Journal of Insect Physiology 59 (2013) 1222–1234

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Journal of Insect Physiology

journal homepage: www.elsevier .com/ locate/ j insphys

Characterization of olfactory receptor neurons for pheromone candidateand plant volatile compounds in the clover root weevil, Sitona lepidus

0022-1910/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.jinsphys.2013.10.002

⇑ Corresponding author. Tel.: +64 3 977 7351; fax: +64 3 977 7341.E-mail address: kpark@plantandfood.co.nz (K.C. Park).

Kye Chung Park a,⇑, Mark McNeill b, C. Rikard Unelius a,c, Hyun-Woo Oh d, David M. Suckling a

a The New Zealand Institute for Plant & Food Research, Lincoln 7608, New Zealandb AgResearch Limited, Private Bag 4749, Christchurch 8140, New Zealandc Faculty of Health and Life Sciences, Linnaeus University, 391 82 Kalmar, Swedend Korea Research Institute of Bioscience & Biotechnology, Daejeon 305-806, South Korea

a r t i c l e i n f o a b s t r a c t

Article history:Received 10 June 2013Received in revised form 9 October 2013Accepted 12 October 2013Available online 23 October 2013

Keywords:Clover root weevilHost-plant volatileOlfactory receptor neuronOlfactory sensillaPheromoneSingle-sensillum recording

Antennal olfactory receptor neurons (ORNs) for pheromone and plant volatile compounds were identifiedand characterized in male and female clover root weevil, Sitona lepidus (Gyllenhal), using the single sen-sillum recording technique with five pheromone-related compounds, and 40 host and non-host plant vol-atile compounds. Overall, seven different types of olfactory sensilla containing specialized ORNs wereidentified in each sex of S. lepidus. Among them, three different types of sensilla in the males and twotypes in the females housed ORNs specialized for pheromone-related compounds. The ORNs in maleswere specialized for 4-methyl-3,5-heptanedione or one or more of four stereoisomers of 5-hydroxy-4-methyl-3-heptanone. In contrast, female sensilla did not contain ORNs sensitive to 4-methyl-3,5-heptanedione while they contained ORNs sensitive to and specialized for the stereoisomers of (4S,5S)-5-hydroxy-4-methyl-3-heptanone. In addition to the pheromone-related ORNs, four types of olfactorysensilla contained ORNs responsive to plant volatile compounds in male S. lepidus, and five types infemales. Most of the ORNs identified in S. lepidus showed a high degree of specificity to specific volatilecompounds although some of the active compounds showed overlapping response spectra in the ORNsacross different types of sensilla. The most active plant volatile compounds were the four green leaf vol-atile compounds, (E)-2-hexenol, (Z)-2-hexenol, (Z)-3-hexenol and (E)-2-hexenal, and isomers of twomonoterpenols, (±)-linalool and (±)-a-terpineol, all eliciting strong responses from relatively large num-bers of ORNs in male and female S. lepidus. Our study indicates that S. lepidus has a set of highly sensitiveand selective ORNs for pheromone and plant volatile compounds. Further work is needed to elucidate thebehavioral implications of these findings.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Olfactory receptor neurons (ORNs) on the insects’ antennae playan essential role in detecting and discriminating important odor-ants for locating their mate and food. Phytophagous insects havespecies-specific sets of antennal ORNs and many of these ORNsare specialized for detecting a narrow range of volatile compounds(Barata et al., 2002; Larsson et al., 2001; Stensmyr et al., 2001).Often, the profiles of ORNs are directly related to the behavioralactivities of their corresponding active compounds (Ache andYoung, 2005; Bargmann, 2006). In pheromone communication, anumber of sex-specific ORNs in insect antennae are specializedfor detecting sex pheromone or its behavioral antagonist com-pounds (Baker, 2008; Baker et al., 2004; Larsson et al., 1999). Inhost location, the presence of ORNs specialized for detecting

kairomones released from host and non-host plants has been re-ported in a wide range of insect groups (Andersson et al., 2012;Larsson et al., 2001; Olsson et al., 2006). Sensory physiologicaland olfactory receptor protein studies indicate that the profiles ofthe ORNs in insects are species-specific (Ache and Young, 2005;Andersson et al., 2012). Furthermore, the specialization of ORNsplays critical parts of species-isolation and host-specificity in in-sects (Andersson et al., 2009; Baker, 2008).

Electrophysiological recording can be used to identify the activecompounds on the specific ORNs and to examine the responsespectra of the ORNs. Single sensillum recording (SSR) measures ac-tion potentials from individual ORNs in olfactory sensilla, provid-ing valuable information on the specificity and sensitivity of eachORN to different volatile compounds. The information obtainedcan often be used to develop effective synthetic semiochemicalbaits to manage the behavior of insects. Indeed, synthetic blendsof volatile compounds identified by the headspace sampling ofhost plants and subsequent electrophysiological recordings have

K.C. Park et al. / Journal of Insect Physiology 59 (2013) 1222–1234 1223

been proven to be behaviorally active in various insects (Bruce andPickett, 2011; Olsson et al., 2009).

Sitona (Coleoptera: Curculionidae) is a relatively large genus ofweevils, containing around 100 species. Sitona weevils are phy-tophagous, feeding exclusively on plants of the family Fabaceae,with their larvae being subterranean feeders (Danthanarayana,1967). Several species of Sitona are important agricultural pestsof legumes (Petrukha, 1970). The clover root weevil, Sitona lepidus,is of European origin but has spread to North America and NewZealand, being a destructive pest of white and red clover in tem-perate grasslands (Barratt et al., 1996; Bright, 1994; Murray andClements, 1994). Sitona species exhibit considerable species-specificity in host selection. For example, Sitona lineatus feedsand oviposits around pea (Pisum sativa), bean (Vicia faba) and vetch(Vicia sativa) (Landon et al., 1997), S. regenstenensis feeds on broom(Cytisus scoparius) (Danthanarayana, 1969), Sitona discoideus andSitona humeralis feed on lucerne (Medicago spp.) (Aeschlimann,1984; Phillips and Barratt, 2004), Sitona crinitus attacks lentils (Lensspecies) (El-Bouhssini et al., 2008), and S. lepidus feeds on clover(Trifolium spp.) with a preference for white clover (Trifolium repens)(Johnson et al., 2004; Phillips and Barratt, 2004). It is likely thatolfactory cues are involved in host location in Sitona weevils, asshown in behavioral observations (Hardwick and Harens, 2000).Therefore, it could be hypothesized that different species of Sitonawould have species-specific sets of ORNs for discriminatingbetween their host plants and non-host plants. However, it is notyet well understood how S. lepidus locate their host plants andmates, and what kind of olfactory cues are involved.

A diketone, 4-methyl-3,5-heptanedione, is an aggregation pher-omone in S. lineatus (Blight et al., 1984; Blight and Wadhams,1987; Toshova et al., 2009). Recently, it has been reported thatthe males of another Sitona species, S. discoideus, produce a sex-specific monoketone, (4S,5S)-5-hydroxy-4-methyl-3-heptanone,as well as the diketone (Unelius et al., 2013). A diastereomer of thismonoketone, (4S,5R)-5-hydroxy-4-methyl-3-heptanone, has beenknown as an aggregation pheromone compound in several weevilspecies in the related genus Sitophilus, Curculionidae (Phillips et al.,1985; Schmuff et al., 1984; Walgenbach et al., 1987). It is commonin insects that taxonomically related species share structurallysimilar compounds for inter- and intra-specific olfactory commu-nication (Cossé et al., 1998; Domingue et al., 2007). It appears thatsome Sitona species share 4-methyl-3,5-heptanedione as a com-mon compound for their pheromone communication, since fieldstudies showed several species were attracted to traps baited withthis compound in Hungary and Bulgaria (Toshova et al., 2009; Tothet al., 1998). In this case, species-specific sets of ORNs for this andother pheromone compounds would facilitate differentiatingsemiochemical signals among different Sitona species, as in othergroups of insects. The strong preference of S. lepidus to white clover(Johnson et al., 2004) also indicates that this weevil uses chemicalsignals to locate its host plant.

In this study, we have investigated the profiles of antennalORNs in male and female S. lepidus, using the SSR technique, witha hypothesis that S. lepidus has a specialized intra- and inter-specific peripheral olfactory sensory system for mate and hostlocation. Specialized ORNs for pheromone-related and plant-volatile compounds have been identified and their responseprofiles characterized.

Fig. 1. A diketone, 4-methylheptane-3,5-dione, and the four isomers of a monok-etone, 5-hydroxy-4-methylheptan-3-one, pheromone-related compounds used inour study. Note that the diketone is in equilibrium with an enol form (ca 10% enol).

2. Materials and methods

2.1. Insects

Adults of S. lepidus used in the experiments were collected fromT. repens stands in the Canterbury region of New Zealand. The age

of the weevils at the time of collection was indeterminable, but atleast 7 days old. Males and females were distinguished based onthe shape of the ventrite (Bright, 1994) and kept in separate con-tainers with fresh-cut T. repens.

2.2. Microscopy

Adult males and females of S. lepidus were individually fixed inPBS-buffered 2.5% paraformaldehyde–glutaraldehyde solution indistilled water for at least 2 days. For scanning electron microscope(SEM) observation the fixed antennae of six males and five femaleswere mounted on aluminum stubs and gold-coated with a sputtercoater (SC502, Polaron). The antennae were then observed with aSEM (FEI Quanta 250 FEG). For light microscopy (LM) and trans-mission electron microscopy (TEM) the fixed antennae from onemale and one female were further fixed with 1% osmium tetroxidein water, embedded in epoxy resin (Epon 812), sectioned at 1 lmthick for LM or at <150 nm thick for TEM with an ultra-microtome(Leica, Austria). The sections for LM were stained with methyleneblue and the sections for TEM with uranyl acetate and lead citrate.The sensilla on the antennae of S. lepidus were observed with alight microscope (Elipse Ci-L, Nikon, Japan) and a transmissionelectron microscope (CM20, Philips, the Netherlands).

2.3. Test compounds and odor presentation

Five pheromone-related compounds (4-methyl-3,5-heptanedi-one and the four stereoisomers of (4S,5S)-5-hydroxy-4-methyl-3-heptanone) (Fig. 1) and 40 host or non-host plant volatilecompounds were used as stimuli in the single sensillum recording(SSR) (Table 1). At least 12 of the plant volatile compounds inves-tigated for SSR activities are present in red and white clover(Buttery et al., 1984; Figueiredo et al., 2007; Kigathi et al., 2009),the host plant of S. lepidus. The rest of the test compounds are com-mon volatiles across many plant species. The source, purity andpresence in clovers of the test compounds are shown in Table 1.The plant volatile compounds were purchased from commercial

Table 1Test compounds for the SSR study of Sitona lepidus and their source, purity and the presence in clovers.

Mixture group Compound Planta Purityb (%) Source

Mix-P 4-Methylheptane-3,5-dione (diketone) 90c Synthesized(4S,5S)-5-Hydroxy-4-methylheptan-3-one (SS) >98 (97 ep) Synthesized(4S,5R)-5-Hydroxy-4-methylheptan-3-one (SR) >98 (95 ep) Synthesized(4R,5S)-5-Hydroxy-4-methylheptan-3-one (RS) >98 (96 ep) Synthesized(4R,5R)-5-Hydroxy-4-methylheptan-3-one (RR) >98 (99 ep) Synthesized

Mix-GLV Hexane 99 Aldrich1-Hexanol R4 99 Aldrich(E)-2-Hexen-1-ol 96 Aldrich(Z)-2-Hexen-1-ol R1 95 Aldrich(Z)-3-Hexen-1-ol R1, W2 98 AldrichHexanal 98 Aldrich(E)-2-Hexenal R1, W2 98 AldrichHexyl acetate 99 Aldrich(Z)-3-Hexenyl acetate R1,3 98 Aldrich2-Heptanone 99 Aldrich

Mix-A 1-Nonanol 98 FlukaEthyl (2E,4Z)-2,4-decadienoate 98 Bedoukian(E)-b-Farnesene R1,3,4 98 BedoukianCaryophyllene R1, 4 98.5 SigmaGermacrene-D 40 Treat & Co

Mix-B (±)-Limonene R3, W2 97 MerckMyrcene 95 Aldrich(E)-b-Ocimene R1,3 70 Fluka(±)-a-Pinene 99 Aldrich

Mix-C Geraniol 98 Aldrich(±)-Linalool R3, W2 97 AldrichNerol 96 Aldrich2-Phenylethanol R4, W2 99 Fluka(±)-a-Terpineol 90 Aldrich

Mix-D Benzaldehyde R4, W2 99.5 AldrichCitral (geranial + neral) 96 AldrichPhenylacetaldehyde 90 Aldrich

Mix-E Benzyl acetate 99 AldrichDiethyl malonate 99 AldrichGeranyl acetate 98 AldrichIsobutyl phenylacetate 98 AldrichMethyl benzoate 99 AldrichMethyl phenylacetate 99 AldrichNeryl acetate 96 Aldrich

Mix-F 1,8-Cineole (Eucalyptol) 98 Aldrich(±)-Citronellal 95 Aldrich(±)-a-Phellandrene 95 Aldrich(±)-b-Pinene 99 Aldrichc-Terpinene 97 Aldrich(±)-a-Terpinyl acetate 90 Aldrich

a Plants producing the corresponding compounds as a major volatile emanation: R (red clover, Trifolium pratense); W (white clover, T. repens).b Chemical purity (ep: enantiomeric purity).c The diketone is in equilibrium with its enol tautomer (ca. 10% according to GC).1 Buttery et al. (1984).2 Kicel et al. (2010).3 Kigathi et al. (2009).4 Figueiredo et al. (2007).

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sources, and the pheromone-related compounds were synthe-sized as described in Bohman and Unelius (2009). Each com-pound was dissolved in hexane as a 50 ng/ll solution, exceptthe green-leaf volatile compounds (Table 1) that were preparedin mineral oil at the same concentration. The test compoundswere divided into eight groups (Table 1), and the mixture solutionof each group was also prepared in hexane (or in mineral oil forGroup Mix-GLV) at a concentration of 50 ng/ll for each com-pound in the group. Hexane or mineral oil was used as the solventcontrol stimulus. Serial dilutions (0.5 pg/ll–50 ng/ll) of somecompounds were also prepared in hexane or mineral oil for mea-suring dose–responses of ORNs.

Presentation of test chemicals to the insect antennae was simi-lar to previous studies (Park and Baker, 2002; Park and Hardie,

2004). A 20 ll aliquot of each test solution was applied onto a piece(5 � 30 mm) of filter paper (Whatman No 1, USA), and the filter pa-per strip was inserted into a glass Pasteur pipette (146 mm, FisherScientific, USA) after being evaporated for 10 s in air. The tip of thepipette was inserted into a small hole (2 mm diameter, 10 cm fromthe outlet to the antennae) in a glass main airflow tube with a con-tinuous, charcoal-filtered and humidified airflow (600 ml/min)over the antennal preparation. A 0.1-s pulse of charcoal-filteredairflow (10 ml/s) was injected through the wide end of the Pasteurpipette odor cartridge for stimulation, using an electronic airflowcontroller (CS-55, Syntech, Hilversum, The Netherlands). The wideend of the Pasteur pipette was covered with a piece of aluminumfoil when not in use to reduce evaporation. Each odor stimulus car-tridge was used less than 10 times.

K.C. Park et al. / Journal of Insect Physiology 59 (2013) 1222–1234 1225

2.4. Single sensillum recording

A weevil was mounted on a Plasticine� block with U-shapedthin copper wire restraints, and each antenna was further fixedusing fine copper wires. Then, the preparation was positioned inthe middle of the charcoal-filtered and humidified main airstream.A fine tip (tip diameter <10 lm) glass electrode (0.86 mm ID, A-MSystems Inc., USA) filled with 0.1 M KCl was inserted into a mem-branous part of abdomen to serve as the reference electrode. Anelectrochemically sharpened tungsten electrode (tip diameter<0.1 lm) was used as a recording electrode and the position ofthe electrodes was controlled with micromanipulators (Leitz, Ger-many). An Ag–AgCl junction was used to maintain electrical conti-nuity between the reference electrode and the ground input of ahigh input impedance headstage preamplifier (Syntech, Hilversum,The Netherlands). The AC signals through the preamplifier werefurther amplified, digitized at 12,000/s sampling rate, and pro-cessed with a PC-based signal processing system (IDAC-4, Syntech,The Netherlands) and software (Autospike 32, Syntech, Hilversum,The Netherlands).

Once a stable electrical contact was made between the elec-trodes and a sensillum, showing spontaneous firing of actionpotentials, the antenna was stimulated with a series of eight mix-tures of test compounds (Table 1). If any electrophysiological re-sponse was observed after the stimulation with mixtures, theantenna was further stimulated with the individual compoundscontained in the mixture eliciting responses. The order of testingchemicals was made at random. The time interval between succes-sive stimulation was approximately 30 s. When a response lastedfor a long time (e.g. >30 s), sufficient time was allowed until spon-taneous activity returned to background levels before re-stimula-tion. Sensilla on the three circumferential sensory band regionson the club-shaped terminal flagella segment (Fig. 2 Sb) weremainly investigated from 12 females and 15 males in this study.

The responsiveness of ORNs was analyzed by comparing thenumber of action potentials before and after odor stimulation. Ineach recording, the number of action potentials for 1 s after odorstimulation was subtracted by the average number of action

Fig. 2. Antennal morphology of female (A–F) and male (G, H) Sitona lepidus. The seven-seat the terminal club (A, G). A number of basiconic sensilla (F, arrows) are visible at the circthe terminal club (E, H). Cell bodies (cb) connected to the sensilla and their nuclei are sebundles (ab) run inside along the flagella segments. Scales: 100 lm (A, G, H), 50 lm (B)

potentials for 1 s before the stimulation. Then, the averaged num-bers (n = 3–47 depending on sensilla types) of the subtracted val-ues were classified into seven different categories of ORNresponse strength (details described in Tables 3 and 4). The ORNswere then classified into different types using an algorithm withstep-by-step analysis according to their response profiles acrossthe test compounds (Table 2). The number of co-compartmental-ized ORNs in a sensillum was determined by comparing the sizeof action potentials generated in each sensillum. Separate ORNsin a sensillum were recognized when the amplitudes of actionpotentials fell into two or more distinctive groups in their histo-gram distribution (Autospike, Syntech, The Netherlands). Statisti-cal analysis was carried out using ANOVA followed by Fisher LSDtest when necessary, with a software package DSAASTATver.1.0192 (University of Perugia, Italy).

3. Results

The club-shaped antennae of male and female S. lepidus containa number of sensilla (Fig. 2). Almost all of the antennal sensilla areeither chaetica, trichodea or basiconica in their shape, and theclub-shaped terminal (7th) flagella segment, which is composedof four subsections, contains the most of trichoid and basiconicsensilla. The most of basiconic sensilla appeared to be located atthe circumferential bands of the first three subsections where themajority of ORNs were identified. Two axon bundles from the sen-sory neurons run inside along the antenna (Fig. 2B and C). A num-ber of cell bodies were seen inside the 1st–6th flagella segments,where these cells appeared to be innervated to the sensilla struc-ture (Fig. 2).

The ORNs observed in S. lepidus showed spontaneous firing ofaction potentials at 19.5 ± 1.90 spikes/s (mean ± SE, n = 37, mini-mum 0, maximum 35) and 13.4 ± 1.93 spikes/s (mean ± SE,n = 48, minimum 0, maximum 50) in pheromone-related ORNs ofmales and females, respectively, and at 6.3 ± 2.18 spikes/s(mean ± SE, n = 20, minimum 0, maximum 32) and 6.3 ± 2.17spikes/s (mean ± SE, n = 21, minimum 0, maximum 33) in plant-volatile related ORNs of males and females, respectively. Mineral

gment antennal flagella bear sensilla, with the majority of the sensilla being locatedumferential regions (sb: ‘sensory band’) of the first three (1–3) in four subsections ofen in the LM (B) and TEM (D) of cross section of flagella (A. vertical line). Two axon, 10 lm (C, D, F) and 30 lm (E).

Table 2An algorithm for classifying the types of sensilla and ORNs (PM, PT and PP, in bold) in Sitona lepidus. The types of sensilla and ORNs were systematically classified according totheir electrophysiological responsiveness to different groups of test compounds.

Step Grouping principle�� Resulting groups

1 Show spontaneous activity Go to 2No spontaneous activity Discard (no ORNs)

2 Respond to pheromone mixture Go to 3 (Type PM)Respond to plant volatile Go to 4 (Type PL)No response to pheromone mixture or plant volatile mixtures Non-responsive ORNs

3 Response to 4-methylheptane-3,5-dione: >50 Hz Go to 5Response to 4-methylheptane-3,5-dione: >20, <50 Hz This type not foundResponse to 4-methylheptane-3,5-dione: <20 Hz Go to 6

4 Respond to a specific group of compounds (<10 compounds) Go to 7Respond to a broad range of compounds (P10 compounds) This type not found

5 Fair or strong response to RR isomera Type M-PM-ANo or weak response to RR isomera Type M-PM-B

6 Strong response to SS isomera, male Type M-PM-CStrong response to SS isomera, female Type F-PM-ANo response to SS isomera, female Type F-PM-B

7 Fair or strong response to green leaf volatiles only Go to 8Fair or strong response to green leaf volatiles and other compounds Go to 9No or weak response to green leaf volatile Go to 10

8 Male Type M-PL-AFemale Type F-PL-A

9 Strong response to Z3-6:OH; Weak or no response to E2-6:Ald, male Type M-PL-BStrong response to Z3-6:OH; Weak or no response to E2-6:Ald, female Type F-PL-BNo response to Z3-6:OH; Strong response to E2-6:Ald Type M-PL-C

10 Fair or strong response to (±)-linalool Go to 11Weak or no response to (±)-linalool Go to 12

11 No response to benzyl acetate, male Type M-PL-DNo response to benzyl acetate, female Type F-PL-C

12 Strong response to (±)-citronellal; no response to benzyl acetate Type F-PL-DStrong response to benzyl acetate; no response to (±)-citronellal Type F-PL-E

� Increase of the number of spikes by >10 spikes/s after the stimulation was regarded as a ‘response’.� Weak response (<20 spikes/s); Fair response (P20 and <50 spikes/s); Strong response (P50 spikes/s).

a RR: (4R,5R)-5-hydroxy-4-methylheptan-3-one; RS: (4R,5S)-5-hydroxy-4-methylheptan-3-one; SR (4S,5R)-5-hydroxy-4-methylheptan-3-one; SS (4S,5S)-5-hydroxy-4-methylheptan-3-one.

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oil used as a solvent for green leaf volatiles showed no activities inany of the ORNs tested. In contrast, hexane used as a solvent for allother test compounds elicited significant responses from someORNs.

In the S. lepidus antennae examined, 94 sensilla (46 in males and48 in females) were found to contain ORNs responsive to phero-mone-related compounds, and 110 sensilla (50 in males and 60in females) contained ORNs for plant-volatile compounds. The sen-silla containing ORNs responsive to pheromone-related com-pounds were classified into five types (Table 3) and the sensillacontaining ORNs responsive to plant volatiles into nine types (Ta-ble 4), based on the response spectra of the ORNs housed in thesesensilla to the test compounds. Although we separated phero-mone-related ORNs and plant-volatile related ORNs in our results,both types of ORNs were co-compartmentalized in a number ofsensilla (Table 5). There were a few sensilla in both males and fe-males showing no responses to any of the compounds tested, butshowed spontaneous firing of action potentials.

Three distinct types of sensilla containing ORNs responsive topheromone-related compounds were identified in the males whileonly one distinct type of such sensilla was observed in the females(Table 3). One sensillum (F-PM-B) in a single female containedORNs showing different response profile from that of the majortype (F-PM-A) sensilla to the pheromone-related compounds.ORNs in two types of sensilla (M-PM-A and M-PM-B) in the malesshowed strong responses to 4-methyl-3,5-heptanedione as well asto the RS-, SR- and SS-isomers of 5-hydroxy-4-methyl-3-heptanone

(Table 3; Fig. 3). While ORNs in type M-PM-A sensilla in males alsoshowed relatively strong responses to the RR-isomer, ORNs in typeM-PM-B sensilla in the males showed only weak responses to thiscompound (Table 3; Fig. 3). In contrast to these two types ofsensilla in the males, ORNs in type M-PM-C sensilla in malesand all the pheromone-related ORNs in females (type F-PM-A)showed no responses to 4-methyl-3,5-heptanedione, except onesensillum (F-PM-B) containing ORNs, showing mild responses tothis diketone (Table 3; Fig. 3).

All the ORNs identified in the 110 plant-volatile responsive sen-silla showed a high degree of specialization in their responsiveness,showing responses only to a narrow range of plant volatile com-pounds. Overall, four different types (M-PL-A–D) of sensilla con-taining ORNs responsive to plant volatile compounds wereidentified in male S. lepidus and five different types (F-PL-A–E) ofsensilla in the females (Table 4). Among 40 plant volatile com-pounds tested, 14 compounds (Table 4) elicited significant re-sponses from the ORNs in these sensilla. The responses of theseORNs to 26 other compounds were not significantly different fromthe responses to the solvent control. Approximately 86% (M-PL-A,M-PL-B and M-PL-C) of plant-volatile responsive sensilla in malesand 67% (F-PL-A and F-PL-B) in females contained ORNs that werehighly sensitive to specific green leaf volatiles (GLVs) (Table 4).Two types (M-PL-B, M-PL-C) of sensilla in males and one type(F-PL-B) in females contained ORNs responsive to both GLVs andnon-GLVs (Table 4; Fig. 4), comprising 35–38% of entire plant-vol-atile responsive sensilla in each sex. An aldehyde, (E)-2-hexenal

Table 3Types of sensilla containing ORNs responsive to pheromone-related compoundsidentified in males and females of Sitona lepidus and their responsiveness to fivepheromone-related compounds.��

Sex Male Female

Type M-PM-A M-PM-B M-PM-C F-PM-A F-PM-B2

N 23 5 18 47 1

Diketone +++++++a +++++++a +c +d ++RR ++++bc +bc +++++a ++++++a +++RS ++++c +++++ab ++bc ++++bc +SR ++++++b +++++++a ++b +++c +++SS +++++bc ++++++a +++++a +++++ab +Hexane1 +d +c +c +d +

� The number of ‘+’ indicates the responsiveness of each type of ORNs for thecorresponding pheromone-related compounds. The average number of spikes: +(<10 spikes/s); ++ (<20 spikes/s); +++ (<30 spikes/s); ++++ (<40 spikes/s); +++++(<50 spikes/s); ++++++ (<60 spikes/s); +++++++ (P60 spikes/s).� Diketone: 4-methylheptane-3,5-dione; RR: (4R,5R)-5-hydroxy-4-methylheptan-

3-one; RS: (4R,5S)-5-hydroxy-4-methylheptan-3-one; SR: (4S,5R)-5-hydroxy-4-methylheptan-3-one; SS: (4S,5S)-5-hydroxy-4-methylheptan-3-one. Twenty-eightsensilla in female Sitona lepidus showed various responsiveness that were notclassified into distinct types and are not shown in this table.Different letters indicate that means (mean number of spikes/s after stimulation)are significantly different within a column (Fisher LSD, p = 0.05).

1 Hexane: solvent blank control.2 The number observed was not enough to carry out statistical analysis.

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(E2-6:Ald), and three alcohols, (E)-2-hexenol (E2-6:OH), (Z)-2-hex-enol (Z2-6:OH) and (Z)-3-hexenol (Z3-6:OH), elicited the strongestresponses from these ORNs in both males and females. Two six-carbon acetates and a seven-carbon ketone elicited no or verysmall responses from these ORNs. There was a clear difference inthe responsiveness of GLV-responsive ORNs to Z3-6:OH andE2-6:Ald between type M-PL-A and M-PL-B sensilla in the malesand between type F-PL-A and F-PL-B sensilla in the females (Table 4;Fig. 4). Z3-6:OH as well as two other alcohols, E2-6:OH and Z2-6:OH,elicited strong responses from the ORNs in type M-PL-B sensilla. Incontrast, Z3-6:OH did not elicit significant response from any ORNsin type M-PL-A sensilla while E2-6:OH and Z2-6:OH elicited strongresponses from ORNs in the same sensilla.

Among the plant-volatile responsive ORNs, approximately 52%of sensilla identified in males (M-PL-B, M-PL-C and M-PL-D) and68% in females contained ORNs responsive to some plant volatilecompounds that are not GLVs (Table 4). Only 9 ((±)-linalool, 2-phenylethanol, (±)-a-terpineol, benzaldehyde, citral, phenylacetal-dehyde, benzyl acetate, methyl phenylacetate and (±)-citronellal)of 30 non-GLVs tested elicited significant responses from theseORNs. Among them, (±)-linalool and (±)-a-terpineol elicited thestrongest responses from the ORNs in two types of sensilla (F-PL-C, F-PL-D) in females. Females of S. lepidus have more diverse andsensitive ORN populations for the non-GLVs than males have. Sixdifferent non-GLVs ((±)-linalool, 2-phenylethanol, (±)-a-terpineol,benzyl acetate, methyl phenylacetate and (±)-citronellal) elicitedrelatively strong, significant responses in females while only fourcompounds ((±)-linalool, benzaldehyde, citral and phenylacetalde-hyde) elicited significant, but mild responses in males.

Although the most of the active compounds on the ORNs iden-tified in our study increased the number of spikes after stimulationin the corresponding ORNs, some ORNs showed inhibitory re-sponses. The four isomers of the monoketones significantly inhib-ited the spontaneous firing activities of some ORNs, decreasing thenumber of action potentials after stimulation (Fig. 5).

The number of co-compartmentalized ORNs and correspondingactive compounds for each ORN could be determined in approxi-mately half of the sensilla examined in this study by comparingthe size of action potentials generated in each sensillum. In theother half of sensilla examined, however, the number of ORNs ina sensillum could not be determined since the size of action

potentials was not clearly distinguishable. Both ORNs responsiveto pheromone-related compounds and ORNs responsive to plantvolatile compounds were co-compartmentalized in the same sen-sillum in several different types of sensilla in male and female S.lepidus (Table 5). The co-compartmentalization of more than oneORN in a sensillum could be observed in all types of sensilla iden-tified in our study except three plant-volatile responsive sensillumtypes: M-PL-D in male and F-PL-A and F-PL-E in female S. lepidus(Table 5). One ORN was specialized for 4-methyl-3,5-heptanedioneand another for at least one of the stereoisomers of 5-hydroxy-4-methyl-3-heptanone in two types (M-PM-A and M-PM-B) of pher-omone-related compound responsive sensilla in males (Table 5).One type (M-PM-C) of sensilla in males and some of another type(F-PM-A) in females contained an ORN specialized for the RR-iso-mer and another ORN for the SS-isomer (Table 5). However, someof type F-PM-A sensilla in females appeared to contain two phero-mone-related ORNs, one specialized for the RR- and SS- and theother for RS- and SR-isomer (Table 5), which indicated that thetype F-PM-A sensilla could be further classified into two sub-groups. At least six types of sensilla containing ORNs responsiveto plant volatile compounds housed more than one ORN special-ized for plant volatile compounds (Table 5). Among these sensilla,type M-PL-A sensilla in males contained two ORNs, one specializedfor E2-6:OH and Z2-6:OH and the other for E2-6:Ald. Similarly,type M-PL-B sensilla in males contained one ORN for Z3-6:OHand another for (±)-linalool. In females, specialized ORNs forZ3-6:OH, (±)-linalool and (±)-a-terpineol were the most distin-guishable among the plant-volatile responsive ORNs that areco-compartmentalized in the same sensilla. Type F-PL-B sensillacontained an ORN specialized for Z3-6:OH and another for (±)-a-terpineol. Likewise, type F-PL-C sensilla contained one ORN for(±)-linalool and another for (±)-a-terpineol, respectively, and typeF-PL-D sensilla contained one for (±)-linalool and another for(±)-citronellal, respectively. Type F-PL-C sensilla in female ap-peared to co-compartmentalize two different ORNs for (±)-linalool,one responsive only to relatively large amount (10 lg) of (±)-linal-ool while the other responsive to a much lower dose, 10 ng (Fig. 6).

Most of the ORNs responsive to pheromone-related compoundsshowed relatively phasic responsiveness although temporal re-sponse profiles varied considerably among different recordings(Fig. 7). Similarly, most of the green-leaf volatiles showed phasicresponse profiles in the corresponding ORNs in both males and fe-males. The ORNs in male and female S. lepidus showed dose-depen-dent response profiles to the corresponding active compounds(Fig. 8). The diketone elicited significant responses from the ORNin type M-PM-A sensilla at 40 ng dose. Some ORNs in S. lepidus ap-peared to have extremely high sensitivity to some GLVs. For exam-ple, less than 100 pg of Z3-6:OH was enough to elicit significantresponses from the ORN in type M-PL-B sensilla in males.

4. Discussion

4.1. Pheromone ORNs in Sitona

Our study demonstrates that both male and female S. lepidus pos-sess a set of antennal ORNs for 4-methyl-3,5-heptanedione as well asto all four stereoisomers of 5-hydroxy-4-methyl-3-heptanone, andthat they can differentiate between the diketone and monoketoneand between the stereoisomers of the monoketone. Our study alsoindicates that the profiles of ORNs responsive to pheromone-relatedcompounds are different between males and females, and this sex-ual dimorphism in ORN profiles may imply the use of a sex phero-mone in S. lepidus. Males of S. lepidus have ORNs highly sensitive tothe diketone while females do not, which suggests that male-specificbehavioral activity of the diketone or structurally similar

Table 4Types of sensilla containing ORNs responsive to plant volatile compounds identified in males and females of Sitona lepidus and their responsiveness to 40 plant volatilecompounds.�

Sex Male Female

Type M-PL-A M-PL-B M-PL-C M-PL-D F-PL-A F-PL-B F-PL-C F-PL-D F-PL-EN 24 11 8 7 19 21 12 5 3

Hexane1 +d +d +c +b +d +cd +b +b +bMineral oil1 +cd +d +cdHexane2 +cd +d +cd1-Hexanol +++c +++bc +++c(E)-2-Hexen-1-ol +++++++a +++++b +++++++a(Z)-2-Hexen-1-ol +++++b +++++++a +++++b(Z)-3-Hexen-1-ol ++cd +++++++a ++cd ++++aHexanal ++cd +d ++cd(E)-2-Hexenal +++++++a +d ++++a +++++++aHexyl acetate +cd +cd ++cdZ3-Hexenyl acetate ++cd ++cd ++cd2-Heptanone +cd +d +cd1-NonanolEthyl decadienoate3

(E)-b-FarneseneCaryophylleneGermacrene-D

(±)-LimoneneMyrcene(E)-b-Ocimene(±)-a-PineneGeraniol +cd +b +cd ++b(±)-Linalool +++bcd ++a ++b +++++++a +++++++aNerol +++cd +b +cd ++b2-Phenylethanol +cd +b +++ab ++b(±)-a-Terpineol +++cd +b +++a +++++++aBenzaldehyde ++bCitral +++abPhenylacetaldehyde ++bBenzyl acetate +++ab ++++aDiethyl malonate +cd +bGeranyl acetate +cd +bIsobutyl phenylacetate +d +bMethyl benzoate +c ++abMethyl phenylacetate +++ab ++++aNeryl acetate +cd +b1,8-Cineole +b(±)-Citronellal ++++++a(±)-a-Phellandrene ++b(±)-b-Pinene ++bc-Terpinene ++b(±)-a-Terpinyl acetate +b

� The number of ‘+’ indicates the responsiveness of each type of ORNs for the corresponding pheromone-related compounds. The average number of spikes: + (<10 spikes/s);++ (<20 spikes/s); +++ (<30 spikes/s); ++++ (<40 spikes/s); +++++ (<50 spikes/s); ++++++ (<60 spikes/s); +++++++ (P60 spikes/s).Different letters indicate that means (mean number of spikes/s after stimulation) are significantly different within a column (Fisher LSD, p = 0.05).

1 Hexane and mineral oil were solvent blank.2 Hexane was dissolved in mineral oil and used as a stimulus.3 Ethyl decadienoate: Ethyl (2E,4Z)-2,4-decadienoate.

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compounds exist in S. lepidus. The diketone, 4-methyl-3,5-heptane-dione, was first identified in S. lineatus as an aggregation pheromone(Blight et al., 1984), and subsequently found to be behaviourallyattractive to several other Sitona species (Toshova et al., 2009; Tothet al., 1998). Therefore, taken together with our results, it is likelythat S. lepidus also use this diketone or a structurally relatedcompound as a pheromone. However, it is unclear how S. lepiduscould use this compound as an aggregation pheromone sincefemales do not appear to have well-developed ORNs for this com-pound. Apparently, male S. lepidus can discriminate between thediketone and the stereoisomers of the monoketone using theirantennal ORNs. Likewise, antennal ORNs in males enable themto discriminate between RR- and SS-, between RR-/SS- and RS-/SR-isomers of the monoketone. Such stereo-specific responsivenessof ORNs has been observed in some other coleopteran species(Larsson et al., 1999). However, it is unclear if males can discriminatebetween SR- and RS-isomers with their ORNs. Similarly, females

appear to be able to distinguish between RR-/SR- and RS-/SS-isomersof the monoketone with their ORNs. Our results also clearly showthat females of S. lepidus can discriminate between RR- and SS- or be-tween RR- and SR-isomers of the monoketone using a pair of ORNsco-compartmentalized in the same sensillum. The presence of ste-reo-specific ORNs for different monoketones may imply the pres-ence of stereo-specific behavioral activities of these monoketonesor structurally related compounds in S. lepidus. The enantio-specificproduction and attraction of an aggregation pheromone, (2S,3R)-1-ethylpropyl 2-methyl-3-hydroxypentanoate, in a weevil, Sitophilusgranarius (Phillips et al., 1989), and different responsiveness of anten-nal ORNs to different stereoisomers of the aggregation pheromonecompounds in three Sitophilus weevil species, Sitophilus oryzae,Sitophilus zeamais and Sitophilus granarius (Levinson et al., 1990), aswell as our observation in S. lepidus suggest that the stereo-specificpheromone communication system may be common in Sitona/Sitophi-lus weevils.

Table 5Co-compartmentalized ORNs identified in Sitona lepidus antennae. The ORNs were distinguished by comparing the amplitude of action potentials generated by the correspondingstimulus volatile compounds.

Sensillum type ORN Active compound�,a Other sensillum type housed together in the same sensillum

M-PM-A I SR M-PL-BII Diketone

M-PM-B I SR M-PL-BII Diketone

M-PM-C I RR Not foundII SS

M-PL-A I E2-6:OH, Z2-6:OH Not foundII E2-6:Ald

M-PL-B I Z3-6:OH M-PM-A, M-PM-BII (±)-Linalool

M-PL-C I Benzaldehyde Not foundII CitralIII E2-6:Ald

F-PM-A I RR F-PL-B, F-PL-C, F-PL-DII SS

F-PM-B I RR F-PL-BII SR

F-PL-B I (±)-a-Terpineol, (±)-linalool, methyl benzoate F-PM-A, F-PM-BII Z3-6:OH, benzyl acetate

F-PL-C I (±)-Linaloolb F-PM-AII (±)-a-Terpineol, (±)-linaloolb

F-PL-D I (±)-Linalool F-PM-AII (±)-Citronellal

� Diketone: 4-methylheptane-3,5-dione; RR: (4R,5R)-5-hydroxy-4-methylheptan-3-one; RS: (4R,5S)-5-hydroxy-4-methylheptan-3-one; SR: (4S,5R)-5-hydroxy-4-methyl-heptan-3-one; SS: (4S,5S)-5-hydroxy-4-methylheptan-3-one.

a Only the compounds showing clear differences in the size of the spikes elicited by them are listed here in the ‘active compound’ column although some other compoundscould elicit responses from the corresponding ORNs as shown in Table 4.

b In this sensilla, 10 lg of (±)-linalool elicited responses from both large-spike generating ORN and small-spike generating ORN while 10 ng of (±)-linalool elicited responsesonly from the small-spike generating ORN.

Fig. 3. Responses of ORNs housed in four different types of sensilla in Sitona lepidus to the diketone, 4-methylheptane-3,5-dione, and the four isomers of the monoketone, 5-hydroxy-4-methylheptan-3-one, pheromone-related compounds used in our study. Red bars indicate stimulation for 0.1 s. (For interpretation of the references to colour inthis figure legend, the reader is referred to the web version of this article.)

K.C. Park et al. / Journal of Insect Physiology 59 (2013) 1222–1234 1229

4.2. Plant volatile ORNs in Sitona

Our study shows that different types of ORNs with relativelynarrow response spectra across 40 different plant volatile

compounds are present in both male and female S. lepidus. Sevencompounds among the 14 plant volatiles eliciting significant re-sponses from ORNs in S. lepidus are major volatile compounds ema-nated by clovers (Buttery et al., 1984; Figueiredo et al., 2007; Kicel

Fig. 4. Responses of ORNs housed in four different types of sensilla in Sitona lepidus to some plant volatile compounds, the diketone and the SR isomer of the monoketone. TheORNs were starting to respond (red arrows) before the onset of stimulation (red bar) appeared to have the high sensitivity to the corresponding stimuli. Note that SR-isomerof the monoketone inhibited the spontaneous activity of ORNs in the Type M-PL-C sensillum. Red bars indicate stimulation for 0.1 s. (For interpretation of the references tocolour in this figure legend, the reader is referred to the web version of this article.)

Fig. 5. Inhibition against ORN activities observed in Sitona lepidus. The two syn-isomers (SR, RS) of the monoketone suppressed the spontaneous firing of actionpotentials almost completely in a type F-PL-C sensillum while a plant volatile,E2-6:Ald elicited excitatory responses of an ORN in this sensillum. The two othermonoketone anti- isomers (RR, SS) showed similar inhibitory activities in thissensillum (data not shown). Red bars indicate stimulation for 0.1 s. (For interpre-tation of the references to colour in this figure legend, the reader is referred to theweb version of this article.)

1230 K.C. Park et al. / Journal of Insect Physiology 59 (2013) 1222–1234

et al., 2010; Kigathi et al., 2009), suggesting that they are key com-pounds relating to the attraction of S. lepidus to its host plants.

We found that S. lepidus has a well developed ORN system thatcan discriminate between different GLVs. The largest proportion ofplant-volatile responsive ORNs in S. lepidus appeared to be special-ized for detecting green leaf volatiles (GLVs), with three alcohols(E2-6:OH, Z2-6:OH and Z3-6:OH) and an aldehyde (E2-6:Ald) beingmost active. The alcohols and the aldehyde are detected by differ-ent ORNs in S. lepidus, which enables the discrimination betweenthe alcohols and the aldehyde. Although it is unclear whether thesethree alcohols are detected by the same ORN in a sensillum it isapparent that both males and females of S. lepidus can discriminateZ3-6:OH from two other alcohols (E2-6:OH and Z2-6:OH) with thecombinational input from the ORNs in different sensilla that aredifferentially tuned to these alcohols. Two of these alcohols (Z2-6:OH and Z3-6:OH) and the aldehyde are the major volatiles pro-duced by clovers, the preferred host plants of S. lepidus (Buttery

et al., 1984; Kicel et al., 2010). Although Z3-6:Ac has been reportedas another major GLV produced by clovers (Buttery et al., 1984;Kigathi et al., 2009) no ORNs responsive to this compounds werefound in S. lepidus in our study. The sensitivity of some GLV-responsive ORNs to specific GLVs is extremely high in S. lepidus.The presence of specialized and extremely sensitive ORNs for spe-cific GLVs that are produced by its host plants suggests the behav-ioral significance of these compounds in S. lepidus. These ORNs forGLVs may also be related to the host-specificity of S. lepidus,since different groups of plants produce different sets of GLVs(Chamberlain et al., 2006). Using GLVs as a key component for hostlocation and recognition may be widespread in weevils and beetlessince the presence of highly sensitive and selective ORNs for GLVsappeared to be common in this group of insects (Andersson et al.,2009, 2012; Hansson et al., 1999; Larsson et al., 2001).

Our study also showed that several different types of ORNs spe-cialized for non-GLV volatiles are present in S. lepidus although theORNs for non-GLV compounds seem to be less abundant than thosein moths or other beetles (Shields and Hildebrand, 2001). In partic-ular, female S. lepidus appear to have more developed ORNs forthese non-GLV compounds. Among them, two compounds, (±)-lin-alool and (±)-a-terpineol, are the most active compounds in termsof sensitivity and the number of ORNs, which is more prominent infemales S. lepidus. It is unclear whether these two compounds arerelated to female-specific behavior in S. lepidus. It can be noted that(±)-linalool is a major volatile in the odor of clovers, while (±)-a-terpineol is not (Kicel et al., 2010; Kigathi et al., 2009). Likewise,benzyl acetate, methyl phenylacetate and (±)-citronellal are notthe major volatile emanations of clovers although specialized ORNsfor these compounds are present in S. lepidus. (±)-Linalool and afew GLVs may not be complete signature signal for clovers sincemany other plants also produce these compounds. However, therelative ratio of these compounds and other olfactory active com-pounds can be important for discriminating between host andnon-host plants by adults of S. lepidus, and the combinational inputfrom the ORNs specialized for the major volatiles produced by clo-vers and the ORNs specialized for non-host volatiles probably plays

Fig. 6. Responses of ORNs in two different types of antennal sensilla in Sitona lepidus to some plant volatile compounds, showing co-compartmentalization of ORNs in thesame sensilla. Different ORNs were distinguished based on the size of action potentials generated (L: large spike; S: small spike). In this sensillum, linalool elicited responsesfrom two different ORNs in the same sensillum (Type F-PL-C), generating large and small spikes from these ORNs, respectively. Note that the number of small spikes started toincrease (red arrow in C, S⁄ in E) before the onset of stimulation puff (red bar) was introduced, probably due to the high sensitivity of the small spike ORN to this compound.Red bars indicate stimulation for 0.1 s. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

K.C. Park et al. / Journal of Insect Physiology 59 (2013) 1222–1234 1231

a critical role in host-plant location in this species. Our studyshows that some sensilla co-compartmentalize two different ORNsresponsive to (±)-linalool, but with different sensitivities. Thismight be simply due to the different sensitivity of each ORN to(±)-linalool. However, it is also possible that each of these ORNshas different sensitivity to the enantiomers in the racemic linaloolthat we used in our study.

4.3. Implication to the biology of Sitona and other weevils

The behavioral mechanism of S. lepidus relating to pheromonecommunication and host location is not well understood althoughtheir attraction and preference to the host plants is relatively wellknown. We do not have evidence that 4-methyl-3,5-heptanedioneor any of the stereoisomers of 5-hydroxy-4-methyl-3-heptanoneis produced by S. lepidus. The diketone and SS-isomer of the

monoketone is produced by the males of a closely related species,S. discoideus and the males were attracted to conspecific females(Unelius et al., 2013). In addition, attraction of several Sitona spe-cies to the traps baited with the diketone in fields (Toshovaet al., 2009; Toth et al., 1998), and the presence of highly sensitiveand selective ORNs observed in our study implies the presence of apheromone communication system in S. lepidus. Although most ofthe pheromones identified in Curculionidae weevils are aggrega-tion pheromones, some weevil species in Curculionidae appear touse sex pheromone. For example, a mixture of two components,(Z)-3-dodecen-1-ol and (E)-2-butenoate, has been reported as fe-male-produced sex pheromone in the sweetpotato weevil, Cylasformicarius (Coffelt et al., 1978; Heath et al., 1986). The presenceof specialized ORNs responding to the diketone and the stereoiso-mers of the monoketone and their sexual dimorphism in S. lepidusmay imply that some of these compounds are used as aggregation

Fig. 7. Temporal response profiles of ORNs to various volatile compounds in Sitonalepidus. A, B and D show phasic responses to two GLV and a monoketonecompounds, C tonic response to a GLV compound, and E phasic-tonic response tohigher dose of the monoketone. Red bars indicate stimulation for 0.1 s. (Forinterpretation of the references to colour in this figure legend, the reader is referredto the web version of this article.)

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pheromone, while some others could play a role as sex pheromone.It is also possible that some of the ORNs for the diketone and themonoketone isomers play a role in detecting behavioral antagonistcompounds for species isolation, as shown in heliothine moths inwhich antennal sensilla contain co-compartmentalized ORNs spe-cialized for sex pheromone and its antagonist.

S. lepidus favors white clover (T. repens L.) with a preference toseedlings over mature plants (Hardwick and Harens, 2000), and thedistinct behavior of ‘antennal movement’ displayed by the adultsof S. lepidus towards white clover (Hardwick and Harens, 2000)indicates the involvement of olfaction in their host location. Simi-larly, the relationship between distinct antennal movement and

Fig. 8. Dose–responses of ORNs in two different types of pheromone-related compoundand those of two different types of plant-volatile responsive sensilla (bottom) to Z3-6:(n = 4–6).

behavioral attraction to volatile compounds has been shown in an-other Sitona species, namely S. lineatus and S. discoideus (Landonet al., 1997; Unelius et al., 2013). It is likely that volatile com-pounds from intact clover are involved in the host location by S.lepidus adults since root and leaf damage did not affect their behav-ioral responses to the host plant (Hardwick and Harens, 2000). Thepresence of specialized ORNs for clover volatiles, together with theolfactory attraction to clover (Hardwick and Harens, 2000) is evi-dence that olfactory signals are the critical cues for locating hostplants in S. lepidus. In a polyphagous Curculionidae weevilDiaprepes abbreviates (tropical root weevil), a mixture of threeplant volatile compounds, (±)-linalool, Z3-6:OH and carvacrol,was behaviorally attractive to females but repellent for males(Otálora-Luna et al., 2009). Likewise, some of the plant volatilecompounds activating ORNs in S. lepidus may have different behav-ioral roles between males and females.

4.4. Interaction between pheromone and plant volatiles and co-compartmentalization of ORNs in olfactory sensilla

The behavioral influence of plant volatile compounds on phero-mone communication has been reported in a number of insects.Synergism of plant volatiles and pheromones has been reportedin several coleopteran species (Deglow and Borden, 1998; Dickenset al., 1990; Reinecke et al., 2002), and in some lepidopteran spe-cies (Reddy and Guerrero, 2004). Some GLVs enhanced or syner-gized the attraction of synthetic sex pheromone in the cornearworm, Helicoverpa zea, and codling moth Cydia pomonella (Lightet al., 1993), and two GLVs, (Z)-3-hexenyl acetate and (E)-2-hexe-nyl acetate, in the tobacco budworm, Heliothis virescens (Dickenset al., 1993). A strongly synergistic effect of pheromone and food

responsive sensilla to the diketone and four stereoisomers of the monoketone (top),OH and linalool (bottom) in Sitona lepidus. Bars indicate standard errors of means

K.C. Park et al. / Journal of Insect Physiology 59 (2013) 1222–1234 1233

volatiles was also observed in the attraction of a weevil Sitophilusoryzae (Trematerra and Girgenti, 1989). In the boll weevil, Anthon-omus grandis, addition of boll weevil-induced volatiles from repro-ductive cotton plants to aggregation pheromone gave increasedattraction, relative to the pheromone alone (Magalhães et al.,2012) and it was found that antennal ORNs for both pheromonecomponents and host plant odors were associated with the sametype of sensilla (Dickens, 1990). The highly sensitive and selectiveORNs for host plant volatiles identified in S. lepidus may enhancethe efficiency of pheromone attraction, if any, in this species byattracting them to the vicinity of their host plants.

Fine spatio-temporal resolution of the synchronous firing ofORNs tuned to specific compounds enables insects to pick out rel-evant host odor cues against high background noise (Bruce andPickett, 2011). In this regard, the co-compartmentalization of dif-ferentially tuned ORNs in the same sensillum may play an impor-tant role in the location of host plants and mates by increasingspatio-temporal resolution in responding to odorants. Our resultsshow that at least two different ORNs for pheromone-related com-pounds are co-compartmentalized in the same sensillum, and thiswas consistent in all sensillum types identified. Each of these sen-silla contains two ORNs, specialized for either the diketone or oneof the monoketones, or two monoketones, respectively. Thesepaired-ORNs may play a critical role in allowing potential matesto distinguish S. lepidus from other Sitona species and the relativeratios of the active compounds on these ORNs may be importantin their pheromone communication. Furthermore, we also foundthat many of the sensilla housing pheromone-related ORNs containORNs specialized for plant volatile compounds in both males andfemales of S. lepidus. The co-compartmentalization of phero-mone-related ORNs and plant volatile ORNs in the same sensillumin S. lepidus implies that these ORNs and their active compoundsmay have an interactive role in the behavior of S. lepidus. In thiscase, the aggregation of S. lepidus around their host plants wouldrequire both pheromone and plant volatile compounds that arelikely to include GLVs.

The likelihood of using olfactory cues in S. lepidus is also well sup-ported by the antennal morphology. Similar to other weevil species(Andersson et al., 2012; Dickens, 1990; Mustaparta, 1975), malesand females of S. lepidus contain a number of sensilla (mostly basi-conic and trichoid) concentrated at three circumferential regions,so-called ‘sensory band’, in the terminal club of the antenna.Although the morphology and distribution of antennal sensilla inS. lepidus showed no sexual dimorphism between males and females,similar to the feature reported in other weevils such as the palmweevil, Rhychophorus palmarum (Said et al., 2003), the profiles ofORNs show differences between males and females in both phero-mone-related compounds and plant volatile compounds. The pres-ence of cell bodies underneath the antennal cuticle in the 1st–6thflagella segments implies that the hairs (chaetica and trichodea)on these segments would have sensory functions although their roleis yet to be elucidated. More behavioral studies are also required tounderstand the full ecological implications of this study.

Acknowledgements

We thank B. Bohman for the synthesis of 4-methyl-3,5-hep-tanedione and 5-hydroxy-4-methyl-3-heptanone used in thesestudies. This research was funded by the Ministry of BuildingInnovation and Employment through the Bio-Protection ResearchCentre (LINX0304, contract 25949). The Linnaeus University,Kalmar, Sweden, is gratefully acknowledged for financial supportof C.R. Unelius.

References

Ache, B.W., Young, J.M., 2005. Olfaction: diverse species, conserved principles.Neuron 48, 417–430.

Aeschlimann, J.P., 1984. Distribution, host plants, and reproductive biology of theSitona humeralis Stephens group of species (Coleoptera, Curculionidae). Journalof Applied Entomology 98, 298–309.

Andersson, M.N., Larsson, M.C., Schlyter, F., 2009. Specificity and redundancy in theolfactory system of the bark beetle Ips typographus: single-cell responses toecologically relevant odors. Journal of Insect Physiology 55, 556–567.

Andersson, M.N., Larsson, M.C., Svensson, G.P., Birgersson, G., Rundlöf, M., Lundin,O., Lankinen, Å., Anderbrant, O., 2012. Characterization of olfactory sensoryneurons in the white clover seed weevil, Apion fulvipes (Coleoptera: Apionidae).Journal of Insect Physiology 58, 1325–1333.

Baker, T.C., Ochieng, S.A., Cosse, A.A., Lee, S.G., Todd, J.L., Quero, C., Vickers, N.J.,2004. A comparison of responses from olfactory receptor neurons of Heliothissubflexa and Heliothis virescens to components of their sex pheromone. Journalof Comparative Physiology A 190, 155–165.

Baker, T.C., 2008. Balanced olfactory antagonism as a concept for understandingevolutionary shifts in moth sex pheromone blends. Journal of Chemical Ecology34, 971–981.

Barata, N., Mustaparta, H., Pickett, J.A., Wadhams, L.J., Araujo, J., 2002. Encodng ofhost and non-host plant odours by receptor neurons in the eucalyptuswoodborer, Phoracantha semipunctata (Coleoptera: Cerambycidae). Journal ofComparative Physiology A 188, 121–133.

Bargmann, C.I., 2006. Comparative chemosensation from receptors to ecology.Nature 444, 295–301.

Barratt, B.I.P., Barker, G.M., Addison, P.J., 1996. Sitona lepidus (Coleoptera:Curculionidae), a potential clover pest new to New Zealand. New ZealandEntomologist 19, 23–30.

Blight, M.M., Pickett, J.A., Smith, M.C., Wadhams, L.J., 1984. An aggregationpheromone of Sitona lineatus. Naturwissenschaften 71, 480.

Blight, M.M., Wadhams, L.J., 1987. Male-produced aggregation pheromone in peaand bean weevil, Sitona lineatus (L.). Journal of Chemical Ecology 13, 733–739.

Bohman, B., Unelius, C.R., 2009. Synthesis of all four stereoisomers of 5-hydroxy-4-methyl-3-heptanone using plants and oyster mushrooms. Tetrahedron 65,8697–8701.

Bright, D.E., 1994. Revision of the genus Sitona (Coleoptera: Curculionidae) of NorthAmerica. Annals of the Entomological Society of America 87, 277–306.

Bruce, T.J.A., Pickett, J.A., 2011. Perception of plant volatile blends by herbivorousinsects-finding the right mix. Phytochemistry 72, 1605–1611.

Buttery, R.G., Kamm, J.A., Ling, L.C., 1984. Volatile components of red clover leaves,flowers, and seed pods: possible insect attractants. Journal of Agricultural andFood Chemistry 32, 254–256.

Chamberlain, K., Khan, Z.R., Pickett, J.A., Toshova, T., Wadhams, L.J., 2006. Dielperiodicity in the production of green leaf volatiles by wild and cultivated hostplants of stemborer moths, Chilo partellus and Busseola fusca. Journal ofChemical Ecology 32, 565–577.

Coffelt, J.A., Vick, K.W., Sower, L.L., McClellan, W.T., 1978. Sex pheromone of thesweetpotato weevil, Cylas formicarius elagantulus: laboratory bioassay and evidencefor a multiple component system. Environmental Entomology 7, 756–758.

Cossé, A.A., Todd, J.L., Baker, T.C., 1998. Neurons discovered in male Helicoverpa zeaantennae that correlate with pheromone-mediated attraction and interspecificantagonism. Journal of Comparative Physiology A 182, 585–594.

Danthanarayana, W., 1967. Host specificity of Sitona beetles. Nature 213, 1153–1154.

Danthanarayana, W., 1969. Population dynamics of the weevil Sitona regenseinensis(Hbst.) on broom. Journal of Animal Ecology 38, 1–18.

Deglow, E.K., Borden, J.H., 1998. Green leaf volatiles disrupt and enhance responseto aggregation pheromones by the ambrosia beetle, Gnathotrichus sulcatus(Coleoptera: Scolytidae). Canadian Journal of Forest Research 28, 1697–1705.

Dickens, J.C., 1990. Speicialized receptor neurons for pheromones and host plantodors in the boll weevil, Anthonomus grandis Boh. (Coleoptera: Curculionidae).Chemical Senses 15, 311–331.

Dickens, J.C., Jang, E.B., Light, D.M., Alford, A.R., 1990. Enhancement of insectpheromone responses by green leaf volatiles. Naturwissenschaften 77, 29–31.

Dickens, J.C., Smith, J.W., Light, D.M., 1993. Green leaf volatiles enhance sexattractant pheromone of the tobacco budworm, Heliothis virescens (Lep.:Noctuidae). Chemoecology 4, 175–177.

Domingue, M.J., Musto, C.J., Linn Jr., C.E., Roelofs, W.L., Baker, T.C., 2007. Evidence ofolfactory antagonistic imposition as a facilitator of evolutionary shifts inpheromone blend usage in Ostrinia spp. (Lepidoptera: Crambidae). Journal ofInsect Physiology 53, 488–496.

El-Bouhssini, M., Sarker, A., Erskine, W., Joubi, A., 2008. First sources of resistance toSitona weevil (Sitona crinitus Herbst) in wild Lens species. Genetic Resources andCrop Evolution 55, 1–4.

Figueiredo, R., Rodrigues, A.I., Costa, M.C., 2007. Volatile composition of red clover(Trifolium pratense L.) forages in Portugal: the influence of ripening stage andensilage. Food Chemistry 104, 1445–1453.

Hansson, B.S., Larsson, M.C., Leal, W.S., 1999. Green leaf volatile-detecting olfactoryreceptor neurons display very high sensitivity and specificity in a scarab beetle.Physiological Entomology 24, 121–126.

Hardwick, S., Harens, B., 2000. The behavior of adult Sitona lepidus Gyllenhal(Coleoptera: Curculionidae) in response to white clover. New Zealand PlantProtection 53, 415–419.

1234 K.C. Park et al. / Journal of Insect Physiology 59 (2013) 1222–1234

Heath, R.R., Coffelt, J.A., Sonnet, P.E., Proshold, F.I., Dueben, D., Tumlinson, J.H., 1986.Identification of sex pheromone produced by female sweetpotato weevil, Cylasformicarius elegantulus (Summers). Journal of Chemical Ecology 12, 1489–1503.

Johnson, S.N., Gregory, P.J., Murray, P.J., Zhang, X., Young, I.M., 2004. Host plantrecognition by the root feeding clover weevil, Sitona lepidus (Coleoptera:Curculionidae). Bulletin of Entomological Research 94, 433–439.

Kicel, A., Wolbis, M., Kalemba, D., Wajs, A., 2010. Identification of volatileconstituents in flowers and leaves of Trofolium repens L. Journal of EssentialOil Research 22, 624–627.

Kigathi, R.N., Unsicker, S.B., Reichelt, M., Kesselmeier, J., Gershenzon, J., Weisser,W.W., 2009. Emission of volatile organic compounds after herbivory fromTrifolium pretense (L.) under laboratory and field conditions. Journal of ChemicalEcology 35, 1335–1348.

Landon, F., Ferary, S., Pierre, D., Auger, J., Biemont, J.C., Levieux, J., Pouzat, J., 1997.Sitona lineatus host-plant odors and their components: effects on locomoterbehavior and peripheral sensitivity variations. Journal of Chemical Ecology 23,2161–2173.

Larsson, M.C., Leal, W.S., Hansson, B.S., 1999. Olfactory receptor neurons specific tochiral sex pheromone components in male and female Anomala cuprea beetles(Coleoptera: Scarabaeidae). Journal of Comparative Physiology A 184, 353–359.

Larsson, M.C., Leal, W.S., Hansson, B.S., 2001. Olfactory receptor neurons detectingplant odours and male volatiles in Anomala cuprea beetles (Coleoptera:Scarabaeidae). Journal of Insect Physiology 47, 1065–1076.

Levinson, H.Z., Levinson, A., Ren, Z., Mori, K., 1990. Comparative olfactory perceptionof the aggregation pheromones of Sitophilus oryzae (L.), S. zeamais (Motsch.) andS. granarius (L.), as well as the stereoisomers of these pheromones. Journal ofApplied Entomology 110, 203–213.

Light, D.M., Flath, R.A., Buttery, R.G., Zalom, F.G., Rice, R.E., Dickens, J.C., Jang, E.B.,1993. Host-plant green-leaf volatiles synergize the synthetic sex pheromones ofthe corn earworm and codling moth (Lepidoptera). Chemoecology 4, 145–152.

Magalhães, D.M., Borges, M., Laumann, R.A., Sujii, E.R., Mayon, P., Caulfield, J.C.,Midega, C.A.O., Khan, Z.R., Pickett, J.A., Birkett, M.A., Blassioli-Moraes, M.C.,2012. Semiochemicals from herbivory induced cotton plants enhance theforaging behavior of the cotton boll weevil, Anthonomus grandis. Journal ofChemical Ecology 38, 1528–1538.

Murray, P.J., Clements, R.O., 1994. Investigations of the host feeding preferences ofSitona weevils found commonly on white clover (Trifolium repens) in the UK.Entomologia Experimentalis et Applicata 71, 73–79.

Mustaparta, H., 1975. Responses of single olfactory cells in the pine weevil Hylobiusabietis L. (Col.: Curculionidae). Journal of Comparative Physiology 97, 271–290.

Olsson, S.B., Linn, C.E., Roelofs, W.L., 2006. The chemosensory bases for behavioraldivergence involved in sympatric host shifts. I. characterizing olfactory receptorneuron classes responding to key host volatiles. Journal of ComparativePhysiology A 192, 279–288.

Olsson, S.B., Linn, C.E., Feder, J.L., Michel, A., Dambroski, H.R., Berlocher, S.H., Roelofs,W.L., 2009. Comparing peripheral olfactory coding with host preference in theRhagoletis species complex. Chemical Senses 34, 37–48.

Otálora-Luna, F., Hammock, J.A., Alessandro, R.T., Lapointe, S.L., Dickens, J.C., 2009.Discovery and characterization of chemical signals for citrus root weevil,Diaprepes abbreviates. Arthropod–Plant Interactions 3, 63–73.

Park, K.C., Baker, T.C., 2002. Improvement of signal-to-noise ratio inelectroantennogram responses using multiple insect antennae. Journal ofInsect Physiology 48, 1139–1145.

Park, K.C., Hardie, J., 2004. Electrophysiological characterization of olfactory sensillain the black bean aphid, Aphis fabae. Journal of Insect Physiology 50, 647–655.

Petrukha, O.I., 1970. Sitona weevils. Zashchita Rastenii 15, 24–26.Phillips, C.B., Barratt, B.I.P., 2004. A guide to assist detection of newly arrived Sitona

species (Coleoptera: Curculionidae) in New Zealand and Australia. Proceedingsof the Eighth Australasian Grassland Invertebrate Ecology Conference, pp. 22–33.

Phillips, J.K., Walgenbach, C.A., Klein, J.A., Burkholder, W.E., Schmuff, N.R., Fales,H.M., 1985. (R⁄, S⁄)-5-hydroxy-4-methyl-3-heptanone: a male-producedaggregation pheromone of Sitophilus oryzae (L.) and S. zeamais Motsch. Journalof Chemical Ecology 11, 1263–1274.

Phillips, J.K., Chong, J.M., Andersen, J.F., Burkholder, W.E., 1989. Determination ofthe enantiomeric composition of (R⁄, S⁄)-1-ethylpropyl 2-methyl-3-hydroxypentanoate, the male-produced aggregation pheromone of Sitophilusgranarius. Entomologia Experimentalis et Applicata 51, 149–153.

Reddy, G.V.P., Guerrero, A., 2004. Interactions of insect pheromones and plantsemiochemicals. Trends in Plant Science 9, 253–261.

Reinecke, A., Ruther, J., Hilker, M., 2002. The scent of food and defense: green leafvolatiles and toluquinone as sex attractant mediate mate finding in theEuropean cockchafer Melolontha melolontha. Ecology Letters 5, 257–263.

Said, I., Tauban, D., Renou, M., Mori, K., Rochat, D., 2003. Structure and function ofthe antennal sensilla of the palm weevil, Rhynchophorus palmarum (Coleoptera,Curculionidae). Journal of Insect Physiology 49, 857–872.

Schmuff, N.R., Phillips, J.K., Burkholder, W.E., Fales, H.M., Chen, C.W., Roller, P.P., Ma,M., 1984. The chemical identification of the rice weevil and maize weevilaggregation pheromone. Tetrahedron Letters 25, 1533–1534.

Shields, V.D.C., Hildebrand, J.G., 2001. Responses of a population of antennalolfactory receptor cells in the female moth Manduca sexta to plant-associatedvolatile organic compounds. Journal of Comparative Physiology A 186, 1135–1151.

Stensmyr, M.C., Larsson, M.C., Bice, S., Hansson, B.S., 2001. Detection of fruit- andflower-emitted volatiles by olfactory receptor neurons in the polyphagus fruitchafer Pachnoda marginata (Coleoptera: Cetoniinae). Journal of ComparativePhysiology A 187, 509–519.

Toshova, T.B., Subchev, M.A., Atanasova, D.I., Velazquez de Castro, A.J., Smart, L.,2009. Sitona weevils (Coleoptera: Curculionidae) caught by traps in alfalfa fieldsin Bulgaria. Biotechnology & Biotechnology Equipment, 132–135 (specialedition).

Toth, M., Smart, L.E., Szarukan, I., Imrei, Z., 1998. Preliminary observation on speciesspecificity of Sitona lineatus (L.) pheromone traps in Hungary (Coleoptera:Curculionidae). Acta Phytopathologica et Entomologica Hungarica 33, 349–356.

Trematerra, P., Girgenti, P., 1989. Influence of pheromone and food attractants ontrapping of Sitophilus oryzae (L.) (Col., Curculionidae): a new trap. Journal ofApplied Entomology 108, 12–20.

Unelius, C.R., Park, K.C., McNeill, M., Wee, S.L., Bohman, B., Suckling, D.M., 2013.Identification and electrophysiological studies of (4S,5S)-5-hydroxy-4-methyl-3-heptanone and 4-methyl-3,5-heptanedione in male lucerne weevils.Naturwissenschaften 100, 135–143.

Walgenbach, C.A., Phillips, J.K., Burkholder, W.E., King, G.G.S., Slessor, K.N., Mori, K.,1987. Determination of chirality in 5-hydroxy-4-methyl-3-heptanone, theaggregation pheromone of Sitophilus oryzae (L.) and S. zeamais Motschulsky.Journal of Chemical Ecology 13, 2159–2169.