GFRAENKELANDHSHOPFmeltingpointofprotoplasmicfatsapointwhichhaslaterbeenenlargeduponbyBelehradek1931Thelatterformulatingthelipoidliberationtheorylinksuptheheatadaptabilityoftheprotoplasmic ID: 522683
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I3I.THEPHYSIOLOGICALACTIONOFABNORMALLYHIGHTEMPERATURESONPOIKILOTHERMICANIMALSI.TEMPERATUREADAPTATIONANDTHEDEGREEOFSATURATIONOFTHEPHOSPHATIDESBYG.FRAENKELDH.S.HOPFDepartmentsofZoologyandBiochemistry,ImperialCollege,London,S.W.7(Received23May1940)ATtemperaturesintheregionof4045°mostanimalsdiewithinafewhours.Thisappliesequallytohot-andtocold-bloodedanimals.Often,however,temperatureslethaltocold-bloodedanimalsareconsiderablylowerthanthatlimit.Thecauseofdeathbyheatandthemechanismofheatinjuryattempera-turesbelowthosewhereavisibledestructionofthetissues(e.g.coagulationofprotein)takesplacehavegivenrisetoaconsiderableamountofspeculationandnumeroushypotheseshavebeenadvancedtoexplainthephenomenaobserved.Intheolderliteratureonthesubjectdeathbyheatwaaattributedmostlytothecoagulationofproteins;morerecentlyotherhypotheses[recentlyreviewedbyBelehradek,1935]havebeenputforward,suchasdestructionofenzymes,asphyxiationorotherdisturbance'intheequilibriumofprotoplasmthroughaccumulationofwasteproducts.Whilenoneofthesehypothesesisyetprovedexperimentallytheonemostdeservingfurtherinvestigationisthe"lipoidliberationtheory"sinceitistheonlyonewhereevidencehasbeenprovidedlinkingthephenomenonofheatadaptation-i.e.thecapabilityofanorganismtoincreaseitsheatresistance-withdataontheconstitutionofanimportantcomponentoftheprotoplasm.Ithasbeenshownbyvariousauthorsthatthemeltingpointoffatsofmicro-organismsandofhigherplantsandanimalsvarieswiththetemperatureatwhichtheyhavebeenlaiddown.Whenexaminedatagiventemperaturefatformedatahighertemperatureisfoundtobemoresolidthanfatformedatalowertemperature.Sincethemeltingpointofafatdependslargelyonitsdegreeofsaturation,theiodinevalue(number)ofafatasameasureofdoublebondsinthefattyacidchainsisagoodmeasureforitsmeltingpoint.Thustheiodinenumberofthevisceralfatofamammalislowerthanthatofthesub-cutaneousfat.Thenumberisalsolowerinoilsfromtropicalplantsthanfromplantsgrowninacoolerclimate.(NumerousotherexamplesinBelehradek[1935].)Variousworkersobservedchangesoccurtinginthelipoidconstituentsofthecellonheating.Faur6-Fremiet[1924]foundintheeggsofSabellariagranulesappearingonheatingwhichseemedtobeoflipoidcharacter.Asimilar"libera-tion"oflipinswasfoundbyHeilbrunn[1924]whocentrifugedeggsofArbaciaafterheattreatment.Koeppe[1903]andRywosch[1911]attributedhaemolysisoferythrocytestoameltingofthesuperficiallipoidmembrane.Heilbrunn[1924]firstdrewspecialattentiontothecorrelationbetweenheatresistanceandthe(1085)69-2 G.FRAENKELANDH.S.HOPFmeltingpointofprotoplasmicfats,apointwhichhaslaterbeenenlargeduponbyBelehradek[1931].Thelatter,formulatingthe"lipoidliberationtheory",linksuptheheat"adaptability"oftheprotoplasmicfats,asexpressedbythecorrela-tionbetweenthemeltingpoints-degreeofunsaturation-offatsandthetem-peratureoftheirformation,withtheadaptabilityofthewholeorganismtohightemperatures.Accordingtohimheatinjury,whetherreversibleorirreversible,iscausedbythemeltingoflipoid'constituentsinthecellorinthecellularmem-branes.Fatformedatahighertemperatureandconsequentlyofahighermeltingpointwouldrenderanorganismmoreresistanttothedamagingeffectofhightemperatures.Asalreadymentioned,therearewell-establishedexamplesavailableaboutthecomparativedegreeofsaturationoflipinsfoundinplantsandanimalsfromdifferentclimatesorofdifferenthabitat.Theliteraturecontainsonlyafew,butwell-establishedexamplesinwhichchangesintheiodinenumberoffatshavebeendemonstratedbygrowinganorganismatdifferenttemperatures.[Terroineetal.1927;1930]onbacteria,Pearson&Raper[1927]onAspergillus.]Althoughthephenomenonofadaptabilityofthelivingorganismtothetemperatureofitsenvironmenthasbeenregardedalmostasanaxiombyecologists,itissupportedbysurprisinglyfewcertainexamplesintheliterature.ThemoststrikingexamplesoftemperatureadaptationarefoundintheworkofHathaway[1927],Wells[1935,1,2],andSumner&Doudoroff[1938],allrelatingtofishes.Thephenomenaofheatadaptationontheonehandandchangingmeltingpointoflipinsontheotherhave,however,neverbeeninvestigatedinthesameorganismandtheircombinationinthelipoidliberationtheoryisbasedonmereanalogy.Theobjectofourworkistofillthisgapinourknowledgebybreedinganorganismatdifferenttemperaturesandsubsequentlytestingfordifferencesinitsheatresistanceandinthesaturationdegreeoflipinsofindividualsbredunderotherwisecomparableconditionsatdifferenttemperatures.AdaptationtohightemperaturesThelarvaeoftwospeciesoffleshflies,CalliphoraerythrocephalaMeigenandPhormiaterra-novaeR.D.,differwidelyintheirtoleranceofhighandlowtem-peratures.Calliphoradevelopsfromtheeggtothefullygrownlarvaatanytemperaturebetween12and310,thecloselyrelatedPhorrniaonlydevelopsattemperaturesbetween18and360.Toattainfullgrowththelarvaeofbothspeciesrequireabout3daysatthehigherand16daysatthelowerendoftherespectiveranges.Thelarvae,thusbredatdifferenttemperaturestotheirfullsize,weresubmittedsimultaneouslytoatestfortheirresistancetoaconstanthightemperature.ThemethodadoptedwassimilartothatdescribedbyMansbridge[1936].Thelarvaeweredroppedsuddenlyintothetemperaturerequiredandthetimeafterwhichapproximately50%ofthemwerestillun-damaged,i.e.wouldpupateanddeveloptonormalffies,wasdetermined.Thefollowingtablespresentthesummaryoftheexperiments.Table1.HeatresistanceofPhormiaPeriodinhr.afterwhich50%werekilledTestedat(Rearedat180)(Rear,dat360)4602-453�7,447-11,1515,184319241086 BASISOFTEMPERATUREADAPTATIONTable2.HeatresistanceofCalliphoraPeriodinhr.afterwhich50%werekilledTestedat(Rearedat120)(Rearedat30°)4101i40it2i393538�413FromthefiguresinTables1and2itisclearthatlarvaewhichwerebredatahighertemperaturecouldresistagivenhightemperatureforaconsiderablylongerperiodthanlarvaewhichwerebredatalowertemperature.Or,con-sideringthetemperaturewhichheat-adaptedlarvaeandnon-adaptedspecimenscouldtolerateforagiventime,itappearsthatthedifferenceisapproximately10.Thus,forPhormia,rearedat360,atemperatureof460for2ihr.hasaboutthesameeffectasatemperatureof450forthesameperiodforlarvaebredat180.ThecorrelationbetweentheiodinevaluesofthefattyacidsofthephosphatidesandthetemperatureofbreedingFortheseexperimentsthesamespeciesoflarvaewerebredunderidenticalconditionsasintheheatresistanceexperimentsdescribed.Ithadbeenproposedatfirsttoworkwiththetotalfatsofthebodyfortheseinvestigations.This,however,wasabandonedforthefollowingreasons.TheworkofEvans[1932]hasshownaselectiveutilizationofthereservefatsinblowflylarvaeintheperiodprecedingpupation,whentheinsectshavestoppedfeeding(so-calledprepupae),anditwasfearedthatthisselectiveutilizationwouldconstituteaconsiderablesourceoferror,astheselarvaeareatthatstageoftheirlifehistoryentirelydependentuponthereservematerialsoftheirbody.Thissourceoferrorwouldbeespeciallylarge,as.theknowndifferenceindurationofthelifehistoryatdifferenttemperaturesmakesitevidentthat,forinstance,1hr.at360doesnotatallcorrespondto1hr.at180,andthateventhesmallestdifferencesin"physiological"agewouldgreatlyaddtothepossibleerror.Thephosphatides,ontheotherhand,arenowgenerallyconsideredasthe"vital"'fats,asopposedtothereservefats.Thoughverylittleisknownabouttheirfunctionsofar,wedoknowthattheyoccurineverycellandthattheirpresenceisapparentlyessentialtothecontinuedexistenceofprotoplasm.Itisthereforeimprobablethattheywouldbeutilizedasreservefats,thoughacertainproportionmightalwaysbefoundinatransitstageengagedinmetabolicprocesses.But,whatevertheiralleged"vital"rolemaybe,itismostlikelythattheywouldbeinvolvedintheprotoplasmicprocessesconnectedwithheatinjury.InthemethodofanalysistheprocedureofTerroineetal.[1930]wasfollowedwithcertainmodifications.Atleast500fullygrownlarvae,withtheirgutsempty,wereextractedfor48hr.inaSoxhletapparatuswithabsolutealcohol.Fromtheextractobtained,thealcoholwasevaporatedoff,andtheresidueextractedseveraltimeswithetherofsp.gr.0-720,towhich,afterconcentration,4vol.acetonewereadded.Theresultingprecipitatewasallowedtostandfor15min.,centrifuged,theliquiddecantedoff,andthenredissolvedwithether.Theprecipitationwasrepeatedfourtimesintheearlypartofthework,whenitwasassumedthatthematerialthusobtainedwouldbefairlypurephosphatide,oratleastphosphatideofacomparablestandardofpurity.Theresultsofthedeterminationsobtainedwith1087 G.FRAENKELANDH.S.HOPFmaterialpreparedbythismethodaregiveninTable3,undertheheading"Iodinevaluesbyfirstmethod".Acomparativelylargedivergenceinthepropertiesofmaterialthusobtained,ledtoaninvestigationofitspurity,anditwasfoundthatthephosphorus/nitrogenratiostillvariedconsiderablyinmaterialspreparedintheaboveway.Itwasthereforedecidedtoworkinfutureonmaterialshowingaconstantpercentageofphosphorus,andtocontinueprecipitationuntilthiswasobtained.PhosphoruswasestimatedbythecolorimetricmethodofMartland&Robison[1926],measuringthetotalphosphorusasinorganicphosphatetowithin+0-005mg.Theresultsforthehighestdegreeofpurityobtainablewereasfollows:Theoreticalvalue,purelecithin+kephalinActualvaluePhosphorus4.83.4-3-6Nitrogen2.42-8-3-1Thematerialfinallyworkedwithwasthereforebynomeanspurephosphatide,butwasinallprobabilitystilladulteratedwithproteinorurea.However,thedegreeofpurityobtainedneednotberegardedasunsatisfactory,ascommercialtechnicallecithin,whichwastestedforcomparison,onlycontainedabout28%phosphorus.Forthesecondseriesofestimations(called"secondmethod"inTable3),thephosphorusintheacetoneprecipitationwasestimatedandprecipitationre-peateduntilthephosphoruspercentageremainedconstantattheabovevalue(approx.3-5%).The"phosphatide"thusobtainedwasthensaponifiedwith10%alcoholicKOHonaboilingwaterbathunderreflux.ThefurtherprocedurewasthenasusedbyEvans[1932]andRainey[1938]intheirworkonblow-flylarvae.Saponificationwascarriedoutfor11hr.atleast,afterwhichtimemostofthealcoholwasboiledoffunderreducedpressureandtheresultantsyrupysolutionwastakenupwithalittlewater,fromwhichtheunsaponifiablematterwasremovedbyshakingfivetimeswithetherinaseparatingfunnel.Fromtheetherealsolutionthusobtainedanyremainingsoapswereshakenoutwithwater,whichwasunitedwiththeoriginalsoapsolution.Fromthisthefattyacidswereprecipitatedbyaddinghydrochloricacid(1partofconc.HCItoonepartofwater).Thesewereremovedbyshakingoutfivetimeswithether.Thesolventwasremoved,theresidue,whichstillcontainedacertainamountofimpurity,wastakenupwithlightpetroleum(B.P.40-60o),andthefattyacidsrecoveredbyevaporationofthesolventinaweighedflask.TheiodinevalaesweremeasuredbythemethoddescribedbyDam[1924],andtheresultsareasfollows:Table3.IodinevalueofphosphatidesIodinevaluesbyTemp.of,AKSpeciesbreedingFirstmethodSecondmethodPhormia18°88,89,92,8691,922764*5,84,76,7871,72,73.53669,60.5,65*8,66.769,62,63Calliphora1278,88,89,88,82-7,84,8515-96,95-82772,76,72-5,84,83,75*572-7,7530-3165-2,66*2,7070,70,671088 BASISOFTEMPERATUREADAPTATION1089Theresultsobtainedbythe"firstmethod"showmorescatterthanthosefromdeterminationofphosphatideswithconstantphosphoruscontent,butthereisnosignificantdifferencebetweenthetwosets.TheresultsaregraphicallyrepresentedinFig.1.Itisapparentthatthedegreeofsaturationofthephosphatidesisdirectlydependentuponthetem-110oCalliphoraxPhormia100909400\-7060-I152025303540°C.TemperatureofbreedingFig.1.TherelationoftheiodinevaluesofthephosphatidesofthelarvaeofCaaUiphoraandPhormiawiththetemperatureofbreeding.peratureofbreedinginthetwocloselyalliedspeciesinvestigated.Thefattyacidsofthephosphatideslaiddownatthehighertemperatureinbothspeciesaremoresaturatedthanthoseproducedatlowertemperatures,provedbyaloweriodinevalue.Thedifferencefortheextremetemperaturesofbreeding,atemperaturedifferenceofabout18°,isabout26unitsineachspecies.DiscussionItisshownthatblow-flylarvae,rearedathighertemperatures,notonlybecomemoreresistanttohightemperaturesthanlarvaerearedatalowertemperature,butthatthemeltingpointoftheircellularfatsisalso"adapted".Inbothspeciesofinsectstherangeofchangeoftheiodinevalueisaboutequal.ThemeandifferencebetweenthevaluesatthehighestandlowesttemperaturesofbreedinginPhormiaisabout26units,andinCalliphoraalso26units,witharangeofbreedingtemperatureof180,inbothcases.Thisamountofadaptationmayappearratherhigh,butthevaluesarewellinagreementwiththosefoundbyotherworkers.Pearson&Raper[1927],workingonthetotalfatoftwospeciesoffungus,foundthemeaniodinevalueofAspergillusnigertobe149ifbredat180,129at250and95ifthecultureswerekeptat35°.InRhizopusnigricansthecorrespondingfigureswere8$for120and78for250.Terroineetat.[1930]foundthatthemicro-organismSterigmatocystisnigrainculturesgrownat180hadaniodinevalueof135forthefattyacidsofthephosphatides,andthesameorganismgrownat38gaveameanof99."BacilledelaFleole"gavemeanvaluesof51and31forculturesbredat18and380respectively.Thevaluesforthetwospeciesofflylarvaecanthereforeberegardedaswellwithintherangetobeexpectedfromotherwork.Nofiguresareavailableforthedegreeofsaturationoffattyacidsofthephosphatidesinotherinsects.Ackerman[1926]cultivatedtheaphidRhopalo-siphumprunifoliaatdifferenttemperaturesandfoundthatthesolidification G.FRAENKELANDH.-S.HOPFpointoffattyglobulesinthebodyfluidincreasedwiththetemperatureofcultivation.Rainey[1938]rearedthelarvaofanotherfleshfly,Luciliasericata,whichiscloselyrelatedtothespeciesusedbyus,attemperaturesof15,25and350andfoundtheiodinevaluesofthetotalfattyacidstobe75-8,72-2and71-0respectively.Thesefigures,althoughshowingacleartendencytofallingiodinenumberwithincreasingtemperature,arenotregardedassignificantbyRainey,thedifferenceiniodinevaluebeingjustoutsidetheexperimentalerrorandthelarvaehavingbeenusedforthedeterminationsatadifferentphysiologicalage.Asthephysiologicalroleofthephosphatidesissodifferentfromthatofthegreaterpartofthetotalfat,whichconstitutesstoragematerial,noconclusionscanbedrawnfromacomparisonofRainey'sfiguresandourown.Themanydatanowavailableofwhichthosequotedaboveconstituteonlyafractionmakesitclearbeyonddoubtthattherulethattheconstitutionoffatsdependsonthetemperatureatwhichtheyarelaiddownappliestoalllivingmatter.Leathes&Raper[1925]andPearson&Raper[1927]pointoutthatlipoidsubstancesareformednotbyasinglechemicalprocessbutbyacatenaryseriesofchanges.Theunsaturatedcompoundsareformedfirst,andthesaturatedonesariseinturnfromthese.Atlowtemperaturethisprocesscannotbecompletedbutstopsatahigherdegreeofunsaturationthanwouldhavebeenthecaseiftheformationhadtakenplaceatahighertemperature.Table3andFig.1showthatthemeaniodinevaluesforthetwofliesusedbyusvaryonlyaccordingtotemperatureandnottospecieswithinthesamerange.Eowever,itisevidentthatthisisnotapplicabletoalllivingmatterandthatcompletelydifferentsetsofresultsareobtainedwithdifferentsortsoforganisms.ThisisshownclearlybycomparingourresultswiththoseofTerroine,quotedabove.Whetherthesimilarityinthetwocloselyrelatedinsectsexaminedismerelyaccidentalorwhetherphylogeneticrelationshipswillalsoleadtoasimilarityinthisrespectisaquestiondemandingfurtherinvestigation.Thatfatsderiveddirectlyfromthefoodareoftenlaiddownunchangedasreservematerialisawell-knownfactinvertebrates.Yuill&Craigshowed[1937]thatlarvaeofLuciliasericatarearedondifferentdietsshowedadifferenceintheiodinevalueoftheirfatsaccordingtothefoodofferedtothem.However,astheflylarvaeinourownexperimentsweregrownatdifferenttemperaturesonthesamefood(leanmeat),thedifferencesfoundintheconstitutionofthephosphatidescannotbeduetothefood.Itisgenerallyassumedthatphosphatides,e.g.lecithin,arenotobtaineddirectlyfromthefoodmaterialinunchangedformbutareproductsofsynthesiswithinthebodyitself.TheresultsobtainedforPhormiaandCalliphorasupportthelipoidliberationtheoryinsofarthatforthefirsttimetherehasbeenfoundadirectcorrelationbetweenthesaturationdegreeofthephosphatidesandtheheatresistanceofthewholeorganism.However,theresultsobtainedmakeitveryunlikelythatdeathatmoderatelyhightemperaturesisduesimplytoameltingoftheprotoplasmicfats.Otherwisetherewouldbenoexplanationofthefactthatthelowesttemperaturewhicheachofthetwospeciesofflylarvaecantolerateforagivenperiodisapproximately7degreeslowerforCalliphorathanforPhormia.Adaptationconsistsonlyofpushingthelowestlethaltemperatureforagivenperiodupbyapproximately1degree.CalliphoraandPhormia,bredat270,arekilledatanexposureof5hr.at39and45brespectivelyandyettheiodinevaluesofthephosphatidesofbothspeciesarethesame,namely75.Fromthistheconclusionmaybedrawnthatitisunlikelythatabreakdownoflipinsinapurelyphysicalway,causingforinstanceachangeincellper-meability,isthecauseofheatinjuryatthecomparativelylowtemperatures1090 BASISOFTEMPERATUREADAPTATIONrequired.Thelipoidliberationtheoryofadaptationtohightemperatures,whilecertainlysubstantiatedbythepresentworkinsofarassuchstrikingdifferencesintheconstitutionoflipinsbearout'thepredictionsmadeby'it,cannotexplainfullyandsatisfactorilythephenomenaofheatinjuryandheatadaptation.Otherfactorsmustplaytheirrole,butthestrikingadaptationofthesaturationdegree'oflipinsdescribedinthisworkleadstothetentativesuggestionthatthephysicalnatureofthesefatsmayhaveadecisiveinfluenceonthechainsofphysiologicalprocessesinvolved.SUMMARYTheadaptabilitytohightemperatureandthenatureofthephosphatideshavebeenstudiedinfullygrownlarvaeoftheblow-fliesCalliphoraerythrocephalaMeig.andPhormiaterra-novaeR.D.Theresultsareasfollows:1.Inbothspeciesofinsecttheamountofheatadaptationissuchthatlarvaebred180higherthanotherswillbeabletowithstandtheinjuriouseffectsofhightemperatureforonedegreemoreforthesametimeofexposurethanthosebredatthelowerendofthetemperaturerange.2.Thedegreeofunsaturationofthephosphatidesoftheselarvae,measuredbytheiriodinevalues,isdependentuponthetemperatureofbreedingonly,andwasfoundtoshowadifferenceof26unitsinbothspeciesforadifferenceinthebreedingtemperatureof180.3.Theseresultsarediscussedinthelightofthe"lipoidliberationtheory",asadvancedbyHeilbrunnandBelehradek.Whilethestrongadaptationofthephosphatidestothetemperatureatwhichtheyhavebeenlaiddownsuggestsaroleforthesesubstancesinthemechanismofheatadaptation,thefactthattwocloselyalliedspeciesofinsects,bredatthesametemperatureandhavingthesameiodinevalueforthefattyacidsoftheirphosphatideshavedifferentresistancestoheat,showsthataphysicalbreakdownofthefattysubstancescannotbethedirectcauseofheatinjury.TheauthorsaregreatlyindebtedtotheAgriculturalResearchCouncilforagrantdefrayingthecostsofthisinvestigation,andtoProfs.J.W.MunroandA.C.ChibnallwhoprovidedthefacilitiesfortheworkinthedepartmentsofZoologyandAppliedEntomologyandBiochemistryrespectively;theyalsowishtothanktheabovenamedandDrH.W.Bustonformuchvaluableadviceandsuggestions.REFERENCESAckerman(1926).J.exp.Zool.44,1.Belehradek(1931).Protoplsnma,12,405.-(1935).Temperatureandlivingmatter.Protopla.snaMonographien,8.Berlin.Dam(1924).Biochem.Z.152,101.Evans(1932).J.exp.Biol.9,314.Faure-Fremiet(1924).Arch.Anat.micr.20,211.Hathaway(1927).BuU.U.S.Bur.Fi8h.43,169.Heilbrunn(1924).Amer.J.Phy8iol.69,190.(1937).Outlineofgeneralphysiology.Philadelphia.Koeppe(1903).Pftig.Arch.ges.Phy8iol.99,33.Leathes&Raper(1925).Thefats.London.Mansbridge(1936).Ann.appl.Biol.23,IC03.1091 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