n ol-limte fish (Pörtner, 2002; Guerley, 2004). Although mussels n oysters overlp in their istriution, the isrepny in their TAG metolism my reflet the ft tht mussels grow etter in oler hitts thn oysters o. Alterntively, ifferenes in TAG metolism etween the two speies my reflet ifferenes in their reproutive yle t low tempertures. The synthesis, storge n use of TAG often show pronoune sesonl yles in ivlves: TAG re sequestere uring perios of high foo vilility in lte summer n fll, n re susequently use for mintenne metolism uring perios of reue feeing in the winter n for initition of gmetogenesis (De Zwn n Mthieu, 1992; Thompson et l., 1996; Brer n Blke, 2006 n referenes therein). It is therefore likely tht the TAG store in igestive glns of mussels were trnsferre to the eveloping gons uring overwintering. By ontrst, oysters tht showe low n onstnt levels of TAG uring overwintering my e quiesent, witing for the spring revivl to initite gmetogenesis. Aoring to their speifi environment, ivlves my support gmetogenesis using reently ingeste foo, store reserves or omintion of the two (Brer n Blke, 2006). Disrepnies in TAG ynmis etween mussels n oysters uring overwintering prtly reflet ifferenes in feeing rte. At 9 C, the temperture t whih nimls were mintine for 8 weeks efore the experiment egn, the CR of mussels ws higher (~2.8 l h 1 g 1 ry mss) thn tht of oysters, where the CR ws well elow <0.5 l h 1 g 1 ry mss (Fig. 2A). Therefore, mussels likely umulte more lipis from phytoplnkton prior to overwintering thn i oysters, explining the higher TAG levels in mussel igestive glns t the onset of the experiment. While the CR of mussels inrese with overwintering temperture, s foun y Cusson et l. (Cusson et l., 2005) n referenes therein, tht of oysters mintine t 0 C, 4 C n 9 C ws similr. Although few stuies hve speifilly exmine the effet of temperture on feeing in C. virgini, one pper reports tht this speies oes not fee elow 5 C (Loosnoff, 1958), in greement with our results. Not only were mussels hrterise y greter pity for TAG umultion ompre to oysters, ut they lso seeme etter le to seletively inorporte. This essentil ftty i ws mrkely higher in mussel thn in oyster TAG n lrgely exeee ietry levels. Although the ftty i omposition of the TAG in ivlve igestive glns generlly reflets tht of their iet (Sount et l., 1999), sllop lrve fe iet rih in preferentilly umulte t the expense of in their TAG, suggesting regultion of the inorportion/utilistion of essentil PUFA in TAG (Deluny et l., 1993). M. eulis my eposit more into TAG thn C. virgini, thus inresing the vilility of storge fts t low tempertures (Flornt, 1998). During overwintering, mussels erese the proportion of ertin unsturte ftty is in TAG, with n 18:4n- 3 elining mrkely uring the stuy, wheres oysters showe fewer hnges (Figs 3 n 4). The Antrti fish Tremtomus newnesi preferentilly oxiises unsturte ftty is (Lun n Siell, 1992), with the hormone-sensitive lipse (HSL) of its ipose tissue preferring sustrtes in the orer PUFA>monoenes>sturtes (Hzel n Siell, 2004). Mmmlin HSL shows similr sustrte preferenes (Rlot, 2003). Although this lipse hs not een stuie in ivlves, the erese in n from the TAG in mussel igestive glns suggests similr seletivity. The miniml hnges of oyster TAG uring overwintering my reflet the limite TAG eposition t the strt of the stuy more thn ifferenes in the HSL properties. Despite the ft tht n erese mrkely in mussel TAG uring overwintering, the more onstnt levels of, n NMI ftty is n the erese of mintine the % PUFA n the unsturtion inex onstnt uring overwintering. The mrke erese in in TAG of mussel igestive gln uring overwintering oul reflet utilistion of this PUFA for mintenne uring perios of reue feeing or for initition of gmetogenesis. In support of the ltter, reent stuy showe tht the femle gon of the sllop Noipeten sunoosus speifilly umultes uring gmetogenesis (Plios et l., 2005). If is lso umulte in the eveloping gons of mussels, it must ome from ietry soures or from store lipis sine ivlves hve limite pity for e novo synthesis of long-hin PUFA (DeMoreno et l., 1976; Lngon n Wlok, 1981; Deluny et l., 1993; Cers et l., 2003). The rte of hnge of levels in igestive gln TAG fell s funtion of overwintering temperture n then rose uring the spring summer simultion (Fig. 4). At first glne, reue moilistion of t low tempertures woul seem to help mintin fluiity, n thus the vilility of the TAG, t low tempertures. However, the unsturtion inex of the TAG, whih is goo initor of the melting point, ws similr mong mussels overwintere t 0 C, 4 C n 9 C. Therefore, it is unlikely tht reue moilistion of ounterte n orering effet of low tempertures on the TAG. The funtionl role of reue moilistion of t low tempertures remins unler. Memrne lipis: HVA n metolism The inrese in the phospholipi to sterol rtio uring the erly stge of wrming in mussels n oysters (Fig. 5) suggests tht sterols were not use for HVA (Roertson n Hzel, 1995; Crokett, 1998; Zehmer n Hzel, 2003). If the istriution of sterols in ellulr memrnes is similr to tht in vertertes (Lnge et l., 1989; Lnge n Stek, 1996) hnges in the reltive importne of plsm n orgnelle memrnes oul e the use of this pttern. HVA my hve een omplishe y the erese unsturtion of phospholipi yl hins with inresing temperture. Furthermore, the sterols of mrine ivlves inlue omplex mixture of phytosterols (Knuer et l., 1998) tht hve less of n orering effet on memrnes thn holesterol (Sukling et l., 1979). The higher phospholipi to sterol rtio of igestive glns ompre to gills grees with ptterns in rinow trout, in whih the rtio of holesterol to phospholipi is mrkely higher in

14 3012 F. Pernet n others gills thn in other tissues (inluing liver) (Roertson n Hzel, 1995). The high holesterol levels in gills my enhne their permeility. The higher phospholipi to sterol rtio in igestive glns oul reflet greter reltive importne of orgnelles. Aoringly, in C. virgini, the mitohonril volume frtion ws twie s high in igestive gln s in gills (Cherksov et l., 2006). The unsturtion inex erese uring the eginning of wrming in mussel n oyster gills n igestive glns, priniplly ue to n (Fig. 5). Although memrne fluiity ws not mesure in our stuy, these temporl vritions in n my ountert the isorering effet of rising tempertures. Aoringly, is thought to e importnt in ontrolling memrne fluiity uring ol limtion of fish (Dey et l., 1993; Bu et l., 1994; Tiku et l., 1996; Logue et l., 2000). Furthermore, 45% inrese in in gill memrnes of the se sllop P. mgellnius fter 21 ys of limtion t 5 C orreltes with n inrese in memrne fluiity (Hll et l., 2002). For oth mussels n oysters, the unsturtion inex of igestive gln phospholipis ws not orrelte with limtion temperture, wheres perfet negtive reltionship ws oserve in gills s preite y HVA. Thus the unsturtion inex ttine threshol vlue t 9 C in igestive glns while it ontinue to inrese with flling tempertures in gills. The miroenvironments of gills n igestive glns my require ifferent physil responses y the memrnes. In support of this hypothesis, the solterl n rush orer memrnes from intestinl epitheli of ol- n wrm-limte rinow trout respon to therml limtion in opposite fshions (Crokett n Hzel, 1995). Bsolterl memrnes exhiit perfet homeovisous effiieny ue to n inrese in the unsturtion inex, wheres rush orer memrnes from ol-limte fish re more orere thn those from wrm-limte fish. These uthors suggest tht ile n igestive lipse, two onstituents of the rush orer memrne miroenvironment, my impose unusul physil requirements. The ft tht the unsturtion inex of igestive gln phospholipis i not inrese eyon threshol vlue t 9 C in oth oysters n mussels suggests nee for stility of the memrne to ountert regulr exposure to igestive enzymes n is. The inverse reltionship etween the unsturtion inex of gill phospholipis n limtion temperture ws priniplly ue to hnges in n, ut the mgnitue of the response of these ftty is vrie etween oysters n mussels (Fig. 6B). The erese of with rise in temperture ws muh stronger in mussels thn oysters. Reline on 20:5n- 3 for remoelling memrne phospholipis in mussels goes hn in hn with the importne of this ftty i in TAG metolism. As the melting point of is C lower thn tht of, effiieny of HVA in mussels is likely higher thn tht of oysters. HVA effiieny vries mong speies, rnging from 20 to 0% for therml limtion n verging 70% for therml pttion (Hzel, 1995). Both oxygen uptke n memrne unsturtion were higher in mussels thn oysters, irrespetive of temperture, time or tissue (Fig. 2B). Similrly, stnr V O 2 n phospholipi unsturtion show positive orreltion in omprisons of wil n seletively re hr lms (Pernet et l., 2006). The unsturtion of memrne phospholipis is positively orrelte with sl metoli rte (Hulert n Else, 1999; Hulert n Else, 2005) in llometri omprisons of mmmls n irs. The positive reltionship etween stnr V O 2 n phospholipi unsturtion in mussels n oysters suggests tht Hulert s theory of memrnes s metoli pemkers my e pplile to invertertes. Memrne lipis: why o n NMI ftty is not follow HVA? One of the most importnt funtions of C 20 PUFA is s preursors of eiosnois, group of hormones tht inlues prostglnins, leukotrienes n hyroxyeiostetrenoi is (Smith n Murphy, 2003). The inrese in in mussels uring the spring summer simultion perio (Fig. 7) my ompenste for the onomitnt erese in. Eiosnoi proution is ssoite with stressful or energetilly expensive situtions, suh s gmetogenesis n spwning in ivlves or stimultion of immune funtion in other invertertes (Os et l., 1989; Stnley n Howr, 1998). Sine eiosnois proue from re generlly more tive thn those proue from, the replement of y in mussels my hve signifint impts. Levels of in mussels my hve risen uring the spring summer simultion through the stimultion of immune funtion ue to inrese teril los in the sewter s temperture rose. Similrly, n inrese in in sllop n hok lrve oinie with elevte mortlity n exposure to pthogeni n opportunisti miroes (Pernet et l., 2005; Plnte et l., 2007). Bivlve hemoyte memrne lipis ontin elevte mounts of, presumly to regulte immune responses (Delporte et l., 2003). We suggest tht the inrese in levels uring the spring summer simultion my reflet n inrese emn for -rih immune ells to ontrol teril prolifertion in mussels (Delporte et l., 2006). In ontrst to our previous work on juvenile hr lms, where we oserve n inrese in 22:2 NMI uring erese in temperture (Pernet et l., 2006), NMI ftty is in mussel n oyster tissues showe no onsistent response to temperture hnge (Fig. 8). In ontrst to essentil PUFA, NMI ftty is re synthesise e novo y ivlves (Zhukov, 1991). Therefore, the synthesis of NMI ftty is ws suggeste s n lterntive to the seletive inorportion of essentil PUFA t low tempertures, prtiulrly when phytoplnkton onentrtions re low or when feeing hs ese (Pernet et l., 2006). When extene, this rgument woul suggest tht the synthesis of NMI ftty is oul e importnt in nimls tht o not hve ny soure of PUFA, either from exogenous feeing or enogenous reserves. Although enogenous TAGs re generlly onsiere s energy reserves, they lso onstitute PUFA reservoir tht oul e use for remoelling memrne lipis s the nee rises. This hypothesis suggests tht oysters h more hnges in NMI thn mussels euse they h fewer TAG reserves t the onset of overwintering. In onlusion, we foun mjor ifferenes etween speies in lipi ynmis tht orrelte with the speies therml hitts, with mussels pprently mintining the fluiity of