Ethicalgao41.com

RESEARCHOpenAccessSusceptibilityofAnophelessinensistoPlasmodiumvivaxinmalarialoutbreakareasofcentralChinaGuodingZhu1,2,3,HuiXia4,HuayunZhou1,2,JulinLi1,2,FengLu1,2,YaobaoLiu1,2,JunCao1,2,QiGao1,2,3*andJetsumonSattabongkot5*AbstractBackground:Anophelessinensis,Anophelesanthropophagus,AnophelesminimusandAnophelesdirusarethemajorvectorsofmalariatransmissioninChina.
Anophelessinensisisconsideredasecondaryvectorduetoitsrelativelylowmalaria-transmissionability.
However,in2005,anoutbreakofover40,000PlasmodiumvivaxmalariacaseswasreportedinareaswhereAnophelessinensiswastheonlymajorvector.
Therefore,itisnecessarytoreassessthemalariatransmissionabilityofthisvectorspeciesinChina.
Methods:LaboratorycoloniesofAn.
sinensisandAn.
anthropophagus,andfirst-generationprogeny(F1)ofAn.
sinensisthathadbeencollectedincentralChina,wereinfectedbydirectmembranefeedingassaywithmono-vivaxgametocyte-containingbloodcollectedfromvivax-infectedpatients.
Themosquitoeswerekeptfor7to14dayspost-bloodfeedingtoallowparasitestodevelopintooocystsandsporozoites.
Infectivitywasmeasuredbydissectingmidgutsandsalivaryglands.
Thepresenceofoocystsandsporozoiteswasdeterminedbymicroscopyat7and14dayspost-bloodfeeding,andthenumbersofgametocytesandasexualparasites,aswellasmosquitoparasiteinfections,weredetermined.
Results:Thepositiveoocystandsporozoitefeedratesofthe142pairsoflab-colonyAn.
sinensisandAn.
anthropophaguswerenotsignificantlydifferent,andthesameresultswerefoundwiththe10pairsoflaboratoryandF1An.
sinensis.
An.
sinensishadmoreoocysts/midgutat7dayspost-feedingthanAn.
anthropophagus,butthegametocytemia,asexualparasitemia,andratioofmacrogametocytestomicrogametocytes,didnotcorrelatewitheitheroocystorsporozoiteinfection.
However,intheoocyst-positivemosquitoes,therewasacorrelationbetweengametocytemiaandtheaverageoocystnumber/midgut.
Conclusions:ThesusceptibilityofAn.
sinensis(bothlaboratoryandF1)toP.
vivax-infectedbloodissimilartoAnophelesanthropophagus,whenevaluatedbymembranefeedingassayunderlaboratoryconditions.
Inrecentyears,incentralChina,thevivaxmalariatransmissionabilityofAn.
sinensishasprobablybeenunderestimated.
Furtherstudiesofthisspeciesinotherregionsareneeded.
An.
sinensiscouldalsobeagoodcandidatevectorforevaluatingcandidatemalariatransmission-blockingvaccines(TBV).
Keywords:Membranebloodfeeding,Plasmodiumvivax,Anophelessinensis,Anophelesanthropophagus*Correspondence:gaoqi54@hotmail.
com;jetsumon@hotmail.
com1KeyLaboratoryofParasiticDiseaseControlandPrevention,MinistryofHealth,JiangsuInstituteofParasiticDiseases,Wuxi,JiangsuProvince,China5MahidolVivaxResearchUnit,FacultyofTropicalMedicine,MahidolUniversity,Bangkok,ThailandFulllistofauthorinformationisavailableattheendofthearticle2013Zhuetal.
;licenseeBioMedCentralLtd.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense(http://creativecommons.
org/licenses/by/2.
0),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
Zhuetal.
Parasites&Vectors2013,6:176http://www.
parasitesandvectors.
com/content/6/1/176BackgroundAnophelessinensis,An.
anthropophagus,An.
minimus,andAn.
dirusarethemajorvectorsofmalariatransmissioninChina[1],andspeciesintheAn.
maculatuscomplexmaybemajortransmissionvectorsinTibetAutonomousRegion[2].
Amongthesespecies,An.
minimusandAn.
dirusaremostlydistributedinsouthernChina(YunnanandHainanprovinces),wherethegeograph-icalenvironmentismarkedlydifferentfromthecentralregion.
An.
sinensisandAn.
anthropophagusarerela-tivelymorewidelydistributedinChina.
AccordingtotheupdateddistributionrecordsoftheAn.
hyrcanusspeciesgroup(Diptera:Culicidae),An.
sinensisisfoundinover29provincesandregionsinChina[3].
Itisnote-worthythatAn.
sinensishasbecometheonlymajorvectorincentralChina,wherePlasmodiumvivaxistheonlyprevalent,locallytransmitted,malariaparasite;however,afewimportedfalciparummalariacaseshavebeenreportedamongtravellers[4].
An.
sinensisandAn.
anthropophagusarebothmem-bersoftheAn.
hyrcanuscomplex,sharingsimilarmor-phologicalcharacteristics,andaribosomalDNA-internaltranscribedspacer2(rDNA-ITS2)-basedmethodisre-quiredtodistinguishthetwospecies[5,6].
However,inadditiontothedistinctdistributionsofAn.
sinensisandanthropophagus,thespeciesdifferstronglyinhostpref-erence,restinghabitat,andotherfeaturesinvolvedinmalariatransmission.
First,An.
anthropophaguspreferstobitehumansratherthananimals,whereasAn.
sinensisisamorezoophilicmosquitoanddemonstratesamarkedpreferenceforcattleandotherwarm-bloodedanimals.
Sec-ond,An.
anthropophagusprefersindoorrestingafterbloodfeeding;residualinsecticidesprayinginareasofcentralChinawhereAn.
anthropophaguspredominatedasthemajorvectorforfalciparumeffectivelyreducedmalariamortalityandmorbidity,from1980to1990.
Asaresult,falciparummalariahasbeeneliminatedincentralChina.
However,An.
sinensistendstowardoutdoorrestingafterindoorbloodfeeding,whichhasmadevectorcontrolofthisspeciesmoredifficult.
Third,An.
anthropophagusismuchmoresusceptibletovivaxmalariaparasites[7].
TheregionsinChinacontainingbothAn.
anthropophagusandAn.
sinensishavesufferedmoreseriousmalariaepidemicsthanthoseareaswhereAn.
sinensisistheonlyvector[8].
Takingtheabove-mentionedfactorsintoconsider-ation,onepossibleconclusionisthatAn.
sinensisplaysalessimportantroleinmalariatransmissionthanotherspeciesincentralChina.
Unexpectedly,frequentout-breaksofvivaxmalariastartedappearinginareaswhereAn.
sinensiswasthemainvector,withover40,000reportedvivaxcasesin2005,accountingfor67%ofallcasesinChina[9];thissuggeststhatthesusceptibilityandotherfeaturesofAn.
sinensisthataffectitsinteractionwithvivaxparasiteshavechanged.
Thus,thecomparativemalaria-transmissionabilityofAn.
sinensiswithothermajorvectorsshouldbereassessed.
Inthisstudy,weassessedthesusceptibilityofAn.
sinensistoP.
vivaxincentralChinabymembrane-feedingassayandcom-paredtheresultswithAn.
anthropophagusandafieldstrainofAn.
sinensis.
ThisstudywillhelptoexplainthevivaxepidemicsituationincentralChinabetter,andim-provethecurrenteliminationprogrammesofthisspeciesinChina.
MethodsStudysiteandpatientsThestudywasconductedinBengbu,AnhuiProvince,centralChina(Figure1).
P.
vivaxistheonlymalariaparasiteinthisregion.
In2004,thetotalnumberofmal-ariacasesinAnhuireached8,909,whichwas22.
9%ofallcasesinChina(Figure2).
Patientsaged18yearsofageormore,whosoughtclinicaltreatmentformalaria,wereincludedinthisstudy.
Thickandthinbloodsmearswerepreparedfromeachindividualandstainedwith10%GiemsabyexperiencedmicroscopiststoexcludemixedinfectionwithP.
falciparum.
Inaddition,gameto-cyteandasexualparasitedensitiesweredeterminedforallP.
vivax-positivepatientsbycountingthenumberofparasitesper500leukocytesinathickbloodsmearunderoilimmersionmicroscopy.
Therawcountswereconvertedintoparasites/microliterbyassumingacountof8,000leukocytes/μL.
Ifgametocyteswerepresent,thepatientwasaskedtoenrolinthestudy.
Afterthepa-tientswerebriefedontheprojectandcompletedcon-sentforms,approximately5mLofbloodwascollectedbyvenepunctureandusedformembranebloodfeeding(detailedbelow)ofstarvedmosquitoes.
Aftermosquitofeeding,thevolunteerswerereleasedfromthestudyandreceivedantimalarialtreatment.
MosquitoesAn.
sinensisandAn.
anthropophagushavebeenmaintainedintheinsectaryoftheKeyTechnicalLaboratoryforPreventionandControlofParasiticDiseasesoftheMinistryofHealth(MOH)intheJiangsuInstituteofParasiticDiseases(JIPD),Wuxi,China,forover30years.
Themosquitoeswererearedat27±1°Cwitharela-tivehumidityof70–80%andprovidedwith10%(w/v)glucoseinwater.
OnehundredmosquitoesineachcarryingcageweretransportedfromJIPDtothefieldsitesinacoolerbox.
Next,6-to8-day-oldmosquitoeswereprovidedonlywaterfor12hourspriortobloodfeeding.
Inthisstudy,engorgedfemaleanophelinemosqui-toesfromBengbu,Anhui,werealsocollected,andtheirprogeny(F1)wereidentifiedviabothmorphologicalchar-acteristicsandanrDNAITS2-basedmethod[5]toconfirmspecies.
Themosquitoesweremaintainedasdescribedabove,priortobloodfeeding.
Zhuetal.
Parasites&Vectors2013,6:176Page2of9http://www.
parasitesandvectors.
com/content/6/1/176MembranefeedingFivehundredmicroliters(500μL)ofwholebloodfromeachpatientwerecentrifugedat5,000gfor5minutes.
Theserumwasthenremovedandreplacedwithap-proximately300μLofABserumfromaP.
vivax-naivedonor.
Thepackedredbloodcellsanddonorserawerecarefullymixedandaddedtothemembranefeeder.
Aconstant-temperature(37°C)circulating-watersystemwasusedtopreventexflagellationofmicroga-metocytes[10].
Thebloodfeedinglastedfor30minutes,afterwhichtheglassmembranefeederwasremovedfromthetopofthecartonandalltheunengorgedmosquitoeswereremovedandfreeze-killed.
Afterfeed-ing,allengorgedmosquitoesweretransportedbacktoJIPD'sinsectaryinWuxi,wheretheywereprovidedwitha12-hlight/darkcycleanddailysugarsolutionbe-foredissection.
MosquitodissectionOnday7post-bloodfeeding,themosquitoeswereaspiratedintoglasstubesandimmobilizedbypla-cingthetubeonice.
Atleast10midgutsofeachspeciesweredissectedinadropofmercurochromeinphosphate-bufferedsaline,andthenumberofoo-cystspermidgutwascountedunder10*or40*microscopicexamination.
Onday14post-feeding,ifthemosquitoeswereoocyst-positive,another10mos-quitoesofeachspeciesweredissected,andthenumberofoocystspermidgutwasfirstcounted,asabove.
Then,theinfectingsporozoitelevelwasrecordedafterdirectdeterminationunderphasecontrastmi-croscopy(LeicaDM2500,US)withoutstaining.
Thelevelwasrecordedasfollows:"+",1–10sporozoites;"++",11–100sporozoites;101–500sporozoitesand500sporozoites.
Figure1Studyareaforvivaxmalariapatientrecruitmentfrom2005to2007.
Zhuetal.
Parasites&Vectors2013,6:176Page3of9http://www.
parasitesandvectors.
com/content/6/1/176StatisticalanalysisThechi-squaretestwasusedtocomparetheproportionofmosquitoesinfectedwithoocysts,theproportionofmosquitoesinfectedwithsporozoites,andthepropor-tionofinfectedmosquitoesperpositivefeeding,betweenpairedlab-colonyAn.
anthropophagusandAn.
sinensis,andbetweenpairedF1andlab-colonyAn.
sinensis.
PairedTtestswereusedtocompareoocystloads(meanoocystnumberperinfectedmidgut)betweenthefeedinggroups.
Aregressiontestwasusedtodetectanylinearcorrelationbetweenparasiteloadandinfectionrate.
EthicalapprovalAllhuman-subjectresearchconductedinthisstudywasreviewedandapprovedbytheInstitutionalEthicsCommit-teeoftheNationalInstituteforParasiticDiseases(NIPD),ChineseCenterforDiseaseControlandPrevention.
Allthepatientsinvolvedinthisstudyreadandsignedthein-formedconsentforms.
ResultsPatientdataFrom2005to2007,over200symptomaticmalariapa-tientscametotheclinicinBengbu,Anhui.
Intotal,142volunteerswerefinallyenrolledinthisstudyafterex-cludingsubjectsagedlessthan18yearsandthosewithmixedinfectionswithfalciparummalaria,orzerogame-tocytesbythickblood-smearcount.
PatientageandparasitedensitydataareshowninTable1.
MembranefeedingIntotal,thebloodof142vivaxpatientswasfedviamem-branefeedingtothelaboratorycoloniesofAn.
sinensisandAn.
anthropophagus.
Amongthese142patients,bloodfrom10patientswasalsofedtolab-colonyandF1An.
sinensismosquitoes.
TheengorgedfeedingpercentagesofthepairedlaboratorystrainAn.
sinensisandAn.
anthro-pophaguswere64.
86%(9210/14200)and62.
86%(8926/14200),respectively.
TheF1An.
sinensishadthelowestengorgedfeedingrate,at16.
5%(165/1000)(χ2=934.
05,p0.
05,χ2=3.
22,P>0.
05,respectively).
Likewise,thepositivesporozo-itefeedrateandpositivemosquitoinfectionrateatday14post-bloodfeedingdidnotdiffer(χ2=0.
09,P>0.
05,χ2=0.
21,P>0.
05,respectively)(Table2).
In10pairedcases,boththeF1andlaboratorystrainhadthesameposi-tiveoocystfeed(80%)andsporozoitefeed(30%)rates.
Inthe10pairedmembranefeedingtests,thelab-strainAn.
sinensishadahigheroocystinfectionrateatday7thanF1(Figure4),asdidthelaboratorystrainAn.
sinensisinthe142pairedfeedingswithla-boratorystrainAn.
anthropophagus(Figure4).
However,Table1Ageandparasitedensityofthestudypatients(n=142)MinimumMaximumAverageAgeofpatients(years)187134.
6±3.
1No.
ofasexualparasites(/μL)0216964332.
8±371.
2No.
ofmacrogametocytes(/μL)3265601057.
6±88No.
ofmicrogametocytes(/μL)644800486.
4±51.
2YearMalariacases0100002000030000400005000060000700002001200220032004200520062007200820092010TotalCentralChinaAnhuiFigure2MalariatransmissioninChinafrom2003to2010.
Zhuetal.
Parasites&Vectors2013,6:176Page4of9http://www.
parasitesandvectors.
com/content/6/1/176interestingly,thetwolaboratorystrainshadsimilarsporo-zoitelevelsatday14post-feeding(z=0.
866,p=0.
38,Table3).
CorrelationofparasitemiaandinfectionIntotal,41/103and38/96infectedlab-colonyAn.
sinensisandAn.
anthropophagus,respectively,developedspo-rozoitesinthesalivaryglandsat14days'post-bloodfeeding.
Theother62and58respectivecasesonlyhadoocystsinthemidgutatday7post-feeding,and32of142caseswerenegativeforbothoocystandsporozoiteinfectioninbothAn.
sinensisandAn.
an-thropophagus(Table4).
Theeffectsofparasitedens-ity(macrogametocyte,microgametocyte,asexual-stageparasite)andratioofmacrogametocytestomicroga-metocytes,inthefivegroupsreferredtoabove,wereevaluated.
Theparasitedensityortheratioofmacro-gametocytetomicrogametocytehadnoeffectonpara-siteinfectionasthedatashowedthattherewasnosignificantdifferencebetweenthenegativeandpositivecases.
However,incasesofpositiveinfection,regres-sionanalysisrevealedasignificantlinearcorrelationbetweenbloodgametocytedensityandmidgutparasiteinfectionloadinbothAn.
sinensisandAn.
anthropopha-gus.
Thecaseswithmoreoocystsorsporozoiteshadhighergametocytemialevels,particularlyinthesporozoite-positivecases(Figure5).
DiscussionThisisthefirststudytoevaluatethesusceptibilityofAn.
sinensistovivaxparasitesincentralChinabymem-branefeeding,afterthere-emergenceofmalariaincen-tralChina.
Inthisstudy,An.
sinensis(bothlaboratorycolonyandF1)wereequallysusceptibletovivaxmalariaparasitesasAn.
anthropophagus,whichwasbelievedformanyyearstobethemajorvectorincentralChina.
Des-pitethatbelief,thelaboratorycolonyofAn.
sinensishadahigheroocystinfectionrate.
AlthoughthesamewasnotfoundintheF1mosquitoes,theirlowobservedvivaxsusceptibilitycouldhavebeenduetotheirlowengorgedfeedingrate,causedbytheswitchofemer-gentenvironmentfromfieldtolaboratory.
Inaddition,thedifficultyofmaintainingtheengorgedmosquitoesunderlaboratoryconditionscannotbeignored[11],asthisdefinitelyreducedthequantityofmosquitoesdis-sectedatday7post-feeding.
Furthermore,inthe10pairedcases,bothlaboratoryandfieldAn.
sinensismosquitoespresentedthesameinfectionrateand100%concordancewiththepositivecaseselection(bothpositiveoocystandsporozoitefeedings),suggestingthelab-colonyAn.
sinensisinthisstudycouldwellrepresentactualcurrentvectorsus-ceptibilitytoparasitesinthefield.
Inthiscase,An.
sinensiswasmoreabletocarryP.
vivaxinthemidgutstagethanAn.
anthropophagus,givenanequalopportunitytofeedonmalariapatients.
ThiscontrastswithpreviousresultsinFigure3Oocystandsporozoiteinfectioninmidgutandsalivaryglands.
(A)Oocystsinthemidgutwerecountedusinganormalmicroscopeat10*objectivemagnificationwithmercurochromestaining.
(B)Sporozoitesinthesalivaryglandswereassessedusing40*objectivemagnificationandaphase-contrastmicroscopewithoutstaining.
Table2Comparisonofbloodfeeding,andoocystandsporozoiteinfection,forAn.
sinensisandAn.
anthropophagusSpecies%offeedsinfectingmosquitoes%ofmosquitoesthatfedonallinfectiouspatientsthatdevelopedparasiteinfectionMeannumberofoocystsperpositivemosquito(Positivefeeds/totalfeeds)(Positivemosquitoes/totalmosquitoes)Dayspost-feeding7147147An.
sinensis(Lab)72.
5(103/142)28.
9(41/142)45.
7(15536/340)11.
1(135/1216)45.
7(15536/340)An.
sinensis(F1)80.
0(8/10)30.
0(3/10)13.
4(281/21)20.
0(9/45)13.
4(281/21)An.
anthropophagus(Lab)67.
6(96/142)26.
8(38/142)21.
0(6437/306)11.
84(96/811)21.
0(6437/306)Zhuetal.
Parasites&Vectors2013,6:176Page5of9http://www.
parasitesandvectors.
com/content/6/1/176centralChina[12].
Indeed,onlymosquitoeswithsporozo-itesinthesalivaryglandscaninfecthumans,althoughbothAn.
sinensisandAn.
anthropophagushadsimilarlylowsporozoiteinfectionratesinthisstudy,whichshouldraisesomesuspicion[13].
However,itisreasonabletonotethatmalariaparasitesalsoreducemosquitosurvivalrates[14].
Inaddition,ourobjectivewastoevaluatethesusceptibilityofAn.
sinensiscomparedwithAn.
anthropophagusandnottocountsporozoitequantitieslong-term.
Bothmosquitospecieshadmoresporozoitesafter14days'post-feedingthanat14days.
Thus,oocystinfectioninthemidgutstagecouldreflectpotentialtransmissioncapacity.
Awell-knownpreferenceforhumanbiting,atendencytorestindoors,andgreatsusceptibilitytoparasiteswithsufficientlongevity,areessentialcriteriaforevaluatingvectorsformalariatransmissioncapacity.
InChina,al-thoughAn.
sinensisisthemostwidelydistributed,withalargepopulationinmostmainlandregions,thespecieshadfordecadesbeenjudgednottobethepredominantvectorformalariaduetoitsexophilicandexophagicfeatures,andrelativelylowsusceptibilitytoparasitescomparedwithothervectors.
Nevertheless,thevivaxmalariaoutbreakin2005incentralChina,inwhichAn.
sinensisservedasthemainvector,suggestedanupdatedevaluationofthevectorcapacityandtrans-missionroleofthisspecieswasnecessary[15].
AlongwithagriculturalandindustrialprogressinChina,fre-quentlymovingpopulationshavebecomeanimportantgroupatriskofcarryingparasitesfrommalaria-epidemicareastomalaria-freeorlow-transmissionregions.
Duringthemalariatransmissionseason,fromJunetoSeptember,farmersandconstructionworkershabituallysleepintheopenwithoutnetprotection,whichincreasesthechanceforAn.
sinensis,whichinthisstudyhadastrongpropen-sitytodevelopvivaxmalariaparasitesfollowingblood-feedingfrominfectedhumans,tobiteseveraldifferentpeople.
DuetotheexophilicnatureofAn.
sinensisandcontinuouslyincreasinginsecticideresistance[16-21],theregularinsecticideresidualspray(IRS)methodsusedinmalaria-transmissionregionsdonotkillallmosquitoes[22].
AnotherpossiblereasonforthemalariaoutbreakincentralChinaisclimaticandenvironmentalchange[10].
IfAn.
sinensismosquitoesareunabletofindtheirusualani-malbloodfeedingtargets,becauseoftheconstructionofbuildingsorothersuchchanges,theymayresorttobitinghumans[23].
Onlymacrogametocytesandmicrogametocytescande-velopinthemosquitomidgut;allotherasexualparasitesaredigestedafterbloodfeeding.
Thisstudysupportsthepreviousfindingthatalthoughtheaverageasexualparasit-emiacountsinthenegativefeedgroupswerelowerthanthepositivefeedgroups,nosignificantdifferenceswerefound[24].
Thiswasalsosupportedbysomepositivefeedswithzeroasexualparasitessomehowachievingahighoo-cystinfectionnumber;however,theoocystswereabsentinthemidgutinsomecaseswithhighasexualparasitecounts.
Althoughthesamephenomenonwasnotedwiththerelationshipofgametocytemiatomidgutinfectioninthenegativeandpositivegroups,oocystload(oocystsperOocyst/midgutAn.
sinensis(Lab)*An.
anthropophagus*An.
sinensis(Lab)**An.
sinensis(F1)**050100150200200300400P<0.
0001P=0.
0139Figure4Scatterplotsoftheresultsofthespeciescomparisons.
ThemedianoocystloadofAnophelesisshownbyahorizontalblackline;*:rangeofoocysts/midgutbetweenthe142pairsoflab-colonyAn.
sinensis(lab)andAn.
anthropophagus,**:rangeofoocysts/midgutbetweenthe10pairsoflab-colony(Lab)andfirst-generation(F1)An.
sinensis.
Table3SporozoiteinfectionofAn.
sinensisandAn.
anthropophagus14days'post-feedingSporozoiteinfectivelevelNo.
ofsporozoitesAn.
sinensis(Lab)An.
anthropophagus+1210++4224+++3119++++5043Total13596*:"+",1–10sporozoites;"++",11–100sporozoites;101–500sporozoitesand500sporozoites.
Zhuetal.
Parasites&Vectors2013,6:176Page6of9http://www.
parasitesandvectors.
com/content/6/1/176positivemosquito)increasedwithgametocytedensityinallinfection-positivegroups,suggestingthatthebloodofpa-tientswithhighlevelsofgametocytemiahadagreaterpotentialtoinducemosquitoes,post-feeding,todevelopoocystsinthemidgutandtherebybeathigherriskoftransferringparasitestootherhumans.
Althoughthere-sultsfromthisstudyconfirmthepreviousfindingthatoocystsdevelopedwellinthemosquitomidgut,witharatioofmacrogametocytestomicrogametocytesoflessthan4[25],thenumberofnegative-infectioncasespro-ducingoocystsinthisstudydemonstratedthatthepres-enceofoocystsinthemidgutfollowingfeedingisnotagoodindicatorofinfectivity,aconclusionsupportedbyastudyinwestThailand[26].
Themalariaparasiteisunderintenseattackfromthemosquito'sinnateimmunesystemduringitsdevelopmentinthemidgutandsalivaryglands[27].
Severalmosquitoimmunegenesplayimportantrolesintheparasiteevasionstagebyinfluencingparasite-mosquitointeractions[28-30].
Inotherwords,thesuscep-tibilityofmosquitoestomalariainfectioncouldberelatedtoanenhancedorweakenedimmuneresponseofmosqui-toestoparasiteinfection[31].
Additionally,thegenotypesoftheinvadingparasitesplayanimportantrole,i.
e.
,parasiteswithVK210andVK247,twomaingenotypesofcircumsporozoiteprotein(CSP),haveanobviousprefer-enceforinfectingmosquitoes[32].
Therefore,furtherstudyofthesusceptibilityofAn.
sinensistoparasitesfromvari-ousgeographicareasinChinaisnecessary.
Table4ParasitemiaandoocystandsporozoiteinfectioninAn.
sinensisandAn.
anthropophagusSpeciesAn.
sinensisAn.
anthropophagusAn.
sinensis/An.
anthropophagusOocyst(+)/Sporozoite()Oocyst(+)/Sporozoite(+)Oocyst(+)/Sporozoite()Oocyst(+)/Sporozoite(+)Oocyst()/Sporozoite()Cases6241583832Meanoocysts/midgut20.
8158.
0318.
3653.
680Meangametocytedensity(/μL)1579.
6±154.
81682.
3±255.
41618.
2±163.
41652.
2±267.
91386.
5±254.
0Meanasexualparasitedensity(/μL)4308.
2±444.
45061.
8±709.
64325.
0±456.
64329.
7±663.
23781.
5±714.
2Femalegametocytes/malegametocytes3.
24±0.
253.
41±0.
463.
31±0.
263.
07±0.
353.
98±0.
920200040006000800010000050100150200250300350400Gametocytemia0200040006000800010000050100150GametocytemiaF=14.
10,P=0.
00030200040006000800010000050100150200250300350400Gametocytemia0200040006000800010000050100150GametocytemiaF=27.
65.
10,P<0.
0001F=11.
19,P=0.
0019F=5.
207,P=0.
00248Oocyst/midgutOocyst/midgut(/L)(/L)(/L)(/L)ABCDFigure5CorrelationbetweennumberofPlasmodiumvivaxgametocytespermicroliterofbloodandnumberofoocystspermidgut.
(A)Lab-colonyAn.
sinensis(103oocyst-positivecases),(B)Lab-colonyAn.
sinensis(41sporozoite-positivecases),(C)Lab-colonyAn.
anthropophagus(96oocyst-positivecases)and(D)Lab-colonyAn.
anthropophagus(38sporozoite-positivecases).
Zhuetal.
Parasites&Vectors2013,6:176Page7of9http://www.
parasitesandvectors.
com/content/6/1/176Althoughthedirect-feedingmethodmoreaccuratelyreflectsepidemiologicreality[33],mostvolunteersprefertoprovidebloodbyvenepunctureratherthanallowingmosquitoestobitetheirskindirectly[34].
Inthemembrane-feedingassayusedinthisstudy,patientserawerereplacedbynaivemalaria-freehumanABserum,toavoidinterferencefromvaryingantibodylevelsinpatientbloodsamples[35].
Furthermore,theconstant-temperaturecyclingsystemallowedunlimitedmainten-anceofparasiteactivityandequalisedblood-feedingconditionsamongthemosquitogroups[36].
Themem-branefeedingassayisavaluabletoolfortheevaluationandvalidationofcandidatemarkersoftransmission-blockingvaccine(TBV)followingthemodificationoftargetgenes[37,38].
BecauseAn.
sinensisisthelargestofthefourmajorvectorsinChina,aswellbeingrela-tivelyeasiertomaintainunderlaboratoryconditions,andwithhighsusceptibilitytovivaxparasites,itcouldbeusedasavaluablecandidatespeciestoevaluateTBV,inparticular.
Comparedtothepreviousstudies[39,40]theAnsinensisfromthisstudyshowedhighersusceptibilityratestoP.
vivaxisolatesinCentralChina,althoughitisknownthattheAn.
sinensisstrainfromKorea,ChinaandJapanwascompatiblegeneticallyand/ornearlyiden-ticaltothatfromThailand,basedonthecrossingex-perimentsandcomparativesequenceanalysesoftheribosomalDNA(rDNA)internaltranscribedspacer2(ITS2)[41].
Thegeneticdiversityoftheparasitesandtheircompatibilitytothevectorsineachlocationmaycon-tributetothedifferenceinvectorsusceptibility.
Inthisstudywedidnotanalysethegeneticdiversityofthepara-sitesasthestudyaimedtocomparethesusceptibilityofAn.
sinensisandAn.
anthopophagusinCentralChinatothesameparasiteisolatescollectedinthisregion.
ConclusionsToourknowledge,thisisthefirstreportofthesusceptibil-ityofthewidelydistributedmalariavectorAn.
sinensistoP.
vivaxfollowingartificialmembranefeedingafterthere-emergenceofmalariaincentralChina.
TheAn.
sinensismosquitoesinthelaboratorymaintainedasimilarcapacitytobecomeinfectedwithvivaxparasitesasthefieldmos-quitoes,andtheirparasite-carryingabilitywasalsosimilartothatofAn.
anthropophagus.
ThevectorcapacityofAn.
sinensisformalariatransmissionduringthevivaxre-emergenceperiod,particularlyincentralChina,hasprobablybeenunderestimated.
Duetoitsmorpholo-gicalcharacteristicsandhighsusceptibilitytoparasites,An.
sinensiscouldbeagoodvectorcandidateforvivaxmalariaTBVevaluation.
CompetinginterestsTheauthorsdeclarethattheyhavenocompetinginterests.
Authors'contributionsGD,QG,andJScontributedequallytothestudydesignanddataanalysis.
HX,HY,JL,FL,YBandJCmanagedtheparasitesandmosquitoesinthefield.
GD,HY,JLandYBcontributedtothedissectionofmosquitoes.
GDdraftedthemanuscript.
QGprovidedscientificsupervision.
JSrevisedthemanuscript.
Allauthorsreadandapprovedthefinalmanuscript.
AcknowledgementsWeacknowledgetheoutstandingtechnicalsupportoftheentomologyteamsoftheArmedForcesResearchInstituteofMedicalSciences(AFRIMS,Bangkok,Thailand).
WearegratefultoQiangFang,ZhiyongTaoandKaimingHufromBengbuMedicalCollegefortheirassistanceinfieldworkandaregratefultoMing-chiehLeefromthePrograminPublicHealth,UniversityofCaliforniaatIrvine,USA,fordraftingthemap.
ThisresearchwassupportedbytheUSMilitaryInfectiousDiseasesResearchProgram,JiangsuProvince'sConstructionproject(BM2009902)andJiangsuProvince'sMedicalHighTechPlatform(ZX201108).
Authordetails1KeyLaboratoryofParasiticDiseaseControlandPrevention,MinistryofHealth,JiangsuInstituteofParasiticDiseases,Wuxi,JiangsuProvince,China.
2JiangsuProvincialKeyLaboratoryofParasiteMolecularBiology,JiangsuInstituteofParasiticDiseases,Wuxi,JiangsuProvince,China.
3DepartmentofParasitology,MedicalCollegeofSoochowUniversity,Suzhou215123,China.
4DepartmentofParasitology,BengbuMedicalCollege,2600DonghaiDadaoRoad,Bengbu233030,China.
5MahidolVivaxResearchUnit,FacultyofTropicalMedicine,MahidolUniversity,Bangkok,Thailand.
Received:13February2013Accepted:10June2013Published:14June2013References1.
ChenB,HarbachRE,ButlinRK:MolecularandmorphologicalstudiesontheAnophelesminimusgroupofmosquitoesinsouthernChina:taxonomicreview,distributionandmalariavectorstatus.
MedVetEntomol2002,16:253–265.
2.
WuS,PanJY,WangXZ,ZhouSS,ZhangGQ,LiuQ,TangLH:AnophelespseudowillmoriisthepredominantmalariavectorinMotuoCounty.
TibetAutonomousRegion.
MalariaJ2009,8:46.
3.
RuedaLM,ZhaoTY,MaYJ,GaoQ,ZhuGD,KhuntiratB,SattabongkotJ,WilkersonRC:UpdateddistributionrecordsoftheAnopheles(Anopheles)hyrcanusspecies-group(Diptera:Culicidae)inChina.
Zootaxa2007,1407:43–55.
4.
ZhouSS,WangY,FangW,TangLH:[MalariasituationinthePeople'sRepublicOfChinain2007].
ZhongguoJiShengChongXueYuJiShengChongBingZaZhi2008,26(6):401–403.
5.
GaoQ,BeebeNW,CooperRD:MolecularidentificationofthemalariavectorsAnophelesanthropophagusandAnophelessinensis(Diptera:Culicidae)incentralChinausingpolymerasechainreactionandappraisaloftheirpositionwithintheHyrcanusgroup.
JMedEntomol2004,41(1):5–11.
6.
GaoQ,ZhouHY,CooperRD,LiFH,SuYP,ZhuGD,CaoJ,BeebeNW:AsimpletechniqueforthegeneticidentificationofdifferentAnophelinemosquitoesamongtheAnopheleshyrcanuscomplex.
ChinJZoonoses2005,21(3):193–196.
7.
LiJL,ZhouHY,ShenBX:ThesusceptibilityinvestigationofAnophelessinensisandAnophelesanthropophagustoPlasmodiumvivax.
ChinJSchistoControl1996,8(4):1.
8.
LiuCF,QianHL,GUZC,PanJY,ZhenX:ThecurrentcomparsionofmalariatransmissioneffectofAnophelessinensisandAnophelesanthropophagusinChina.
JMedRes1991,20(5):2.
9.
ZhouSS,WangY,TangLH:[MalariasituationinthePeople'sRepublicofChinain2006].
ZhongguoJiShengChongXueYuJiShengChongBingZaZhi2007,25(6):439–441.
10.
BillkerO,ShawMK,MargosG,SindenRE:Therolesoftemperature,pHandmosquitofactorsastriggersofmaleandfemalegametogenesisofPlasmodiumbergheiinvitro.
Parasitol1997,115(Pt1):1–7.
11.
BangsMJ,SoelartoT,Barodji,WicaksanaBP,BoewonoDT:ColonizationofAnophelesmaculatusfromCentralJava,Indonesia.
JAmMosqControlAssoc2002,18(4):359–363.
Zhuetal.
Parasites&Vectors2013,6:176Page8of9http://www.
parasitesandvectors.
com/content/6/1/17612.
RongYQ,FangHY,YangZY,ZhouHY,MeiHG:ThesusceptibilitystudyofAnophelessinensisandAnophelesanthropophagustoPlasmodiumvivaxandPlasmodiumfalciparum.
JiangSuJiFang1986,3:4.
13.
SolarteY,ManzanoMR,RochaL,HurtadoH,JamesMA,Arevalo-HerreraM,HerreraS:PlasmodiumvivaxsporozoiteproductioninAnophelesalbimanusmosquitoesforvaccineclinicaltrials.
AmJTropMedHyg2011,84(2Suppl):28–34.
14.
HeatherM,FergusonAFR:WhyistheeffectofmalariaparasitesonmosquitosurvivalstillunresolvedTrendsParasitol2002,18(6):256–261.
15.
ZhouSS,ZhangSS,WangJJ,ZhengX,HuangF,LiWD,XuX,ZhangHW:Spatialcorrelationbetweenmalariacasesandwater-bodiesinAnophelessinensisdominatedareasofHuang-Huaiplain,China.
ParasitVectors2012,5:106.
16.
LehmannT,HumeJC,LichtM,BurnsCS,WollenbergK,SimardF,RibeiroJM:MolecularevolutionofimmunegenesinthemalariamosquitoAnophelesgambiae.
PLoSOne2009,4(2):e4549.
17.
TanWL,WangZM,LiCX,ChuHL,XuY,DongYD,WangZC,ChenDY,LiuH,LiuDP,etal:Firstreportonco-occurrenceknockdownresistancemutationsandsusceptibilitytobeta-cypermethrininAnophelessinensisfromJiangsuProvince.
ChinaPLoSOne2012,7(1):e29242.
18.
VerhaeghenK,VanBortelW,TrungHD,SochanthaT,KeokenchanhK,CoosemansM:KnockdownresistanceinAnophelesvagus,An.
sinensis,An.
paraliaeandAn.
peditaeniatuspopulationsoftheMekongregion.
ParasitVectors2010,3(1):59.
19.
CuiF,RaymondM,QiaoCL:InsecticideresistanceinvectormosquitoesinChina.
PestManagSci2006,62(11):1013–1022.
20.
WangJ:ResistanceandresponsetoselectiontodeltamethrininAnophelessinensisfromZhejiang,China.
JAmMosqControlAssoc2000,16(1):9–12.
21.
ZhangZ,YangC:Applicationofdeltamethrin-impregnatedbednetsformosquitoandmalariacontrolinYunnan,China.
SoutheastAsianJTropMedPublicHealth1996,27(2):367–371.
22.
ThemalERAConsultativeGrouponVectorControl:Aresearchagendaformalariaeradication:vectorcontrol.
PLoSMed2011,8(1):e1000401.
23.
TadeiWP,DutaryThatcherB:MalariavectorsintheBrazilianamazon:AnophelesofthesubgenusNyssorhynchus.
RevInstMedTropSoPaulo2000,42(2):87–94.
24.
ZollnerGE,PonsaN,GarmanGW,PoudelS,BellJA,SattabongkotJ,ColemanRE,VaughanJA:PopulationdynamicsofsporogonyforPlasmodiumvivaxparasitesfromwesternThailanddevelopingwithinthreespeciesofcolonizedAnophelesmosquitoes.
MalariaJ2006,5:68.
25.
LiuD,LuoSH,ShuHP,FuRD:ResearchonthefactorsinfluencingthesporogonicmultiplicationofPlasmodiumvivaxinthemosquitovector.
ZhongguoJiShengChongXueYuJiShengChongBingZaZhi1995,13:21–24.
26.
NacherM,SilachamroonU,SinghasivanonP,WilairatanaP,PhumratanaprapinW,FontanetA,LooareesuwanS:RiskfactorsforPlasmodiumvivaxgametocytecarriageinThailand.
AmJTropMedHyg2004,71(6):693–695.
27.
CirimotichCM,DongY,GarverLS,SimS,DimopoulosG:MosquitoimmunedefensesagainstPlasmodiuminfection.
DevCompImmunol2010,34(4):387–395.
28.
HughesGL,KogaR,XueP,FukatsuT,RasgonJL:WolbachiainfectionsarevirulentandinhibitthehumanmalariaparasitePlasmodiumfalciparuminAnophelesgambiae.
PLoSPathog2011,7(5):e1002043.
29.
ParmakelisA,MoustakaM,PoulakakisN,LouisC,SlotmanMA,MarshallJC,Awono-AmbenePH,Antonio-NkondjioC,SimardF,CacconeA,etal:Anophelesimmunegenesandaminoacidsitesevolvingundertheeffectofpositiveselection.
PLoSOne2010,5(1):e8885.
30.
CrawfordJE,GuelbeogoWM,SanouA,TraoreA,VernickKD,SagnonN,LazzaroBP:DenovotranscriptomesequencinginAnophelesfunestususingIlluminaRNA-seqtechnology.
PLoSOne2010,5(12):e14202.
31.
BahiaAC,KubotaMS,TemponeAJ,PinheiroWD,TadeiWP,SecundinoNF,Traub-CsekoYM,PimentaPF:AnophelesaquasalisInfectedbyPlasmodiumvivaxdisplaysuniquegeneexpressionprofileswhencomparedtoothermalariavectorsandplasmodia.
PLoSOne2010,5(3):e9795.
32.
Gonzalez-CeronL,RodriguezMH,NettelJC,VillarrealC,KainKC,HernandezJE:DifferentialsusceptibilitiesofAnophelesalbimanusandAnophelespseudopunctipennistoinfectionswithcoindigenousPlasmodiumvivaxvariantsVK210andVK247insouthernMexico.
InfectImmun1999,67(1):410–412.
33.
M-TRC:ThemalarialinfectivityofanAfricanvillagepopulationtomosquitoes(Anophelesgambiae).
AmJTropMedHyg1957,6:971–979.
34.
Awono-AmbeneHP,DiawaraL,RobertV:ComparisonofdirectandmembranefeedingmethodstoinfectAnophelesarabiensiswithPlasmodiumfalciparum.
AmJTropMedHyg2001,64(1–2):32–34.
35.
SattabongkotJ,TsuboiT,HisaedaH,TachibanaM,SuwanabunN,RungruangT,CaoYM,StowersAW,SirichaisinthopJ,ColemanRE,etal:BlockingoftransmissiontomosquitoesbyantibodytoPlasmodiumvivaxmalariavaccinecandidatesPvs25andPvs28despiteantigenicpolymorphisminfieldisolates.
AmJTropMedHyg2003,69(5):536–541.
36.
SattabongkotJ,ManeechaiN,PhunkitcharV,EikaratN,KhuntiratB,SirichaisinthopJ,BurgeR,ColemanRE:ComparisonofartificialmembranefeedingwithdirectskinfeedingtoestimatetheinfectiousnessofPlasmodiumvivaxgametocytecarrierstomosquitoes.
AmJTropMedHyg2003,69(5):529–535.
37.
TachibanaM,SatoC,OtsukiH,SattabongkotJ,KanekoO,ToriiM,TsuboiT:PlasmodiumvivaxgametocyteproteinPvs230isatransmission-blockingvaccinecandidate.
Vaccine2012,30(10):1807–1812.
38.
TonwongN,SattabongkotJ,TsuboiT,IrikoH,TakeoS,SirichaisinthopJ,UdomsangpetchR:NaturalinfectionofPlasmodiumfalciparuminducesinhibitoryantibodiesagainstgametocytedevelopmentinhumanhosts.
JpnJInfectDis2012,65(2):152–156.
39.
JoshiD,ChoochoteW,ParkMH,KimJY,KimTS,SuwonkerdW,MinGS:ThesusceptibilityofAnopheleslesteritoinfectionwithKoreanstrainofPlasmodiumvivax.
MalariaJ2009,8(12):42.
40.
JoshiD,KimJY,ChoochoteW,ParkMH,MinGS:PreliminaryvivaxmalariavectorcompetenceforthreemembersoftheAnopheleshyrcanusgroupintheRepublicofKorea.
JAmMosqControlAssoc2011,27(3):312–314.
41.
ParkMH,ChoochoteW,KimSJ,SomboonP,SaeungA,TuetanB,TsudaY,TakagiM,JoshiD,MaY,MinGS:NonreproductiveisolationamongfourallopatricstrainsofAnophelessinensisinAsia.
JAmMosqControlAssoc2008,24(4):489–495.
doi:10.
1186/1756-3305-6-176Citethisarticleas:Zhuetal.
:SusceptibilityofAnophelessinensistoPlasmodiumvivaxinmalarialoutbreakareasofcentralChina.
Parasites&Vectors20136:176.
SubmityournextmanuscripttoBioMedCentralandtakefulladvantageof:ConvenientonlinesubmissionThoroughpeerreviewNospaceconstraintsorcolorgurechargesImmediatepublicationonacceptanceInclusioninPubMed,CAS,ScopusandGoogleScholarResearchwhichisfreelyavailableforredistributionSubmityourmanuscriptatwww.
biomedcentral.
com/submitZhuetal.
Parasites&Vectors2013,6:176Page9of9http://www.
parasitesandvectors.
com/content/6/1/176