Olfaction in the Queensland fruit fly, Bactrocera tryoni. I: Identification of olfactory receptor...

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Journal of Chemical Ecology [joec] PP127-299584 April 12, 2001 21:36 Style file version Nov. 19th, 1999

Journal of Chemical Ecology, Vol. 27, No. 5, 2001

OLFACTION IN THE QUEENSLAND FRUIT FLY,Bactroceratryoni. I: IDENTIFICATION OF OLFACTORY RECEPTORNEURON TYPES RESPONDING TO ENVIRONMENTAL

C. D. HULL∗ and B. W. CRIBB

Department of Zoology and Entomology and the Centre for Microscopy and MicroanalysisThe University of Queensland, 4072

Queensland, Australia

(Received April 19, 2000; accepted January 1, 2001)

Abstract—The electroantennogram method was used to investigate the numberof distinct olfactory receptor neuron types responding to a range of behaviorallyactive volatile chemicals in gravid Queensland fruit flies,Bactrocera tryoni.Three receptor neuron types were identified. One type responds to methyl bu-tyrate, 2-butanone, farnesene, and carbon dioxide; a second to ethanol; and athird to n-butyric acid and ammonia. The receptor neuron type responding tomethyl butyrate, 2-butanone, farnesene, and carbon dioxide consists of threesubtypes. The presence of a limited number of receptor neuron types respond-ing to a diverse set of chemicals and the reception of carbon dioxide by a receptorneuron type that responds to other odorants are novel aspects of the peripheralolfactory discrimination process.

Key Words—Olfaction, electroantennogram,Bactrocera tryoni, Diptera, Tephri-tidae, receptor type, environmental odorants, carbon dioxide.

INTRODUCTION

Bactrocera tryoni, a serious agricultural pest, has been shown to use environmen-tal odorants in orientation behavior. Solvent extracts of the skin of fruits containvolatile chemicals that stimulate orientation to their source and elicit ovipositorprobing and egg-laying (Pritchard, 1969; Eisemann, 1980). Olfactometer experi-ments have also been used to identify various chemicals that elicit positive orien-tation of femaleB. tryoni to the source (Eisemann and Rice, 1992). The flies areknown to respond to stimulation with carbon dioxide (Rice, 1989; Stange, 1999),∗ To whom correspondence should be addressed. e-mail: c.hull@ento.uq.edu.au

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which may be part of the ovipositional orientation process. FemaleB. tryonialsorespond positively to ammonia (Bateman and Morton, 1981), which may be relatedto their feeding behavior. From these experiments, we have selected chemicals thathave a proven behavioral effect, and therefore must be detectable by the olfactorysystem, in order to undertake physiological investigations of the olfactory system.

The antennae are the principal organs for detecting olfactory signals in insects.The recording of electrical activity across the whole antenna in response to stimu-lation with odors is known as an electroantennogram (EAG). An EAG is thoughtto be the summed receptor potentials of the olfactory receptor neurons (Schneider,1962), but the exact electrical processes are not known (Park and Hardie, 1998).The electrical potential may result from many “leakage” current loops aroundindividual sensilla. Because an EAG indicates activity of the receptor potentialsproduced by olfactory receptor neurons, by using mixtures of chemicals the EAGtechnique can be used to show whether two chemicals are accepted on the sameor different receptor neuron types. Such EAG methods have been used to indicateseparate receptor neuron types for various chemicals inHelicoverpa zea(Lep-idoptera) (Christensen et al., 1990), various species ofGlossina(Diptera) (DenOtter, 1991),Argyrotaenia velutinana(Lepidoptera) (Baker and Roelofs, 1976),Psila rosae(Diptera) (Guerin et al., 1983a), andDrosophila (Diptera) (Borst,1984). Knowing the number of olfactory receptor neuron types responding to arange of chemicals indicates how specific the receptors neurons are and providesinformation on how environmental information is processed by the nervous system.

Among the Tephritidae, EAG studies have been used to investigate the chemo-receptor response to plant volatiles forRhagoletis pomonella(Fein et al., 1982;Averill et al., 1988),Ceratitis capitata(Light et al., 1988, Hernandez et al., 1996,Cosse et al., 1995),B. dorsalis(Light and Jang, 1987),B. oleae, andR. cerasi(Guerin et al., 1983b). However, none of these studies investigated the potentialnumber of receptor neuron types. In this paper, EAGs are recorded from femaleB.tryoni in order to determine the number and specificity of olfactory receptor neurontypes responding to a range of behaviorally significant, environmental odorants.These experiments also serve to increase our understanding of the peripheral pro-cessing of nonpheromonal semiochemicals and confirm the usefulness of the EAGtechnique for studying such processes.

METHODS AND MATERIALS

Insects. Bactrocera tryoni(Froggatt) were cultured at the Department ofEntomology, University of Queensland, from flies originally obtained from theDepartment of Primary Industries, Brisbane, Australia. The larval flies were cul-tured as per Neale (1989). Adults were given unlimited access to sugar and waterand regularly provided with protein hydrolysate. Only gravid flies were selected

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B. tryoni OLFACTION I 873

for experimentation. Intact flies were wrapped in Blu-tack [Bostik (Australia) PtyLtd] adhesive to constrain head movements and manipulated so that only the an-tennae were left protruding. We have found that Blu-tack has no discernible effecton the olfactory system of insects when used in electrophysiological experiments.

Electrophysiological Recording Technique. Ag–AgCl electrodes were usedfor both recording and indifferent electrodes. They were placed in disposable glasscapillaries, about 1 cm from the tip. The glass capillaries, drawn to a point on avertical pipet puller, were cut to a diameter of about 25µm at the tip and filledwith blowfly saline (Chen and Friedman, 1975).

The indifferent electrode was inserted at the base of the antenna, throughthe circumantennal sulcus. The recording electrode was inserted in the tip of thefuniculus. The electrode output was first transmitted to a preamplifier (Grass P16Dmicroelectrode DC amplifier), then to a differential amplifier (Tektronix 5A22N).Signals were displayed on a Tektronix 5103N storage oscilloscope and were alsostored as a strip chart using the Delta program (Digital Solutions Pty Ltd). Somesignals were collected by using a Syntech probe and data acquisition system andanalyzed with the Syntech EAG 2.4 program on a personal computer. Electricalshielding was provided by a Faraday cage, with all metal surfaces within it beinggrounded.

Test Chemicals. Chemicals were selected because they elicit behavioral ac-tivity in B. tryoni [see Bateman and Morton (1981), Eisemann and Rice (1992)and Rice (1989)]: 2-butanone (Aldrich Chemical Company, claimed purity 99%),methyl butyrate (Aldrich Chemical Company, claimed purity 99%),n-butyric acid(British Drug Houses Ltd, claimed purity 99%), a mixture of isomers of farne-sene (TCI Ltd, purity unknown), ethanol (Ajax Chemicals Pty Ltd, claimed purity99.5%) and ammonia (Ajax Chemicals Pty Ltd 28.0–30.0% w/w), and carbondioxide (taken from a cylinder of compressed food grade purity, provided byCommonwealth Industrial Gases, Brisbane, Australia).

Odorant Delivery. Humidified analytical grade compressed air was continu-ously blown over the fly at a rate of 500 ml/min, with the nozzle for the airstreamplaced 1 cm from, and directly in front of, the fly. The tube carrying the airflowwas 3-mm-ID teflon tubing and was connected to a glass nozzle (same internaldiameter). Test odorant samples were taken as saturated vapor, at room temper-ature, using gas-tight syringes. They were manually injected into the airstreamthrough a rubber septum, 50 cm from the delivery point. Injection time was 0.5–1.0 secs. All samples were injected at the same time segment after the recordingprogram had been activated, so that onset of stimulation could be easily identi-fied. For carbon dioxide, a 100-ml flask was filled directly from a gas cylinderand then sealed with Parafilm (American National Can). A sample was then takenup by a gas-tight syringe inserted through the Parafilm. Preliminary experimentsconfirmed the method of odorant delivery for carbon dioxide and the other chem-icals to be a reliable way of presenting specific quantities of chemicals to the

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antenna. The relationship between the syringe volume and the actual concentrationof the chemical at the preparation was not established because we only needed toknow that the receptors were saturated. This was achieved by establishing dose–response curves (see below). For the mixture experiments, two chemicals werecollected into one syringe and then injected into the continuous airstream. Stimu-lus delivery controls (see below) were undertaken to ensure volume artefacts wereexcluded.

Experimental Procedure. In all experiments the responses were compared toa standard of 50µl methyl butyrate vapor. The standard was tested, followed bythree injections of the test sample, then the standard again. At least 2 mins was leftbetween each injection to allow disadaptation of the antenna. Responses from thethree sample injections were averaged and expressed as a percentage of the stan-dard. Standardizing each test vapor allowed comparison between individual fliesand between different test chemicals and corrected for time dependent variabilitywithin preparations (Roelofs and Comeau, 1971; Light and Birch, 1979; Light,1983). The EAG responses were initially measured in millivolts, recorded as theheight of the initial depolarization.

Establishing Saturation Volumes of Test Chemicals. Saturation volumes aredefined as the volume of vapor at which the standard error of the mean responseoverlapped the standard error of the mean response of the highest volume (Dickens,1984). The saturating volumes had to be established, as it is a requirement of mix-ture experiments that all the receptor neuron types be fully activated. Differencesin volatility, and therefore the number of molecules of each chemical in the testsample, will affect the size of the EAG (Brockerhoff and Grant, 1999). Applyingenough of each chemical to maximally stimulate the receptor neurons will avoidthis problem. To find the saturation points, the antennae were stimulated withvolumes of vapor ranging between 5µl and 400µl.

Mixture Experiments. Chemicals were tested as binary mixtures to determinehow many different receptor neuron types were present. First, the antennal responsewas determined for two individual chemicals at a volume in excess of the saturationvolume (for all chemicals 400µl was used). Second, a mixture containing 400µlof each of the two chemicals was applied. Chemicals were always presented at avolume in excess of the saturation volume to ensure that the sensory dendrites forthat chemical were maximally activated. The order of presentation of the mixtureseries and the chemicals within a mixture series were randomized.

Before statistical analysis, the mean result of stimulation with a control in-jection of clean air (see stimulus delivery controls below) was subtracted from theantennal response of each of the samples in the mixture experiments. The mixtureexperiments were then analyzed using paired samplet tests. Initially a one-way testwas conducted to determine if the response to the mixture of chemicals was greaterthan the response of the largest of the two individual chemicals (i.e., a summatingresponse). If the response to the mixture was summating, then a two-way test was

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conducted to determine if the response to the mixture was equal to the calculatedadditive response of the two individual chemicals (i.e., fully summating).

Control Stimuli. Each test chemical used in the mixture experiments wasapplied using 400µl and 800µl of vapor to determine whether any of the responsesto the binary mixtures were the result of artifacts of the stimulus delivery system. Alarger EAG at 800µl would indicate a stimulus delivery artifact, as the chemicalshave been shown to saturate the antenna at volumes below 400µl. To see if thedelivery of a burst of odor produced a response in the nervous system, separatefrom the effect of the odor itself, 400µl and 800µl of clean air were tested. AnEAG response to clean air was found and was therefore subtracted from all ofthe responses of the test chemicals and mixtures. This ensured that the calculatedadditive response for each mixture accurately reflected the EAG response to theindividual chemicals. Consideration of extraneous elements in EAGs is particularlyimportant when dealing with small EAG responses.

Local Potential Controls. Three of the mixtures were tested with the record-ing electrode in a different position to see if the results of the mixture experimentswere influenced by local voltage potentials from specific sensillar fields at the tipof the antenna. The mixtures tested were methyl butyrate with ammonia, methylbutyrate with 2-butanone, and ethanol with 2-butanone. In these mixture experi-ments the recording electrode was inserted into the basal region of the funiculus,with all other parameters remaining as before.

Electrode Potential Controls. To determine whether any of the responseswere electrode potentials (artifacts), methyl butyrate, 2-butanone, farnesene,ethanol,n-butyric acid, and ammonia were tested on recently killed flies. Flieswere killed by freezing at−20◦C, but were tested at room temperature. This tech-nique shows how much of the EAG is the result of physiological activity withinthe antenna, and how much (if any) is the result of an interaction of the chemicalwith the electrodes. All other parameters were as previously mentioned.

RESULTS

Saturation Volumes of Test Chemicals. The largest responses were obtainedwith 2-butanone, followed by methyl butyrate,n-butyric acid, ethanol, carbondioxide, ammonia and farnesene (Table 1). All the chemicals had antennal responsevalues at 400µl that were significantly larger than the response to stimulation withclean air, indicating a chemoreceptor response within the antenna, rather than anartifact. The actual EAG responses ranged from 0.2 to 4.0 mV.

Mixture Experiments. The results of the mixture experiments are displayedaccording to the outcome of the statistical tests, i.e., divided into fully summat-ing, nonsummating, and partly summating groups (Figures 1 and 2). Ethanol wasfully summating with methyl butyrate (two-way paired samplet test;t = 0.836)

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TABLE 1. SATURATION VOLUMES AND EAG RESPONSES ATPOINT OF SATURATION FOR

CHEMICALS USED IN MIXTURE EXPERIMENTS

Saturation volume EAG response at saturationChemical (µl) (mean± 1 SE)

Methyl butyrate 100 119± 32-Butanone 250 151± 7Farnesene 250 35± 9

n-Butyric acid 150 61± 10Ammonia 250 40± 6Ethanol 300 59± 3

Carbon dioxide 15 33± 11

(Figure 1A), 2-butanone (two-way paired samplet test;t = 0.311) (Figure 1D),farnesene (two-way paired samplet test;t = 2.055) (Figure 1G), carbon dioxide(two-way paired samplet test; t = 0.574) (Figure 1L),n-butyric acid (two-waypaired samplet test;t = 2.323) (Figure 1J) and ammonia (two-way paired sam-ple t test;t = 1.946) (Figure 1K). Ammonia also had fully summating responseswhen mixed with farnesene (two-way paired samplet test;t = 1.650) (Figure 1I),methyl butyrate (two-way paired samplet test;t = 1.483) (Figure 1C), 2-butanone(two-way paired samplet test;t = 0.827) (Figure 1F) and carbon dioxide (two-way paired samplet test; t = 0.288) (Figure 1N). Similarly,n-butyric acid wasfully summating with farnesene (two-way paired samplet test;t = 0.858) (Fig-ure 1H), methyl butyrate (two-way paired samplet test;t = 0.055) (Figure 1B),2-butanone (two-way paired samplet test; t = 0.714) (Figure 1E) and carbondioxide (two-way paired samplet test;t = 0.942) (Figure 1M).

The combination of ammonia andn-butyric acid gave a response that was notlarger than the largest response of the individual chemicals (produced by the ammo-nia) (one-way paired samplet test;t = −3.490) (Figure 2F), that is nonsummating.Methyl butyrate was nonsummating when presented in combination with farne-sene (one-way paired samplet test;t = 0.612) (Figure 2B), 2-butanone (one-waypaired samplet test;t = 1.133) (Figure 2A) and carbon dioxide (one-way pairedsamplet test;t = 0.263) (Figure 2C). 2-Butanone was nonsummating when pre-sented in combination with farnesene (one-way paired samplet test;t = −2.498)(Figure 2D) and carbon dioxide (one-way paired samplet test; t = −3.259)(Figure 2E).

The combination of farnesene with carbon dioxide resulted in a partly sum-mating response. The mixture of these two chemicals gave a bigger response thanthe largest response of the two individual chemicals (produced by the carbon diox-ide) (one-way paired samplet test;t = 3.529), but the mixture was not equivalentto the calculated additive response of the two individual chemicals (two-way pairedsamplet test;t = 4.038) (Figure 2G).

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FIG. 1. Results of the mixture experiments showing those mixtures of chemicals that pro-duced additive EAG responses. The EAG responses represent the mean response± 1 SE(N = 6). MB = methyl butyrate, 2-But= 2-butanone, Farn= farnesene, Eth= ethanol,nBa= n-butyric acid, Amm= ammonia, CO2= carbon dioxide, and C.A.R.= calculatedadditive response.

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FIG. 2. Results of the mixture experiments showing those mixtures of chemicals that pro-duced nonadditive or partially additive EAG responses. The EAG responses represent themean response± 1 SE (N = 6). MB= methyl butyrate, 2-But= 2-butanone, Farn= far-nesene, Eth= ethanol, nBa= n-butyric acid, Amm= ammonia, CO2= carbon dioxideand C.A.R.= calculated additive response.

Control Stimuli. We compared the mean responses from stimulating with400µ and 800µl for each of the odors. The use of 800µl of a single chemicalsimulates the presentation of a binary mixture, but with only one chemical presentany increase in response compared to 400µl represents a stimulus delivery artifact.The two means were compared by using two-way paired samplet tests. In all casesthe null hypothesis (that the difference between the two means was equivalent to

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zero) was accepted at the 95% significance level (ammonia,t = 0.085; 2-butanone,t = 1.432; carbon dioxide,t = 0.553; ethanol,t = 0.775; farnesene,t = 0.990;methyl butyrate,t = 0.202; n-butyric acid,t = 0.119). The means for the con-trol stimulus experiments using 400µl and 800µl of analytical grade air werecompared using a two-samplet test, and found to be equal (t = 0.616).

Local Potential Experiments. Three of the mixtures were tested with therecording electrode in different positions, to see whether the position of the elec-trode had any effect on the types of responses (i.e., nonsummating, partly sum-mating, or fully summating). The mixture of ethanol with 2-butanone showed afully summating response (two-way paired samplet test,t = 0.562). The mixtureof methyl butyrate with 2-butanone showed a nonsummating response (one-waypaired samplet test,t = −0.227). The mixture of methyl butyrate with ammo-nia showed a response that was fully summating (two-way paired samplet test,t = 2.370). In all three cases the types of summating and nonsummating responsesare the same as for the initial mixture experiments, indicating that the position ofthe recording electrode in the funiculus was not a contributing factor.

Electrode Potential Experiments. In the experiments with recently killedflies, the EAGs were eliminated for most chemicals (Figure 3). In comparison,the responses from live flies showed large, negative potentials. Only ammoniashowed any potential deflection. However, the potential generated for ammoniawas small in comparison to those obtained from living flies and was a positivepotential. The positive potential of approximately 0.1 mV compares to the averagenegative deflection obtained from living flies of−1.8 mV.

DISCUSSION

Number of Olfactory Receptor Neuron Types. The results of the mixtureexperiments indicate that at least three types of receptor neurons are being used bygravidB. tryoni in detecting the seven tested chemicals. One receptor neuron typedetects ethanol, another type detects ammonia andn-butyric acid, and the thirdtype detects farnesene, methyl butyrate, 2-butanone and carbon dioxide.

The division of the mixtures into fully, partly, or nonsummating categorieswas based solely on the results of the statistical tests. Ethanol, therefore, appearsto be detected by a different receptor neuron type than the other six chemicals.The remaining chemicals can be divided into two groups based on the types ofresponses they elicited when presented in combinations. Ammonia is detected bythe same receptor neuron type asn-butyric acid because a mixture of these twochemical vapors was not summating, not larger than the largest of the individualchemicals. However, these two chemicals showed fully summating responses withall other chemicals, indicating separate receptor neuron types. The nonsummatingresponse to the mixture of ammonia andn-butyric acid is not likely to be due to an

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FIG. 3. Comparison of electroantennogram traces between standard and control prepa-rations. The control preparation used a recently killed specimen; therefore any resultantpotentials were artifacts. (a) 400µl of methyl butyrate, (b) 400µl of ethanol, (c) 400µl ofn-butyric acid, (d) 400µl of ammonia. The arrow represents the point of stimulus injection.

interaction between the two chemicals in the syringe. We believe that significantassociation between the weak base ammonia and the weak acid butyric acid in thevapor phase can be ruled out on the grounds that there is no visible evidence ofsolid ammonium butyrate. This contrasts with the situation where an ammoniumchloride “smoke” forms between hydrochloric acid vapor and ammonia.

Farnesene, methyl butyrate, 2-butanone, and carbon dioxide are likely to bedetected by a separate, third type of receptor neuron, but the results are not assimple to interpret as the previous two groups. Methyl butyrate and 2-butanoneare nonsummating when presented together, indicating common receptor neurontypes. Farnesene is also nonsummating when tested in combination with eithermethyl butyrate or 2-butanone, showing that it is detected by the same receptorneuron types. Similarly, carbon dioxide is nonsummating when tested in combi-nation with methyl butyrate or 2-butanone. If carbon dioxide and farnesene both

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FIG. 4. Model of the olfactory receptor cell types. The receptors are divided into three broadtypes. The ethanol cell types only receives ethanol (Eth). The ammonia cell type receivesn-butyric acid (nBa) and ammonia (Amm). The methyl butyrate receptor cell type is dividedinto three subgroups, comprising one that receives methyl butyrate (MB), 2-butanone (2-But), and farnesene (Farn); a second subgroup that receives MB, 2-But, Farn and carbondioxide (CO2), and the third subgroup receives MB, 2-But, and CO2.

use the same receptor neuron types as methyl butyrate and 2-butanone, we wouldexpect the mixture of farnesene and carbon dioxide to also produce a nonsummat-ing response. Instead, a partly summating response was detected. This partly sum-mating response for farnesene and carbon dioxide suggests that different receptorneuron types are used for these two chemicals but that there is some cross-reactivitybetween the two.

How, then, are farnesene and carbon dioxide both detected by the same recep-tor neuron types that detect methyl butyrate and 2-butanone? Rather than havingone highly selective receptor mechanism, as was found for ethanol and for ammo-nia andn-butyric acid, we propose that farnesene, methyl butyrate, 2-butanone,and carbon dioxide are detected by a group of receptor neurons that show variationin their acceptance of odor ligands. The receptor neuron type for these four chem-icals consists of three subgroups of the one basic receptor type. Figure 4 shows amodel of these receptor subgroups that is consistent with the experimental results.One subgroup detects methyl butyrate, 2-butanone, and farnesene. A second sub-group detects methyl butyrate, 2-butanone, farnesene, and carbon dioxide; and thethird subgroup detects methyl butyrate, 2-butanone, and carbon dioxide. In thismodel, when methyl butyrate or 2-butanone are tested, all three subgroups wouldbe activated. There would therefore be nonsummating responses when farneseneor carbon dioxide were added to either methyl butyrate or 2-butanone, as all ofthe receptors would already be activated. When either farnesene or carbon dioxidewas presented individually, only two of the three subgroups would be activated.When farnesene and carbon dioxide were presented together, however, all threesubgroups would be activated, which would give a response that is larger thanthe response to farnesene or carbon dioxide on their own, but would not equalthe calculated additive response for farnesene with carbon dioxide. The calculatedadditive response would be the equivalent of adding the two receptor groups forfarnesene to the two receptor subgroups for carbon dioxide, assuming no cross-reactivity between the two. The larger EAG responses to methyl butyrate and

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2-butanone compared to farnesene and carbon dioxide may also indicate that morereceptor neurons respond to methyl butyrate and 2-butanone than for the other twochemicals, which supports our hypothesis.

Furthermore, if each of the subgroups of receptor neuron types for farneseneand carbon dioxide produced approximately the same amount of electrical potentialwhen stimulated, then the model suggests that the response to the mixture offarnesene with carbon dioxide should be about 50% larger than the responses toindividual treatments of farnesene or carbon dioxide. The experimental resultscome close to matching that prediction. The EAG response to farnesene was 32%± 6, the response to carbon dioxide was 35%± 3, while the response to the mixtureof the two was 46%± 3. These antennal responses to carbon dioxide are of greatinterest on their own (especially the nonsummating responses with methyl butyrateand 2-butanone) and will be investigated in more depth in a separate study.

The EAG results seem to support our model; however, they should not be seento be conclusive. The EAG responses to some of the chemicals (e.g., farneseneand carbon dioxide) were quite small, which makes interpretation of the resultsdifficult. While our hypotheses were based on the results of statistical tests, single-unit electrophysiology will be needed to confirm the specificity of these receptorneuron types. Single-unit electrophysiology will also allow us to test whether thehigh stimulus dosages affect the responses of the receptor neurons. For instance,there may be inhibition of nerve impulses at high stimulus doses. High stimulusconcentrations can also affect the selectivity of sensory cells, for example, thepheromone sensitive cells ofT. ni lost some of their selectivity when high stimu-lus concentrations were applied (Mayer and Mankin, 1987). Finally, the receptorneuron types indicated in these experiments should not be seen as the only typespresent. Only a limited number of chemicals were tested, and it is likely that thereare many more receptor neuron types on the antenna.

Comparison with Behavioral Experiments. Eisemann and Rice (1992) lookedat behavioral responses ofB. tryonito some of the chemicals we tested and postu-lated that at least three olfactory receptor neuron types were responding. However,their predictions were based on assigning receptor neuron types according to grossdifferences in the chemical structure of the odors. They postulated that the long-chain hydrocarbons (such as farnesene) would be detected by separate receptorneuron types, compared to the short-chain aliphatic acids, alcohols, esters, andketones, and thatn-butyric acid would activate the same receptor neuron typesas methyl butyrate and 2-butanone. However the electrophysiological results indi-cate that farnesene activates the same receptor neuron types as methyl butyrate and2-butanone and thatn-butyric acid activates separate receptor neuron types fromthe other short chain chemicals.

The chemicals for this EAG study were selected because they are known tohave a behavioral effect on the flies. All the chemicals (except for ammonia) arevolatile chemicals that have been found to be emitted from fruits (Nursten, 1970).

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Ammonia is a common emission from bacterial activity and the attractancy ofB.tryoni to it is most likely a feeding response (see Courtice and Drew, 1984; Drewet al., 1983; Lloyd, 1991). 2-Butanone is also produced by bacterial cultures (Drew,1987). However, the concentrations of chemicals used may be much greater thannormally experienced. As such they may negate or even reverse the behavioralaction of the chemicals. Therefore, this study should not be taken out of context.Our experiments are looking at the physiological mechanisms associated with theinitial reception and transduction of the odorants.

The EAG responses have provided us with valuable information on the initialreception of nonpheromonal chemicals inB. tryoni. By examining the antennalresponses when stimulated with mixtures of behaviorally active chemicals, wehave shown that three basic receptor neuron types are likely to be present and thatthere is little cross-reactivity between them. The use of these olfactory receptorneuron types represents an important part of the peripheral discrimination processand results in a selective neural input to the central nervous system. The results havealso raised some important questions, particularly concerning the response of theolfactory receptor nurons that detect carbon dioxide as well as other environmentalodorants. Carbon dioxide receptors are generally thought to be a specialist classof receptors (Stange, 1996). The responses of the carbon dioxide receptors areinvestigated in more detail in the accompanying paper.

Saturation Volumes of Test Chemicals. The saturation points for most of thechemicals were between 100µl and 300µl. Carbon dioxide saturated at only15 µl; however, the figure is not directly comparable with the other chemicals,as the concentration of carbon dioxide in the syringe was close to 100%. Theconcentration of the other chemicals would have been lower, depending on thevapor pressures. Nevertheless, the EAG response to carbon dioxide was smallerthan for methyl butyrate and 2-butanone, suggesting that there are fewer dendriteson the antenna sensitive to carbon dioxide compared to methyl butyrate or 2-butanone. Alternatively the receptor potentials produced by the dendrites receivingcarbon dioxide may be small compared to the receptor potentials produced for theother chemicals.

Control Experiments. There was only one artifact identified from the controlexperiments: a small EAG response was found when clean air was injected, mean-ing that there is an antennal response in the absence of test odor stimulation. Theresponse to the control stimulus air treatment was a stimulation technique artifactand was taken into account when interpreting the results. The antennal response tothe air treatments may come from more than one source. First, mechanoreceptors,located solely on the scape and pedicel (Giannakakis and Fletcher, 1985; Hulland Cribb, 1997), may have been stimulated by the mechanical effect of the puffof air from the syringe. The response may also come from delivery-line contami-nants, most likely Teflon plasticizers (Mayer et al., 1984). However, the continuousflushing of the stimulus delivery system would have negated the build-up of such

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contaminants. The continuous air flow would also have reduced the chance ofodorants from previous stimulations adhering to the tube walls and being releasedat a later time. The comparatively small response to the air treatments comparedto the EAGs of the test chemicals shows that the larger part of the antennal re-sponses resulted from an interaction of the chemicals with the olfactory receptormechanisms of the antennae.

We found no local potential effect. Other researchers have found that localpotentials (EAG responses that are more strongly influenced by sensilla in theimmediate vicinity of the recording electrode) can be recorded from the antennae oftephritid flies (Crnjar et al., 1989; Bigiani et al., 1989). In the above cases the EAGresponse depended on the position of the recording electrode. Because sensilla arenot equally distributed over the surface of the antenna, different responses occurredas a result of different recording sites. Our results showed that the types of EAGresponses to the mixtures were consistent and did not depend on the position of therecording electrode. The difference between these findings is likely to be due tothe recording method used: the authors referred to above gently pressed the tip ofthe recording electrode against the surface of the antenna. The recording electrodewould only have been in contact with the external surface of the antenna. This mayincrease the resistance of the electrical connection, thus restricting the recordingrange of the electrode. Bigiani et al. (1989) estimated that the effect of localpotentials was limited to a radius of about 30µm around the recording electrode.Inserting of the electrode does have an effect on the dynamics of the potentialsrecorded. With a deep insertion, Kay (1971) found that some transient elements ofthe EAG response were eliminated. Nearly equal EAG responses were also foundbetween different regions of the antenna ofTrichoplusia niwhen the electrodeswere implanted through the cuticle (Payne et al., 1970). In our experiments, therecording electrode was inserted into the antenna. Therefore, we suggest that thedifferent electrical contact achieved with our methods accounts for the absence oflocal potentials.

One potential source of error in EAG experiments is an interaction of thechemicals with the electrodes, resulting in artificial electrical potentials (so-calledelectrode potentials). Kafka (1970) recorded electrode potentials that were the re-sult of interactions between chemicals and the electrodes, upon stimulation withboth acid and alkaline substances. The aim of the control experiments with thedead fly was to see if significant electrode potentials were produced. The potentialsfrom living flies had an initial negative deflection. With the dead fly the negativepotentials were eliminated, showing that the chemicals were not significantly in-teracting with the electrodes. A small, positive electrode potential was producedwith ammonia, but the effect of this potential with a live fly is minimal compared tothe normal negative antennal response. The negative potentials recorded from thelive flies in the dose–response and mixture experiments were therefore the resultof physiological activity within the antenna.

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Acknowledgments—The authors wish to thank Mr. Andrew Noskoff for technical assistance;Ms. Joan Hendrikz, the Faculty of Biological and Chemical Sciences, for assistance in the statisticalanalyses; Dr. Chris Moore, Department of Primary Industries, for help in interpreting some of theresults and Dr. E. Rumbo for his constructive criticism and advice.

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Olfaction in the Queensland fruit fly, Bactrocera tryoni. I: Identification of olfactory receptor...

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