PLOSONE|https://doi.o r g/10.13 7 1/journal.p o ne.0177 8 2 7 Ma y23,2017 1/12
RESEARCHARTICLE
Moreoxygenduringdevelopmentenhancedflig htperformancebutnotthermaltoleranceofDros ophilamelanogaster
ShayanShiehzadegan1,JacquelineLeVinhThuy1,NataliaSzabla2,MichaelJ.Angilletta,Jr.1,J ohnM.VandenBrooks3*
1SchoolofLifeScience,ArizonaStateUniversity,Tempe,Arizona,UnitedStatesofAmerica,2InstituteofEnviron mentalStudies,JagiellonianUniversity,Krako
´w,Poland,3DepartmentofPhysiology,MidwesternUniversity,Glendale,Arizona,UnitedStatesofAmerica
*jvande n brooks@ m idwestern. e du
Abstract
Hightemperaturescanstressanimalsbyraisingtheoxygendemandabovetheoxygensup- ply.Consequently,animalsunderhypoxiacouldbemoresensitivetoheatingthanthose OPENACCESS
Citation:ShiehzadeganS,LeVinhThuyJ,Szabla N,AngillettaMJ,Jr.,VandenBrooksJM(2017)More oxygenduringdevelopmentenhancedflightperfor mancebutnotthermaltoleranceofDrosophilamela nogaster.PLoSONE12(5):e0177827.https:// d oi.o rg/10.1371/ j ournal. pone.01778 2 7
Editor:MichaelSears,ClemsonUniversity,UNITEDST ATES
Received:February9,2017 Accepted:May3,2017 Published:May23,2017
Copyright:©2017Shiehzadeganetal.Thisisano penaccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense,whichper mitsunrestricteduse,distribution,and
exposedtonormoxia.Althoughsupportforthismodelhasbeenlimitedtoaquaticanimals,oxygen supplymightlimittheheattoleranceofterrestrialanimalsduringenergeticallydemandingactivitie s.Weevaluatedthismodelbystudyingtheflightperformanceandheattoleranceofflies(Drosophil amelanogaster)acclimatedandtestedatdifferentconcentra-
tionsofoxygen(12%,21%,and31%).Weexpectedthatfliesraisedathypoxiawoulddevelopintoa dultsthatweremorelikelytoflyunderhypoxiathanwouldfliesraisedatnor-
moxiaorhyperoxia.Wealsoexpectedfliestobenefitfromgreateroxygensupplyduringtesting.Th eseeffectsshouldhavebeenmostpronouncedathightemperatures,whichimpairlocomotorperf ormance.Contrarytoourexpectations,wefoundlittleevidencethatfliesraisedathypoxiaflewbett erwhentestedathypoxiaortoleratedextremeheatbetterthandidfliesraisedatnormoxiaorhyper oxia.Instead,fliesraisedathigheroxygenlevelsperformedbetteratallbodytemperaturesandoxy genconcentrations.Moreover,oxygensupplyduringtestinghadthegreatesteffectonflightperfor manceatlowtemperature,ratherthanhightemperature.Ourresultspoorlysupportthehypothesi sthatoxygensupplylimitsperformanceathightemperatures,butdosupporttheideathathyperoxi aduringdevelop-mentimprovesperformanceofflieslaterinlife.
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
DataAvailabilityStatement:Datacanbeaccessedthr oughOpenScienceFrameworkathttps://osf. i o/ fvw7g/
withaDOIof10.17605 / OSF.IO/FVW7G .
Funding:ThisresearchwasfundedbytheSchoolof LifeSciences,theBarrettHonorsCollegeatArizona StateUniversity,andMidwesternUniversity.N.Sza blawassupportedbyprojectco-
foundedfromEUresourcesundertheEuropeanSoc ialFund(POKL04.01.01-00-053/09).The fundershadnoroleinstudydesign,datacollection
Introduction
Despitedecadesofresearch,thequestionofwhatcausesanimalstodieathightemperaturesremainsu nresolved.Severalmechanismshavebeenproposed,includingthedestabilizationofproteinsormem branesandthefailureofcellsignaling[1–
4].Acriticalflawintheseproposalsisthatanimalsexposedtothermalstressoftendieattemperaturesw ellbelowthosepredictedbythesemechanisms[5].Toaddressthisproblem,Po¨rtnerproposedamode linwhich
Oxygensupplyandflightperformance
andanalysis,decisiontopublish,orpreparationofth emanuscript.
Competinginterests:Theauthorshavedeclaredth atnocompetinginterestsexist.
animalsfailathightemperaturesbecauseoxygendemandexceedsthesupply[5–
7].Thismodelassumesthathightemperaturesrequireanimalstorelyonanaerobicmetabolism,whic hprovidesinsufficientenergyandthusimpairsthefunctionofnervesandmyocytes.IfPo¨rtner’smodel iscorrect,ananimal’ssusceptibilitytohypoxiaanditsabilitytoacquireoxy-
genshoulddetermineitsheattolerance[8,9].Withoutacompensatorychangeintherespira-
toryandcardiovascularsystem,adecreasingsupplyofoxygeninanenvironmentshouldultimatelyma keananimalmoresusceptibletooverheating.
Studiesofaquaticanimalstendtosupporttheoxygen-limitationhypothesis[9–
12],butstudiesofterrestrialanimalshaveyieldedlesssupport.Recently,lizardswerefoundtopref erlowertemperatures[13]orlosemobilityatlowertemperatureswhenexposedtohypoxiacom- paredtonormoxia.However,mostterrestrialanimals,includinglizards,willneverexperiencethee xtremelylowlevelsofoxygenusedintheseexperiments.Moreover,thevastmajorityofanimalsarei nsects,whichpossessasophisticatednetworkoftracheaeandtracheolesthatdeliveroxygendirectlyt omitochondria[14].Obviously,anyunifyingmodelofhowoxygensupplylimitsthermaltolerancew ouldneedtoconsiderthephysiologyofinsectsunderrealisticlevelsofoxygen.Asoxygensupplydecr eases,insectscanopentheirspiraclesmorefrequently,ventilatetheirtrachealsystemmorerapidly,a ndmodulatetheirfluidstoenhancediffusion[15].Athightemperatures,suchresponsesmustenable someinsectstomeetthegreaterdemandforoxygen,becausehypoxiadidnotreduceheattolerance sofdragonflies,cock-
roaches,orfliesinpreviousexperiments[8,16,17].Eveninspeciesthattoleratedheatworseunder hypoxia,hyperoxiafailedtoenhanceheattolerance,asonemightexpect[reviewedby9].Sofar,ox ygensupplyseemstolimitheattolerancesofterrestrialanimalsonlyduringtheembryonicstage[1 8,19],whentherespiratorysystemisstilldevelopingandoxygendeliverydependsondiffusionacro ssaneggshell[20].
Despitetheseresults,thegeneralityofPo¨rtner’smodelmighthavebeenunderestimatedbecauser esearchershavetesteditspredictionsmainlybystudyinganimalsatrest.Restingani-
malsconsumefarlessenergythandoactiveanimals.Thecombinedstressofheatandhypoxiashouldc hallengeanimalsmostduringstrenuousactivities,suchasrunningorflying.Duringflight,themusclesof insectsdemandmoreoxygenthanjustaboutanytissue[21,22].Inprevi-
ousexperiments,hypoxiareducedthemaximalaerobicmetabolismofinsects[15],andhyper- oxiaincreasedaerobicmetabolismandflightperformance[23].Thus,thethermaltoleranceofinsectflig htisalogicalsubjectforevaluatingthegeneralityofPo¨rtner’soxygenlimitationhypothesis.
Wetestedtheoxygenlimitationhypothesisbyquantifyingchronicandacuteeffectsofoxy-
gensupplyontheflightperformanceandthermaltoleranceofflies(Drosophilamelanogaster).Oneofth eadvantagesofworkingwithinsectsisthatithasbeenshownthattheirrespiratorysystemcanbemanipu latedduringdevelopmentbyalteringoxygensupply.Developmentalplasticityinthenumberandsizeof tracheaehasbeenobservedinavarietyofinsects[24,25],includingD.melanogaster[26,27].Thisplasti citycouldpotentiallyenhancetheheattoleranceofanadultinsectinahypoxicenvironment.Ifthisreaso ningholds,exposuretohypoxiadur-
inglarvaldevelopmentshouldenhancetoleranceofhypoxiaandheatingasanadult.There-
fore,wepredictedthatdevelopmentathypoxiawouldleadtogreatertoleranceofhypoxiaandheatdurin gadulthood.Theseeffectsshouldbestrongerunderenergeticallydemandingactivi-
ties,suchasflight,thanatrest.Asatradeoff,however,amoreelaboratenetworkoftracheaecouldleave aninsectmoresusceptibletooxidativedamageorwaterlossinanormoxicorhyperoxicenvironment[15 ].Additionally,atradeoffbetweeninvestingintrachealsystemsandothertissues,suchasflightmuscles andrelatedtissues,wouldincreasetheflightperfor-manceofinsectsrearedunderhyperoxia[15,28].
Materialsandmethods
Originandmaintenanceofisofemalelines
WestudiedtheacclimationoffliesfromanaturalpopulationofD.melanogastercollectedinDanvil le,Indiana,USA.Isofemalelinesderivedfromthispopulationwereraisedat20.5˚Candnormoxiaf orfivegenerationsbeforeourfirstexperiment.Duringthistime,linesweremaintainedin25x90m mvials(GeneseeScientific,SanDiego,USA)on~3–4cmoftheBloomingtonStandardcornmeal- cornsyrupdiet.Adultsfromeachlineweretransferredtofreshvialsevery3–
4weekstominimizeoverlapbetweengenerations.Priortoeachexperi-
ment,wecontrolledthedensityofeachisofemalelinefortwogenerationsbytransferringonlytwoa dultsofeachsexintonewvialstoreproducefor48h.
Atthebeginningofeachexperiment,pairsofadultfliesfromeachisofemalelineweretransferredton ewvialskeptatcertainlevelsofoxygen(seedetailsofeachexperimentbelow).Oxygenconcentrations weremaintainedbyacommercialoxygencontroller(ROXY-
8;SableSystemsInternational,LasVegas,Nevada,USA).Temperaturewasmaintainedat20.5˚Cbya programmableincubator(DR-
36VL;PercivalScientific,Perry,Iowa,USA).Adultsthatemergedundertheseconditionswereusedino urstudiesofflightperformanceandheattolerance.
Acclimationofflightperformance
Weraisedfliesfromsixisofemalelinesateachofthreeconcentrationsofoxygen(12%,21%,and31%)a ndstudiedtheirflightperformanceasadults.Seventotendaysaftereclosion,fliesweretestedatallcom binationsofthreeairtemperatures(37˚C,39˚C,and41˚C)andthreeoxygenconcentrations(12%,21
%,and31%).Additionally,wetestedasubsetofflies,raisedundernormoxia,at25˚Ctotestwhetherperf ormanceswerelesssensitivetooxygensupplyatabenigntemperature.Twenty-
fourhoursbeforetesting,femaleswereanesthetizedwithCO2,transferredtonewvials,andreturnedto anincubatorsetat20.5˚C.Topreventeffectsofanes-
thesiaonflight[29],eachflywastransferredwithoutanesthesiatoanemptyvialjustbeforetesting.
Wequantifiedflightperformancesinacustomchamber[30].Theflightchamber(30.5x
30.5 x30.5cm)wasconstructedfromclearacrylic,withacircularopeningatthetop.Thisope ningwastemporarilysealedbyamovableplate.Asavialwithaflywasinvertedontopofthisplate,ho lesintheplateenabledthetemperatureandoxygenconcentrationofthevialtoequilibratewithlevel sinthechamber.Afaninsidetheboxcirculatedairfromacommercialoxygencontroller(ROXY- 1,SableSystemsInternational,LasVegas,Nevada,USA).Prelimi-
narytestsshowedthattemperatureandoxygenconcentrationofthevialmatchedthoseofchambe rwithin3.5min.Afterthisperiod,theplatewasslidasideandtheflywastappedintothechamber.Flie seitherfelluncontrollablyorflewtoasurface.Toensureobjectivity,afallwasscoredwhenaflyland edonthefloorwithin10cmofthepointbelowthevial.Theorderoftestingateachtemperatureandox ygenconcentrationwasrandomizedamongdevelopmen-taltreatmentsandisofemalelines.
Acclimationofheattolerance
Weraisedfliesfromfourisofemalelinesateachofeightconcentrationsofoxygen(10%,12%,15%,18
%,21%,24%,27%,and30%)andmeasuredtheirheattoleranceasadults(N2:30foreachtreatment).H eattolerancewasestimatedasthetimerequiredforaflytolosemotorcon-
trolat39.5˚Cinanormoxicatmosphere(21%),alsoknownasknockdowntime[31].Eachday,newlyem ergedfliesfromeachisofemalelinewereisolatedinnewvials.Between5and9
daysafteremergence,eachflywastransferredwithoutanesthesiaintoaglassvial(10mL)withastopper .Flieswerekeptinvialsforfewerthan5minbeforeheattolerancewasmeasured.
Wemeasuredknockdowntimeinvialssuspendedinacustomwaterbath.Thebathcon- sistedofaclearacrylicbox(28x4x7.5cm)withasealed,watertightlid.Eightvialsweresus-
pendedfromholesinthelid.Waterflowedintooneendoftheboxandouttheotherend,suchthatvialswer ecompletelysubmergedinwaterwhentheboxwassealed.Thetemperatureofthewaterwascontrolle dat39.5˚C(±0.1˚C)byacommercialcirculator(Model11505,VWR,USA).Thistemperaturewasbase donpreviousstudies[32,33],inwhichfliessuc-
cumbedquicklyenoughtominimizeeffectsofenergyorwaterlossbutslowlyenoughtorecordtheknoc kdowntimesprecisely.Ineachtrial,thetimeuntilaflycollapsedwasrecordedastheknockdowntime.Ti meswererecordedwithbytheJWatchercomputersoftware[34].
Statisticalanalyses
Wemodeleddependentvariablesusingthelme4library[35]oftheRStatisticalPackage[36].Forflight performance,wefitalinearmixedmodelwithbinomialdistributionoferrorandfixedeffectsofoxygend uringdevelopment,oxygenduringtesting,andtemperatureduringtesting.Forknockdowntime,weu sedalinearmixedmodelwithanormaldistributionoferrorandfixedeffectsofsexandoxygenduringde velopment.Ineachanalysis,theeffectofisofemalelinewasmodeledasarandomfactor.
FollowingBurnhamandAnderson[37],weusedmulti-
modelaveragingtoestimatethemostlikelyvaluesofmeans.First,weestimatedthemostlikelyrandom effectsaccordingtoZuurandcolleagues[38].Then,weusedtheMuMInlibrary[39]tofitallpossiblesets offixedeffectstothedata.Finally,wecalculatedtheAkaikeinformationcriterion(AICC)andAkaikeweig htofeachmodel(Tables1and2).Tocalculatethemeanforeachgroup,weusedtheweightedaverageof eachparameter,includingestimatesfromallmodels.Thisapproach
Table 1. Alllikelymodelsincludedaneffectoftemperatureonflightperformance.Thetwomostlikelymodelsalsoincludedaneffectofdevelopmentaltemperatu re.Foreachmodel,weprovidetheAkaikeinformationcriterion(AICc)andtheAkaikeweight,whichequalstheprobabilitythatthemodeldescribesthedatabetterthanot hermodels.Allmodelscontainedaninterceptandanerrortermassociatedwithisofemaleline.
Model Parameters Loglikelihood AICc ΔAICc Akaikeweight
1)sex+dev[O2]+temp 6 -124.0 260.2 0 0.19
2)dev[O2]+temp 5 -125.2 260.6 0.34 0.16
3)sex+temp 4 -126.4 261.0 0.79 0.13
4)temp 3 -127.5 261.1 0.85 0.13
5)dev[O2]+temp+test[O2]+(dev[O2]·test[O2]) 11 -3119.9 262.7 2.46 0.06
6)dev[O2]+temp+test[O2]+(dev[O2]·test[O2])+(temp·test[O2]) 15 -115.8 263.2 3.00 0.04
7)sex+dev[O2]+temp+test[O2]+(dev[O2]·test[O2]) 12 -119.2 263.5 3.27 0.04
8)sex+dev[O2]+temp+test[O2] 8 -123.6 263.8 3.52 0.03
9)dev[O2]+temp+test[O2] 7 -124.8 264.0 3.75 0.03
10)temp+test[O2] 5 -127.1 264.4 4.19 0.02
11)sex+temp+test[O2] 6 -126.1 264.5 4.22 0.02
12)sex+dev[O2]+temp+test[O2]+(dev[O2]·test[O2])+(temp·test[O2]) 16 -115.4 264.6 4.33 0.02
13)dev[O2]+temp+test[O2]+(temp·test[O2]) 11 -120.8 264.6 4.34 0.02
14)sex+dev[O2]+temp+test[O2]+(temp·test[O2]) 12 -119.9 264.8 4.52 0.02
15)temp+test[O2]+(temp·test[O2]) 9 -123.3 265.2 5.00 0.02
16)dev[O2]+temp+(dev[O2]·temp) 9 -123.6 265.7 5.44 0.01
17)sex+temp+test[O2]+(temp·test[O2]) 10 -122.5 265.7 5.44 0.01
https://d o i.org/10.1371/ j ournal.pon e .0177827.t001
Table 2. Themostlikelymodelofknockdowntimeincludedonlyaneffectofsex.Allothermodelswerepoorlysupported(AICc>6).Foreachmodel,weprovideth eAkaikeweight,whichequalstheprobabilitythatthemodeldescribesthedatabetterthanothermodels.Allmodelscontainedaninterceptandanerrortermassociated withisofemaleline.
Model Parameters Loglikelihood AICc ΔAICc Akaikeweight
1)sex 4 -648.5 1305.2 0 0.95
2)sex+test[O2]+(sex·test[O2]) 18 -636.4 1311.4 6.17 0.04
3)sex+test[O2] 11 -645.7 1314.4 9.20 0.01
https://d o i.org/10.1371/ j ournal.pon e .0177827.t002
focusesonestimatesofeffectsizeandeliminatestheneedforPvalues,becauseallmodels(includingth enullmodel)contributetothemostlikelyvalueofeachmean[37].
Results
Flightwasaffectedmorebytemperaturethanbyoxygenconcentration,duringdevelopmentandd uringtesting(Table3).Alllikelymodels,summingtoanAkaikeweightof1,includedaneffectoftemp erature(seeTable 2 ).Whentheseeffectswereaveragedacrossmodels,theproba-
bilityofflightdecreasedastemperatureincreasedfrom37˚to41˚C.Thiseffectwassostrongthatflie sgenerallyflewwhentestedat37˚Cbutrarelyflewwhentestedat41˚C(Fig1).
Flightdependedmoreonoxygenconcentrationduringdevelopmentthanonoxygencon- centrationduringtesting(Table 3 ).Atalltemperatures,fliesthatdevelopedathigherconcen- trationsofoxygenweremorelikelytofly(Fig1).Aweakbutinterestinginteractionemergedwhenfliesw eretestedat39˚C.Fliesthatdevelopedatnormoxiaorhyperoxiaflewmoreoftenwhentestedathigherc oncentrationsofoxygen.Bycontrast,fliesthatdevelopedathypoxiaweremorelikelytoflywhentested atlowerconcentrationsofoxygen.At37˚C,fliesfromalldevelopmentaltreatmentsflewmostoftenwhe ntestedatnormoxia,althoughtheprobabilityofflightexceeded75%inallgroups.Theseinteractiveeffe ctswereeitherrelativelyunimpor-
tantortoovariabletodetectwithoursamples,asevidencedbythelowlikelihoodofmodelswiththeseint eractions(seeTable 1 ).However,temperaturelikelyinfluencestheresponsetooxygen,becauseamu chstrongereffectofoxygenwasobservedwhenfliesweretestedat25˚C.Atthistemperature,bothhyp oxiaandhyperoxiagreatlyreducedtheprobabilityofflight(Fig2).Infact,fliesat25˚Cperformedabouta swellorbetteratnormoxiaasdidfliestestedat37˚C.
Theheatresistanceofrestingflieswasunrelatedtotheiroxygensupplyduringdevelopment(Table 3 ).
Themostlikelymodelofknockdowntimeincludedonlythefixedeffectofsex(see
Table 3. Theimportanceoffactorsinourmodelsofflightperformanceandknockdowntime.ImportanceequalsthesumofAkaikeweightsformodelsthatincludet hefactor(ortheprobabilitythatthefactorwouldoccurinthebestmodel).Adashindicatesthatafactorwasnotconsideredinthesetofmodels.
Factor Importanceforflightperformance Importanceforknockdowntime
Sex 0.49 1.00
Oxygenconcentrationduringdevelopment 0.67 —
Oxygenconcentrationduringtesting 0.36 0.05
Sex·testoxygen — 0.04
Temperatureduringtesting 1.00 —
Developmentaloxygen·testoxygen 0.17 —
Testoxygen·temperature 0.15 —
Developmentaloxygen·temperature 0.05 —
Developmentaloxygen·testoxygen ·temperature <0.01 —
https://d o i.org/10.1371/ j ournal.pon e .0177827.t003
Fig1.Flightperformancedependedonbodytemperatureandoxygensupply.At37˚C(left)and39˚C(center),fliesperformedbetteriftheyhaddevelope dwithagreatersupplyofoxygen.At41˚C(right),fliesperformedpoorlyoverall.Thecolorofeachbardenotestheoxygenlevelatwhichfliesweretested(lightgra y=12%,darkgray=21%,black=31%).Themostlikelyprobabilityofflightundereachconditionwascomputedbymultimodelaveraging.Thenumberofobserv ationsusedtoestimatethemeanismarkedatthetopofeachbar.
https://d o i.org/10.1371/ j ournal.pon e .0177827.g0 0 1
Table 2 ).Thismodelwas95%likelytobethebestmodelintheset,andwas22timesaslikelyasthesecon d-rankedmodel(ΔAIC=6.2).Althoughfemalestookabout1.5minlongertosuc-
cumbtoheatstressthanmalesdid,meanknockdowntimesforbothsexeswerevirtuallyiden- ticalamonggroupsthatdevelopedindifferentoxygentreatments(Fig3).
Fig2.At25˚C,fliesraisedatnormoxiaperformedbestwhentestedatnormoxia.Themostlikelyprobabilityoffli ghtundereachconditionwascomputedbymultimodelaveraging.Fiftyfliesweretestedateachconcentrationofoxy gen.
https:// d oi.org/10.1371/ j ournal.po n e.0177827.g 0 02
Fig3.Oxygenduringdevelopmenthadnoeffectonafly’sabilitytoresistknockdownat39.5˚C.Large,solidsymbolsdenotethemostlikelymeansestimated bymultimodelaveraging.Samplessizeswereasfollows:23,16,17,11,21,16,17,and17femalesraisedat10%,12%,15%,18%,21%,24%,
27%,and30%oxygen,respectively;and21,18,16,13,23,16,18,and16malesraisedat10%,12%,15%,18%,21%,24%,27%,and30%oxygen, respectively.
https:// d oi.org/10.137 1 /journal.pone . 0177827.g003
Discussion
Ourresultspoorlysupporttheoxygenlimitationhypothesis.Raisingtheatmosphericconcen- trationofoxygenfrom12%to31%hadweakeffectsonflightperformanceatthestressfultem-
peratureof39˚C(seeFig1).Althoughfliesraisedinnormoxiaandhyperoxiamighthavebenefittedfrom hyperoxia,fliesraisedathypoxiawerelesslikelytoflyathyperoxia.Yet,wehesitatetogeneralizethesep atternsgivenhowlittlevariationwasexplainedbythisinteraction(seeTable 1 ).Furthermore,fliesraise dathypoxiawereunabletoresistknockdownat39.5˚Canylongerthanwerefliesraisedathyperoxia(se eFig3).Thus,wedetectedlittleornoevi-
dencethatheattoleranceacclimatedtooxygensupply,regardlessofwhetherheattolerancewasmeas uredduringactivityoratrest.Thisresultagreeswiththoseofpreviousstudiesinwhichthelethaltemperat ureofrestinginsectswereunrelatedtooxygensupply[8,16,40–42].
Ingeneral,developingatahigherlevelofoxygenconferredamajorbenefittoaerobicpeformancedu ringadulthood,whichhasbeenobservedrarelyininsects[15,43].Atboth37˚and39˚C,fliesraisedathyp eroxiaweremostlikelytoflywhentestedatanyconcentrationofoxygen.Interestingly,thisdevelopment alacclimationtohyperoxiaimposednolossofperfor-
manceathypoxia,whichweexpectedasatradeoff.Thispatternaccordswithrecentevidencethatoxyg endoesnotaffectcriticalpO2ortrachealconductanceofflies[44].Fliesraisedathyperoxiacouldhavere ducedtheirinvestmentinthetrachealsystemtosuchadegreethattheywereunabletodeliversufficient oxygenunderhypoxia;ifso,theseflieswouldhaveper-
formedworsethanotherflieswhentestedathypoxia[24–27,45].Instead,thesefliesper-
formedbetterthanotherfliesatalloxygenconcentrations.Thisgreaterperformancecouldcouldhaveb eenasimplebenefitofenhancedgrowthorreducedstressduringdevelopment.Inpreviousexperiment s,fliesdevelopingathyperoxiareachedalargersizeatmaturitythanthosedevelopingatnormoxiaorhy poxia[24,27,46–
49].Ifflieswerelargerinhyperoxia,theirsizemighthaveimpartedanadvantageduringflight.Alternative ly,orinconjunctionwiththishypothesis,byreducingtheneedtoinvestinanenergeticallycostlytracheal system,theseinsectscouldincreasetheirinvestmentinflightmusclemassandotherrelevanttissues[2 8,
50].Theseadvantagescouldhaveresultedinagreaterratioofflightmuscletobodysize,whichtendstosca lehypermetrically,enhancingpowerandagility[51–55].
Becausefliesdesiccatemorequicklyathighertemperatures,weshouldconsiderwhether
theknockdowntimesinourexperimentwereinfluencesbyhydricstressmorethanthermalstress.Thek nockdownassaylastedabout11minonaverage(Fig3),duringwhichflieswereinsealedvialswitharelati vehumidityequaltothatoftheroom(50%-
60%).Basedonpreviousexperiments,thisdegreeofhydricstressseemstoobenigntocauseaknockdo wnresponse.IndividualsofD.melanogasterresisteddesiccationat25˚Cand0%humidityforanaverag eof10to80hours[56,57].Morerelevanttoourstudyofheatstress,fliesresisteddesiccationat37˚Cand0
%humidityforanaverageof48–
53minutes[58].Althoughthetemperatureinourexperimentwasslightlyhigher,aknockdowntimeof11 mininanenvironmentwithmuchgreaterhumidityseemslikelytooshorttoattributetodesiccation.There fore,thepatternofknockdowntimereflectsheatstressratherthanhydricstress.
Atlowertemperatures,weoberservedacleardisadvantagetoflyingateitherhypoxiaorhyperoxia.
Weweresurprisedbythisresult,havinghypothesizedthathyperoxiawouldenhanceaerobicmetabolis mduringactivity.Althoughwecanonlyspeculateaboutthecauseofthispattern,fliesathyperoxiamight havesacrificedtheirpotentialforaerobicmetabolismtolimittheproductionofreactiveoxygenspecies.
Duringabriefexposuretohyperoxia,asinourexperiment,aninsectcouldeitherreduceitsexposuretore activeoxygenspeciesorsufferdamagetocells[15,59–
61].Inresponsetohyperoxia,somespeciesofinsectsareknowntoclosetheirspiracles[62–
64].Infact,researchershavearguedthattherespiratorypatternsofinsectsevolvedtoavoidoxidativeda mage[65].Contrarytothishypothesis,theoxygenlevelinthetrachealsystemoflocustsdirectlymatche dthatoftheambientairwhenexposedtohyper-
oxia[66].Althoughthishypothesisremainsunresolved,themechanismthatweproposewouldreducefl ightperformancebylimitingtheoxygensupplytocellsdespitetheabundanceofoxygenintheenvironm ent.Thishypotheticalmechanismhastheadvantageoffittingobser-
vationsathighertemperaturesaswell.Asbodytemperatureincreased,thedemandforATPmighthavei ncreasedtothepointthatonlyatinyfractionofoxygenwasreducedtoformsuperoxideradicalsinsteadof water[67,68].Inlinewiththisreasoning,thedifferencebetweenperformanceatnormoxiaandperforma nceathyperoxiadependedonthetemperature:nor-
moxiaconferredalargeadvantageat25˚C,amildadvantageat37˚C,andaslightdisadvantageat39˚or4 1˚C.Ifourhypothesisholds,ahyperoxicenvironmentwouldimposeacostwhenfliesdemandlessoxyg en.
Wehaveonlybeguntoexploretheinteractionbetweenheatandoxygenstresseswhenanimalse ngageinenergeticallydemandingyetecologicallyrelevantactivities.Additionalexperimentsare neededtosupportorrefutetheideathatoxygencanlimitheattoleranceinterrestrialanimals[9].Alth oughresearchhasfocusedonafewspecies,whichoftenresideinoxygen-
richenvironments,manyanimalsliveinsoilsthatbecomehypoxic[69].Some
insectspassthroughlarvalstagesthatexperienceperiodsofhypoxiainrottingfruit,meat,orfeces[7 0].Otherinsectspassthroughaquaticstagesbeforebecomingterrestrialadults.Thepotentialforoxyg enlevelsduringthesestagestoinfluencetolerancetohypoxiaorheatatlaterstagesremainslargelyu nexplored.Futureresearchshouldfocusonheattolerancedur-
ingaerobicallychallengingactivitiesattheoxygenlevelsencounteredatspecificstagesofthelifecycl e.
Acknowledgments
WethankMarcinCzarnoleskifordiscussionsrelatedtotheideasinthispaper.Thisresearchwasfund edbytheSchoolofLifeSciences,theBarrettHonorsCollegeatArizonaState
University,andMidwesternUniversity.N.Szablawassupportedbyprojectco-
foundedfromEUresourcesundertheEuropeanSocialFund(POKL04.01.01-00-053/09).
AuthorContributions
Conceptua lization:SSJLVTJMVMJA.Datacuratio n:JMVMJA.Formalanalysis:JMVMJA.Funding acquisition:JMVMJA.Investigatio n:SSJLVTNS.Methodology:SSJM VMJA.Projectadministration:JMV MJA.Resources:JMVMVA.Supervi sion:JMVMJA.Validation:SSJLVT.
Visualization:JMVMJA.
Writing–originaldraft:SSJMVMJA.
Writing–review&editing:SSJLVTNSJMVMJA.
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