and K with ahigher but therelationshipdiffered betweenthediets( to K . natural dietsforthemostaccuratecalibrationsfree-ranging rhinoceros 6005, Australia. Botanic Gardens, The BotanicGardensandParks Authority, West WA Perth, the K ( different ambienttemperaturestoinducedifferences inmetabolicrate was higherat higher ambienttemperature( V University of Western Australia, Western Crawley,University of WA 6009,Australia. 1040 Received 26August 2013;Accepted5December2013 1 (Quinlan andGibbs,2006).Metabolicratecanbereadilymeasured BMR, andtheSMRisusuallyusedforcomparativepurposes living (HulbertandElse,2004).Ininsectsthereisnodefinition for is atrest,andbroadlyconsideredtorepresenttheminimalcost of (SMR) ismeasuredunderlessstringentconditionswhenananimal (Ricklefs etal.,1996;McNab,1997).Thestandardmetabolic rate absorptive andnon-reproductivewithinthethermoneutral zone measured underdefinedconditionswhenananimalisinactive, post- metabolic rateofendothermstoeachother, beingthemetabolicrate The basalmetabolicrate(BMR)isgenerallyusedtocompare the expenditure andreflectsthecostofliving(HulbertElse,2004). The metabolicrateofananimalisameasurethatanimal’s energy Metabolic rate KEY WORDS: between metabolism, andthatthisisthemechanismofrelationship ( The measurementof Sean Tomlinson rubidium ( Special K:testingthepotassiumlinkbetweenradioactive METHODS &TECHNIQUES © 2014.PublishedbyTheCompanyofBiologistsLtd|JournalExperimentalBiology(2014)217,1040-1045doi:10.1242/jeb.096222 *Author ([email protected]) for correspondence 6009, Australia. Australia, Western Crawley,Life andPhysical Sciences, The University of WA jam dietatallT We investigatedaproposedmechanismof a usefulmethodformeasuringfieldmetabolicrateinsmallanimals. diet (F ( INTRODUCTION ABSTRACT relationship between jam enrichedwithK School of AnimalBiology, Natural andAgriculturalSciences, TheSchool of Faculty of V · F F CO · CO 1,11 1,8 2 2 =12.34; + =35.00; ) whilefeedingthemthejamandK + , bycomparingtheturnoverof should maintaindietary[K + 2,311 and theRb ( F 1,8 86 =32.4; Rb turnoverandV =15.39; P 86 3 P School of Anatomy,School of Physiology andHumanBiology, Faculty of 86 =1.0×10 Rb turnoverinbeetlesontheK =0.008) dietarygroups.Therewasasignificant Rb k a . We concludethat P + gideon P =1.58×10 diets washigherthanthatforbeetlesonthejam + b 1,2, =0.004) dietarygroups,andtheturnoverof yorpsgideon,,Isotopeturnover, , Xylotrupes 86 T or Rb 86 − a 86 Rb turnoverrecentlyhasbeensuggestedas *, PriyaD.Mathialagan 3 for bothjam(F ) andtheK Rb turnoverandV Rb) turnoverandmetabolicrate + . Theturnoverof T − · CO 13 a ) forbothjam(F ). We alsoexposedthebeetlesto 2 + . Studiesrelating ] asclosepossibletothatof + , fedadietofplumjamor ( 86 F 86 1,8 Rb turnoverisrelatedtoK 1,11 Rb inamodelinsect,the + =64.33; =10.80; diets. 86 Rb turnover, itsanalogy · CO 86 + F -enriched thanonthe 1,19 Rb inthebeetleson 1,11 2 V · P for boththejam =14.07; CO =14.56; 2 P=0.007) andK =4.3×10 Kings Park and 86 3 2 Rb turnoverto was higherat and ShaneK.Maloney P P − =0.001), =0.003) 5 ) diets, 86 Rb + + production, 1996), andalso torecapturetheanimalswithin thebiologicalhalf- King andHadley, 1979;Yokota, 1979;Cooper, 1983; Wolf etal., to invertebratesystemsdifficult andlimited(Buscarletetal., 1978; body fluidsfromthesubjectanimal, whichhasmadeitsapplication aspect totheDLW method,however, istherequirement tosample Bevan etal.,1995b;Speakman, 1998;Jonesetal.,2009).A key 1984; Williams, 1985;Nagyetal.,1990;Tiebout andNagy, 1991; living animals(SchoellerandWebb, 1984;Williams andNagy, precision oftheDLW methodmakeitideal forstudiesinsomefree- Westerterp andPlasqui, 2004).TheDLW methodcalculatesCO water method(DLW) (Lifsonetal.,1955;Schoeller, 1988; turnovers havebeenused,inwhatisknownasthedoublylabelled Bevan etal.,1994;1995a).Moreoften,stableisotope rate withmetabolism,convertedtoDEE(BevanandButler, 1992; been measuredinfree-livinganimalsand,usingcorrelationsof heart pursues allitsnaturalfunctionsinthatenvironment. measure ofmetabolicrateundernaturalconditionswhenananimal metabolism forecologicalstudiesastheycomenearesttothereal and ADMRareconsideredtobethemostrelevantmeasureof expenditure (DEE)oraveragedailymetabolicrate(ADMR).DEE average dailyrateofmetabolismisoftenreferredtoasenergy digestion (Nagy, 1987).AsFMRismeasuredoverseveraldays,the locomotion, feeding,predationavoidance,alertness,postureand The FMRincludesthecostsofBMR,SMR,thermoregulation, summary costoflivingforthatanimalinitsnaturalenvironment. in theirnaturalenvironment(Ricklefsetal.,1996)andreflectsthe metabolic rate(FMR)ismeasuredinanimalsthatarefree-ranging with laboratoryconditions(Nagy, 1987).Incontrast,thefield conditions suchasfoodsupply, qualityoflifeandweathercompared been questioned,especiallywhenthelatterexperiencedifferent metabolic ratedatafromcaptiveanimalstofree-livinghas in dynamicecologicalscenarios.Theutilityofextrapolating these measurementsundercontrolledconditionstothecostofliving and Harvey, 1987;McNab,1997),itisoftendifficult toextrapolate comparative context(BrodyandProcter, 1932;Kleiber, 1932;Elgar known. respiratory exchangeratio(RER)orquotient(RQ)is relatively straightforwardconversionstocalorimetry, providingthe calorimetric representationsofenergy expenditurebyvirtueof (Frappell, 2006).Theseareoftenusedasproxiesfortrue most accurateapproachestothemeasurementofmetabolism (eitheroxygenconsumption, through respirometry, which measuresthegasmetabolismofan 1987). Morecommonlyusedisindirectcalorimetry, includingflow- the heatproducedbyananimal(Kleiber, 1961;McLeanandTobin, in thelaboratorybydirectcalorimetry, whichinvolvesmeasuring hydrogen (i.e. production bymeasuringthefractionalturnoverofisotopes of There areseveralwaysthatDEEcanbemeasured.Heartratehas While laboratorymeasuresofmetabolicrateareinformativeina 3 V · CO 2 2 H or , orboth)andisrecognisedasoneoftheeasiest 3 H) andoxygen(i.e. V · O 18 2 , orcarbondioxide O). Theaccuracyand 2

The Journal of Experimental Biology turnover ( increase intheactivityofNa turnover maysimplybeameasureofK 2013). Themeasureof that maintainshighintracellular[K metabolic rateviasomemechanismthatisrelatedtoK rate butmayalsobeinfluencedbyK activity ofthepump,thenmeasurewillberelatedtometabolic to oxidativemetabolism,andifRb 2013). ItishypothesisedthatthebodyhandlesRb FMR is,however, alimitationofthetechnique(Tomlinson etal., intake upontheassociationof taxa; secondly, totesttheeffects ofincreaseddietary potassium concept thatthetechniquecouldbeusedtomeasureFMRininsect gideon Xylotrupes et al.,1995),however, presentedevidencethat experiments ( ( 86 There wasasignificanteffect ofdietontheexponentialdecay of gfortheK 4.15±0.43 gforthejamdiet, mean (±s.e.)bodymasswas3.84±0.43 There wasnosignificantchangein turnover, andbecause[K fairly wellestablishedthattheturnoverofrubidium-86( 1956; OdumandGolley, 1963; Argoud etal.,1987).Ithasnowbeen radio-isotopic turnovershavebeenusedtomeasureFMR(Relman, animals (Speakman,1997;BradshawandBradshaw, 2007). life oftheisotopes,whichisoftenprohibitivelyshortinsmall METHODS &TECHNIQUES ATP utilisationbytheubiquitousNa between simply ameasureofK table belowK electron initsouterorbital(groupI),andappearstheperiodic indicating aslower rateofdecay. TheK for foodintake.Theotherpossibilityisthat way ithandlesK to measuringFMRininsects(Tomlinson etal.,2013). Tomlinson etal.,2013).Themethodmaybeparticularlyapplicable life oftheisotopesislonger(BradshawandBradshaw, 2007; beyond thatofDLW insmallanimalsbecausethebiologicalhalf- for measuringFMR,andonethatextendstherecaptureperiodfar Furthermore, radio-isotopeenrichmentprovidesasimpleravenue sub-sampling ofthebodypool(BradshawandBradshaw, 2007). can bemeasuredbywhole-bodycounting,obviatingtheneedfor 2013). Radio-isotopeenrichment,particularlyofgammaemitters, relationship thatspanallofthetaxastudiedsofar(Tomlinson etal., al., 2013),andthattherearesomegeneralpatternstothis 1995; Peters,1996;BradshawandBradshaw, 2007;Tomlinson et 1968; BakerandDunaway, 1975; Williamson, 1975;Petersetal., in vertebrates(MishimaandOdum,1963;FairbanksBurch, gamma andbetaradio-emitter, correlateswellwithmetabolicrate The jamdiethadasignificantly lowerdecayexponentthantheK the useof Mass measurements RESULTS The effect of dietary K dietary of effect The t 12 Rb overtime( As analternativetothemeasurementofstableisotopeturnover, Our lackofaquantifiedmechanismthatlinks The purposeofthepresentstudywastwofold;firstly, tovalidate =2.35; 86 86 P Rb 86 A Rb =0.049) andRb Rb ND F k + k b 2,24 b (Bradshaw andBradshaw, 2007;Tomlinson etal., k F and metabolicrateremainedevenafteraccounting , wherek DISCUSSION b 2,311 + =0.196; to measuretheDEEofaninsect(inthiscase, Guérin-Méneville 1830)toprovideaproofof because Rb + =32.4; 86 itad44±. 3gforthe Rb diet and4.47±0.4 + Rb intake inlizards,becausetherelationship + b + and Rb + is thebiologicaleliminationconstant)to P ] istightlyregulatedinthebody, the k ( =0.824; P b t 12 is probablyanindirectmeasureofK =1.58×10 86 =2.70; + + Rb /K is analkalimetalwithasingle + + + + ] andlowintracellular[Na .gideon X. + k turnover capturesthechangein -ATPase shouldbeproportional on /K b + N with P intake. + + jam 86 =0.020) experimentaldiets, -ATPase incellmembranes − intake. Petersetal.(Peters 13 Rb washout + =11, ; and Rb V N · CO body massduringthe 86 jam 2 N Rb . =6, K =9, + 86 + k 86 similarly tothe Rb N b diets werenot Rb biological is measuring K N + =5, k Rb diet. b + =4). The , suchas was not 86 N Rb), a Rb + ]. An =5). + + Metzler, 1977).Theincreased processes (Ringer, 1884;Shoemakeretal.,1972;Lehninger, 1975; of allthreedietswasfasterthanthephysicaldecay to Rb Rb lines arenon-linear leastsquaresmodelsofexponential decayforeachdiet. mean (±s.e.)percentage ofinitialenrichment,uncorrected forphysicaldecay; Burch, 1968),implyingthat,although consistent withdatareportedbyFairbanksandBurch(Fairbanks excreted however, alsopossiblethatthebeetlesmanagedtore-ingestsome impacts onactivitycountsatlowlevelsofenrichment.Itis, as smallshiftsinthepostureofbeetleshavehigherproportional enrichment. Thiswasprobablyaresultofmeasurementvariability, occasionally plateau,orsometimesincreaseatlowlevelsof 1),wherefromdaytosomemeasurestended (Fig. turnover ratesofboththeK trials, especiallyontheRb enrichment onthejamdiet.Therewasnaturalattritionduringthese enrichment ontheenricheddiets,andbetweendays2425post- were belowthedetectionlimitatapproximatelyday14post- 2008), andK O’Donnell, 1992;Wieczorek, 1992;Zeiske,O’Donnell, et al.,1989;Klein1991;Wieczorek etal.,1991;Maddrelland aspect ofinsectiono-regulation(Schweikletal.,1989;Wieczorek significantly different ( Rb i.1. Fig. indicates that increases inK ANOVA oftheln-transformedlinearregression.The where 15 beetleswereanalysed. field, whereexcretedisotopeshoulddissipaterapidly. contaminated environments,itisunlikelytobeaprobleminthe Although thisisadifficulty ofusingradioisotopesincontainedand excretory processesasK aincie(i.1).Theexponentialdecayrelationshipswere: radionuclide (Fig. % Jamremaining=0.926e % Rb % K There wassomevariabilityaroundthegeneralexponentialdecline The rapidbiologicaldecayof + +

diet groupsandthephysicaldecay of 86 Rb enrichment (%) experimental dietscomparedwiththejamdietsuggeststhat + 100 86 + , itisalsoananalogueofK 10 20 30 40 50 60 70 80 90 + t Rb washoutcurvesoftheXylotrupesgideon control, K remaining =0.958e 0 remaining =0.958e is thetimesinceenrichment,andstatisticsrepresent 86 01 025 20 15 10 5 0 Rb isotope,andthattheirenrichmentgenuinelyrose. The JournalofExperimentalBiology(2014)doi:10.1242/jeb.096222 + 86 and Rb + Rb wasbeingexcreted,possiblythroughthesame intake increasethe + t 12 are excretedthroughthesamehomeostatic + =0.469; . TheeliminationofK + –0.203t –0.106t –0.234t + experimental diet,anddatafromonly and Rb 86 Time (days) Rb 86 ( P ( ( F

+ Rb inX.gideon 86 F F =0.886), andthebiologicaldecay . 1,105

k Rb physical decay physical Rb 1,130 1,76 b + in beetlesonboththeK 86 =99.4; P =927; P diet groupswerethesame, 86 86 =177; P Rb isobviouslyanalogous boe 5days. Rb over25 Rb k b =2.20×10 + =1.90×10 . Furthermore,the =2.20×10 is awell-described on allthreediets ᮀ ᭺ ᭹ Rb K Control + + 86 − Rb counts − Data are − 16 15 16 + (2) , ) (3) , ) (1) , ) and 1041 + 86 and Rb

The Journal of Experimental Biology al., 2001;Woodman etal.,2007;GibbsandHoshizaki,2008),DEE ectotherm (Lighton,1996;Komai,1998;Zhouetal.,2000;Acar nrae o01±.0 day l increased to0.16±0.005 .500 day l 0.15±0.01 between DEEand P ambient temperature( 0.964±0.078 1042 86 of metabolicrate. 2.Interrelationshipsofisotopicandindirect calorimetricmeasures Fig. METHODS &TECHNIQUES was 1.22±0.108 The rateofCO 0.536±0.098 2.24±0.276 control diet.Linear trendlinesareshown. K and therewasnoeffect ofdiet( and 86 diet. At P 30°C ontheK T a Rb k =3.03×10 =6.10×10 Rb + =20°C theV Although thewashouttrialsestablishedalinkbetweenK The -supplemented dietthanonthejamateach 86 k b . Rb b g (B).(C)Therelationship between 86Rb k g 86Rb k g V (l day–1) 86 b b CO2 rate andmetabolic , temperature Rb 0.10 0.20 k 1.0 2.0 T 1.0 2.0 3.0 b − − 0 0 a , thisisnotinformativeofDEE,noranyassociation k 0 k =20°C theV 8 4 52 25 20 15 b b k i.2B).AtT ; Fig. ; k .501 .502 0.25 0.20 0.15 0.10 0.05 0 b g at30°ContheK b increased significantlywithincreasing + N 2 − C B A ,wihicesdt .5±.1 k g, whichincreasedto0.656±0.119 · g at30°Conthejamdiet.At k diet. 1 CO jam The effect ofambienttemperature( production increasedsignificantlywithincreasing b t2° n .800 day l at 25°Cand0.18±0.01 g, whichincreasedto2.14±0.129 2 =5, 86 Rb a .100 day l was 0.11±0.01 T 86 N a ) (F k K Rb k b · =4), andwassignificantlyhigheronthe . Consistentwithourexpectationsforan CO ᭺ 1,19 2 b K Control V g=14.1ϫV + − . =30.1, r was 0.11±5.0×10 CO 1 2 F =0.89 + t2° n .800 day l at 25°Cand0.18±0.02 T 1,19 2 (lday a diet. a (°C) =20°C the =0.031; P V 86 · CO =2.71×10 . Rb k –1 CO 2 ) and − 2 –0.27 1 , whichincreasedto b P g=7.1ϫV − 86 T 035 30 082 i.2A).At =0.862; Fig. 1 r Rb k T a 2 at 30°Conthejam =20°C the a =0.76 − ) onV − 5 4 ; 86 k b b day l N T b g forK Rb T g at25°Cand . a g at25°Cand jam · a CO CO ( ( F =5, 2 F + 2 − –0.32 1,19 (A), and 1 1,19 excretion k + , which b 86 and N =73.2; =19.4, Rb K − =4), was 1 k at b the twogroups,presumablyeffect wasrelatedtoK expectation that enriched dietcomparedwiththejam( was notsupportedbythemeta-analysis,whereK k The datafor slope inthebeetlesreceivingK al., 2013), al., 1995;Peters,1996;BradshawandBradshaw, 2007;Tomlinson et rate (V P with thewashouttrials,relationshipof higher slope,resultingin additional K ( independent oftheroleK group thaninthejamdietgroup.As i.3 Resultsofameta-analysis of 3. Fig. for boththejam( There wasasignificantlinearrelationshipbetween increased withincreasing our jamdiethadalower[K group conformedtoexpectations.Thisraisestwopossibilities:either (Tomlinson etal., 2013)andreferencestherein.Theresultsfor established patterncouldbeanartefactofelevatedK 3).Deviationsfromthe congruent withthosepreviousfindings(Fig. control dietandtheK consistent withthese relationships. CO registering atalowerelevation,with maintain ionicbalanceonthehigher[K analysis of for thecontroljamdietwerenotcongruentwithapreviousmeta- Bradshaw andBradshaw, 2007;Tomlinson etal.,2013).Theresults at thesame the jamdiet.A moreintuitivewayofinterpretingthesedataisthat and thatthemeasureischangedwhendietary[K metabolism, andgoodcorrelationsbetweenmetabolicrate life ofradioisotopesininsectsobeysthesamelawsasthoseregulating Buscarlet etal.(Buscarletal.,1974)statedthatthebiologicalhalf- The effect of metabolic rateon metabolic of effect The relationship; theresults forthosebeetlesontheK the jamdiet(filledcircles)werenotconsistent withtheestablished F b =7.95×10 As therewasnodifference inthe We haveshownthat 1,8 have beenreportedinvertebrates(Petersetal.,1995;Peters,1996; 2 86

production inarangeofectothermic species.SeeTomlinson etal. Rb k g

b =64.33; 2.0 4.0 6.0 1.0 3.0 5.0 · CO 0 . . . . 0.5 0.4 0.3 0.2 0.1 0 2

Bufo terrestris ). 86 − 86 The JournalofExperimentalBiology(2014)doi:10.1242/jeb.096222 T 12 Rb + a X. gideon Rb P i.2C). ; Fig. there wasahigher intake hadnoeffect onDEE.However, consistent =4.3×10 k 86 k b b Rb also increasedwithincreasing for ectotherms(Tomlinson et al., 2013)(

Xylotrupes gideon F k 86 1,11 b on theK − Rb should increasesimilarlyto 5 + =35.00; ). Therelationshiphadasignificantly experimental diet,itisapparentthat + k + ] thanisnormalinthenaturaldietof T b in whatwemeasuredasthemetabolic a is relatedtometabolicrateinaninsect, . Furthermore,consistentwiththe V . CO 86 + 86 86 Rb experimental diet,however, were 2 P Rb (lday Rb biologicalturnoverversus 86 + =1.0×10 V Rb washout · k diet tothatinthosereceiving CO k b + b V ] diet,butthisinterpretation for agiven 2 · in theK –1 CO was notdifferent between 86 ) + Diposaurus dorsalis 2 86 Rb diet (opencircles) were − of thebeetleson Rb 3 ) andtheK k T b + + k ] intakeisaltered. a to b experimental diet . V V · at agiven · + CO T CO + V · -enriched diet elimination to a + CO 2 X. gideon F 2 had ahigher elimination, , ontheK and 2 1,19 (Peters et =215.8; i.3), Fig. + 86 Rb V diets 0.6 86 · CO on Rb X. k + 2 b - .

The Journal of Experimental Biology ATPase thattransportsK ATPase andmetabolicrate. to metabolicrate,theexcretionof fjmwssbtnilylwr(.5m g mg of jamwassubstantiallylower(0.75 proportional toK increases whenK speculated tobethesubstitutionof intake withfood,andisonlytangentially influencedbytheNa the extracellularbodypoolthat isregulatedinresponsetoionic Na of thecellshouldbedirectlyproportionaltoactivity Na here, wespeculatethat,afterthebolusofinitialenrichment, (Peters etal.,1995),withsomeinferencesfromourdatapresented DEE (Petersetal.,1995).FollowingtheconclusionsofPetersal. entering anintracellularpool.ThemovementofK energetic link betweenaspeciesanditsenvironment. Itcanbeused FMR isausefulecologicalmeasurement becauseitquantifiesthe 86 receiving K gideon, orthepreviouspublishedliteraturewasbasedonanimals itwssmlrt h .101 gg mg diet wassimilartothe1.81±0.14 METHODS &TECHNIQUES the intracellularpoolviaNa of probability ofbeingexcreted.Whilethereisaconstantmovement extracellular poolsisaltered.Whilethetransferof influenced byfoodintake, the Na will beexcreted,whilesomereturntotheintracellularfluidvia rate ofexcretionK from thecellataratethatisproportionaltometabolicrate,and although comparative interpretationstrengthensourrecommendationthat, controlled orreportedinotherstudies.Suchaconfoundingof in thewild. (Peters etal.,1995)extensivelyexploredthepossibilitiesthat Bradshaw andBradshaw, 2007; Tomlinson etal.,2013).Petersal. process thatconsumesATP (Petersetal.,1995;Peters,1996; rvosfnig Fg 3).ThedietaryK previous findings(Fig. control. Undersuchascenarioourdatawouldbeconsistentwith the Na for eachisotopeusingstatisticalmethods.While the dietaryintakeofions,andmanagedtoseparateeffectors balance of jam dietprovidedlessthanthenormaldietaryK probability ofagivenmolecule intracellular totheextracellularpoolremainsconstant,moreof that proportionally withmetabolicrate.AnincreaseindietaryK 1992), therateofthisexchangeshouldincreaseordecrease produce excreta.Themechanismsuggestedisthat extracellular fluidthatisfilteredbytheMalpighiantubulesystemto dietary K the extracellularfluidincreaseswhenK of thetechniquetomimic[K result inanincreasetheexcretionofK while ourK k (USDA, 2004)fornaturallyoccurringlevelsofK food intakeprobablyresultsfrom Conclusion b Rb The mechanismofassociationbetween and 86 + + /K /K Rb fromtheintracellulartoextracellularfluidinproportion + + + will beexcretedfromtheextracellularpool,reducing + + 22 -ATPase transportsmostoftheinjected -ATPase, asintracellularK /K /K Na + 86 + + should becarefullycontrolledinanylaboratoryvalidations Rb -ATPase comprisesalarge componentofDEE(Withers, -ATPase. IftherateofexcretionK + k + -enriched diets.Whilethe[K b 86 experimental dietinfactrepresentsamoreaccurate k were co-correlatedwithboththemetabolicrateand b Rb can beusedtoinferFMRinabroadrangeoftaxa, + + + intake. When intake increases.Theassociationof exchange betweentheintracellularand + (and + 86 into, andNa Rb 86 Rb k + b + /K ] thataspecieswillbeconsuming was moststronglyassociatedwith + 86 22 + 86 ) fromtheextracellularfluidis 86 + Rb Na beingmaintainedmostlyin is regulated.Astheactivityof Rb fromtheextracellularfluid -ATPase, andincreasing its Rb 86 + Rb + + + − leaves thecell,someofit + + 1 86 being transportedbackto − + intake increases,thenthe out of,cellsinanactive + 1 ] ofourK (and has notpreviouslybeen Rb ). Thissuggeststhatour reported bytheUSDA for K k 86 b 86 22 Rb + and DEEhasbeen + Rb + Na + (and + 86 in fruit,the[K + by theNa (and for thebeetles, 86 Rb + + into cells,thus ), anditisthe k Rb experimental b + 86 was mostly is released + Rb 86 22 from the Rb Na with + ) from + + + + ) out 86 /K /K will Rb + + + - - ] different diets,presumablyasaresultoftheinfluenceK ecologically relevantasameasureofFMR. de o35mlofplum jam.Thefinal[K added to375 gof either KCl(Merck)orRbCl(BDHChemicalsLtd) enriched with0.53 The othergroupsreceivedthesame diet,exceptthattheplumjamwas onthejamdietreceivedfresh mashedpeachesandplumjamdaily. commercially availableplumjam(IXL,Australia). libitum organic mulch(RichgroEzi-Wet Organic Mulch,Australia)andfedan maintained at23.5±0.1°Cconstantly. Thebeetleswerekeptonabedof length) keptinalaboratoryroomnearwindows.The was cm cmwidth×30 cmheight×15 were maintainedinPerspextanks(25 dayspriortothebeginningofexperiments.Thebeetles maintained for15 overnight totheU.W.A. CrawleycampusinWestern Australia,and from theAustralianInsectFarm(Innisfail,QLD,Australia),shipped beetles (15maleand16female)werepurchasedintwoseparateshipments by injection,andalsoavailablethroughapettradeinAustralia.Mature gmass,andthuslarge enoughtoenrichwithradioisotope approximately 4 regions ofthePacific(ZelaznyandAlfiler, 1987).Theseinsectsare of coconutpalmplantationsinthesouthernregionsAsiaandseveral Islands andtheNewHebrides(Bedford,1975).Theyareregardedaspests to India,south-eastAsia,PapuaNewGuinea,Australia,theBritishSolomon rhinoceros beetles( . We foundthatthe technique tomeasureDEEandFMRinsmallvertebrates contention. turnover, andwehereprovidesomeevidencetosupportthat 2013). Thebiologicalturnoverofradioactive are notsuitableforthetechnique(forreview, seeTomlinson etal., has theoreticalandpracticallimits,wheresomespecieshabitats has broadsupportandapplicationformeasuringFMR,thetechnique disturbance uponaspeciesofinterest.AlthoughtheDLW method 2007), dispersal(ClusellaTrullas etal.,2006)andanthropogenic Bradshaw, 1999;Bradshawand Bradshaw, 2007;Bradshawetal., Jodice etal.,2003),ecosystemproductivity(Bradshawand Jetz, 2005),reproduction(Fyhnetal.,2001;Bevan2002; to understandtheimpactsoftemperatureandclimate(Anderson We testedtheassociationbetween 86 elimination upon .5m g mg 0.35 accuracy oftherelationshipbetween Tomlinson etal.,2013).A problemwithusing (Peters etal.,1995;Peters,1996;BradshawandBradshaw, 2007; DLW, andmaybeapplicabletotaxathatareunsuitableforDLW proposed asanalternativethatavoidssomeofthelimitations affect of K to control[K experiments, thebeetles wereweigheddailytomonitor theirconditionusing unchanged, andwaterfoodwere provided measured byflamephotometry. Allotherelementsofthedietwere Na mechanistic linkbetweenmetabolismandtheturnoverof of ananimal’s metabolismisalackofunderstandingthe V suggested mechanisticpathwayisthatRb (Bradshaw andBradshaw, 2007; Tomlinson etal.,2013).One Animal source and maintenance Animal source MATERIALS ANDMETHODS · CO Rb The beetleswereassignedrandomlytothreetreatmentgroups. The Measurement of + 2 /K + k and and Rb + b V · -ATPase. Futurecalibrationsof CO is relatedtoDEEandFMRviatheactionofubiquitous control dietofmashedpeaches(Ardmona,Australia)and − 86 1 2 , whiletheK . Althoughthereweresignificantrelationshipsbetween Rb The JournalofExperimentalBiology(2014)doi:10.1242/jeb.096222 + + , butthatchangesintheintakeoftheseionsdidnot ] intaketonaturallevelsinordermaximisethe k b , therelationshipwasdifferent inbeetlesonthe X. gideon 86 86 Rb Rb + k and Rb b k . Thesedatasupportthelikelihoodthat , Coleoptera;Scarabidae;),native b 86 is acheap,non-invasiveandpractical Rb 86 + Rb k it eeerce o14 gg mg diets wereenrichedto1.42 b was influencedbydietarylevels k b and thedietaryintakeofK 86 86 Rb Rb + ad libitum turnover isrelatedtoK k + k b ] ofthejamdietwas b 86 and DEEtomakeit with Rb 86 k . Throughoutthe V b Rb hasbeen · CO as ameasure 2 will need + 1043 using − 86 1 , as Rb ad + +

The Journal of Experimental Biology Each respirometry systemwasmaintainedatconstant nominaltemperatures within acylindricalleadshield.Eachtimethe To reducetheeffects ofexternalsourcesradiation,thevialwasenclosed by astyrofoamplugthesamediameterasvialplacedabovebeetle. mmdiameterplasticvialandrestrainedfrommoving were enclosedina50 mmsodiumiodidecrystal.Duringthecountingprocedure,beetles 76×76 using anAmpTek GammaRadV (AmpTek, USA)gammacounterwitha sodium iodidecrystal,whilecountsduringtherespirometrytrialweremade mm using aportablegammacounter(PSR8,Nucleonics,UK)with50×50 hequilibrationperioduntiltheexperimentalmeasurementsbegan. a 24 the beetleswerereturnedtotheirrespectivetankswheretheyremainedfor the injectateentereddirectlyintohaemocoel.Followinginjections, integument betweenabdominalsegmentsoneandtwo.Itispresumedthat needle (Terumo, Japan).Thebeetleswereinjectedthroughtheventral mlsyringe(Livingstone,Australia),andadministeredviaa27G disposable 1 Intravenous InfusionBP, Baxter).Dilutionsweremadeinasterile, where beetle ( the countsweremadeeachday. Thebiologicaleliminationconstantforeach where enrichment. isotope remainingineachbeetlewascalculatedwithreferencetoits initial reached backgroundlevelsineachbeetle.Eachdaythepercentage of the daysuntil theenrichment the beetlesweremeasureddailyforatleast26 respectively, and over time(henceforthreferredtoasthewashout).Afterinjectionof 1044 METHODS &TECHNIQUES otolr(alogDC1,UA tart f20m min ml controller (Aarlborg DFC-17, USA)atarateof200 Withers (Withers, 2001).Compressedairwaspassedthroughamassflow A four-channel flow-throughrespirometrysystemwasconstructedfollowing experimental groupwasremovedfromthenextphaseofexperimentation. Following thewashouttrial,wherenodifference wasfoundin To testwhetherincreaseddietaryintakeofK MBq of a0.05 09/07/01. This researchwasconductedunderUWA RadiationSafetyApproval declined bymorethan10%,thefoodquantityoffered wasincreased. mg.Incaseswherebodymass a balance(Sartorius)withprecisionof0.1 between theK White etal.(Whiteal.,2006)to minimiseCO A. HammondCompanyLtd,USA), usedinlinewithrecommendationsby excurrent airstreamwasdriedwith Drierite (anhydrouscalciumsulphate,W. which thebeetlewashousedfor thedurationofexperiment.The mmdiameter) beddedwithorganic mulch, in mmlength×70 chamber (85 temperature andpressuredry).The airwaspassedintoacylindricalglass isotope bydividingthecountradioactivedecayexponent: each beetle. background countwassubtractedfromtheaveragedradioactivityof when thevariationbetweenthreecountswaslessthan2%.Theaveraged beetle inthechamber. Eachsetofcountswasaveraged,andonlyaccepted scountsweretakenwiththe background counts,aminimumofthree60 sbackgroundcountswererecorded.Followingthe was measured,three60 86 86 through aQubit S151 infraredCO biological eliminationof Isotope enrichment andcountingprotocols Rb turnover, rate andmetabolic temperature Rb washout trials Whole-body radioactivitycountsduringthewashoutstudyweremade ttebgnigo aheprmn,ec etewsijce ih01ml At thebeginningofeachexperiment,beetlewasinjectedwith0.1 Gamma emissioncountswerecorrectedforthephysicaldecayof C C k t b 1 ) wascalculatedas: is theradioactivitycountand and Corrected biologicaldecay= C 86 + 2 RbCl solution(PerkinElmer)insterilesaline(0.09%NaCl, t are thefirstandlastradioactivitycountsforeachbeetle, and Rb 1 and t 2 86 k + are thetimesthat Rb, wemeasuredandcomparedtherateofdecay b diets (seeResultsandDiscussion),theRb = () n()–1 ) ( 1n – ) ( 1n CC 2 12 tt analyser (Qubitsystems Inc.,USA). 21 t – is thetimefrominjectionatwhich C t C / [e + 1 and Rb and 86 ( − 0.693 /18.66)t Rb remaininginabeetle ,(5) 2 C absorption, andpassed 2 + were measured. changed therateof (4) , ] 86 − Rb washout 1 (ambient 86 Rb, + provided withtheirexperimentaldietandwater to baseline.Whiletheywereintherespirometrychamber, thebeetleswere removed fromthechamber, therespirometrysystemswereallowedtoreturn weighed daily, andtheirradioactivitycounted.Whilethebeetleswere days.Duringtherespirometrytrials,beetleswere a randomorderfor2 radioactivity counted.Thebeetleswerethenexposedtoeachtemperaturein here. 1992), thisisprobablyasafeassumptionwiththecontrolleddietspresented proportional representationofdietarylipidsandcarbohydrates(Withers, treatment totreatment,butasthisusuallychangesinresponsethe rate dependsontheassumptionthatRERdoesnotchangefrom including themulchbedding.Using was usedtocharacterisetherelationshipsbetween each respirometrytrial,recordingthebackground[CO hbaselinewasrunbeforeandafter acquisition program.A minimum12 collected everyminuteusingacustom-writtenVisual Basicversion6.0data interfaced toacomputerviaDataQ710dataacquisitionboard,and repeated measuresANOVA. Exponentialeffects oftimeon Body masswascomparedbetweendietsandovertimeusingatwo-way ( restoration’. Grant LP110200304 ‘Thespatialenergeticsofpollinationfailureinhabitat This researchwaspartiallyfundedundertheAustralianResearchCouncilLinkage supervised byS.K.M.andS.T. and executionoftheexperiments.P.D.M. wasaresearchtrainingstudentco- All authorsmadesubstantialandequivalentcontributiontotheconception,design The authorsdeclarenocompetingfinancialinterests. analysis usingRisgratefullyacknowledged. equipment andextensivetechnicaladvice.DrMurrayLogan’s adviceonstatistical counter, andtoProfessorPhilWithersHaiNgofortheuseofrespirometry Thanks gotoEmeritusProfessorDonBradshawfortheuseofhisPSR8gamma the the different diets.Non-linearleastsquaresmodelswereusedtocharacterise nested bydietweretestedANOVA withTukey and ‘biology’ (Logan,2010).Dataarepresentedasmeans±s.e. ‘nlme’,‘multcomp’ performed inRversion2.15.2usingthepackages‘car’, between experimentalgroupsusingANCOVA. Statisticalanalyseswere dietary groups.Theserelationshipswerecomparedforslopeandintercept ea,R .adBte,P. J. Butler, and R.M. Bevan, ae,C .adDnwy P. B. Dunaway, and C.E. Baker, R.P. Eaton, and D.S. Schade, G.M., Argoud, cr .B,Sih .N,Hne,L .adBoh G.M. W. Booth, Jetz, and and K.J. Anderson, L.D. Hansen, B.N., Smith, E.B., Acar, used tocharacterisetherelationshipsbetween transformed priortoparametricanalysis.Linearmixedeffects modelswere and Hui,2011), thepercentageofcorrectedbiologicaldecaywasnotarcsin transformed decay. FollowingthediscussionbyWarton andHui(Warton significance oftheserelationshipswastestedbylinearregressionusingln- k Statistical analyses References Funding Author contributions Competing interests Acknowledgements efr,G.O. Bedford, T b of endotherms. insect. Environ.Entomol. calorespirometry todetermineeffects oftemperatureonmetabolicefficiency ofan yhafuligula Aythya consumption, heart rate anddeepbodytemperatureduring diving inthetuftedduck, : Dynastinae)inMelanesia. relationship tometabolicratesofwildsmall rodents. E615. glucose productionbyradioactiveand stable tracers. a The beetleswereweighedpriortorespirometrytrials,andtheir and ) of20,25or30°C.Analogdatasignalsfromalltheequipmentwere 86 Rb exponentialdecayrelationshipsofeachdietovertime.The T a , nestedwithindietarytreatments.Reducedmajoraxisregression The JournalofExperimentalBiology(2014)doi:10.1242/jeb.096222 (1975). Obsevrationsonthebiologyof . J.Exp.Biol. Ecol. Lett. 30, 811-816. 8 , 310-318. 163 (2005). Thebroad-scaleecologyofenergyexpenditure (1992). Theeffects ofthetemperatureonoxygen , 139-151. (1975). Eliminationof V · J. Aust.Entomol.Soc. CO 2 as thesolemeasureofmetabolic (1987). Underestimationofhepatic V · CO J. Exp.Zool. yorpsgideon Xylotrupes ad libitum. 2 post hoc and Am. J.Physiol. 86 Rb 137- 2 T ] ofablankchamber a Cs and , andbetween k b 14, 213-216. 192 tests tocompare and (2001). Useof , 223-236. V 86 59- · (Coleoptera: CO 252 Rb decay Fe andits 2 , E606- within 86 Rb

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