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BioMedCentralPage1of5(pagenumbernotforcitationpurposes)MalariaJournalOpenAccessResearchAnophelespseudowillmoriisthepredominantmalariavectorinMotuoCounty,TibetAutonomousRegionWuSong1,3,PanJia-Yun1,WangXue-Zhong2,ZhouShui-Sen1,ZhangGuo-Qing1,LiuQian3andTangLin-Hua*1Address:1NationalInstituteofParasiticDiseases,ChineseCenterforDiseaseControlandPrevention,Shanghai,PRChina,2YunnanInstituteofParasiticDiseases,Puer,PRChinaand3SchoolofIntegratedTraditionalandWesternMedicine,AnhuiCollegeofChineseTraditionalMedicine,Hefei,PRChinaEmail:WuSong-jswu167@126.
com;PanJia-Yun-jypan15@yahoo.
com.
cn;WangXue-Zhong-xuezhongw@hotmail.
com;ZhouShui-Sen-ccdczss@sh163.
net;ZhangGuo-Qing-ahmuzgq@163.
com;LiuQian-lq254@126.
com;TangLin-Hua*-ipdtlh@sh163.
net*CorrespondingauthorAbstractBackground:MalariaisendemicinLinzhiPrefectureintheTibetAutonomousRegion(TAR),butthevectorformalariatransmissionhadneverbeenidentified.
Methods:AdultAnophelesspp.
werecollectedinMotuoCounty,LinzhiPrefectureontheSino-IndianborderinJulyandAugust,2007.
MultiplexPCRwasadoptedforspeciesidentification,andanestedPCRapproachwasusedtodetectsporozoitesinthesalivaryglandsofthemosquitoes.
Results:3,675mosquitoesoftheAnophelesmaculatusgroupwerecollectedandprocessedforspeciesidentification.
Amongthem,3,602(98.
0%)wereAnophelespseudowillmoriand73(2.
0%)wereAnopheleswillmori.
ThePlasmodiumvivaxSSUrDNAfragmentwasamplifiedintwoof360pooledAn.
pseudowillmorisamples.
Conclusion:ThelocalAn.
maculatusgroupcomprisesthespeciesAn.
pseudowillmoriandAn.
willmori.
AnophelespseudowillmoriisconsideredthesolemalariavectorinMotuoCountyinLinzhiPrefecture.
BackgroundLinzhiPrefectureislocatedinthesouth-easternpartoftheTibetAutonomousRegion(TAR)ofChina.
MotuoCountyinsouthofLinzhiPrefecturehadatotalpopula-tionof10,019in2006andsharesborderswithbothIndiaandMyanmar.
Atotalof2,459malariacaseswerereportedfromLinzhiPrefecturebetween1986and2004.
MostoftheseinfectionswereattributedtoPlasmodiumvivax,and2,441(99.
3%)ofthecasesoriginatedfromMotuoCounty[1].
In2005and2006,theannualmalariaincidencerates(IR)inMotuoCountywere56.
8and69.
4casesper10,000persons,respectively.
Alsoin2005,amalariaoutbreakwasreportedfromBayiTownoutofMotuoCounty,indicatinganincreasingmalariathreat.
Uptothepresentday,MotuoCountyisinaccessiblebycar,andmostvillagescanonlybereachedbyfoot.
AlthoughmalariaappearstobeaconsiderablepublichealthprobleminMotuoCounty,onlyfewstudiesfocusedonthisarea,andthelocalmalariavector(s)Published:16March2009MalariaJournal2009,8:46doi:10.
1186/1475-2875-8-46Received:9October2008Accepted:16March2009Thisarticleisavailablefrom:http://www.
malariajournal.
com/content/8/1/462009Songetal;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
MalariaJournal2009,8:46http://www.
malariajournal.
com/content/8/1/46Page2of5(pagenumbernotforcitationpurposes)remainedelusive.
Thiscanbeattributedbothtothelackoflocalhealthworkers,andtothechallenginggeograph-icalconditions.
Studiesconductedin2006establishedthatAnophelesmac-ulatuss.
l.
isthedominantAnophelestaxoninMotuoCounty(unpublished).
TheAn.
maculatusTheobaldgroupbelongstotheNeocelliaseriesofthesubgenusCelliaandisdistributedthroughouttheOrientalregion[2].
Glo-bally,ninemembersoftheAn.
maculatusgrouphavebeendescribed[3,4].
FiveamongthemoccurinChina.
TheseareAn.
maculatuss.
s.
,An.
willmori,An.
pseudowillmori,AnophelessawadwongporniandAnophelesdravidicus[5,6].
Anophelesmaculatuss.
l.
andrelatedspecieshavelongbeenrecognizedasimportantmalariavectorsinMalaysia[7],Thailand[8]andthePhilippines[9].
Anophelesmaculatuss.
l.
infectedwithPlasmodiumwerealsodetectedinYunnanProvince,south-westChina[10].
Theaimofthepresentstudywastoelucidatethesignifi-canceofAn.
maculatuss.
l.
formalariatransmissioninMotuoCounty,thusprovidingthebasisforthedevelop-mentandimplementationofalocallyadaptedintegratedmalariacontrolstrategy.
MethodsStudysitesandperiodMotuoCountystretchesacrossthelowerreachesoftheBrahmaputraRiverbetween27°36'and29°50'Nlatitudeand93°42'and96°36'Elongitude.
Thealtitudeofthemountainouscountyrangesbetween700and2,100mabovesealevel(mean:1,200m)andtheaverageannualtemperatureis16.
1°C.
Almostallmalaria-endemicvil-lagesarescatteredalongtheBrahmaputraRiver.
Thesevil-lagesaremostlyinhabitedbymembersoftheZang,MenbaandLuobanationalities.
ThestudywasimplementedduringthepeakmalariatransmissionseasonfromJulytoSeptember2007inthreevillages(Figure1)wherethemalariaincidencewashighinpreviousyears,namelyYadong(29°32'N,95°33'E),Dexin(29°33'N,95°30'E)andMadi(29°37'N,95°41'E)neartheSino-Indianborder.
Theinhabitantsofthesevil-lagesliveinhousesandhutsgroupedintohamletsscat-teredacrossthevillageterritory,usually1–3kmapartfromeachotherandonlyaccessiblebyfoot.
SamplecollectionCow-baitedtraps(CBT),human-baitednettraps(HBNT)andCDClighttraps(NewStandardMiniatureLightTraps5126V150mm)weresetuptocollectadultmosquitoesbetween21:00and01:00,orbetween21:00to06:00.
Eachmorning,thetrappedmosquitoeswerecountedandidentifiedaccordingtomorphologicalcriteriausingthekeydevelopedbyLuBL[5].
AccordingtoManguinetal.
[2],themorphologicalidentificationofadultmembersoftheAn.
maculatusgroupiserror-proneduetooverlappingcharacteristics;therefore,thecollectedsamplesweremor-phologicallyclassifiedbyspeciescomplexratherthanspe-cies.
Followingtheclassification,themosquitoeswerekilledbychloroformanddriedonsilica-gel,andsubse-quentlytransportedtothelaboratorywheretheywerestoredat-20°CpendingDNAextraction.
DNAextractionAllAn.
maculatuss.
l.
adultswereindividuallyregistered.
Subsequently,about70%An.
maculatuss.
l.
DNAwasextractedfromthelegsofeachindividualforspeciesiden-tificationbymultiplexPCR,andpooledsampleseachcomprising10thoraxesofthesamespecieswerehomog-enizedtoextractDNAforsporozoitesidentificationasdescribedbyLietal.
[11]andSunetal.
[12].
DNAextrac-tionfollowedtheprotocolpresentedbyCollinsetal.
[13],andthequalityoftheisolatedDNAwasassessedbymeas-uringtheproportionalopticaldensity(OD)at260/280nm.
SpeciesidentificationbymultiplexPCR3,675An.
maculatuss.
l.
specimensunderwentspeciesidentificationbymultiplexPCR.
AprotocolcapableofdistinguishingfivemembersoftheAn.
maculatuss.
l.
wasadaptedfromMaetal.
[6],withprimersdesignedbasedonsequencevariationsinthesecondinternaltranscribedspacer(ITS2)oftheribosomalDNA(rDNA).
Anophelespseudowillmori,An.
maculatuss.
s.
,An.
willmori,An.
dravidi-cusandAn.
sawadwongporniarecharacterizedby119bp,186bp,231bp,327bpand406bpfragments,respec-tively.
Well-characterizedDNAsampleswereusedaspos-itivecontrols.
Theamplifiedproductswerespreadbyelectrophoresisona2%agarosegel,stainedwithethid-iumbromide,andvisualizedusingtheGelImagingSys-ThemapofrelativestudysitesFigure1Themapofrelativestudysites.
MalariaJournal2009,8:46http://www.
malariajournal.
com/content/8/1/46Page3of5(pagenumbernotforcitationpurposes)tem(AlphaImagerHP,USA).
AsubsampleofthePCRproductswasclonedandsequencedforconfirmation.
DetectionofP.
vivaxbynestedPCRTheDNAof3,600An.
pseudowillmori(=360pooledsam-ples)and70An.
willmori(=7pooledsamples)thoraxeswasextractedaccordingtotheCollinsmethod[13].
ForthenestedPCR,thefollowingprimersdescribedbySnou-nouetal.
[14]wereused:thePlasmodiumspecificSSUrDNAprimersrPLU5(5'-CCTGTTGTTGCCT-TAAACTTC-3')andrPLU6(5'-TTAAAATTGTTGCAGT-TAAAACG-3'),andthePlasmodiumvivaxspecies-specificSSUrDNAprimersrVIV1(5'-CGCTTCTAGCTTAATCCA-CATAACTGATAC-3')andrVIV2(5'-ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA-3').
DNAamplificationfol-lowedtheprotocolofTassanakajon[15].
Specifically,thefirstamplificationcyclewasperformedusingtheprimersrPLU5andrPLU6in50μlreactionmixturecontaining1μlDNAtemplate.
Forthesecondcycle,1μlofthefirst-roundproductwasusedastemplatefortheprimersrVIV1andrVIV2.
Pooledsampleswereconsideredpositiveifa121bpfragmentwasobtainedinthesecondPCRcycle.
Allproductswerecloned,sequencedandblastedinNCBIBLASThttp://blast.
ncbi.
nlm.
nih.
gov/Blast.
cgi.
ResultsAnophelinespeciescomplexandspeciesidentificationDuringthestudyperiod,atotalof5,345adultanophelinemosquitoeswerecollectedbyHBNTs,CBTsandCDClighttraps.
Amongthem,An.
maculatuss.
l.
wasthepre-dominanttaxonrepresenting97.
1%(5,190)ofthetotalnumberofcollectedanophelinemosquitoes.
An.
peditae-niatusaccountedfor2.
9%(155).
Atotalof3,675morpho-logically-identifiedAn.
maculatuss.
l.
wererandomlyselectedforspeciesidentificationbymultiplexPCR.
The231bpfragmentcorrespondingtoAn.
willmoriandthe119bpfragmentassociatedwithAn.
pseudowillmoriwereamplified3,602(98.
0%)and73(2.
0%)times,respec-tively(Figure2).
Cloningandsequencingofthe231bpfragmentyieldedthefollowingsequence:5'-CTGCAGGGCACATGAACACCGTTGAACGCATATTGCGCATCGGACGCTTCAACCCGACCGATGCACACATCCTTGAGTGCCTACCAAGTTATCTATTTTCTCCTACCAAACTGACCGTCCCATCCCCGTGATGGGCTGTCGCAGCATGGCGTGCTCGGACCCGCATCTGTCGGGACCGTGGGCGTTGATAGTGAGAGTGCTATTATAACGAATGGGGTTACACTATGGGGC-3'.
Thesequenceofthe119bpfragmentwas:5'-GAACTGCAGGACACATGAACACCGTTGAACGCATATT-GCGCATCGGACGATTCACCCGACCGATGCACACATC-CTTGAGTGCCTACTCAGTTATCTTATATGCCCATACCAGACTAGAC-3'.
TheNCBIBlastrevealedahomologyof98%betweenthe231bpfragmentsandtheAn.
will-mori-specificsequenceAF512552.
1.
Therespectivevalueforthe119bpfragmentandAn.
pseudowillmorisequenceAF512550.
1was97%.
SporozoitedetectionA121bpfragmentwasamplifiedfromtwoofthe360pooledAn.
pseudowillmorisamples(Figure3).
CloningandsequencingoftheproductconfirmedthepresenceoftheP.
vivaxSSUrDNAfragmentwhichwasfoundtobeidenticalwiththepreviouslyreportedsequence:5'-ACTTCCAAGCCGAAGCAAAGAAAGTCCTTAAAAAGAATCATTTTAATTAAAAGAACACATAATAGCAAAATGCGCACAAAGTCGATACGAAGTATCAGTTATGTGGATTAAGCTAGAAGCG-3'(AF145335,nt520–640).
Theamplifica-Anophelesmaculatuss.
l.
speciesidentificationbyPCRFigure2Anophelesmaculatuss.
l.
speciesidentificationbyPCR.
Ethidiumbromide-stainedPCRproductsfromfivespeciesbelongingtoAn.
maculatuss.
l.
aswellaspositivesamplescol-lectedinMotuoCounty,TARafterelectrophoresisonagar-osegel.
LaneM:100bpladder;lane1:An.
pseudowillmori;lane2:An.
maculatuss.
s.
;lane3:An.
willmori;lane4:An.
dravidicus;lane5:An.
sawadwongporni;lanes6–8:Anophelesspp.
col-lectedinMotuoCounty,TAR;lane9:negativecontrol.
SporozoitedetectionbyPCRFigure3SporozoitedetectionbyPCR.
Ethidiumbromide-stainedPCRproductsfromthenestedPCRapproachforsporozoitedetection.
LaneM:100bpladder;lane1:positivecontrol;lanes2–3:pooledsamplesfromMotuoCounty,TAR;lane4:negativecontrol.
MalariaJournal2009,8:46http://www.
malariajournal.
com/content/8/1/46Page4of5(pagenumbernotforcitationpurposes)tionofsevenpooledAn.
willmorisamplesyieldednopos-itiveresults.
DiscussionThisisthefirstinvestigationaimingatidentifyingthemalariavector(s)inMotuoCountyofLinzhiPrefecture,TAR.
Accordingtothepresentresults,theAnophelesspp.
populationinMotuoCountyalmostexclusivelyconsistsofAn.
maculatuss.
l.
withAnophelespeditaeniatusaccount-ingfortheremainder.
Apreviousstudy[16]reportedthatAnophelessinensis,oneofthemajormalariavectorsincen-tralChina,alsooccurredintheTAR.
However,thepresentstudyfailedtoconsolidatethisclaim.
Althoughanexper-imentalstudy[17]hadconfirmedthatthesporozoitesofPlasmodiumcynomolgicoulddevelopinAn.
peditaeniatusandthatthismosquitocouldalsotransmitthemtomon-keys,therecurrentlyisnodirectevidenceforthetransmis-sionofhumanmalariabythisspecies.
Thus,An.
maculatuss.
l.
canberegardedasthesolelocalmalariavector.
ThestudyfurthershowedthattheAn.
maculatusgroupinMotuoCountycomprisesthetwospeciesAn.
willmoriandAn.
pseudowillmori.
AnopheleswillmoriisoneoftheprimarymalariavectorsinNepal[18]andhaspreviouslybeenreportedtooccurintheTAR[5].
However,itslocalrele-vanceformalariatransmissionmightbelimitedregardingitssmallerabundancecomparedtoAn.
pseudowillmori.
ThelatterhasbeenestablishedasamalariavectorinnorthwestThailandalongtheborderwithMyanmar[19,20]buthasnotbeenrecordedintheTARbefore.
Dongetal.
[21],however,reportedaclearrelationshipbetweentheseasonalabundancevariationofAn.
pseu-dowillmoriandmalariaIRinamountainousareaofYun-nanProvinceinthesouthoftheTAR.
ThedetectionofsporozoitesinthesalivaryglandsofAnophelesspp.
conclusivelyidentifiesthemasmalariavec-tors.
Traditionally,sporozoiteinfectionratesweredeter-minedbydissectionandexaminationofsalivaryglandsofindividualmosquitoesunderalightmicroscope,atimeconsumingandlaborintensiveapproach.
Therefore,alter-nativesporozoitedetectionmethodshadbeendevelopedinrecentyears:enzyme-linkedimmunosorbentassay(ELISA)[7,9,22-24]detectingthecircumsporozoitepro-tein(CSP),anddifferentapproachesbasedonthePCRtechnique[25,26]aimingatamplifyingspecificDNAsequences.
ItappearsthatthedetectionofPlasmodiumfal-ciparumandP.
vivaxspecificDNAsequencesinmosqui-toesbyPCRhasahighersensitivitycomparedtoELISAtests[27].
Inthisstudy,anestedPCRapproachaimingattheidenti-ficationoftheSSUrDNAofP.
vivaxsporozoiteshadbeenselectedtoidentifymalariavectors.
AconventionalPCRtestcandetectasfewas10sporozoitespersalivarygland,makingitausefultoolforscreeningsmallnumbersofAnophelesspp.
[15].
ThesensitivityoftheemployednestedPCRtestisaslowasthreesporozoites[11],andthepool-ingofsamplesallowsscreeningoflargesamples[28].
Inourstudy,poolingsamples[11,12]andscreeningthembynestedPCR[14,15]resultedintheidentificationofP.
vivaxSSUrDNAinsalivaryglandsoftwopooledAn.
pseu-dowillmorisamples,thusestablishingthatAn.
pseudowill-moriisthemainmalariavectorinthisarea.
ConclusionThecompositionofthelocalanophelinepopulationinMotuoCountyattheSino-IndianborderconsistsmostlyofmembersoftheAn.
maculatusgroup,withAn.
pseudowi-llmoribeingmuchmoreabundantthanAn.
willmori.
Anophelespseudowillmorihasbeenidentifiedasthelocalmalariavector.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsWSperformedfieldandlaboratoryworkandwrotethemanuscript.
PJY,WXZandZSSperformedthefieldwork.
ZGQ,LQperformedlaboratorywork.
TLHwasinvolvedinallstagesofthisstudy.
AcknowledgementsTheauthorsthankProf.
MaYa-JunfromtheSecondMilitaryMedicalUni-versityofChinainShanghaiforprovidingconfirmedDNAsamplesoffivespeciesoftheAn.
maculatusgroup.
ThesmoothcollaborationwiththestaffoftheLinzhiPrefectureCDCandtheMotuoCountyCDCisacknowl-edged,andwearegratefultoZhuomaYang-jin,HuYong-Hong,JiangWei-Kang,HuSong-Lin,XuHui-Mei,CirenQuzhandZhangWeifortheirassist-anceinthefield.
WethankPeterSteinmannfromSwissTropicalInstituteforassistancewiththeEnglish.
References1.
LuoS,HuYH,HuSL,ZhenN,LiCC:[Anepidemiologicalanaly-sisonmalariainLinzhiDistrictofTibetin1986–2004].
ChinJParasitolDis2005,12:457-459.
2.
ManguinS,GarrosC,DusfourI,HarbachRE,CoosemansM:Bio-nomics,taxonomy,anddistributionofthemajormalariavectortaxaofAnophelessubgenusCelliainSoutheastAsia:Anupdatedreview.
InfectGenetEvol2007,8:489-503.
3.
HarbachRE:TheclassificationofgenusAnopheles(Diptera:Culicidae):aworkinghypothesisofphylogeneticrelation-ships.
BullEntomolRes2004,94:537-553.
4.
WaltonC,SomboonP,O'LoughlinSM,ZhangS,HarbachRE,LintonYM,ChenB,NolanK,DuongS,FongMY,VythilingumI,MohammedZD,TrungHD,ButlinRK:Geneticdiversityandmoleculariden-tificationofmosquitospeciesintheAnophelesmaculatusgroupusingtheITS2regionofrDNA.
InfectGenetEvol2007,7:93-102.
5.
LUBL:FaunaSinaca,Insecta,Diptera:CulicidaeIIVolume9.
Beijingchina,SciencePress;1997.
6.
MaYJ,LiSZ,XuJ:MolecularidentificationandphylogenyoftheMaculatusgroupofAnophelesmosquitoes(Diptera:Culicidae)basedonnuclearandmitochondrialDNAsequences.
ActaTrop2006,99:272-280.
7.
ReidJA:AnophelinemosquitoesofMalayaandBorneo.
StudInstMedResMalaysia1968,31:1-520.
PublishwithBioMedCentralandeveryscientistcanreadyourworkfreeofcharge"BioMedCentralwillbethemostsignificantdevelopmentfordisseminatingtheresultsofbiomedicalresearchinourlifetime.
"SirPaulNurse,CancerResearchUKYourresearchpaperswillbe:availablefreeofchargetotheentirebiomedicalcommunitypeerreviewedandpublishedimmediatelyuponacceptancecitedinPubMedandarchivedonPubMedCentralyours—youkeepthecopyrightSubmityourmanuscripthere:http://www.
biomedcentral.
com/info/publishing_adv.
aspBioMedcentralMalariaJournal2009,8:46http://www.
malariajournal.
com/content/8/1/46Page5of5(pagenumbernotforcitationpurposes)8.
RattanarithikulR,KonishiE,LinthicumKJ:DetectionofPlasmo-diumvivaxandPlasmodiumfalciparumcircumsporozoiteanti-geninanophelinemosquitoescollectedinsouthernThailand.
AmJTropMedHyg1996,54(2):114-121.
9.
TorresEP,FoleyDH,SaulA:RibosomalDNAsequencemarkersdifferentiatetwospeciesoftheAnophelesmaculatus(Dip-tera:Culicidae)complexinthePhilippines.
JMedEntomol2000,37:933-937.
10.
ZhouHN,ZhangZX,CurtisC,HillN,LiCF,ChaoW:[Evaluationoftheenzyme-linkedimmunosorbantassayindetectingcir-cumsporozoiteproteinofanophelinevectorsinYunnan].
ChinJParasitolParasitDis2004,22:227-230.
11.
LiFW,NiuC,YeBH:NestedpolymerasechainreactionindetectionofPlasmodiumvivaxsporozoitesinmosquitoes.
ChineseMedicalJournal2001,114:654-657.
12.
SunQW,ZhuHM,LuL,GuZC,ChengX,FangY:[Restudyonamodelforestimatingthesporozoiteinfectionrateinmos-quitoesusingpooledsampling].
ChinJParasitolParasitDis2002,20:351-353.
13.
CollinsFH,MendezMA,RasmussenMO,MehaffeyPC,BesanskyNJ,FinnertyV:AribosomalRNAgeneprobedifferentiatesmem-berspeciesoftheAnophelesgambiaecomplex.
AmJTropMedHyg1987,37(1):37-41.
14.
SnounouG,PinheiroL,GoncalvesA,FonsecaL,DiasF,BrownKN,doRosarioVE:Theimportanceofsensitivedetectionofmalariaparasitesinthehumanandinsecthostsinepidemi-ologicalstudies,asshownbytheanalysisoffieldsamplesfromGuineaBissau.
TransRSocTropMedHyg1993,87:649-653.
15.
TassanakajonA,BoonsaengV,WilairatP,PanyimS:PolymerasechainreactiondetectionofPlasmodiumfalciparuminmosqui-toes.
TransRSocTropMedHyg1993,87:273-275.
16.
EditorialCommitteeofmalariacontrolandresearchinChina:MalariacontrolandresearchinChinaBeijing,People'sMedicalPublishingHouse;1991.
17.
CollinsWE,WarrenM,GallandGG:StudiesoninfectionswiththeBerokstrainofPlasmodiumcynomolgiinmonkeysandmosquitoes.
JParasitol1999,85:268-272.
18.
PradhanSP,ShresthaSL,VaidyaRG:MalariatransmissioninhighmountainvalleysofwestNepalincludingthefirstrecordofAnopheleswillmori(James)asathirdvectorofmalaria.
NepalMedAssoc1970,8:89-97.
19.
GreenCA,RattanarithikulR,PongparitS,SawadwongpornP,BaimaiV:Anewly-recognizedvectorofhumanmalarialparasitesintheOrientalregion,Anopheles(Cellia)pseudowillmori(Theobald,1910).
TransRSocTropMedHyg1991,85:35-36.
20.
GreenCA,RattanarithikulR,CharoensubA:PopulationgeneticconfirmationofspeciesstatusofthemalariavectorsAnoph-eleswillmoriandAn.
pseudowillmoriinThailandandchromo-somephylogenyoftheMaculatusgroupofmosquitoes.
MedVetEntomol1992,6:335-341.
21.
DongXS,ZhouHL,BiY,DongLM:[TherelationshipbetweenAnophelesmaculatusgeographicdistribution,ecologicalfeaturesandmalariainYunnanprovince].
ActaParasitolMedEntomolSin1996,3:100-105.
22.
TrungHD,VanBortelW,SochanthaT,KeokenchanhK,QuangNT,CongLD,CoosemansM:MalariatransmissionandmajormalariavectorsindifferentgeographicalareasofSoutheastAsia.
TropMedIntHealth2004,9:230-237.
23.
BenetA,MaiA,BockarieF,LagogM,ZimmermanP,AlpersMP,ReederJC,BockarieMJ:PolymerasechainreactiondiagnosisandthechangingpatternofvectorecologyandmalariatransmissiondynamicsinPapuaNewGuinea.
TheAmericanjournaloftropicalmedicineandhygiene2004,71(3):277-284.
24.
CuambaN,ChoiKS,TownsonH:MalariavectorsinAngola:dis-tributionofspeciesandmolecularformsoftheAnophelesgambiaecomplex,theirpyrethroidinsecticideknockdownresistance(kdr)statusandPlasmodiumfalciparumsporozoiterates.
MalarJ2006,5:2.
25.
StoffelsJA,DoctersvanLeeuwenWM,PostRJ:DetectionofPlas-modiumsporozoitesinmosquitoesbypolymerasechainreactionandoligonucleotiderDNAprobe,withoutdissec-tionofthesalivaryglands.
MedVetEntomol1995,9:433-437.
26.
MorenoM,CanoJ,NzamboS,BobuakasiL,BuaticheJN,OndoM,MichaF,BenitoA:MalariaPanelAssayversusPCR:detectionofnaturallyinfectedAnophelesmelasinacoastalvillageofEquatorialGuinea.
MalarJ2004,3:20.
27.
VythilingamI,NitiavathyK,YiP,BakoteeB,HugoB,SinghB,WirtzRA,PalmerK:Ahighlysensitive,nestedpolymerasechainreactionbasedmethodusingsimplednaextractiontodetectmalariasporozoitesinmosquitos.
SoutheastAsianJTropMedPublicHealth1999,30(4):631-635.
28.
ZhouHY,ZhuGD,JinXL,LiJL,WangWM,CaoJ,GuYP,GaoQ:[DetectionofPlasmodiumsporozoitesinmosquitoesbynested-PCR].
ChinJSchistoControl2006,18:365-368.