DTIC ADA578685: Mini Review: Mode of Action of Mosquito Repellents

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PesticideBiochemistryandPhysiologyxxx(2013)xxx–xxx
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Pesticide Biochemistry and Physiology
journal homepage: www.elsevier.com/locate/pest
Mini review: Mode of action of mosquito repellents
⇑
Joseph C. Dickens , Jonathan D. Bohbot
UnitedStatesDepartmentofAgriculture,AgriculturalResearchService,HenryA.WallaceBeltsvilleAgriculturalResearchCenter,PlantSciencesInstitute,InvasiveInsectBiocontrol
andBehaviorLaboratory,Beltsville,MD,USA
a r t i c l e i n f o a b s t r a c t
Articlehistory: Themodeofactionofmosquitorepellentsremainsacontroversialtopic.However,electrophysiological
Availableonlinexxxx studiesandmolecularapproacheshaveprovidedabetterunderstandingofhowrepellentsexerttheir
effects. Here, webriefly discuss various theories ofrepellentaction and present the current status of
Keywords: knowledge of the effects of repellents on olfactory and gustatory processes. These findings provide a
Insectrepellent framework for further development of existing repellents and the discovery of new compounds with
Feedingdeterrent novelmodesofaction.
Olfaction
PublishedbyElsevierInc.
Gustation
Modulation
1.Introduction availableforsinglecellrecordingsfromORNsontheantennaeof
mosquitoesandanumberofrepellentcompoundsweretestedfor
Mosquitoes vector numerous diseases including malaria, den- theiractivityonthesecells.Basedmostlyontheseelectrophysio-
gue,westnilevirusandyellowfever.Evenintheabsenceofdisease, logicalstudies, Davis and his colleagues hypothesized that repel-
mosquitoesare anannoyance that can disrupt outdoor activities. lentshadtheireffectbymodifyingorblockingresponsesofORNs
Theuseofrepellentsdecreasescontactsbetweenmosquitoesand normallysensitive toattractants.Thisideawas supportedby the
theirhosts,andmayevenlowertherateofdiseasetransmissionin observationthatDEETdecreasedthesensitivityofbothlacticacid
manyinstances[1].Themostcommonlyusedmosquitorepellent, sensitive ORNs to lactic acid, a component of human sweat [5],
DEET (N,N-diethyl-3-methylbenzamide), was discovered over and an ORN sensitive to an oviposition attractant, ethyl proprio-
60yearsagoandhasbeeninusesincethe1950’s[2].Manyother nate [6].
compounds have been characterized as having repellent activity
formosquitoesaswellasotherarthropodvectorsbasedonlabora- 2.2.DEETexertsitseffectsbyactivatingspecificORNsorspecific
torybehavioralbioassaysortopicalapplicationofthecompounds odorantreceptors(ORs)
totheskinforfieldandlaboratorytesting[3].
Here,webrieflyoutlinevarioustheoriesonthemodeofaction Boeckhandhiscolleagues[7]showedthattwoORNs(basedon
ofrepellents.Thenwepresentrecentstudiesmostlyfromourlab, differentactionpotentialamplitudes)associatedwithA-2sensilla
whichprovideinsightintosomeoftheearlytheoriesonthemode ontheantennaofAedesaegyptiwereactivatedbyDEET.Theypos-
of action of insect repellents, and a model for future research tulatedthatsincetheseneuronswerenotactivatedbyattractants
aimedatdiscoveryofnewcompoundswithrepellentaction. thatamessagemaybesenttothecentralnervoussystemwhich
counteractstheperceptionofattractantsbyotherneurons.How-
ever,theydidnotruleoutdirectinhibitionofanattractantrecep-
2.Theoriesofrepellentaction
tor neuron as Davis and his colleagues had shown earlier [5,6].
Syed and Leal (2008) showed that DEET activated a specific ORN
2.1.DEETmasksresponsesofolfactoryreceptorneurons(ORNs)to
in a trichoid sensillum on the antennae of Culex quinquefasciatus
attractants
[8]. The demonstration that DEET activated a specific odorant
receptor(OR)inlarvalAnophelesgambiaeprovidedadditionalsup-
Thefirstdetailedinvestigationsofthemodeofactionofrepel-
portforthistheory[9].
lentsweresummarizedbyDavis[4].Atthetime,techniqueswere
⇑ 2.3.DEETsequestersanattractant
Correspondingauthor.Address:USDA,ARS,BARC,PSI,IIBBL,10300Baltimore
Avenue,Beltsville,MD20705,USA.Fax:[phone].
Syed and Leal [8] showed that when DEET was released from
E-mail addresses: [email protected], [email protected] (J.C.
Dickens). odor cartridges with the attractant component, octenol, the
0048-3575/$-seefrontmatterPublishedbyElsevierInc.
http://dx.doi.org/10.1016/j.pestbp.2013.02.006
Pleasecitethisarticleinpressas:J.C.Dickens,J.D.Bohbot,Minireview:Modeofactionofmosquitorepellents,Pestic.Biochem.Physiol.(2013),http://
dx.doi.org/10.1016/j.pestbp.2013.02.006
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Mini review: Mode of action of mosquito repellents
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14. ABSTRACT
The mode of action of mosquito repellents remains a controversial topic. However, electrophysiological
studies and molecular approaches have provided a better understanding of how repellents exert their
effects. Here, we briefly discuss various theories of repellent action and present the current status of
knowledge of the effects of repellents on olfactory and gustatory processes. These findings provide a
framework for further development of existing repellents and the discovery of new compounds with novel
modes of action.
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Fig.1. Modesofactionofinsectrepellents.A.FixativeeffectofDEETontheattractant,octenol.B.Interactionofcitronellalwithareceptorassemblagethroughanallosteric
siteonDrosophilaOrco.C.ActivationofamosquitoTRPA1channelbycitronellal.D.ActivationofOR8-OrcobyinteractionofoctenolwiththeorthostericsiteonOR8.E.
InhibitionofoctenolresponsebyinteractionofDEETwithanallostericsiteonOR8.F.ActivationofOR2-OrcobyinteractionofindolewiththeorthostericsiteonOR2.G.
ActivationofOR2-OrcobyinteractionofDEETwiththeorthostericsiteonOR2.
amount of octenol released from the cartridge was reduced. This 2.5.Abotanicalrepellent,citronellal,interactswithtwodistinct
effectledtosmallerresponsesofoctenolORNsinAe.aegypti.They molecularpathwaystomediaterepellency
alsoshowedthatDEETappliedtotheskinchangedthe‘‘chemical
profile’’ofvolatilesbeingreleased,perhapsdecreasingtheattrac- Kwon et al. [12] showed that citronellal interacted with the
tivenessoftheskin.However,thisfixativeeffect(Fig.1A)wasre- olfactoryco-receptorOrcoandwithTRPA1channelsinAn.gambiae
futedbyanotherstudybyPellegrinoetal.[10]. and D. melanogaster (Fig. 1B and C). In An. gambiae, the TRPA1
channelisdirectlyactivatedbycitronellal,whereasinDrosophila,
citronellalmayregulatetheactivityofaCa2+-activatedK+channel
byinteractingwithTRPA1.
2.4.DEETstimulatesagustatoryreceptorneuron(GRN)sensitiveto
bitteraversivecompoundsinDrosophila 2.6.DEETmodulatesresponsesofspecificORNsandORstotheir
ligands
Leeetal.[11]showedthatDEETsuppressesthefeedingbehav-
iorofthevinegarfly,Drosophilamelanogaster.GRNshousedinthe Bohbot and Dickens [13] used Xenopus oocytes as an ex vivo
short sensilla on the outer labellum of the fly responded to both expressionsystemtoexplorethemolecularreceptiverangeofAe.
DEETandotherbitterfeedingdeterrentssuchasquinine.Theseef- aegyptiORs.Thesepharmacologicalstudiesrevealedthattheactiv-
fects were determined to be mediated by direct interactions be- ityofORscouldbemodulatedbyavarietyofinsectrepellents.This
tweenDEETandseveralgustatoryreceptors(GRs). ideaprovidedsupportforanearlierstudythatshowedthatDEET
Pleasecitethisarticleinpressas:J.C.Dickens,J.D.Bohbot,Minireview:Modeofactionofmosquitorepellents,Pestic.Biochem.Physiol.(2013),http://
dx.doi.org/10.1016/j.pestbp.2013.02.006
J.C.Dickens,J.D.Bohbot/PesticideBiochemistryandPhysiologyxxx(2013)xxx–xxx 3
andotherrepellentsmaystimulatespecificORs(Fig.1DandE)or odorcartridgeandreachingthesurfaceoftheORNcannotbeprac-
inhibitresponsesofORstoattractantsinAe.aegypti(Fig.1FandG). ticallymeasured.
Thus, messages received by the CNS were scrambled resulting in Octenol (=1-octen-3-ol) was identified as a natural attractant
disorientationoftheinsect. for mosquitoes in 1989 [22]. A study using single-cell recordings
in Culex pipiens demonstrated the selectivity of the ‘‘C’’ neuron,
housed in basiconic sensilla on the maxillary palps, toward the
2.7.Oldinsightsstimulatenewquestionsandanswers
(R)-enantiomerofoctenol[23].Thesameyear,two-electrodevolt-
ageclampofXenopusoocytesexpressingtheAn.gambiaeoctenol
Afterthenumerouseffortsover60yearssincethediscoveryof
receptor(OR8-Orco),revealedincreasedresponsestothe(R)-enan-
DEET,themodeofactionofDEETandotherrepellentsremainsa
tiomerofoctenol[21](Fig.1D).Thisinformation,incombination
matter of debate. In the last 5years a handful of reports have
with expressiondata [21,24], strongly suggested that the natural
implicated olfactory, gustatory and temperature signaling path-
odorantligandforOR8was(R)-octenolandpavedthewayforade-
ways.Whilethesestudieshavebeguntouncoverthemechanisms
tailedpharmacologicalanalysiswiththeAe.aegyptiOR8ortholog
ofactionofinsectrepellents,theyhavealsogeneratednewques-
[25].
tions.HowcanDEETandotherrepellentsinteractwithunrelated
receptorproteins?Doesthisquestionrelatetothebroadspectrum
3.2.TheodorantreceptiverangeofORNsandORs
activityofrepellentsonphylogeneticallydiversearthropods?How
doestheactivationandinhibitionofORsbyDEETrelatetoitsef-
Usingvoltageclamprecordings,OR8-Orcoresponseswereeval-
fectsoninsectbehavior?Thefollowingsectionsfocusonthenewer
uatedwhenexposedtooctenolandvariousanalogswithminimal
discoveries regarding the physiological and molecular effects of
structural modifications (Fig. 2) [25]. OR8-Orco was estimated to
repellentsonORsandGRsinanattempttoaddresstheseintrigu-
be 100-fold more sensitive to the (R) enantiomer than to the (S)
ingquestions.
form,adifferencelikelyunderestimatedconsideringthepresence
oftraceamountsoftheoppositeenantiomerineachtestedsample.
3.ActionofrepellentsonORNsandORs PerhapsmoreremarkablewasthatOR8-Orcoaffinitywasstronger
for other octenol analogs than for (S)-octenol. This experiment
3.1.Electrophysiologicaltechniquesusedtostudyligand-gatedORs demonstratedthatanORwasabletoachieveremarkablesensitiv-
ityandspecificitytowardanon-pheromonalcompound.
ElectrophysiologicalrecordingsfromORNsinvivoandORsex- Resultsoftheexvivostudiesweretestedinvivousingsingle-
pressedheterologously(=exvivo)offerapowerfulcombinationof cellrecordingsfromoctenolsensitiveORNsinsensillaonthemax-
tools for the study of the mode of action of mosquito repellents. illarypalps[26].UsingCO freeair,the‘‘C’’neuronwaschallenged
2
ThebiochemicalenvironmentofORsremainsanactivefieldofre- with the same octenol analogs and the two octenol enantiomers
searchaswehaveonlyapartialunderstandingofthecomponents tested in the heterologous expression system. While the actual
involved. Insect ORs are heteromeric ligand-gated ion channels. concentration of odorant reaching the ORN could not be deter-
They are comprised of a variable odorant-sensing subunit (ORx) mined,thedataconcurredwiththoseobtainedfromvoltageclamp
andanobligatoryandinvariableORco-receptor(Orco)thatform recordings from Xenopus oocytes expressing OR8-Orco. Increased
ORx-Orco[14–16]. WhileORsplayapivotalroleinolfactorysig- differencesinthesensitivityto(R)-octenolrelativetoothercom-
naling,otherfactorsinthelymphofanolfactorysensillum,includ- poundsobservedinvivoweredifficulttointerpretduetoinherent
ing odorant-binding proteins (OBPs), sensory neuron membrane differencesinstimulusdeliverybetweenvoltageclampandsingle-
proteins (SNMPs), and odorant-degrading enzymes (ODEs) may cellrecordingtechniques.Thus,aroleforaccessoryproteinssuch
influencetheirfunction. as OBPs could not be excluded in enhancing the sensitivity of
Inordertoeliminatethesefactors,ORsmaybeexpressedout- OR8-Orco in vivo; many studies have suggested that OBPs might
sidethesensillum,invariouscellexpressionsystemssuchasthe influencethe solubilityofodors withinthe sensillumlymphsur-
oocytesofthefrogXenopuslaevis.Thispharmacologicaltechnique roundingtheneuron.
allows for the establishment of precise concentration–response TwootherORsprovidedpharmacologicalprospectstostudythe
relationships,asthequantityofthestimulustowhichtherecep- effectofmosquitorepellents.OR2andOR10areparalogousmem-
torsareexposedisknown.PriorknowledgeoftheORanditsnat- bersofaconservedgroupofindolereceptorspresentinboththe
ural ligand is strongly recommended. ORs, like other classes of aedine and anopheline mosquitoes [27]. Using voltage clamp
receptors,arelikelytopossessmultiplerecognitionsitesincluding recordings from Xenopus oocytes expressing OR2-Orco, indole
aputativeprimary‘‘orthosteric’’siteinteractingwithevolutionary was identified as a biologically meaningful ligand for OR2
selectedsemiochemicalsandsecondary‘‘allosteric’’siteswhichre- (Fig. 1F). Due to the protein sequence identity between OR2 and
act with chemicals with no specific biological meaning (e.g. syn- OR10,itwaspredictedthatthelatterwouldspecificallyrecognize
theticcompounds)[17]. anindoleanalog[27].Subsequently,3-methyl-indole(skatole)was
Inordertotestthepharmacologicalinformationobtainedusing showntobethecognateligandforOR10[28].Theseexperiments
Xenopus oocytes, these same experiments may be conducted led to three important conclusions: (1) ORs are capable of a
in vivo on basiconic sensilla, which house three ORNs [18] that remarkablelevelofdiscriminationwithouttheassistanceofother
can be functionally distinguished based on the shape and ampli- factorssuchasOBPs,(2)non-pheromonereceptorsexhibitligand
tude of their action potentials. The ‘‘A’’ neuron characterized by specificity and sensitivity on par with pheromone receptors, and
the largest amplitude action potential responds to CO [19]. The (3)themoleculargeometryofsemiochemicals areadeterminant
2
‘‘B’’ neuron has no known effective stimulus and constitutively factorforproperligandrecognition.
generatesmediumsizedactionpotentials.Finally,the‘‘C’’neuron, Theresultsoftheaforementionedstudiesagreedwiththethe-
withthesmallestamplitudeactionpotential,respondstooctenol ory that ORs possess a highly specific primary recognition site
[20]andisthoughttoexpresstheOR8-Orcoreceptorassemblage (orthostericsite)forabiologicallymeaningfulsignal(semiochem-
[21].Whilesingle-cellrecordingisapowerfultechniquetostudy ical)and‘‘specialist’’ORsnowincludednon-pheromonalreceptors.
neuron physiology in vivo, the establishment of dose–response Theseideasrequiredare-examinationoftheconceptof‘‘general-
relationshipsbetweenORNactivityandodorantsordrugspresents ist’’ORs,i.e.,receptorsactivatedbyligandswithvariouschemical
onenotablelimitation:theexactquantityofstimulusleavingthe geometry[17].TheabilityofanORtorecognizeavarietyofligands
Pleasecitethisarticleinpressas:J.C.Dickens,J.D.Bohbot,Minireview:Modeofactionofmosquitorepellents,Pestic.Biochem.Physiol.(2013),http://
dx.doi.org/10.1016/j.pestbp.2013.02.006
4 J.C.Dickens,J.D.Bohbot/PesticideBiochemistryandPhysiologyxxx(2013)xxx–xxx
Fig.2. Structureof(R)-and(S)-enantiomersof1-octen-3-olandanalogssupposedlyinteractingwithorthostericsitefor(R)-1-octen-3-olonOR8.Bothoctenolenantiomers
andeachofthecompoundsshownweretestedonoctenolreceptorneuronsinvivoandOR8-OrcoexpressedheterologouslyinXenopusoocytes.Therelativesensitivitiesof
OR8-Orcotoward(R)-1-octen-3-ol(s)andoctenolanalogs(d)weretranslatedfromEC50valuesintosurfaceareas.
requires either a single but variable-geometry ligand recognition DEETreducedORsensitivityandthemaximumeffectoftheago-
site(singlebindingdomain)ormultiplerecognitionsites(multiple nistodorants[31].Asurveyofthepotentialeffectsofvariousin-
bindingdomains).Growingexperimentalevidencesupportthelat- sect repellents on OR8-Orco and OR2-Orco confirmed that these
terideawithoutexcludingsomelimiteddegreeofligandpromis- compoundseitherelicitedspecific,dominantorunspecificagonist
cuity[25]. or antagonist effects depending on the ORx tested [30]. More
importantly, these results were largely corroborated by in vivo
studiesshowingthatDEETalteredthefine-tuningoffunctionally
3.3.DEETmodulatestheactivityofORs
diverseORNs[10].However, theexactoperatingmechanismsfor
themodeofactionofinsectrepellents,viaorthostericorallosteric
SeparatestudieshaveshownthatDEET,aknowninsectrepel-
sites,werenotconclusivelydemonstratedandthemultimericnat-
lent, selectively activates [9] or inhibits ORs [29]. This synthetic
ureofORscompoundedtheinterpretationsoftheseresults.
compound,notencounteredinnaturebyinsects,wasnonetheless
Usinghigh-throughputscreenstosearchfornovelmodulators
abletointeractwithORs,albeitathighconcentrations.Howcould
of An. gambiae ORs expressed in human embryonic kidney cell
this compound exert agonist and antagonist effects on different
lines, a new class of synthetic chemicals, known as VUAA were
ORs? We used our knowledge of OR-ligand pairs to study this
shown to activate Orco alone [31]. These results were the proof
question. OR2, OR8 and OR10 exhibit a wide range of sequence
ofconceptfortheexistenceofallostericsitesconsideringthatOrco
similarities [24]. OR2 and OR10 share 69% amino acid identity,
isthenon-sensingsubunitoftheORx-Orcocomplex.Anadditional
and14%identitywithOR8.Theseproteinsequencevariationspro-
studyontheactivationofOrcobyaVUAAanalogwasconsistent
videdacontroltotestthemolecularbasisofthepossiblemodeof
withtheideathatOrcoformsmultimericassemblages[32].
actionofinsectrepellents.Wealsoexpandedourstudiestoinclude
structurally different insect repellents including both synthetic
compoundsandnaturallyoccurringchemicals[30]usingXenopus 4.ActionofrepellentsonGRNsandGRs
oocytes heterologously expressing OR2, OR8 or OR10 along with
Orco. While mosquito repellents have mostly been studied in an
Thesestudiessuggestedthatmosquitorepellentsexertmultiple olfactory context, several reports have indicated that repellents
effectsonORs.Forexample,whileDEETaloneactivatedOR2-Orco maybedetectedbyviagustatoryreceptors(=contactchemorecep-
(Fig.1G),itinhibitedOR8-Orcoresponsetooctenol[13](Fig.1E). tors) on the labella (Fig. 3A and B) and function as feeding
Pleasecitethisarticleinpressas:J.C.Dickens,J.D.Bohbot,Minireview:Modeofactionofmosquitorepellents,Pestic.Biochem.Physiol.(2013),http://
dx.doi.org/10.1016/j.pestbp.2013.02.006
J.C.Dickens,J.D.Bohbot/PesticideBiochemistryandPhysiologyxxx(2013)xxx–xxx 5
deterrents.Christophers[33]suggestedthatcompoundswithlow potentialsrecorded,atleastthreeGRNsarehousedwithinindivid-
volatility acted as contact repellents. Bar-Zeev and Schmidt [34] ual sensilla on the labella (Fig. 3B). The neuron with the largest
usedaradiotracertopresentevidencesuggestingthatlowconcen- amplitudeactionpotentialrespondedtoincreasingconcentrations
trationsofDEETweredetectedbycontactchemoreceptorsonthe ofNaCl(Fig.3C).Asomewhatsmalleramplitudeactionpotential
labellaofAe.aegypti.MorerecentlyDEETandotherrepellentswere was activated by sucrose (Fig. 3D), while the smallest amplitude
showntoactasfeedingdeterrentsinbehavioralbioassays[35,36]. action potential was activated by the feeding deterrent, quinine
The suggestion that DEET and other repellents acted as feeding (Fig.3E).Thisneuronwiththesmallestamplitudeactionpotential
deterrents indicates that gustatory receptors might be targeted wasalsoactivatedinadosedependentmannerbyDEET,andre-
byinsectrepellents. sponses were elicited by Picaridin, IR3535, and citronellal
We have recently used single-cell recordings from sensilla on (Fig. 3E). GRNs in the fruit fly, D. melanogaster are also activated
thelabellaofAe.aegyptitodemonstratethepresenceofaGRNthat byDEET[11].
respondstoDEETandotherrepellentsincludingPicaridin,IR3535, ThediscoveryofaGRNthatrespondsreliablytoDEETandother
and citronellal [37]. Based on the size and shape of the action repellents is a first for mosquitoes or, for that matter, any
Fig.3. A.LocationofsensillaonthelabellaofAe.aegypti.B.Electrophysiologicalrecordingsfromsensillaonthelabellarevealedactionpotentialsfromatleast3gustatory
receptorneurons(GRNs).C.ThelargestamplitudeactionpotentialrespondedtoincreasingconcentrationsofNaCl.D.Asomewhatsmalleramplitudeactionpotential
respondedtosucrose.E.Thesmallestamplitudeactionpotentialwasactivatedbyquinine,afeedingdeterrent,andtheinsectrepellentsDEET,IR3535,Picaridin,and
citronellal.
Pleasecitethisarticleinpressas:J.C.Dickens,J.D.Bohbot,Minireview:Modeofactionofmosquitorepellents,Pestic.Biochem.Physiol.(2013),http://
dx.doi.org/10.1016/j.pestbp.2013.02.006
6 J.C.Dickens,J.D.Bohbot/PesticideBiochemistryandPhysiologyxxx(2013)xxx–xxx
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