DTIC ADA535504: Mode of Action for Natural Products Isolated From Essential Oils of Two Trees Is Different From Available Mosquito Adulticides

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VECTORCONTROL,PESTMANAGEMENT,RESISTANCE,REPELLENTS
Mode of Action for Natural Products Isolated From Essential Oils of
Two Trees Is Different From Available Mosquito Adulticides
JANETC.MCALLISTER1ANDMARYF.ADAMS
DivisionofVector-BorneInfectiousDiseases,CentersforDiseaseControlandPrevention,
3150RampartRoad,FortCollins,CO80521
J.Med.Entomol.47(6):1123Ð1126(2010);DOI:10.1603/ME10098
ABSTRACT Insecticidal properties of natural products may present alternatives to the use of
synthetic molecule pesticides that are of diminishing effectiveness due to resistance. Three com-
pounds, thymoquinone, nootkatone, and carvacrol, components of Alaska yellow cedar, Chamae-
cyyparisnootkatensis(D.Don)Spach,andincensecedar,Calocedrusdecurrens(Torr.),essentialoils,
havebeenshowntohavebiologicalactivityagainstavarietyofmosquitoandtickspecies.Although
these components act as both repellents and insecticides, how they function in either capacity is
unknown.Theiruseasmosquitocontrolinsecticideswouldbegreatlyincreasediftheirmodeofaction
isnotthesameasthatofcurrentlyusedcommercialproducts.Thisstudycomparedthelethaldosages
fornootkatone,carvacrol,andthymoquinonebyusingcolonystrainsofAnophelesgambiaeGileswith
knownmutationsatthreedifferenttargetsites.Thealteredtargetsitesevaluatedwerethesodium
channelpara-locusmutation(L1014FKDR)thatconferspermethrinresistance,theACE-1genethat
confersorganophosphateandcarbamateresistance,anda(cid:1)-aminobutyricacidreceptormutationof
theRdllocusconferringdieldrinresistance.SigniÞcantincreasesinlethaldosewerenotobservedin
anyofthemosquitostrainsforanyofthecompoundstestedcomparedwiththedosesrequiredof
chemicalswithknownmodesofactionatthemutatedsites.Althoughthemodeofactionwasnot
determined,thisscreeningstudyindicatesthatnoneofthesecompoundsinteractatthetargetsites
representedinthetestmosquitostrains.Thesecompoundsrepresentadifferentmodeofactionthan
existingchemicalscurrentlyusedinmosquitocontrol.
KEYWORDS modeofaction,mosquitoes,nookatone,thymoquinone,carvacrol
Commercial insecticides used in mosquito control trol. Finding suitable chemicals with alternative
have traditionally come from those developed for modes of action is of urgent concern among vector
agriculture.IntheUnitedStates,insecticidesused controlpersonnel.
foradultmosquitocontrolcomefromtwoclassesof Essentialoilsofmanyplantspecieshavebeenin-
chemicals,pyrethroidsandorganophosphates.Out- vestigatedaspossiblealternativesoftraditionalchem-
side of the United States, additional classes may icalsbasedontheassumptiontheyarenaturalandthus
sometimes be used such as organochlorines, car- safer(Coats1994,Isman2006).Essentialoilsoftwo
bamates, and cyclodienes. All of these chemical
North American tree species, Alaska yellow cedar,
classesrelyonthreeprimarytargetsiteswithinthe
Chamaecyyparisnootkatensis(D.Don)Spach,andin-
insect vector: the gated sodium channel (pyre-
censecedar,Calocedrusdecurrens(Torr.),havebeen
throids and DDT), inhibition of (cid:1)-aminobutyric
investigatedfortheirinsecticidalandacaricidalprop-
acid (GABA) receptors (cyclodienes), and the in-
erties (Panella et al. 2005, Dolan et al. 2007). Their
hibitionofacetylcholinesterase(organophosphates
resultshaveshownessentialoilsofbothspecieshave
andcarbamates)(Ware1991).
insecticidal and repellent properties, albeit at lower
Resistance in vector mosquitoes has been docu-
levelsthancurrentlyavailablecompounds.Ourstudy
mentedtooccurthroughouttheworldinavarietyof
wasconductedtoevaluatewhetherthreecomponents
importantspecies(HemingwayandRanson2000,Xu
etal.2006).Withonlytwoprimarymodesofaction ofthesetreeessentialoils,thymoquinone,nootkatone,
availableforvectorcontrolofadultmosquitoesinthe and carvacrol, have similar modes of action to cur-
UnitedStates(i.e.,thegatedsodiumchannelandin- rentlyavailablemosquitoadulticides.Possessingadif-
hibitionofacetylcholinesterase),thedevelopmentof ferentmodeofactionwouldmakethesecompounds
resistanceinanareacancriticallyaffectvectorcon- morevaluableforfurthercommercialdevelopmentas
they would be particularly beneÞcial in areas with
1Correspondingauthor,e-mail:[email protected]. documentedinsecticideresistance.
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1. REPORT DATE 3. DATES COVERED
2010 2. REPORT TYPE 00-00-2010 to 00-00-2010
4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER
Mode of Action for Natural Products Isolated From Essential Oils of Two
5b. GRANT NUMBER
Trees Is Different From Available Mosquito Adulticides
5c. PROGRAM ELEMENT NUMBER
6. AUTHOR(S) 5d. PROJECT NUMBER
5e. TASK NUMBER
5f. WORK UNIT NUMBER
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION
Centers for Disease Control and Prevention,Division of Vector-Borne REPORT NUMBER
Infectious Diseases,3150 Rampart Road,Fort Collins,CO,80521
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S)
11. SPONSOR/MONITOR’S REPORT
NUMBER(S)
12. DISTRIBUTION/AVAILABILITY STATEMENT
Approved for public release; distribution unlimited
13. SUPPLEMENTARY NOTES
14. ABSTRACT
Insecticidal properties of natural products may present alternatives to the use of synthetic molecule
pesticides that are of diminishing effectiveness due to resistance. Three compounds thymoquinone,
nootkatone, and carvacrol, components of Alaska yellow cedar, Chamaecyyparis nootkatensis (D. Don)
Spach, and incense cedar, Calocedrus decurrens (Torr.), essential oils have been shown to have biological
activity against a variety of mosquito and tick species. Although these components act as both repellents
and insecticides, how they function in either capacity is unknown. Their use as mosquito control
insecticides would be greatly increased if their mode of action is not the same as that of currently used
commercial products. This study compared the lethal dosages for nootkatone, carvacrol, and
thymoquinone by using colony strains of Anopheles gambiae Giles with known mutations at three different
target sites. The altered target sites evaluated were the sodium channel para-locus mutation (L1014 F
KDR) that confers permethrin resistance, the ACE-1 gene that confers organophosphate and carbamate
resistance, and a -aminobutyric acid receptor mutation of the Rdl locus conferring dieldrin resistance.
Signi?ant increases in lethal dose were not observed in any of the mosquito strains for any of the
compounds tested compared with the doses required of chemicals with known modes of action at the
mutated sites. Although the mode of action was not determined, this screening study indicates that none of
these compounds interact at the target sites represented in the test mosquito strains. These compounds
represent a different mode of action than existing chemicals currently used in mosquito control.
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON
a. REPORT b. ABSTRACT c. THIS PAGE Same as 4
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Prescribed by ANSI Std Z39-18
1124 JOURNALOFMEDICALENTOMOLOGY Vol.47,no.6
Table1. LethaldosesforcomponentsoftreeessentialoilstestedonAn.gambiae
Chemical Msotsrqauinito N Slope(SE) (95%CLID,5(cid:2)0g/mg) (95%CLD,(cid:2)95g/mg) (cid:3)2 RR
Thymoquinone G3 370 0.002(0.00021) 1.425(1.277Ð1.570) 2.812(2.546Ð3.198) 14.92
RSPÐST 413 9.79(1.00) 0.933(0.875Ð0.995) 1.865(1.634Ð2.259) 12.68 0.65
P(cid:1)DLRC(cid:1)R(cid:1) 447 0.004(0.0004) 1.170(1.083Ð1.253) 1.950(1.808Ð2.149) 7.99 0.82
AKRON 381 0.0024(0.0004) 1.507(1.311Ð1.691) 2.712(2.377Ð3.357) 13.57 1.06
Nootkatone G3 441 4.88(0.47) 1.460(1.190Ð1.726) 5.861(4.785Ð7.676) 13.23
RSPÐST 421 3.17(0.33) 1.633(1.335Ð1.954) 13.900(9.655Ð23.705) 16.18 1.12
P(cid:1)DLRC(cid:1)R(cid:1) 382 1.53 2.089(1.802Ð2.344) 5.039(4.565Ð5.713) 11.98 1.47
AKRON 406 0.0009(0.0001) 2.502(2.236Ð2.775) 5.636(4.046Ð6.498) 7.08 1.71
Carvacrol G3 308 0.0011(0.0001) 2.649(2.378Ð2.905) 5.283(4.731Ð6.171) 8.18
RSPÐST 547 8.15(0.72) 1.791(1.675Ð1.908) 4.117(3.631Ð4.888) 14.65 0.68
P(cid:1)DLRC(cid:1)R(cid:1) 421 12.28(1.21) 2.031(1.925Ð2.141) 3.526(3.195Ð4.060) 5.84 0.78
AKRON 368 9.57(0.94) 2.088(1.950Ð2.237) 4.238(3.715Ð5.112) 14.60 0.79
Permethrin G3 485 2.03(0.33) 0.000214(0.000130Ð0.000291) 0.00598(0.00312Ð0.0203) 12.02
RSPÐST 365 2.72(0.35) 0.00358(0.00285Ð0.00442) 0.0432(0.0260Ð0.0987) 16.48 16.42
Propoxur G3 496 2.82(0.24) 0.000719(0.000590Ð0.00874) 0.00797(0.00546Ð0.0132) 11.77
AKRON 379 3.79(0.47) 3.628(3.088Ð4.246) 25.079(16.830Ð47.619) 17.29 5,046
Dieldrin G3 485 0.47(0.04) 0.00455(0.00404Ð0.00503) 0.0108(0.00981Ð0.0121) 12.33
P(cid:1)DLRC(cid:1)R(cid:1) 536 3.10 6.104(4.926Ð7.622) 151.470(91.920Ð296.225) 11.45 1,318
MethodsandMaterials either0.2(cid:2)lofeachtestcompoundor0.2(cid:2)lofacetone
foracontrolusingasyringeandrepeatingdispenser
Mosquitoes.FourstrainsofAnophelesgambiaeGiles
(Hamilton,Reno,NV).Fiveconcentrationsdilutedin
wereprocuredfromthemalariaresearchandrefer-
acetoneproducingarangeof0Ð100%mortalitywere
encereagentresourcecenter(MR4)program,partof
used for each compound. Twenty-Þve mosquitoes
the American Type Culture Collection (Manasses,
wereusedforeachconcentration.Assayswererep-
VA). Three strains have insecticide resistance char-
licated four times. After all individuals were treated
acterizedbyalteredtargetsitesandthefourthisin-
foraparticularconcentration,theywereweighedand
secticidesusceptible.TheP(cid:1)DLRC(cid:1)R(cid:1)strainhasan
anaverageweightpermosquitowascalculated.Mos-
alanine-to-glycine mutation in the Rdl locus coding
quitoesweremaintainedon10%sucrosefor24hby
for a (cid:1)-aminobutyric acid (GABA) receptor confer-
using the same conditions as the colonies. Mortality
ring resistance to dieldrin (Du et al. 2005). The
wasrecordedafter24h.Thecompoundstestedwere
RSP-STstraincontainsaleucine-phenylalaninesub-
nootkatone(Frutarom,NorthBergen,NJ),thymoqui-
stitutionatposition1014(L1014FKDR)ofthevolt-
none (TCI America, Portland, OR), and carvacrol
age-gatedsodiumchannelallele(Ransonetal.2000)
(TCIAmerica).
andotherunidentiÞedresistancemechanisms(Vulule
To conÞrm the level of resistance in the resistant
et al. 1999) conferring pyrethroid resistance. The
colonies,permethrin,dieldrin,andpropoxur(Chem-
AKRONstrainhasresistanceconferredtocarbamates
Service,WestChester,PA)weretestedontheRSP-
byaninsensitiveACE-1mutationandalsopossesses
ST,P(cid:1)DLRC(cid:1)R(cid:1),andAKRONstrains,respectively,
L1014FKDR.TheG3wildtypeisasusceptiblestrain
andcomparedwithresultsfromtestingeachchemical
incolonysince1975.
on G3 (susceptible colony). Control mortality was
Allmosquitocoloniesweremaintainedat27Ð28(cid:2)C,
correctedusingAbbottÕsformulaasmodiÞedandpro-
85Ð90%RH,andaphotoperiodof12:12(L:D)h.All
videdbytheWHO(1998).Lethaldosageswerecal-
adultswereallowedtofeedona10%sucrosesolution
culatedandchi-squaregoodness-of-Þttestswereper-
adlibitum.AdultfemaleswereprovideddeÞbrinated
formed using SAS software (PROC PROBIT, SAS
calfblood(ColoradoSerum,Denver,CO)byusinga
Institute2003).
Hemotek membrane feeding system (Discovery
Workshops, Accrington, United Kingdom) once a
week.MoistÞlterpaperwasplacedincages3dafter
ResultsandDiscussion
the bloodmeal for the collection of eggs. Eggs were
ßoatedonwaterwithonegrainofbakerÕsyeast(Uni- Table1summarizestheresultsforallthetests.The
versalFoods,Milwaukee,WI)inplasticpans(34.3by LD value for thymoquinone ranged from 0.933
50
25.4 cm, 3.8 cm in depth). Larvae were fed daily (cid:2)g/mg (conÞdence interval [CI], 0.875Ð0.995 (cid:2)g/
startingonday3withÞnelygroundTetraMinÞshfood mg)inRSP-STto1.507(cid:2)g/mg(CI,1.311Ð1.691(cid:2)g/
(TetraHolding,Blacksburg,VA).Pupaeproducedin mg)inAKRON.TherangeofLD valuefornootka-
50
asingledaywereseparatedfromlarvaeincohortsof tone was 1.460 (cid:2)g/mg (CI, 1.190Ð1.726 (cid:2)g/mg) in
600 and put into 35- by 35- by 35-cm experimental G3-2.502 (CI, 2.236Ð2.775 (cid:2)g/mg) in AKRON. The
cages. carvacrolLD valuerangedfrom1.791(cid:2)g/mg(CI,
50
Assays. Three- to 7-d-old females were subdued 1.675Ð1.908 (cid:2)g/mg) in RSP-ST to 2.649 (cid:2)g/mg (CI,
withCO andplacedonachilltable(BioQuipProd- 2.378Ð2.905 (cid:2)g/mg) in G3. The LD value for per-
2 50
ucts, Rancho Dominguez, CA) maintained at 4(cid:2)C. methrinwas0.000214(cid:2)g/mginG3and0.00358(cid:2)g/mg
Each female was treated on her dorsal thorax with in RSP-ST. Propoxur had an LD value of 0.000719
50
November2010 MCALLISTERANDADAMS:MODEOFACTIONFORESSENTIALOILCOMPONENTS 1125
(cid:2)g/mg in G3 and 3.628 (cid:2)g/mg in AKRON, whereas Development of these compounds into commer-
fordieldrintheLD valuewas0.00455(cid:2)g/mginG3 cially available products would give vector control
50
and6.628(cid:2)g/mginP(cid:1)DLRC(cid:1)R(cid:1). newproductswithalternativemodesofactionfrom
Resistance ratios (RRs) were calculated for each thosecurrentlyavailable.Theseproductsalsowould
compoundtested.Aratioof(cid:3)1indicatesthe“resis- benovelinthattheyweredevelopedforvectorcon-
tant”strainismoresusceptiblethanthe“susceptible” trol and not adapted from existing agriculture uses.
strain;however,inthisstudyallRRswerecloseto1 Furthermore,theyhavethepotentialofbeingmore
for the tree oil components. The thymoquinone RR readilyacceptablebythepublicbecauseoftheiror-
was 0.65, 0.82, and 1.06 in RSP-ST, D(cid:1)DLRC(cid:1)R(cid:1), iginsfromnaturalsources.
andAKRON,respectively.NootkatoneRRwas1.12,
1.47,and1.71,andcarvacrolRRwas0.68,0.78,and0.79
Acknowledgments
forthesamemosquitostrainsinthesameorder.
ThiscontrastsdramaticallywiththeRRscalculated WethankMariahScott,ErinBorland,AndreaPeterson,and
for the resistant strains and insecticides known to MichaelHayashiforhelpwithrearingmosquitoesandBrad
BiggerstaffandMarkDeloryforhelpwiththestatisticalanalysis.
affect the target sites containing the mutations con-
Wealsothankthefollowingindividualsfordonatingmosquito
ferringresistance.Propoxur,acarbamate,inhibitsace-
strainstotheMR4program,partofAmericanTypeCulture
tylcholinesterase; dieldrin, a cyclodiene, acts by in-
Collection: F. Collins and H Ranson, RSP-ST; M. Benedict,
hibitingGABA;andpermethrin,apyrethroid,works P(cid:1)DLRC(cid:1)R(cid:1);M.Akogbeto,AKRON;andW.Collins,G3.The
onthegatedsodiumchannel(Ware1991).TheRRfor DepartmentofDefenseprovidedfundingforthisstudythrough
permethrin in the RSP-ST strain with resistance to theDeployedWarFighterProtectionProgram.
pyrethroidswas16.42.TheRRforpropoxurwas5,046
in the AKRON strain with organophosphate and
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