The molecular basis of CO2 reception in DrosophilaJae Young Kwon, Anupama Dahanukar, Linnea A. Weiss, and John R. Carlson*

Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06520-8103

Communicated by Sydney Kustu, University of California, Berkeley, CA, January 4, 2007 (received for review December 15, 2006)

CO2 elicits a response from many insects, including mosquitovectors of diseases such as malaria and yellow fever, but themolecular basis of CO2 detection is unknown in insects or otherhigher eukaryotes. Here we show that Gr21a and Gr63a, membersof a large family of Drosophila seven-transmembrane-domainchemoreceptor genes, are coexpressed in chemosensory neuronsof both the larva and the adult. The two genes confer CO2 responsewhen coexpressed in an in vivo expression system, the ‘‘emptyneuron system.’’ The response is highly specific for CO2 anddependent on CO2 concentration. The response shows an equiva-lent dependence on the dose of Gr21a and Gr63a. None of 39 otherchemosensory receptors confers a comparable response to CO2.The identification of these receptors may now allow the identifi-cation of agents that block or activate them. Such agents couldaffect the responses of insect pests to the humans they seek.

chemoreceptors � insect � Gr genes

Ever since the classic experiments of Joseph Priestley in the18th century, the role of CO2 in the natural world has been

a subject of great interest. However, despite its ubiquity andcentral role in the metabolism of living organisms, some of themost fundamental questions about how CO2 interacts withbiological systems remain unanswered. One such question is howCO2 is detected in the animal world.

CO2 elicits behavioral responses in many insects that seekhuman hosts, including tsetse flies (1), which carry Africansleeping sickness; Aedes mosquitoes (2), which carry dengue andyellow fever; and Anopheles mosquitoes (3), which transmithundreds of millions of cases of malaria each year. CO2 also actsas an attractive cue for many insects that seek plants as foodsources and oviposition sites (4–6). In Drosophila, high concen-trations of CO2 evoke an avoidance response (7, 8).

CO2-sensitive neurons have been identified in many insectspecies (9) and in most cases are dedicated to the detection ofCO2. In adult Drosophila, odors are detected by olfactoryreceptor neurons (ORNs) that are housed in sensilla on theantenna and the maxillary palp (10). One class of antennalORNs, the ab1C class, detects CO2 (11). Axons of these CO2-sensitive neurons project to a single glomerulus in the antennallobe of the brain, the V glomerulus, which has been shown to beresponsive to CO2 (8).

Drosophila contains a family of 60 Odor receptor (Or) genes(12–14), and a related family of 60 Gustatory receptor (Gr) genes(14, 15), both of which encode seven-transmembrane-domainproteins. In most ORN classes, a single Or gene defines theodorant response profile (16–18). Typically, the Or gene iscoexpressed with the noncanonical receptor Or83b, an atypicalfamily member that is required for efficient localization of thecanonical Or receptor to the dendrites (19). CO2-sensitiveneurons are unique in that they do not express an Or gene (20,21). Instead, a Gr gene, Gr21a, has been shown to be expressedin this class of neurons (8). Genetic ablation of Gr21a-positiveneurons results in defects in the behavioral avoidance responseto CO2 in adults (8) as well as in larvae (7). However, there hasbeen no evidence that Gr21a acts in CO2 detection.

Here we show that another Gr gene, Gr63a, is coexpressedwith Gr21a in larvae as well as in the adult. Coexpression ofGr21a and Gr63a in an in vivo expression system confers a CO2

response. The response depends on the presence of both Grgenes; neither gene alone confers a CO2 response. The responseis highly specific for CO2 and depends on the concentration ofCO2. Our results suggest that Gr21a and Gr63a form a het-erodimeric receptor for the detection of CO2.

ResultsGr21a and Gr63a Are Coexpressed in the Larva and the Adult. In alarge-scale study of Gr gene expression in the Drosophila larva,we generated a number of Gr promoter-GAL4 lines and foundthat the Gr21a and Gr63a promoters each drive expression of aGFP reporter in a single neuron in the terminal organ, a larvalchemosensory organ (Fig. 1 A and B). When the two Grpromoter-GAL4 drivers were introduced into the same animal,the expression patterns were not additive; rather, a single neuronwas again labeled (Fig. 1C). The simplest interpretation of thisresult is that Gr21a and Gr63a are coexpressed in the same larvalchemosensory neuron.

In the adult, previous studies have shown that Gr21a isexpressed in one class of ORN in the antenna, ab1C (20, 21). ThisORN is housed in the ab1 sensillum with three other neuronsdesignated ab1A, ab1B, and ab1D (Fig. 1D) (11, 22). ORNs ofthe ab1C class send axons to the V glomerulus in the most ventralportion of the antennal lobe of the brain (Fig. 1E) (8, 23). Wetested the hypothesis that Gr21a and Gr63a are also coexpressedin the adult olfactory system. We found that the Gr21a andGr63a promoters each drive expression of GFP in a subset ofantennal ORNs that project to the V glomerulus (Fig. 1 E andF). The simplest interpretation of these results is that Gr21a andGr63a are coexpressed not only in the larva but also in the ab1Cneurons of the adult.

Gr21a and Gr63a Confer a CO2 Response When Coexpressed. Theab1C neuron has been shown to respond with high sensitivity andspecificity to CO2 (11). The V glomerulus has also been impli-cated in CO2 response (8). However, the role of Gr21a in CO2response has not been elucidated. Our results suggested thatGr21a and Gr63a might function together in imparting a CO2response. We tested this possibility by using an in vivo expressionsystem, the ‘‘empty neuron’’ system (16), to ask directly whetherthese receptors, either singly or in combination, conferred a CO2response. This system is based on the ab3A ORN, which in thewild-type antenna responds strongly to several volatile com-pounds but not to CO2 (11). In the empty neuron system theendogenous receptors in this ORN, Or22a and Or22b, have beengenetically removed, thereby eliminating the normal odorantresponses of the ORN (16). Gr21a and Gr63a were expressed inthis mutant via an Or22a promoter and the GAL4-UAS system(Fig. 2A).

Author contributions: J.Y.K., A.D., and L.A.W. designed research; J.Y.K., A.D., and L.A.W.performed research; J.Y.K., A.D., L.A.W., and J.R.C. analyzed data; and J.Y.K., A.D., L.A.W.,and J.R.C. wrote the paper.

The authors declare no conflict of interest.

Abbreviation: ORN, olfactory receptor neuron.

*To whom correspondence should be addressed. E-mail: john.carlson@yale.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0700079104/DC1.

© 2007 by The National Academy of Sciences of the USA

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We found that expression of either Gr21a or Gr63a alone didnot confer a response to CO2, but that the two when coexpressedproduced a physiological response to CO2 in the ab3A neuron(Fig. 2 B and C). Moreover, the magnitude of the response toCO2 depended on the dosage of each gene: CO2 response wasstronger when we increased the dosage of either Gr21a or Gr63aby introducing an additional copy of the respective transgene(Fig. 2 B and C). These results show that the CO2 responsedepends on both Gr21a and Gr63a.

We note that the level of spontaneous activity also increasedwith increasing dosage of Gr genes. The empty neuron, withouttransgenes, produces a spontaneous action potential frequencyof 0.6 � 0.3 spikes per second (SEM; n � 20), and adding eithera single copy of Gr21a or a single copy of Gr63a alone did notaffect this rate. When one copy of Gr21a was expressed togetherwith one copy of Gr63a, the level of spontaneous firing was 2.3 �0.4 spikes per second (n � 14). When one copy of Gr21a and twocopies of Gr63a were expressed, the frequency increased to 26 �5.7 spikes per second (n � 8); when two copies of Gr21a and onecopy of Gr63a were expressed, the frequency was 22 � 5.7 spikesper second (n � 8). These results show that the spontaneous ratedepends on the dosage of these receptors, and that Gr21a andGr63a are functionally equivalent in their effects on this rate.

The CO2 Response Is Dose-Dependent and Highly Specific. We thenexamined whether the CO2 response was dose-dependent. Wetested a wide range of CO2 concentrations (0.1–100%) andfound that responses conferred by Gr21a and Gr63a show a steepdose-dependence over this range (Fig. 3). The responses to0.035% CO2, a level typically found in air, are low, but theresponses to 5% CO2, the level in exhaled breath, are near thehalf-maximal responses observed for each of the three genotypeswe examined in the empty neuron system. Although none ofthese responses is as strong as those measured in the endogenousab1C neuron, the response strength clearly depends on the copynumber of Gr21a and Gr63a transgenes (Fig. 3). The increaseconferred by adding a second copy of Gr21a is the same as theincrease conferred by adding a second copy of Gr63a. It istechnically difficult to generate a mutant ab3A neuron express-

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Fig. 2. Gr21a and Gr63a receptors together confer an electrophysiologicalresponse to CO2 in the mutant ab3A neuron. (A) Schematic illustrating theempty neuron system used to test electrophysiological responses of receptorcombinations in the mutant ab3A neuron. (B) Representative traces showingthe electrophysiological responses of ab3 ORNs. Flies contain Or22a promoter-GAL4 and UAS-Gr transgenes encoding the indicated receptors. Spikes of twoamplitudes can be distinguished; the large spikes represent the activity of theab3A neuron and are counted; the small spikes represent the activity of theneighboring ab3B neuron (16). Bars indicate a 0.5-sec stimulus period of 100%CO2. (C) Mean responses to 100% CO2 of the mutant ab3A neurons expressingthe indicated receptors. Error bars indicate SEM; n � 8–20.

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Fig. 1. Gr21a and Gr63a are coexpressed in larval and adult Drosophila sensoryneurons. (A–C) Expression patterns driven by Gr21a-GAL4 (A), Gr63a-GAL4 (B),and Gr21a-GAL4�Gr63a-GAL4 (C) in larvae were visualized by using a UAS-GFPreporter. Pictures show only one side of the larval head region, and a singlelabeled neuron is visible in each case. (D) The ab1 sensillum, which houses foursensory neurons as indicated (A, B, C, and D). In the adult fly, Gr21a (E) and Gr63a(F) promoters drive expression of the GFP reporter in neurons that project to theV glomerulus (green) of the antennal lobe. Brain neuropil is labeled with anti-body nc82 (magenta). One antennal lobe is shown in each case.

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ing two copies of each transgene, and we do not know whethersuch a transgenic neuron would show wild-type responses.

We next examined the specificity of the response conferred bycoexpression of Gr21a and Gr63a. We used an odorant panelthat included esters, alcohols, ketones, an aldehyde, and anaromatic compound, all of which were used to categorize theantennal basiconic sensilla into functional classes (11), as well aspropionic acid and ammonia, which elicit responses in coelo-conic sensilla (24). We found that the response elicited by CO2was much greater than the response to any other compound(Fig. 4A).

To determine whether the specificity of the response con-ferred by coexpression of Gr21a and Gr63a recapitulated that ofthe ab1C neuron, we reexamined the specificity of ab1C. Acomplication in analyzing ab1C has been that it shares a sensil-lum with three other ORNs: the responses of ab1C to manyodorants can be difficult to evaluate because the other ORNsrespond strongly to them (11), yielding a complex pattern ofaction potentials. To increase the sensitivity and resolution ofour analysis, we therefore measured ab1C responses in flies thatwere mutant for Or83b (19). In ab1 sensilla of mutants lackingOr83b, only the ab1C neuron retains its responses (19), thusallowing a sensitive examination of its odorant response profile.Consistent with previous studies, the ab1C neuron in thesemutants has a very strong response to CO2, and its response, likethat conferred by coexpression of Gr21a and Gr63a, is highlyspecific (Fig. 4A).

Does CO2 elicit a strong response only from cells coexpressingGr21a and Gr63a? Of the 39 Or receptors that have been testedwith CO2 in the empty neuron system (refs. 17 and 25; S. A.Kreher and J.R.C., data not shown), none confers a responseapproaching that conferred by Gr21 and Gr63a (Fig. 4B).Although some of the Or receptors appear to confer a weak ormodest response, much of this response can be attributed toother factors: of the five Or genes that confer the strongestresponses, all five were found to give strong responses to diluentcontrols. When these control values were subtracted, the singularnature of the Gr21a-Gr63a response is even more striking (Fig.4C). Taken together, our results support the possibility thatGr21a and Gr63a together are the primary receptors for CO2detection in Drosophila, a possibility that is consistent withbehavioral and imaging analysis of the cells that express them inwild type (7, 8).

DiscussionWe have provided evidence that Gr21a and Gr63a, members ofa large family of chemoreceptors, together function as a CO2

receptor in Drosophila. Both Gr21a and Gr63a are expressed ina single class of olfactory neurons that is uniquely and exquisitelytuned to CO2. When expressed in an ‘‘empty’’ olfactory neuron,

0.1 1 10 100

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Fig. 3. CO2 response of Gr21a and Gr63a is dose-dependent. Error barsindicate SEM and are too small to be seen in some cases; n � 8–9.

pentyl acetate

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Fig. 4. Gr21aandGr63aresponse isnarrowly tunedtoCO2. (A)Responsesof theempty neuron expressing either Gr21a�Gr63a�Gr63a (Left), Gr21a�Gr21a�Gr63a (center), and of the ab1C neuron, which was analyzed in Or83b2

flies (right). 100% CO2 was used to test the responses of Gr21a and Gr63acombinations in the empty neuron system (Left and Center), and 1% was used totest responses of the ab1C neuron. Error bars indicate SEM; n � 8–10. (B and C)CO2 responses of Gr21a�Gr21a�Gr63a and of 39 odorant receptors (refs. 17 and25; S. A. Kreher and J.R.C., data not shown) in the mutant ab3A neuron (B). (C)Responses after subtraction from values in (B) of control responses of the 39odorant receptors to paraffin oil or water. All odorant receptors were stimulatedwith a 500-ms puff of 100% CO2 as described (17); Gr21a�Gr21a�Gr63a wasstimulated for 500 ms with a stream of 100% CO2 as described in Materials andMethods. Some receptors were tested with both delivery methods, which gaveidentical results. All receptors are arranged along the abscissa according to thestrengthsoftheir responses.Receptorswiththehighestresponsesareplacednearthe center of the distribution; those that have the lowest responses are placednear the edges. The order of receptors is thus different for B and C and is availablein the supporting information (SI) Text.

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Gr21a and Gr63a confer a narrowly tuned and dose-dependentresponse to CO2. In addition, the level of the response dependson the dosage of both genes. Other receptors tested in this systemconfer little, if any, response to CO2.

The simplest interpretation of our results is that Gr21a andGr63a form a heterodimer that responds to CO2. There isprecedence for heterodimerization of Or proteins (26, 27), butnot Gr proteins. Most receptors of the Or family determine theligand-specificity of the ORN in which they are expressed(16–18) but are believed to require Or83b as a coreceptor forefficient transport and/or stabilization at the membrane (19, 26,27). Based on our results we cannot determine whether bothGr21a and Gr63a act directly in ligand binding and/or signaling,or whether one of them acts as a cofactor in a manner analogousto Or83b. We note that the two genes were functionally equiv-alent in terms of the effects of their dosage on both CO2 responseand spontaneous firing rate.

There is no precedence for seven-transmembrane-domainproteins that act as receptors for CO2 or any other gas to ourknowledge. Previously described gas sensors include solubleguanylate cyclases that have been implicated in responses to NOand CO (28, 29), atypical guanylate cyclases that have beenimplicated in responses to O2 (30), and a heme-binding nuclearreceptor that has been implicated in the response to NO and COin Drosophila (31).

We do not know whether CO2 acts on the Gr receptor via theextracellular lymph that surrounds the dendrites of ORNs. Onealternative possibility is that CO2 enters the cell by an indepen-dent mechanism and activates the receptor via the cytoplasm.There is abundant genetic and physiological evidence in unicel-lular organisms that Amt proteins and Rh proteins act aschannels for NH3 and CO2, respectively (32–34). It is possiblethat a similar channel facilitates entry of CO2 into the ab1C cellof Drosophila.

We do not know whether CO2 binds directly to the receptor. CO2is readily hydrated to HCO3

� (bicarbonate), which may bind to thereceptor; it is also possible that CO2, a very small molecule, bindsto a larger soluble factor that activates the receptor. We note finallythat CO2 lowers the pH of water by forming a weak solution ofcarbonic acid, H2CO3. Such pH changes contribute to responses incentral respiratory chemosensory cells as well as in acid-sensingtaste cells in vertebrates (35), and could also play a role inCO2-sensing cells of Drosophila.

The responses to CO2 conferred by Gr21a and Gr63a in theempty neuron are lower than that of the ab1C neuron. The lowerresponse may be primarily a result of lower gene dosage.However, the ab1C neuron is unique among ORNs in itsdendritic morphology (22), which may be specialized to enhanceCO2 reception. The ab1C neuron might also contain solublefactors that optimize CO2 sensing and that are not present in theempty neuron.

Gr21a and Gr63a are among a small number of Gr genes thathave orthologs in the malaria vector mosquito Anopheles gam-biae (36). We have also identified orthologs in the dengue andyellow fever vector mosquito Aedes aegypti, the silk moth Bombyxmori, and the flour beetle Tribolium castaneum. Interestingly,genes closely related to either Gr21a or Gr63a have not beenidentified in the honey bee Apis mellifera (37), suggesting thatbees employ a different receptor for CO2.

Our finding that Gr21a and Gr63a confer a response to CO2suggests the possibility of screening for compounds that inhibitor activate these proteins. Such compounds could affect theresponse of insect disease vectors, which are responsible forhundreds of millions of infections each year, to CO2 emanationsfrom the human hosts they seek.

Materials and MethodsDrosophila Stocks and Transgenes. Wild-type flies were Canton-S.All transgenic constructs were injected into w1118 f lies. ForGr63a-GAL4 f lies, �0.8 kb of DNA upstream of the ATG wasused to generate the promoter-GAL4 construct. Primers used toamplify the promoter region were 5�-TGGGAGTGCGCCAAT-TGTGG-3� and 5�-TCCGGAGAGACTGTGTCCGG-3�.UAS-Gr constructs were created as described (17). Briefly,coding regions of Gr21a and Gr63a were amplified from Can-ton-S genomic DNA and inserted into the UAS expressionvector, pUAST. Chromosomes bearing multiple copies of UAS-Gr21a or UAS-Gr63a transgenes were obtained by standardrecombination techniques and confirmed by PCR. Or83b2 andGr21a promoter-GAL4 lines were gifts from Leslie Vosshall(The Rockefeller University, New York, NY) and Kristin Scott(University of California, Berkeley, CA), respectively.

Immunohistochemistry. Anterior regions of larvae were dissected(Institut fur Zoologie, Regensburg, Germany) and mounted inPBS containing 50% glycerol. Adult brains were dissected andprepared as described (38). Samples were immunostained withnc82 monoclonal antibody (a gift from Alois Hofbauer). Alltissues were visualized by using a Bio-Rad (Hercules, CA) 1024laser-scanning confocal microscope.

Electrophysiology. Extracellular recordings were performed onfemale antennal sensilla, and the action potentials of ORNswere quantified as described (16). All odor stimuli, with theexception of CO2, were puffed in a 500-ms pulse into acontinuous airstream directed at the antenna (16). All odor-ants in the odor panel were dissolved in paraffin oil exceptpropionic acid and ammonia, which were dissolved in water,and the responses to the diluent were subtracted from theresponses to the odorants.

For CO2 responses, the flow rate for a CO2 gas stream wascalibrated to match that of the continuous airstream (37.5ml/sec). A stimulation box was devised such that the airstreamdirected at the antenna could be diverted and replaced for 500ms by a steady stream from tanks containing various concen-trations of CO2 (0.1%, 0.5%, 1%, 5%, 20%, and 100%, dilutedwith N2) (Airgas). For all CO2 recordings, the number of spikesin 500 ms of prestimulus spontaneous activity was subtractedfrom the number in the 500 ms after the onset of CO2 stimulationunless indicated otherwise.

Supporting Text. The orders of receptors along the abscissas inFig. 4 B and C are listed in SI Text.

Note Added in Proof. Similar results were obtained in ref. 39.

We thank Wynand van der Goes van Naters for help with electrophys-iology and suggestions and Scott Kreher for sharing unpublished data.This work was supported by National Institutes of Health GrantsGM63364, DC04729, and DC02174.

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