bioRxiv preprint first posted online Apr. 8, 2016; doi: http://dx.doi.org/10.1101/047704. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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Physiologicalandbiochemicalchangesassociatedwithexperimentaldehydrationinthe
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desertadaptedcactusmouse,Peromyscuseremicus
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LaurenKordonowy1,KaelinaLombardo1,HannahGreen2,EviceBolton1,SarahLaCourse3,Matthew
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MacManes1
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1
University of New Hampshire, Department of Molecular Cellular and Biomedical Sciences
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2
University of New Hampshire, Department of Biological Sciences
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3
University of New Hampshire, Department of Psychology
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Abstract
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Characterizingtraitscriticalforadaptationtoagivenenvironmentisanimportantfirststepin
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understandinghowphenotypesevolve.Howanimalsadapttotheextremeheatandaridity
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commonplacetodesertsrepresentsisanexceptionallyinterestingexampleoftheseprocesses,andhas
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beenthefocusofstudyfordecades.Incontrasttothosestudies,whereexperimentsareconductedon
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eitherwildanimalsorcaptiveanimalsheldinnon-desertconditions,thestudydescribedhereleverages
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auniqueenvironmentalchamberthatreplicatesdesertconditionsforcaptivePeromyscuseremicus
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(cactusmouse).Hereweestablishbaselinevaluesfordailywaterintakeandforserumelectrolytes,as
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wellastheresponseofthesevariablestoexperimentaldehydration.Inbrief,P.eremicus’dailywater
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intakeisverylow.It’sserumelectrolytesaredistinctfrommanypreviouslystudiedanimals,andits
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responsetodehydrationifprofound,thoughnotsuggestiveofrenalimpairmentinthefaceofprofound
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dehydration,whichisatypicalofmammals.
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bioRxiv preprint first posted online Apr. 8, 2016; doi: http://dx.doi.org/10.1101/047704. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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Introduction
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Understandingtheevolutionofadaptivetraitshaslongbeenoneoftheprimarygoalsinevolutionary
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biology.Thestudyoftherelationshipsbetweenfitnessandphenotype,oftenpoweredbymodern
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genomictechniques(Vignierietal.2010),hasprovidedresearcherswithinsightintothemechanistic
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processesthatunderlieadaptivephenotypes(Castoeetal.2013;Huerta-Sánchezetal.2014).Systems
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inwhichthepowerofgenomicscanbecombinedwithanunderstandingofnaturalhistoryand
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physiologyarewellsuitedforthestudyofadaptation(Mullenetal.2009;BedfordandHoekstra2015)
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especiallywhenresearchershavetheabilitytoassaythelinkbetweengenotypeandphenotypeinwild
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animalsandthenconductcomplementaryexperimentsusingrepresentativeanimalsincarefully
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controlledlaboratoryenvironments.Thestudydescribedhere,characterizingthephysiologyandserum
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biochemistryofPeromyscuseremicusisthefirststepinalargerstudyaimedatunderstandingthe
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genomicsarchitectureofadaptationtodesertenvironments.
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Desertadaptationhassignificantecological,evolutionary,andbiomedicalsignificance.Incontrastto
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humansandothermammals,desertrodentscansurviveinextremeenvironmentalconditionsandare
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resistanttotheeffectsofdehydration.Physiologicaladaptionstodesertshavebeencharacterizedin
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severalrodents.Specifically,renalhistologyhasbeenstudiedinmultipleHeteromyidrodents(Altschuler
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etal.1979),andthegeneralconclusionisthatthesedesertadaptedanimalshaveevolvedelongate
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LoopsofHenle(Barrettetal.1978;Mbassa1988;Beuchat1996)thatarehypothesizedtooptimize
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waterconservation.Inadditiontostudiesofrenalhistology,severalstudieshavecharacterized
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pulmonarywaterloss(Schmidt-NielsenandSchmidt-Nielsen1950;Hayesetal.1998),watermetabolism
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(HowellandGersh1935),andwaterconsumption(MacMillenandLee1967;Bradford1974;Mares
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1977;Nagy1988;MerktandTaylor1994)indesertrodents.Whiledesertanimalspossessspecialized
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physiologythatisefficientwithregardstowatermetabolismandloss,whetherornotspecialized
bioRxiv preprint first posted online Apr. 8, 2016; doi: http://dx.doi.org/10.1101/047704. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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genomicadaptationexistsisanactiveareaofresearch(Marraetal.2012;MacManesandEisen2014;
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Marraetal.2014).
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Althoughthecactusmouse(Peromyscuseremicus)hasnotbeenaparticularfocusforthestudyof
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desertadaptation(butsee(al-Kahtanietal.2004;MacManesandEisen2014),thisCricetidrodent
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nativetothearidregionsoftheSouthwesternUnitedStatesandNorthernMexico(VealandCaire2001)
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offersauniqueopportunitytounderstandphysiologicaladaptationstodeserts.P.eremicusisa
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memberofalargergenusofanimalsknowncolloquiallyasthe“Drosophilaofmammals”(Bedfordand
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Hoekstra2015),andPeromyscusspecieshavebeenthefocusofextensivestudy(Hoekstraetal.2001;
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Steineretal.2007;MacManesandLacey2012;Shorteretal.2012).P.eremicusisasisterspeciestothe
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non-desertadaptedP.californicus(Bradleyetal.2007),anditiscloselyrelatedtoP.crinitus,thecanyon
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mouse,whichisanotherdesertadaptedrodentnativetoSouthwesterndeserts.
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Criticaltodesertsurvivalistheabilitytomaintainwaterbalanceevenwhenthelossofwaterexceeds
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dietarywaterintake(Heimeieretal.2002).Indeed,themammaliancorpusconsistsof60%water
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(JéquierandConstant2009).Farfromastaticreservoir,properphysiologicfunctionrequireswaterfor
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numerousprocesses,includingnutrienttransport(Haussinger1996),signaltransduction,pHbalance,
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thermalregulation(Montainetal.1999)andtheremovalofmetabolicwaste.Toaccomplishthese
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functions,anearlyconstantsupplyofwaterisrequiredtoreplacewaterloss(JéquierandConstant
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2009),whichoccursmainlyviathegastrointestinalandgenitourinarysystems,andevaporativeloss,
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whichisgreatlyacceleratedinextremeheatandaridity(Cheuvrontetal.2010).Becausethebody
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possesseslimitedreserves,whenlossexceedsintakeduringevenashortperiodoftime,dehydration
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anddeathcanoccur.Mammalsareexquisitelysensitivetodehydrationandpossesslimited
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compensatorymechanisms.
bioRxiv preprint first posted online Apr. 8, 2016; doi: http://dx.doi.org/10.1101/047704. The copyright holder for this preprint (which was not
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Characterizingdesertadaptationrequirescarefulandintegrativephysiologicalstudies,whichshould
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includeadetailedcharacterizationofwaterintake,responsestodehydration,andthemeasurementof
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bloodelectrolytes.Indeed,quantifyingthesemetricsisoneofthefirststepsinunderstandinghow
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animalssurviveintheextremeheatandaridityofdeserts.Inparticular,theelectrolyteschlorideand
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sodiumareimportantmarkersofdehydration(Costilletal.1976).Thesemoleculesplayessentialroles
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inmetabolicandphysiologicalprocesses,andtheyareintegraltothefunctionallyofavarietyof
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transmembranetransportpumps(BlausteinandLederer1999;Jentschetal.2002),neurotransmission
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(YuandCatterall2003),andmaintenanceoftonicity(FeigandMcCurdy1977).Furthermore,
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hypernatremiacausesrestlessness,lethargy,muscleweakness,orcoma(AdroguéandMadias2000).
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Bicarbonateion,incontrast,isprimarilyresponsibleforaidinginthemaintenanceoftheacid-base
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balanceandisresorbedintherenaltubules(McKinneyandBurg1977).Bloodureanitrogen(BUN)isa
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testthatassaystheabundanceofurea–theendproductformetabolismofnitrogencontaining
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compounds.Ureaisresorbedintheglomerulus,andrenalimpairmentisofteninferredwhenBUN
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becomeselevated(Baumetal.1975).Importantly,thecanonicalmodelofurearesorptionisdependent
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onurinevolume,whichismarkedlydiminishedindesertrodents,thuslimitingtheutilityofusingBUN
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asanindicatorofrenalfunction.Lastly,creatinine,aproductofmusclebreakdown,whosemeasured
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leveldoesnotdependonurinevolumeisusedasameasureofrenalfunction(Baumetal.1975).
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Genesmostfrequentlyimplicatedindesert-adaptationincludemembersoftheaquaporinfamily(Huang
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etal.2001).However,previousworksuggeststhatanalternativegenefamily,thesolutecarriers,are
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morerelevantfordesert-adaptationinthecactusmouse(MacManesandEisen2014).Asafirststep
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towardsfullyelucidatingthepatternsofadaptiveevolutiontodesertsinP.eremicus,wecharacterized
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thenormalpatternsofwaterintakeandelectrolytelevelsaswellasthephysiologicresponseto
bioRxiv preprint first posted online Apr. 8, 2016; doi: http://dx.doi.org/10.1101/047704. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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experimentaldehydration.Assuch,thisstudyprovidescriticalphysiologicalandbiochemical
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informationaboutP.eremicusanditsresponsetodehydrationandisgenerallyusefulasresearchers
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begintoleveragelarge-scalegenomedataagainstclassicquestionsregardingtheevolutionofadaptive
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phenotypes.
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MaterialsandMethods
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WeusedcaptiveP.eremicusthatwerepurchasedfromtheUniversityofSouthCarolinaPeromyscus
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GeneticStockCenterin2013.Theseanimals,whicharedescendantfromwildcaughtanimalsfroma
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dry-desertpopulationinArizona,havebeenbredincaptivityattheUniversityofNewHampshire.
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Animalsarehousedinalargewalk-inenvironmentalchamberbuilttoreplicatetheenvironmental
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conditionsinwhichthispopulationhasevolved.Specifically,theanimalsexperienceanormaldiurnal
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patternoftemperaturefluctuation,rangingfrom90Fduringthedaytimeto75Fduringthenight.
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Relativehumidity(RH)rangesfrom10%duringthedayto25%duringthenight.Animalsarehousedin
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standardlabmousecageswithbeddingthathasbeendehydratedtomatchdesertconditions.Theyare
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fedastandardrodentchow,whichhasalsobeendehydrated.Waterisprovidedadlibduringcertain
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phasesofexperimentationandwithheldcompletelyduringothers.Allanimalcareproceduresfollow
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theguidelinesestablishedbytheAmericanSocietyofMammalogy(Sikesetal.2011)andhavebeen
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approvedbytheUniversityofNewHampshireAnimalCareandUseCommitteeunderprotocolnumber
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103092.
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Allanimalsincludedinthisstudyweresexuallymatureadults.Aslightbiasfortheinclusionofmales
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exists,asaconcurrentstudyofmalereproductivegenomicswasoccurring.Preliminaryanalyses
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conductedsuggestthatnosignificantdifferencesinanyofthephysiologicalmeasures,andasaresult,
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malesandfemaleswereanalyzedasonegroup.Forasubsetofanimals,waterintakewasmeasured,
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whichwasaccomplishedviatheuseofcustomized15mlconicaltubes,whereinwaterintakewas
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measuredevery24hoursforaminimumof3consecutivedays(range3-10days).Animalsselectedfor
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thedehydrationtrialwereweighedonadigitalscale,housedwithoutwaterforthreedays,thenre-
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weighedtodeterminethechangeinbodymassduetodehydration.Attheconclusionofwater
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measurementorafterathree-daydehydrationanimalsweresacrificedviaisofluraneoverdoseand
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decapitation.Immediatelyafterdeath,a120uLsampleoftrunkbloodwasobtainedforserum
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electrolytemeasurement.ThiswasaccomplishedusinganAbaxisVetscanVS2machinewithacritical
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carecartridge,whichmeasurestheconcentrationofseveralelectrolytes(Sodium,Chloride,Bicarbonate
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ion,Creatinine,andBloodUreaNitrogen(BUN))relevanttohydrationstatusandrenalfunction.Lastly,
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thekidney,spleen,liver,lung,hypothalamus,testes,vasdeferensandepididymisweredissectedout
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andstoredinRNAlater(AmbionInc.)forfuturestudy.Allstatisticalanalyseswerecarriedoutinthe
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statisticalpackage,R(RCoreDevelopmentTeam2011).
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Results
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Wemeasuredthedailywaterintakefor22adultcactusmiceforbetweenthreeand10consecutive
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days.Meanwaterintakewas0.11mLperdaypergrambodyweight(median=0.11,SD=0.05,min=
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0.033,max=0.23).WemeasuredlevelsofserumSodium,Chloride,Bicarbonateion,Creatinine,and
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BloodUreaNitrogen(BUN)for44adultmice,therebyestablishingnormal(baseline)valuesforP.
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eremicus(Figure1andTable1).
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Acomparisonofmiceprovidedwithwateradlibitumtomicethatexposedtoexperimentalwater
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deprivationforthreedaysrevealedthatthedehydratedmicelostanaverageof23.2%oftheirbody
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weight(median=23.9%,SD=5.3%,min=12.3%,max=32.3%).Despitethissubstantialweightloss,
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anecdotally,miceappearedhealthy.Theywereactive,eating,andinteractingnormally.Theamountof
bioRxiv preprint first posted online Apr. 8, 2016; doi: http://dx.doi.org/10.1101/047704. The copyright holder for this preprint (which was not
peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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weightlossdidnotdependondailywaterintake(p=0.63,R2= 0.03),thoughthetrendsuggeststhat
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animalsthatdrinkmorewaterlostmoreweight).Furthermore,bodyweightdidnotstronglyinfluence
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thepercentlossofbodyweight(Figure2;p=0.68,R2= 0.02).
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Inadditiontoasubstantiallossinbodyweight,dehydrationwasassociatedwithdifferencesinserum
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electrolytes(Figure3;n=19dehydrated,n=24hydrated).Thesechangesweresubtle,butsignificant
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usingatwo-samplet-test(p<0.008inallcases).
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Lastly,thelevelsofserumelectrolytesweretightlycorrelatedwithpercentbodyweightloss(Figure4).
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Indeed,therelationshipbetweenthelevelofserumsodiumandweightlosswaspositiveandsignificant,
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(ANOVA,F-statistic:12.85,11DF,p=0.004),aswastherelationshipbetweenBUNandweightloss
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(ANOVA,F-statistic:9.089,11DF,p=0.012).Therelationshipsbetweenweightlossandchlorideand
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bicarbonatelevelsrespectively,werepositivebutnotsignificant.
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Discussion
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Desertsareamongsttheharshestenvironmentsontheplanet.Indeed,animalslivingintheseareas
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mustbehighlyadaptedtotheuniquecombinationofextremeheatandaridity.Giventhatour
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understandingofthephysiologyofdesertadaptedanimalsislimitedlargelytostudiesinrenalhistology
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(Mbassa1988)andonwaterintakeandoutput(MacMillenandLee1967;TracyandWalsberg2001),an
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enhancedunderstandingofserumelectrolytechangesduetodehydrationisinformative.Becausemany
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oftheharmfuleffectsofdehydrationresultfromelectrolyteabnormalities,characterizingnormalvalues
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andtheelectrolyteresponsetodehydrationrepresentsacriticalfirststepingarneringadeeper
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understandingofhowdesertanimalssurvivedespitesevereandprolongeddehydration.
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Inthisstudy,normal(baseline)valuesforserumSodium,Chloride,BicarbonateIon,Creatinine,and
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BloodUreaNitrogenwereestablishedinacaptivecolonyoflabanimalshousedindesertconditions.
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Althoughthesemeasuresmaydifferinwildanimals(see(CalisiandBentley2009)forabriefreviewof
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suchdifferences),establishingnormalvaluesincaptiveanimalsiscrucial,thoughfuturestudiesaimto
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understandthepatternsofelectrolytevariationinwildanimals.InP.eremicus,wedefinethenormal
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rangesforeachelectrolyteasthosevaluesfallingbetweenthe1stand3rdquartile.SerumChlorideand
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Sodiumweresignificantlyhigherthaninpublishedrangesforothermammals,includinghumans,a
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marsupial(ViggersandLindenmayer1996),Cricetomys(Nssienetal.2002), andtheporcupine(Moreau
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etal.2003).However,serumchlorideandsodiumlevelsinourstudywerequitecomparabletoanother
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wildrodent,Neotimafuscipes(Weberetal.2002),aMustelid(Thorntonetal.1979),andtheHyrax
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(Arochetal.2007).ValuesforBUNaregenerallyhigherinthisstudy;unfortunately,adirectcomparison
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isnotpossible,asmeasuredvaluesaredependentonthevolumeofurineproduced.SerumCreatinineis
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low,largelyresultingfromthegenerallackofmusclemassinP.eremicusrelativetoothermammals.
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However,becausetheequipmentusedtoanalyzethiselectrolytedoesnoteffectivelycapturethelower
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endofthebiologicalrange,directcomparisonsarenotmadeforthismetric.
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Inadditiontocharacterizingbaselineelectrolytesandtheirresponsetoexperimentaldehydration,the
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normativevaluefordailywaterintakewasestimatedtobe0.11mLperdaypergrambodyweight.
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Thoughcomparablemeasuresofwaterconsumptionarescarce,onestudyintwoaridadaptedLimoys
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(L.pictusandL.irroratus)housedinnon-desertcaptivesettingswereestimatedtobe0.18and0.17mL
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perdaypergrambodyweightrespectively(Christianetal.1978)–avaluemuchgreaterthaninP.
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eremicus.
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
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Animalsthatwereexposedtoexperimentaldehydrationlostasubstantialamountofbodyweight.
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Dehydrationinhumans,resultinginlossofevenafractionofthisamountresultsincardiovascular
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collapseanddeath.Indeed,evenadehydration-relatedlossofafewpercentofbodyweightmaycause
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seriousrenalimpairmentorrenalfailure.Thatthecactusmousemaylosesomuchweightasaresultof
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dehydrationandremainactive,andapparentlyhealthy,withoutrenalimpairmentisatestamentto
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theirdesertadaptation.Yet,whileanecdotallymiceappearwell,theymaybeexperiencingsubstantial
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cognitiveimpairment,asisthecasewithmild-humandehydration(Armstrongetal.2012).Future
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studiesinthelabaimtounderstandthecognitiveeffectsofdehydrationincactusmouse.
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Inadditiontoweightloss,dehydratedanimalsdemonstratedbiochemicalevidenceofphysiological
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stress,intheformofincreasedSodium,Chloride,BUN,andBicarb.Therewerenosignificant
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relationshipsbetweenanyphysiologicalvalueandCreatinine,suggestingthatdehydrationrelatedstress
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doesnotresultinrenalimpairmentordamage.Indeed,thisisincontrasttohumansandother
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mammalswhereacutedehydrationofthenatureimposedontheseanimalsisuniversallyrelatedto
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renalfailureandsubsequentdeath.ThatP.eremicuscanwithstandthislevelofdehydrationisa
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testamenttotheprocessesinvolvedinadaptation.Studiesinprogressaimtolinkpatternsof
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physiologicalchangeofthetypesdescribedheretopatternsofgeneexpressioninbothcaptiveandwild
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animals,furtherinformingourunderstandingofrenalfailureduetodehydrationinmammals.
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Table1
346
Normal
Min
Max
Mean
Sodium(mMol/L)
148-158
144
170
153
Chloride(mMol/L)
110-115
105
126
113
BUN(mg/dL)
29-46
22
64
37
Bicarb(mMol/L)
19-25
15
26
22
Creatinine(mg/dL)
>0.2-0.3
>0.2
0.4
0.22
347
348
Table1.Normalvaluesforserumelectrolytes.Normalvaluesaredefinedasthosevaluesfalling
349
betweenthe1stand3rdquartile.Ofnote,theAbaxisVS2electrolyteanalyzerdoesnotmeasure
350
Creatininebelow0.2mg/dL;therefore,therangefornormalCreatinineistruncatedatthisvalue.
351
352
353
354
355
356
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peer-reviewed) is the author/funder. It is made available under a CC-BY 4.0 International license.
358
comparisonindottedredlines.Ofnote,theAbaxisVS2electrolyteanalyzerdoesnotmeasure
359
Creatininebelow0.2mg/dL,andthereforetherangefornormalCreatinineistruncatedatthisvalue.
360
Na
361
362
363
364
365
366
367
368
0.8
0.4
15
95
130
10
0.2
100
20
105
20
30
110
0.6
40
115
25
50
1.0
60
1.2
70
30
130
125
120
160
150
140
1.4
Figure1.Normalvaluesforserumelectrolytes.Humannormalvalues(fromMedline)areplottedfor
170
357
Cl
Bicarb
BUN
Creatinine
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370
Nosignificanttrendexists.
371
R 2 = −0.0738
0.15
0.20
0.25
0.30
Figure2.Percentbodyweightlossasafunctionofinitialbodyweightduetoexperimentaldehydration.
Percent loss body weight
369
20
22
24
26
28
30
32
Initial body weight
372
373
374
375
376
377
378
379
380
381
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bicarbonateion.Reportedp-valuesarefromatwo-tailedt-test(n=19dehydrated:DRY,n=24hydrated):
384
WET.
385
16
Wet
P − val ue = 0.00871
Wet
30
40
50
60
Dry
Blood Urea Nitrogen
125
115
105
Serum Chloride
386
Wet
P − val ue = 0.000207
Dry
20
Serum Bicarb
155
145
P − val ue = 1.2e−07
Dry
24
383
165
Figure3.Experimentaldehydrationresultedinincreasesinserumsodium,chloride,BUNand
Serum Sodium
382
P − val ue = 0.00221
Dry
Wet
387
388
389
390
391
392
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394
(F-statistic:12.85,11DF,p-value:0.004283)andBUN(F-statistic:9.089,11DF,p-value:0.01177)
0.15
145
150
155
160
165
R 2 = 0.222
0.25
0.25
R 2 = 0.539
0.15
Percent loss body weight
Figure4.TherelationshipbetweenserumelectrolytesispositiveinallcasesandsignificantforSodium
Percent loss body weight
393
170
16
18
110
115
Serum Chloride
395
396
24
26
120
125
0.25
R 2 = 0.452
0.15
Percent loss body weight
0.25
R 2 = 0.0419
105
22
Serum Bicarb
0.15
Percent loss body weight
Serum Sodium
20
30
40
50
60
Blood Urea Nitrogen