Elizabeth R.Gillies University, Brook,NLA2H5G4,Canada. Corner ON N6A5B7,Canada. Canada. Toronto, Toronto, ONM5G1L7,Canada. 1580 Received 9November 2013;Accepted20January2014 § 1 organisms, includingtheantlers ofJapanesedeer( Trace amountsofacTAGs havebeendescribedinabroadrangeof density ofacTAGs compared withlcTAGs (Durrettetal.,2010). results inthereducedviscosityandsignificantlylowerenergy at the3-positionofglycerolbackbone.Thisdistinctstructure acetylated triacylglycerols(acTAGs) haveanacetylgroupesterified lipid hasbeenreportedasastoragemoleculeinterrestrialanimals. in (Williams etal.,2011; Yen etal.,2008).Nootherneutral energy density, andareconsequentlythemostabundantlipidclass are generallyusedasfuelstoresbyanimalsbecauseoftheirhigh Reue, 2011; Yen et al.,2008).Long-chaintriacylglycerols(lcTAGs) structure, signallingandenergy storage(Canavosoetal.,2001; Lipids fulfilldiversebiologicalfunctionsincludingcellmembrane solidaginis,Freezetolerance KEY WORDS:Lipid,Triglyceride, Acetylatedtriacylglycerol, lcTAGs duringdevelopmentandinresponsetostress. the firstevidenceofdynamicinterconversionbetweenacTAGs and is thefirstobservationofaccumulationacTAGs byananimal,and overwintering andtheseasonalacquisitionoffreezetolerance.This that accumulationofacTAGs coincideswithpreparationfor aqueous solutionsinamannerunusualforneutrallipids.We note solidaginis lcTAGs, andthusremainsliquidattemperatureswhichE. in tolerant ,norintheplanthost.ThemixtureofacTAGs found synthesized byE.solidaginis concentrations ofacTAGs, presentonlyduringwinter, appeartobe neutral lipidpoolismadeupoftypicallcTAGs. Thesehigh the goldenrodgallfly, Eurostasolidaginis comprise 36%oftheneutrallipidpooloverwinteringprepupae have lowerenergydensitythanlcTAGs. HerewereportthatacTAGs reported inanimals,butarenotaccumulated,likelybecausethey Trace amountsofacetylatedtriacylglycerols (acTAGs) havebeen Most animalsstoreenergyaslong-chaintriacylglycerols(lcTAGs). Katie E.Marshall freeze-tolerant Seasonal accumulationofacetylatedtriacylglycerolsbya RESEARCH ARTICLE © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,1580-1587doi:10.1242/jeb.099838 Health Network, Toronto,Health Network, ONM5G1L7,Canada. Vancouver, BCV6T1Z4,Canada. Zoology, BritishColumbia, of University of *Present address:Department Ontario, Western London,ONN6A5B7,Canada. University of INTRODUCTION ABSTRACT Author ([email protected]) for correspondence Biology, Ontario, Western London,ONN6A5B7, of University of Department E. solidaginis Instead ofhavingthreelong-chainfattyesters(asinlcTAGs), 2 Department of Psychology, Ontario, Western London, of University of Department is frozeninthefield,anddepressesmeltingpointof has asignificantlylowermeltingpointthanequivalent 3 Department of Pharmaceutical Sciences, University Pharmaceutical of of Department 1, *, RaymondH.Thomas 5 and BrentJ.Sinclair ‡ Present address:Grenfell Campus, Memorial and arenotfoundinotherfreeze- 4 Ontario CancerInstitute, University 5 Department of Chemistry, of Department , whileonly17%ofthe 1,§ Cervus nippon) 2,‡ , ÁronRoxin 3,4 lcTAGs andfreefattyacids(FFAs) hadnoeffect. pool; and(3)acTAGs reducedthemeltingpointof salinewhile solidaginis lcTAGs duringoverwintering conditions;(2)repeatedfreezingof that: (1)acTAGs remained liquidattemperaturesthatwouldsolidify decline totracequantitiesduringmetamorphosis.We alsofound species accumulatesacTAGs beforewinter, andthattheseacTAGs solidaginis, weexaminedtheneutrallipidpoolandfoundthatthis freeze (Mugnanoetal.,1996;Salt,1959). that wouldnormallycauselcTAGs tosolidifyandthecytoplasm droplets thatremainliquidwhenthecellsarechilledtotemperatures winter, Lee, 2003;Williams andLee,2005;Williams etal.,2004).Over exclusively ongallsitinduces converts acTAGs intolcTAGs inthespring. acTAG concentrationfollowingrepeatedfreezingbouts,and rather thantypicallcTAGs –priortooverwintering,increases 1855), accumulatessignificantquantitiesoftheserareacTAGs – Here wereportthatthegoldenrodgallfly, hypotheses foraphysiologicalroleofacTAGs inbiologicalsystems. are presentinafewanimals,wenotawareofanyclear diacylglycerol (DAG)(Durrettetal.,2010).AlthoughtraceacTAGs an acetylgroup,ratherthanathirdlong-chainfattyacid,to via anunusualdiacylglycerolacyltransferase(DGAT) byesterifying cryoprotection isnotknown, et al.,1993;Mugnano1996).Althoughthemechanismof this cells nucleateiceformationandsurviveintracellularfreezing(Lee during thegrowingseason(IrwinandLee,2003).Thesefatbody fuelled bylipidsandcarbohydratesaccumulatedinthefatbodycells Overwinter metabolismandsubsequentspringreproductionareboth phosphatidylcholine (Bennettetal.,1997;PruittandLu,2008). temperatures for winter, resultinginincreasedmembranefluidityatlow remodel theirmembranephospholipidsandlcTAGs inpreparation 1997; Leeetal.,1995;Salt,1959). hardiness hasbeeninvestigatedforover50 seed oils(Durrettetal.,2010). Yang etal.,2004)],butpredominate(92%bymoles)in investigated [<0.1%bymass(Hanetal.,1999;Limb small proportionofthetotallipidpoolintwomammalspecies ( (Limb etal.,1999)andtheseedsofburningbushplant (Yang etal.,2004),theuddersofdomesticcows( (TLC-FID) revealed thatoverwintering Thin-layer chromatographycoupled toflameionizationdetection acTAGs lipidpoolof predominate intheneutral RESULTS Euonymus alatus)(Durrettetal.,2010).acTAGs constituteavery , EricK.Y. Chen In theprocessofinvestigatingoverwinterenergy storagein Eurosta solidaginis E. solidaginis increased theamountofacTAGs intheneutrallipid via increased abundanceofunsaturatedfattyacidsand 5 pre-pupae arefreezetolerant,andtheircold , JasonC.L.Brown is anobligateplantparasite,feeding E. solidaginis Euonymus alatus canadensis Eurosta solidaginis E. solidaginis Eurosta solidaginis fat bodycellscontain

years (Collinsetal., synthesises acTAGs 1 , Bos primigenius) E. solidaginis E. pre-pupae had (Irwin and pre-pupae E. alatus (Fitch E. E.

The Journal of Experimental Biology 721 of severalsimilarlipidswithpeaksthatrangedbetween654.5and (ESI-MS) showedthattheseisolatedsamplescomprisedamixture (Pruitt andLu,2008).Electrosprayionization-massspectrometry (C16:1) andpalmiticacid(C16:0;supplementarymaterialFig. predominantly oleicacid(C18:1),followedbypalmitoleic contained fattyacidspreviouslyreportedin spectrometry (GC-MS)revealedthatitwasaclassoflipid chromatography purification,andgaschromatography-mass elute withcholesterol(supplementarymaterialFig. samples withcholesteroltoconfirmthattheunknownlipiddidnotco- of storagelipidsin cholesterol concentrationisthreeordersofmagnitudelowerthanthat because ofthedilutionrequiredtoresolvestoragelipidpeaks(e.g. solidaginis, suchascholesterolanddiacylglycerol,werenotdetected The two oleicacidesters(Fig. ammonium adductof3-acetyl-1,2-diacyl-sn-glycerol(acTAG) with ( eluted betweenFFAs andcholesterol[retardationfactor neutral lipidpoolwascomprisedofanunknowncompoundthat contributed anadditional25%(bymass).Theremaining46%ofthe comprised only29%ofneutrallipids(bymass)whileFFAs an unusualneutrallipidcomposition.Inmid-December, lcTAGs acTAGs, withthefollowing peaks[deuteratedchloroform(CDCl supported theidentificationbyESI-MS/MSofunknownlipid as methyl, andcorroboratedtheresultsofESI-MS/MS.ProtonNMR 2.08 et al.,1999)characterizedbythepresenceofasingletpeak at sample exactlymatchedthatpreviouslyreportedforacTAGs (Limb RESEARCH ARTICLE 600 at 680.56m (Kim etal.,1999).TheESI-MSspectrumshowedadominantpeak ESI-MS/MS analysis afterpurifyingacTAGs byTLC,oleicacid Fig. 1H), 4.28(t,J positions oftheglycerolbackbone (Fig. distinct long-chainfattymoieties esterifiedatthe mixture ofanunusualclass neutrallipid,acTAG, withseveral GC-MS. Thus,theunknowncompound detectedbyTLC-FIDisa contained avarietyoffattyesters andcorroboratedtheresultsof proton integrationsconfirmedthatthepurifiedacTAG samples 4H), 1.40–1.22(br, m,42H)and0.92–0.86 (m,6H).Fractional 2.36–2.29 (m,4H),2.08(s,3H),2.05–1.986.4H),1.67–1.58 (m, R f The unknowncompoundwasisolatedbypreparativethin-layer L-I thin-layerchromatographycoupledtoflameionization nuclearmagneticresonance TLC-FID R NMR MgSO long-chaintriacylglycerol(moleculeswiththreefattyacids freefattyacid gas-chromatographycoupledtomassspectrometry lcTAG GC-MS FFA electrosprayionizationmassspectrometry/massspectrometry ESI-MS/MS CDCl acetylatedtriacylglycerol(moleculeswithtwofattyacids acTAG List of symbolsand abbreviations List of )=0.18–0.25; f MHz]: S2A). Basedonthefattyacidprofiles identifiedbyGC-MSand 1 m ppm correspondingtotheterminalprotonsonacetyl H nuclearmagneticresonance(NMR)spectrumofthispurified / z 3 4 (as ammoniumadducts,supplementarymaterialFig. δ / 5.37–5.32(m,2.8H),5.30–5.241H),4.32(t, z retardation factor magnesium sulfate deuterated chloroform =4.3, 1H),4.17(d,J ([M+NH i.1A].Otherneutrallipidslikelypresentin Fig. detection backbone) greater than12carbonslongesterifiedtoaglycerol position) backbone, withanacetylgroupesterifiedtothethird greater than12carbonslongesterifiedtoaglycerol E. solidaginis 4 ] +

1B; supplementarymaterialFig. ), whichESI-MS/MSindicatedwasthe ) (Yi andLee,2005),wespiked =6.1, 1H),4.14(d,J

1B; supplementarymaterial

S1B). E. solidaginis sn-1- and-2- =6.1, 1H),

S2B,C). J

S2A) S2B) =4.3, 3 E. ), : acTAGs detectedwithin acTAGs werenotpresent inanyoftheseotherinsects,norwere feeds onE.solidaginis caterpillar thatalsoformsagallon of accumulationandphysicalproperties ofacTAGs in conditions, andpromptedfurther investigationsofthelocation,pattern even comparedwithotherinsects capableofsurvivingcold suggested thatthepresenceofacTAGs in remaining polarlipids,whichwillnotmigrateinthesolventsystem. in 70:30:05benzene:chloroform:formicacid(v/v/v).Theoriginincludesany free fattyacids(FFA) andacetylatedtriaclyglycerols(acTAG) afterseparation components includedmostly18:1,16:1and16:0fattyacids. solidaginis To examinewhetheracTAGs arefoundinthemembraneof cold-hardy lepidopteranlarvae,aparasitoidwaspof neutral lipidsfromotherDiptera,freeze-tolerantbeetlesandwasps, these animalsalsocontainedthisrareclassoflipid.We extracted of severalcold-hardyandfreeze-tolerantinsectstoinvestigatewhether pool ofE.solidaginis,wecharacterizedtheneutrallipidcompositions Once thepresenceofacTAGs wasidentifiedwithintheneutrallipid material Fig. palmitic (C16:0)andpalmitoleic(C16:1)acids(supplementary (C18:1) isthepredominantesterifiedfattymoietyfollowedby composition of Eurosta solidaginis. Fig. ESI-MS/MS and retardation factor. (B)GeneralstructureofacTAGs asdeterminedbyGC-MS, acTAGs intheplasmamembrane notfound are preferentially insects acTAGs orfreeze-tolerant notfound are inothercold-hardy

.Characterizationofthemajorneutral lipidclassesfoundin 1.

A B and thusmayplay aroleinmembranefluidity, weused The JournalofExperimentalBiology(2014)doi:10.1242/jeb.099838

−2 Signal (mV)

E. solidaginis S2A) (Bennettetal.,1997). 0 2 4 6 1 H NMR.ESI-MS/MSandGC-MSindicatedfattyacid 1.0 (A) ChromatogramfromTLC-FIDoftheneutrallipid in thegallduring the summer(Tables . . . . 0 0.2 0.4 0.6 0.8 showing: long-chaintriaclyglycerols(lcTAG), S. canadensis R S. canadensis,andabeetlethat O f lcTAG O O gall tissue.Theseresults E. solidaginis O FFA O O acTAG E. solidaginis

E. solidaginis Origin + polar lipids was unique, R f ,

1, 2). 1581 , a E. .

The Journal of Experimental Biology 1582 RESEARCH ARTICLE neutral lipidsoverthistimeperiod(FFAs: We foundsignificantchangesinthemolarquantityofallthree from Augustuntilmid-MarchandquantifiedacTAGs byTLC-FID. We collected localized inthemembrane enriched homogenate,indicatingthatacTAGs arenotselectively fraction comparedwiththeabundanceoftheselipidsinun- acTAGs relativetoFFAs orlcTAGs inthemembrane-enriched fraction. However, therewasnoincreaseintheabundanceof enrichment ofplasmamembraneinthemembrane-enriched fraction. Plasmamembranemarkersindicateda9.2-fold differential centrifugationtoproduceaplasmamembrane-enriched lcTAGs: acTAGs accumulated are priortowinter onset offreezetoleranceinearlyautumn(Fig. 3A).Thelargest accumulationofacTAGs coincidedwiththe Fig. fluidity. that theylikelydonotcontributetomodifyingcellmembrane acTAGs arestoredwithinothercellularcompartmentsand proportion oftheneutrallipidpool( significant interactionbetweenlipidtypeandmonthofcollection on E. solidaginis (lcTAGs, FFAs and acTAGs), ‘+’ indicatescl (lcTAGs, FFAsand acTAGs), temperatures withoutfreezing(freeze avoidi surviveextracellular freezingofbodyfluids(freezetolerant)Cold tolerancestrategyindicateseither thenaturalabilityto Table detected, and‘Trace’indicates<10%ofthetotalneutrallipidpool. [long-chain triacylglycerols(lcTAG),freefattyacids(FFA)and temperatures withoutfreezing(freezeavoidi surviveextracellularfreezingofbodyfluids(freezetolerant)Cold tolerancestrategyindicateseitherthenaturalabilityto galls Table Mordellistina convicta Mordellistina

Eurytoma Pyrrharctia isabella Gryllus pennsylvanicus Drosophila melanogaster Eurosta solidaginis Solidago canadensis Species Chrysomela aenicollis Chrysomela Choristoneura fumiferana Species Epiblema scudderiana obtusiventris   2. Neutrallipidcomponentsofseveral 1. Neutrallipidcomponentsofti F 9,39 E. solidaginis =4.48, synthesized acTAGs seasonally. We alsofounda aliqiie oepea odlia Lat Coleoptera, Gall inquiline Parasitoid of Obligate NA

Obligate A S. canadensis ssociation with P E. solidaginis parasitoid parasitoid <0.001; acTAGs: F pre-pupae fromthefieldeverymonth Fg 2). (Fig. Order, Family eiotr,Acide ia ntrlra Freezetolerant (Layne Finalinstarlarva Lepidoptera, Arctiidae Orthoptera, Gryllidae Diptera, lra Freezeavoiding(Hanand Coleoptera, 2ndinstarlarva Lepidoptera, Tortricidae Chrysomelidae re,Fml Lf tg sae Co Lifestageassayed Order,Family F 2,24 ssue fromgallsontheplant ng), oraverylimitedabilitytosurvive ng), oraverylimitedabilitytosurvive ear presenceofthe lipid, ‘–’indicatesnot detected,and ‘Trace’ indicates <10%of Hymenoptera, itr,Tprtde r-ua Freezetolerant Pre-pupa Diptera, Lepidoptera, A

These resultssuggestthat trls seaee n.a. sterales, Asteraceae =9.11, Eurytomidae Olethreutidae insectspeciesnotassociatedwith F 9,39

9,39 3A), indicatingthat P =0.001), whereby =9.16, =8.25, acetylated triacylglycerols (acTAG)], ‘+’ indicates clear presence of thelipid, acetylated triacylglycerols(acTAG)],‘+’indicatesclearpresence ‘–’ indicatesnot Life stageassayed A A A dult Chill dult Chill dult Freeze P P

<0.001; <0.001; aeisa av Freezetolerant(Baust Late instarlarva Pupa Freeze avoiding (Baust A

dult ntrlra Freezetolerant(Baust e instarlarva Solidago canadensis F prolonged freeze(Tukey’s and inducedanear-significant increaserelative toasingle with unfrozencontrolindividuals(Tukey’s freezing significantlyincreasedtheamountofacTAGs compared experiment, soallcontrolindividualswerepooled.Repeated control individualssampledatthebeginningorendof pooled intheanalysis.Therewasalsonodifference between exposures every5 (Fig. abundance concomitantlydecreasedinanequimolarratio lcTAG abundanceincreasedinthespring,whileFFA andacTAG of lcTAG remainedunchanged(Tukey’s increased significantlybetweenlatesummerandwinter, theamount proportion (Fig. lcTAGs andFFAs eitherremainedconsistentordecreasedin acTAGs increasedinproportionfromAugusttoSeptemberwhile neutral lipidcompositionamongindividualsthatreceived12 synthesized followingcoldexposure.Therewerenodifferences in with overwintering,weinvestigatedwhetheracTAGs were Because increasedacTAG concentrationappearedtobeassociated acTAGs inquantitywithrepeated freezing increase sub-zero temperature(chillsusceptible). sub-zero temperature(chillsusceptible). 2,26 =7.559, 3A,C). odtlrnesrtg lTG F acTAG FFA lcTAG Cold tolerancestrategy et al.,1999) Bauce, 1995) 2011) (MacMillan andSinclair, (MacMillan etal.,2009) 2012) Solidago canadensis susceptible susceptible Chill susceptible n.a. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.099838 et al.,1979) et al.,1979) 2005) Williams andLee, and Storey,2008; freezing) (McMullen (including intracellular et al.,1979) Storey, 2008) (McMullen and tolerant dtlrnesrtg lTG F acTAG FFA lcTAG ld tolerancestrategy P =0.003, 3B). WhilethequantityofbothFFAs andacTAGs , andseveralinsectspeciesassociatedwiththe

(Boychuk, days orevery10 i.4). Fig. post hoc

There wasnosignificantdifference , theabilitytosurvivelow , theabilitytosurvivelow – + – – + – + – – + – – + Trace Fortheneutrallipidcomponents Fortheneutrallipidcomponents rc – + Trace rc – + Trace + + rc – + Trace + Trace Trace

– – days, sothesesampleswere thetotalneutrallipid pool. test, post hoc post hoc P =0.064; ANOVA, test, test, P P =0.085). =0.002), –

h

The Journal of Experimental Biology ( FFA contentinpre-pupaethatreceivedrepeatedfreezing quantity remainedunchanged,therewasatrendtowardsdecreased prolonged freeze(Tukey’s in acTAG quantityrelativetocontrolvaluesafterasingle increase inacTAGs. (B) Lipidprofileof9.2-foldenrichedmembranefraction,showingnorelative pre-pupae, showinglipidcomponentsincludinglcTAGs, FFAs andacTAGs. acTAGs inE.solidaginis Fig. freezing withcontrolvalues, RESEARCH ARTICLE Proportion of neutral F 2,26

lipid pool Amount of lipid (µmol –1) Differentialcentrifugationshowsno membraneassociationwith 2. =2.976, 10 12 0.1 0.2 0.3 0.4 0.5 0.6 0 2 4 6 8 0 August A B August Signal (mV) * lcTAG acTAG FFA −2 −2 P September A c 0 2 4 6 0 2 4 6 =0.069, Tukey’s lcTAG acTAG FFA 1.0 1.0 B A * , *** a B October . (A) Lipidprofileofrawhomogenatefromwhole . . . . 0 0.2 0.4 0.6 0.8 α . . . . 0 0.2 0.4 0.6 0.8 September ** post hoc a,b , November B *** P =0.056). post hoc * B,C a,b , *** R December test, f IcTAG IcTAG Absolute change in lipid P test comparingrepeated =0.669). WhilelcTAG µ –1 * ( mol fly ) January

, FFA

a,b FFA −2 −4 *** * B 0 2 4 acTAG acTAG C B,C , a ** February FFA Neutral lipidclass Origin + polar lipids Origin + polar lipids * acTAG , a,b *** B March

* , a,b *** B β lcTAG April A,C b,c c ** acTAGs (1 had thepotentialtointeractwithaqueoussolutions.We foundthat solutions usingnanolitreosmometrytodeterminewhetheracTAGs explored theeffects ofacTAGs onthemelting point ofsaline could havefurther, potentiallycryoprotective,properties.We solidaginis the seasonalaccumulationofacTAGs infieldsamplesof (Pruitt andLu,2008). together makeup80%ofthelcTAG compositionof (5°C) andtripalmitin(66°C)(KnotheDunn,2009),which mixture issubstantiallylowerthanthemeltingpointsoftriolein within low meltingpointscomparedwithnaturalmixturesoflcTAGs individual acTAG compoundswithinthepurifiedmixtureeachhave no effect (F lcTAGs (depressionof0.02±0.05°C)andFFAs (0.02±0.02°C)had point ofsalineby1.21±0.03°C,whilethesameconcentrations endotherm between acTAGs isolatedfrom calorimetry toinvestigatethethermalprofileofmixture 2009; Ribeiroetal.,2009).We useddifferential scanning individual componentsofthesemixturesmelt(KnotheandDunn, therefore, showmultipleendothermsbetween+5and+35°Cas mixtures oflcTAGs containavarietyoffattyestermoietiesand, with thatoflcTAG mixturespresentin our purifiedacTAG sampleandcomparedthismeltingpointprofile unusual thermalproperties,wecharacterizedthemeltingpointof To explorethepossibilitythatacTAGs in Here weshowthatoverwintering reported astracecomponents(<1%bymass)ofneutrallipid mass) andFFAs (bymoles).AlthoughacTAGs havepreviouslybeen unusual neutrallipidcomposition,predominatedbyacTAGs (by point of salinesuspensions point of acTAGs have low themelting meltingpointsanddepress DISCUSSION , A γ c ***

The relativelylowmeltingpointrangeofthepurifiedacTAGs and E. solidaginis.Specifically, themeltingpoint oftheacTAG in eachneutrallipidclass(µmol Data aremeanproportions±s.e.m.; neutral lipidclasscompositionbetweenAugustandSeptember. solidaginis. Fig. (*P for FFA, uppercaselettersforacTAG, andasterisksforlcTAG test. Differences amongmonths aredenotedbylowercaseletters statistically different quantities inanANOVA withTukey’s same neutrallipidclass,pointswithdifferent symbolsindicate pupation; tolerance accordingtoBennettetal.(Bennettal.,1997); relation tolifestage[indicatedbyarrows: dramatically fromlatesummeruntilearlyspring(2011–2012) in April. Dataaremeans±s.e.m.; <0.05; ** mol

during wintermonthsledustohypothesizethatacTAGs The JournalofExperimentalBiology(2014)doi:10.1242/jeb.099838 .SeasonalchangesinacTAG concentrationofE. 3. 2,16 =387, l − γ 1 , eclosion].Dataaremeans±s.e.m.; P in 1 (A) Theneutrallipidpoolof <0.01; *** P − <0.001). 35 and− Osm NaCl E. solidaginis, P <0.001). (B)Proportionalchangesin 3C(i.5).Thissuggeststhat 13°C (Fig. aq E. solidaginis

n fly solution) depressedthemelting =5. − 1 ) betweenmid-Marchandearly n and foundasinglebroad =5. (C)Quantitativechanges E. solidaginis E. solidaginis E. solidaginis α , onsetoffreeze pre-pupae havean n =5. Withinthe E. solidaginis shifts may have post hoc . Natural β , 1583 E.

The Journal of Experimental Biology * 1584 RESEARCH ARTICLE indicates significantdifference relativetocontrol(Tukey’s square meansincorporatingtheeffect oftotallipidmassareplotted.Asterisk (Fig. properties oftheresultinglipid fromtypicallcTAGs orDAGs glycerol moiety. This3-acetylesteristhekeymoietythatalters the sn-2 positionsandacetate-esterified atthe during iceformationatlowtemperatures. interactions betweenacTAGs andthewatermolecules involved the lowmeltingpointrangeofacTAGs, couldallow hypothesize thatthisincreasedhydrophilicity, inconjunctionwith similar tothatofaphospholipid(HondaandKozawa,1992). We direction tothetwofattymoietiesandresultingstructure is acTAGs indicatesthat theacetylgrouporientsinanopposite moiety. A modellingstudyofthethree-dimensionalstructure presence oftheacetylgroupratherthanathirdlong-chainfatty ester hydrophilicity ofacTAGs, comparedwithlcTAGs, becauseofthe saline solutionswaslikelyduetotherelativelyincreased solidaginis observation ofclearlipiddropletswithinfrozenfatbodycells of low meltingpointprofileofacTAGs explainsSalt’s early at whichstructurallysimilarlcTAGs wouldsolidify. Therefore,the these lipidscouldremainintheliquidstate may havebeencatabolizedtoproduceATP. (Churchill andStorey, 1989),sowesuspectthatadditionalFFAs stress inE.solidaginis increase inFFAs. Repeatedfreeze–thawcyclesleadtoenergetic following repeatedfreezing,wedidnotobserveaconcomitant lcTAGs. ItiscuriousthatwhileweobservedanincreaseinacTAGs freezing stress,andthatacTAGs aresynthesizedfromavailable acTAG synthesisisinduced,evenduringmid-winterdiapause,by content increasedafterrepeatedfreezing,whichsuggeststhat diet. Undertemperature-controlledconditions,wefoundthatacTAG appears thatthepre-pupaedonotacquireacTAGs directlyfromthe acTAGs areabsentfromthegallsthathouse ubiquitous featureoffreeze-tolerantorcold-hardyinsects.Because dense lcTAGs forthispurpose(Canavosoetal.,2001). winter; anunusualstrategy, asanimalsgenerallyusemoreenergy- accumulated large quantitiesofacTAGs forenergy storageover acTAGs. We observedthatfield-collected other reportofananimalwithaneutrallipidpooldominatedby 1999; Kimetal.,Limb1999),weareunawareofany fractions isolatedfromdeerantlersandbovineudders(Hanetal., heachat− 12 Fig. P <0.05). Dataaremeans±s.e.m.; acTAGs havetwolong-chainfattyacidsesterifiedat the The lowmeltingpointrangeoftheacTAG mixturesuggeststhat We alsodeterminedthatthepresenceofacTAGs isnota

.FreezingE.solidaginis 4. 1B) (Kimetal., 1999;Limbetal.,1999).Incontrast toplants, (Salt, 1959).Finally, thedepressioninmeltingpointof

20°C) inducesadditionalproductionofacTAGs. Amount of lipid (µmol fly–1) 10 0 2 4 6 8 Control (as measuredbyATP andglycogencontent) Experimental treatment pre-pupae repeatedly(10exposuresof 10 ×12h n =5–10. * 1 ×120h in vivo sn-3 positionofthe lcTAG acTAG FFA E. solidaginis post hoc E. solidaginis at temperatures Least test, sn-1 and in vivo , it E. body cells(Salt, 1959)facilitateslipid–cytosolic interactions,and freeze. We alsonotethatthepresenceofliquidlipid droplets infat components suchasintracellular membranesas could preventcontactbetween icecrystalsandkeycellular and canmodulateicecrystalformation, wehypothesizethatacTAGs solidaginis lcTAGs co-localiseinthelipiddropletsfatbody cellsof localized withincellmembranes,weexpectthatacTAGs and of theacTAGs, theirnon-polar nature,andthefactthattheyarenot not yetunderstood(SinclairandRenault,2010).Giventhequantity described mainlyinfatbodycells,butthemechanismsinvolved are this toxicityisunknown. triolein (lcTAG with18:1fattyacids esterified;dottedline). neutral lipidchangessuggeststhat which synthesizeacTAGs supercooling pointof Vertical linesindicatemeltingpointofacTAGs (dashedline);whole-body Fig. are toxictoinsects(Haradaetal.,2000).How toxicological riddle:inlarge quantity, FFAs (particularlyoleicacid) The large quantityofFFAs foundin point ofsaline,whichreducesthelikelihoodacryoprotective role. backbone oflcTAGs. In addition,FFAs didnotdepressthemelting after hydrolysisoffattyestersatthe accumulation mayresultfromthesynthesisofacTAGs fromlcTAGs accumulation isrelatedtofreezetolerance.Instead,FFA observed FFA accumulationinautumn,wedonotbelievethatthis mass), withonlysmallquantitiesofFFAs andDAGs.Whilewe compositions aretypicallydominatedbylcTAGs (over90%by in additiontothatofenergy storage.Insectneutrallipid energy storageforE.solidaginis 2010). Thus,acTAGs arenotanenergetically favourable modeof of theinclusiononlytwolong-chainfattymoieties(Durrettetal., conflated acTAGs andlcTAGs. species (Williams etal.,2011). Thus,previous workhaslikely nonpolar compoundsandthereforedonotdistinguishamonglipid glycerolipids. Gravimetrictechniques,bycontrast,dissolveall backbone (Williams etal.,2011), maskingthe presenceofother almost alwaysinvolvehydrolysisoffattyacidsfromtheglycerol vanillin orenzymaticassays)assumethatlcTAGs predominate,and because thetechniquesforstudyinglipids(i.e.gaschromatography, of theseabundantacTAGs. We suspectthat thisoversightarises et al.,2004)andthereforeitissurprisingthatthisthefirstreport decades (Collinsetal.,1997;Lee1995;Salt,1959;Williams produced byexistingoilseedcrops(Durrettetal.,2010). synthesize low-viscosityacTAGs fromthehigher-viscosity lcTAGs pathway responsibleforthisconversioncouldyieldamethodto from existinglcTAGs. We suggestthatidentifyingthemetabolic Survivable intracellulariceformationininsectshasbeen acTAGs containlessenergy perunitmassthanlcTAGs because The biologyof

.HeatflowduringmeltingofacTAGs extractedfrom 5.

–1 . BecauseacTAGs remainfluidatsubzerotemperatures Heat flow (W g ) The JournalofExperimentalBiology(2014)doi:10.1242/jeb.099838 −0.15 −0.10 −0.05 0 4 3 2 1 02 30 20 10 0 −10 −20 −30 −40 E. solidaginis E. solidaginis de novo,stoichiometryoftheseasonal Temperature (°C) (dot-dash line);andmeltingpointof , whichsuggestsaroleforacTAGs has beeninvestigatedformany E. solidaginis sn-3-position ontheglycerol E. solidaginis E. solidaginis produces thislipid E. solidaginis does presenta E. solidaginis resists E. .

The Journal of Experimental Biology seasonal profile,pre-pupaewere collected andthenincubatedin0.2 2 0.2 quantity followingrepeatedfreeze–thaw cycles’,below)werekeptin − liquid nitrogenandstoredin0.2 from gallswithin2 for theseasonalprofile.Pre-pupaeprofilewereextracted autumn 2009fortherepeatedfreezingexperiment,andwinter2011–2012 directly fromthefieldwasperformed.Collectionstookplacein late A seasonalprofileconsistingofmonthlycollections plants inoldfieldhabitatsLondon,ON,Canada(43°00′ Spherical gallscontaining stress. quantities ofacTAGs areproducedfollowingrepeatedfreezing utility inlow-temperatureenvironments,andthatincreased have unusualpropertiesforastoragelipidthatmayincreasetheir acquisition andlossoffreezetolerance.We alsoshowthatacTAGs acTAGs, andthatthetimingoftheseeventscoincidewith terrestrial animalidentifiedtodateaccumulateandconsume Here wehaveshownthatoverwintering accumulation ofacTAGs afterrepeatedfreezingstress(Fig. frozen duringwinter. However, wesuggestthattheincreased such asmaintenanceofaliquidlipidpooloraccesstoFFAs while may accumulateseasonallyforapurposeotherthancryoprotection the large accumulationofacTAGs in lipids inE.solidaginis of increased abundanceofacTAGs duringtheonsetoffreezetolerance cryoprotection. BasedonthephysicalpropertiesofacTAGs andthe may thereforefavourtheevolutionoflipid-basedintracellular RESEARCH ARTICLE individuals within 7 Eppendorf tubes inconstantlightat20°C.Pupation occurredinall Field collection of Field collectionof MATERIALS ANDMETHODS Conclusions cell linesderivedfrom difficult. However, itcouldbepossibletodevelopandmanipulate manipulation ofintracellularlipidpools further exploration.Asaunivoltine,gall-dependentspecies, biochemistry inanimals. acTAG biosynthesisinE.solidaginis quantities ofunusuallipidsisunlikely, detailedinvestigationof unknown metabolicpathwaythatproducesmilligram-scale physiological stressesoffreezing.Whiletheexistencean process duringpreparationforwinter, andinresponsetothe accumulate asabyproductofsomeother, unknownmetabolic metabolism (StoreyandStorey, 1985).Second,acTAGs may pre-pupae likelyrelyoncarbohydrate(ratherthanfattyacid) inconsistent withthishypothesis.Inaddition,frozen vitro. acTAGs onindividualcellularcomponentscouldbeexplored be identified.We alsosuggestthatthecryoprotectiveeffects of solidaginis be usedtoknockdownacTAG accumulationin be exploredinmorereadilymanipulatedorganisms, orforRNAito accumulation couldallowthecryoprotectiveeffects ofacTAGs to identification ofthemetabolicpathwaysassociatedwithacTAG role ofacTAGs insurvivalofintracellulariceformation.Alternately,

80°C. Pre-pupaedestinedforlaboratory manipulations(see‘acTAG months priortoexperimentation. Forthespringcollectionsin We identifytwonon-cryoprotectivealternativeexplanationsfor Our hypothesisforacryoprotectiveroleacTAGs requires E. solidaginis,wehypothesizeacryoprotectiverolefortheserare ml Eppendorftubes(withanairhole) inconstantdarknessat0°Cfor larvae intheautumnifappropriateenzyme(s)could

days, andthenpupae weresnap-frozenafter2 h ofcollection,snap-frozenbydirectimmersionin E. solidaginis E. . E. solidaginis E. solidaginis

ml Eppendorfmicrocentrifugetubes at were collectedfrom fat bodycellstodeterminethe could yetidentifynovellipid E. solidaginis E. solidaginis E. solidaginis E. solidaginis . First,acTAGs E. solidaginis N, 81°15′ Solidago ex situE. is thefirst will be in vivo

weeks. 1C) is taken spp. W).

ml in (Williams etal.,2011). layer chromatographycoupledtoflameionizationdetection(TLC-FID) were snap-frozen.TheacTAGs of eachindividualwerequantifiedbythin a mass/volumebasis. purified, pooled,andthenweighed.A standardcurvewasthenproducedon at 80°C(heldfor2 resolution column,andrunonthe following temperatureprofile:injection equipped withaFinniganMAT 8400MSfittedwitha DB-23high and organic, 1 μleach)wereinjectedinsplitlessmodeonaVarian GC3500 and theresiduewasresuspendedin50 extracted with3×500 distilled water(0.8 (4 HCl (Sigma,StLouis,MO,USA)wasaddedtothedriedacTAG samples 3.0 cms scudderiana, hydroxytoluene (v/v).Next,0.25%KCl in 2.5 Folch extractionswereconductedbygrindingwholebodiesof All individualseclosedwithin28 Bicalho etal.(Bicalhoal.,2008).Briefly, 200 according tothecombinedmethodsofChowetal.(Chowal.,1989) and Gas chromatographycoupledtomassspectrometry(GC-MS)wasconducted During collectionsof removed at plates elutedwiththedescribedsolventsystem.A bandofsilicawas The acTAGs werepurifiedfromFolchlipidextractsusing200 assessed usingTLC-FIDasabove. acTAGs weredriedundernitrogenwhileheatingat70°C.Puritywas atmospheric air, 160 Supelco FAME mixC8-C24). commercial standards (Supelco37componentmix, SupelcoPUFA No.3, 2008) andbycomparisonofretention timesandmassspectraobtainedfrom The resolvedpeakswereidentified fromliteraturereferences(Bicalhoetal., quantify acTAGs, theacTAGs from10individual 60% oleicacid,20%palmiticpalmitoleicacidforFFAs). To standards (60%triolein,20%tripalmitin,tripalmitoleinforlcTAGs, area andstandardcurvesofknownconcentrationslcTAG andFFA Quantification ofneutrallipidcomponentswasbasedonintegratedpeak unknown lipidpeakwasnotcholesterol(supplementarymaterialFig. E. solidaginis times toknownstandards(supplementarymaterialFig. Analyzer (ShellUSA)atthefollowingsettings:flowrateof2 2011). TLC-FIDwasperformedusingan Iatroscan MK-6TLC-FID with 70:30:0.5benzene:chloroform:formicacid(v/v/v)(Williams etal., chloroform andthecomponentswereseparatedonchromarods(ShellUSA) while heatingat70°C.Thecrudelipidextractswereresuspendedin1.7 heated (to70°C).Theorganic layerwascollectedanddriedundernitrogen to 200°Cat1.5°C was removedwithMgSO stage. for obtained fromalaboratorycolonymaintainedat22°C.Inallcasesexcept controlled incubatorinconstantdarknessat2°Cfor1 Forest Service,SaultSte.Marie,Ontario,Canada)andkeptinatemperature- (diapausing strain)wasobtainedfromInsectProductionServices(Canadian immediately assayedforlipidcontent. Pyrrharctia isabella TLC-FID Gas chromatography coupled tomassspectrometry Gas chromatography Other insectspeciesassayed acTAGsPurification of were measured. mg), andthesolutionwasheatedat90°Cfor30 G. pennsylvanicus ml of2:1chloroform:methanol(v/v)containing0.1%butylated −1 , andneutrallipidswereidentifiedbycomparingpeakretention R The JournalofExperimentalBiology(2014)doi:10.1242/jeb.099838 f =0.18–0.25 andwassuspendedinchloroform.Trace water lipids wasalsospikedwithcholesteroltoconfirmthatthe Eurytoma obtusiventris Drosophila melanogaster

min

min), temperaturerampedto180°C at5°C

ml) wasadded,andthenon-polarcomponentswere were collectedfromthesameoldfieldhabitatsand μl ofhexane.Thehexanewasdriedundernitrogen,

−1 , theassayedlifestagewasoverwintering ml , thento240°Cat10°C E. solidaginis min 4 . Thesuspensionwasfilteredandthepurified − 1 for hydrogen,andscanningspeedof

days ofincubation,atwhichtimeadults μl ofhexane.Samples(bothaqueous (aq) , in September2011, and was addedandthesolution Mordellistina convicta Gryllus pennsylvanicus μl of0.5 Choristoneura fumiferana

min −1

E. solidaginis

min. Aftercooling, month beforelipids and heldfor3

mol S1A). A sampleof l − E. solidaginis 1

methanolic- min

l μm silica

Epiblema min − 1

1585 S1B). , then −

were were 1 min. and

for ml

The Journal of Experimental Biology rate of0.1°C 3530, Wurzburg, Germany)with1:1methanol:water (v/v)withacooling freezing experimentswereconductedinrefrigeratedbaths(LaudaProline 1970) withfourdensitylayers(1.02,1.08,1.14and1.18 further purifiedusingadiscontinuoussucrosegradient(McKeelandJarett, expressed relativetoproteinconcentrationmeasuredbyBradfordassay. measured accordingtoTaussky andShorr(Taussky andShorr, 1953),and a Varian Inova600 centrifugation at28,000 purified plasmamembrane.Thebandat1.14 [NaCl error waslessthan0.05 1586 solvent signalofCDCl chemical shiftsarereportedinppmandcalibratedagainsttheresidual RESEARCH ARTICLE three individualsusingTLC-FID. nitrogen andstoredat for 24 using dropletsof 1 Melting pointdepressionwasdetermined inaCliftonnanolitreosmometer to +40°Cat5°C using a822eMettlerToledo DSC basedonduplicatemeltcurvesof The meltingpointofa10 12 subjected tooneoftwoexperimentalfreezingregimes:frozenat Freezing experimentscommencedinJanuary2010.Pre-pupae were (Graham, 2002) homogenization buffer) werepurifiedbydifferential centrifugation Crude subcellularfractionsof The positive ionmode,ataflowrateof50 Waters, Milford,MA,USA)equipped withaZ-spraysourceoperatedinthe with atriplequadruplemassspectrometer(MicromassQuattroMicro; The structureofpurifiedacTAGs (inmethanol)wasexaminedbyinfusion m adducts ofacTAG speciesweredetermined usingfullscanmode(200–1800 at 2×e collision energy at30-50 to theliterature(Kimetal.,1999).Theconevoltagewassetat30 spectrometry/mass spectrometry(ESI-MS/MS;80–1000 full scananalysiswasdeterminedviaelectrosprayionizationmass solution of200 1981), byincubating50 plasma membranemarker(ButtersandHughes,1980;NorrisCary, above. described Folchmethod.SampleswereprofiledbyTLC-FID,as the rawhomogenateand1.14 suggesting considerableenrichmentoftheplasmamembrane.Lipidsfrom 1970), showed5′ membrane shouldaccumulatebasedonitsdensity(McKeelandJarett, centrifuged at1000 mixture wasremoved,addedto10%trichloroaceticacid(w/v),and 1 66 ESI-MS/MS Nanolitre osmometry Nanolitre scanningcalorimetry Differential acTAG quantity following cycles repeated freeze–thaw acTAGsSubcellular localization of buffer, andcentrifugedtwiceat8700 at eachdensityboundarywereremoved,resuspendedinhomogenization in Hz. H NMRcharacterization / The fractionwiththehighestenrichmentof5 z h every5or10 mmol ). ThefattyacidcompositionofeachacTAG speciesidentifiedfromthe 1 H NMRspectrumofthepurifiedacTAG mixturewasobtainedusing (aq) − h at0°C,beforebeingsnap-frozenbydirectimmersioninliquid 3 ] in20–30 Mbar. Calibrationwasperformedwithsodiumiodideandmass

l − 1 5 min ′ -AMP for60 .

min − All purifiedfractionswereassayedfor5 mmol -nucleotidase activity9.2-foldhigherthanhomogenate, 1

days, orfrozenat− mol . Afterthefreezingtreatment,pre-pupaewereincubated nl dropletssuspended inType Bimmersionoil. Samples

MHz NMRwithacTAGs dissolved inCDCl −

g 1 − (Ribeiro etal.,2009). 3 l

80°C. TheacTAGs werethenquantifiedinpoolsof for 15 g μl aliquotsofcrudefraction(andhomogenate)ina − l Da.

− at V, sourcetemperatureat80°Candthecollisiongas 1

for 90 1 mg sampleofthepurifiedacTAGs wasmeasured triolein, oleicacid oracTAG mix in 1 δ glycine (pH 72 ppm).Coupling constantsareexpressed =7.26 min at37°C.Analiquotoftheincubation E. solidaginis min. Inorganic phosphateproductionwas min at4°C.Followingcentrifugation,bands

g

ml 20°C forasingleperiodof120

g −

μl 8.5), 200 1 for 10 band wereextractedusingthe

min (15–30 individualsin15 ′ -nucleotidase (~3-fold),was − 1 min at4°Ctopelletthe . Ammoniumorsodium

g mmol

ml m − 1

/ , whereplasma l z ′ − -nucleotidase, a ) withreference 1 MgSO

g

Osm saline

ml − 20°C for 3 − − (NMR 1

100°C V, the 4

) and h. All ml of and and DougHairsinefortechnicalassistance. equipment; andEvelynBoychuk,AnnegretNicolai,RyanGuterman,PaulaPittiock paper; ChrisGuglielmoandJimStaplesforuseoflaboratoryspace numerous anonymousrefereesforconstructivecriticismofearlierversionsthe We thankCarolineWilliams, MarkBernards,JohnSteele,DavidWhartonand overwintering lipid class,andinthesameproportionsaspreviouslyreportedwhole- assuming thatrelativefattyacidcompositionswereidenticalineachneutral The averagemolarmassofeachneutrallipidclasswasestimatedby solution meltedaftersnap-freezingandslowheating(0.11°C point wasreportedasthetemperatureatwhichfinalicecrystalineach were vigorouslyvortexedimmediatelypriortoloadinginwells.Melting http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.099838/-/DC1 Supplementary materialavailableonlineat grants toB.J.S.andE.R.G. Council (NSERC)post-graduatescholarshiptoK.E.M.,andNSERCDiscovery This researchissupportedbyaNaturalSciencesandEngineeringResearch B.J.S. analyzedthedata.K.E.M.andwrotepaper. reagents oranalytictools.K.E.M.,R.H.T., A.R.,E.K.Y.C., J.C.L.B.,E.R.G.and J.C.L.B. performedtheresearch.R.H.T., J.C.L.B.andE.K.Y.C. contributednew K.E.M., A.R.andB.J.S.designedtheresearch.R.H.T., A.R.,E.K.Y.C. and The authorsdeclarenocompetingfinancialinterests. aaoo .E,Jui .E,Kra,K . enntn .E n el,M.A. Wells, and J.E. Pennington, K.J., Karnas, Z.E., Jouni, L.E., Canavoso, R.C. Hughes, and T. D. Butters, oln,S . lesah .L n e,R.E. Lee, and A.L. Allenspach, S.D., Collins, K.B. Storey, and M. T. H. Churchill, Jondal, and I. Björkhem, L., Sisfontes, S.C., Chow, as,J,Gade . odn .adMrisy R. Morrissey, and G. Condon, R., Grandee, J., Baust, tests, alphawassetto0.05. popMeans functioninthedoBypackageR(Højsgaardetal.,2012).Inall Population means(least-squaresmeans)wereobtainedforplottingusingthe experimental groupsbyANOVA withtwo-tailedTukey’s total lipidmassandtheneutralwereobtainedcomparedbetween covariate doesnotaltertheconclusions).Residualsofaregressionbetween (minus thecomponentofinterest)asacovariate(usingtotallipidmass mass, soallanalyseswereconductedasANCOVAs withtotallipidmass a large (butnon-significant)effect ofexperimentaltreatmentontotallipid linearity inresponse.Intherepeatedfreezingexperiment,becausetherewas with monthtreatedasafixedfactoritwasexpectedtherewouldbenon- among monthsbyANOVA, followedbytwo-tailedTukey’s posthoc Acknowledgements Statistical analyses References Supplementary material Funding Author contributions Competing interests concentration (µmol 20% palmitoleicacid)(Bennettetal.,1997).Intheseasonalprofile, ocu,E. Boychuk, P. Sandra, and E. Kindt, K., Rumplel, F., David, B., Bicalho, ent,V . rit .L n e,R.E. Lee, and N.L. Pruitt, V. A., Bennett, Biochem. Soc.Trans. temperature change. gall fly, freezing onbrain,muscle andMalpighiantubulesfromfreeze-tolerant larvaeofthe (2001). Fatmetabolismininsects. of Western Ontario,London,ON,Canada. Beetle, ChrysomelaAeneicollis(Coleoptera:Chrysomelidae) 700-704. growth inaleukemicT celllinebyn-3andn-6polyunsaturatedfatty acids. Zool. overwintering strategiesutilizedbyseparatepopulationsofgallinsects. Chromatogr. A high resolutioncapillarygaschromatographyandmassspectrometry. acid methylesterdatabaseforlipidprofilinginasingledropofhumanbloodusing solidaginis composition associatedwithcold-hardeninginthirdinstarlarvaeof 52, 572-580. Eurosta solidaginis . J.Comp.Physiol.B (2012). The JournalofExperimentalBiology(2014)doi:10.1242/jeb.099838 1211 Cold Tolerance ofEachLifeStagetheSub-AlpineWillow Leaf , 120-128. J. InsectPhysiol. fly 8 E. solidaginis , 170-171. −1 . J.InsectPhysiol. ) ofeachneutrallipidcomponentwascompared 167 (1989). Metabolicconsequencesofrapid cyclesof Annu. Rev. Nutr. , 249-255. (1980). Effects oftunicamycinoninsectcells. 35, 579-585. (60% oleicacid,20%palmiticacidand (1997). Seasonalchangesinfattyacid 43, 39-45. (1997). Ultrastructuraleffects oflethal 21, 23-46. (1989). Suppressionof . MScthesis,University (2008). Creatingafatty (1979). Diversityof post hoc

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