Transcriptomic and metabolomic profiles of Chinese citrus fly, Bactrocera minax

20 Pages·2017·4.11 MB·English

RESEARCHARTICLE
Transcriptomic and metabolomic profiles of
Chinese citrus fly, Bactrocera minax (Diptera:
Tephritidae), along with pupal development
provide insight into diapause program
JiaWang*,HuanFan,Ke-CaiXiong,Ying-HongLiu
InstituteofEntomology,CollegeofPlantProtection,SouthwestUniversity,Chongqing,China
a1111111111
*[email protected]
a1111111111
a1111111111
a1111111111
Abstract
a1111111111
TheChinesecitrusfly,Bactroceraminax(Enderlein),isadevastatingcitruspestinAsia.
Thisunivoltineinsectentersobligatorypupaldiapauseineachgeneration,whilelittleis
knownaboutthecourseandthemolecularmechanismsofdiapause.Inthisstudy,the
OPENACCESS
courseofdiapausewasdeterminedbymeasuringtherespiratoryratethroughoutthepupal
Citation:WangJ,FanH,XiongK-C,LiuY-H(2017) stage.Inaddition,thevariationoftranscriptomicandmetabolomicprofilesofpupaeatfive
Transcriptomicandmetabolomicprofilesof
developmentalstages(pre-,early-,middle-,late-,andpost-diapause)wereevaluatedby
Chinesecitrusfly,Bactroceraminax(Diptera:
next-generationsequencingtechnologyand1Hnuclearmagneticresonancespectroscopy
Tephritidae),alongwithpupaldevelopmentprovide
insightintodiapauseprogram.PLoSONE12(7): (NMR),respectively.Atotalof4,808genesweresignificantlyalteredintenpairwisecom-
e0181033.https://doi.org/10.1371/journal. parisons,representingmajorshiftsinmetabolismandsignaltransductionaswellasendo-
pone.0181033
crinesystemanddigestivesystem.GeneexpressionprofileswerevalidatedbyqRT-PCR
Editor:J.JoeHull,USDAAgriculturalResearch analysis.Inaddition,48metaboliteswereidentifiedandquantifiedby1HNMR.Nineof
Service,UNITEDSTATES
whichsignificantlycontributedtothevariationinthemetabolomicprofiles,especiallyproline
Received:February28,2017 andtrehalose.Moreover,thesamplescollectedwithindiapausemaintenance(early-,mid-
Accepted:June26,2017 dle-,andlate-diapause)onlyexhibitedmarginaltranscriptomicandmetabolomicvariation
witheachother.ThesefindingsgreatlyimproveourunderstandingofB.minaxdiapause
Published:July12,2017
andlaythefoundationforfurtherpertinentstudies.
Copyright:©2017Wangetal.Thisisanopen
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited.
Introduction
DataAvailabilityStatement:Thecleanreaddata
areavailableintheGeneBankSequenceRead TheChinesecitrusfly,Bactroceraminax(Enderlein),isadevastatingoligophagouspestofcit-
Archive(AccessionNo.SRP083788).Otherdata
rusplantsinthetemperateareasofAsia,especiallyinChina[1–3].Larvalfeedingcancause
arewithinthepaperanditsSupportingInformation
seriousyieldlosses[4–6],assuchthispesthasbecomeafocusofinterestincitrus-growing
files.
regionsinChina.GiventheeconomicimportanceofB.minax,anumberofpriorstudieshave
Funding:Thisstudywasfinanciallysupportedby
beencarriedout[7–13].However,thelong-lastingpupalstageinwhichdiapauseoccurshas
theNationalNaturalScienceFoundationofChina
severelyhinderedthelaboratory-rearingofthispestandrestrictedfurtherscientificresearch.
(31401742),theChinaPostdoctoralScience
Diapauseisalifehistorystagethatallowsinsectstomitigateacuteenvironmental
Foundation(2014M552307),andtheChongqing
ResearchprogramofBasicResearchandFrontier stresses[14,15]. Someunivoltineinsectsenterobligatorydiapauseatspecificstagesin
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 1/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
Technology(cstc2016jcyjA0203).Thefundershad eachgeneration,butrequirenotokenstimulifordiapauseinductionandpreparation
noroleinstudydesign,datacollectionand [14,16]. Similarly,univoltinetephritidflyB.minaxalsoentersthepupaldiapausetoover-
analysis,decisiontopublish,orpreparationofthe
winter.However,thecourseofdiapausehasyettobereported.Generally,diapausein
manuscript.
insectsfeaturesintensemetabolicdepression.Respiratoryrateisthereforeausefulmarker
Competinginterests:Theauthorshavedeclared fordeterminingdiapauseinitiationandtermination[17].Forexample,theprecisetimeof
thatnocompetinginterestsexist.
diapauseterminationofRhagoletispomonellawasidentifiedbasedonthefittedtrajectory
ofmetabolicrateestimatedbyCO production.Subsequently,samplesduringthedia-
2
pauseterminationwerecollectedtoinvestigatethemolecularmechanismsunderlyingdia-
pauseterminationandresumptionofdevelopment[16].Bydeterminingthecourseof
diapauseinB.minaxthroughrespiratoryratemeasurement,severalotherinvestigations
intodiapausecanbeconducted.
Next-generationsequencinghaswidelybeenusedtocharacterizegenomesandtran-
scriptomes.Thisapproachhasfacilitatedstudiesonbiologicalprocessesinorganisms,
suchasthediscoveryofdifferentiallyexpressedgenes(DEGs)[18–20]. AB.minaxtran-
scriptomethatwaspreviouslyassembledandannotatedcanprovideafoundationfor
furtherDEGanalysis[21].Similarly,metabolomicprofilinghasincreasinglybeenused
worldwidetoinvestigatethequalitativeandquantitativevariationofmetabolitesintissues
andbiofluidsinresponsetobioticorabioticfactors[22].Themostwidelyusedmethods
formetabolomicanalysisare1Hnuclearmagneticresonancespectroscopy(NMR),gas-
chromatographycoupledwithmassspectrometry(GC-MS),andliquidchromatography
coupledwithmassspectrometry(LC-MS)[23,24]. Ofthesemethods,NMRhassignificant
advantages,includingnear-universaldetectionofmetabolites,easyquantitation,high
reproducibility,minimalsamplepreparation,andrapidity[25,26]. Sofar,metabolomic
analysishasbeenperformedinmanyfieldssuchasdiagnostics[27],pharmacology[28],
microbiology[29],andnutrition[30].Recently,alimitednumberofmetabolomicanaly-
seshavealsobeenconductedoninsectdiapause[31–33].
Inthisstudy,therespiratoryrateofB.minaxwasperiodicallymeasuredthroughoutthe
pupalstageinordertoclarifythecourseofdiapause.Tobetterunderstandthemolecular
mechanismsunderlyingdiapause,high-throughputnext-generationsequencingtechnology
and1HNMRanalysiswereperformedtoanalyzerespectivelythetranscriptomicandmetabo-
lomicprofilesofB.minaxatfivepupaldevelopmentalstages.Theresultshaverevealedvaria-
tionsinthephysiologicalpathwaysutilizedthroughoutthecourseofdiapauseandprovided
newinsightsintodiapauseprogramming.
Materialsandmethods
Ethicsstatement
TheowneroftheorchardinWulongCounty,ChongqingMunicipality,China,providedper-
missiontocollectthesamplesforourscientificresearch.
Insectrearing
Fallenorangesinfestedwithlarvaewerebroughtbacktothelabfromanorchard(N29o
34.373’,E107o54.564’)inWulongCounty,ChongqingMunicipality,China.Third-instar
larvae(15~18mminlength)collectedfromtheorangeswereplacedoversandinplastic
dishesandallowedtopupate.Alldisheswereplacedoutdoorsinthenyloninsectrearing
cageundernaturaltemperatureandlight/darkcycleintheBeibeidistrict,Chongqing
Municipality,China.Thesandwaschangedweeklyandregularlywateredtomaintain
moisture.
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 2/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
Measurementofrespiratoryrateofpupae
Therespiratoryrateofeachgroupwasmeasured46timesfrompupationtothefirstadult
emergence,usingaself-designedportablerespirometrysystemwithahigh-accuracyinfrared
CO detector(S1FigWosaite,Shenzhen,China).Priortomeasuringthepupalrespiratory
2
rate,thirtypupaewererandomlyselectedanddividedintothreegroups.Eachgroupwas
weighedandplacedina50mLsealedchamberfor2handtheCO concentrationinthecham-
2
berwasrecorded.Therespiratoryrateofpupaewascalculatedby:
C:V
R¼ ð1Þ
M:T
WhereRisthecalculatedrespiratoryrateinμLCO /mg/h,CistheraisedCO concentration
2 2
inμL/L,VisthevolumeofthesealedchamberinL,Mistheweightofthepupaeinmg,andT
isthedurationofCO measurementinh.Visualinspectionofrespiratoryratedatastrongly
2
suggestedanexponentialdecayfollowedbyalogisticincreaseandanexponentialincrease,
basedonwhichaneightparameternon-linearmodeldescribingthistrajectorywascon-
structed:
g
R¼ða(cid:0) bÞe(cid:0) ktþ þcebtþb ð2Þ
1þeða(cid:0) rtÞ
WhereRistherespiratoryrate,tisthetimeindayssinceinvestigation,αistherespiratoryrate
atthestartingpoint,βistherespiratoryrateatthetransitionbetweenexponentialdecayand
logisticincrease,kistherateconstant,γistherespiratoryrateatthetransitionbetweenlogistic
andexponentialincrease,aistheconstant,cisthescalingparameter,randbareparameters
thatdeterminethetimingoftwotransitions,respectively.Thenon-linearmodelwasfitted
usingGraphPadPrism5andMicrosoftExcel2010,andthegoodnessoffitwasdeterminedby
χ2testusingExcel2010.Theinitiation,maintenance,andterminationofpupaldiapausewere
determinedinrelationtothefittedmodel.
Insectsamplecollection
Sampleswerecollectedatfivetimepoints,pre-diapause(PreD),early-diapause(ED),middle-
diapause(MD),late-diapause(LD),andpost-diapause(PD)(Fig1),asdeterminedbyrespira-
toryrate.Priortosamplecollection,allpupaewererearedseparatelyin15dishes,threedishes
foreachofthefivedevelopmentalstages,undertheconditionsdescribedabove.Ateachsam-
plingpoint,allofthepupaeinthefourdisheswerecollectedandstoredseparatelyinliquid
nitrogenforsubsequenttranscriptomicandmetabolomicanalysis.
RNAisolation,libraryconstructionandIlluminasequencing
TotalRNAfromeachpupawasisolatedusingTRIZOLReagent(Lifetechnologies,Carlsbad,
CA,US)accordingtothemanufacturer’sinstructions.ThequantityandqualityoftotalRNA
wasassessedwithaNanoVuespectrophotometer(GEHealthcareBio-Science,Uppsala,Swe-
den)and1%agarosegelelectrophoresis,respectively.ThecDNAlibrarieswereconstructed
usingTruSeqRNASamplePreparationKit(Illumina,SanDiego,CA,USA)followingthe
manufacturer’sprotocol.Briefly,oligo(dT)magneticbeadswereusedtopurifypoly(A)
mRNA.TheresultingmRNAwasmixedwithfragmentationbufferandcleavedintoshort
fragments.Thefirst-strandcDNAsweregeneratedwithrandomhexamerprimers.Second-
strandcDNAsweresynthesizedusingDNApolymeraseI(NewEnglandBioLabs,Ipswich,
MA)andRNaseH(Invitrogen,Carlsbad,CA).ThesecDNAfragmentswereend-repaired,fol-
lowedbysinglenucleotideA(adenine)additionandlitigationofadaptors.Afterpurification
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 3/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
Fig1.Non-linearregressionofrespiratoryrateofBactroceraminaxpupae.Arrowsindicatethetimepointswhenthesampleswere
collectedfortranscriptomicandmetabolomicanalysis.
https://doi.org/10.1371/journal.pone.0181033.g001
withAMPureXPbeads,theligatedproductswereamplifiedbyPCRtogeneratecDNA
libraries,whichweresequencedonanIlluminaNextSeq500(Illumina).Therawreadswere
filteredtoremoveadaptorsequences,low-qualitysequenceswithunknownnucleotidesN,
readsshorterthan50bp,andreadswithmorethan20%lowqualitybases,usingtheNGS
QCtoolkitpackage[34].ThecleanreadswereassessedforqualityusingFastQC(http://
www.bioinformatiH2.babraham.ac.uk/projects/fastqc/)andthenmappedtoourpreviously
generatedtranscriptomereferencedatabase[21].Threebiologicalreplicatesweregenerated
foreachdevelopmentalstage.
DiscoveryofdifferentiallyexpressedgenesandKEGGpathwayanalysis
Geneexpressionlevelsweredeterminedbyreadsperkbofexonmodelpermillionmapped
reads(RPKM)values[35],whichwerecalculatedbasedonthenumberofreadsmappingto
eachunigeneobtainedpreviously[21].TheDEseqpackagewasusedtoidentifytheDEGs
[36].UnigeneswithPvalue<0.05andfoldchangevalue>2ineachcomparisonwereconsid-
eredtobesignificantlydifferentiallyexpressedgenes.Benjamini-Hochbergcorrectionwas
thenconductedtoreducethefalsediscoveryrate(FDR).DEGclusteranalysiswasperformed
usingcluster[37]andvisualizedbyJavaTreeview[38].Principalcomponentanalysis(PCA)
wascarriedoutusingpcaMethods[39]toevaluatethevariationingeneexpressionprofiles
amongsamplesandvisualizetheclusteringofsamples.KyotoEncyclopediaofGenesand
Genomes(KEGG)pathwaysenrichmentanalyseswereconductedusingKEGGmapperto
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 4/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
categorizetheDEGs(http://www.genome.jp/kegg/tool/map_pathway2.html),andtheP
value<0.01wassetasathresholdtodeterminethesignificantlyregulatedpathwayineach
comparison.
Quantitativereal-timePCR(qRT-PCR)verification
qRT-PCRwasperformedtoverifytheaccuracyoftheDEGanalysis.TotalRNAfromthefive
pupalstagesdescribedabovewasextractedusingTRIZOLReagent.Thefirst-strandcDNA
wassynthesizedusingPrimeScript™RTMasterMix(PerfectRealTime)Kit(Takara,Shiga,
Japan).Twenty-onepairsofspecificprimersweredesignedtoamplifythegenesselectedfrom
multiplecomparison(S1Table).Ubiquitinwasusedasareferencegenefornormalization
[40].qRT-PCRwasconductedin25μLvolumescontaining12.5μLSYBR1PremixExTaqII
(Takara),2μLprimers(10μM),1μLcDNA,and9.5μLddH O,usingaCFX96™Real-Time
2
PCRDetectionSystemthermalcycler(BIO-RAD,Hercules,CA,USA).Amplificationcondi-
tionswereasfollows:initialdenaturationat95˚Cfor30s;followedby40cyclesofdenaturation
at95˚Cfor5s,60˚Cfor30s.Pearson’srcorrelationcoefficientwascalculatedtoevaluatethe
correlationbetweentheqRT-PCRandDEGdata.Threebiologicalandthreetechnicalrepli-
cateswereperformedforeachgene.
1HNMRspectroscopy
About500mgpupaeweregroundinliquidnitrogenandlyophilizedinavacuumfreezedryer.
Morethan50mglyophilizedsamplewasweighedandresuspendedin1mlwater.Aftervortexing
for1min,samplesweresonicatedandcentrifugedat13000rpmat4˚Cfor10min.Thesuperna-
tantwasfilteredusing3-kDamicrocentrifugefilterstoremoveproteinsandinsolubleimpurities.
A450μLfiltratewasthenmixedwith50μLDSSstandardsolution(4.088mM),aninternalNMR
chemicalshiftstandard,forsubsequentNMRanalysis.One-dimensionalNMRspectraofsamples
wereacquiredusingaBrukerAVIII600MHzspectrometer(BrukerBiospinLtd.,Coventry,UK)
equippedwithaninversecryoprobe,operatingatanNMRfrequencyof600.13MHz,andadata
acquisitiontemperatureof298K.Atotalof64transientswereacquiredin32,768datapoints
usingaspectralwidthof8000Hz.Thefreeinductiondecay(FID)signalwasmultipliedbyan
exponentialwindowfunctionwith1HzlinebroadeningfactorbeforeFouriertransform.Metabo-
liteswereassignedbyChenomx(ChenomxInc.,Edmonton,Canada)onthebasisofchemical
shifts,couplingconstants,andrelativeintensities,againstaChenomxdatabasethatcontainedthe
uniqueNMRspectraofeachstandardcompound.Theabsoluteconcentrationofeachcompound
wasnormalizedtothesampleweight.Fivebiologicalreplicatesweresetforeachselecteddevelop-
mentalstage.PCAandpartial-leastsquaresdiscriminantanalysis(PLS-DA)[41]werecarriedout
toevaluatethevariationinmetaboliteprofilesamongdevelopmentalstagesandtovisualizesam-
pleclustering.BasedonPLS-DAanalysis,theVariableImportanceinProjection(VIP)scores
wereobtainedtoindicatethemetabolitesthatsignificantlycontributedtotheintergroupdifferen-
tiation.Theconcentrationsofnineidentifiedmetabolites(VIPscore>1)atfivedevelopmental
stageswerecomparedusinganon-parametricKruskal-Wallistest,followedbyBonferronicorrec-
tion[42].TheanalyseswereperformedusingthestatisticalpackageSTATISTICAversion10
(StatSoftInc.2011,Tulsa,OK).
Results
Respiratoryratetrajectory
ThemodeldescribingtherespiratoryratetrajectorywasfittedwellaccordingtoChi-square
test(χ2=0.0824,d.f.=44,P>0.05).Theexponentialdecay,logisticincrease,andexponential
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 5/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
increaseinthismodelrepresentperiodsofdiapausepreparation,diapausetermination,and
preparationforadultemergence,respectively(Fig1).Accordingtothismodel,respirationwas
suppressedafterpupationandreacheditslowestlevelapproximately40dayslater,when
pupaehadcompletelyentereddiapause.Approximatelytwomonthsafterinitiation,diapause
terminatedandpupaldevelopmentresumedwiththerespiratoryrateincreasingby39-fold.
Post-diapausedevelopmentlastedforoveramonthuntiladultsstartedtoemerge.
Identificationofdifferentiallyexpressedgenes(DEGs)amongthe
differingdevelopmentalstages
FifteenmRNAlibraries,threereplicatesforeachdevelopmentalstage,weresequenced.At
least18millionrawreadsweregeneratedineachlibrary(S2Table).Allrawdatahavebeen
depositedintheGeneBankSequenceReadArchive(Accessionnumber:SRP083788).After
removinglowqualityreads,thenumberofcleanreadsrangedfrom18.37millionto51.87mil-
lion,andtheproportionofusefulreadsexceed97%inalllibraries.Geneexpressionprofilesat
differentpupaldevelopmentalstageswerecalculatedtoidentifyDEGs.Atotalof4,808genes
weresignificantlydifferentiallyexpressedintenpairwisecomparisons(Fig2andS2Fig),
whereasthenumberdecreasedto3,290afterBenjamini-HochbergcorrectiontoreduceFDR
(S3Fig).Interestingly,samplescollectedwithinthemaintenanceofdiapause(ED,MD,and
LD)didnotshowintensevariationingeneexpressionprofilescomparedtoeachother,with
onlyafewgenesaltered.ThelargeoverlapofED,MD,andLDinthePCAplotalsoshowedno
obviousdifferenceingeneexpressionprofilesamongthesethreestages(Fig3).However,adis-
tinctseparationinthePCAplotandanumberofDEGsbetweenPreD/PDandtheothertime
pointsrevealedlargevariationsinthegeneexpressionprofilesbetweenthesetwostagesand
others(Figs2and3).AllDEGsweredividedinto6groupswitheachexhibitingarepresenta-
tiveexpressionpattern.Genesincluster1and3werehighlyexpressedinPreDandPD,
respectively,whereasgenesincluster4werelowlyexpressedinPreD.Throughoutdiapause
maintenance,theexpressionofgenesincluster2wassuppressed,whereasthoseincluster5
and6wereactivated(Fig4).
TounderstandthepotentialfunctionsofidentifiedDEGs,KEGGpathwayenrichmentfor
eachpairwisecomparisonwasperformedatPvalue<0.01.Pathwaysinvolvedinhumandis-
easewereexcludedafteranalysis.TheresultsshowedthatnoKEGGpathwaywasenrichedin
pairwisecomparisonsamongthethreegroupsfrommaintenanceofdiapause,exceptfor“Ala-
nine,aspartate,andglutamatemetabolism”betweenMDandLD.Mostofthesignificantly
alteredpathwaysinothercomparisonswererelatedto“Carbohydratemetabolism”,“Lipid
Fig2.Numberofsignificantlydifferentiallyexpressedgenes(DEGs)ineachpairwisecomparisonof
differentBactroceraminaxpupalstages.PreD,pre-diapause.ED,early-diapause.MD,middle-diapause.
LD,late-diapause.PD,post-diapause.
https://doi.org/10.1371/journal.pone.0181033.g002
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 6/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
Fig3.Principalcomponentanalysis(PCA)plotofgeneexpressionprofileatdifferentBactrocera
minaxpupalstages.PreD,pre-diapause.ED,early-diapause.MD,middle-diapause.LD,late-diapause.PD,
post-diapause.
https://doi.org/10.1371/journal.pone.0181033.g003
metabolism”,“Aminoacidmetabolism”,“Biosynthesisofothersecondarymetabolites”,
“Metabolismofcofactorsandvitamins”,“Xenobioticsbiodegradationandmetabolism”,
“Signaltransduction”,“Endocrinesystem”,and“Digestivesystem”(Table1).
Validationofgeneexpressionprofiles
Theexpressionlevelsofthe21selectedgenesweremeasuredbyqRT-PCRtovalidatetheDEG
analysis.TheresultsshowedastrongcorrelationbetweentheqRT-PCRandDEGdatawith
Pearson’scorrelationcoefficient>0.95(Fig5),indicatingthereliabilityofusingDEGdatato
investigatetemporal-specificgeneexpressionprofilesthroughoutthepupalstage.
MetabolomicvariationamongBactroceraminaxdevelopmentalstages
Atotalof49metaboliteswereidentifiedacrossallsamples,includingaminoacidsandtheir
derivatives,organicacids,nucleicacidcomponents,sugars,andothers(S4Fig).Afterremov-
ingcitrate,whichcannotbepreciselyquantifiedduetotheeffectofpHvalue,theother48
metaboliteidentitieswereconfirmedforsubsequentanalysis.BoththePCAandPLS-DAplots
showedcloseoverlapbetweentheED,MD,andLDgroups,butalsoshowedclearseparation
betweenPreDandPD(Fig6),indicatingthemetabolomicprofileschangedmarginallywithin
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 7/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
Fig4.Clusterofdifferentiallyexpressedgenes(DEGs)amongthedifferentBactroceraminaxpupal
stages.Eachcolumnrepresentsasample,andeachrowrepresentsadifferentiallyexpressedgene.Greenand
redcolorgradientsindicateadecreaseorincreaseinexpression,respectively.PreD,pre-diapause.ED,early-
diapause.MD,middle-diapause.LD,late-diapause.PD,post-diapause.
https://doi.org/10.1371/journal.pone.0181033.g004
diapausemaintenance,butvarieddramaticallywithinitiationandterminationofdiapause.
AfterVIPscorecalculation,thetop9metabolitesthatcontributedthemosttothevariationof
metabolomicprofiles(VIPscore>1)wereidentified(Fig7A).Significantdifferenceswere
foundintheconcentrationofninemetabolitesacrossthefivedevelopmentalstageswithnon-
parametricKrustal-Wallistest(Table2).Ofalltheseninemetabolites,proline,trehalose,N-
acetylglutamate,succinate,glutamate,alanine,andsn-glycero-3-phosphocholinesawsignifi-
cantaccumulationswithinmaintenanceofdiapause;theglutamineconcentrationwashigher
inPreD;andthe2-oxoglutarateconcentrationwashigherinPreDbutgraduallydecreased
alongwithdevelopment(Fig7B).
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 8/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
Table1. KEGGpathwayanalysisofdifferentiallyexpressedgenesamongcomparisons.
Comparison KEGGpathway Comparison KEGGpathway
EDvsPreD Pentoseandglucuronateinterconversions* PDvsPreD Pentoseandglucuronateinterconversions*
Galactosemetabolism* Galactosemetabolism
Ascorbateandaldaratemetabolism Ascorbateandaldaratemetabolism*
Starchandsucrosemetabolism* Starchandsucrosemetabolism
Glycerolipidmetabolism Steroidhormonebiosynthesis
Etherlipidmetabolism Glycerolipidmetabolism
Arginineandprolinemetabolism Retinolmetabolism
Glutathionemetabolism* MetabolismofxenobioticsbycytochromeP450*
Retinolmetabolism Drugmetabolism—cytochromeP450*
Porphyrinandchlorophyllmetabolism DNAreplication
MetabolismofxenobioticsbycytochromeP450* Celladhesionmolecules(CAMs)
Drugmetabolism—cytochromeP450* PPARsignalingpathway*
Proteinprocessinginendoplasmicreticulum Proteindigestionandabsorption
Antigenprocessingandpresentation EDvsPD Biosynthesisofaminoacids*
Estrogensignalingpathway Ascorbateandaldaratemetabolism
Proteindigestionandabsorption Starchandsucrosemetabolism*
Fatdigestionandabsorption Glycine,serineandthreoninemetabolism*
MDvsPreD Biosynthesisofaminoacids* Retinolmetabolism
Pentoseandglucuronateinterconversions* Porphyrinandchlorophyllmetabolism
Galactosemetabolism Antigenprocessingandpresentation
Starchandsucrosemetabolism* MDvsPD Carbonmetabolism
Aminosugarandnucleotidesugarmetabolism Biosynthesisofaminoacids*
Nitrogenmetabolism Pentosephosphatepathway*
Fattyaciddegradation Ascorbateandaldaratemetabolism
Glycerolipidmetabolism Starchandsucrosemetabolism
Glycerophospholipidmetabolism Steroidhormonebiosynthesis
Alanine,aspartateandglutamatemetabolism* Glycine,serineandthreoninemetabolism
Arginineandprolinemetabolism* Glutathionemetabolism
Glutathionemetabolism Retinolmetabolism
Streptomycinbiosynthesis Porphyrinandchlorophyllmetabolism
Aminobenzoatedegradation MetabolismofxenobioticsbycytochromeP450
Two-componentsystem Drugmetabolism—cytochromeP450
Hipposignalingpathway-fly MAPKsignalingpathway
Peroxisome Hipposignalingpathway-fly
OvarianSteroidogenesis Celladhesionmolecules(CAMs)
Estrogensignalingpathway Antigenprocessingandpresentation
Fatdigestionandabsorption* LDvsPD Purinemetabolism*
LDvsPreD Pentoseandglucuronateinterconversions* Glutathionemetabolism
Galactosemetabolism* Wntsignalingpathway
Starchandsucrosemetabolism* Hipposignalingpathway-fly
Nitrogenmetabolism Insulinsecretion
Fattyaciddegradation Adrenergicsignalingincardiomyocytes
Glycerolipidmetabolism* Pancreaticsecretion
Glycerophospholipidmetabolism* MDvsLD Alanine,aspartateandglutamatemetabolism
Glycosaminoglycandegradation
Onecarbonpoolbyfolate
Retinolmetabolism
(Continued)
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 9/20
TranscriptomicandmetabolomicprofilesofBactroceraminax
Table1. (Continued)
Comparison KEGGpathway Comparison KEGGpathway
Caffeinemetabolism
Drugmetabolism—otherenzymes
Neuroactiveligand-receptorinteraction
Peroxisome*
PPARsignalingpathway*
Proteindigestionandabsorption
Fatdigestionandabsorption
*Pvalue<0.001.ForKEGGpathwayswithoutasterisks,Pvalue<0.01.
https://doi.org/10.1371/journal.pone.0181033.t001
Discussion
Respiratoryratetrajectory
Metabolicdepressionisfrequentlyconsideredauniversalcharacteristicofdiapausinginsects
[16,17,43],hence,therespiratoryrateofB.minaxpupaewasmeasuredfirsttodeterminethe
courseofdiapause.ThesensitivityoftheCO detectorprecludedmonitoringrespiratoryrate
2
ofindividualB.minax.However,monitoringagroupofpupaeprovedtobefeasibleasall
adultsemergedwithin8days(datanotshown),indicatingsynchronizationofpupaldevelop-
ment.Thegeneratedcurveidentifiedimportantdevelopmentallandmarksthroughoutthe
pupalstageandguidedoursamplecollection(Fig1).Toourknowledge,thisisthefirst
Fig5.CorrelationanalysisofqRT-PCRanddifferentiallyexpressedgene(DEG)dataforselected
genesofBactroceraminax.
https://doi.org/10.1371/journal.pone.0181033.g005
PLOSONE|https://doi.org/10.1371/journal.pone.0181033 July12,2017 10/20