Corrections

CELL BIOLOGYCorrection for “Spinster is required for autophagic lysosomereformation and mTOR reactivation following starvation,”by Yueguang Rong, Christina McPhee, Shuangshen Deng, LeiHuang, Lilian Chen, Mei Liu, Kirsten Tracy, Eric H. Baehreck,Li Yu, and Michael J. Lenardo, which appeared in issue 19, May10, 2011, of Proc Natl Acad Sci USA (108:7826–7831; first pub-lished April 25, 2011; 10.1073/pnas.1013800108).The authors note that, due to a printer’s error, the author name

Eric H. Baehreck should have appeared as Eric H. Baehrecke.Additionally, the author name Christina McPhee should haveappeared as Christina K. McPhee. The corrected author lineappears below. The online version has been corrected.

Yueguang Rong, Christina K. McPhee, ShuangshenDeng, Lei Huang, Lilian Chen, Mei Liu, Kirsten Tracy,Eric H. Baehrecke, Li Yu, and Michael J. Lenardo

www.pnas.org/cgi/doi/10.1073/pnas.1108410108

DEVELOPMENTAL BIOLOGYCorrection for “Tissue-specific roles of Axin2 in the inhibitionand activation of Wnt signaling in the mouse embryo,” by LihuiQian, James P. Mahaffey, Heather L. Alcorn, and KathrynV. Anderson, which appeared in issue 21, May 24, 2011 of ProcNatl Acad Sci USA (108:8692–8697; first published May 9,2011; 10.1073/pnas.1100328108).The authors note that on page 8693, left column, first para-

graph, line 9, “T-to-C” should instead appear as “T-to-A.” Theauthors note that on page 8693, right column, first paragraph,lines 3 and 4, “Axin2canp/Axinnull mice (n=15)” should insteadappear as “Axin2canp/Axin2null mice (n=15).”

www.pnas.org/cgi/doi/10.1073/pnas.1108478108

NEUROSCIENCECorrection for “Antidepressant effects of selective serotoninreuptake inhibitors (SSRIs) are attenuated by antiinflammatorydrugs in mice and humans,” by Jennifer L. Warner-Schmidt,Kimberly E. Vanover, Emily Y. Chen, John J. Marshall, and PaulGreengard, which appeared in issue 22, May 31, 2011, of ProcNatl Acad Sci USA (108:9262–9267; first published April 25,2011; 10.1073/pnas.1104836108).The authors note that the following acknowledgment was

omitted from the article: “This work was supported by NIHGrant MH090963.”

www.pnas.org/cgi/doi/10.1073/pnas.1109215108

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NEUROSCIENCECorrection for “Functional agonism of insect odorant receptorion channels,” by Patrick L. Jones, Gregory M. Pask, DavidC. Rinker, and Laurence J. Zwiebel, which appeared in issue 21,May, 24, 2011, of Proc Natl Acad Sci USA (108:8821–8825; firstpublished May 9, 2011; 10.1073/pnas.1102425108).The authors note that Figures 2, 3, and 4 appeared incorrectly.

The corrected figures and their legends appear below.

www.pnas.org/cgi/doi/10.1073/pnas.1108343108

Fig. 4. 8-Br-cAMP and 8-Br-cGMP did not elicit currents in AgOrco orAgOrco+AgOr10 cells. (A) Representative trace from whole-cell recordingsfrom cells expressing AgOrco with application of 8-Br-cAMP, 8-Br-cGMP, andVUAA1. (B) Representative trace from cells expressing AgOrco+AgOr10 withapplication of 8-Br-cAMP, 8-Br-cGMP, BA, and VUAA1. (C) Representativetrace from cells expressing rCNGA2 with application of 8-Br-cGMP. Holdingpotentials for all recordings were −60 mV. (D) Histogram of normalizedcurrents from cyclic nucleotide and control responses (n = 4). All currentsnormalized to VUAA1 responses.

Fig. 2. RR blocks inward currents of AgOrco alone and in complex. (A–C)Representative traces of RR-blocked inward currents in AgOrco (A) andAgOrco+AgOr10 (B and C) cells. Holding potential was −60 mV for A–C. (D)Analysis of RR blockage of VUAA1 and BA-induced currents from A (n = 5),B (n = 5), and C (n = 4).

Fig. 3. AgOrco is a functional channel and responds to VUAA1 in outside-out membrane patches. (A) Single-channel recording from an outside-outexcised patch pulled from a cell-expressing AgOrco. (B–D) Expansions oftrace A before (B), during (C), and after (D) a 5-s application of −4.0 logMVUAA1. All-point current histograms of trace expansions are on the rightsides of B–D. Excised membrane patch was held at −60 mV.

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Functional agonism of insect odorant receptorion channelsPatrick L. Jonesa,1, Gregory M. Paska,1, David C. Rinkerb, and Laurence J. Zwiebela,b,c,2

aDepartment of Biological Sciences, Vanderbilt University, Nashville, TN 37235; bCenter for Human Genetics Research, Graduate Training Program; andcDepartment of Pharmacology, Center for Molecular Neuroscience, Institutes of Chemical Biology and Global Health and Program in Developmental Biology,Vanderbilt University Medical Center, Nashville, TN 37235

Edited* by John G. Hildebrand, University of Arizona, Tucson, AZ, and approved April 4, 2011 (received for review February 11, 2011)

In insects, odor cues are discriminated through a divergent familyof odorant receptors (ORs). A functional OR complex consists ofboth a conventional odorant-binding OR and a nonconventionalcoreceptor (Orco) that is highly conserved across insect taxa.Recent reports have characterized insect ORs as ion channels,but the precise mechanism of signaling remains unclear. We reportthe identification and characterization of an Orco family agonist,VUAA1, using the Anopheles gambiae coreceptor (AgOrco) andother orthologues. These studies reveal that the Orco family canform functional ion channels in the absence of an odor-bindingOR, and in addition, demonstrate a first-in-class agonist to furtherresearch in insect OR signaling. In light of the extraordinary con-servation and widespread expression of the Orco family, VUAA1represents a powerful new family of compounds that can be usedto disrupt the destructive behaviors of nuisance insects, agricul-tural pests, and disease vectors alike.

olfaction | mosquito | malaria | electrophysiology

In insects, olfactory cues are in part sensed through the activationof a family of cell-surface odorant receptors (ORs). In vivo, in-

sect ORs form heteromeric complexes of unknown stoichiometryconsisting of a conventional OR, and a universal coreceptor, nowcollectively referred to as OR coreceptor (Orco) (1, 2). In thismodel, highly divergent conventional ORs provide coding speci-ficity to the complex and have broad ligand specificities (3–5). Incontrast, the functionally requisiteOrco has not been shown to bindodorants and is extremely well conserved across taxa (6). Orco isrequired for neuronal cell-surface trafficking and proper signaltransduction, and it has been demonstrated that orthologues arefunctionally equivalent in vivo and in vitro (7). Although it is knownthat Orco is essential for OR-mediated chemoreception, the pre-cisemechanismof signaling has remained unclear.Recent evidencefrom two studies supports two alternative—and not necessarilyexclusive—models for insect olfactory signal transduction. In bothmodels, the OR complex signals ionotropically through odorant-gated ion channels; however, one study demonstrates complexgating through cyclic nucleotides, whereas the other does not (8, 9).ORs from the principal Afro-tropical malaria vector, Anopheles

gambiae, which partially dictate their host-seeking behavior, wereused to examine the function of insect ORs. In An. gambiae, 78conventional Agam\Ors (hereafter referred to as AgOrs) havebeen described and their coding specificities have been exten-sively characterized (5, 10–13). A single conventional AgOr isexpressed in every olfactory receptor neuron (ORN) in con-junction with the An. gambiae Orco orthologue Agam\Orco,hereafter referred to as AgOrco (2, 14).

ResultsIn the context of a larger effort to identify broadly effective insectrepellents, we carried out high-throughput, calcium-imagingscreens for novel modulators of the AgOrco+AgOr10 complexexpressed in human embryonic kidney (HEK293) cell lines.AgOr10 was chosen in particular on the basis of its molecular andfunctional conservation across multiple mosquito species (15).

Unlike the many novel agonists identified in our small-mole-cule screens against AgOrco+AgOr10-expressing cells, only oneof the 118,720 compounds tested (denoted here as VUAA1; Fig.1A) elicited activity consistent with allosteric agonism. Classifi-cation as an allosteric agonist was based on VUAA1’s intrinsicefficacy and capacity to potentiate the complex’s response to anatural ligand. The chemical identity of VUAA1 was verifiedusing high-resolution MS as well as 1H and 13C NMR (Methods).VUAA1 was revalidated against AgOrco+AgOr10 cells and eli-cited concentration-dependent responses that were not seen incontrol cells (Fig. 1B). In addition, VUAA1 activated severalother AgOrco+AgOrx cell lines in the context of other, ongoinghigh-throughput screens. We pursued VUAA1 on the basis of itsnovelty, as a probe for AgOR pharmacology, and in light of itspotential role as a modulator of olfactory driven behaviors inAn. gambiae.As AgOrco was the common element among the functional

responses of numerous AgOrco+AgOrx cell lines, we postulatedthat VUAA1 was a potential AgOrco agonist. To test this hy-pothesis, whole-cell patch-clamp responses were examined inAgOrco+AgOr10-expressing cells and HEK293 cells stably ex-pressing AgOrco alone. In these experiments, VUAA1 elicitedconcentration-dependent inward currents in both AgOrco- andAgOrco+AgOr10-expressing cells (Fig. 1C andD) demonstratingthat VUAA1 is anAgOrco agonist and that currents wereAgOrco-dependent. The VUAA1-induced currents in AgOrco+AgOr10cells resembled those resulting from application of benzaldehyde,an AgOr10 agonist (Fig. 1E). AgOrco+AgOr10 cells were moresensitive to VUAA1 than AgOrco cells, producing inward currentsat −5.0 logM, a concentration at which AgOrco had no response.All currents induced by VUAA1 were AgOrco-dependent; noresponses were observed in control cells (Fig. S1).To further investigate the specificity of VUAA1 agonism, and

to determine if it was capable of activating related orthologs, wetested Orco orthologues across Dipteran, Lepidopteran, and Hy-menopteran taxa. When we transiently transfected HEK cells withthe Orco orthologues of Drosophila melanogaster (Dmel\Orcohereafter referred to DmOrco), Heliothis virescens (Hvir\Orcohereafter referred to HvOrco), and Harpegnathos saltator (Hsal\Orco hereafter referred to HsOrco), VUAA1 elicited robust in-ward currents similar to AgOrco-expressing cells (Fig. 1F). Theseresults demonstrate that VUAA1 is a broad-spectrumOrco familyagonist capable of activating Orcos within and across multipleinsect orders. This is consistent with the high sequence identity that

Author contributions: P.L.J., G.M.P., D.C.R., and L.J.Z. designed research; P.L.J., G.M.P., andD.C.R. performed research; P.L.J., G.M.P., D.C.R., and L.J.Z. analyzed data; and P.L.J.,G.M.P., D.C.R., and L.J.Z. wrote the paper.

Conflict of interest statement: Preliminary patents have been filed on the use of thiscompound for insect applications.

*This Direct Submission article had a prearranged editor.1P.L.J. and G.M.P. contributed equally to this work.2To whom correspondence should be addressed. E-mail: l.zwiebel@vanderbilt.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1102425108/-/DCSupplemental.

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is characteristic ofOrco familymembers (76% toDmOrco, 67% toHvOrco, and 62% to HsOrco), as well as their previously dem-onstrated functional overlap (6).It has been previously demonstrated that insect OR com-

plexes function ionotropically, so we set out to characterize theconductive properties of the anopheline complex in response toVUAA1 (8, 9). By using whole-cell patch-clamp experiments, wedetermined the current–voltage relationships of AgOrco on itsown and in complex with AgOr10. Currents induced by VUAA1in AgOrco-expressing cells as well as those induced by VUAA1 orbenzaldehyde in AgOrco+AgOr10 cells were all nearly symmet-rical (Fig. S2 A–C). The reversal potential of AgOrco alone in thepresence of VUAA1 was −4.74 ± 3.17 mV, whereas AgOrco+AgOr10 reversal potentials were +0.18 ± 0.02 mV with VUAA1and −0.81 ± 2.12 mV with benzaldehyde (mean ± SEM; Fig.S2D). These current–voltage relationships do not indicate anyvoltage-dependent gating, and the near-zero reversal potentialsare consistent with previous reports that described nonselectivecation conductance (8, 9). We next examined whether VUAA1-induced responses could be attenuated by ruthenium red (RR),a general cation channel blocker previously found to inhibit insectOR currents (9). Application of RR reduced the VUAA1-elicitedcurrents of AgOrco cells by 79.4 ± 4.0%, whereas RR reducedVUAA1 and benzaldehyde responses of AgOrco+AgOr10 cells

by 68.3 ± 2.8% and 87.8 ± 1.8%, respectively (Fig. 2). Taken to-gether, these data indicate that AgOrco exhibits channel-like prop-erties consistent with a nonselective cation channel.In the next set of studies, outside-out membrane patches were

excised from AgOrco-expressing cells to examine single-channelcurrents evoked by VUAA1 (Fig. 3A). Here, spontaneous chan-nel opening was observed before VUAA1 stimulation, but withvery low probability (Po = 0.02; Fig. 3B). During a 5-s applicationof VUAA1, channel opening probability increased to Po = 0.38(Fig. 3C). Subsequent to agonist washout, channel opening prob-ability decreased to 0.00 (Fig. 3D). The average unitary current ofAgOrco was 1.3 ± 0.3 pA (mean ± SD; Fig. 3C, Inset), which iscomparable to previous single-channel studies of insect ORs (9).Taken together, these data support the hypothesis that VUAA1agonizes AgOrco in the absence of other intracellular compo-nents and provide additional support for the ionotropic nature ofthis channel as well as the role of VUAA1 as a direct agonist ofAgOrco and other Orco family members.We then investigated whether activation of AgOrco involves

second-messenger–based signaling, which has been reported tocontribute to insect olfactory signaling (8). In these studies, whichare consistent with a previously published report, two cyclic nu-cleotide analogues (8-Br-cAMP and 8-Br-cGMP) were unableto evoke whole-cell currents in AgOrco or AgOrco+AgOr10cells (Fig. 4 A and B) (9). Importantly, Rattus norvegicus cyclic-nucleotide gated channel A2 (rCNGA2) demonstrated robust

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imaging with AgOrco and AgOrco+AgOr10 cell lines in response to VUAA1.(C and D), Whole-cell patch-clamp recordings of concentration-dependentresponses to VUAA1 in cells stably expressing AgOrco alone (C) and AgOrco+AgOr10 (D). (E) Benzaldehyde (BA), an AgOr10 agonist, elicits concentration-dependent responses in AgOrco+AgOr10 cells. (F) Whole-cell current re-sponses to VUAA1 in HEK293 cells expressing DmOrco, HvOrco, and HsOrco.Holding potentials of −60 mV were used in C–F.

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responses to 8-Br cGMP (Fig. 4C) (16). These data support ahypothesis in which an ionotropic mechanism is the principal, ifnot sole, signaling mechanism of functional AgOR complexes.In addition to demonstrating that AgOrco alone and AgOrco+

AgOr10 complexes act as functional, ligand-gated ion channels,these studies have shown that VUAA1 elicits AgOR currentssimilar to those evoked by odorants. As an additional indicatorof the specificity of VUAA1 for Orcos, we tested VUAA1 onanother nonselective cation channel, the rat transient receptorpotential vanilloid receptor 1 (TRPV1) (17, 18). In these controls,VUAA1 failed to evoke any response, whereas capsaicin elicitedrobust responses in TRPV1-expressing cells, thereby demonstrat-ing that VUAA1 does not act as a general cation channel agonist(Fig. S2F).We next performed single-unit, extracellular electrophysiolog-

ical recordings on the maxillary palp of adult female An. gambiaeto determine whether VUAA1 could activate AgOrco-expressingORNs in vivo. We have previously demonstrated that the maxil-lary palp, an elongate olfactory appendage emanating from thehead, contains only a single sensilla type, the capitate peg (Cp),and that all Cps contain three chemosensory neurons (10). Thehighly stereotypic Cp sensilla contain two AgOrco/conventionalOR-expressing ORNs (CpB and CpC), as well as a CO2-sensitiveneuron (CpA), which does not express AgOrco. Single sensillumrecordings (SSRs) involve puncturing the sensillum wall with aglass electrode, which enables the passive sampling of all sensil-lum neurons simultaneously. The activities of individual neuronsare discriminated from each other based on compound responseprofiles and action potential amplitudes. In these preparations,CpA spike activity is clearly distinguished from those of CpB/Cby its large action potential amplitude. CpB/C spikes were muchsmaller, and, in some preparations, indistinguishable from eachother. Consequently, the spike activities of CpB/C neurons werebinned for data analysis. Accordingly, we reasoned that if VUAA1acts as a specific AgOrco agonist, we would expect it to selectivelyincrease the spike frequency of the CpB/C neurons, but have noeffect on CpA responses.

Because of its relatively high molecular weight, and despitemultiple attempts, volatile delivery of VUAA1 was not feasible.As a result, VUAA1 was directly introduced to each sensillumvia the glass-recording electrode, rather than through the moreconventional method of volatile delivery. When VUAA1 wasadded to the electrode solution, it increased the spike frequencyof CpB/C neurons in a dose-dependent manner, whereas vehiclealone had no effect (Fig. 5 A, B, andD). Differential CpB/C spikeactivity was observed immediately after puncturing each sensillum,suggesting millisecond compound diffusion rates into the sensil-lum (Fig. 5 A, B, and D). At the completion of each assay, a CO2pulse was delivered to the sensillum to test whether VUAA1 af-fected the CpA neuron; in contrast to the responsiveness of theAgOrco-expressing CpB/C neurons, CpA activity was unchangedin the presence of vehicle and/or VUAA1 (Fig. 5A–C). The abilityof VUAA1 to activate AgOrco-expressing ORNs in vivo furtherdemonstrates that AgOrco is an accessible biological target in An.gambiae, which is not obscured by other proteins or cofactors in-volved in olfactory signal transduction.

DiscussionHere we report the identification and characterization ofVUAA1, an Orco family agonist that is capable of gating ortho-logues across multiple insect taxa. In contrast to previous models,these data support a hypothesis whereby AgOrco and relatedOrco orthologues act as ion channels, which can function in-dependently of their heteromeric partners and are indifferent tocyclic nucleotide modulation (8, 9). Other than the unique activityof VUAA1, there are currently no known natural ligands for Orcofamily members. Therefore, we suggest that AgOrco and otherOrco family members should be recognized as independentlygated ion channels or channel subunits rather than ORs.As Orco functionality is required for OR-mediated chemore-

ception across all insects, an Orco agonist would theoretically becapable of activating all OR-expressing ORNs. Accordingly, Orcoagonism would be expected to severely impact the discrimination

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of odors across all insect taxa, affecting a broad range of che-mosensory driven behaviors. In An. gambiae females, universalORN activation would likely disrupt a variety of olfactory-drivenbehaviors, most notably human host-seeking, which serves as thefoundation for their ability to transmit malaria (19). The discoveryof a universal Orco agonist is also an important step toward thedevelopment of a new generation of broad-spectrum agents forintegrated management of nuisance insects and agricultural pests.The in vivo VUAA1-mediated activation of AgOrco-expressing

cells serves as a proof of principle that targeting AgOrco and otherOrco orthologues is a viable approach for the development of be-haviorally disruptive olfactory compounds to control a wide rangeof insect pests and vectors. Although it is premature to speculate asto the ultimate utility of VUAA1 as an antimalarial behaviorallydisruptive olfactory compound or as a general modulator of insectchemosensory-driven behaviors, VUAA1 nevertheless representsan important tool for the direct study of AgOrco and other Orcoorthologues in insect chemosensory signal transduction.

Materials and MethodsCell Culture and Ca2+ Imaging. Transient transfections of pCI (Promega)-containing OR constructs were performed with FuGENE 6 (Roche) into FLP-INT-REX 293 (Invitrogen) cell lines. TRPV1 cells were a gift from D. Julius(University of California, San Francisco, CA) (17), and the pCIS-rCNGA2 con-struct was a gift from R. Reed (16) (Johns Hopkins, Baltimore, MD). Con-struction of the AgOrco+AgOr10 cell line has been previously described (15).Fluo-4AM dye–loaded cells were assayed for ligand response in an FDSS6000(Hamamatsu) as previously described (15).

Chemicals. Benzaldehyde (CAS 100–52-7) and capsaicin (CAS 404–86-4) werepurchased from Sigma-Aldrich. 8-Br-cAMP and 8-Br-cGMP were obtainedfrom Enzo Life Sciences. VUAA1 (N-(4-ethylphenyl)-2-((4-ethyl-5-(3-pyr-idinyl)-4H-1,2,4-triazol-3-yl)thio)acetamide) was purchased from the Sigma-

Aldrich Rare Chemical Library (CAS no. 525582–84-7; product discontinued asof the time of manuscript submission). To ensure that observed activity waselicited from VUAA1, and not from a contaminant present in the mixture,we performed preparative HPLC. Briefly, 20 mg VUAA1 was dissolved in a 50/50 mixture of methanol and DMSO, and HPLC was performed on a Phe-nomenex Luna 30 × 50-mm C18 prep column with 0.1% TFA in H2O coupledto an acetonitrile gradient. Appropriate fractions were pooled and passedover a TFA scavenger column (StratoSpheres SPE PL-HCO3 MP-resin; PolymerLaboratories). The solvent was removed by rotary evaporation with a Biot-age V10 rotary evaporator, yielding white powder. VUAA1 was subse-quently redissolved in DMSO and assayed as described.

Characterization of Chemical Materials. Hydrogen-1 NMR spectra (400 MHz,DMSO-d6): δ 8.73 (d, J = 1.8 Hz, 1H), 8.65 (dd, J = 1.5, 4.8 Hz, 1H), 7.97(dt, J= 1.9, 8.0 Hz, 1H), 7.49 (dd, J = 2.5, 8.3 Hz,1H), 7.37 (d, J = 8.4 Hz, 2H),7.04 (d, J = 8.4 HZ, 2H), 4.10 (s, 1H), 3.95 (q, J = 7.2 Hz, 2H), 2.43 (q, J =7.6 Hz, 2H), 1.13 (t, J = 8.0 Hz, 3H), 1.04 (t, J = 8.0 Hz, 3H).Carbon-13 NMRspectra (400 MHz, DMSO-d6): δ 165.71, 152.92, 151.32, 150.95, 149.07,139.35, 136.87, 136.33, 128.38, 124.34, 123.90, 119.58, 37.91, 27.97, 16.05,15.42. The high-resolution MS (m/z) [M]+ calculated for C19H22N5OS was368.1544 and 368.1545 was found.

Patch-Clamp Recording in HEK Cells. Currents from OR-expressing HEK293 cellswere amplified using an Axopatch 200b amplifier (Axon Instruments) anddigitized through a Digidata 1322A digitizer (Axon Instruments). Electro-physiological data were recorded and analyzed using pCLAMP 10 (AxonInstruments). Electrodes were fabricated from quartz tubing (Sutter Instru-ments) and pulled to 4 to 6 MΩ for whole-cell recording. Electrodes werefilled with internal solution [120 mM KCl, 30 mM D-glucose, 10 mM Hepes,2 mM MgCl2, 1.1 mM EGTA, and 0.1 CaCl2 (pH 7.35, 280 mOsm)]. External(bath) solution contained 130 mM NaCl, 34 mM D-glucose, 10 mM Hepes, 1.5mM CaCl2, 1.3 mM KH2PO4, and 0.5 MgSO4 (pH 7.35, 300 mOsm). Com-pounds were diluted in external solution and locally perfused to the re-cording cell using Perfusion Pencil (Automate Scientific) and controlled bya ValveLink 8.2 controller (Automate Scientific). Whole-cell recordings were

Vehicle

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Fig. 5. VUAA1 activates AgOrco-expressing neurons in An. gambiae females. (A) Representative traces of SSR recordings from Cp sensilla upon electrodepuncture. VUAA1 or vehicle alone (DMSO) was delivered through the glass recording electrode. CpA is discernible from the smaller CpB/C action potentials.(B) Expansions of traces in A. (C) Activity of CpA neuron in response to VUAA1. Spike frequency was calculated every second for the first 10 s after sensillumpuncture and every 10 s thereafter. After 60 s, the preparation was pulsed for 2 s with atmospheric air to confirm a functional CpA neuron (n = 6). (D) Activityof CpB/CpC neurons in response to VUAA1, as in C.

4 of 5 | www.pnas.org/cgi/doi/10.1073/pnas.1102425108 Jones et al.

sampled at 10 kHz and filtered at 5 kHz. Outside-out patches were obtainedusing 10- to 15-MΩ electrodes pulled from standard glass capillaries (WorldPrecision Instruments) and fire-polished with an MF-830 microforge (Nar-ishige). Single-channel recordings were sampled at 20 kHz. Recordings werereduced to 1 kHz and low-pass filtered at 500 Hz for display and analysisusing QuB (State University of New York at Buffalo).

Cloning of HsOrco. The full-length coding sequence of HsOrco was PCR am-plified from H. saltator antennal cDNA using the primers 5′-ATGATG-AAGATGAAGCAGCAGGGCCT-3′ and 5′-TTTCATGGTGCTGGTACAACTGAAGT-GA-3′. The subsequent PCR fragment was then cloned into pENTR/D-TOPO(Invitrogen) and then subcloned into the pCI mammalian expression vector.

SSRs. SSRs were performed on 4- to 7-d-old, non–blood-fed An. gambiaefemales maintained on 10% sucrose and a 12 h/12 h light/dark cycle. Legs,wings and antennae were removed from cold-anesthetized females thatwere then restrained on double-stick tape with thread. A glass referenceelectrode filled with sensillar lymph Ringer solution was placed in the eye,and the recording electrode filled with DMSO or VUAA1 diluted in sensillarlymph Ringer solution was used to puncture sensilla at their base (20).

Preparations were kept under a steady stream of humidified, synthetic air(21% O2/79% N2) to limit the basal activity of CpA. Sensilla that did notrespond to CO2 or 1-octen-3-ol were excluded from analysis. Responses wererecorded and digitized using a Syntech IDAC-4 and analyzed with AutoSpikesoftware (Syntech). A new glass recording pipette was used for every re-cording. Data were sampled at 12 kHz.

ACKNOWLEDGMENTS.We thank P. Reid, P. Portonovo, G. A. Sulikowski, C. J.Denicola, and D. L. Hachey of the Vanderbilt Institute for Chemical Biologyfor assistance in preparative HPLC purification, MS, and NMR; K. Erreger,C. C. Hernandez, A. Galli, and R. L. Macdonald for assistance and advice inpatch-clamp electrophysiology; D. C. Dorset, E. L. Days, C. Farmer, and C. D.Weaver for high-throughput screening and technical and data analysisassistance; D. Julius for providing TRPV1-expressing HEK293 cells; J. Liebigfor providing H. saltator antennal cDNA; and R. R. Reed for providing therCNGA2 construct. We also thank members of the Zwiebel laboratory forcritical discussions, Z. Li for assistance in mosquito rearing, and L. Sun, J. G.Camp, and M. Rützler for cell culture and screening assistance. This work wassupported by a grant from the Foundation for the National Institutes ofHealth through the Grand Challenges in Global Health Initiative (to L.J.Z.).

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