an entomopathogenic fungus - wur

�� entomologische berichten 68(1)2008

Introduction

Foralongtime,theprincipalchoicetoreducenuisancebitingbymosquitoesortheirtransmissionofparasiticorarboviraldiseaseshasbeentheselectiveapplicationofresidualsyntheticinsecticides.Thepublichealthbenefitdeliveredbythismethod,especiallyintropicalresource-poorsettings,ishuge,savinguncountablehumanlives.Poweredbyastrongindustriallobby,newandmoreenvironmentallyfriendlycompoundsreplaceolder,moreharmfulones.However,beyondgainsineconomicandpublichealthterms,thegrimrealityofenvironmentalimpactandever-developingresistanceremainsanissueofgra-veconcern(Chandreet al.1999,Hemingway&Ranson2000,

Brookeet al.2002).Itisthereforenotsurprisingthatinterestinalternativenon-chemicalstrategieshasincreasedoverthelastdecades.Theuseofbiologicalcontrolagentssuchasprotozoa(Chapman1974,Legner1995),nematodes(Kaya&Gaugler1993),predatoryfish(Legner1995),andbacteria(Becker&Ascher1998)hasshownpromiseasameanstocontrolmosquitopopulations,andsomeoftheseagentsaresuccessfullyusedintoday’smosquitocontrolprogrammesinvariousregionsaroundtheworld(Beckeret al.2003).Unfortunately,intheoneconti-nentwheremalariaaccountsformorethan1milliondeathseachyear,alternativestoinsecticidesarevirtuallynon-existent.Recently,interesthasincreasedinanothergroupofbiological

An entomopathogenic fungus (Metarhizium anisopliae) for control of the adult African malaria vector Anopheles gambiae

Ernst-Jan ScholteBart G.J. KnolsWillem Takken

KEY WORDSinsect pathogenic fungi, biological control, Diptera, malaria control

Entomologische Berichten 68 (�): ��-�6

Current indoor control for malaria vectors in Africa depends on the use of insecticides and/or repellents. Although several biological control agents can reduce larval mosquito populations, none are appropriate for targeting the adult stage. This manuscript describes a series of experiments to evaluate the potential impact of a new method, the use of an insect-pathogenic fungus (Metarhizium anisopliae) against adult Anopheles gambiae sensu stricto, both in the laboratory and in the field. Various experiments describe different aspects of this developing methodology, among which application techniques, sublethal effects, and the impact it may have on malaria transmission. Finally, it is discussed how this method can play a part in an integrated approach of mosquito vector control in the tropics.

Fungus mosquito species affected stage reference

Oomycota1

Leptolegnia2sp.(unidentified) An. gambiae larva Nnakumusana1986 Pythiumsp. An. gambiae larva Nnakumusana1985 Lagenidium giganteum An. gambiae larva Golkaret al.1993Chytridiomycota Coelomomyces indicus An. arabiensis larva Service1977 An. gambiae larva Muspratt1963Zygomycota Smittium (unidentified) An. gambiae larva Coluzzi1966FungiAnamorfici Aspergillus parasiticus An. gambiae larva/adult Nnakumusana1985 Metarhizium anisopliae An. gambiae larva/adult Roberts1967, Scholteet al.2003 Beauveria bassiana An. gambiae adult Scholteet al.2003

1=Phylum,2=Genus

Table �.EntomopathogenicfungithathavebeenreportedtobepathogenictoAfricanmalariavectors.Table �. Entomopathogene schimmels waar-van gerapporteerd is dat ze pathogeen zijn voor Afrikaanse malariavectoren.


controlagentsthatopensupperspectivesforvectorcontrolinAfrica:fungi(Scholteet al.2003a,b, 2004a,b, 2005, 2006, Blanford2003a,b,2004a,b,2005,2006,Blanfordet al.2005,Thomaset al.2005, Knols & Thomas 2006).2005,Knols&Thomas2006).

Manyentomopathogenicfungihaveproventobepatho-genictomosquitoes(Scholteet al.2004b),andsomeofthemha-vealsobeenfoundinAfricanmalariavectors(table1).ThislistislikelytobecomemuchlongeriffungalpathogensofthemalariavectorAnopheles gambiae Giles sensustrictoarespecifi-callybeingsearchedforinthefield,andotherfungiwillbetestedonthisvectorinthelaboratory.Whilesuccessfulmos-quitovectorcontrolinAfricaiscurrentlybasedoncontrollingadultmosquitoes,thevastmajorityofmosquitocidalfungiisaquaticandmayonlybeusedtocontroltheaquaticstagesoftheinsects.However,recentprogresshasbeenmadeinusinganamorphicentomopathogenicfungi(Deuteromycetes)forcon-trollingadultAfricanmosquitoes(Scholteet al.2003a,b,2004a,b,2005,Blanfordet al.2005).Thesefungihaveadistinctadvantageoverthebiologicalcontrolagentsmentionedabove,inthattheydonotneedtobeingestedtoinfectandkilltheinsects–infec-tiontakesplacethroughphysicalcontactoftheinfectivepropa-gules(conidia)withtheinsectcuticle.Assuch,thecontaminati-onmethodissimilartoconventionalindoorresidualsprayingwithinsecticides.Itisgenerallyacceptedthattheuseoftheseinsect-pathogenicfungi,suchasMetarhizium anisopliae,asamy-co-insecticideisnotharmfultotheenvironment(Zimmermann1993,Strasseret al.2000),nortohumans.Thiswasillustratedbythefactthatin2003,anM. anisopliaeisolate(strainF52)wasgrantedregistrationbytheU.S.EnvironmentalProtectionAgencytobeusedagainstinsectpests.Thedossierstatedthat,afterthorough(eco)toxicologicaltesting,noharmisexpectedtobecausedtohumansortheenvironment(EPA2003,Kanzok&Jacobs-Lorena2006).

General life cycle of deuteromycetous entomopathogenic fungi

Deuteromycetousfungiareagroupoffungiofwhichnosexualstagehasbeenreported.Severalinsectpathogenicfungalge-nerasuchasBeauveria,FusariumandMetarhiziumbelongtothisgroup.Thelifecycleofentomopathogenicdeuteromycetousfungistartswithconidiathatattachto,germinateon,andpene-tratetheinsectcuticle.Onceinthehaemocoel,themycelium

growsthroughoutthehost,forminghyphalbodiescalledblasto-spores.Deathoftheinsectisoftenduetoacombinationoftheactionoffungaltoxins,physicalobstructionofbloodcirculati-on,nutrientdepletionand/orinvasionoforgans.Afterthehosthasdied,hyphaeusuallyemergefromthecadaverand,undersuitableabioticconditions,conidiaareproducedontheexteriorofthehost(figure1).Thesearethendispersedbywindorwater(Goettel&Inglis1997).

First screening of candidate fungi

InordertoobtainasuitablefungalcandidateforcontrollingAn. gambiae s.s.,asearchformosquitocidalDeuteromyceteswasconductedinWesternKenya(Scholteet al.2003b).Fourinsect-pathogenicfungiwereisolated.Threespecieswereisolatedfromdeadinsects:Beauveria bassianaandFusariumsp.fromthestemborerBusseola fusca (Fuller),andanM. anisopliaestrainfromthewhitefly Trialeurodes vaporariorum(Westwood);oneM. anisopliaeisolatewasfoundinasoilsample.Afifthfungus(M. anisopliaeisolateICIPE30)wasprovidedbytheInternationalCentreofInsectPhysiologyandEcology(ICIPE),Nairobi,Kenya(courtesyN.K.Maniania).UnformulatedconidiaofthesefungiwerescreenedfortheirvirulenceagainstadultAn. gambiaes.s. mosquitoes.Survivalanalysis(KaplanMeier)showedthatsurvi-valofmosquitoesthatwereinfectedwitheitheroneofthefivefungiwassignificantlylowerthanthatofthecontrolgroup,althoughvirulencevariedbetweenfungi(figure2)(Scholteet al.2003b).Meaninfectionpercentagesaredepictedintable2.Fromthisinitialfungalscreeningexperiment,thehyphomyceteM. anisopliaeisolateICIPE30provedtobethemostvirulentforAn. gambiae,andsubsequentexperimentswerecarriedoutusingthisisolate.

Development of standard infection methodology

Forcontaminationofadultmosquitoeswithfungalconidiaofaknownconcentration,astandardprotocolwasdevelopedsimi-lartoscreeningofresidualpesticidesonmosquitoes:afilterpaperwasimpregnatedononesidewithaknownconcentrati-onofconidia,formulatedwithan8%adjuvantvegetableoilsus-pendedin0.05%Tween-80anddriedslowlyduring48hrsatroomtemperature.Thepaperwasrolledupandinsertedintoa

�.Metarhizium anisopliae,sporulatingfromacadaveroftheAfricanmalariamosquitoAnopheles gambiae s.s.Thegreen-whitishcolouriscausedbymillionsofconidia,producedbyconidiogenouscellsformingonhyphaegrowingoutofthedeceasedmosquito.Photo:E-JScholte(source:Knols&Thomas2006).�. Metarhizium anisopliae, sporulerend op een dood exemplaar van de Afrikaanse malaria-mug Anopheles gambiae s.s. De groen- witachtige kleur wordt veroorzaakt door miljoenen conidiën, die geproduceerd wor-den door conidiogene cellen, groeiend op de schimmelhyphen uit de dode mug.


glasscylinderinsuchawaythatthesidecontainingconidiawasthesurfaceonwhichmosquitoeswouldrestonceinsertedintheglasscylinder.Subsequentlaboratoryexperimentsonfe-maleAn. gambiaes.s.showedthatadoseof1.6x1010conidia/m2resultedininfectionpercentagesbetween83.7and97.7,andLT50-values(i.e.,thetimeittakesfor50%ofagrouptodie)rangingfrom3.2to5.9days,whileuninfectedfemaleslivedupto31dayswithLT50sbetween9.9and18.5days(Scholteet al.2003a,2004a).Thisdosagewasconsideredsuitableforfieldapplication,becausetheexpectedinfectionproportionsintargetedmosquitoeswerehigh,andthequantitiesofrequiredconidiawereofanacceptablemagnitude.

Fungal infection effects on blood-feeding and fecundity of mosquitoes

Inadditiontocausingsignificantmortality,M. anisopliaeisknowntocausereductionsinfeedingandfecundityinarangeofinsects.Sinceachangeinblood-feedingpropensityofmalariavectorscouldhaveimportantimplicationsregardingmalariatransmission,weinvestigatedwhetherbloodfeedingofAn. gambiaes.s.wasalsoaffectedbyinfectionwithM. anisopliae

�.Survivalcurves(fittedonGompertzdistribution),fromthesecondexperimentonblood-feedingpropensity,andproportions(±SE)ofM. anisopliae-infectedfemaleAn. gambiae s.s.thattookbloodmeals,ascalculatedfromthemosquitoesaliveatthetimewheneachsuccessivebloodmealwasdue(source:Scholteet al.2006).�. Overleving (geplot op de ‘Gompertz-overlevingsverdeling’), uit het tweede experiment dat de neiging tot bloedvoeden onderzocht, en de relatieve aantallen (± SE) van M. anisopliae-geïnfecteerde vrouwelijke An. gambiae s.s. die een bloedmaaltijd namen, berekend op basis van de aantallen muggen die een volgende bloedmaaltijd namen.

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�. Survivalcurves(fittedonGompertzsurvivaldistribution)ofadultmosquitoes(Anopheles gambiae s.s.)infectedwithvariousentomo-pathogenicfungi(source:Scholteet al.2003).�. Overleving (geplot op de ‘Gompertz-overlevingsverdeling’) van volwassen Anopheles gambiae s.s., geïnfecteerd met verschillende soorten entomopathogene schimmels.

Fusarium

Metarhizium (soil)

Beauveria

Metarhizium (white fly)

Metarhizium IC30

control

1 6 11 16 Time (days)

Table �.InfectivityandpathogenicityofvariousfungalspeciestoadultAnopheles gambiae s.s.mosquitoes(source: Scholte(source:Scholteet al.2003).Table �. Mate van besmettelijkheid en ziekteverwekkend vermogen van enkele schimmelsoorten tegen volwassen stadia van Anopheles gambiae s.s. muggen.

Fungus % Infected LT�0 ± SE Grouping�

± SE� (days) LT�0

control 8.80±0.66 AM. anisopliae(soil-sample) 46.5±3.6 5.87±0.77 BFusarium sp. 79.0±4.3 4.48±0.21 BCB. bassiana 81.7±0.9 3.49±0.29 CM. anisopliae(whitefly) 83.0±3.4 4.71±0.55 BCM. anisopliae(ICIPE30) 88.7±3.3 3.39±0.37 C

1SE:Standarderrorofthemean,2LT50valueswithoutlettersincom-monaresignificantatp<0.05

TreatmentGonotrophic cycle Control Low fungal dosage High fungal dosage

1 50.97±4.17(34) 50.13±2.72(56) 70.31±4.79(29)2 65.09±5.80(32) 64.31±5.56(39) 36.65±10.1(23)3 71.53±7.27(32) 58.29±7.13(28) 31.69±9.89(16)4 80.96±6.90(27) 58.22±8.93(23) 24.80±18.3(5)5 6.33±9.87(18) 44.91±14.3(11) 40.00±13.3(2)6 59.23±14.2(13) 54.89±15.6(9) 0 (2)7 51.67±22.6(3) 19.60±6.53(5) 0 (2)8 0 0 0average* 65.55±2.96 54.81±2.67 45.48±4.70

*significantoveralleffectoffungalinfectiononaveragenumberofeggsperbloodmeal(ANOVA):F=9.784;p<0.001,df=2.

Table �.Meannumberofeggs(±SE)laidperbloodmeal,ascalculatedfromthefemalemosquitoesaliveatthetimewhenoviposi-tionwasdueineachgonotrophiccycle(n)(source:Scholteet al.2006).Table �. Gemiddelde aantal eitjes (± SE) dat gelegd werd per bloedmaaltijd, berekend op grond van het aantal vrouwtjesmuggen dat levend was op het moment waarop ze, per gonotrofische cyclus, verwacht werden eitjes te leggen (n).


(Scholteet al.2006).Themosquitoeswerecontaminatedwitheitheralow(1.6x108conidia/m2)oramoderatelyhigh(1.6x109conidia/m2)doseofoil-formulatedM. anisopliaeconidia,andof-feredasinglehumanbloodmeal48,72,or96hrslatertoassessfeedingpropensityandindividualbloodmealsize.Femalesinfectedwiththelowdoseofconidiaingestedlessbloodthanuninfectedfemales[3.79±1.34μgvs.7.78±0.99μghematin/bloodmealforlargefemales(wingsize≥3.1mm)and2.88±1.11μgvs.3.90±0.82μghematinforsmallfemales(wingsize<3.1mm)].Thisexperimentalsoshowedthatfe-malestooksignificantlyfewerbloodmealsthanuninfectedfemales72hrsaftertheyhadbeencontaminated.

Inasecondexperiment,individuallykeptfungus-infectedfemaleswereofferedabloodmealeverythirdday(toatotalofeightgonotrophiccycles),andallowedtoovipositaftereachcycleinordertoquantifyfeedingpropensityandfecundity.Aftereachfeedingopportunityitwasscoredwhetherthe

femalehadfedornot.Itwasfoundthatmosquitoes,inoculatedwiththemoderatelyhighdoseofconidia,exhibitedreducedappetite(figure3).Ofthefungus-infectedfemales,theproporti-onofmosquitoestakingasecondbloodmealwasreducedwith51%.Thiswasfurtherreducedto35.3%forthefourthbloodme-al.Duringeightfeedingopportunities,theaveragenumberofbloodmealstakenbyuninfectedfemaleswas4.39,against3.40(lowdose)and2.07(highdose)bloodmealsforthefungus-infectedfemales.Moreover,infectedfemalesproducedfewereggspergonotrophiccycle(table3).

Application in the field – can entomopathogenic fungi reduce malaria transmission?

Fundamentaltothedesignofmalariacontrolprogrammesisabasicunderstandingoftherelationshipbetweenmalariatransmissionbymosquitovectorpopulationsandmalariapre-valence,incidence,morbidityandmortality.OneparameterthatdescribestheintensityofmalariatransmissionistheEntomo-logicalInoculationRate(EIR),whichistherateatwhichsporo-zoite-positivebitesarereceived.Thisfunctionexpressestheim-portanceofmosquitosurvivalandthenumberofbloodmealstaken,reductionsofwhichhavehighimpactontheintensityofmalariatransmission(Charlwood1997,Charlwoodet al.1997,Killeenet al.2000,Drakeleyet al.2003).Inasmall-scalefieldstudyinTanzania,weplaced M. anisopliae-impregnatedclothsindoorsoflocalhouses(figure4)(Scholteet al.2005).OfallAn. gambiae sensulatocollectedinthesehouses,23%werefoundtobeinfectedwiththefungus.Theinfectedmosquitoeshadsignificantlyshorterlifespanscomparedtothoseofnon-infectedones(figure5).LT50-valueswere3.70and3.49daysforinfectedmalesandfemales,respectively,and5.88and9.30daysforuninfectedmalesandfemales.Thereductionsindailysurvi-valratesofwildAn. gambiaes.l.andnumbersofbloodmealstakenbyM. anisopliae-infectedfemalesmaythushaveahighimpactontheEIR,providedthatenoughmosquitoesbecomeinfected.ExtensivefieldstudiesonvillagescalehaverecentlybeguntoassesstheimpactofthisfungusonEIRandmorbidity.

Importantaspectsconcernthesystemofdeliveringthefun-gustothemosquitopopulation,andeffectivecoverageofthe

�.Survivalofuninfected(opensymbols:r=females,®=males)andM. anisopliae-infected(closedsymbols:p=females,n=males)wildAn. gambiae s.l.mosquitoescollectedfromruralTanzanianhouses(source:Scholteet al.2005).�. Overleving van ongeïnfecteerde (open symbolen: r = vrouwtjes, ® = mannetjes) en M. anisopliae-geïnfecteerde (gesloten symbolen: p = vrouwtjes, n = mannetjes) wilde An. gambiae s.l. muggen, ver- zameld van huisjes in een Tanzaniaans dorp.

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�.(a):Positionofblackcloth(dottedline)impregnatedwithconidiaoftheentomo-pathogenicfungusMetarhizium anisopliaeinsideaTanzanianhouse.(b):Blood-fedAn. gambiaefemalesonaM. anisopliae-infectedcloth(source:Scholteet al.2005).�. (a): De positie van de met de entomopatho-gene schimmel geïmpregneerde zwarte doek (gestreepte lijn) in een Tanzaniaans huis. (b): Volgezogen An. gambiae vrouwtjes op een M. anisopliae-geïmpregneerd doek.

ba


targetedinsectpopulationwiththefungus.InthefieldstudyinTanzania,conidiaweresuspendedinan8%adjuvantoilformu-lationandappliedon3-m2blackcottonsheets.Thesesheetswereplacedinsidehousesontheceilingandcontaminatedindoor-restingmosquitoeswiththefungus.Increasedsizeofthese‘restingtargets’andapplicationinlargerpartsofhumanhabitationsmaymaximisecoverage.

Application strategies

Killeenet al.(2004)showedthattheEIRcanbedrasticallyredu-cedwithmethodsthatdonotrequirechemicalsbutarebasedonintegrationofafewmoderatelyefficientvectorcontrolmethodssuchasenvironmentalmanagement-basedlarvalcontrolandzooprophylaxis.Inasimilarwayweenvisageanintegratedvector-controlprogrammewithoutanyuseofchemicals,whereM. anisopliaeisusedagainstadultmosquitovectorsinadditiontobiologicallarvalcontrolwithBacillus thuringiensissubsp.israelen-sis and/orB. sphaericus(Fillingeret al.2003),andapplicationof

untreatedbednets,push-pullmethodswithzooprophylaxis(Seyoumet al.2002)andrepellentplants(Seyoumet al.2003).Larvalcontrolwillreducethenumberofemergingadults,where-ascattleinornearhousesmaypreventsomeofthevectorsfromfeedingonhumans.Repellentodoursfromlive,potted,orburnedplantsaroundhouseswilldiminishthenumbersofmosquitoesenteringthehouses.Mosquitoeswhichareabletoenterdwel-lingswillbepreventedtobitebybednets(Guyatt&Snow2002,Takken2002).AndifsomeofthesemosquitoesthatenteredhouseswillbeinfectedbyM. anisopliaewhentheylandonthe‘restingtargets’,thiswillreducetheirsurvivalrate.Noneofthesetechniques,asstand-alonetools,isperfect,butthecombinationofthesevectorcontrolmethodsmaybeverypowerfulandgreat-lyenhancereductionsoftheEIR.Suchastrategymayresultinamoresustainablevector-bornediseasecontrolthanthecurrentonesbasedonpotentiallyharmfulchemicals.However,inordertobesuccessful,substantialfinancial,politicalandlocalsupportforlong-termandlarge-scaleapplicationisnecessary.

ReferencesBecker N & Ascher KRS 1998. The use of Bacil-

lus thuringiensis subsp. israelensis (Bti) against mosquitoes, with special emphasis on the ecological impact. Israel Journal of Entomology 32: 63-69.

Becker N, Zgomba M, Petric D, Dahl C, Boase C, Lane J & Kaiser A 2003. Mosquitoes and their control. Kluwer Academic / Plenum Publishers.

Blanford S, Chan BHK, Jenkins N, Sim D, Turner RJ, Read AF & Thomas MB 2005. Fungal pathogen reduces potential for malaria transmission. Science 308: 1638-1641.

Brooke BD, Hunt RH, Chandre F, Carnevale P & Coetzee M 2002. Stable chromosomal in-version polymorphisms and insecticide re-sistance in the malaria vector mosquito Anopheles gambiae (Diptera: Culicidae). Journal of Medical Entomology 39: 568-573.

Chandre F, Darriet F, Manga L, Akogbeto M, Faye O, Mouchet J & Guillet P 1999. Status of pyrethroid resistance in Anopheles gambiae sensu lato. Bulletin World Health Organ 77: 230-234.

Chapman HC 1974. Biological control of mosquito larvae. Annual Review of Ento-mology 19: 33-59.

Charlwood JD 1997. Vectorial capacity, species diversity and population cycles of anophe-line mosquitoes (Diptera: Culicidae) from indoor light-trap collections in a house in southeastern Tanzania. African Ento- mology 5: 93-101.

Charlwood JD, Smith T, Billingsley PF, Takken W, Lyimo EOK & Meuwissen JHET 1997. Survival and infection probabilities of an-thropophagic anophelines from an area of high prevalence of Plasmodium falciparum in humans. Bulletin of Entomological Research 87: 455-453.

Drakeley C, Schellenberg D, Kihonda J, Sousa CA, Arez AP, Lopez D, Lines J, Mshinda H, Lengeler C, Armstrong-Schellenberg J, Tanner M & Alonso P 2003. An estimation of the entomological inoculation rate for Ifakara: a semi-urban area in a region of intense malaria transmission in Tanzania. Tropical Medicine & International Health 8: 767-774.

Fillinger U, Knols BGJ & Becker N 2003. Efficacy and efficiency of new Bacillus thuringiensis var. israelensis and Bacillus sphaericus

formulations against Afrotropical anophe-lines in Western Kenya. Tropical Medicine & International Health 8: 37-47.

Goettel MS & Inglis GD 1997. Fungi: Hypho- mycetes. In: Lacey LA (ed) Manual of techniques in insect pathology: 213-249. Academic Press.

Guyatt HL & Snow RW 2002. The cost of not treating bednets. Trends in Parasitology 18: 12-16.

Hemingway J & Ranson H 2000. Insecticide resistance in insect vectors of human disease. Annual Review of Entomology 45: 371-391.

Kanzok SM & Jacobs-Lorena M 2006. Entomo-pathogenic fungi as biological insecticides to control malaria. Trends in Parasitology 22: 49-51.

Kaya HK & Gaugler R 1993. Entomopathogenic nematodes. Annual Review of Entomology 38: 181-206.

Killeen GF, McKenzie FE, Foy BD, Schieffelin C, Billingsley PF & Beier JC 2000. The potenti-al impact of integrated malaria transmis-sion control of entomologic inoculation rate in highly endemic areas. American Journal of Tropical Medicine and Hygiene 62: 545-551.

Killeen GF, Seyoum A & Knols BGJ 2004. Ratio-nalizing historical success of malaria control in Africa in terms of mosquito resource availability management. Ameri-can Journal of Tropical Medicine and Hygiene 71 (suppl): 87-93.

Knols BGJ & Thomas M 2006. Fungal entomo-pathogens for adult mosquito control – a look at the perspectives. Outlooks on Pest Management 17: 1-3.

Legner EF 1995. Biological control of Diptera of medical and veterinary importance. Jour-nal of Vector Ecology 20: 59-120.

Roberts DW 1967. Some effects of Metarhizium anisopliae and its toxins on mosquito larvae. In: Laan PA van der (ed) Insect pa-thology and microbial control: 243-246. North-Holland Publishing Company.

Scholte EJ 2004a. The entomopathogenic fungus Metarhizium anisopliae for mosquito control. Impact on the adult stage of the African malaria vector Anopheles gambiae and filariasis vector Culex quinquefasciatus. PhD-thesis, Wageningen University.

Scholte EJ, Knols BGJ, Samson RA & Takken W 2004b. Entomopathogenic fungi for

mosquito control: A review. Journal of Insect Science 4: 19-42.

Scholte EJ, Knols BGJ & Takken W 2006. Infec-tion of the malaria mosquito Anopheles gambiae with the entomopathogenic fungus Metarhizium anisopliae reduces blood-feeding and fecundity. Journal of Invertebrate Pathology 91: 43-49.

Scholte EJ, Ng’habi K, Kihonda J, Takken W, Paaijmans K, Abdulla S, Killeen GF & Knols BGJ 2005. An entomopathogenic fungus for control of adult African malaria mosquitoes. Science 308: 1641-1642.

Scholte EJ, Njiru BN, Smallegange RC, Takken W & Knols BGJ 2003a. Infection of malaria (Anopheles gambiae s.s.) and filariasis (Culex quinquefasciatus) vectors with the entomo-pathogenic fungus Metarhizium anisopliae. Malaria Journal 2: 29-36.

Scholte EJ, Takken W & Knols BGJ 2003b. Pathogenicity of five East African entomopathogenic fungi to adult Anopheles gambiae s.s. (Diptera: Culicidae) mosquitoes. Proceedings of the Section Experimental and Applied Entomology of the Netherlands Entomological Society (NEV, Amsterdam) 14: 25-29.

Seyoum A, Balcha F, Balkew M, Ali A & Gebre-Michael T 2002. Impact of cattle keeping on human biting rate of anopheline mosquitoes and malaria transmission around Ziway, Ethiopia. East African Medical Journal 79: 485-489.

Seyoum A, Killeen GF, Kabiru EW, Knols BGJ & Hassanali A 2003. Field efficacy of ther-mally expelled or live potted repellent plants against African malaria vectors in western Kenya. Tropical Medicine & Inter-national Health 8: 1005-1011.

Strasser H, Vey A & Butt TM 2000. Are there any risks in using entomopathogenic fungi for pest control, with particular reference to the bioactive metabolites of Metarhizi-um, Tolypocladium and Beauveria species? Biocontrol Science and Technology 10: 717-735.

Takken W 2002. Do insecticide-treated bednets have an effect on malaria vectors? Tropi-cal Medicine & International Health 7: 1022-1030.

Thomas MB, Blanford S, Jenkins NE, Killeen GF, Knols BGJ, Read AF, Scholte E-J & Takken W 2005. Benefits and risks in malaria control: A response. Science 310: 49.

�6 entomologische berichten 68(1)2008

Zimmermann G 1993. The entomopathogenic fungus Metarhizium anisopliae and its potential as a biocontrol agent. Pesticide Science 37: 375-379.

Ontvangen: 26 maart 2007Geaccepteerd 16 december 2007

Samenvatting

Een insectpathogene schimmel (Metarhizium anisopliae) ter bestrijding van volwassen Afrikaanse malariamuggen (Anopheles gambia)BestrijdingvanmalariamuggeninAfrikagebeurtvrijwelgeheelmetchemischeinsectici-den.Hoewelerdiversebiologischebestrijdingsorganismenbestaandielarvalepopulatieskunnendecimeren,zijnergeenorganismendiekunnenwordeningezetvoordebestrijdingvanhetvolwassenstadium.Ditartikelbeschrijfteenserielaboratorium-enveldexperi-mentenwaarineennieuwebiologischebestrijdingsmethodewordtgeëvalueerd.Dezeme-thodegebruikteeninsect-pathogeneschimmel(Metarhizium anisopliae)diewordtingezettegenadultenvandeAfrikaansemalariamugAnopheles gambiaesensustricto.Diverseas-pectenvandezenieuwemethodewordenbesproken,waaronderdebeoogdetoepassings-techniek,sublethaleeffectenvandeschimmelopeengeïnfecteerdemug,deimpactdiege-bruikvandezemethodekanhebbenopmalariaoverdracht,enhoedezenieuwemethodedeelkanuitmakenvaneengeïntegreerdebenaderingvanmuggenbestrijdingindetropen.

Ernst-JanScholteDutch Plant Protection Service

P.O. Box 9102

6700 HC Wageningen

The Netherlands

[email protected]

BartG.J.Knols,WillemTakkenLaboratory of Entomology

Wageningen University and Research Centre

P.O. Box 8031

6700 EH Wageningen

The Netherlands

an entomopathogenic fungus - wur

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