Drosophila TRPA1 Channel I

Current Biology 20, 1672–1678, September 28, 2010 ª2010 Elsevier Ltd All rights reserved DOI 10.1016/j.cub.2010.08.016

Reports Required

to Avoid the Naturally OccurringInsect Repellent Citronellal

Young Kwon,1,4 Sang Hoon Kim,1,4 David S. Ronderos,2,4

Youngseok Lee,1 Bradley Akitake,1 Owen M. Woodward,3

William B. Guggino,3 Dean P. Smith,2 and Craig Montell1,*1Department of Biological Chemistry and Department ofNeuroscience, Center for Sensory Biology, The Johns HopkinsUniversity School of Medicine, Baltimore, MD 21205, USA2Department of Pharmacology and Department ofNeuroscience, University of Texas Southwestern MedicalCenter, Dallas, TX 75390, USA3Department of Physiology, The Johns Hopkins UniversitySchool of Medicine, Baltimore, MD 21205, USA

Summary

Plants produce insect repellents, such as citronellal, whichis themain component of citronellal oil. However, themolec-

ular pathways through which insects sense botanical repel-lents are unknown. Here, we show that Drosophila use two

pathways for direct avoidance of citronellal. The olfactorycoreceptor OR83b contributes to citronellal repulsion and

is essential for citronellal-evoked action potentials. Muta-tions affecting the Ca2+-permeable cation channel TRPA1

result in a comparable defect in avoiding citronellal vapor.The TRPA1-dependent aversion to citronellal relies on a G

protein (Gq)/phospholipase C (PLC) signaling cascaderather than direct detection of citronellal by TRPA1. Loss

of TRPA1, Gq, or PLC causes an increase in the frequency

of citronellal-evoked action potentials in olfactory receptorneurons. Absence of the Ca2+-activated K+ channel (BK

channel) Slowpoke results in a similar impairment in citro-nellal avoidance and an increase in the frequency of action

potentials. These results suggest that TRPA1 is requiredfor activation of a BK channel tomodulate citronellal-evoked

action potentials and for aversion to citronellal. In contrastto Drosophila TRPA1, Anopheles gambiae TRPA1 is directly

and potently activated by citronellal, thereby raising thepossibility that mosquito TRPA1 may be a target for devel-

oping improved repellents to reduce insect-borne diseasessuch as malaria.

Results and Discussion

In Drosophila, a trap-based assay has been employed toinvestigate the mechanism of action of the most commonlyused man-made repellent, N,N-diethyl-meta-toluamide(DEET) [1, 2]. The assay does not monitor behavioral avoid-ance of a volatile repellent; rather, it tests for inhibition ofattraction to food odors.

To measure repulsion to chemical vapors, we developeda binary-choice assay (see Figure S1A available online). Weapplied a repellent and a control chemical to the bottoms oftwo test tubes, inserted screens to prevent physical contactwith the compounds, and introduced w100 flies into the

*Correspondence: cmontell@jhmi.edu4These authors contributed equally to this work

connected tubes. After an incubation period, we counted theflies in the two test zones and calculated the avoidance index(AI). This direct airborne repellent test (DART) has two advan-tages over the trap assay. First, it measures direct repulsion toan airborne chemical vapor. Second, it monitors the responseto a repellent in minutes, compared to 24–72 hr used in the trapassay [1].To evaluate the DART assay, we tested benzaldehyde. This

odorant caused dose-dependent avoidance, whereas thevehicle (DMSO) or a nonvolatile aversive tastant (quinine) didnot (Figure S1C). Thus, the DART assay effectively measuresevasive behavior, rather than the masking of a food odor.Benzaldehyde (0.1% or 1%) elicited avoidance within 15 min,and the AIs peaked between 30 and 60 min (Figure S1D). After4 hr, there was still strong avoidance, which then declined,possibly as a result of desensitization or a reduction in thechemical gradient.Citronellal is used commonly in lotions, sprays, and candles

to repel mosquitoes, fleas, and ticks [3]. Indeed, as observedby using a variation of theDART assay (Figure S1B),Anophelesgambiae avoided citronellal and benzaldehyde (Figure 1A).To address whether citronellal repels Drosophila, we per-formed the DART assay. We found that flies avoided citronellalin a dose-dependent manner (Figure 1B), and there was nodifference between males and females (Figure S1E). As withbenzaldehyde, the aversion to citronellal peaked between 30and 60 min (Figure 1C).The synthetic repellent DEET has been reported to function

through masking the response to attractive odors in a trapassay [2]. Addition of DEET to the entrance of a food-contain-ing vial results in a larger proportion of flies selecting the foodvial without the DEET [2] (Figure S1F). 1% citronellal alsoreduced the attractive response to food in the trap andDART assays (Figures S1F and S1G). As with citronellal,DEET functioned as a direct repellent using the DART assay(Figure S1H).Citronellal avoidancewas through airborne vapor rather than

contact chemosensation, because a screen separated the fliesand the repellent (Figure S1A). The hair-like sensilla that housethe olfactory receptor neurons (ORNs) are located on the mainolfactory organ, the antenna, and the maxillary palp [4, 5].Consistent with the detection of citronellal through the senseof smell, animals with ablated antennae and maxillary palps,or ablated antennae only, showed dramatic decreases inavoidance (Figure 1D). To address a requirement for ORNsfor repulsion tocitronellal,we inactivated theORNsusingeitheran inward-rectifying K+ channel, Kir2.1 (UAS-kir2.1) [6], whichhyperpolarizes neurons and thereby interferes with productionof action potentials, or tetanus toxin (UAS-TNT) [7], whichprevents synaptic transmission. We expressed UAS-kir2.1and UAS-TNT in ORNs under control of the Or83b promoter(Or83b-GAL4) [8]. OR83b is a coreceptor required for detectinga broad range of odorants because it promotes trafficking ofother olfactory receptors to dendrites [8]. We found that inacti-vationofORNsbyexpression ofKir2.1 or tetanus toxin stronglyimpaired citronellal avoidance (Figure 1D).To identify the ORNs mediating citronellal detection, we

surveyed the electrophysiological responses of the olfactory

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Figure 1. Avoidance to Airborne Citronellal Vapor

(A) A variation of the direct airborne repellent test assay (Figure S1B) was used to assay repulsion of female Anopheles gambiae to citronellal and benzal-

dehyde (10% each).

(B) Avoidance to 0.1%–10% citronellal vapor.

(C) Time dependence for avoidance to 1% citronellal.

(D) Requirement of antenna andOr83b-expressing olfactory receptor neurons (ORNs) for citronellal repulsion. *p < 0.05 versus wild-type control by analysis

of variance (ANOVA).

(E) Survey of single-sensillum recording (SSR) responses of the chemosensory neurons in the third antennal segment to 10% citronellal.

(F) Cartoon indicating location of novel ab11 and ab12 sensilla (black box).

(G and H) Representative SSR traces of spontaneous activity demonstrating presence of three ORNs (top) and unique odorant response profiles for ab11 (G)

and ab12 (H) sensilla to 1% odorants (bottom).

Data are shown asmean6 standard error of themean (SEM). The numbers of experiments (n) for the SSRs shown in (E), (G), and (H) are indicated in Table S1.

Flies Use Dual Pathways for Avoiding Citronellal1673

sensilla located on the third antennal segment. Olfactorysensilla are classified into three morphological types, basi-conic, coeloconic, and trichoid, and each contains the odor-sensitive dendrites of one to four ORNs (reviewed in [9, 10]).Using single-sensillum recordings (SSRs), we assayed thesensitivity of previously characterized sensilla to citronellaland found that none of these sensilla responded strongly to

citronellal [11, 12] (Figure 1E). Therefore, we surveyed twopreviously uncharacterized basiconic sensilla, ab11 andab12, located on the lateral surface of the third antennalsegment below the aristae (Figure 1F). The ab11 and ab12sensilla each contained three neurons that had spontaneousactivity (activity in the absence of odorants) with distinct spikeamplitudes. We named these neurons ab11a–c and ab12a–c,

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Figure 2. Requirement for trpA1 for Avoidance to

Airborne Citronellal Vapor

(A) Avoidance of trp mutants to 1% citronellal.

(B)Wild-type and trpA11 responses to 0.1%–10%

citronellal.

(C) Rescue of the trpA11 phenotype with either

Drosophila (dtrpA1-A and dtrpA1-B) or Anoph-

eles gambiae (agtrpA1-A and agtrpA1-B) trpA1

transgenes using the GAL4/UAS system.

(D)Wild-type and trpA11 responses to 0.1%–10%

benzaldehyde.

(E) Expression of UAS-trpA1 in ORNs via the

Or83b-GAL4 restored citronellal repulsion in

trpA11.

(F–H) Expression of the trpA1 reporter in second

and third antennal segments under control of the

trpA1-GAL4 and UAS-GFP.

(F) Staining in a coronal plane of an antenna from

a late-stage pupa. GFP-positive neurons in the

third antennal segment are indicated with arrow-

heads.

(G)High-magnificationviewof the inset inpanel (F).

(H) Staining in a transverse plane of an antenna

from a late-stage pupa.

Data are shown as mean6 SEM. *p < 0.05 versus

wild-type control by ANOVA; #p < 0.05 between

the indicated flies by unpaired Student’s t test.

Current Biology Vol 20 No 181674

in descending order of relative amplitude (Figures 1G and 1H).ab11 and ab12 contained one olfactory neuron each (ab11aand ab12a, respectively) that was activated strongly bycitronellal (Figures 1G and 1H). Of 11 odorants tested, onlycitronellal induced a high frequency of action potentials inab11 (Figure 1G); ab12 also responded strongly to benzalde-hyde (ab12b) and moderately to methyl salicylate (ab12a;Figure 1H).

To address the identities of proteins required for citronellalavoidance, we considered transient receptor potential (TRP)channels. These cation channels are candidates becausemany are modulated in vitro by noxious compounds, someof which induce pain or have repellent properties [13–22].The Drosophila genome encodes 13 TRP channels, 11 ofwhich are dispensable for adult viability. To address whethera Drosophila TRP channel is essential for citronellal avoidancebehavior, we tested all 11 viable mutants. The responses of 10were indistinguishable from wild-type (Figure 2A). In contrast,the repellent reaction to 1% citronellal was reduced greatly intrpA11 [23] and trpA1ins [24] mutant flies (Figures 2A and 2B).At a concentration of 10%, citronellal aversion was reducedbut not eliminated (Figure 2B). Expression ofUAS-trpA1 underthe control of the trpA1-GAL4 rescued the trpA11 phenotype

(Figure 2C; Figure S2A). The trpA11

defect was not due to a general impair-ment in olfaction, because the avoid-ance to benzaldehyde was unaltered(Figure 2D).To address whether the mosquito

TRPA1 homolog might function in citro-nellal repulsion, we tested for rescue ofthe Drosophila trpA11 phenotype withamosquito trpA1+ transgene.We clonedthe Anopheles gambiae trpA1 gene(Figures S2D and S2E) and introducedthe transgene (UAS-agtrpA1-A) into

the Drosophila trpA11 background. Expression of theUAS-agtrpA1-A transgene in combination with the trpA1-GAL4 rescued the defect in trpA11 (Figure 2C; Figure S2B).During the isolation of mosquito trpA1 cDNAs, we found thatAnopheles expressed an alternative mRNA (agtrpA1-B) en-coding 34 residues distinct from those in TRPA1-A (666–699;Figure S2G). A corresponding mRNA isoform was alsoproduced in Drosophila (Figures S2F and S2G). We expressedUAS-agtrpA1-B or UAS-dtrpA1-B in trpA11 flies under thecontrol of the trpA1-GAL4 and found that either variantrescued the citronellal impairment (Figure 2C). These resultsindicate that a role for TRPA1 in citronellal avoidance isconserved in Anopheles.The finding that inactivation of ORNs with Kir2.1 or tetanus

toxin impaired citronellal avoidance (Figure 1D) raised thepossibility that trpA1 may function in these neurons.To address this possibility, we expressed UAS-trpA1-A underthe control of theOr83b-GAL4 and found that it restored citro-nellal avoidance in trpA11 (Figure 2E). This functional rescuestrongly supports the conclusion that trpA1 is required inORNs for sensing citronellal. Consistent with this finding,trpA1-GAL4, which rescued the trpA11 defect in combinationwith the UAS-trpA1 transgenes, drove expression in two to

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Figure 3. Expression of TRPA1 in Xenopus

Oocytes and Requirement for Phospholipase C

and Gqa for Avoiding 1% Citronellal

(A and B) Expression of Drosophila TRPA1 (A)

and Anopheles TRPA1 (B) in oocytes. Black

bars indicate the duration of a stimulus, and

white bars indicate washouts with stimulus-free

buffers.

(A) Representative effects of 1 mM citronellal

(mean DI 6 SEM = 20.16 6 0.03 mA, n = 6) and

a heat shift (w25�C to 39�C, DI = 211.0 6

2.7 mA, n = 3) on Drosophila TRPA1.

(B) Activation of Anopheles gambiae TRPA1

by increasing concentrations of citronellal

(10 mM, DI = 23.00 6 0.68 mA, n = 7; 100 mM,

DI = 25.48 6 0.78 mA, n = 10; 1 mM,

DI = 212.0 6 3.4 mA, n = 5).

(C) Requirement for norpA and dGqa for citro-

nellal avoidance.

(D) Expression ofUAS-norpA in combination with

the trpA1-GAL4 or the Or83b-GAL4 rescued the

norpAP24 phenotype.

(E) Requirement of norpA and dGqa in ORNs for

citronellal avoidance. We used RNA interference

(RNAi) to knock down norpA and dGqa via the

trpA1-GAL4 or Or83b-GAL4.

Data are shown as mean6 SEM. *p < 0.05 versus

wild-type control (except where indicated by

brackets) by ANOVA; #p < 0.05 between the indi-

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t test.

Flies Use Dual Pathways for Avoiding Citronellal1675

six neurons in the third antennal segment, in addition toseveral neurons in the second antennal segment (Figures2F–2H). Moreover, expression of UAS-kir2.1 via the trpA1-GAL4 caused a reduction in citronellal avoidance (Figure S2C).

To address whether TRPA1 might be modulated directly bycitronellal, we expressed wild-type Drosophila TRPA1 in Xen-opus oocytes. Consistent with a previous report, TRPA1 wasactivated transiently by a temperature increase from 25�C to39�C (Figure 3A) [25] andmore slowly by addition of increasingconcentrations of allyl isothiocyanate (AITC; Figure S3A) [26].However, concentrations of citronellal as high as 1 mMinduced little current (Figure 3A), and citronellal did not inhibittemperature-induced activity (Figure S3B). A higher concen-tration (2 mM) induced a relatively small current in TRPA1-injected oocytes, but not in controls injected with the vehicle(Figures S3C and S3D). This current was reversed rapidly ina citronellal-free buffer (Figure S3C). Thus, citronellal activatedDrosophila TRPA1 directly, but with low potency. In contrast,we found that 10 mM citronellal robustly activated TRPA1from Anopheles gambiae (Figure 3B).

Because Drosophila TRPA1 was activated poorly by citro-nellal, we considered the possibility that it might function

downstream of a G protein (Gq) andphospholipase C (PLC) signaling cas-cade. Multiple TRP channels are linkedto Gq/PLC signaling [18], and TRPA1contributes to the avoidance of somenoxious tastants [27] and to larvaltemperature discrimination in the 18�C–24�C range through a Gq/PLC signalingcascade [23]. Of significance here, theevasive behavior induced by citronellalwas reduced significantly in the dGqa1

and norpAP24 mutants (Figure 3C). The defect in norpAP24

was rescued by expressing UAS-norpA in combination witheither the trpA1-GAL4 or the Or83b-GAL4 (Figure 3D). RNAinterference (RNAi) knockdown of dGqa (UAS-dGqa-RNAi)and norpA (UAS-norpA-RNAi) under the control of either thetrpA1-GAL4 or the Or83b-GAL4 significantly reduced citro-nellal avoidance (Figure 3E). These results indicate that dGq,PLC, and TRPA1 function in the same ORNs and supporta model in which they act in a common signaling pathway.To characterize the consequences on citronellal-induced

ORN activity resulting from loss of TRPA1, we performedSSRs. For nearly all ORN types, there were no significantdifferences in citronellal responses between wild-typecontrols and trpA11 mutants, including the ab12a neurons(Figure S4A; data not shown), which also responded to benz-aldehyde (Figure S4B). However, rather than being reduced,citronellal-evoked action potential frequencies in trpA11

ab11a neurons were significantly higher than in controls(Figure 4A; Figure S4C). The increased activity in trpA11

ab11a neurons was reversed by expressing a wild-typetrpA1 transgene under control of either the trpA1-GAL4 orthe Or83b-GAL4 (Figure 4A; Figure S4C).

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Figure 4. Increased Action Potential Frequency in trpA11

(A and B) Representative SSR traces from ab11 sensilla stimulated with 10% citronellal.

(C) Quantification of deactivation defect observed in ab11a ORNs during 200 ms immediately following citronellal stimulus (n = 10).

(D) Impairment of citronellal avoidance by mutation or RNAi knockdown of slowpoke (slo).

(E) Reduction of citronellal avoidance in Or83b1 flies.

(F) Dose response to benzaldehyde in Or83b1 flies.

(G) Representative SSR traces from ab11 sensilla in control and Or83b1 neurons.

Data are shown as mean 6 SEM. *p < 0.05 versus wild-type control by ANOVA; #p < 0.05 between the indicated flies by unpaired Student’s t test.

Current Biology Vol 20 No 181676

We found two features of the citronellal responses that wereabnormal in the trpA11 ab11a neurons. First, therewas a highercitronellal-evoked action potential frequency than in wild-type(Figure 4A; Figure S4C). Second, there was a defect indeactivation in trpA11 ab11a neurons (Figures 4A and 4C).We observed the same two defective phenotypes in ab11neurons in the dGqa1 and norpAP24 mutants (Figure 4B),although only the increase in the evoked responses wasclearly different when testing significance by analysis of vari-ance (Figure S4D). These results support the conclusion thatthe dGqa1, norpAP24, and trpA11 mutations affect the citro-nellal response in an ORN in ab11.

The finding that there were increases in the frequency ofcitronellal-evoked action potentials was unexpected andraised a question as to the basis for these defects. TRPA1 is

a Ca2+-permeable channel, and because reduced activity ofCa2+-activated K+ channels (BK channels) increases thefrequency of action potential firing [28, 29], we wonderedwhether loss of TRPA1 caused reduced BK channel activity.If so, then a mutation in the gene (slowpoke, slo) encodingthe fly BK channel might phenocopy the trpA1 phenotype.In support of this model, the slof05915 mutation caused anincrease in the frequency of citronellal-evoked action poten-tials (Figures 4B; Figure S4D) and impaired citronellal avoid-ance (Figure 4D). Introduction ofUAS-slo-RNAi in combinationwith either the trpA1-GAL4 or the Or83b-GAL4 resulted ina similar defect in citronellal avoidance (Figure 4D).We propose that TRPA1 is required for activity of Slo, which

in turnmodulates citronellal-induced firing of action potentials.Slo might be required in many ORNs and be regulated by

Flies Use Dual Pathways for Avoiding Citronellal1677

additional TRP channels. In support of this proposal, ab12 alsoresponded to citronellal and displayed a higher frequency ofaction potentials in slof05915 but did not function througha Gqa/PLC/TRPA1 pathway (Figure S4A). Knockout ofa mammalian TRP channel, TRPC1, also disrupts the activityof a Ca2+-activated K+ channel (KCa) in salivary gland cells,and mutations affecting either TRPC1 or KCa result in similardefects in salivary gland secretion [30, 31]. Thus, a role forTRP channels in activating Ca2+-activated K+ channels mightbe a common but poorly appreciated general phenomenonthat is evolutionarily conserved.

The finding that loss of TRPA1 causes an increase ratherthan a decrease in citronellal-induced action potentialssuggests that there might be a TRPA1 independent-pathwayrequired for generating action potentials in response to citro-nellal. OR83b is a candidate for functioning in such a pathway,because mutation of Or83b interferes with the ability of thesynthetic repellent DEET to inhibit the attraction to food odors[2]. We found that Or83b1 mutant flies, or Or83b1 in trans witha deficiency that uncovers the locus, exhibited an impairmentin citronellal avoidance similar to that in trpA1 mutant flies(Figure 4E). An Or83b1 defect in the DART assay was notspecific to citronellal, because these flies were also impairedin the response to benzaldehyde (Figure 4F). We testedwhether the frequency of citronellal-induced action potentialswas altered in Or83b1 ab11 sensilla. In contrast to the trpA1mutant phenotype, none of the mutant Or83b1 ab11 neuronsresponded to citronellal (Figure 4G).

Our data indicate that there are dual pathways required forthe response to citronellal. OR83b is necessary for producingcitronellal-induced action potentials, and a Gq/PLC/TRPA1pathway appears to function in the modulation of actionpotential frequency by activating BK channels. We suggestthat an abnormally high frequency of action potentials maylead to rapid depletion of the readily releasable pools of neuro-transmitter, thereby muting the citronellal response. Interest-ingly, a loss-of-function mutation affecting a worm BK channelalso results in a behavioral phenotype—increased resistanceto ethanol [32]. Although Drosophila TRPA1 functions down-stream of a Gq/PLC signaling pathway, citronellal can alsodirectly activate TRPA1, but with low potency. Nevertheless,because Anopheles gambiae TRPA1 is also expressed in theantenna [33] and is activated directly by citronellal with highpotency, we suggest that mosquito TRPA1 represents a newpotential target for in vitro screens for volatile activators thatmight serve as new types of insect repellents.

Supplemental Information

Supplemental Information includes four figures, one table, and Supple-

mental Experimental Procedures and can be found with this article online

at doi:10.1016/j.cub.2010.08.016.

Acknowledgments

We thank the Bloomington Drosophila Stock Center for fly stocks and

FlyBase for critical information. We also thank P. Garrity for the UAS-trpA1

flies and trpA1 plasmid constructs, S. Das and G. Dimopoulos for the

Anopheles cDNA pool and providing female Anopheles, H.S. Shim for fly

husbandry, K. Venkatachalam for suggesting that loss of a BK channel

(Slowpoke) might phenocopy the trpA1 phenotype, and M. Fowler for help

with functional expression of the Anopheles TRPA1. S.H.K. was supported

in part by a Samsung Fellowship, and B.A. was supported by National Insti-

tute of Neurological Disorders and Stroke postdoctoral fellowship

NS064684. W.B.G. was supported by National Institute of Diabetes and

Digestive and Kidney Diseases grant DK032753. This work was supported

by grants to D.P.S. from the National Institute on Deafness and Other

Communication Disorders (DC02539) and to C.M. from the National Institute

of General Medical Sciences (GM085335) and theBill &MelindaGates Foun-

dation (53126).

Received: February 9, 2010

Revised: July 21, 2010

Accepted: August 10, 2010

Published online: August 25, 2010

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