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Articlehistory:
Received16November2010 Receivedinrevisedform15March2011 Accepted15March2011
Hypertrehalosemichormone(HTH)isaneuropeptidewithintheadipokinetichormone(AKH)family thatinducesareleaseoftrehalosefromfatbodyintohemolymphinanumberofinsects.Inthisstudy,we firstshowedthatfemaleadultGermancockroach,Blattellagermanica,displayedacyclicfluctuationof hemolymphtrehaloselevelscorrelatedtothematurationofoocytesinthereproductivecycle.After cloningtheHTHcDNAfromtheGermancockroach(Blage-HTH),expressionstudiesindicatedthat Blage-HTHmRNAshowedthecyclicchangesduringthefirstreproductivecycle,wherepeakvaluesoccurredin 8-day-oldvirginfemalecockroaches,whichweregoingtoproduceoothecae.ThefunctionsofBlage-HTH werestudiedusingRNAinterference(RNAi)toknockdownitsexpression.AdultvirginfemalesofB.
germanicainjectedwithBlage-HTHdsRNAincreasedhemolymphtrehaloselevelsinthelateperiodof vitellogenesismoreslowlythancontrol.Furthermore,RNAiofBlage-HTHdelayedovipositiontimeand some(10%)individualsdidnotproducethefirstoothecauntil15daysaftereclosion,whereasthecontrol groupproducedoothecabefore9daysinallcases.
CrownCopyrightß2011PublishedbyElsevierLtd.Allrightsreserved.
* Correspondingauthor.Tel.:+886223636581;fax:+886223636581.
E-mailaddress:m480@ntu.edu.tw(H.-J.Lee).
ContentslistsavailableatScienceDirect
Journal of Insect Physiology
j our na l ho me pa ge : w ww . e l se v i e r . com / l oca t e / j i ns phy s
0022-1910/$–seefrontmatter .CrownCopyrightß2011PublishedbyElsevierLtd.Allrightsreserved.
doi:10.1016/j.jinsphys.2011.03.012
Appendix 1
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nematode Caenorhabditis elegans are involved in egg-laying behavior through a structurally vertebrate-like gonadotropin-releasinghormone(GnRH)receptor(Lindemansetal.,2009).
FemalesoftheGermancockroach,Blattellagermanica,follow several reproductive cycles in their adult life and produce an oothecaenclosingthematureeggs,eitherfertilizedorunfertilized, ineachreproductivecycle(RothandStay,1962).Virginfemalesof B.germanicadisplaybothcallingbehaviorandrobustlocomotor activityforseveraldaysbeforeoviposition(LeeandWu,1994;Lin andLee,1996).Increasedlocomotionisrelatedtomate-searching behavior, since the females of B. germanica reduce locomotion immediatelyafterasuccessfulmating(LinandLee,1998).Wecan presumethatahighenergydemandisneededtosupportrobust mate-searchingbehavior,andtrehaloseisthemajorsugarinthe hemolymph that is used as an energy source by tissues like muscles and ovaries(Thompson, 2004).Moreover, the involve-mentoftrehaloseinvitellogeninintakebydevelopingoocyteshas beendemonstratedincockroaches(Konoetal.,2001).Therefore, mobilizationoftrehaloseshouldbeexpectedtoincreasebefore ovipositioninthefemaleB.germanica.
In the present study, the hemolymph trehalose titers of B.
germanicaweredeterminedduringreproductivecycles,andresults showthatthefluctuationsofhemolymphtrehaloselevelsparallthe ovarian growth. Then, the Blage-HTH gene was cloned in B.
germanica,anditsphysiologicalfunctionwasinvestigatedbyRNA interference(RNAi)towhichthiscockroachspeciesisparticularly sensitive(Belle´s,2010).RNAi-mediatedknockdownofBlage-HTH geneexpressiondelayedtheincreaseofhemolymphtrehaloseand theproductionofthefirstoothecainvirginfemaleB.germanica.
2. Materialsandmethods 2.1. Insects
TheGermancockroach,B.germanica(L.),colonywasrearedin environmentalchambersunder288CandL:D=16:8hconditions.
Dogchowand waterwereprovidedad libitum.Newlyemerged adultswerecollectedwithin24handseparatedbysex.Detailed informationaboutrearinghasbeengivenpreviously(LeeandWu, 1994).
2.2. CloningofBlage-HTHinB.germanica
Total RNA was isolated with TRIzol1 reagent (Invitrogen, Carlsbad,CA)frombrain–corporacardiaca–corporaallata(Br–CC–
CA) complexes of adult B. germanica females, following the manufacture’sprotocol.WeusedSuperscriptIIIreverse transcrip-tase (Invitrogen)and theoligo(dT)20primer togeneratecDNAs.
ThefirstprimersetwhichwasdesignedbasedontheknownAKH sequencesofotherinsects,includingBlaberusdiscoidalis(U35277), P.americana(AY622321),andBombyxmori(AB298930),andwas asfollows:forward,50-TGTGTGAGGCTCAGGTGAACTT-30;and reverse, 50-TGA GAA TTT TTC ACA TTC CA-30. The amplified fragment (159bp) was subcloned into the pGEMT-easy vector (Promega) and sequenced. To complete the Blage-HTH cDNA sequence, GeneRacerTMKit (Invitrogen)wasappliedto accom-plishrapidamplificationofcDNAends(RACE),accordingtothe manufacturer’sprotocol.For50-RACE,thereverseprimerwas50 -CAC ATTCCAGCAGTTTCTGA-30,and for30-RACE,theforward primerwas50-GTACATGTACAGTGCAATGC-30.AllPCRproducts were subcloned into pGEMT-easy vector (Promega) and se-quenced. BLAST analyses indicated that the sequence obtained in B. germanica was B. discoidalis prepro-hypertrehalosemic hormonehomologue(GenBank:U35277.1)andwecalleditasB.
germanica prepro-hypertrehalosemichormone(Blage-HTH, Gen-Bankaccessionno.FJ943774).
2.3. QuantitativerealtimePCRanalysis
To quantify the mRNA levels of Blage-HTH, we followed a quantitative real-time PCR (qRT-PCR) approach. TotalRNA was extractedfrom3adultfemalebrain–CC–CAcomplexeswithTRIzol1 reagent(Invitrogen,Carlsbad,CA).DNaseI(Promega)wasusedto removethegenomicDNAcontamination.About1
m
goftotalRNAof eachsamplewasreverse transcribedintocDNAwith oligo(dT)20primer using Superscript III reverse transcriptase (Invitrogen).
Expressionofthehousekeepinggene,actin(AJ862721),wasusedas areference,andtheprimerpairswereasfollows:forward,50-TTG TGCTGGACTCTGGTGAC-30,andreverse,50-ACGATTTCTCGCTCT GCTGT-30.TheprimerstostudytheBlage-HTHgenewere:forward, 50-TTGGTAGTTGTGGTGGCTCTAGCA-30,andreverse,50-CCAGCA GTTTCTGAGCTTCACTCT-30.Theefficiencyofeachprimersetwas firstvalidatedbyconstructingastandardcurvethroughfourserial dilutions.TheqRT-PCRreactionswerecarriedoutintriplicateina Bio-RadiCycleriQ5Real-TimePCRdetectionsystem(Bio-Rad),using SYBR Green Realtime PCR Master Mix (#QPK-201; Toyobo Co., Osaka, Japan). A control without template was included in all batches.ThePCRprogrambeganwith asinglecycleat958Cfor 3min,40cyclesat958Cfor15sand608Cfor60s.Afterwards,the PCRproductswereheatedto958Cfor15s,cooledto558Cfor15s and increasing temperature 0.58C/min in order tomeasure the dissociationcurvesanddetermineauniquePCRproductforeach gene. We followed a method based on Ct (threshold-cycle) to measure the gene expression levels according to the Pfaffl mathematicalmodel(Pfaffl,2001).Theresultswereanalyzedusing the software associated with the instrument. The values were normalizedwiththevaluesofactinincontrasttothevaluesof 1-day-oldsamples.Resultsof6biologicalindependentsampleswereused tocalculatethemeanSEM.
2.4. RNAiandsemiquantitativeRT-PCRanalysis
The Blage-HTH double-stranded RNA fragment(dsRNA) was preparedfollowingthePCR-templatemethodusingMEGAscript RNAi Kit (Ambion, Inc.). The Blage-HTH cDNA of the German cockroachwasfirstclonedintothepGEMTeasyvector(Promega, Madison,WI),thena330basepair(bp)fragmenttargetingtothe wholeORFofBlage-HTHmRNA(Fig.2A)wasamplifiedbyPCR.The primers, which contained theT7 promoter sequence (TAATAC-GACTCACTATAGGGAGACCAC),usedinthisPCRreactionwereas follows:forwardprimer,50-AGCTCCTACATCCCACGTGTT-30,and reverse primer, 50-TGT ACA TGT ACT GTG CAA TGC A-30. The MEGAscriptRNAiKit(Ambion)wasusedtogeneratethedsRNA following themanufacturer’sprotocol.The dsRNAsolution was storedat808Cuntiluse.Adoseof 3
m
gofthe332-bpdsRNA targetingtheBlage-HTHgenewasinjectedintotheabdomenofthe newlyemergedfemaleadults.AsthedsRNAcontrol,weusedthe enhanced green fluorescence proteingene applied of thesame dose.TheBlage-HTHgeneexpressioninthebrain–CC–CAcomplex wasdeterminedbysemiquantativeRT-PCRwithinthefollowing daysafterdsRNAtreatment.ToinvestigatetheeffectofRNAi,Blage-HTHexpressioninthe cockroacheswasdeterminedbysemiquantativeRT-PCRbasedon theORFoftheBlage-HTHgene.Thus,the25
m
lPCRmixturefor amplifying the Blage-HTH fragment included 1m
l of cDNA, 10pmolofforwardprimer(50-CTGCCATTCAACTGGAAGACG A-30),10pmolofreverseprimer(50-AGCATTCCCCACTCATAC ATACAAAATC-30),and5m
lofTaqpolymerasemixture(Protech).ThePCR(30 cycles)wasperformedasfollows:denaturationat 948Cfor30s,annealingat588Cfor30s,extensionat728Cfor 30s. In PCRs for amplifying the actin gene fragment were performed in parallel. The PCR products were separated and visualizedona1.5%agarosegel.
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2.5. Trehalosedetermination
Trehaloseinthehemolymphwasdeterminedwiththemethod ofParrouandFranc¸ois(1997),slightlymodified.Hemolymphwas obtained fromthe hind leg coxa and a 5
m
l volume from two individuals was collected using a pipetman. The hemolymph samplewasplacedinapolyethylenecentrifugetubecontaining 200m
l0.25MNa2CO3buffer,itwasthenvortexedfor1min,and incubatedat968Cfor2htoinactivateallenzymesandtoconvert glucoseintoitsreductiveform.Then,120m
lof1Maceticacidand 480m
l0.25MNa-acetate(pH5.2)wereadded,andthesolution wascentrifugedat 12,000rpm, 248Cfor10min.Anamountof 100m
lofsupernatantwasincubatedovernightat378Cwith2m
l porcine kidney trehalase (Sigma, #T8778) in order to convert trehaloseintoglucose.Theamountofglucosein50m
loftheabove solutionwasmeasuredusingtheGlucoseAssayKit(Sigma, GAGO-20).Glucoseconcentrationwascorrectedbydeductingtheglucose amount of the same supernatant prepared under the same conditionsintheabsenceoftrehalase.2.6. Statisticalanalysis
The results in the graphs represent the means of measur-ementsSEM.Statisticalanalysiswasperformedusingthe comput-ing environment R (The R Foundation for Statistical Computing, 2009).Significancesoftheresultswereevaluatedusingunpaired t-test(Figs.1Aand4B),one-wayANOVA(a=0.05)withFisher’sLSD multiplecomparisonspost-test(Fig.3B),andWilcoxontest(Fig.4C).
Correlation betweenthe hemolymph trehalose andbasal oocytes lengthinvirginfemaleB.germanicawasanalyzedbylinearregression usingbasicregressioncommand,GLM,inR.
3. Results
3.1. Dailyfluctuationofhemolymphtrehalose
FluctuationofthetrehalosetiterinthehemolymphinadultB.
germanicaafterimaginalmoltisshowninFig.1A.Trehalosetiter inthevirginfemaleswasabout4
m
g/m
lafterimaginalmoltand steadilyincreasedalmost2-fold(7.5m
g/m
l)onday8,whenthe oothecawasformed.Whentheunviableoothecaeweredropped onday9,trehalosetiterbecamesimilartothatofindividualsat the early stage of the reproductive cycle. Then, hemolymph trehalosetiterofvirginfemalesincreasedagainwhilethenext reproductive cycle proceeded. Regression analysis showed a positive correlation between hemolymph trehalose and basal oocyte length (Fig.1B) in virgin femaleB. germanica(r=0.98, P<0.0001). Interestingly, a successful mating advanced the decreased response of the trehalose titer in the hemolymph (Fig. 1A). When virgin females were mated on day 5, the hemolymphtrehalosetitershowedsignificantdifferenceswith thatof virgin females onday7 (t-test, P<0.05)and8 (t-test, P<0.001).Moreover,thehemolymphtrehaloseof thefemales carrying an ootheca did not increase, and showed significant differenceswiththatofvirginfemaleswhowereintothenext reproductive cycle on days 17 and 19 (t-test, P<0.001). In contrast,theadultvirginmaleB.germanicadisplayedarelative lowandconstanttrehaloselevelafteremergenceinthefirstdays ofadultlife(Fig.1A).3.2. CloningandcharacterizationofBlage-HTH
Blage-HTH cDNA (GenBank accession no. FJ943774) of B.
germanica was cloned and sequenced following a two-steps PCRstrategy,firstamplifyingafragmentofthespecificsequence with primers based on the conserved motifs of other HTH
sequences,andthenusing50-and30-RACEapproachtocomplete the sequence (Fig. 2A). The Blage-HTH mRNA contained 77 nucleotidesinthe50untranslatedregion(UTR)beforetheopen readingframe(ORF),whichextendsfromthestartcodonATGat position78tothestopcodonTGAatposition296.TheORFwas followedbya30-UTRof189nucleotidesincludingthepotential polyadenylationsignalAATAAA(Beaudoingetal.,2000)foundat position439, and a 20-nucleotide poly (A)tail. The predicted aminoacidsequenceofBlage-HTHpeptidewasQVNFSPGWGT, whichwasidenticaltotheHTHpeptidepreviouslyisolatedfrom the cockroach(Ga¨deandRinehart, 1990).The HTH-precursor-related-peptide (HPRP) for the putativeBlage-HTH C-terminal peptide hadtwo cysteine residues(Fig. 2B) predictedto form disulfidebondsforhomodimerizationduringstorageintheCC (O’SheaandRayne,1992).Inaddition,alignmentamongknown AKHprepropeptidesequencesintheOrderBlattariashowedthat Blage-HTHsharedahighpercentageofidenticalresidueswithB.
discoidalisHTH(72.2%;U35277)andP.americanaAKH-II(57.7%;
AY622321)(Fig.2B).
3.3. ExpressionofBlage-HTH
TheexpressionofBlage-HTHwasstudiedindifferenttissues includingbrain–CC–CAcomplex,ventralnervecord,midgut,fat body,ovary, oviduct,andaccessory gland of 6-and 7-day-old adultvirginfemaleB.germanica.Thebrain–CC–CAcomplexwas the only tissue expressing the Blage-HTH gene (Fig. 3A). The expressionpatternoftheBlage-HTHgeneinvirginfemalesofB.
germanicaafter imaginal molt is shown in Fig. 3B.Expression graduallyincreasedafteremergenceandreachedatapeakonday 8(one-wayANOVA,F66,4.606=6.0588,P=0.01646;LSDposthoc test).Whentheunfertilizedootheca wasformedby thevirgin female, the highest expression level of the Blage-HTH gene immediately dropped to the same level as the beginning of reproductivecycle.
Fig.1.(A)DailychangesofhemolymphtrehaloselevelsinadultsofBlattella germanica.Thecurvewithsolidsquaresrepresentsthehemolymphtrehalose concentrationofvirginfemales(n=10–16)andthesolidarrowindicatesthe timeofovipositionformorethan50%ofthevirginfemales.Thecurvewithopen trianglesrepresentsthehemolymphtrehaloseconcentrationsofmatedfemales whomatedwithsexuallymaturemaleadults(10-day-old)onday5(marked withagraytriangle),andtheopenarrowindicatesthetimeofovipositionfor more than 50%of the matedfemales (n=12). Thecurve withopen circles representsthehemolymphtrehaloseconcentrationofmales(n=10).(B)The growthofbasaloocytelengthinvirginfemalesofBlattellagermanica(n=10).All valuesrepresentthemeanSEM;significantdifferencesofhemolymphtrehalose titerbetweenvirginandmatedfemalesareindicatedbyasterisks(t-test,*P<0.05;
**P<0.001).
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3.4. FunctionalstudyingofBlage-HTH
ThestudyofthefunctionalrolesofBlage-HTHwasapproached withRNAiexperiments.Concerningtranscriptlowering,theless expression of Blage-HTH was observed one day after dsRNA injection,butitbecameundetectablefromday2afterthedsRNA treatmentto,atleast,day12(Fig.4A).
OncetheBlage-HTHexpressionwasknockeddown,trehalose levelsinthehemolymphweremonitored(Fig.4B).Ondays5and6, thehemolymphtrehalosetiterofdsHTH-treatedfemalesremained at the same level as the controls (dsEGFP-treated). On day 7, however,thetiterincreasedincontrolsbutremainedatthesame levelsindsHTH-treatedspecimens;thus,onthatday,hemolymph trehalosetiterwassignificantlylowerindsHTH-treatedspecimen thanincontrols(t-test,P=0.003).Onday8,thelevelsin dsHTH-treatedincreased,andthoseofcontrolstabilized.Thedataindicate that theincreaseof hemolymphtrehaloselevelincreasedmore slowlyindsHTH-treatedthanincontrols.
ThesilencingeffectofBlage-HTHgeneonoothecaproductionis shown in Fig.4C. Mostof thecontrol(dsEGFP-treated)females started to produce an ootheca on day 8, whereas the treated
(dsHTH-treated)femalesdelayedthetimeofoviposition signifi-cantly(Wilcoxontest,P<0.01).Some(10%)ofthedsHTH-treated females did not oviposit until day 15. Furthermore, a few individuals(7.9%)treatedwithdsHTHproducedshortdeformed oothecae(Fig.4D).
4. Discussion
Given that reproductive processes requirehuge amounts of energy,theenergymetabolismmustbeanimportanttraitinthe life history of adultinsects (Lorenz and Ga¨de, 2009). We have monitored thefluctuations of hemolymphtrehalose titersafter imaginalmoltandcharacterizedfunctionsoftheBlage-HTHgene during the reproductive cycle in female B. germanica. In B.
germanica, adult females undergo several gonadal maturation cycles accompanied with vitellogenesis and sexual receptive behaviorsincludingsexpheromonereleaseandrobustlocomotion in contrast to the adult males, which display no such cyclic reproductiveactivities(LeeandWu,1994;Schaletal.,1997).The fluctuationofhemolymphtrehalosebetweenfemaleandmaleB.
germanica also shows this sexual dimorphism (Fig. 1A). The dynamics of hemolymph trehalose in the female B. germanica suggeststhat energydemandincreasesduringthereproductive cycle and that trehalose supplies, at least in part, thisenergy.
Moreover,theparallelrelationshipbetweenhemolymphtrehalose and ovarian development was observed in virginfemales of B.
germanica,whereasmatedfemalesdidnotincreasetheir hemo-lymphtrehaloseduringtheperiodofcarryinganootheca,which inhibits ovarian growth (Schal et al., 1997) (Fig. 1A). During oogenesis,theinsectoocytesaccumulatevariousnutrientsfrom hemolymphsuchascarbohydrates,lipids,andvitellogenin(Vg)in particular (Raikhel and Dhadialla, 1992; Martin et al., 1996;
Raikheletal.,2005).TheVguptakebyoocytescouldbesuppressed byinjectionofthetrehalaseinhibitor,validoxylamineA(VAA),as shown in severalinsect species includingB. germanica (Tanaka etal.,1998;Konoetal.,1999).TheuptakeofVgbythematurating oocytesisconsideredasanenergy-demandingprocessaccording Fig.2.(A)NucleotidesequenceanddeducedaminoacidsequenceforBlattella
germanicaHTH(Blage-HTH)withflanking50-and30-UTRsequences.Theupper diagramshowstheBlage-HTHgeneorganizationwhichconsistsofa77-nucleotide 50-untranslatedregion,219nucleotidesencodingtheHTHpolypeptideprecursor, HTH-precursor-related-peptide(HPRP)a189-nucleotide30-untranslatedsequence, andpolyadenylatedtail.Intheprepropeptidesequence,signalpeptideisinitalics, andBlage-HTHisshowninboldtypewiththeunderlinedglycineresiduerequired foramidationandthepotentialdibasiccleavageresiduesboxed,followedbythe HPRP.*Thestopcodon.Apossiblepolyadenylationsignalisshowninbothboldtype andunderlinedinthenucleotidesequencesat30-UTR.(B)Comparisonofdeduced amino acid sequences on known HTH peptide among different species of cockroaches.Thelevelofsequencesimilarityisindicatedbyblackshadingfor4 identity,grayshadingwithwhiteletterfor3identity,andgrayshadingwithblack letterfor2identity.ThetwoconservedCysresiduesshownbyarrowsarethoughtto beinvolvedininter-moleculardisulfidebondsforhomo-orheterodimerization betweenthesepeptidesduringstorageincells(O’SheaandRayne,1992). Peram-AKH-I: Periplanetaamericana AKH I (P84259), Bladi-HTH: Blaberus discoidalis hypertrehalosemic hormone (U35277), Blage-HTH: Blattella germanica hypertrehalosemichormone(FJ943774),andPeram-AKH-II:P.americanaAKHII (AY622321).
Fig.3.ExpressionofBlage-HTHtranscriptinfemaleadultBlattelagermanica.(A) RT-PCRanalysisofBlage-HTHexpression(BgHTH,232bp)indifferenttissues:brain–
CC–CAcomplex(B–CC),ventralnervecord(VNC),midgut(Mgt),fatbody(FB),ovary (O),oviduct(Ovd),andaccessorygland(Asg)of6-or7-day-oldadultfemales.The actingene(BgActin,409bp)wasusedascontrol.M:100bpDNAmarker;NTC: non-templatecontrol.(B)ExpressionpatternofBlage-HTHmRNAinthebrain–CC–CA complexduringthefirstreproductivecycleoffemaleB.germanica.Quantitative Real-timeRT-PCRanalysisofBlage-HTHexpressionlevelwasnormalizedagainst actingene.ThedatarepresentthemeanSEM(n=6)ofrelativetranscriptionlevels normalizedincomparisonto1-day-oldfemalefortheBlage-HTHgeneinthedifferent ages.Differentlettersonthefigureindicatesignificantdifferences(ANOVA,P<0.05 andfollowedbyLSDposthoctest).
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tothefurtherdemonstrationofreducingVgcontentbyimpairing theactivityoftrehalaseintheovaryofthecockroachP.americana (Konoetal.,2001).Thesteadilyincreaseofhemolymphtrehalose during the reproductive cycle in the female B. germanica can thereforebeassumedasanenergyfuelfortheprocessofoocyte maturation.
The manifest function of HTH is to trigger biosynthesis of trehalose from glycogen of the fat body in theOrder Blattaria (Scarboroughetal.,1984;Ga¨de,1989)andlikelyinresponsetothe increase of hemolymph trehalose in the virgin female of B.
germanica. In order to study thefunction of Blage-HTHas the upstreamregulatoroftrehaloseonreproduction,wesuccessfully
germanica. In order to study thefunction of Blage-HTHas the upstreamregulatoroftrehaloseonreproduction,wesuccessfully