Pêches & Océans Canada, Mont-Joli, Québec, Canada, G5H 3Z4. *Author for correspondence ( Accepted 25 January 2008

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1 969 The Journl of Experimentl Biology 211, Pulished y The Compny of Biologists 28 doi:1.122/je.1639 Physiologil nd iohemil trits orrelte with differenes in growth rte nd temperture dpttion mong groups of the estern oyster Crssostre virgini Frie Pernet 1, *, Réjen Tremly 2, Iften Redjh 1,2, Jen-Mrie Sévigny 3 nd Chntl Gionet 1 1 Institut de Reherhe sur les Zones Côtières, 232B rue de lʼéglise, Shippgn, Nouveu-Brunswik, Cnd, E8S 1J2, 2 Institut des Sienes de l Mer, 31 llée des Ursulines, Rimouski, Quée, Cnd, G5L 3A1 nd 3 Institut Murie-Lmontgne, Pêhes & Oéns Cnd, Mont-Joli, Quée, Cnd, G5H 3Z *Author for orrespondene (e-mil: frie.pernet@ifremer.fr) Aepted 25 Jnury 28 SUMMARY We tested two hypotheses in this study: first, tht intrspeifi growth vritions in mrine ivlve re orrelted with physiologil (sl metoli rte nd sope for growth) nd iohemil (memrne lipids) hrteristis, nd, seond, tht this ivlve shows intrspeifi vritions in physiologil nd iohemil dpttions to temperture. To test these hypotheses, five genetilly distint groups of juvenile oysters Crssostre virgini tht showed differenes in their growth rtes were mintined in the lortory (1) for further mesurements of growth nd stndrd metoli rtes nd (2) sujeted to limtion t C, 12 C nd 2 C nd further exmined for sope for growth nd determintion of memrne lipid omposition. Our results show tht lower sl metoli rte nd lower unsturtion index of memrne lipids oinides with higher growth rtes nd higher sope for growth in oysters. We provide evidene tht intrspeifi differenes in sl metoli rte in oysters re relted to memrne unsturtion s predited y Hulertʼs theory of memrnes s metoli pemkers. Furthermore, our results suggest tht the theory of memrnes s metoli pemkers is relted to intrspeifi differenes in growth. A perfet negtive reltionship ws oserved etween the limtion temperture nd the unsturtion index of memrne lipids in oysters, s predited y the homeovisous dpttion theory. However, hnges in the unsturtion index in response to temperture were minly due to vritions in the eiospentenoi (2:5n-3) ftty id in fst-growing oysters, wheres slowgrowing nimls hnged oth dooshexenoi id (22:6n-3) nd 2:5n-3. Thus, the pttern of iohemil ompenstion in response to temperture in this speies shows intrspeifi vrition. Key words: genetilly sed intrspeifi vrition, growth, temperture dpttion, sope for growth, lipid remodelling, homeovisous dpttion, mollus, quulture. INTRODUCTION The physiologil omponents regulting intrspeifi growth differenes mong individuls living in the sme environment my e ffeted y differenes in energy quisition (food onsumption nd ssimiltion), differenes in the llotion of energy mong mintenne, growth, reprodution nd other onsuming tivities, nd differenes in the metoli ost of growth (Byne, 1999). The energy udget or sope for growth provides mens of integrting the si physiologil proesses into n index of energy ville for growth nd reprodution. In ivlves, the sope for growth hs proved to e n urte preditor of totl prodution, whih inludes growth rte nd gmete prodution (for review, see Byne et l., 1985) (see lso Toro et l., 1996; Lrt et l., 1997; Sml nd Vonk, 1997; Byne, 1999; Byne et l., 1999; Pouvreu et l., 2; Rued nd Sml, 2). The stndrd metoli rte, defined s the minimum energy requirement for the mintenne of ll essentil funtions within n intive niml, is n importnt omponent of totl prodution (Byne et l., 1985). Interestingly, the unsturtion of memrne phospholipids (the numer of doule onds per 1 ftty id hins) is positively orrelted with the stndrd metoli rte in llometri omprisons of mmmls nd irds (Couture nd Hulert, 1995; Hulert et l., 22; Hulert et l., 22). Memrne ilyers in metolilly tive systems re more polyunsturted nd less monounsturted thn those in systems tht re metolilly less tive (Hulert nd Else, 1999; Hulert nd Else, 2). Suh polyunsturted memrnes hve een proposed to result in n inresed moleulr tivity of memrne proteins; in this mnner, the mount of memrne nd its omposition n t s pemker for metolism. We hve shown tht intr- nd interspeifi differenes in sl metoli rtes in ivlves relte to memrne unsturtion s predited y Hulert s theory of memrnes s metoli pemkers (Pernet et l., 26; Pernet et l., 27). The estern oyster Crssostre virgini is eurytherml suspension-feeding ivlve distriuted round the est ost of North Ameri from the Gulf of St Lwrene to the Gulf of Mexio (Gltsoff, 196). In the Gulf of St Lwrene, C. virgini is restrited to the wrm shllow ys nd esturies in the southwestern prt. In these res, wter temperture inreses from elow zero during the winter to C during the summer. Crssostre virgini is of primry interest for old-wter quulture euse of its high ommeril vlue. However, vriility in growth rtes mong individuls nd spordi juvenile overwintering mortlities omplite the ommeril exploittion of this speies (Lvoie, 1995). Therefore, there is some interest in estlishing seletive reeding progrmme for old-wter performne in C. virgini, onsidering tht ioenergeti prmeters (Hwkins et l., 1989; Tremly et l., 1998; Pernet et l., 26), survivl nd growth [see

2 97 F. Pernet nd others Dégremont et l. (Dégremont et l., 27) nd referenes therein] re genetilly orrelted in mny ivlve speies. However, little is known out the physiologil nd iohemil responses of C. virgini to temperture. We first hypothesized tht physiologil (metoli rte nd sope for growth) nd iohemil (memrne lipid) trits would orrelte with intrspeifi growth differenes in C. virgini. We predited tht fst-growing oysters llote smller proportion of their eroi pity to mintenne requirements thn do slow-growing nimls. We lso predited tht differenes in metoli rte mong oyster groups orrelte with the unsturtion index of their memrne lipids. Finlly, we predited tht the sope for growth of these oysters orreltes with their growth rte s the fst- nd slow-growing nimls differ in their pity to exploit food. Our seond hypothesis ws tht genetilly distint groups of oysters exhiit different degrees of dpttion to temperture. The underlying premise ws tht physiologil rtes in C. virgini would show mrked degree of temperture dependene even fter period of limtion (Newell et l., 1977; Shumwy nd Koehn, 1982). Furthermore, we predited tht juvenile C. virgini would ountert the therml effets on memrne fluidity y hnging their memrne ftty ids s predited y the theory of homeovisous dpttion. Briefly, the theory of homeovisous dpttion sttes tht the visosity of the lipid ilyer is djusted to offset hnges imposed y temperture (Sinensky, 197; Hzel, 1995; Hywrd et l., 27). An importnt omponent of the old response is the inrese in memrne lipid unsturtion, nd this hs een linked to n enhned resistne to old (Hywrd et l., 27). Given tht memrne dpttion is now viewed s entrl ontriutor to low temperture survivl of ll orgnisms (Hywrd et l., 27), this trit my e of prtiulr interest for the oldwter quulture of mrine invertertes. MATERIALS AND METHODS Animls Oysters Crssostre virgini (Gmelin 1791) were olleted in the field or produed in the hthery. Wild juvenile oysters were initilly olleted etween 21st July nd 12th August 2 on mteril suspended in the wter olumn in Bie de Mirmihi, Négu, NB, Cnd (7 N, 65 W). They were immeditely trnsferred to n quulture grow-out site loted in Bie de Sint Simon (7 2 N; 6 5 W, lese MS-176), Gulf of St Lwrene, NB, Cnd. Oysters were ultured following usul lol prties: they were seprted from olletors during the fll, pled in mesh gs tthed to ottom tles tht were rised slightly off the sustrte for overwintering under the ie-over nd then pled in floting gs t the se surfe the following spring (Lvoie, 1995). Hthery oysters otined from the Costl Zone Reserh Institute (CZRI, Shippgn, NB, Cnd) were rered following stndrd proedure (Dégremont, 23). Briefly, 1 dults olleted in Ferury 25 t Boutouhe (NB, Cnd) were onditioned in the CZRI hthery. The sewter temperture ws grdully inresed from 1 C to 2 C over 15 dy period nd mintined for 35 dy onditioning period. A ultured phytoplnkton diet of Isohrysis gln, Chetoeros grilis nd Pvlov lutheri ws dded to the sewter t totl onentrtion of ~1 ells l 1. After the onditioning period, five mles nd 2 femles were rndomly seleted for the prodution of juveniles. Spermtozoids or ooytes were olleted y stripping the gond. Sperm from eh mle ws omined with the ooytes from four different femles to produe 2 full-si fmilies on 7th April 25. Three-million ooytes per femle were fertilized t rtio of 2 spermtozoids per ooyte. Additionlly, ooytes from eh femle were mixed together nd fertilized with mixture of spermtozoids from eh mle to produe pooled offspring (Dégremont, 23). Lrve were rered in 115 l tnks t 21 C in.5 m-filtered sewter nd fed dily with the diet used for roodstok t ~ ells l 1. Wter nd food were renewed three times per week. Oyster lrve ttined the pediveliger stge 22 dys post-hthing nd settled on olletors tht were suspended in the lrvl tnks for 1 dys. When hthery-rered oysters rehed t lest 8 mm in shell length (9th August), they were pled in floting gs nd trnsferred to the quulture grow-out site longside the wild juvenile oysters. Oysters originting from the field, the pooled group, nd three full-si fmilies, designted F 2, nd, were hrvested on 12th Otoer 25 nd trnsferred to the Sttion Aquiole de Pointe-u-Père (Institut des Sienes de l mer, ISMER, QC, Cnd). We hose these five groups of oysters for their differenes in growth rte, s lulted from settlement until olletion. Oyster hrteriztion Growth nd stndrd metoli rtes Upon rrivl t ISMER, ~8 oysters from eh group were numered with ee tgs nd pled into two 15 l quri (~ oysters per group per qurium, slinity ws 28 nd temperture ws 2±.8 C). Temperture, slinity nd oxygen were monitored dily with multiprmeter proes YSI 85 (Yellow Springs Inorported, OH, USA). Oysters were fed ontinuously with mixed suspension of I. gln, P. lutheri nd Nnnohloropsis sp. t 1 ells l 1. The shell length of 68 7 individul oysters from eh group ws reorded monthly for 9 months. In the mentime, 5 oysters from eh group were numered with ee tgs nd pled into 15 l qurium. After 21 dys of limtion to lortory onditions, the minimum oxygen onsumption (V O 2 min or stndrd metoli rte) ws mesured fter hving strved the oysters for 1 dys. Oysters were kept individully in 5 ml metoli hmers for 6 min efore strting the mesurement. Empty shells were used s ontrol. Six hmers were used simultneously, whih llowed us to mesure five oysters nd one ontrol t time. Animls tht remined losed in the hmer were exluded from physiologil nlysis. Oxygen onsumption for n individul oyster ws determined y seling the hmer nd mesuring the redution in %O 2 with Strthkelvin hnnel dissolved oxygen system using mirothode 132 eletrode (Strthkelvin Instruments, Glsgow, Sotlnd, UK). Sewter ws well mixed with mgneti stirrer. The output signl ws monitored ontinuously on omputer until derese in O 2 of t lest 2% ws rehed. After the oxygen uptke mesurement, eh set of oysters ws frozen t 8 C for lter determintion of dry mss, whih ws mesured fter drying t 7 C for 72 h. Respirtion rte is expressed s the rte expeted for stndrd oyster in ml 1 g 1 dry mss h 1 y pplition of llometri orretion (Widdows nd Johnson, 1988). Genetis Upon rrivl t ISMER, 5 oysters from eh group were stored t 8 C until geneti nlyses. DNA extrtion ws rried out using DNesy tissue kits (Qigen, Mississug, ON, Cnd). Geneti vrition ws determined t the polymorphi mirostellite loi Cvi7, Cvi8, Cvi 9, Cvi12, Cvi13, Cvi1 nd Cvi23 (Brown et l., 2; Reee et l., 2). Amplified frgments were seprted y pillry eletrophoresis using n ABI PRISM 31 utomted geneti nlyser (Applied Biosystems, Foster City, CA, USA) nd nlysed

3 Growth omponents nd temperture dpttion in oysters 971 with the Genesn nd Genotyper softwre pkges (Applied Biosystems). Temperture experiment Experimentl design Upon rrivl t ISMER, 23 nimls from eh group were eqully distriuted into nine 37 l quri (6-Pk Arti model, Aquioteh, Cotiook, QC, Cnd). Eh qurium hd its own filtrtion, ertion nd wter temperture ontrol unit. Oysters were limted to lortory onditions for 5 dys prior to strting the experiment. The slinity ws 28, the nturl photoperiod ws followed, nd the temperture ws mintined t 12 C (Fig. 1). The five groups of oysters were mintined together in these quri over the entire experiment. Animls were fed diet of I. gln, P. lutheri nd Nnnohloropsis sp. t totl onentrtion of ~1 ells l 1 twie dy. Temperture ws monitored every dy in eh tnk throughout the experiment. On 17th Otoer 25, the temperture of three quri ws grdully deresed (~1.5 C dy 1 ) to C while the temperture of three other quri ws grdully inresed (~1.5 C dy 1 ) to 2 C. The temperture of the remining quri ws mintined t 12 C s ontrol (Fig. 1). All nimls rehed the desired temperture y 2th Otoer fter whih these tempertures were mintined for 6 weeks. Physiologil rtes nd lipid omposition of oysters were mesured on 2th Otoer fter ttining the desired temperture (ute response) nd gin on 5th Deemer (long-term limtion; Fig. 1). Physiologil rtes A pool of five oysters per qurium ws used for physiologil mesurements in 5 ml hmers efore the oysters were killed for dry mss determintion to lulte mss-stndrdized physiologil rtes y llometri eqution. Temperture ( C) 2 12 A 2 12 B C Tnk Oyster origin Hthery Pre-limtion phse in the field Pooled group Fmilies 1 Aug. 22 Aug. 12 Sep. 3 Ot. 2 Ot. 1 Nov. 5 De. Dte Dte 2 Ot. (ute) 5 De. (limtion) Experimentl phse in the l Fig. 1. (A) Shemti digrm of the split split plot experimentl design. (B) Experimentl protool for the temperture experiment. Strs indite dtes of physiologil mesurements nd lipid smpling. F 2 Clerne rte, defined s the volume of wter lered of suspended prtiles per unit time nd iomss, ws quntified using stti system (Pernet et l., 27). Briefly, nimls were provided with I. gln t n initil onentrtion of 1 ells l 1 nd food prtiles were ounted every 15 min for 6 min using n eletroni prtile ounter (Bekmn Coulter Counter Z2, Mississug, ON, Cnd). The lerne rte (l h 1 ) ws then used in onjuntion with the lgl iomss (mg ml 1 ) to estimte the mount of ingested energy, ssuming tht the energy ontent of the diet ws 23.5 J mg 1 (Widdows nd Johnson, 1988). Oxygen onsumption (V O 2) for n individul niml ws determined y seling the hmer nd mesuring the redution in %O 2 s desried in the previous setion. Respirtion ws then expressed s ml O 2 g 1 tissue dry mss h 1 nd then onverted into energy equivlents using the onversion ftor 1 ml O 2 =2.33 J (Widdows nd Johnson, 1988). Assimiltion, defined s the produt of ingested energy nd sorption effiieny (Widdows nd Johnson, 1988), ws estimted using the Conover rtio [see Conover, 1966 in Widdows nd Johnson (Widdows nd Johnson, 1988)]. Food nd fel smples were filtered onto pre-omusted pre-weighed 7 mm GFC filters tht were rinsed with isotoni mmonium formte (3.2%), dried t 8 C for 8 h, ooled to room temperture in desitor, nd re-weighed. Afterwrds, they were omusted overnight t 5 C, ooled to room temperture in desitor, nd finlly weighed gin. This proedure provided estimtes of the orgni nd inorgni frtions ontined in the food nd fees. The sope for growth ws estimted y sutrting the energy lost through respirtion nd exretion from the energy otined y food ssimiltion (Widdows nd Johnson, 1988). As exretion represents <5% of the energy udget in most ivlves, it ws ignored. Lipid nlysis Five oysters from eh group were rndomly smpled in eh qurium on 2th Otoer nd 5th Deemer in eh temperture tretment for the determintion of lipid omposition. Tissues of different oysters from the sme qurium were pooled together to otin 1 mg wet mss nd stored in lipid-free mer glss vils with Teflon-lined ps under nitrogen in 1 ml dihloromethne t 8 C. Lipids were extrted (Folh et l., 1957), nd lsses determined s previously desried (Pernet et l., 27). Lipids were seprted into neutrl lipids (inluding triglyerides nd sterols) nd polr lipids (inluding minly phospholipids) using olumn hromtogrphy on sili gel hydrted with 6% wter (Pernet et l., 27). Ftty id methyl esters (FAME) from polr lipids were prepred using 2% H 2 SO in methnol (Lepge nd Roy, 198). Gs hromtogrph prmeters nd the proedure for FAME identifition nd nlysis hve een desried previously (Pernet et l., 27). Sttistil nlyses The men numer of lleles per lous, nd oserved nd expeted heterozygosities were lulted using the softwre Genetix.5 (Belkhir et l., 1998). The softwre FSTAT (Goudet, 21) ws used to lulte the fixtion index F IS nd F ST. The F IS vlues were tested (1 rndomiztions) for signifint differenes from zero to ssess the ompline with the Hrdy Weinerg equilirium. F ST vlues were used s n index of geneti differentition mong oyster groups. All proility vlues were djusted for multiple omprison tests using sequentil Bonferroni djustments (Rie, 1989). Three-wy split split plot ANOVAs were onduted to determine differenes in the physiologil rtes nd the ftty id omposition

4 972 F. Pernet nd others Tle 1. Summry of the split split plot three-wy ANOVAs on the effet of temperture, oyster origin nd smpling time on physiologil rtes nd lipid omposition in juvenile oysters Crssostre virgini Soure of vrition d.f. Min plot nlysis Temperture 2 Error A 6 Suplot nlysis Origin vs hthery 1 Pooled group vs fmilies 1 vs F 2, 1 F 2 vs 1 Temperture origin 8 Error B 2 Su-suplot nlysis Time 1 Time temperture 2 Time origin Time temperture origin 8 Error C 3 Physiologil rtes: sope for growth, lerne rte nd oxygen onsumption. Lipid omposition: unsturtion index, totl polyunsturted ftty id (PUFA) nd mjor individul PUFA of polr lipids. Error A: tnk (temperture). Error B: origin tnk (temperture). Error C: time tnk (temperture) + origin time tnk (temperture). Independent vriles were temperture ( C, 12 C nd 2 C), oyster origin (field or hthery; in the hthery: pooled group or fmilies F 2, nd ) nd time (ute nd limtion response). of the polr lipids in oysters, i.e. the unsturtion index nd the mjor polyunsturted ftty ids (PUFA), nmely dooshexenoi id (22:6n-3) nd eiospentenoi id (2:5n-3), s funtion of overwintering temperture, oyster origin nd smpling time (Tle 1). The unit of replition ws the qurium in whih the temperture ws pplied. The min plots were temperture levels, suplots were oyster origin, nd su-suplots were smpling time. Here we used mixed liner model, whih models not only the mens of our dt ut lso their vrines nd ovrines. The repeted option ws pplied to the term time to tke into ount temporl dependene (SAS Help nd doumenttion; SAS Institute In., Cry, NC, USA). Where differenes were deteted, lest-squre mens multiple omprison tests were used to determine whih mens were signifintly different. When differenes mong groups of oysters were deteted without intertion with other ftors (min effet), plnned ontrsts etween group mens were used (Tle 1). Residuls were sreened for normlity using the expeted norml proility plot nd further tested using the Shpiro Wilk test. Dt on sope for growth, V O 2 nd 2:5n-3 were log+1 trnsformed to hieve normlity of residuls nd homogeneity of vrines. Homogeneity of vrine ovrine mtries ws grphilly ssessed. Anlyses were rried out using SAS RESULTS Oyster hrteristis Although oysters originting from the field were igger thn those produed in the hthery t the time of olletion, they showed redued growth rte ompred with tht of hthery-rered nimls (Tle 2). The growth rte of oysters olleted in the field from settlement to 12th Otoer 25 (72.6 m dy 1 ) ws lmost hlf Tle 2. Chrteristis of juvenile oysters Crssostre virgini Hthery (ge +) Fmilies Vrile (ge 1+) Pooled group F 2 Generl hrteristis t the onset of the experiment Mting sheme ND f 2 39 m 5 9 f 2 m 8 f 26 m 9 f 35 m 7 Shell length (mm) 31.3± ± ± ± ±1.9 Growth rte ( m dy 1 ) Before experiment 72.6± ± ± ± ±9.8 After experiment 32.2± ±.3 6.2± ± ±. Bsl metoli rte (ml O 2 g 1 tissue dry mss h 1 ) V O 2 min.3±.9.27±.8.19±..18±.7.27±.1 Geneti hrteristis (verge t 7 loi using DNA mirostellite mrkers) N Men no. lleles per lous H o H e F IS Oysters were olleted in the field or produed in the hthery. Five mles (m) nd 2 femles (f) were mted to produe nimls in the pooled group wheres F x represents full-si fmilies. Shell length ws mesured on 1 nimls upon rrivl t ISMER on 12th Otoer, t the onset of the temperture experiment. Growth rtes were lulted from settlement to rrivl t ISMER, t the onset of the temperture experiment (Before experiment), nd in the lortory t 2 C from the end of the experiment (Novemer 2) to July 25 (After experiment, N=68 73 nimls). Bsl metoli rte ws lulted for individul juvenile oysters mintined t 2 C for 6 weeks (N=23 59 nimls). N, smple size; H o, oserved proportion of heterozygotes; H e, Hrdy Weinerg proportions of heterozygotes; F IS, devition from Hrdy Weinerg equilirium. Loi tht show signifint devition from Hrdy Weinerg equilirium re in old. Dt re mens (±s.d. where pproprite).

5 Growth omponents nd temperture dpttion in oysters 973 Tle 3. Mtrix of index of geneti differentition (F ST ) mong the five groups of juvenile oysters Crssostre virgini Pooled group F Pooled group F All pirwise omprisons were signifint (P<.5). Oysters were olleted in the field or produed in the hthery. Five mles nd 2 femles were mted to produe nimls in the pooled group wheres F x represents fullsi fmilies. tht of nimls produed in the hthery, whih verged m dy 1. Furthermore, the verge growth rte of oysters olleted in the field nd then exposed to lortory onditions for 9 months t 2 C ws only 32.2 m dy 1 ompred with 2.2 m dy 1 for nimls produed in the hthery. Oysters from fmilies F 2 nd showed higher growth rtes thn other htheryrered groups for the two time periods. The minimum oxygen onsumption or stndrd metoli rte (V O 2 min ) of oysters olleted in the field (.3 ml O 2 g 1 h 1 ) showed n inrese of 9.5% ompred with tht of hthery-rered nimls, 1 where V O 2 min for the four groups verged.23 ml O 2 g 1 h (Tle 2). It is noteworthy tht the fst-growing oysters from fmilies F 2 nd showed lower V O 2 min thn the other group of oysters. Interestingly, the V O 2 min of oysters exposed to lortory onditions deresed linerly with inresing growth rte (V O 2 min =.1 growth rte+.65; r 2 =.998, N=5, P=.1; Tle 2). The verge numer of lleles ws muh higher in oysters from the field ompred with those from the pooled group or the fmilies (Tle 2). Interestingly, oysters from F 2 nd showed no devition from Hrdy Weinerg equilirium wheres the overll F IS vlues for oysters from other groups indited signifint heterozygote defiienies. The F ST vlues mong the five groups of oysters vried from.9 to.3, nd ll pirwise omprisons were signifint (P<.5), thus inditing high level of geneti differentition (Tle 3). Temperture experiment Physiologil rtes The overll sope for growth vried s funtion of oyster origin (Fig. 2A). Fst-growing oysters produed in the hthery showed n verge sope for growth of 6.6 J h 1 g 1 ompred with only 18.7 J h 1 g 1 in nimls from the field (ontrst: field vs hthery, P<.1). Although oysters originting from the hthery showed similr sope for growth, it is noteworthy tht fst-growing nimls from showed the highest sope for growth. More rodly, the sope for growth of oysters inresed linerly with inresing growth rte mesured under lortory onditions t 2 C for 9 months (y=2.75 growth rte 68.5; r 2 =.956, N=5, P=.). Differenes in sope for growth mong groups of oysters were minly due to lerne rte (Fig. 2A nd B, inset). Overll, lerne rte inresed with temperture (min effet, P=.25), lthough it lso vried s funtion of temperture origin (Fig. 2B). While lerne rte ws similr mong groups of oysters t C nd 12 C (P=.785), it inresed y 2.7 times for fst-growing oysters from with the temperture inrese from 12 C to 2 C (P<.1). Although oysters from other groups showed similr trend for inresing lerne rte with inresing temperture from 12 C to 2 C, the mens were not signifintly different Sope for growth (J h 1 g 1 ) Clerne rte (l h 1 g 1 ) Oxygen onsumption (ml g 1 h 1 ) A B 6 2 C Pool F 2 Origin of oysters, Origin, P=.5, Pool F 2 F 2 Pool 12 2 Temperture ( C) Temp. origin, P=.3 Temp., P< Temperture ( C) Time, P=.9 2 Ot De Dte Fig. 2. (A) Sope for growth s funtion of oyster origin. (B) Clerne rte of oysters s funtion of temperture origin. The inset shows lerne rte s funtion of oyster origin only (min effet). (C) Oxygen onsumption rte of oysters s funtion of temperture (left) nd dte (right). Oysters were olleted in the field or produed in the hthery. Five mles nd 2 femles were mted to produe nimls in the pooled group (Pool) wheres F x represents full-si fmilies. Dt re mens ± s.e.m., n=3 quri. Dt from different tempertures nd times were pooled together when these effets were not signifint. Different letters indite signifint differenes.

6 97 F. Pernet nd others Unsturtion index A Origin, P <.1 Time temp., P <.1,, Pool F 2 Oyster origin 12 2 Temperture ( C) (.1<P<.5 for F 2 nd the pooled group, P>. for others). The sorption effiieny ws unffeted y ny of the tested ftors. Oxygen onsumption rte remined unffeted y oyster origin (origin effet: P=.131; temperture origin effet: P=.898; time origin effet: P=.326; time temperture origin effet: P=.935). However, oxygen onsumption inresed with inresing temperture nd deresed during limtion (Fig. 2C). Ftty id omposition of polr lipids The unsturtion index, whih is the numer of doule onds per 1 moleules of ftty ids, ws notiely higher in nimls B, Aute response Alimtion response Fig. 3. Unsturtion index of polr lipids in oysters s funtion of (A) their origin nd (B) time temperture. Oysters were olleted in the field or produed in the hthery. Five mles nd 2 femles were mted to produe nimls in the pooled group (Pool) wheres F x represents full-si fmilies. Dt re mens ± s.e.m., n=3 quri. Different letters indite signifint differenes. d olleted in the field (266.1) ompred with tht of fst-growing oysters produed in the hthery, where it verged only 255. (ontrst: field vs hthery, P<.1; Fig. 3A). The unsturtion index lso vried s funtion of time temperture (Fig. 3B), deresing with inresing temperture. However, the unsturtion index of nimls mintined t C remined onstnt irrespetive of smpling time wheres oysters exposed to higher tempertures showed mjor derese in their unsturtion index with limtion. A stepwise multiple regression model using groups of ftty ids s explntory vriles nd the unsturtion index s the response vrile showed tht the unsturtion index ws positively orrelted with PUFA (y=7. PUFA 111.7; r 2 =.916, N=85, P<.1). A seond regression model using individul PUFA s explntory vriles showed tht vritions in the unsturtion index were minly ttriutle to 22:6n-3 nd 2:5n-3 (y=3.6 22:6n :5n-3+9.3; r 2 =.958, N=85, P<.1). These two PUFA ounted for ~6% of the totl PUFA in oyster polr lipids. Overll, the level of 22:6n-3 in the fst-growing hthery oysters (18.5% of totl ftty id) ws 35.6% lower thn tht of oysters from the field, where it verged 25.1% (Fig. A). The level of 22:6n-3 in nimls olleted in the field inresed y 25% with temperture derese from 12 C to C, wheres it remined onstnt (pooled nd ) or inresed only mrginlly (y 8% nd 13% for F 2 nd, respetively) in oysters produed in the hthery. The level of 22:6n-3 vried s funtion of time temperture (Fig. B). Indeed, nimls mintined t C showed similr 22:6n-3 levels irrespetive of smpling time wheres oysters exposed to higher tempertures showed mjor derese in 22:6n-3 following limtion (Fig. B). The effets of time, temperture nd oyster origin interted on 2:5n-3 (Fig. C). The level of 2:5n-3 in the fst-growing oysters 22:6n-3 (mol %) A Temp. origin, P=.3 Pool F 2 B, Time temp., P<.1 Aute response Alimtion response Fig.. Dooshexenoi id (22:6n-3; mol %) in polr lipids s funtion of (A) temperture oyster origin nd (B) time temperture. (C) Eiospentenoi id (2:5n- 3; mol %) in the polr lipids s funtion of time temperture oyster origin. Oysters were olleted in the field or produed in the hthery. Five mles nd 2 femles were mted to produe nimls in the pooled group (Pool) wheres F x represents full-si fmilies. Dt re mens (± s.e.m. for B ut omitted for lrity in A nd C), n=3 quri. Different letters indite signifint differenes. 16 d e C 16 Time temp. origin, P=.37 2:5n-3 (mol %) 12 8 Aute response Alimtion response Temperture ( C)

7 Growth omponents nd temperture dpttion in oysters 975 produed in the hthery (13.6%) ws 5.8% higher thn tht of oysters originting from the field, where it verged 8.8% efore limtion (Fig. C, left side). The 2:5n-3 level in oysters produed in the hthery deresed with inresing limtion temperture from C to 2 C (Fig. C, right side). However, 2:5n-3 in hthery-rered oysters mintined t C remined onstnt irrespetive of smpling time, wheres those exposed to tempertures of 12 C nd 2 C showed mrked derese in 2:5n- 3 following limtion. In ontrst, 2:5n-3 in oysters from the field mintined t C nd 12 C remined onstnt irrespetive of smpling time. DISCUSSION Components of growth For the first time, we hve shown tht redution in the stndrd metoli rte (V O 2 min ) nd in the unsturtion index of memrne phospholipids oinides with higher growth (s expressed y growth rte nd sope for growth) in hthery oysters. Stndrd V O 2 nd memrne unsturtion were lower in fst-growing hthery oysters ompred with tht of slow-growing oysters from the field. Similrly, stndrd V O 2 nd phospholipid unsturtion showed positive orreltion in omprison to wild nd seletively red hrd lms (Pernet et l., 26) nd in omprison to mussels nd oysters (Pernet et l., 27). Here we provide evidene tht intrspeifi differenes in sl metoli rte in ivlves relte to memrne unsturtion s predited y Hulert s theory of memrnes s metoli pemkers (Hulert nd Else, 1999; Hulert nd Else, 25). The higher unsturtion index oserved in memrne lipids of oysters from the field ws minly due to higher 22:6n-3 levels ompred with those of fst-growing oysters from the hthery. The importne of 22:6n-3 in regulting n niml s metoli rte ws previously emphsized y others (Hulert nd Else, 25). Mmmlin phospholipids showed sttistilly signifint llometri deline in the unsturtion index with inresing ody size (Hulert et l., 22). This deline in the phospholipid unsturtion index ws predominntly due to the ft tht the 22:6n- 3 ontent of tissue phospholipids deresed s the mmml speies inresed in size. These results re very similr to those reported in irds (Hulert et l., 22; Turner et l., 26). The funtionl signifine of the elevted 22:6n-3 levels in the memrnes of smll mmmls nd irds is potentilly relted to their high mss-speifi metoli rte ompred with lrger endotherms. Beuse sustntil proportion of the sl metolism is ssoited with memrne-linked proesses [mitohondril proton lek, N + nd C 2+ yling together ount for pproximtely hlf of stndrd metoli rte (Rolfe nd Brown, 1997)], it ws suggested tht memrne lipids, prtiulrly 22:6n-3, my ply role in determining the metoli rte of different speies vi n influene on the moleulr tivity of memrne-ound enzymes (Hulert nd Else, 1999; Hulert nd Else, 2). Differenes in 22:6n-3 levels etween oysters from the field nd those from the hthery my reflet differenes in their pity for seletive inorportion of dietry PUFA into memrne lipids. Indeed, long-hin PUFA suh s 2:5n-3 nd 22:6n-3 hve een reported to e essentil for sustining optiml growth in severl ivlve speies (DeMoreno et l., 1976; Lngdon nd Wldok, 1981). Although the iosyntheti prodution of these PUFA is rther low or sent, mening tht the ftty id omposition of lipids in ivlve tissues generlly reflets tht of the diet, severl studies hve shown tht the ftty id omposition in ivlve memrne lipids is regulted y seletive inorportion or elimintion of ftty ids (e.g. Deluny et l., 1993). It is unlikely tht differenes in 22:6n- 3 etween groups of oysters reflet differenes in their diet s these nimls were kept in the sme onditions for more thn 2 months efore the experiment. Fst-growing oysters from the hthery showed (1) lower stndrd metoli rte nd (2) higher sope for growth due to higher food onsumption ompred with slow-growing nimls from the field. Similrly, Pifi oysters Crssostre gigs from fstgrowing line showed higher rte of ingestion nd sorption nd lower metoli rte t mintenne thn did those from slowgrowing line (Byne, 1999). A relted field experiment with the Sydney rok oyster Sostre glomert (formerly S. ommerilis) showed tht seletion for fst-growing nimls leds to fster rtes of feeding ross wide rnge of food onentrtions (Byne et l., 1999). Finlly, the superior growth in C. gigs ompred with tht of S. glomert mintined in the sme onditions lso oinided with fster rte of feeding in C. gigs (Byne, 1999; Byne, 22). Therefore, our results on the ioenergetis of juvenile C. virgini re in good greement with previously pulished dt on other oyster speies. Finlly, there ws smller defiieny in heterozygote frequenies in fst-growing oysters from F 2 nd ompred with tht of nimls from other groups. Severl studies hve shown negtive orreltions etween heterozygote defiieny nd fitness-relted trits in mrine ivlves, ttriuted to lower metoli requirements for heterozygous individuls (Hwkins et l., 1989; Tremly et l., 1998; Byne et l., 1999). In our study, higher metoli demnds in oysters from the field, the pooled group nd the ssoited with higher heterozygote defiieny proly impose supplementry stress tht results in redution of growth rte in these nimls. Temperture dpttion A perfet negtive reltionship ws oserved etween limtion temperture nd the unsturtion index of polr lipids in juvenile oysters, s predited y the homeovisous dpttion theory (Hzel, 1995). Interestingly, hnges in the unsturtion index in response to temperture were minly due to 2:5n-3 levels in fst-growing oysters from the hthery, wheres slow-growing nimls hnged levels of oth 22:6n-3 nd 2:5n-3, s previously oserved in dult C. virgini (Pernet et l., 27; Pernet et l., 27). This pttern my reflet differenes in the proportions nd thus the vilility of 22:6n-3 nd 2:5n-3 mong groups of oysters (see previous setion). In previous study, we showed tht the inverse reltionship etween the unsturtion index of gill phospholipids nd limtion temperture ws priniplly due to hnges in 22:6n-3 nd 2:5n-3 levels, ut tht the mgnitude of the response of these ftty ids vried etween oysters nd mussels (Pernet et l., 27). Indeed, the derese of 2:5n-3 with rise in temperture ws muh stronger in mussels thn in oysters. Although speultive, the effiieny of homeovisous dpttion in fstgrowing oysters from the hthery my e higher thn tht of slowgrowing nimls from the field, sine the melting point of 2:5n- 3 is 1 C lower thn tht of 22:6n-3. The unsturtion index nd the ftty id omposition of oysters showed only smll differenes mong temperture tretments on 2th Otoer ompred with those oserved on 5th Deemer, fter 6 weeks of limtion. Therefore, the ftty id remodelling in the memrne lipids of juvenile oysters my e viewed s longterm djustment to temperture. It is generlly epted tht hnges in the ftty id omposition of memrne lipids usully our fter period of limtion, vrying in durtion from 1 week

8 976 F. Pernet nd others in the se of wrm limtion to severl weeks during old limtion (Hzel nd Willims, 199). A previous study on rinow trout showed tht exposure to low tempertures led to grdul inrese in PUFA during old limtion (Hzel nd Lndrey, 1988). Likewise, hrd lms exposed to lowering of environmentl temperture showed grdul inrese in the unsturtion index tht ws viewed s long-term djustment to winter temperture (Pernet et l., 26). However, few studies showed tht hnges in ftty id omposition ould our in the short term. For exmple, in oysters, the unsturtion index of gills sujeted to dily flututions in temperture etween 12 C nd 25 C for 7 dys vried in wy onsistent with memrne homeovisous dpttion, minly due to rpid hnges in 22:6n-3 nd 2:5n-3 levels (Pernet et l., 27). There ws no signifint temperture effet on the sope for growth of juvenile oysters mintined t C, 12 C nd 2 C, refleting the ft tht oth energy expenditure (respirtion) nd quisition (ingestion) inresed with temperture. This result ontrsts with severl pulished studies on other ivlve speies (for review, see Byne et l., 1985) (see lso MDonld nd Thompson, 1986; Beirs et l., 1995; Yukihir et l., 2; Cusson et l., 25). For exmple, the sope for growth of juvenile oysters O. edulis tht were initilly mintined t 2 C nd further exposed to 1 C, 2 C or 26 C for 3 weeks inresed with temperture (Beirs et l., 1995). Likewise, the sope for growth of mussels M. edulis limted t 8 C ws found to e higher thn tht of mussels kept t 1 C (Cusson et l., 25). Furthermore, onsidering tht sope for growth is preditor of totl prodution (growth rte in juveniles), our result suggest tht growth rte in oysters would e similr over tempertures rnging from C to 2 C, whih is very unlikely. The lk of temperture effet on the sope for growth of oysters in our study my reflet n inility of the sttistil model to detet the effet of temperture due to the low numer of replite tnks (n=3) oupled with high vrition: nimls limted t C, 12 C nd 2 C showed sope for growth of 2.5±15.3, 3.6±17.5 nd 59.3±35.8, respetively. For the first time, we hve shown intrspeifi vritions in the effet of temperture on lerne rte. Overll, the lerne rte of nimls exposed to C ws lower thn tht of oysters kept t 2 C (P=.1). Although very few studies hve speifilly exmined the effet of temperture on feeding in C. virgini, one pper reported tht the pumping rte in this speies inresed stedily s temperture rose from 8 C to 28 C (Loosnoff, 1958). More reently, we showed tht dult C. virgini limted to overwintering tempertures (<9 C) exhiited redued lerne rte ompred with tht of nimls limted to spring summer onditions t 2 C (Pernet et l., 27), whih greed well with the pttern oserved in. Oxygen onsumption inresed with temperture irrespetive of smpling time, thus suggesting tht the respirtion rte of these nimls did not limte even fter 6 weeks. We know from the physiologil literture tht therml limtion is not universl feture of ivlve metolism: the ility of ivlves to lter metolism following temperture hnge is speies speifi [see for exmple Beirs et l. (Beirs et l., 1995) nd referenes therein]. More prtiulrly, the oxygen onsumption in oysters C. virgini nd Ostres edulis remins strongly dependent on the mient temperture even fter 3 weeks, thus suggesting tht these speies hve limited ility to limte to temperture hnge (Newell et l., 1977; Shumwy nd Koehn, 1982). Therefore, the mrked therml dependene of the respirtion rtes found in this work on juvenile C. virgini is onsistent with previous studies on oyster speies. The uthors re grteful to the stff from IRZC for oyster prodution nd lipid nlysis, ISMER for physiologil mesurements, nd Ministère des Pêhes ex Oéns Cnd (MPO)/Institut Murie Lmontgne for geneti nlysis. Thnks re ddressed to Pierre Boudry (IFREMER) for ritil disussions nd Lure Devine (MPO) for linguisti revision. This study ws supported y grnts from the Atlnti Innovtion Fund to F.P. nd disovery grnts from the Nturl Sienes nd Engineering Reserh Counil of Cnd nd Réseu Aquole du Quée to F.P. nd R.T. REFERENCES Byne, B. L. (1999). Physiologil omponents of growth differenes etween individul oysters (Crssostre gigs) nd omprison with Sostre ommerilis. Physiol. Biohem. Zool. 72, Byne, B. L. (22). A physiologil omprison etween Pifi oysters Crssostre gigs nd Sydney rok oysters Sostre glomert: food, feeding nd growth in shred esturine hitt. Mr. Eol. Prog. Ser. 232, Byne, B. L., Brown, D. A., Burns, K., Dixon, D. R., Ivnovii, A., Livingstone, D. R., Lowe, D. M., Moore, M. N., Steing, A. R. 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