Plant odour processing in the antennal lobe of male and female grapevine moths, Lobesia botrana (Lepidoptera: Tortricidae)

Journal of Insect Physiology 48 (2002) 1111–1121www.elsevier.com/locate/jinsphys

Plant odour processing in the antennal lobe of male and femalegrapevine moths,Lobesia botrana (Lepidoptera: Tortricidae)

I. Masante-Rocaa, C. Gadennea,∗, S. Antonb,1

a Institut National de la Recherche Agronomique, Unite Mixte de Recherche en Sante Vegetale, Centre de Recherche de Bordeaux, BP81, 33883Villenave d’Ornon Cedex, France

b Department of Ecology, Lund University, S-223 62 Lund, Sweden

Received 28 August 2002; accepted 2 September 2002

Abstract

Moths of Lobesia botrana (Lepidoptera: Tortricidae) are confronted with different volatiles emitted from the host plant duringthe different seasons. To test the hypothesis of plasticity of central plant odour processing in moths of different generations in thefuture, we first investigated the responses of antennal lobe (AL) interneurons of laboratory-reared virgin and mated males andfemales. We used intracellular recording and staining techniques while stimulating the antenna with a range of host and non-hostplant odours. The AL structure ofL. botrana is similar to that found in other Lepidoptera species studied. The most frequentphysiological responses for all types of moths were obtained with (E)-2-hexenal, and with thujyl alcohol andβ-thujone, componentsof tansy, a behaviourally attractive non-host plant. Some broadly responding neurons were capable of distinguishing between differ-ent compounds through different response patterns (excitation/inhibition) and/or different dose–response characteristics. Responsecharacteristics (response spectra, threshold and specificity) of neurons were similar, independent of sex or mating status of themoths. Significant differences between the groups were, however, found in the proportion of responding neurons for a few testedcomponents. 2002 Elsevier Science Ltd. All rights reserved.

Keywords: Lobesia botrana; Plant volatiles; Central nervous processing; Antennal lobe; Electrophysiology

1. Introduction

Olfaction is one of the most important insect senses,guiding them towards their host plants, feeding sites, andmating partners. Behaviourally active plant compoundshave been found for many insect species of differentorders (Bernays and Chapman, 1994). The peripheraldetection of behaviourally active plant odours was inves-tigated in several species by electro-antennography(EAG) and single cell recordings (Visser, 1986; Burgui-ere et al., 2001, and references therein). The central pro-cessing of behaviourally relevant odours has mainlybeen studied by elucidating the sex pheromone pathway

∗ Corresponding author. Tel.:+33-5-57-12-26-42; fax:+33-5-57-12-26-32.

E-mail address: [email protected] (C.Gadenne).

1 Also at: SLU, Department of Crop Sciences, Chemical Ecology,P.O. Box 44, 23053 Alnarp, Sweden

0022-1910/02/$ - see front matter 2002 Elsevier Science Ltd. All rights reserved.PII: S0022-1910 (02)00204-4

in the primary olfactory centre, the antennal lobe (AL),of male moths (Hansson and Anton, 2000). Only a fewstudies concerning the central processing of plant vol-atiles have been performed on noctuid and sphingidmoths. Anton and Hansson investigated the central pro-cessing of plant odours in females (Anton and Hansson,1994) and males (Anton and Hansson, 1995) ofSpodop-tera littoralis, a noctuid moth. In both sexes, they foundspecialised and unspecialised multiglomerular localinterneurons and projection neurons (PNs) with mainlyuniglomerular arborisations sending their axons to thecalyces of the mushroom body and to the lateral horn ofthe protocerebrum. Similar observations were made in alater study ofS. littoralis females (Sadek et al., 2002)and in males of the noctuid moth,Agrotis ipsilon(Greiner et al., 2002). In females of the sphingid mothManduca sexta, intracellular recordings from AL PNsshowed that (+/�)-linalool was strongly excitatory inPNs that arborised exclusively in the lateral large femaleglomerulus (Roche King et al., 2000). In Tortricidae,

1112 I. Masante-Roca et al. / Journal of Insect Physiology 48 (2002) 1111–1121

however, nothing is known regarding central processingof either pheromones or plant related volatiles.

Odour-guided behaviour is dependent on the environ-ment and on the physiological state of an animal. Behav-ioural responses to odours can vary depending on thedifferent processing of these odours in the central ner-vous system. Plasticity of odour-guided behaviour andmatching plasticity of central nervous processing ofpheromones have been shown to occur in the moth, A.ipsilon (Anton and Gadenne, 1999; Gadenne and Anton,2000). Sensitivity of AL interneurons to sex pheromoneswas found to be age- and hormone-dependent, whereascentral processing of plant volatiles occurring in foodsources was age-independent (Greiner et al., 2002).

The European grapevine moth, Lobesia botrana(Lepidoptera: Tortricidae) is a good model to studyenvironmentally induced plasticity of central processingof plant odours. Different generations of this species areconfronted with different volatiles emitted from the mainhost plant, grapevine, during different seasons. This pol-yphagous insect develops on plants from different famil-ies (Bovey, 1966; Stoeva, 1982; Gabel et al., 1992). Itis one of the most serious pests of vineyards in the pale-artic area. The larvae feed on flower buds, ruining themor inducing lesions, and on grapes, damaging them andfacilitating infection by the gray mould Botrytis cinerea(Fermaud and Le Menn, 1989).

Although single components present in grapevine elic-ited EAG responses in L. botrana females (Masante-Roca, personal observation), nothing is yet known aboutthe effect of host plant odours on the behaviour of L.botrana. However, in females, volatiles emitted fromtansy flowers (Tanacetum vulgare), a non-host plant, areknown to be behaviourally attractive (Gabel et al., 1992).Some components like d-limonene, α and β-thujone andthujyl alcohol emitted from tansy induced an EAGresponse in L. botrana females (Gabel et al., 1992, 1994;Masante-Roca, personal observation). Tansy odourtested in wind tunnel experiments increased the pro-portion of mated females that initiated flight, the durationof flight and reduced the latency of takeoff (Hurtrel andThiery, 1999). Field experiments showed that tansyodour inhibited mating and oviposition behaviour andreduced adult longevity (Gabel and Thiery, 1994).

In insects, intersexual differences have been revealedin antennal sensitivity (Palaniswamy and Gillott, 1986;Hansson et al., 1989) and in behaviour. Stronger attrac-tion to host plant volatiles in mated females comparedto males has been found in different moth species, Trich-oplusia ni (Landolt, 1989), Pectinophora gossypiella(Wiesenborn and Baker, 1990), Heliothis subflexa(Tingle et al., 1989) and Heliothis virescens (Tingle andMitchell, 1992), unveiling the possibility of a differentialprocessing of plant odours as a function of sex and/ormating status.

In the present study, we have investigated the general

anatomy and the physiological characteristics of ALinterneurons in virgin and mated laboratory-rearedfemale and male L. botrana. We used intracellular rec-ordings and stainings to reveal the response character-istics of AL interneurons to host and non-host plant vol-atiles and to describe the arborisation patterns ofphysiologically identified AL neurons. The physiologicaland anatomical data are discussed in their behaviouralcontext.

2. Materials and methods

2.1. Insects

Larvae of L. botrana, originating from a laboratoryculture in Bordeaux, France, were reared on a semi-arti-ficial diet under a 16-L/8-D photoperiod and 22 ± 1°Ctemperature (Stockel et al., 1989). Male and femalepupae were kept separately in glass tubes(8 × 0.5 cm2) until emergence with similar conditions.Three-day old male and female moths were used forintracellular recordings. Mated moths were obtained bypairing one male with one female in a glass tube on theday of emergence and tested one or two days after mat-ing. Mating was confirmed by checking the presence ofeggs in the glass tube (we confirmed in a control experi-ment that only mated females lay eggs; all 50 femalesused in the control experiment that laid eggs had aspermatophore).

Preparations for intracellular recordings were doneaccording to standard methods for moths (Kanzaki et al.,1989). Briefly, an insect was mounted in a plastic pipettetip with the head protruding. The pipette tip had beencut beforehand to allow the passage of the head, whichwas fixed by using dental wax (Kerr). The cuticle wasremoved from the front of the head and tissue overlyingthe brain was removed. The sheath overlying the ALswas taken away manually to facilitate the penetration ofthe microelectrode. The preparation was perfused withsaline solution at pH 6.9 (Christensen and Hildebrand,1987) and the antenna was exposed to a continuous airstream (0.5 m/s) (Anton and Hansson, 1994).

2.2. Intracellular recording and stimulation

AL neurons were randomly penetrated by a glassmicroelectrode filled with 2 M KCl or with the tip filledwith 4% Lucifer yellow (LY) CH (Sigma) and backfilledwith 2 M LiCl2. In males, the electrode was placed out-side the macroglomerular structure. When intracellularcontact was established, the antenna was stimulated witha 0.5 s air pulse (4 ml/s) containing clean air, the solventor the different test compounds. The stimuli werepresented in a random order separated by interstimulus-intervals of at least 10 s. For each neuron, the number

1113I. Masante-Roca et al. / Journal of Insect Physiology 48 (2002) 1111–1121

of stimuli that could be tested varied depending on theduration of the recording. If possible, each stimulus wastested more than once to verify the reproducibility ofthe responses.

Thirteen single components present in grapevine(Schreier et al., 1976) and in tansy flowers (Gabel et al.,1992), and one tansy extract were tested during intra-cellular recordings (Table 1). The individual componentswere obtained from Sigma (1-heptanol, 1-octene-3-ol, α-terpineol, (+/�)-linalool, heptaldehyde, (E)-2-hexenal,(E)-2-heptenal, α-farnesene, 2-heptanone and hexylacetate), Fluka (d-limonene), INRA Dijon, France(thujyl alcohol), and from the Pharmaceutical Faculty,Bratislava, Slovakia (β-thujone). The tansy extract wasa gift by D. Thiery (INRA, Bordeaux). It was obtainedby distillation of tansy flowers according to the methoddescribed by Gabel et al. (1992).

The compounds were diluted in hexane and the tansyextract in dichloromethane. In preliminary experiments,the threshold range of AL neuron responses was firstassessed using different dilutions of plant odours.According to the most frequent response threshold, vir-gin and mated male and female moths were then testedwith 10 µg of the 13 plant odours. In order to determinedose–response relationships for AL neurons, five com-pounds (thujyl alcohol, β-thujone, (E)-2-hexenal, (+/�)-linalool, 1-heptanol) and the plant extract were chosenbecause they frequently elicited responses in intracellu-lar recordings. Ten microlitres of dilutions containingfrom 0.1 to 100 µg/µl were applied on a piece of filterpaper in a Pasteur pipette. A Pasteur pipette containingsolvents (hexane or dichloromethane) was used as ablank.

Table 1List of compounds used as stimuli. G: leaf, grape and immature grapecomponents (in Schreier et al., 1976); T: tansy flower components(Gabel et al., 1992)

Chemical class Compound Formula Origin Abbreviation

Alcohol 1-Heptanol C7H16O G hpolAlcohol 1-Octene-3-ol C8H16O G 1oct3Alcohol Thujyl alcohol C10H18O T thjyAldehyde Heptaldehyde C7H14O G hpdeAldehyde (E)-2-heptenal C7H14O G E2hpAldehyde (E)-2-hexenal C6H10O G E2hxMonoterpene d-limonene C10H16 T limoMonoterpene β-Thujone C10H14O T thjoMonoterpene α-Terpineol C10H18O G terpMonoterpene (+/�)-Linalool C10H18O G linaSesquiterpene α-Farnesene C15H24 G farnKetone 2-Heptanone C7H14O G 2hepEster Hexyl acetate C3H11O G hexaExtract Tansy T tans

2.3. Data analysis

The delay, the duration and patterns of excitatory andinhibitory parts of the responses were analysed manuallyin detail. Excitatory responses were quantified asdescribed previously (Gadenne and Anton, 2000).Briefly, the net number of spikes (number of spikes dur-ing a 600 ms period after the stimulus minus the numberof spikes counted during the preceding 600 ms rep-resenting spontaneous activity) produced in response tothe blank stimulus was substracted from the net numberof spikes produced in response to an odour stimulus toquantify the response to a specific stimulus. A neuronwas classified as responding to a stimulus when theodour response exceeded the blank response by atleast 10%.

Differences in dose-dependent responses between thegroups of experimental animals were analysed using theKruskal–Wallis test. Response spectra of L. botrana ALinterneurons for plant odours were analysed by compar-ing the percentage of responding neurons to each odourbetween virgin and mated males and females. As inhibi-tory responses for each odour were rare, they were nottaken into account for statistics. The sensitivity of ALneurons for the six selected plant odours was analysedby comparing the percentage of AL neurons with spe-cific thresholds between virgin and mated males andfemales. Response specificity of AL neurons for the 13plant odours and the extract (tansy) was analysed bycomparing the percentage of AL neurons responding todifferent numbers of stimuli. Response spectra, sensi-tivity and specificity were analysed using the G-test(Sokal and Rohlf, 1995).

2.4. Anatomy

To investigate the general anatomy of the L. botranaAL, male and female brains were dissected and fixed inglutaraldehyde, washed in buffer, dehydrated andembedded in Fluoromount (Sigma). Optical sectionswere taken with a confocal microscope (Leica TCS NT).The spatial sampling frequency was 0.19 µm/voxel inthe image plane (1024 × 1024pixels2) and 1 µm/voxel inthe axial direction. The stacks of images were used toreconstruct the AL on a Silicon Graphics computer workstation using imaris (software package by Bitplane, Zur-ich, Switzerland) to estimate the number of glomeruli ina female and a male brain.

To stain individual neurons after physiological rec-ordings, standard staining methods were used (Antonand Hansson, 1994). The neurons were stained with 4%LY (Sigma) by passing ca. 0.5 nA of constant hyperpol-arising current through the LY filled recording electrodefor more than 5 min. After the recording, the brains weredissected, fixed in formaldehyde solution, dehydrated,and first examined as wholemounts in the confocal


microscope. Subsequently, brains were embedded inSpurr’s resin and sectioned at 10 µm. Sections were thenre-examined in the confocal microscope and details ofthe arborisations in the AL were visualised. The spatialsampling frequency was 0.12 µm/voxel in the imageplane (1024 × 1024pixels2) and 0.48 µm/voxel in theaxial direction. In addition, sections were photographedin a fluorescence microscope and neurons were recon-structed from slides.

3. Results

3.1. Anatomical characteristics of the AL and PNs

The AL of both male and female L. botrana containedapproximately 62 glomeruli, which were arranged inseveral layers around a small central fibre core. In males,a macroglomerular structure could be identified close tothe entrance of the antennal nerve in the AL. Cell bodiesof AL neurons were mainly situated in a large anterior-ventral cell cluster (Fig. 1).

Out of 36 attempts to stain AL neurons intracellularly,eight AL neurons identified as PNs were stained in sixbrains. In four preparations, at least one PN was stainedcompletely, leaving the AL through the inner antenno-cerebral tract (IACT) and exhibiting arborisations in thecalyces of the mushroom bodies and in the lateral proto-cerebrum (Fig. 2). In one out of these four preparations,two PNs were stained completely, in another preparationa second PN was stained incompletely, with arboris-ations only visible in the AL. In two additional brains,one PN was stained incompletely in each. Each PNarborised very densely in only one glomerulus (Fig. 2).Two neurons had their arborisations in a central ordinaryglomerulus and three neurons each arborised in a lateralor a medial glomerulus, respectively. The cell bodies of

Fig. 1. AL morphology of (A) male and (B) female L. botrana. Fron-tal optical sections through the central part of the AL. AN: antennalnerve, MG: macroglomerular structure, F: central fibre core, CB: cellbody cluster.

Fig. 2. Morphology of a LY-filled PN. Reconstruction from 10 µmfrontal sections of LY-stained preparations. The axonal branches inthe calyces of the mushroom bodies and in the lateral protocerebrumare characterised by a high number of large varicosities. Inset: opticalsection through the very dense dendritic arborisations in the innervatedglomerulus. Note that details of the arborisations are hardly visible.AL: antennal lobe, cmb: calyces of the mushroom bodies, lp: lateralprotocerebrum.

all stained PNs were situated in the anterior-ventralcell cluster.

3.2. General physiological characteristics of ALneurons

A total of 184 neurons in 83 L. botrana male andfemale moths were recorded from in this study. Another200 tested neurons did not respond to any of the testedstimuli. The spontaneous activity for odour respondingAL neurons ranged between 5 and 60 Hz and was mostfrequently between 20 and 30 Hz. The amplitude of therecorded spikes ranged between 10 and 30 mV (Fig. 3).Recordings were held up to 10 min with an average of5 min. For the same neuron, a component tested morethan once during different times within the experiment,elicited the same response pattern (data not shown).

Approximately 20% of the neurons responded purelyexcitatory to the stimuli tested, characterised by anincrease in spike frequency between 20 and 70 Hz abovespontaneous activity (Fig. 3(A)). All other neuronsresponded either with a combined excitatory/inhibitory


Fig. 3. Response characteristics of two AL neurons stimulated withplant odours. (A) Purely excitatory responses of a generalist neuronwith high spontaneous activity. The neuron did not respond to 1-octene-3-ol and hexyl acetate in addition to 2-heptanone (not shown).(E)-2-hexenal, α-farnesene and the tansy extract were not tested in thisexperiment. (B) Different response patterns of a generalist neuron todifferent compounds. (+/�)-linalool elicited an inhibitory response, β-thujone a purely excitatory response and thujyl alcohol and 1-heptanola combined response, consisting of an initial excitation, followed byan inhibitory period of different durations. Bar beneath registrationindicates stimulus time. Vertical scale bar 10 mV, horizontal scale bar250 ms. The contact quality changed during recording and for con-venience the spikes were cut in some of the traces.

response to all stimuli (ca. 35%) or with combinedexcitatory/inhibitory (rarely also pure excitatory)responses to some and with inhibitory responses to otherstimuli (ca. 45%) (Fig. 3(B)). Inhibitory responses,characterised by a hyperpolarisation of the neuron andthe absence of spikes for a certain time after stimulation,occurred, however, for only few stimuli in each neuron.For each single tested component, the number of neuronswith an inhibitory response was very low. The threetypes of responses occurred in all four groups of mothsand were found for all stimuli tested except for the tansyextract, which never elicited an inhibitory response inany neuron.

The delay of excitatory and inhibitory responses was

between 300 and 400 ms after the onset of stimulationin the vast majority of the experiments (Fig. 3) (note thatthe airstream needs that time to reach the antenna). Forsome excitatory responses, however, a delay of up to700 ms after the onset of stimulation was observed (datanot shown). These long delays occurred mainly in neu-rons which exhibited combined excitatory/inhibitoryresponses and independently of the odour tested and thetype of moth.

The duration of the excitatory period in a responsewas mostly between 300 and 600 ms, but very shortexcitatory responses (100–200 ms) also occurred (Fig.3(A)). Purely excitatory responses could, on the otherhand last for up to 5 s. Inhibitory responses lastedbetween 300 ms and 1.4 s (Fig. 3(B)). No correlationwas found between the duration of the responses and theodours tested or the type of moth examined.

3.3. Response spectra of AL neurons

Intracellular recordings were performed on all testedneurons in the four types of moths using single compo-nents at a 10 µg dose and the tansy extract as stimuli(Fig. 4). Fifty-two neurons in 23 mated females, 63 neu-rons in 31 unmated females, 39 neurons in 16 matedmales and 30 neurons in 13 unmated males were tested.Table 2 shows the total number of replicates for eachsingle stimulus tested for each group of neurons. Theproportion of neurons responding to each compoundvaried. The stimuli that generated the most frequentresponses for all the groups of moths were (E)-2-hex-enal, β-thujone and thujyl alcohol. Responses to each ofthe tested compounds were found within the 184 neuronsthat responded to at least one compound.

There was no significant difference in the proportionof responding neurons between virgin and mated femalesto the different plant odours tested. However, for 10 outof 14 tested compounds, mated females seemed torespond more frequently than unmated females (Fig.4(A)). In males, significant differences were found forthree odours (1-octen-3-ol, G � 5.270; thujyl alcohol,G � 5.867 and α-farnesene, G � 6.530; df � 1; P �0.05) between mated (n � 24,34,21, respectively) and

virgin males (n � 14,21,3, respectively) (Fig. 4(B)). Thecomparison between mated males and females revealeda significant difference for five compounds (thujyl alco-hol: G � 3.879; 2-heptanone: G � 4.309; α-terpineol:G � 6.219; heptaldehyde: G � 7.336; (E)-2-hexenal:G � 7.085 between n � 34,23,27,26,30 neurons ofmated males and n � 41,26,30,31,41 neurons of matedfemales, respectively) for which the percentage ofresponding neurons was higher in mated females. A sig-nificant difference existed only for α-farnesene (G �7.926; df � 1; P � 0.05) between unmated males

(n � 6) and females (n � 22).


Fig. 4. Response representation of the tested compounds in (A)females and (B) males of L. botrana AL interneurons. All single stim-uli were tested at a 10 µg dose. An asterisk (∗) indicates a significantdifference between two groups. Statistical treatment between groupsof moths was performed using the G-test (P�0.05). The comparisonbetween mated males and mated females reveals a significant differ-ence for five compounds (thujyl alcohol, α-terpineol, heptaldehyde,(E)-2-hexenal and 2-heptanone) whereas a significant difference existsjust for one compound (α-farnesene) between unmated males andunmated females. See Table 2 for number of neurons tested for eachstimulus.

3.4. Response threshold of AL neurons

Intracellular recordings from neurons of the fourgroups of moths were performed using four dilutions ofthe selected compounds to determine response thresh-olds and dose–response relationships (Figs. 5 and 6).There was no significant difference in the proportion ofneurons with different threshold between groups for anytested compound. Unmated males showed a low thres-hold for tansy (Fig. 5(F)), β-thujone (Fig. 5(B)) and(+/�)-linalool (Fig. 5(C)) as compared to the othergroups of moths whereas, mated females, in general,exhibited a relatively high threshold for most tested com-pounds. Comparison of absolute threshold levels oftested neurons is not easy due to differences in vapourpressures between the different compounds.

Different dose–response curves were obtained,depending on the tested compounds and tested neurons.

Table 2Number of L. botrana AL neurons tested in the different groups ofmoths for each stimulus

Compound Mated Unmated Mated Unmatedmales males females females

1-Heptanol 36 25 45 431-Octene-3-ol 24 14 30 27Thujyl alcohol 34 21 41 38Heptaldehyde 26 15 31 34(E)-2-heptenal 23 15 27 33(E)-2-hexenal 30 15 41 38d-limonene 24 13 29 30β-Thujone 35 23 44 44α-Terpineol 27 15 30 27(+/�)-Linalool 33 23 45 51α-Farnesene 21 3 21 222-Heptanone 23 13 26 33Hexyl acetate 24 14 30 26Tansy 16 6 20 19

Some neurons revealed an increase followed by adecrease in the number of spikes, others showed noincrease in spike frequency with increasing stimulusamounts at all (Fig. 6(A) and (B)). Within the same neu-ron, different shapes of dose–response curves could beobtained for different stimuli tested (Fig. 6(A) and (B)),or the shape of the curves varied for different neuronsduring application of the same stimulus (Fig. 6(C)). Nosignificant difference was revealed in the dose–responserelationships between male and female L. botrana(P � 0.05 for each selected compound, data not shown).

3.5. Response specificity of AL neurons

Neurons with different specificity for the tested com-pounds were found in all groups of moths (Fig. 7). Circa20% of the neurons responded to only one stimulus.More than half of the neurons were considered as gen-eralist neurons, responding to more than three differentstimuli. For the great majority of these neurons, the stim-uli that elicited responses belonged to different chemi-cal classes.

Compound-specific neurons responded frequentlyonly to (E)-2-hexenal and always at the same threshold(100 µg) (four neurons for mated females and forunmated females, one for mated males and three forunmated males). Some neurons responded to all testedcompounds (five neurons in mated females).

No significant difference in response specificity wasfound between neurons of mated and unmated male andfemale moths (G � 3.841; a � 0.05, NS).

4. Discussion

In this study, we describe the general anatomy of theAL and physiological characteristics of AL interneurons


Fig. 5. Response thresholds of L. botrana AL interneurons to plant odours: (A) thujyl alcohol, (B) β-thujone, (C) (+/�)-linalool, (D) 1-heptanol,(E) (E)-2-hexenal and (F) tansy. Percentage of tested AL neurons responding to the different plant odours at different thresholds in virgin andmated males and females. Similar thresholds were found in the four groups of moths for all tested compounds. n: number of neurons tested.

in male and female L. botrana responding to host plantand non-host plant odours. General anatomical andphysiological characteristics of AL interneurons in thisspecies are similar to what was found in other mothspecies earlier, but we also found species-specificcharacteristics.

4.1. Anatomy

The AL shows the same general organisation as thatobserved in other species studied so far (for a review, see

Anton and Homberg, 1999). The number of glomeruli(approximately 60–65) and their spatial arrangementaround a central fibre core are similar to what was foundin other moth species (Rospars, 1983; Rospars and Hild-ebrand, 1992, 2000; Sadek et al., 2002). However, ourresults showed that glomeruli of L. botrana are arrangedin several layers around the small central core, not inone layer like in most other moth species (Anton andHomberg, 1999). Moreover, an extensive cell body clus-ter is situated in the anterior-ventral part of the AL ascompared with two or three separate smaller clusters in


Fig. 6. Examples of dose–response curves obtained from L. botranaAL interneurons in response to plant odours. Responses of one individ-ual neuron to different stimuli (A), (B). Responses of three differentneurons to the same stimulus ((+/�)-linalool) (C). Various shapes ofcurves were observed ranging from dose independent to highly dosedependent.

Fig. 7. Response specificity of L. botrana AL interneurons. Percent-age of tested AL neurons that responded to a varying number of com-pounds. A large proportion of neurons responded specifically to onlyone of the tested compounds, but also a large proportion of generalistneurons was found.

other species. The organisation of the AL showed a sex-ual dimorphism, characterised by the presence of oneenlarged glomerulus in males, which is most likely a sexpheromone-specific macroglomerulus, as found in manyother insect species (Anton and Homberg, 1999 and ref-erences therein). The structure of the stained PNsresembled also that of PNs in other species. The func-tional significance of the extremely dense arborisationswithin the AL and the protocerebrum, however, is notknown so far.

4.2. General physiology

All tested compounds elicited a response in at leastsome AL neurons. Many neurons responded to tansy-specific odours (β-thujone, thujyl alcohol) and (E)-2-hexenal. β-thujone is the main component of tansyflowers, a non-host plant, and induced a high EAGresponse in L. botrana females (Gabel et al., 1992,1994). (E)-2-hexenal is a green leaf volatile and wasfound to induce responses in a large proportion of ALinterneurons in A. ipsilon (Greiner et al., 2002), and inS. littoralis (Anton and Hansson, 1994, 1995). Manyneurons responded also to (+/�)-linalool, a compoundthat is commonly emitted by floral and vegetative partsof many plants including grapevine (Schreier et al.,1976; Knudsen et al., 1993). Intracellular recordingsrevealed that PNs of an identified sexually dimorphicolfactory glomerulus responded preferentially to (+/�)-linalool in females of M. sexta (Roche King et al., 2000)but its effect on female-specific behaviour is unknown.The different response patterns for different compounds(delays, excitation/inhibition pattern, response duration)in some neurons might serve as a means for dis-tinguishing different compounds. Neurons exhibiting


long delays in their responses might be higher order neu-rons.

The amount of plant volatile needed to obtain a neuronresponse varied, but ranged often between 10 and 100µg on a filter paper for different compounds. Thesevalues are similar to plant volatile thresholds of ALinterneurons in M. sexta females (Roche King et al.,2000), in S. littoralis males and females (Anton andHansson, 1994, 1995) and in A. ipsilon males (Greineret al., 2002). A direct comparison of thresholds for dif-ferent compounds cannot, however, be made, due to dif-ferences in volatility of the used odours and solvents.Dose–response relationships in AL neurons varieddepending on the applied volatiles and on the neurontypes and were often different from typical dose–response curves found in olfactory receptor neurons(Anderson et al., 1995; Hansson et al., 1999), indicatingthat integration processes take place at the level of theAL.

Response specificity of the tested neurons varied to alarge extent. Although most neurons responded to manyplant odours, 20% of them were found to respond toonly one of the compounds tested, as found in othermoth species, e.g. in A. ipsilon males (Greiner et al.,2002). These results indicate that information on plantodours is subject to parallel processing, like sex phero-mone information in moths. Some specific informationon single compounds could reach the AL through labeledlines and would be preserved until the protocerebrum,whereas the same information is in parallel highly inte-grated already in the AL (see Hansson and Chris-tensen, 1999).

4.3. Neuron responses as a function of sex and matingstatus

In general, similar response characteristics of AL neu-rons were found in the different groups of moths forthe stimuli used. Unfortunately, only few behaviourallyactive compounds are known so far and other volatilesmight have elicited clearer differences in responsesbetween either males and females or virgin and matedinsects. Possibly, volatiles produced by grapevine plantsare not the main attractant for L. botrana, as indicatedby field studies (Geisler, 1959).

Neuron responses to all tested plant odours wereobtained for both males and females of L. botrana, butthere was a tendency towards a lower proportion ofresponses in males. Response thresholds for the sameodours (10–100 µg) were similar in both sexes and com-parable to thresholds in male and female S. littoralis(Anton and Hansson, 1994, 1995).

Plant odours attract females to oviposition sites andcould be important for males in addition to female sexpheromones in finding a mating partner. Plant odoursmight also indicate food sources for both sexes. Mated

L. botrana females were observed in the field to feed onnectar and pollen of tansy (Gabel, 1992). A high pro-portion of responses to rather specific tansy odours wasobtained in interneurons of female and male L. botrana.β-thujone and thujyl alcohol which elicited frequentresponses in neurons of L. botrana are not restricted totansy odours but do not seem to be present in grapevine(Schreier et al., 1976; Buchbauer et al., 1994). It thusappears that tansy flowers could be of behaviouralimportance for both sexes. Although the biological sig-nificance of tansy attraction is not known, our resultsconfirmed previous electrophysiological and behaviouraldata (Gabel et al., 1992, 1994; Gabel and Thiery, 1994).

Response delay, duration, and excitation/inhibitionpatterns, as well as response specificity of neurons forplant odours were similar in mated and unmated mothsof both sexes. Differences in response thresholds and theoccurrence of responses were, however, obtained for afew compounds. There was a tendency towards morefrequent responses in mated females and unmated malescompared to unmated females and mated males. If theserelatively subtle differences could be enough to explainbehavioural changes after mating needs to be proven.In addition, we only investigated specific elements of acomplex neuronal network treating odour information.The effects could of course be enhanced at higher levelsin the central nervous system and we might have misseddifferences occurring in other elements of the odour pro-cessing pathway. The physiological results fit in any casewith the general idea that mated females would look foroviposition sites and that unmated males would searchfor sites where females are present. Possibly, matingcould have a suppressing effect on plant odour respon-siveness in males and an enhancing effect in females.A transient inhibition of central nervous processing andbehavioural response to sex pheromone has recentlybeen demonstrated in newly mated A. ipsilon males, sug-gesting that such mechanisms could also possibly occurfor plant odours (Gadenne et al., 2001).

The present study describes response characteristicsof AL interneurons to plant volatiles in laboratory-rearedL. botrana. These results will serve as a basis for thecomparison of central nervous processing in differentgenerations of field-collected moths, which are exposedto very different odours depending on the developmentalstatus of their host plants. These future experiments willallow us to test the hypothesis that plasticity exists notonly in sex pheromone processing but also in centralplant odour processing.

Acknowledgements

We thank M.C. Dufour for insect rearing, D. Thieryfor supplying us with tansy extract and B.S. Hansson,M. Carlsson and two anonymous referees for comments


on the manuscript. This work was supported by Frenchand Swedish councils (INRA, Formas), Comite Interpro-fessionnel des Vins de Bordeaux (CIVB) and by theConseil Regional d’Aquitaine. I. Masante-Roca ben-efited from travel grant by INRA and University Bor-deaux II.

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Plant odour processing in the antennal lobe of male and female grapevine moths, Lobesia botrana (Lepidoptera: Tortricidae)