ExposuretoLcycloserineincurssurvivalcosts Aedesaegypti females VirginiaBelloniandPatriciaYScaraffia Abstract Background ItwaspreviouslydemonstratedthatalanineaminotransferaseALATEC2612partic ID: 509342
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RESEARCHOpenAccess ExposuretoL-cycloserineincurssurvivalcosts Aedesaegypti females VirginiaBelloniandPatriciaYScaraffia * Abstract Background: Itwaspreviouslydemonstratedthatalanineaminotransferase(ALAT,EC2.6.1.2)participatesin maintainingthealanine-prolinecyclebetweenflightmusclesandfatbodyduring Aedesaegypti flight.ALATisalso activelyinvolvedinthemetabolismofammoniain A.aegypti .Here,weinvestigatedthesurvivalandbehavioral costsofALATinhibitionin A.aegypti femalestobetterunderstandtheroleofALATinblood-fedmosquitoes. Methods: Weanalyzedhow A.aegypti femalemosquitoesrespondtobloodmealssupplementedwith0,2.5,5 and10mML-cycloserine,awell-knowninhibitorofALATinanimals.Mosquitoeswerealsoexposedtobloodmeals supplementedwithL-cycloserineanddifferentconcentrationsofglucose(0,10and100mM).Additionally,the effectsofALATinhibitorandglucoseinmosquitoesstarvedfor24or48hwereinvestigated.Survivaland behavioralphenotypeswereanalyzedduringatimecourse(1,2,4,6,12,24,48and72hafterfeeding). Results: L-cycloserineat10mMresultedinhighmortalityrelativetocontrol,withanacuteeffectduringthefirst 6haftertreatment.Asignificantdecreaseinthenumberofactivemosquitoescoincidingwithanincreaseinfutile wingfanningduringthefirst24hwasobservedatallinhibitorconcentrations.Ahighoccurrenceofknockdown phenotypewasalsorecordedatthistimeforboth5and10mML-cycloserine.Thesupplementationofglucosein thebloodmealamplifiedtheeffectsoftheALATinhibitor.Inparticular,weobservedahighermortalityrate concomitantwithanincreaseintheknockdownphenotype.Starvationpriortobloodfeedingalsoincreasedthe effectsofL-cycloserinewitharapidincreaseinmortality. Conclusions: OurresultsprovideevidencethatexposureofhighdosesofL-cycloserineduring A.aegypti blood feedingaffectsmosquitosurvivalandmotoractivity,suggestinganinterferencewithcarbohydrateandammonia metabolisminatime-dependentmanner. Keywords: Mosquitobehavior,Motoractivity,Wingfanning,Knockdownresponse,Mortality,Aminoacids, Mosquitometabolism,Survival Background pyruvictransaminase(EC2.6.1.2),isresponsibleforabi- molecularping-pongreaction,wherethe -aminogroup ofalanineistransferredto -ketoglutarate,leavingbe- hindpyruvateandglutamate.Thisreversiblereaction synthesizesalaninefrompyruvateaswell[1].Theenzyme containstheprostheticgrouppyridoxalphosphate,which actsasacoenzymeincatalyticreactions. Invertebrates,ALATactivityhasbeendescribedinsev- eraldifferentorganismssuchasfishes[2],amphibians[3], birds[4],andmammals[5-8].ALATislocalizedtoboth thecytosolandmitochondria[8,9]andiswidelydistrib- utedinseveralorgans,withhighlevelsintheliverand kidney[10-12].Inparticular,ALATisexpressedingluco- neogenictissues[8,13].Inmuscle,theenzymesynthesizes alaninefrompyruvate,whichisproducedbyglycolysis. Alanineisshuttledtotheliver,whereitisconvertedback topyruvateandusedforgluconeogenesis.Theglucose *Correspondence: pscaraff@tulane.edu DepartmentofTropicalMedicine,Vector-BorneInfectiousDiseaseResearch Center,SchoolofPublicHealthandTropicalMedicine,TulaneUniversity, 1430TulaneAve.,SL-17,NewOrleans,LA70112,USA ©2014BelloniandScaraffia;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/4.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublic DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis article,unlessotherwisestated. BelloniandScaraffia Parasites&Vectors 2014, 7 :373 http://www.parasitesandvectors.com/content/7/1/373 producedisthendeliveredtothemuscletocontinuethecycle[13].Moreover,ALATplaysanimportantroleinthebraincontributingtoitsenergysupply[14].Ininvertebrates,ALATactivityhasbeenobservedincrustacea[15],mollusks[16],andseveralinsects,includ-inglocusts,tsetseflies,cockroaches,bees,moths[17],andmosquitoes[18].Insomeinsects,ALATparticipatesinthemetabolismofprolineduringflight[18-21],andisinvolvedinthemetabolismofammonia[22-24].Inaddition,ALATactivityispresentinthenervoussystemofsomeinsects,suchasbees[25,26],waterbugs,cock-roaches[27]andfruitflies[28],andprovidesenergytothenervecells.Forexample,inthebeesretina,glycogenaccumulatedinthegliaistransferredtotheneuronsaspyruvateandalanine[25,26].Overthelastfewdecades,agrowinginteresthasde-velopedintheeffectsofALATinhibition,inbothverte-bratesandinvertebrates.Severalpotentialinhibitors[2,6,11,29]havebeentestedincluding-chloro-L-alanine[30-32]andL-cycloserine(LCS),anL-isomerof4-amino-3-isoxazolidinone[33].LCSisoneoftheALATinhibitorsmostcommonlyusedbothinvivo[30,34]andinvitro[7,11,16,29,30,35].Inthepresentstudy,weevaluatedthepotentialeffectsofALATinhibitioninA.aegypti,awidelydistributedspeciesofmosquitoandaprimaryvectorofviraldis-easessuchasdengue,yellowandchikungunyafever[36].WeinvestigatedhowA.aegyptifemalesrespondtodifferentconcentrationsofLCSthroughoutaperiodofthreedayspostbloodmeal,consideringbehavioralend-pointsandmortalityasindicesofmetabolicalterations.WealsoanalyzedtheeffectsofLCSandglucoseinnon-starvedandstarvedA.aegyptifemales.Theresultspre-sentedinthismanuscriptdemonstratethathighdosesofLCSinterferewithA.aegyptibloodmetabolismcaus-inganimpairmentofimportantbehavioralphenotypesandahighmortality.ChemicalsL-cycloserine(LCS),D-glucose(Glc)andsucrosewerepur-chasedfromSigma-Aldrich(St.Louis,MO).BovinebloodwasobtainedfromPel-FreezBiologicals(Rogers,AR).Aedesaegypti(NIHRockefellerstrain,[37])mosquitoeswererearedatstandardconditionsaspreviouslydescribed[38].Newly-eclosedmosquitoeswererandomlyassignedtodifferentcontainers.Femalemosquitoeswerefedon3%sucroseadlibitumuntilbloodfeedingorstarved24or48hwithaccesstowaterpriortoabloodmeal(BM).MosquitoeswerekeptinaCaron6015InsectGrowthChamber,connectedtoaCaronCRSY102con-densaterecirculatingSystem(CaronProducts&Services,Inc.,Marietta,OH)at28°C,75%relativehumidityandalight:darkcycleof16h:8huntiltheendoftheexperiments.LCStreatmentsFour-day-oldfemalemosquitoesofthesamesizewereallowedtofeedfor15minonbloodmeals(seebelowTreatment1,2and3)throughanartificialbloodfeederconnectedtoa37°Cwaterbath[38].Largegroupsoffe-malemosquitoeswerefedatthesametime.Afterfeed-ing,eachfemalewascarefullyinspectedandonlyfullyengorgedmosquitoeswereindividuallytransferredto20mlpolyethylenevials(onefemalepervial).Eachvialwascoveredwithnylonmeshandsecuredwitharubberband.Mosquitoeswereprovidedwateradlibitumthrough-outthestudyperiodandmaintainedinanInsectGrowthChamber,asdescribedabove.Mosquitoesunderwentdifferentexperimentaltreatments:Treatment1:BMsupplementedwithLCS(0,2.5,5,10mM).Theexperimentwasreplicatedfivetimeswithfiveseparatecohortsofmosquitoes,withatotalsamplesizeof250foreachconcentration.Treatment2:BMsupplementedwithLCS(0,10mM)andGlc(0,10,100mM).AllthecombinationsofLCSandGlcweretested.Theexperimentwasreplicatedthreetimeswiththreeseparatecohortsofmosquitoes,withatotalsamplesizeof75foreachconcentration.Treatment3:mosquitoeswerestarvedfor24or48hpriortotheBMsupplementedwithLCS(0,10mM)andGlc(0,100mM).AllthecombinationsofLCSandGlcweretested.Theexperimentswerereplicatedthreetimeswiththreeseparatecohortsofmosquitoes,withatotalsamplesizeof75foreachconcentration.MortalityandbehavioralphenotypesDuringa15secwindow,mortalityrateandbehavioralobser-vationswererecordedat1,2,4,6,12(onlyinTreatment1),24,48and72hpostbloodmeal(PBM).Presenceorabsenceofthefollowingbehavioralend-pointswasindividuallyscored:Active:mosquitoshowsnormalbehaviorsuchaspropercoordinationandtheabilitytostand,walk,andfly.Wingfanning:mosquitoshowsapersistentwingfanningbehaviorinafutileattempttofly.Wingfanningwaspreviouslydescribedinotherinsects(reviewedinHaynes,1988[]).Itcanbeaccompaniedbylossofcoordination.Knockdown:mosquitoshowsinabilitytostand,walkorfly,accordingtotheethologicalprofiledescribedinmosquitoes[BelloniandScaraffiaParasites&VectorsPage2of9http://www.parasitesandvectors.com/content/7/1/373 Inthismanuscriptthetermswingfanningandknock- downrefertoimpairedmotoractivity. Statisticalanalysis SurvivalanalysiswasperformedusingLog-rankandTarone- Waretests.Bonferronicorrectionwasappliedwhenmul- tiplecomparisonswereperformed.Theanalysiswas carriedoutusingGraphPadPrismversion6.0forMac OSX(GraphPadSoftware,SanDiego,CA).Behaviorwas categorizedintothreelevels(active,wingfanningand knockdown).Generalizedestimatingequation(GEE)re- gressionmethodswereusedtoexaminetheimpactof treatment,andanyinteractionwithtime,onbehavior.Be- havioraldatawereanalyzedusingPROCGENMOD,SAS version9.3(SASInstituteInc.,Cary,NC)withacumula- tivelogitlinkandanindependentcorrelationstructure.A p -valuelessthan0.05wasconsideredsignificant.Dataare presentedasmean±standarderror(SE). Results LCSimpairsmotoractivityandsurvival TobetterunderstandtheroleofALATinblood-fed A.aegypti metabolism,weexperimentallyassessedthe survivalcostsofLCS-dependentinhibitionin A.aegypti femalesandanalyzedtheirbehavioralphenotypesduring 3daysposttreatment.Thiswasdonebycomparingout- comesbetweenmosquitoesthatwerefedaBM(control group)andmosquitoesfedBM swithLCSatvaryingcon- centrations(Treatment1). AsshowninFigure1,only5(2%±1)oftheindivid- ualsfedwithbloodalone(BM)diedduringthe72hof observation,whilemosquitoesexposedto10mMLCS (BM+10mMLCS)hadasignificantlyhighermortality (35%±6)[Log-Rank, 2 3 =259.2, p =0.0001]thanthe otherthreegroups(BM=2%±1;BM+2.5mMLCS= 0.4%±0.4;BM+5mMLCS=2%±1).Moreover,at 10mMLCSmostmortalityoccurredwithinthefirst hoursPBM,reaching24%by6h,whileonly11%ofthe mosquitoesdiedbetweenday1andday3.Nodifference inmortalitywasobservedamongtheothergroupsof thistreatment[Log-Rank, 2 2 =3.55, p =0.17]. Whenweexaminedbehavioralphenotypes(Figure1; Additionalfile1:VideoS1)ofallfourgroups,motorac- tivitywasimpairedinatime-dependentmannerbyLCS administration[GEE,Time, 2 1 =507.24, p =0.0001], resultinginagreateroccurrenceofwingfanningand knockdownbehaviorrelativetocontrol.TheLCSeffect wasdose-dependent,wherethenumberofactivemosqui- toesdecreasedwiththeincreaseofLCSconcentrationac- companiedbyahigherprobabilityofknockdown response[GEE,Dose, 2 1 =273.54, p =0.0001].Wingfan- ningwasobservedatallLCSconcentrations,whileknock- downwasalmostabsentat2.5mMLCS(Figure1A-D; Additionalfile2:TableS1).Impairmentofmotoractivity Figure1 EffectofLCSonthebehavioralphenotypesandmortalityin A.aegypti .A) BM; B) BM+2.5mMLCS; C) BM+5mMLCS; D) BM+ 10mMLCS.BM:bloodmeal;LCS:L-cycloserine.FemalemosquitoeswerefedabloodmealsupplementedwithdifferentconcentrationsofLCS (0,2.5,5and10mM).Dataareexpressedasmeanpercentage±SE. BelloniandScaraffia Parasites&Vectors 2014, 7 :373 Page3of9 http://www.parasitesandvectors.com/content/7/1/373 occurredwithinthefirsthoursPBM.However,aclearre- coverywasobservedovertime(Figure1B-D). LCSandGlcincreasethebehavioralresponseand mortality Sincepyruvaterequiredfortransaminationcanbepro- ducedbytheoxidationofGlc,wespeculatedthattheef- fectsofLCSonmosquitoeswouldbemodifiedinthe presenceofGlc.TodeterminatewhetherGlcalterstheef- fectsofLCSonmosquitoes,weinvestigatedthesurvival costsoftheALATinhibitorin A.aegypti femalesafter theywereprovidedbloodmealssupplementedwithLCS andGlc(Treatment2).AswithTreatment1,weevaluated theirbehavioralresponsesduring72hPBM. Duringthecourseoftheexperimentnomosquitoes diedinthegroupsexposedtoBM+10mMGlcorBM+ 100mMGlcwhencomparedtoBM(Figure2).Whenthe effectof10mMLCSwastested,mortalitywassignifi- cantlyhigherthanintheBMcontrol[Log-Rank, 2 3 = 89.76, p 0.0001;BM:4%±2;BM+10mMLCS:52%±0; BM+10mMLCS+10mMGlc:77%±7;BM+10mM LCS+100mMGlc:67%±7](Figure2).Inthepresence ofGlc,theeffectofLCSincreased[Log-Rank, 2 2 =12.37, p =0.0021;BM+10mMLCS;BM+10mMLCS+ 10mMGlc;BM+10mMLCS+100mMGlc],reducing thepopulationbyhalfat4hPBM.WhenBM+10mM LCS+10mMGlcandBM+10mMLCS+100mMGlc werecompared,nodose-dependenteffectwasobserved (Figure2). AsshowninFigure2A-C,nodifferenceinthebehavioral phenotypeswasobservedamongtheBM,BM+10mM GlcandBM+100mMGlcgroups,whileLCSsignificantly affectedmosquitoactivityinatime-dependentway[GEE, Time, 2 1 =40.61, p =0.0001].Whenwecomparedtheef- fectoftheinhibitorunderGlcsupplementation(0,10and 100mM),Glcdecreasedtheprobabilityofactivebehavior [GEE,DoseGlc: 2 2 =34.39, p =0.0001]duetoanincrease ofknockdownresponse(Figure2).However,theeffectwas independentfromtheGlcconcentration(Additionalfile3: TableS2),andovertimeBM+10mMLCS+100mMGlc grouprecovered,reachinganumberofactivemosquitoes similartoBM+10mMLCSgroup.Aslightrecoverywas observedinBM+10mMLCS+10mMGlcgroup[GEE, Time*DoseGlc: 2 2 =14.64, p =0.0007;Figure2D-F]. StarvationpriortoLCS-treatmentseverelyincreasesthe LCSeffects Itiswellknownthatstarvationmobilizesthenutritional reservesinbothvertebratesandinvertebrates.Toex- plorewhetherstarvationimpactsthephenotypesob- servedafterLCStreatment,wetestedthesurvivalcosts oftheALATinhibitoronmosquitoesstarvedfor24or 48h(Treatment3).Wealsoexaminedtheirbehavioral responsesduring3daysposttreatment. Figure2 EffectofLCSandGlconthebehavioralphenotypesandmortalityin A.aegypti .A) BM; B) BM+10mMGlc; C) BM+100mM Glc; D) BM+10mMLCS; E) BM+10mMLCS+10mMGlc; F) BM+10mMLCS+100mMGlc.BM:bloodmeal;LCS:L-cycloserine;Glc:glucose. FemalemosquitoeswerefedabloodmealsupplementedwithdifferentconcentrationsofLCS(0,10mM)andGlc(0,10,100mM).Dataare expressedasmeanpercentage±SE. BelloniandScaraffia Parasites&Vectors 2014, 7 :373 Page4of9 http://www.parasitesandvectors.com/content/7/1/373 Starvationfor24hsignificantlyaffectedmosquitosur- vivalinthepresenceofLCS(BM+10mMLCS:93%±1; BM+10mMLCS+100mMGlc:76%±2)relativetothe controlgroups(BM:3%±3;BM+100mMGlc:3%±3) [Tarone-Ware, 2 3 =219.1, p 0.0001](Figure3).Al- thoughasimilartrendwasobservedinLCS-treated groups,therateofmortalityincreasedfasterinthepres- enceofGlcduringthefirst24h,where50%mortalitywas reachedby4hPBM.Theeffectwasstrongerlaterinthe absenceofGlcwithahighertotalmortalityat72hPBM (Figure3). AsshowninFigure3andAdditionalfile4:TableS3,the highmortalityrateinLCSgroupswasassociatedwitha reducednumberofactivemosquitoesandagreaterknock- downresponse,comparedtocontrolgroups(BMandBM+ 100mMGlc).Wingfanningwasalsoobservedshortly afterfeedinginLCSgroups.Timewasnotasignificant factor[GEE,Time: 2 1 =0.01, p =0.9]withfewactivemos- quitoesintheLCSgroups.However,Glcsupplementation resultedinahigherprobabilityofactivebehaviorand lowerknockdownresponseassociatedwithagreater percentageofsurvival[GEE,TreatmentGlc, 2 1 = 13.73, p =0.0002;Time*TreatmentGlc, 2 1 =5.04, p = 0.0248;Figure3C-D]. Whenmosquitoeswerestarvedfor48hpriortoblood feeding(Figure4),asimilarresponsepatternto24h starvationwasobserved;LCStreatmentresultedinsig- nificantmortality(BM+10mMLCS:96%±4;BM+ 10mMLCS+100mMGlc:100%)comparedtothe controlgroups(BM:11%±1;BM+100mMGlc:3%±1) [Log-Rank, 2 3 =319.9, p 0.0001],withafasterincrease inthemortalityrateinthefirst24hPBMinthepres- enceofGlc(mediansurvival=2hPBM). TheLCS-treatedgroupsshowedalowoccurrenceof activebehaviorandahighoccurrenceofknockdownre- sponse,whichreacheditshighestlevelwithinthefirst hoursPBMwithrespecttothegroupsnotexposedto LCS(Figure4andAdditionalfile5:TableS4).Further- more,thesamemosquitoesshowedwingfanningbehav- ioronlyshortlyafterfeeding(1 2h)(Additionalfile5: TableS4).WhentheeffectofGlcwasconsidered(BM+ 10mMLCS+100mMGlc),norecoverywasobserved.In absenceofGlc(BM+10mMLCS),LCS-survivedmos- quitoesrecoveredslightlyovertime[GEE,TreatmentGlc, 2 1 =17.39, p 0.0001;Time*TreatmentGlc, 2 1 =11.75, p =0.0006;Figure4C-D]. Discussion Alanineaminotransferase(ALAT)playsanimportant roleinmaintainingthealanine-prolinecyclebetween flightmusclesandfatbodyduring A.aegypti flight[18]. Additionally,theALATenzymerespondsefficiently when A.aegypti mosquitoesfaceanammoniachallenge [22-24].Thus,anyinterferencewithALATactivitycould compromisetheefficiencyofthepathwaysinvolved inammoniametabolism[22-24,38,42],resultingin deleteriouseffectsonblood-fed A.aegypti females. Inthestudyperformedhere,wetreated A.aegypti Figure3 EffectofLCSonthebehavioralphenotypesandmortalityin24hstarved A.aegypti .A) BM; B) BM+100mMGlc; C) BM+10 mMLCS; D) BM+10mMLCS+100mMGlc.BM:bloodmeal;LCS:L-cycloserine;Glc:glucose.Femalemosquitoeswerestarvedfor24hpriorto theadministrationofabloodmealsupplementedwithLCS(0,10mM)andGlc(0,100mM).Dataareexpressedasmeanpercentage±SE. BelloniandScaraffia Parasites&Vectors 2014, 7 :373 Page5of9 http://www.parasitesandvectors.com/content/7/1/373 mosquitoeswithL-cycloserine(LCS),awell-known inhibitorofALAT. Inanimals,LCSisabletostronglyorcompletelyin- hibitALATactivity[7,11,16,29,30,34].Whenincreasing concentrationsofLCS(0,2.5,5and10mM)inblood mealsweretestedin A.aegypti females(Treatment1, MethodsSection),onlythehighestdoseaffectedfemale survivalwithanacuteeffectduringthefirsthourspost treatment.However,motorbehaviorwasaffectedinall theLCS-groupsfollowingatime-dependentpatternwith aclearrecoveryovertime.Allthreeconcentrationsof LCSshowedahighoccurrenceofwingfanning,butonly 5and10mMLCSwereassociatedwithasignificant numberofmosquitoesexhibitingtheknockdownbehav- ior.Mortalitywasobservedonlyat10mMLCS,suggest- ingadose-dependentincreaseintheseverityofthe motordisruptionwithdeathastheultimateimpairment. Interestingly,theadditionofglucoseintobloodmeals (Treatment2,MethodsSection)orstarvation(Treat- ment3,MethodsSection)increasedtheLCSeffectson mosquitoes.Ourdataalsoindicatethatbehavioralend- pointsareausefultooltoinvestigatetheeffectofenzym- aticinhibitorsabletointerferewithmosquitometabolism. Motoractivityimpairmentcausedbytheexposuretoche- micalsinnaturalconditionscouldresultinacompro- misedseekingandbitingbehavior,andthereforeaffect diseasetransmission[43,44]. InLCS-bloodfed A.aegypti (Treatmentswith10mM LCS),theearlypeakobservedinbothmortalityand impairedbehaviorsuggeststhatLCSstronglyinteracts withALATsoonafterbloodfeeding.EarlyLCSresponse wasalsodescribedinrats,whereinhibitionofALATac- tivityrapidlyreachedapeakmerely30 60minutespost LCSinjection[34].OnehourLCS-perfusionalsore- sultedinafastdecreaseofALATinrats[45];whereas inmiceanintra-peritonealinjectionoftheinhibitoraf- fectedALATandinducedamoderateimpairmentof motorperformancethreehoursafteradministration [46].TherecoveryobservedinsurvivingLCS-bloodfed mosquitoesmightindicatethattheinhibitoractsrapidly ontissues,butsomehowitisthendetoxifiedorremoved fromthesystem.Theunderlyingmechanismsofmos- quitorecoveryareunknownatthepresent.However,it ispossiblethatthesurvivingmosquitoesusealternative pathwaystodealwithALATinhibition,aspreviouslyre- portedinblood-fed A.aegypti femaleswhenglutamine synthetasewassilencedbyspecificinhibitors[22-24]. Thephenotypesdescribedherein A.aegypti mosqui- toestreatedwithhighdosesofLCScouldbeassociated withanalterationofammoniametabolicpathways.In A.aegypti ,inhibitionofenzymesinvolvedinfixation andassimilationphasesofammoniametabolism[22-24] resultedinahighmortality,precededbythesuppression oflocomotoractivity[24].Inmammals,anexcessofam- moniainthebrainaffectedtheactivityofneurotransmitters involvedintheregulationofmotoractivitysuchasacetyl- choline[47],glutamateanditsproduct -aminobutyricacid [48-50].Highlevelsofammoniainneuronalandother Figure4 EffectofLCSonthebehavioralphenotypesandmortalityin48hstarved A.aegypti .A) BM; B) BM+100mMGlc; C) BM+10mM LCS; D) BM+10mMLCS+100mMGlc.BM:bloodmeal;LCS:L-cycloserine;Glc:glucose.Femalemosquitoeswerestarvedfor48hpriortothe administrationofabloodmealsupplementedwithLCS(0,10mM)andGlc(0,100mM).Dataareexpressedasmeanpercentage±SE. BelloniandScaraffia Parasites&Vectors 2014, 7 :373 Page6of9 http://www.parasitesandvectors.com/content/7/1/373 tissueshavebeenassociatedwithdeleteriouseffectsanddeathinseveralanimalspecies[48,51-53]owingtoanincreaseinoxidativestress,energydeficiency,andalter-ationofneurotransmissionsystemsinaconcentrationandtime-dependentmanner[48,51,54].Recently,Beusteretal.,[30]foundthatALATinhibitionwithLCScorre-lateswithseveralalterationsinmammaliancancercells,suchasenergydeficiency,increasedrespirationrates,andmitochondrialproductionofreactiveoxygenspecies.Moreover,inhibitionofALATbyLCSwasassociatedwithadecreaseinglucoseuptake,andthusasuppressionoftheoverallglucosemetabolisminrodentcelllines[30].InA.aegyptimosquitoes,sugarfeedingplaysanimportantroleinthemetabolismofaminoacidsandenergysupply[55].InTreatment2,alltheLCS-groupsshowedasimilartime-dependentpatternassociatedwithrecoveryovertime,althoughlowerinthepresenceofglucose.These-verityofLCSeffectsafterglucosesupplementationob-servedherecorrelatescloselywithadisruptionofglucoseandaminoacidmetabolisminA.aegyptifemales.Itcouldbeinterestingtoexplorewhetheranyofthesecellularef-fectscausedbyLCSinvertebratescanbealsocorrelatedtothebehavioralalterationsobservedinmosquitoestreatedwiththehigherdosesofLCS.A.aegypti,starvationstronglyimpactsenergysupplyandresultsinlowflightpotential[56].Moreover,whenaegyptiaresugar-starvedfor24hpriortofeedingonaproteinmeal,prolineandalaninelevelsaresignificantlyin-creasedrelativetothelevelofnon-starvedmosquitoes[57].Inrodents,48hstarvationinducesanincreaseinala-nineproduction,whilealaninereleaseisdecreasedbyLCSperfusion[58].Thesefindingscouldsupporthowstarva-tioncanresultinthegreatermortalityandbehavioralal-terationsobservedinA.aegyptimosquitoesexposedtoALATinhibitor.TheLCSadministrationinducedarapidincreaseinmortalityinA.aegyptifemalesstarvedfor24h.Theeffectwasamplifiedinfemalesstarvedfor48h.Inaddition,thesupplementationofglucoseresultedinafas-terincreaseinthemortality,butonlyinthefirst4hposttreatment.Finally,aslightrecoveryandreducednumberofknockdownphenotypeoccurredinthepresenceofglu-coseinmosquitofemalesstarvedfor24h,whilenorecov-erywasobservedinfemalesstarvedfor48h.DespitehighspecificityofLCSforALAT,itisknownthatLCScaninteractwithothertransaminasesinanimals[11,34,46].Therefore,wecannotexcludethepossiblesideeffectsoftheLCStreatmentonadditionaltargetsinmos-quitoes.Toovercomethisproblem,wearecurrentlyin-vestigatingtheeffectsofsilencingALATinA.aegyptifemalesthroughRNAinterference.ThisstudydemonstratesthatexposuretohighLCSdosesincurssurvivalcostsandbehavioralalterationsinA.aegyptifemales.TheLCSeffectswereamplifiedwhenmosquitobloodmealsweresupplementedwithglucoseorwhenfemaleswerestarvedpriortobloodfeeding.Takentogether,ourdatasuggestthatLCSinterfereswithcarbohydrateandammoniametabolisminatime-dependentmannerinblood-fedA.aegyptifemales.AdditionalfilesAdditionalfile1:VideoS1.EffectofLCSonwingfanningbehaviorinA.aegyptifemale.MosquitobehaviorwasvideorecordedinanA.aegyptifemaleexposedtoabloodmealsupplementedwithLCS(10mM).Theobservationwasrecordedat4hpostbloodmeal.Additionalfile2:TableS1.ComparisonofbehavioralphenotypesinA.aegyptiexposedtoLCS(0,2.5,5and10mM)(Treatment1,MethodsSection).Dataareexpressedasmeanpercentage(±SE)calculatedfromthesurvivingmosquitoes.Additionalfile3:TableS2.ComparisonofbehavioralphenotypesinA.aegyptiexposedtoLCS(0,10mM)andGlc(0,10,100mM)(Treatment2,MethodsSection).Dataareexpressedasmeanpercentage(±SE)calculatedfromthesurvivingmosquitoes.Additionalfile4:TableS3.ComparisonofbehavioralphenotypesinA.aegyptistarvedfor24handexposedtoLCS(0,10mM)andGlc(0,100mM)(Treatment3,MethodsSection).Dataareexpressedasmeanpercentage(±SE)calculatedfromthesurvivingmosquitoes.Additionalfile5:TableS4.ComparisonofbehavioralphenotypesinA.aegyptistarvedfor48handexposedtoLCS(0,10mM)andGlc(0,100mM)(Treatment3,MethodsSection).Dataareexpressedasmeanpercentage(±SE)calculatedfromthesurvivingmosquitoes.Alanineaminotransferase;LCS:L-cycloserine;Glc:Glucose;BM:Bloodmeal;PBM:Postbloodmeal.CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.Conceivedanddesignedtheexperiment:PYS.Performedtheexperiments:VB,PYS.Analyzedthedata:VB,PYS.Wrotethepaper:VB,PYS.Bothauthorsreadandapprovedthefinalversionofthemanuscript.AcknowledgmentsTheauthorswouldliketothankDr.StacyMazzalupoforhercriticalreadingofthemanuscriptandvaluablecomments.ThisworkwasfinanciallysupportedbyNationalInstitutesofHealth(NIH)grantR01AI088092toPYS.Received:3March2014Accepted:17July2014Published:16August20141.BergJM,TymoczkoJL,StryerL:Thefirststepinaminoaciddegradationistheremovalofnitrogen.5thedition.NewYork:FreemanWH;2002.Series23.23.2.GonzálezJD,CaballeroA,ViegasI,MetónI,JonesJG,BarraJ,FernándezF,BaananteIV:EffectsofalanineaminotransferaseinhibitionontheintermediarymetabolisminSparusauratathroughdietaryaminooxyacetatesupplementation.BrJNutr3.AlekhovaT,Sof'inA,KobelkovaT,MarcoR,DournonC:Sex-linkeddifferencesinactivityofenzymesinthebloodoftheurodeleamphibianPleurodeleswaltlCompBiochemPhysiolAMolIntegrPhysiol2001,130:819825.4.PoloFJ,PeinadoVI,ViscorG,PalomequeJ:HematologicandplasmachemistryvaluesincaptivePs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Submit your next manuscript to BioMed Central and take full advantage of: Convenient online submission Thorough peer review No space constraints or color gure charges Immediate publication on acceptance Inclusion in PubMed, CAS, Scopus and Google Scholar Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit BelloniandScaraffia Parasites&Vectors 2014, 7 :373 Page9of9 http://www.parasitesandvectors.com/content/7/1/373