discrepancy between antennal and behavioral responses for enantiomers of -pinene:...

P1: GAD

joec2004.cls (04/06/2004 v1.1 LaTeX2e JOEC document class) pp1335-joec-493719 October 20, 2004 17:55

Journal of Chemical Ecology, Vol. 30, No. 10, October 2004 (©C 2004)

DISCREPANCY BETWEEN ANTENNAL ANDBEHAVIORAL RESPONSES FOR ENANTIOMERS

OF α-PINENE: ELECTROPHYSIOLOGY AND BEHAVIOROF Helicoverpa armigera (LEPIDOPTERA)

C. D. HULL,1 J. P. CUNNINGHAM,2 C. J. MOORE,3 M. P. ZALUCKI,2

and B. W. CRIBB1,2,∗

1Centre for Microscopy and MicroanalysisThe University of Queensland

Brisbane, 4072, Australia2Department of Zoology and Entomology, School of Life Sciences

The University of Queensland, Brisbane, 4072, Australia3Animal Research Institute

Queensland Department of Primary IndustriesBrisbane, 4105, Australia

(Received July 25, 2003; accepted June 16, 2004)

Abstract—The ability of adult cotton bollworm, Helicoverpa armigera(Hubner), to distinguish and respond to enantiomers of α-pinene was inves-tigated with electrophysiological and behavioral methods. Electroantennogramrecordings using mixtures of the enantiomers at saturating dose levels, and sin-gle unit electrophysiology, indicated that the two forms were detected by thesame receptor neurons. The relative size of the electroantennogram responsewas higher for the (−) compared to the (+) form, indicating greater affinity forthe (−) form at the level of the dendrites. Behavioral assays investigated theability of moths to discriminate between, and respond to the (+) and (−) formsof α-pinene. Moths with no odor conditioning showed an innate preference for(+)-α-pinene. This preference displayed by naıve moths was not significantlydifferent from the preferences of moths conditioned on (+)-α-pinene. However,we found a significant difference in preference between moths conditionedon the (−) enantiomer compared to naıve moths and moths conditioned on(+)-α-pinene, showing that learning plays an important role in the behavioralresponse. Moths are less able to distinguish between enantiomers of α-pinenethan different odors (e.g., phenylacetaldehyde versus (−)-α-pinene) in learning

∗ To whom correspondence should be addressed. E-mail: [email protected]

2071

0098-0331/04/1000-2071/0 C© 2004 Springer Science+Business Media, Inc.

P1: GAD


2072 HULL, CUNNINGHAM, MOORE, ZALUCKI, AND CRIBB

experiments. The relevance of receptor discrimination of enantiomers and learn-ing ability of the moths in host plant choice is discussed.

Key Words—Heliothis, EAG, electrophysiology, α-pinene, learning, prefer-ence, cotton bollworm, moth.

INTRODUCTION

The peripheral sensory system of an insect initially detects and filters informationfrom the environment. The information is then used to produce an environmentallyrelevant response. Some questions that have arisen recently in the literature arewhether peripheral receptors can distinguish enantiomers of odors, whether theenantiomers are received on the same receptor, and whether this information can beoutput in terms of effecting behaviorally driven choices (see Stranden et al., 2002).

The discrimination of enantiomers of a single chemical is thought to providethe insect with a greater amount of information about its environment, and thusenable more relevant host choices (Wibe et al., 1998). Insects use the ratio ofenantiomers of pheromones as a relevant source of information, as is seen in thecommunication system of bark beetles, Ips pini (see Lanier et al., 1980; Mustapartaet al., 1980), the scarab beetles Anomala osakana and Popillia japonica (see Leal,1996), and the moth, Lymantria dispar (see Dickens et al., 1997). Enantiomershave also been shown to be important in host choice of the moths, Dioryctriaabietivorella (see Shu et al., 1997) and the Eurasian cotton bollworm, Helicoverpaarmigera (see Hartlieb and Rembold, 1996; Bruce and Cork, 2001; Burguiere et al.,2001; Stranden et al., 2002). The sensory basis, and aspects of the behavioraleffect of (−)-germacrene-D have been investigated in H. virescens (Røstelienet al., 2000; Mozuraitis et al., 2002; Stranden et al., 2003). However, the role ofexperience or learning has not been considered. Learning plays an important rolein host plant location by insects (Papaj and Prokopy, 1989). Rather than havingfixed behavioral patterns, the attraction of an insect to a plant can be modifiedaccording to previous experience. Such learning behavior is thought to allow theinsect to respond appropriately in variable environments (Stephens, 1993). In H.armigera, learning in adults has been demonstrated in feeding behavior (Hartlieb,1995; Cunningham et al., 1998a) and in oviposition behavior (Cunningham et al.,1998b). An understanding of the role learning plays in host location is importantif we are to improve control techniques that rely on modifying the behavior of theadult moth (Cunningham et al., 1999). Knowledge of the role of learning behavioralso extends to our understanding of perception of enantiomers.

Here, we investigate how the enantiomers of another common host plantchemical, α-pinene, are detected by the peripheral sensory receptors ofH. armigera. We determine if these can be discriminated behaviorally, and whetherlearning can affect this behavior. We also investigate whether behavioral responses

P1: GAD


DISCREPANCY BETWEEN ANTENNAL AND BEHAVIORAL RESPONSES IN Helicoverpa 2073

to enantiomers reflect intensity of antennal response—an assumption inherent inthe literature (see Stranden et al., 2002).

METHODS AND MATERIALS

Electrophysiology

Insects. Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) were ob-tained as pupae from the Queensland Department of Primary Industries,Toowoomba. The culture has been maintained for over 100 generations, withregular injections of wild stock. The moths were kept at constant temperature (23± 1◦C) and exposed to a natural light cycle. On emergence, moths were individ-ually placed into 50 ml plastic containers and given unlimited access to water.Female moths were tested between 2 and 5 d of age.

Test Chemicals. The (−) and (+) forms of α-pinene were obtained fromAldrich Chemical Company. Our own determination of optical purity (chiral GC)indicated that both were 95% ee (enantiomeric excess). Absolute purity (GC) forboth was greater than 99%.

Electroantennograms (EAGs). This technique was used to assess the summedreceptor potentials of the olfactory receptor neurons and to determine responseprofiles across receptor cell fields. The method used follows Hull and Cribb(2001a) with minor changes: The glass capillaries were filled with a physiologicalsaline (Chen and Friedman, 1975); the antenna was cut off at the base, andsecured on Blu-Tack [Bostik (Australia) Pty. Ltd.]. A drop of physiological salinewas placed over the base of the antenna to prevent desiccation. The indifferentelectrode was inserted into the base of the antenna. The tip of the recordingelectrode was cut so that it could be placed over the tip of the antenna—whichwas not cut—as adequate electrical contact could be made this way.

Odor Delivery. Humidified analytical grade compressed air was continu-ously blown over the moth at a rate of 400 cm3/min, with the nozzle for theair-stream placed 1 cm from, and directly in front of, the antenna. The tube carry-ing the air flow was 3 mm internal diam teflon tubing, connected to a glass nozzle(same internal diameter). Test odor samples were taken as saturated vapor, at roomtemperature, using gas-tight syringes: different volumes of the same concentrationwere tested (25, 100, 400, 800, 1600, 3200 µl). They were manually injected intothe air-stream through a rubber septum, 8 cm from the delivery point. Injectiontime was between 0.5 and 1.0 sec.

Experimental Procedure. In all experiments the responses were compared toa standard of 400 µl saturated hexanol vapor (equating to 12 nmol at 20◦C). Themethod of standardization, as well as the establishment of the saturating volumes,followed that of Hull and Cribb (2001a). Chemicals were tested as binary mixtures,using 800 µl saturated vapor of each form (800 µl saturated vapor equates to

P1: GAD



130 nmol at 20◦C of α-pinene for either enantiomer), to determine receptor neurontypes. The order of presentation of the mixture series and the chemicals within amixture series were randomized.

Mixture experiments were analyzed using paired sample t tests. Initially aone-way test was conducted to determine if the response to the mixture of chem-icals was greater than the response of the larger of the two individual chemicals(i.e., a summating response). If the response to the mixture was summating, thena two-way test was conducted to determine if the response to the mixture wasequal to the calculated additive response of the two individual chemicals (i.e.,fully summating). Before analysis, the mean result of stimulation with a controlinjection of clean air was subtracted from the antennal response in both the doseresponse and mixture experiments. To eliminate the possibility that some or allof the signals were artefacts due to electrode potentials (see Kafka, 1970), con-trol experiments with dead antennae were conducted. The moths were frozen atapproximately (−) 20◦C. After removal from the freezer, they were allowed toreturn to normal room temperature before the antennae were tested as above withboth (+)- and (−)-α-pinene.

Single Unit Electrophysiology. This technique was used to record the nerveimpulses from individual receptor cells and determine their response profiles:responses from two receptors were achieved. Methodology followed Hull andCribb (2001b) with these modifications: the excised antenna was secured onto Blu-Tack adhesive, and a drop of saline placed over the base to prevent desiccation. Theindifferent electrode was inserted into the base of the antenna. The nerve impulseswere counted for the first 0.5 sec of stimulation (using Syntech AutoSpike V. 3.1).

Behavior

Insects. Pupae were sexed and female moths were placed into a separateholding cage (200 × 150 × 150 mm) until eclosion. Newly emerged adult femaleswere transferred to sealed 120 mm diam (×6 cm height) plastic containers 2 hrbefore sunset each day in order to obtain discrete age groups. Moths were deprivedof food until used in experiments. Adult moths were kept in a laboratory at 25◦Cunder ambient light conditions. Female moths were tested between 3 and 5 d ofage.

Wind Tunnel Trials. Dual-choice preference tests were carried out in a windtunnel with a central Perspex flight chamber measuring 1600 × 650 × 650 mm (seeCunningham et al., 2004, for details). A laminar flow of clean air was circulatedthrough the flight chamber at 0.7 m/sec (as measured at the center of the chamberusing a fan system).

Preference Tests. Procedures for conditioning and testing moths in the windtunnel have been described in detail previously (Cunningham et al., 2004). Odorsources (lures hereafter) were created by inserting an absorbent cotton wool plug

P1: GAD



to a depth of 25 mm below the wide end of a 145 mm glass pipette. An amountof 2 µl of either (+)- or (−)-α-pinene were pipetted onto the cotton wool nomore than 15 min before the start of each trial. The narrow end of the pipette waspushed into a block of floral foam (Smithers-Oasis, South Aus.), positioning theodor source at a height of 145 mm above the floor of the wind tunnel. Testingwas conducted immediately after conditioning (i.e., approx 1 min). To test thepreference of moths for either the (+)- or (−)-α-pinene enantiomer, lures wereplaced 300 mm apart at the upwind end of the wind tunnel. Smoke tests (usingtitanium tetrachloride) showed that at a wind speed of 0.7 m/sec these plumesremained separate within the wind tunnel. Two perspex wedges positioned in thedownwind end of the wind tunnel brought the odor plumes together at a distanceof 800 mm from the lures and left a 200 mm gap through which the odors weredirected into the rear portion of the flight chamber.

Moths having previously undergone one of three treatments [associative con-ditioning with either (+)- or (−)-α-pinene, or no conditioning] were allowed torelocate into the downwind end of the wind tunnel before odor lures were placedinto position. Preference for a particular odor was seen as a characteristic upwindflight pattern in the odor plume to within 100 mm of a lure. Once a lure had beenapproached, the odor type was recorded and the test terminated.

If moths failed to approach either lure within a 5 min period, the preferencetest was terminated. The position of the feeding lure and odor source in theconditioning trials (centrally placed, 325 mm from either wall) differed from theposition of either lure in the preference trials (200 mm from either the right-orleft-hand-side wall) so that learning the position of the feeding lure would notinfluence the choice of lure in the test. The position of each lure (i.e. nearest to theright- or left-hand wall of the chamber) was allocated randomly throughout theexperiment to avoid positional biases. The volatile used in conditioning treatmentswas alternated throughout the experiment.

Associative Conditioning Treatments. Associative conditioning trials wereused to determine whether learning of one enantiomer would lead to a prefer-ence for that enantiomer in a dual-choice test. The ability to learn to prefer oneenantiomer would imply that moths can distinguish between the (+)- and (−)-α-pinene forms. Feeding sites were constructed similarly by plugging the end of aglass pipette with a cotton wool wick that had been soaked in 25% w/v sucrosesolution. This second pipette was placed into the same block of floral foam, suchthat the sucrose wick was situated 2 cm downwind from the lure. New feedingsites and lures were used in each experiment.

Conditioning trials commenced by placing an individual moth on the sucrosewick and allowing a 30 sec feeding bout. Feeding was identified as contact of theextended proboscis with the sucrose wick. In this way, the moth fed approximately2 cm downwind from the lure. After 30 sec, the moth was removed with a woodentoothpick and placed 400 mm directly downwind from the lure/feeding site. Moths

P1: GAD



were then allowed to fly freely back to the feeding source. Upon contact with thesucrose wick, the moth was allowed to feed for a further 20 sec, and then returnedto the downwind starting position. This process was repeated until moths had beengiven a total of four feeding visits in the presence of the volatile; one initial 30 secfeed and 3 × 20 sec return feeds. This procedure has previously been shown tolead to associative conditioning in male H. armigera (Cunningham et al., 2004).

Conditioning Trials Using Phenylacetaldehyde. Previous studies using maleH. armigera moths in an identical experimental design have shown that associativelearning of odors leads to a strong preference for the learned odor (Cunninghamet al., 2004). To quantify the ability of moths to learn to distinguish betweenenantiomers of α-pinene, we compared changes in preference for (−)-α-pinenein the above preference test [(−)-α-pinene vs. (+)-α-pinene)] with a learningtrial comparing (−)-α-pinene with an alternative floral odor. In the latter test,we compared the odor preferences of female moths conditioned on (−)-α-pinenewith moths conditioned on the single floral odor phenylacetaldehyde (90% puritySigma-Aldrich reagents) in a dual-choice preference test using (−)-α-pinene andphenylacetaldehyde lures (2 µl per lure). Phenylacetaldehyde is a floral volatile towhich wild H. armigera are naturally exposed and is a well known noctuid attrac-tant that can be learned by male H. armigera moths in the laboratory (Meagher,2001; Cunningham et al., 2004). Conditioning trials and preference testing pro-cedures for this experiment were identical to those described for the α-pineneenantiomer trials.

Innate Preference Treatment. We used unfed female moths with no previousexposure to either α-pinene enantiomer to determine the innate odor preferences.Adult moths were placed into individual sealed (120 mm diam) plastic pots uponemergence and kept until testing at 3 to 4 d old. Preference to (+)- or (−)-α-pinenewas determined using the dual-choice testing procedure described above.

Statistical Analysis. Data were analyzed using generalized linear modellingtechniques (McCullagh and Nelder, 1989) in the GLIM statistical package(Crawley, 1993). Choice test outcomes were analyzed as proportions, with thenumber of moths selecting a particular odor as the response variable and the totalnumber of moths selecting either host as the binomial denominator. Binomiallydistributed error variances were assumed and a logit link function employed.Hypothesis testing was carried out using the χ2 test on differences in deviance.Treatment order was randomized to prevent any biasing that may have related tonight of testing.

RESULTS

Electrophysiology

Dose–Response Curves. For the (+)- and (−) forms of α-pinene, the (−)form gave larger responses at all of the tested concentrations (Figure 1). The

P1: GAD



FIG. 1. Dose–response curves of female H. armigera antennae in response to (+)- and(−)-α-pinene (each data point represents the mean EAG response from 10 individuals ±one standard error).

saturation point for both forms was reached by 400 µl (N = 10). No signals weredetected in the control experiments, showing that the signals recorded were solelydue to physiological activity within the antenna.

Mixture Experiments. The lack of an additive effect (Figure 2) indicatesthat the two isomers are being detected by the same sensory receptor cell. Theresponse to the mixture of the two isomers was compared to the larger responseof the individual chemicals; the (−) form. The response to the mixture was notlarger than the response to the minus form (one-way t test between the mixtureand the (−) form, t = 1.419, df = 9, P = 0.0948, N = 10).

P1: GAD



FIG. 2. Results of the mixture experiments between the (+) and (−) enantiomers of α-pinene. The EAG responses represent the mean response ± one standard error (N = 10).Cal. Add. Res. = calculated additive response.

Single Unit Electrophysiology. Dose responses for both enantiomers wereobtained from two cells. In both cases the cell responded to the (+) and (−)forms: examples are shown in Figure 3. The responses of the cells to the twoenantiomers were similar. The cells responded in a dose-dependent fashion andproduced up to 37 impulses for the (−) form and up to 38 for the (+) form. Oneconsistent spike height was present, indicating one cell type only was responding.There were no instances of double-height spikes, which would have indicated thattwo or more cells with a similar spike height were present.

Behavior

We tested the enantiomer preference of 84 female moths from three treat-ments. Once trained, most moths responded to a lure in preference tests (a total

P1: GAD



FIG. 3. Electrophysiological responses from a female H. armigera sensory cell in responseto stimulation with various concentrations of (+) and (−)-α-pinene vapor. Arrow indicatesthe point of stimulus injection.

P1: GAD



FIG. 4. Percentages of moths selecting either (+)- or (−)-α-pinene in dual-choice pref-erence tests. Treatments with common letters are not significantly different (P > 0.05).Different letters denotes a significant difference (P < 0.01) as determined by G tests.

of 74 moths were trained in order to achieve 60 completed trials). In dual-choicepreference tests (Figure 4), treatment groups showed significant differences inenantiomer preference (G = 7.832, df = 2, P < 0.05). Moths with no odor con-ditioning showed an innate preference for (+)-α-pinene (χ2 = 8.708, df = 1,P < 0.005). This preference displayed by naıve moths was not significantly dif-ferent from the preferences of moths conditioned on (+)-α-pinene (G = 1.057,df = 1, P > 0.05). However, we found a significant difference in preference be-tween moths conditioned on the (−) enantiomer compared to naıve moths andmoths conditioned on (+)-α-pinene (G = 6.776, df = 1, P < 0.01).

Moths were conditioned on either the single floral odor phenylacetaldehyde or(−)-α-pinene in a preference test using these two odors. This test (N = 24 moths)showed a significant difference as a result of odor conditioning using these twoodors (Figure 5). A strong learning effect was seen: all moths (12/12) trainedon phenylacetaldehyde showed a preference for this odor compared with 1/12moths trained on (−)-α-pinene (i.e., 11/12 moths showed a preference for the(−)-α-pinene lure). This significant difference between treatments (G = 26.22,df = 1, P < 0.001) demonstrates that when given two distinct odors, moths showstrong differences in preference.

We compared the changes in preference as a result of experience in(−)-α-pinene vs. (+)-α-pinene trials with tests using phenylacetaldehyde vs.(−)-α-pinene. Moths trained and tested on the latter two odors showed a sig-nificantly stronger learning effect compared to moths trained on two enantiomers

P1: GAD



FIG. 5. Percentages of moths selecting (−)-α-pinene and phenylacetaldehyde in a dual-choice preference test. Significant difference (P < 0.05) as determined by G test.

of the same odor (G = 12.31, df = 1, P < 0.001). This outcome suggests thatalthough learning changed preferences in both these experiments, the ability tolearn the difference between different enantiomers of the same odor was lowerthan when different odors were learned.

DISCUSSION

α-Pinene is a monoterpene (10-carbon) compound given off by several hostplant species of H. armigera (Rembold et al., 1989) and has been shown to play arole in behavioral attraction of the female moths toward artificial lures (Remboldet al., 1991). Our electrophysiological results indicate that the two enantiomersof α-pinene are detected by the same receptor cell dendrites: when mixtures ofchemicals at saturating levels are used, a response to the mixture that is higherthan either of the individual chemicals indicates separate receptor cells are beingused (Borst, 1984; Hull and Cribb, 2001a). Because no additive effect was foundin EAG experiments, the same cells are likely to receive both enantiomers. Singleunit recording confirms this hypothesis. Such a result is consistent with thatof Stranden et al. (2002), who found that the enantiomers of a different plantchemical, the sesquiterpene (15-carbon) germacrene-D, were also received by thesame receptor cells. The initial dose–response EAG experiments showed that the(−) form of α-pinene produced a higher level of activation of the receptor celldendrites. This was not mirrored in the single unit recordings where responsesappeared similar for (−) and (+) forms, however the small number of receptorsdirectly recorded from does not necessarily provide the average response acrossthe sensillar field: for this information the EAG data are more reliable. The mostlikely explanation for a higher average response to the (−) form is that the (+) formdoes not bind as efficiently with the molecular receptor in many of the sensilla;

P1: GAD



although less efficiency in other steps of the transduction process such as transportto the receptor site cannot be discounted. A difference in electrophysiologicalresponse to the enantiomers of germacrene-D was also found (Stranden et al.,2002). However, this difference was seen only in single unit recordings so theaverage response for germacrene-D across the sensillar field of H. armigera is notyet known.

The behavioral experiments demonstrate that associative learning of (−)-α-pinene leads to an increased preference for this enantiomer compared to the (+)enantiomer and moths without experience of these odors. This provides evidencethat H. armigera can distinguish between the enantiomers of α-pinene despite theirbeing received on the same receptor. If moths could not differentiate between thesetwo enantiomers, we would not expect any change in enantiomer preference as aresult of experience. Moths conditioned on (+)-α-pinene did not show a changein preference relative to moths with no experience. The most likely explanationfor this is that the higher innate preference for the (+)-α-pinene enantiomerovershadowed any learning effect. This effect has been seen in studies on odorlearning in male H. armigera (Cunningham et al., 2004).

The innate preference for one enantiomer is greater than for the other, but theresults are counterintuitive. The moths show an innate behavioral preference forthe (+) form of α-pinene that provides the smaller physiological signal in EAGs.This result shows that behavioral decisions are not necessarily based simply onthe largest physiological response of the receptors, and once again indicates thecomplexity of the decision-making process. For the studies using germacrene-D,Mozuraitis et al. (2002) only tested the behavioral response to the enantiomerthat gave the larger electrophysiological response (in single unit recordings). Ourresults suggest that further studies with the (+) form of germacrene-D need to beundertaken.

An important outcome from our study is that moths can change their responseto enantiomers as a result of experience: in other words, they can learn to discrim-inate in favor of an enantiomer. This occurred when moths were able to increasetheir response to the (-) form over the innate response. One possible explanationfor the way in which the same receptor might discriminate different enantiomersis via a concentration effect. This deserves further investigation. Although dis-crimination was achieved, the ability to learn to distinguish between the (+) and(−) enantiomers was low when compared to learning to distinguish (−)-α-pinenefrom the single odor phenylacetaldehyde, suggesting a behavioral interaction inresponse between enantiomers. Stranden et al. (2002) hypothesize that separatereceptor neuron types for enantiomers will be needed if an insect is to be able todistinguish a plant based on differences in enantiomeric composition of specificcompounds. Our data indicate that this is unlikely to be the case for α-pinene.However, response spectra for this single receptor type might vary from neuron toneuron.

P1: GAD



Acknowledgments—We gratefully acknowledge the Queensland Department of Primary Indus-tries (QDPI) for supply of insects. This project was funded by the Australian Research Council andQDPI under Grant C00107108 and Cotton RDC.

REFERENCES

BORST, A. 1984. Identification of different chemoreceptors by electroantennogram-recording. J. InsectPhysiol. 30:507–510.

BRUCE, T. J. and CORK, A. 2001. Electrophysiological and behavioral responses of female Helicoverpaarmigera to compounds identified in flowers of African marigold, Tagetes erecta. J. Chem. Ecol.27:1119–1131.

BURGUIERE, L., MARION-POLL, F., and CORK, A. 2001. Electrophysiological responses of female He-licoverpa armigera (Hubner) (Lepidoptera: Noctuidae) to synthetic host odors. J. Insect Physiol.47:509–514.

CHEN, A. C. and FRIEDMAN, S. 1975. An isotonic saline for the adult blowfly, Phormia regina, and itsapplication to perfusion experiments. J. Insect Physiol. 21:529–536.

CRAWLEY, M. J. 1993. GLIM for Ecologists. Blackwell Scientific, Oxford.CUNNINGHAM, J. P., JALLOW, M. F. A., WRIGHT, D. J., and ZALUCKI, M. P. 1998b. Learning in host

selection in Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). Anim. Behav. 55:227–234.CUNNINGHAM, J. P., MOORE, C. J., ZALUCKI, M. P., and WEST, S. A. 2004. Learning, odor preference,

and flower foraging in moths. J. Exp. Biol. 207:87–94.CUNNINGHAM, J. P., WEST, S. A., and WRIGHT, D. J. 1998a. Learning in nectar foraging behavior in

Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae). Ecol. Entomol. 23:363–369.CUNNINGHAM, J. P., ZALUCKI, M. P., and WEST, S. A. 1999. Learning in Helicoverpa armigera

(Lepidoptera: Noctuidae): A new look at the behavior and control of a polyphagous pest. Bull.Entomol. Res. 89:201–207.

DICKENS, J. C., OLIVER, J. E., and MASTRO, V. C. 1997. Response and adaptation to analogs ofdisparlure by specialist antennal receptor neurons of gypsy moth, Lymantria dispar. J. Chem.Ecol. 23:2197–2210.

HARTLIEB, E. 1995. Odor learning in the moth Helicoverpa armigera in classical conditioning ex-periments, in N. Elsner and R. Menzel (eds.). Learning and Memory. Proceedings of the 23rdGottingen Neurobiology conference 1995, Vol. 1, Abstract 38. Thieme Verlag, Stuggart, Germany.

HARTLIEB, E. and REMBOLD, H. 1996. Behavioral response of female Helicoverpa (Heliothis) armigeraHB. (Lepidoptera: Noctuidae) moths to synthetic pigeonpea (Cajanus cajan L.) kairomine. J.Chem. Ecol. 22:821–837.

HULL, C. D. and CRIBB, B. C. 2001a. Olfaction in the Queensland fruit fly, Bactrocera tryoni. I:Identification of olfactory receptor neuron types responding to environmental odors. J. Chem.Ecol. 27:871–887.

HULL, C. D. and CRIBB, B. C. 2001b. Olfaction in the Queensland fruit fly, Bactrocera tryoni. II:Response spectra and temporal coding characteristics of the carbon dioxide receptors. J. Chem.Ecol. 27:889–906.

KAFKA, W. A. 1970. Molekulare wechselwirkungen bei der erregungeinzelner riechzellen. Z. Vergl.Physiol. 70:105–143.

LANIER, G. N., CLASSON, A., STEWART, T., PISTON, J. J., and SILVERSTEIN, R. M. 1980. Ips pini: Thebasis for interpopulational differences in pheromone biology. J. Chem. Ecol. 6:677–687.

LEAL, W. S. 1996. Chemical communication in scarab beetles: Reciprocal behavioral agonist–antagonist activities of chiral pheromones. Proc. Natl. Acad. Sci. U.S.A. 93:12112–12115.

MCCULLAGH, P. and NELDER, J. A. 1989. Generalized Linear Models. Chapman and Hall, London.

P1: GAD



MEAGHER, R. L. 2001. Collection of soybean looper and other noctuids in phenylacetaldehyde-baitedfield traps. Fla. Entomol. 84:154–155.

MOZURAITIS, R., STRANDEN, M., RAMIREZ, M. I., BORG-KARLSON, A.-K., and MUSTAPARTA, H. 2002.(−)-Germacrene D increases attraction and oviposition by the tobacco budworm moth Heliothisvirescens. Chem. Senses 27:505–509.

MUSTAPARTA, H., ANGST, M. E., and LANIER, G. N. 1980. Receptor discrimination of enantiomers ofthe aggregation pheromone ipsdienol, in two species of Ips. J. Chem. Ecol. 6:689–701.

PAPAJ, D. R. and PROKOPY, R. J. 1989. Ecological and evolutionary aspects of learning in phytophagousinsects. Annu. Rev. Entomol. 34:315–350.

RØSTELIEN, T., BORK-KARLSON, A.-K., FALDT, J., JACOBSSON, U., and MUSTAPARTA, H. 2000. Theplant sesquiterpene Germacrene D specifically activates a major type of antennal receptor neuronof the tobacco budworm moth Heliothis virescens. Chem. Senses 25:141–148.

REMBOLD, H., KOHNE, A. C., and SCHROTH, A. 1991. Behavioral response of Heliothis armigera Hb.(Lep., Noctuidae) moths on a synthetic chickpea (Cicer arietinum L.) kairomone. J. Appl. Ent.112:254–262.

REMBOLD, H., WALLNER, P., NITZ, S., KOLLMANNSBERGER, H., and DRAWERT, F. 1989. Volatilecomponents of chickpea (Cicer arietinum L.) seed. J. Agric. Food Chem. 37:659–662.

SHU, S., GRANT, G., LANGEVIN, D., LOMBARDO, D. A., and MACDONALD, L. 1997. Oviposition andelectroantennogram responses of Dioryctria abietivorella (Lepidoptera: Pyralidae) elicited bymonoterpenes and enantiomers from eastern white pine. J. Chem. Ecol. 23:35–50.

STEPHENS, D. W. 1993. Learning and behavioral ecology: Incomplete information and environmentalpredictability, pp. 195–218, in D. R. Papaj and A. C. Lewis (eds.). Insect Learning: Ecologicaland Evolutionary Perspectives. Chapman and Hall, New York.

STRANDEN, M., BORG-KARLSON, A.-K., and MUSTAPARTA, H. 2002. Receptor neuron discriminationof the germacrene D enantiomers in the moth Helicoverpa armigera. Chem. Senses 27:143–152.

STRANDEN, M., LIBLIKAS, I., KONIG, W. A., ALMAAS, T. J., BORG-KARLSON, A. K., and MUSTAPARTA,H. 2003. (−)-Germacrene D receptor neurones in three species of heliothine moths: Structure–activity relationships. J. Comp. Physiol. A 189:563–577.

WIBE, A., BORG-KARLSON, A.-K., PERSSON, M., NORIN, T., and MUSTAPARTA, H. 1998. Enantiomericcomposition of monoterpene hydrocarbons in some conifers and receptor neuron discriminationof α-pinene and limonene enantiomers in the pine weevil Hylobius abietis. J. Chem. Ecol. 24:273–287.

discrepancy between antennal and behavioral responses for enantiomers of -pinene:...

Documents