© 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,3447-3456doi:10.1242/jeb.095828 Received 10September 2013;Accepted15July2014 *Author ([email protected]) for correspondence Arizona State University, POBox 874701,Tempe, AZ85287,USA. pressure ofoxygenintheatmosphere wouldallowlarger to more challengingforlarger insects;therefore,ahigher partial al., 2010).Onepossiblemechanismisthatoxygendeliverycould be size appeartobecomplexandvariableacrossspecies(Harrison et mechanisms responsibleforoxygen-linkedchangesininsect body competitors [birdsandbats(ClaphamKarr, 2012)].The with decreasesinsizeassociatedtheevolutionofflying then independentofatmosphericoxygenlaterinevolutionary time, rise andfallthatoccursintheCarboniferousPermian,but is strongly withatmosphericoxygenlevelduringthemajor fossilsdemonstratesthatmaximumsizecorrelates tissues (Grahametal.,1995).Recentexaminationofover10,000 constrained bytheabilitytosupplysufficient oxygentoactive hyperoxia; thisfindingstimulatedthehypothesisthatinsectsize is gigantism inthelatePaleozoiccoincidedwithatmospheric output (Marden,1994).Inrecentyears,ithasbeenshownthatinsect enforce anuppersizelimitbecauseofreducedmaximalpower problems (Price,1997),and/orthelackofanaerobiccapacitiesmay of insectsmaynotbeabletosupportlarge bodiesbecauseofscaling mechanistic constraintsoninsectsizemayoccur. Theexoskeletons Clapham andKarr, 2012).Alternatively, somenon-adaptive reptiles andmammals(Damuth,1981;BlackburnGaston,1994; be adaptiveasaresultofcompetitionwithorpredationbybirds, possible explanationshavebeenproposed.Smallerbodysizesmay Why areinsectssosmallcomparedwithvertebrates?Several Respirometry KEY WORDS:, Flight,Hyperoxia,Hypoxia,Insect, maintain anoxygensupply-to-demandbalanceevenduringflight. correlated withbodysize,indicatingthatlargerinsectsareableto oxygen sensitivityofflightmetabolicratesandbehaviorswerenot sensitive todecreasingoxygenlevelsthanflightmetabolicrate.The did inducehigherflightmetabolicrates.Flightbehaviorwasmore metabolic demandsofhoveringflight.Loweredatmosphericdensities density air(nitrogenreplacedwithhelium)inordertoincreasethe oxygen. We alsoassessedtheoxygensensitivityofflightinlow- from 30%to2.5%assessthesensitivityoftheirflightatmospheric and flightmetabolicratesinsevenoxygenconcentrationsranging ranged inmassbyanorderofmagnitude.We measuredflighttimes rates andbehaviorduringhoveringfor11 speciesofdragonfliesthat bymeasuringtheoxygensensitivityofflightmetabolic challenging inlargerinsects.Thisstudytestedthishypothesis levels constraininsectbodysizesbecauseoxygendeliveryismore and theexistenceofgiantfossilinsects,isthatatmosphericoxygen One hypothesisforthesmallsizeofinsectsrelativetovertebrates, Joanna RandylHenry*andJonFewellHarrison Effects ofbodysizeonthe oxygensensitivityofdragonflyflight RESEARCH ARTICLE INTRODUCTION ABSTRACT has testedwhether larger insectsaremoreoxygen sensitiveusinga that matchaerobicneedsregardless ofsize.To date,only one study sufficient timeforevolutiontoselecthighoxygendelivery rates body massonmaximalsize, asacrossspeciestherehasbeen comparisons fortestingpossible physico-chemicalconstraintsof than occursacrossspecies).Interspecific comparisonsarethebest variation amongindividuals(with usuallymuchsmallervariance developmental effects that are independentofbodysize)and variation occursbecauseofontogeny(whichcan have between intra-andinterspecificstudies.Inintraspecificstudies, size Schilder andMarden,2004;Darveauetal.,2005). related patternsofflightphysiology(Marden,1994;Dudley, 2002; species ofdifferent sizeshave beenveryusefulindiscerningsize- rest, andcomparisonsofflightmetabolismkinematicsacross flight doeselicitveryhighratesofaerobicmetabolismrelative to vary withspeed,loadliftingandturning(Dudley, 2002),hovering flight. Althoughpoweroutputandmetabolicrateduringflight can increasing sensitivityoflarger insectstooxygenwouldbeduring 2006), suggestingthattheidealperiodinwhichtotestforan delivery decreases(RascónandHarrison,2005;Harrisonetal., insects increase5-to100-fold,andthesafetymargin foroxygen maximal capacity. Duringflight,oxygenconsumptionratesof metabolism whentheoxygendeliverysystemisoperatingnearits basis thattheydidnotexamineinsectsduringhighratesofaerobic increase inoxygensensitivitywithbodysizecanbecriticizedonthe (Greenlee andHarrison,2005).However, thesepriortestsforan 2005), restingbeetles(Leaseetal.,2012)andfeedingcaterpillars 2004; Greenleeetal.,2007),hoppinggrasshoppers(Kirkton been negativeforrestinggrasshoppers(GreenleeandHarrison, of larger insectsaremoresensitivetodecliningoxygenlevelshave evidence againstthishypothesis. Schistocerca Maddrell, 2005;HarrisonandHaddad,2011) whilethesizeof1 size of1 oxygen relativetosmallerinsects.However, theobservationthat addressed whetherthegrowthoflarger insectsismoresensitiveto atmospheric oxygenongrowthandsizehavenotexplicitly hyperoxia haslittleeffect. Suchexperimentsexaminingtheeffect of although themostcommonfindingisthathypoxiareducessizeand positive correlationbetweeninsectmassandrearingoxygenlevel, 2010; HarrisonandHaddad,2011) haveshownthattherecanbea multi-generation studies(HenryandHarrison,2004;Kloketal., Frazier etal.,2001;PeckandMaddrell,2005;Klok2010) yielded mixedresults.Somesingle-(Greenberg andAr, 1996; that insectbodysizeisconstrainedbyoxygenavailabilityhave delivery, unlessaugmentedby convection. be associatedwithincreasingdiffusive limitationsonoxygen component oftrachealoxygendelivery, andthuslarger sizecould the gasphasethroughtrachealsystem.Diffusion isanimportant exist andfunction(Grahametal.,1995).Insectsdeliveroxygenin Tests foreffects ofbodysizeonoxygensensitivitymaydiffer Direct testsofwhetherthephysiologicalandbehavioralfunctions The experimentsthathavebeenconductedtotestthehypothesis mg Drosophila does not(Harrisonetal.,2006)providessome decreases stronglywithhypoxia(Peckand 3447 g

The Journal of Experimental Biology atmospheres, CO when thedragonflieswereflown inheliox,but2.5%oxygen uain(is4,5, Table duration (Figs significant effect ofairdensityonflightboutdurationortotal flight 3,Table mixtures (Fig. The flightboutswerealsosignificantly morefrequentinhelioxgas al 1).CO Table 2, when hoveringinhypodenseatmospheres(helioxmixtures;Fig. terms wereexcludedfromsubsequentstatisticalanalyses. were significantforanyofthesevariables;therefore,interaction rate andallofourflightbehaviorvariables.Nonetheseinteractions 3448 mass andoxygen(boththree-waypair-wise) on CO We firsttestedforsignificant interactionsbetweenairdensity, body in 21%nitrox.AlthoughCO emission rateassociatedwiththatpeakforindividualanimalsflown the questionofwhetherCO Many oftheflightboutswerequiteshort(afewseconds),raising 30% O durations ofindividualsweremeasuredin2.5,5,7.5,10,15,21and flow-through respirometry, theflightmetabolicratesandhovering dragonflies thatvariedbyanorderofmagnitudeinbodysize.Using in ambientoxygenconcentrationsusingmultiplespeciesof similar oxygensensitivities. plausible thatextantOdonataandthePaleozoicProtodonatahad similar tomoderndragonflies(GrimaldiandEngel,2005),thusitis giant ProtodonataofthelatePaleozoicweremorphologicallyvery oxygen thangroupsthatrelyheavilyonabdominalpumping.The perhaps dragonfliesaremoresensitivetochangesinatmospheric adjustability ofventilationmechanismssuchasabdominalpumping, autoventilation isstrictlycoupledtowing-beatsandlacksthe muscle motionduringwingstrokes,causingairmovement.Because air sacssurroundingtheflightmuscles;thesearedeformedby primarily byautoventilation(Weis-Fogh, 1967).Dragonflieshave 1998). Ventilation duringflightofdragonfliesisthoughttooccur dragonflies arehighlysensitivetooxygen(HarrisonandLighton, found thathyperoxiastimulatedflightmetabolicrate,suggesting level. Theonlypriorstudyofoxygendeliveryinaflyingdragonfly the sensitivityofdragonflyhoveringflighttoatmosphericoxygen of restingmetabolicrates(Greenleeetal.,2007). comparative approach,andthatstudyexaminedthesizesensitivity RESEARCH ARTICLE be morenegativelyaffected bydecliningoxygen. performance (metabolicrate,flightduration)oflarger specieswould changing oxygenlevels,thenwepredictedthattheflight sensitivity offlight.Iflarger dragonflyspeciesaremoresensitiveto 1995; Robertsetal.,2004),andthusmightincreasetheoxygen the powerrequirementsandmetabolicratesduringflight(Dudley, helium asabalancegas(heliox).Theuseofhypodenseairincreases flown invariableoxygenatmosphereshypodenseairusing lgt(i.1). flight (Fig. short flightboutsof1–2 flight boutduration,suggestingthatmetabolicratesduring very when flightboutdurationswereshort,theyindependentof relationship betweenflightboutdurationandthemeasuredCO steady-state conditions.To assessthisquestion,wetestedthe Air density effects on flight metabolism andbehavior onflightmetabolism Air densityeffects flightboutdurationonCO of Effect RESULTS Dragonflies consistentlyproducedcarbondioxideatahigherrate We measuredbehavioralandphysiologicalresponsestochanges The presentinterspecificstudylooksattheeffects ofbodysizeon 2 , balancedwithN 2 emission ratesaveragedapproximately 10%higher 2 emission rateswere75%higher thaninnitrox.

1). However, therewasnoconsistentor s approximatetheratesduringsteady-state

2 1). (nitrox). Thesameindividualswerealso 2 emission ratesweremorevariable 2 emission ratesapproximated 2 emission rates 2 emission 2 b (using allanimals)wasnotsignificantlydifferent fromzero( shown. Eachuniquesymbolrepresentsasingleanimal.Theregression flight boutsindragonflies. Fig. atmosphere (nitrox). each oftheseparametersin21%oxygen,normaldensity atmospheric oxygenlevel,weassessedtheeffect ofbodymasson effects morefully, andindependentlyoftheeffects ofairdensityand emission ratesduringflight(Table duration (Table mass onthenumberofflightbouts,boutdurationandtotal flight as independentfactors,thereweresignificantlineareffects ofbody Within modelscontainingbodymass,oxygenlevelandairdensity relatively constant( to orgreaterthan5%,carbondioxideproductionremained emission rates(Table There wasanoveralleffect ofoxygenconcentrationonCO oxygen, metabolicratesdropped( oxygen concentrationsgreaterthan5%, t mass andmetabolicratetypicallyseenininsects( not significantlydiffer from the2/3powerrelationshipbetween power. Thoughtheregressionmodelwasapoorfit,thisslopedid Body masseffects andbehavior onflightmetabolism Oxygen effects t (Figs positive correlationbetweenflightbehaviorandoxygenlevel species (across all oxygenlevels). levels greaterthan10%)andfewer flightboutspertrialthansmaller bout durationsandlongertotal flightdurationspertrial(inoxygen statistically significant,larger dragonfliesgenerallyhadlonger flight but itdidaffect measuresofflightbehavior(Table did notsignificantlyaffect carbondioxideproductionduring flight, significantly affected byoxygenlevel(Table Fig. =0.071, d.f.=9,P =–5.02, d.f.=11.61, =2.688, F CO Using agenerallinearmodel, the massofdragonflyspecies

.Carbondioxideemissionratewasindependent oftheduration 1. –1 2, Table CO2 emission rate (log µmol h ) 3–5). 2 2.0 2.5 3.0 3.5 emission scalednon-significantlywithmasstothe0.44 1,606 01 02 30 25 20 15 10 5 0 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095828 =0.153, P

1). Allmeasuresofflightbehaviorwerehighly

1), thoughnosignificanteffect ofbodymassonCO ● ● >0.05). P ● =0.696, R

a priori 1). However, atoxygenconcentrationsequal <0.001; heliox,t Flight boutduration(s) To simplifyvisualization,onlyeightanimalsare 2 =0.0002). paired

1). To analyzethesebodymass a priori t =–2.91, d.f.=11.10, -tests versus21%:forall P >0.05); whileat2.5% paired

1), withageneral t a

-test onslopes: 1). Thoughnot =0.001, t -tests: nitrox, − − − − 0.01 0.04 0.13 0.40 P =0.01; Metabolic rate (W) 2 2

The Journal of Experimental Biology flight boutduration(datanotshown). flight durationpertrial(Fig. flight bouts,meanboutdurationortotal RESEARCH ARTICLE There werenosignificant two-wayorthree-wayinteraction termsinanyofthesemodels. Boldindicatessignificancewith CO Dependent variable linear modelstestingforeffects onCO There werenosignificantoxygen×massinteractionsinthegeneral differ intheiroxygensensitivityofCO flight bouts(ANOVAs, allP rates, meanflightboutdurations,totaltimeorthenumberof Perchers andfliersdidnotdiffer intheircarbondioxideproduction energy equivalenceof1 quotients remainedconstantat1.0,metabolicrateswerecalculatedusingan emission ratesandmetabolic(Table species). Thereweresignificanteffects ofairdensityandoxygenonCO represent meanemissionratesinnormodenseair(averagedacrossall White barsrepresentmeanemissionratesinhypodenseairandblack Fig. number offlightbouts( Table Number offlightbouts( metabolism andbehaviormetabolism flight bodymassontheoxygen sensitivityof of Effects Perchers versus fliers Total flightduration (log Mean flightduration(log 2

.Carbondioxideemissionversusoxygen levelindragonflies. 2. –1 emission rate(log CO2 emission rate (log µmol h )

1. ANOVA resultsdemonstratingtheimpactofgasdensity, oxygencontentorspeciesbodymassonlog 0.5 1.0 1.5 2.0 2.5 3.0 0 . . 01 130 21 15 10 7.5 5 2.5 Nitrox Heliox 10 10 √

10 ml N μ s) mol ) s) Oxygen level(%O O √ N 2 =20.1 h ), meanflightduration(log − 1 >0.05). Perchersandfliersalsodidnot )

6B), numberofflightboutsormean J. Dataaremeans±s.e.m.

1). Assumingthatrespiratory 2 oreo aito d.f. Source ofvariation Density Density Density Total Mass Oxygen Oxygen Mass Mass Oxygen Density Total Total Oxygen Mass Total 2 rdcin(i.6A),total production (Fig. 2 emission rate,numberof ) 10 − − − − s) andtotalflightduration(log 0 0.001 0.021 0.126 2 Metabolic rate (W) 1 1 1 149 1 1 1 1 1 1 1 134 135 1 1 150 number offlightboutspertrial(Table Lower oxygenlevelsandhigherairdensitybothsignificantlyreducedthe represent resultsinhypodenseairandblackbarsnormodenseair. Fig. sensitivity ofmetabolicrateandbehaviorduringaerobicflightfor Our studyisthefirsttotestforaneffect ofbodymassontheoxygen if weadjustedtheanalysesforphylogeneticrelatedness. 8). Thisconclusiondidnotchange each ofthetestedvariables(Fig. correlation betweendragonflymassandtheoxygensensitivityof weremoreresponsivetovaryingoxygenlevel. These slopeswerethenplottedversusmasstotestwhetherlarger 7). data separately(slopesfortotalflightdurationareshowninFig. calculated theslopeofalinearregression,assessingnitroxandheliox for eachdependentvariablethatspeciesversusoxygenand assess responsetooxygenlevelswithinspecies,weplottedthevalue was assessedrepeatedlyindifferent oxygenlevels.Therefore,to because itdoesnotconsiderthefactthateachindividualandspecies similarly tooxygen.However, thisapproachhasrelativelylowpower (Table DISCUSSION In bothnormobariaandhypobaria,therewasnosignificant

.Frequencyofflightversusoxygenlevelindragonflies.Whitebars 3. 1), suggestingthatdragonfliesofdifferent massesresponded SS 2.061 0.002 0.025 72.386 0.167 14.897 0.07 0.102 0.031 0.34 5.503 0.714 0.9 46.86 9.951 175.462 Number of flight bouts 10 15 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095828 0 5 10 s) . . 01 130 21 15 10 7.5 5 2.5 Nitrox Heliox MS 2.061 0.002 0.025 0.486 0.167 14.897 0.07 0.102 0.031 0.34 5.503 0.005 46.86 0.007 9.951 1.17 Oxygen level(%O

1). Dataaremeans±s.e.m. P <0.05. 10 CO 4.242 30.663 0.412 3.6937 0.344 13.054 19.091 4.641 50.226 FP 4.705 40.06 8.507 2 2 ) emission rate, 0.522 0.057 0.558 0.041 <0.001 <0.001 <0.001 <0.001 0.033 0.032 <0.001 0.004 3449

The Journal of Experimental Biology integrating CO to maintainlongerflightsinthechamber. Also,ourmethodsrequired most ofthedragonflyspecies(thoughabletohover)werenotwilling 3450 Argonne National Laboratory;forthose few observations,we examined severalX-raysof different speciesofdragonflies at reported measurementsoftracheal volumesfordragonflies.We significant reservoirsofoxygen duringtheseflights.Thereareno to hypoxiawouldbeifoxygen storeswithinthebodyserveas catabolism. Onepossibleexplanation forthelowsensitivityofflight that flightsofseveralseconds weresustainedbynon-aerobic metabolism tofuelflight(Beenakkersetal.,1985),soitisunlikely emission ratescanbemeasuredaccurately. durations werequiteshortinthisstudy(averaging2.9±0.36 limited priorattemptstomeasuredragonflyflightmetabolism.Flight a respirometrychamberprovidesmajortechnicalchallenge,andhas The reluctanceofmostdragonflyspeciestoflyforalongdurationin support limitedflightinallbutthemosthypoxicenvironments. may allowflightmetabolicratestoremainsufficiently highto duration andthenumberofflightattemptsinresponsetohypoxia atmospheric oxygencontent.Thisstrategyofreducingflight metabolic rateofdragonflieswashighlysensitivetochangesin of mass.However, regardlessofsize,theflightbehaviorbutnot sensitivity offlightmetabolicrateandbehaviorwereindependent consistent withpriorresultsusingrestinganimals,theoxygen hypothesis thatlarger insectsareincreasinglyoxygenlimited,but any animalspecies.Incontrasttopredictionsbasedonthe (Table bout durations,butdurationswerenotsignificantlyaffected byairdensity represent flighttimesinnormodenseair. Loweroxygenlevelsreducedflight bars representspeciesmeanflighttimesinhypodenseairandblack Fig. RESEARCH ARTICLE issues raises thequestionofwhetheroxygensensitivityflightand CO short flightdurationsandrelativelylongchamberequilibration times specifically chosenforitslongflightdurationsinarespirometer. The (Harrison andLighton,1998);however, thattarget specieswas measured muchlongerflightdurationsforonedragonflyspecies required relativelylongequilibrationtimes( periods becausetherelativelylarge chambersrequiredtopermitflight theflightCO Reliability of Unlike vertebrates,insectsrelyalmostexclusivelyonaerobic

.Meanflightdurationversusoxygenlevelindragonflies.White 4.

1). Dataaremeans±s.e.m. Mean flight duration (s) 10 0 2 4 6 8 2 peaks thatextendedtemporallywellbeyondtheflight . . 01 130 21 15 10 7.5 5 2.5 Nitrox Heliox Oxygen level(%O 2 emission ratesandsamplesize 2 ) i.9). Fig. One study

s) because 2 conditions. completely responsiblefortheobservedflightunder these 5% oxygen,theuseofoxygenfrominternalstorescannot be addition, astheestimated0.8 conditions, eventhoughtheywereofquiteshortdurations. In (Fig. comparing flightslastingonly1–2 assess thein-flight CO that ourflow-throughrespirometry systemwasabletoaccurately bracketing ourrangeofestimates. Insum,theseanalysessuggest Ellington, 1997),while the dragonfly range ofourvalues.Usingheat lossmodels,themetabolicratefor Erythemis simplicicollis flight metabolicrateof0.12 these metabolicratesrangedfrom0.24to0.43 rates, ourestimatedmetabolicrates(range0.05–0.38 of theCO of internaloxygenstoresmayhavebluntedthesensitivity 2 respectively. Thesedurationsarerelativelylongcomparedwiththe oxygen storescouldmaximallysupport7.4,2.8and0.8 that whendragonfliesareflyingin21,10and5%air, thetracheal low as5 dragonflies tosustainsimilarmetabolicratesinatmosphereswithas duration andflightCO at 2.5%(Figs tracheal oxygenlevelsdropto2%,asflightbehaviorwasminimal minimally 1%belowairvalues,andthatflightmuststopwhen oxygen stores,weestimatedthattracheallevelwouldbe volume. To estimatethedurationofflightpossibleusinginternal visually estimatedthattrachealvolumesapproximated30%ofbody Fig. 907 μmol junius, consumption ratesforfourofthesamespeciesweused( range ofpreviouslyreportedvalues.Polcynoxygen duration, buttherewasnotasignificanteffect ofairdensity(Table represent flighttimesinnormodenseair. Hypoxiareducedtotalflight bars representthetotaltimespentinflighthypodenseairandblack are means±s.e.m.

s flightboutsobservedinmanycases;thusitisplausiblethatuse Also supportingthereliabilityofourmeasuredCO

.Total flightdurationversusoxygenlevelindragonflies. 5. 1), suggestingthatmostofourflightsdidrepresentsteady-state Aeshna multicolor

kPa Total flight duration (s) h 10 15 20 25 30 35 − The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095828 0 5 2 1 , andassumingarespiratoryquotientof1,weestimated O emission rate,perhapsexplainingtheabilityof Sympetrum sanguineum 2 3–5). UsingouraverageCO . However, wefoundnorelationshipbetweenflight . . 01 130 21 15 10 7.5 5 2.5 Nitrox Heliox 2 A. junius emission ratesof thesedragonflies. (Harrison andLighton,1998),falling inthe 2 , emission ratewithinindividuals,even Libellula saturata Oxygen level(%O

s offlightislessthanweobservedin

W wasreportedforthedragonfly had arateof0.37

s withflightslastingover20 was 0.04 and 2

) W (Polcyn,1994).A 2 emission ratesof W (Wakeling and Tramea lacerata

W (May, 1995),

W) wereinthe

2 s offlight,

1). Data White emission Anax );

s

The Journal of Experimental Biology RESEARCH ARTICLE attempted (Fig. (Figs flight durationsdeclined2-to6-foldasoxygenlevel was muchmoresensitivetohypoxiathanflightmetabolicrate. Total grasshoppers (RascónandHarrison,2005),isthatflightbehavior A strikingresult,similartoapriorstudyoftetheredflightin increase inCO by 33%comparedwithflightinnitrox.Inourstudy, theaverage flight inhelioxincreasedpoweroutputby10%andmetabolicrate by 45%whenhoveringinheliox(Dudley, 1995).Incarpenterbees, in hypodenseair(Dudley, 1995).Orchidbeesincreasepoweroutput because oftheincreaseinpowerproductionrequiredtogeneratelift metabolic rateofflyinginsects(Robertsetal.,2004),probably As foundbypreviousresearchers,hypodenseairincreasesthe could causethemtobelessaffected byloweringairdensity. amplitudes andreducedwing-loadingofdragonfliesrelativeto bees dragonflies asbees.Thelowerwingbeatfrequencies,smallerstroke may notaffect thepower requirementforflightasmuchin in helioxthannitrox(Fig. Fig. quite insensitivetooxygenlevels overabroadrange(Fig. Oxygen sensitivity of flight metabolism andbehavior flightmetabolism Oxygen sensitivityof air Inducing maximalflightpower outputbyusinghypodense hypoxia, theCO central nervoussystemdrive(Amann etal.,2006;Noakes,2009). is alsobelievedtobepartiallyexplained byhypoxicsuppressionof motivation. Reducedlocomotor performancebyhumansinhypoxia –1 In contrasttotherelativelystrong sensitivityofflightbehaviorto CO2 emission rate (log µmol h ) 0 1 2 3 4 0 1 2 3 4 6A), withsignificant declinesonlyobserved in 2.5%oxygen A 5, 7),withalarge partofthis effect duetothenumberofbouts ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 10152025305 5 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

2 3). Thesedatasuggestthathypoxiasuppressesflight

10 1520 emission ratewasonlyapproximately10%higher ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 2 emission ratesofflyingdragonflies tendedtobe Percher Flier ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● 2), suggestingthatloweringtheairdensity ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Nitrox Heliox

25 30 Oxygen level(%O ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

Total flight duration (log s) 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0 0 B 2 ● ● ● ● ● ● ● ● ● ● ● ) 5 10152025305 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

10 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

2, Percher

15 20 Flier ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● study todetectchangesinCO these twostudiesistherelativelylowpowerofthiscomparative 18%. Oneobviousexplanationforthedifference inresultsbetween hypoxia (10%O emission ratesduringflightbyapproximately10%,andmoderate 1998). Forthisspecies,itwasfoundthathyperoxiastimulatedCO findings forthedragonflyE.simplicicollis relatively insensitivetoatmosphericoxygenleveldiffers fromprior because oftheirlowerwing-loading. emission rateat7.5%oxygenandbelow(Joosetal.,1997),perhaps bees, whichshoweddecreasedwing-beatfrequencyandCO flights. Dragonfliesseemsignificantlylesssensitivetohypoxiathan metabolism inrelativelyextremehypoxia,atleastduringshort delivery totheirflightmusclessupportmusclebehaviorand (Fig. producing gas exchange indragonflies(Weis-Fogh, 1967),our that autoventilationdueto wing movementpredominatesin oxygen contentoftheatmosphere. Althoughithasbeenreported able tomaintaingasexchange ratesdespitestrongdropsinthe explore themechanismsbywhich dragonfliesandotherinsectsare changes inflightmetabolicrate. Itwouldbeveryinterestingto atmospheric oxygencanbedropped fourfoldwithrelativelyminor In contrast,bothstudiessupportthegeneralobservation that significant, within-specieschangesinflightparameterswithoxygen. rather thanwithinspecies,leadingtoamuchgreaterpowerdetect smaller samplessizes,variationisexpectedtobegreateracross species meansfor11 speciesasourdataset.Inadditiontothe level (HarrisonandLighton,1998);inthepresentstudy, weused The The conclusionthatflightmetabolicrateofdragonfliesis ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● E. simplicicollis 2). Thusdragonfliesseemabletoachieveadequateoxygen

25 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095828

30 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 2 ) significantlydecreasedflightmetabolicrateby CO duration versusoxygenlevelindragonflies.Both Fig. dashed linesindicatehelioxregressions. intercepts. Solidlinesindicatenitroxregressions; fliers didnotdiffer significantlyinregressionslopesor increased withhigheroxygenlevels.Perchersand study measured25individualsateachoxygen 2

.Carbondioxideemissionandtotalflight 6. emission rates(A)andtotalflightdurations(B) 2 emission rateinresponsetooxygen. (Harrison andLighton, 3451 2 2

The Journal of Experimental Biology flight. important becausetheoxygendemandofinsectsishighestduring sensitivity isindependentofsizeduringflightparticularly caterpillars (GreenleeandHarrison,2005).Thefindingthatoxygen (Kirkton etal.,2005),restingbeetles(Lease2012)andfeeding and Harrison,2004;Greenleeetal.,2007)hoppinggrasshoppers which issimilartowhathasbeenobservedwithresting(Greenlee flying dragonflieshavesimilarsafetymargins foroxygendelivery, 3452 smaller thanvertebrates) cannotbeexplained byasimple,direct giant insectsofthePaleozoic (and thefactthatextantinsectsare constrained byatmosphericoxygen. Thesedatasuggestthatthe insects, andthusarenotparticularly physiologicallyorbehaviorally extant insectshaveoxygensensitivities similartothoseofsmaller Our studycontributestotheincreasing bodyofevidencethatlarge The CO al., 1997);perhapsthisisalsotrueindragonflies. in hypoxiasuggestthatconvectionisstimulatedby(Jooset flight. Inhoneybees,increasesinevaporativewaterlossduringflight imaging suggestthatdragonfliesuseabdominalpumpingduring personal observationsofdragonfliesusingX-raysynchrotron RESEARCH ARTICLE rate (Table significant interactionbetweenbodymassandoxygenonmetabolic responded similarlytoatmosphericoxygenlevel;therewas no durations andtotalflightoflarge andsmalldragonflies Oxygen as a constraint oninsectbodysize Oxygen asaconstraint emission ratesandflightbehavior CO bodysize ontheoxygen sensitivityof of Effects Total flight duration (log s) 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 0.5 1.0 1.5 2.0 I E A 2 ● ● 10152025305 ● ● ● ● ● emission rates,numberofflightbouts,mean

1, Fig. ● ● ● ● ● ● ● ● ● ● ● 0.15 g 0.44 g 0.30 g ● ● ● ● ● ● 8). Theseresultssuggestthatlarge andsmall ● ● ● ● ● ● ● ● ● ● ● J F B ● ● Oxygen level(%O 10152025305 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.16 g 0.63 g 0.35 g ● ● ● ● ● ● ● ● ● ● ● 2 ● ● ● ● ● ● ) K G C ● ● ● ● 10152025305 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 2 0.22 g 1.23 g 0.39 g ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● Desert StudiesCenter atZzyzx,CA,USA,whichis locatedatthewestern 2)werecollectedfrom theSodaSprings Eleven speciesofdragonflies(Table distinguish thesealternativehypotheses. major challengeforevolutionary biologistsistofindapproaches 1981; BlackburnandGaston,1994;ClaphamKarr, 2012).A insect sizesaslarger, moresuccessfulvertebratesevolved(Damuth, competition andpredationmayhavealsoledtothereduction of (Kingsolver andHuey, 2008).Fromanecologicalstandpoint,niche contributed totheevolutionoflarger speciesofinsectsinthepast the lateCarboniferous(Royeretal.,2004)couldhave also before thecuticlebuckles(Price,1997).Lowtemperaturesduring may needtoincreasefasterthanitsdiameter, imposingalimit from thePaleozoic.Thewallthicknessofaninsect’s exoskeleton have smallermaximumsizesnowrelativetosomefossilizedinsects insects arefrequentlysmallrelativetootherterrestrialanimals, and oxygen levelsarelower(Harrisonetal.,2010). insects todevotemoreresourcestherespiratorysystemwhen body sizemightbelimitedbytrade-offs associatedwiththeneedfor et al.,2009;Lease2006;Kaiser2007).Thus,insect proportionally moreoftheirbodiestothetrachealsystem(Greenlee Loudon, 1989),andotherstudiesindicatethatlarger insectsinvest number oftracheae(HenryandHarrison,2004;Jareckietal.,1999; atmospheric oxygenlevelisnegativelycorrelatedwithsizeand atmospheric oxygenlevels.However, severalstudiessuggestthat stimulation ofaerobicmetabolismintrachealsystemsbyhigher Animals andstudysites MATERIALS ANDMETHODS H D ● ● ● Oxygen level(%O Other hypothesesunrelatedtooxygenmayalsoexplainwhy ● ● 51015202530 ● ● ● ● ● ● ● ● Nitrox Heliox ● ● ● ● 0.43 g 0.28 g ● ● ● ● The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095828 ● ● ● ● ● ● ● ● 2 ) regressions. nitrox regressions;dashedlinesindicateheliox density andoxygenlevel.Solidlinesindicate density (Table and mass.Therewasnosignificanteffect ofair interaction betweeneachdependentvariable recorded andusedtotestwhethertherewasan junius lacerata comanche hymenaea balteata (B) Pachydiplaxlongipennis different masses. versus oxygenfordragonflyspeciesof species fortotalflightdurationpertrial linear regressionsperformedwithineach Fig.

.Representativegraphsshowingthe 7. . Theresultantslopefromeachgraphwas , (J)Aeshnamulticolor , (D)Libellulaluctuosa , (H)Libellulasaturata , (F)Tramea onusta

1) orinteractionbetweenair (A) Pantalaflavescens , (C) , (E)Pantala , (G)Libellula and (K)Anax , (I)Tramea ,

The Journal of Experimental Biology represented bythe widthofthelightgraybars. by thedarner, RESEARCH ARTICLE Fig. continuously throughoutthedayandwerecategorizedas‘fliers’,whereas Toledo, Columbus,OH,USA). animals wasmeasuredusingananalyticalbalance(MettlerAE100;Mettler located nexttoLakeTuendae. Thewetbodymass(±0.001 within 5 in July2004,oneMay2005andafinaltripAugust2005. during different summermonths,weconductedthreecollectingtrips–one dragonflies (Polcyn,1994).Asdifferent dragonflyspeciesemerge asadults made, spring-fedpond(LakeTuendae) supportsatleast13speciesof end oftheMojaveNationalPreserve(35°08 from theflightchamber. ThisparticularexampleshowstheCO –1 Roughly halfofthespeciescapturedtendedtopatrollake Oxygen effects onflightmetabolicratesandperformanceweremeasured Total flight duration (log s) CO2 emission rate (log µmol h )

.Representative traceoftheCO 9. CO2 (ppm) versus oxygen slope versus oxygen slope 0.5 0.1 0.2 0.3 0.4 10 15 0.02 0.04 0.06 0.08 0.10 0 5 0 min ofcollectingtheanimal,atanoutdoorrespirometrysetup 0 .501 .502 .503 0.35 0.30 0.25 0.20 0.15 0.10 0.05 C .501 .502 .503 0.35 0.30 0.25 0.20 0.15 0.10 0.05 A ● ● 0 ● ● Anax junius ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

● ● 50 100150 ● ● ● ● ● ● ● ● ● ● ● ● ● ● , in30%nitrox.Eachflightboutduration is ● ● ● ● ● ● Time (s) Nitrox Heliox 2 content ofexcurrentairsampled

● ● 200 250300 ● ● ′ 35″ N, 116°06′ Mass (logg)

Number of flight bouts ( N) Mean flight duration (log s)

g) ofallcaptured

15″ versus oxygen slope versus oxygen slope 0.01 0.02 0.03 0.04 0.05 0.10 0.15 0.20 0.25 2 W). A man- 0 produced 0 .501 .502 .503 0.35 0.30 0.25 0.20 0.15 0.10 0.05

B .501 .502 .503 0.35 0.30 0.25 0.20 0.15 0.10 0.05 D 350 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●● ● ● ● ● ● ● ● ● ● ● ● induced bygentlyshakingtheflight chamber. CO recording oftheflightchamberwas initiated,and,ifnecessary, flightwas for thedragonflytoequilibrate theexperimentalatmosphere,avideo Movie did occasionallyflyintothechamber walls(seesupplementarymaterial than tetheredflight(KammerandHeinrich,1978),althoughsomespecies unimpeded freeflight,whichgenerallyproduceshighermetabolic rates through respirometrysystem.A 4 transferred totheclearplasticflightchamber, whichwasalsoaflow- individuals ofthatspecies. means; unlessotherwisestated,reportedvaluesareaverages across per species(Table species andclades,ourstudyhadarelativelylownumberofindividuals collect. Thuswedecidedtofocusonobtainingthemaximumnumber of individual. Inaddition,somespecieswererelativelyrareanddifficult to several hourswererequiredtocompletemeasurementsfora single metabolic ratein14different gasmixesplussomeadditionalcontrols), many measurementsweremadeonindividuals(flightbehaviorand effects, ontherangeofspeciesmasses(HarveyandPagel,1991).Because of thesampledspecies,numberspeciesused,and,forquestionsmass The powerofinterspecificcomparativeanalysesdependsonthephylogeny wing wearindicatedthatallanimalswerematureadults. (Heinrich andCasey, 1978).Activitylevels,colorationandtheamountof vegetation andonlyflewwhenactivelyhuntingorattemptingtomate the remainingspecies,categorizedas‘perchers’,preferredtositon achieved liftduring those2 shaken relativelycontinuouslyfor atleast2 activity wasrecorded(Fig. Experimental design Individual dragonflieswerecapturedwithanetorbyhand, and ● ● ● ● 1 fortypicalflightbehaviorinthe chamber).Afterallowing5 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095828 ● ● ● ●

2). Ingeneral,wecollapsedindividualdatatospecies (A) CO either normobaricorhypobaricatmospheres. were notsignificantlyrelatedtobodymassin rates andflightbehaviorsoflargerdragonflies of theparameteronatmosphericO oxygen sensitivity(slopeofalinearregressionfit Phylogenetically uncorrecteddataareshown,with (C) Total flightduration.(D)Numberofbouts. Fig. ( sensitivity versusmassregressionswerepoor versus speciesmass.Allfitsoftheoxygen R 2

<0.1) andslopesdidnotdiffer fromzero. min, theywerenot recordedasflying.Flight 9). Ifanimalsdidnotfly, thechamberwas

.TheoxygensensitivityofCO 8. 2 emission rate.(B)Meanflightduration.

l chamberwasused,whichallowed

min. Iftheanimalsnever 2 released duringflight 2 ) plotted 2 emission 3453

min

The Journal of Experimental Biology 30 Melville, NY, USA).Video imageswereshot at astandardrateof frame analysisofvideotakenbyadigitalrecorder(ZRseries;Canon, the chamberfloor. Flightboutdurationsweremeasuredusingframe-by- continuous periodoftimewhenthewingswereactiveandanimalsoff perchlorate) andthenpulledsequentially throughaCO trial. evidence fordegradationofflightperformanceindividualsoverthe Anax junius Aeshna multicolor comparing theseanimals’ CO to testfordegradationinperformanceovertime.Althougha of animalswasflowninnormoxiabeforeswitchingtoeachnewtestgas exposed tothediffering oxygenlevelswasrandomlydetermined.A subset mixes wasalternatedthroughthestudy. Theorderinwhichtheanimalwas gas mixtures;whethertheindividualwasfirstflowninnitroxorheliox nitrogen (nitrox)orhelium(heliox).Eachanimalwasflowninallofthe 5, 7.5,10,15,21and30%oxygen,withthebalancegasbeingeither at arateof500 chamber wasdumpedintoamanifold fromwhichtheairwassubsampled readings wereinsteadusedtoconfirmthegasmixes.Excurrentairfrom the precise tomeasureoxygenconsumptionratesduringflight;the could beaccuratelydetermined.Ouroxygenanalyzerwasnotsufficiently Li-Cor, Lincoln,NE,USA),AscariteII(forCO d.f.=39.481, in 21%normoxiashowedthattherewasasignificantdifference ( 3454 rates duringflight werecalculatedbyintegratingthe areaundereachCO digitized andrecordedusingSable SystemsDATACAN. Themetabolic 1; AMETEK,Pittsburg, PA, USA).Theoutput ofbothanalyzerswas analyzer (FOXBOX,SableSystems, LasVegas, NV, USA)bya pump (R- 16.2±0.1 The flowrateofairthroughtheflightchamber(constant at (see supplementarymaterialMovie The videocamera’s angleofviewencompassedtheentireflightchamber performance andCO If multipleanimalsfromthesamespeciesweretested,s.e.m.valuesandrangesareincluded.F, flier;P, percher. Species RESEARCH ARTICLE keeping flowslowenoughsothattheCO temporal resolution(95%equilibrationtimewaslessthan1 USA). Theflowratewaschosentoreducewashouteffects andimprove regulated bymassflowcontrollersandmeters(Omega,Stamford, CT, balanced withnitrogen Table Respirometry behavior of analysis Video Libellula comanche mean CO duration ofallflightboutsinthattestoxygen. flight durationduringthe3 Pantala hymenaea Pantala flavescens Pachydiplax longipennis Libellula saturata Libellula luctuosa Tramea onusta Tramea lacerata frames 2. Meanmasses,CO l 2 s

min − emission ratewashigherforthefinalflight;thusthereno 1 ; thus,measuredflightdurations0.03 P − =0.0002), metabolicrateswerenotreduced.Instead,the

1 ml ) andtheoxygenconcentrationofmixturewere min 2 − emission ratesweremeasuredintestgasesof2.5, 1 . Thesubsampledairwasfirstdried (magnesium aeoyofanimals Category F F P F F P P P P F F

min testboutwascalculatedasthesumof 2 2 emission ratesandbehaviorcharacteristicsforeachspeciesstimulated~3 emissions duringtheirfirstandlastflights

1); aflightboutwasdefinedas Number andsex 1 male 1 male 1 male ae,1fml .6100 7.6157 2.75±1.86 475.96±115.72 1 male 3 males 1.36±0.12 0.1631±0.02 3 males,1female 348.93±127.32 1 male 3 males 0.2847±0.06 2 males,1female 2 males 1 male 2 content oftheexcurrentair 2

s couldbedetected.Total removal) andthenanO 2 analyzer (LI-6252; Mass (g) 1.2329 0.6338 0.3882 .4600 7.1278 1.40±0.18 474.91±207.87 0.2997 0.1496±0.03 4.23±1.82 1210.80±273.15 0.2189 0.4311±0.03 .54003606±9.41.90±0.31 660.64±290.64 0.3534±0.003 0.4387 010–.00 290–6.1 (0.89–4.61) (209.04–765.91) (0.1105–0.2020) (1.12–1.50) (128.66–569.71) (0.1558–0.3597) 001–.90 270–8.9 (1.25–1.75) (207.07–884.19) (0.0913–0.1970) (1.75–7.78) (719.71–1663.60) (0.3767–0.4521) 030–.59 300–5.7 (1.59–2.21) (370.00–951.27) (0.3508–0.3559)

min) while t =–4.153, t -test 2 2 air densityandbodymassasindependentfactors.ForCO duration, totalflightduration)withgenerallinearmodelsusingoxygen, relatedness (Fig. (Fig. 10B). A thirdtreewasconstructedusing randombranchlengthsand other inamonophyleticclade, while keepingtheaeshnidsseparated generated thatassumesalllibellulid specieswereequallyrelatedtoeach strict supertreealgorithm(Sanderson etal.,1998).A secondtreewas by combiningtwoothertrees(Saux etal.,2003;Ware etal.,2007) using a 10A)thatincludedallofthespeciestestedatZzyzxwasconstructed (Fig. package inR(Paradis,etal.,2004;Felsenstein,1985). independent anddependentvariablesusedinthisstudyusingthe ape phylogenetically independentcontrastswerecalculatedforeach of the affected byphylogeneticrelatedness inadditiontophysicalsizedifferences, statistically relatedtobodymassusinglinearregression. slope. We thentestedwhethertheseslopes(oxygenresponsiveness)were dependent variablesversusoxygenforeachspeciesandcalculatedthe linear species mean. then allmeasurementswithinaspecieswerefurthercollapsed into a dependent parameterswerefirstcollapsedintoanindividualmeanand then rerun.To avoidproblemswithoversampling,allmeasurementsofour model wereinsignificant,theydroppedfromthemodel,whichwas and thentwo-wayinteractionterms.Ifthesehigher-level termsofthe reduced forthesevariables.We firsttestedforthree-wayinteractionterms, time, manyanimalsdidnotflyin2.5%oxygen,sothesamplesizeswere oxygen levelsandairdensities.Forflightboutdurationstotal and numberofflightbouts,allspeciesindividualsweretestedat Core Team, 2010;http://www.R-project.org/); graphs weregeneratedusing elevation). 0.76×0.76×0.91 maintained at31.6±0.1°Cbymonitoringthetemperaturewithina to reducetheeffects oftemperatureonmetabolicrate.Thewas video recordingofbehavior. of flight,anddividingbythetimespentinflightasdeterminedusing emission peak(Fig. parameters (flightCO We testedforgeneraleffects ofoxygenandairdensityonourdependent Statistical analyses Zzyzx duringthecourseofexperimentwas101.07±0.04 from anattachedairconditioneraccordingly. Thebarometricpressureat ( CO 1015.44 350.29 1206.54 2909.53 582.10 742.13 To calculatethephylogeneticallyindependentcontrasts,a supertree Because observeddifferences inmetabolicrateandbehaviormaybe To assesstheoxygenresponsivenessofdragonflies,weplotted the All statisticalanalyses werecarriedoutusingRlanguage (RDevelopment The flightchamberwashousedinatemperature-controlledenvironment μ mol 2 msinrt Ma lgtdrto Ttlfih Numberof Total flight Meanflightduration emission rate h − 1 ) The JournalofExperimentalBiology(2014)doi:10.1242/jeb.095828

m wood-framed,Plexiglaschamberandadjustingtheoutput

10C).

9) thatcorrespondedtoaburstorcloselytimed 2 4.77 4.54 2.74 2.57 2.21 per bout(s) 2.95 emission rate,numberofflightbouts,bout

min toflyin21%oxygen 1965 5±1 (5.36–18.44) 11.9±6.54 9±3 (2.9–18.08) 11.49±4.50 (6.26–16.85) (5.24–85.54) 38.13 13.61 (4.42–12.73) 00 34 8±3 10.05 ±3.41 6±2 34.49±24.62 10.95 .841 5±3 8.58±4.16 25.67 11.03 ie()flightbouts time (s) 8.86 2 emission rates

kPa (286 8 3 4 10 5 3 m

The Journal of Experimental Biology RESEARCH ARTICLE noted. parametric tests. All valuesareshownasmeans± s.e.m. unlessotherwise rates andflightbehaviors.Data were logtransformedpriortorunning and linearregressionswereusedin the analysisofmasseffects on metabolic an experimentaltypeIerrorlessthan orequalto5%.Analysisofcovariance were determinedtobesignificantly different fromthenullhypothesis using the ggplot2andlatticepackages(Wickham, 2009; Sarkar, 2008).Results into twoclades,and(C)randombranchlengthsrelatedness. independent contrasts.(A)Supertree,(B)simpletreedividingdragonflies Fig. B

C A 0 Thethreephylogenetictreesusedtocalculatephylogenetically 10. Macrodiplax balteata Anax junius Aeshna multicolor Pachydiplax longipennis Tramea lacerata Tramea onusta Tramea lacerata Pantala hymenaea Anax junius Pachydiplax longipennis Libellula comanche Aeshna multicolor Libellula saturata Libellula luctuosa Pantala flavescens Macrodiplax balteata Tramea onusta Anax junius Aeshna multicolor Tramea onusta Libellula comanche Libellula luctuosa Tramea lacerata Macrodiplax balteata Libellula saturata Pantala flavescens Pachydiplax longipennis Pantala hymenaea Libellula saturata Libellula comanche Libellula luctuosa Pantala flavescens Pantala hymenaea arsn . rze,M . er,J . asr . lk .J n acn B. Rascón, and C.J. Klok, A., Kaiser, J.R., Henry, M.R., Frazier, J., Harrison, J.R.B. Lighton, and J.F. Harrison, arsn .F n add G. Haddad, and J.F. Harrison, M.S. Engel, and D. Grimaldi, J.F. Harrison, and H.A. Woods, M.R., Frazier, rele .J,Hny .R,Krtn .D,Wsna,M . eza . e,W.-K. Lee, K., Fezzaa, M.W., Westneat, S.D., Kirkton, J.R., Henry, K.J., Greenlee, J.F. Harrison, and C. Nebeker, K.J., Greenlee, arsn .F,Kie,A n adnros J.M. VandenBrooks, and A. Kaiser, J.F., Harrison, rele .J n arsn J.F. Harrison, and K.J. Greenlee, J.F. Harrison, and K.J. Greenlee, A. Ar, and C. S. Gans, Greenberg, R.and Dudley, N.M., J.B.,Aguilar, Graham, J. Felsenstein, http://jeb.biologists.org/lookup/suppl/doi:10.1242/jeb.095828/-/DC1 Supplementary materialavailableonlineat 0419704 toJ.F.H.]. This researchwaspartiallysupportedbytheNationalScienceFoundation[IBN analyses. Bothauthorswrotethemanuscriptandapprovedfinalversion. Both authorsdesignedthisproject.J.R.H.collecteddataandperformedall The authorsdeclarenocompetingfinancialinterests. and RobFultonforsupportwiththefieldresearch. assistance withthestatisticalanalysis.Finally, wethanktheDesertStudiesCenter regarding theanalysisandpresentationofdata.We thankMelanieFrazierfor of theHarrisonlabandSocialInsectResearchGroupgavehelpfulsuggestions assistance withrespirometrytechniques.AlexKaiser, RonRutowskiandmembers John Lighton,RobinTurner, MikeQuinlanandBrendaRascónprovidedcrucial Harvey R. Dudley, ava,C . ohck,P . obk .W n urz R.K. Suarez, and D.W. Roubik, P. W., Hochachka, C.A., Darveau, auh J. Damuth, lcbr,T .adGso,K.J. Gaston, and T. M. Blackburn, W. J.A. Van Marrewijk, and D.J. Van der Horst, A.M.T., Beenakkers, and D.F. Pegelow, M.K., Stickland, A.T., Lovering, M.W., Eldridge, M., Amann, enih .adCsy T. M. Casey, and B. Heinrich, References Supplementary material Funding Author contributions Competing interests Acknowledgements uly R. 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