3332

The Journal of Experimental Biology 213, 3332-3339 © 2010. Published by The Company of Biologists Ltd doi:10.1242/jeb.044396

Peripheral modulation of pheromone response by inhibitory host compound in a

Martin N. Andersson1,*, Mattias C. Larsson1, Miroslav Blazenec2, Rastislav Jakus2, Qing-He Zhang1,† and Fredrik Schlyter1 1Chemical Ecology, Department of Plant Protection Biology, Swedish University of Agricultural Sciences, SE-230 53 Alnarp, Sweden and 2Institute of Forest Ecology, Slovak Academy of Sciences, 960 53, Zvolen, Slovakia *Author for correspondence ([email protected]) †Present address: Sterling International, Incorporated, 3808 N. Sullivan Road, Building 16, Spokane, WA 99216, USA

Accepted 30 June 2010

SUMMARY We identified several compounds, by gas chromatographic–electroantennographic detection (GC–EAD), that were antennally active in the bark beetle Ips typographus and also abundant in beetle-attacked spruce trees. One of them, 1,8-cineole (Ci), strongly inhibited the attraction to pheromone in the field. Single-sensillum recordings (SSRs) previously showed olfactory receptor neurons (ORNs) on I. typographus antennae selectively responding to Ci. All Ci neurons were found within sensilla co-inhabited by a pheromone neuron responding to cis-verbenol (cV); however, in other sensilla, the cV neuron was paired with a neuron not responding to any test odorant. We hypothesized that the colocalization of ORNs had a functional and ecological relevance. We show by SSR that Ci inhibited spontaneous activity of the cV neuron only in sensilla in which the Ci neuron was also present. Using mixtures of cV and Ci, we further show that responses to low doses (1–10ng) of cV were significantly reduced when the colocalized Ci neuron simultaneously responded to high doses (1–10mg) of Ci. This indicated that the response of the Ci neuron, rather than ligand–receptor interactions in the cV neuron, caused the inhibition. Moreover, cV neurons paired with Ci neurons were more sensitive to cV alone than the ones paired with the non-responding ORN. Our observations question the traditional view that ORNs within a sensillum function as independent units. The colocalization of ORNs might sharpen adaptive responses to blends of semiochemicals with different ecological significance in the olfactory landscape. Key words: Ips typographus, Coleoptera, Curculionidae, Scolytinae, single-sensillum recording, olfactory receptor neuron, colocalization, co-compartmentalization, host selection, blend discrimination.

INTRODUCTION strict in that specific ORNs are located in the same functional Most essential behaviors, such as mate finding and host sensillum type (de Bruyne et al., 2001; Ghaninia et al., 2007). location, are guided by odors. In nature, rarely encounter Selective ORN pairing might be an adaptation to refine the odors as single compounds. Decisions regarding whether to progress perception of odor mixtures; for instance, both the spatiotemporal towards an odor source, or to abort, are probably based upon a resolution of volatile stimuli (Fadamiro et al., 1999) and compound- balance mechanism in which attractive and anti-attractive/repellent ratio detection would be improved if the olfactory sensors are located inputs in the combined ‘odor bouquet’ are weighed against each at the same point in space. Thus, neurons responding to compounds other. In the insect olfactory system, specific olfactory receptor that together constitute an ecologically important signal should then neurons (ORNs) constitute separate input channels, each detecting often be found paired within the same sensillum. Good examples compounds from different chemical and biological categories are the pheromone ORNs that are colocalized with ORNs that (Andersson et al., 2009; Bengtsson et al., 2009; Larsson et al., 2001; respond to pheromone antagonists (Cossé et al., 1998; Fadamiro et Mustaparta, 1975; Wibe and Mustaparta, 1996). ORN axons project al., 1999; Larsson et al., 2002; Wojtasek et al., 1998). ORN co- to the glomeruli of the primary olfactory center, the antennal lobe, compartmentalization might also provide the means for signal where integration of odor input takes place through lateral modulation in the periphery, in that responses in neighboring neurons interactions between glomeruli (Olsen and Wilson, 2008; Shang et could potentially affect the activity of each other (Getz and Akers, al., 2007; Silbering et al., 2008). 1994). In fact, a theoretical model predicts the existence of passive However, the organization of the insect peripheral olfactory electrical interactions between colocalized ORNs (Vermeulen and system allows for integration also at the level of ORNs or sensilla. Rospars, 2004), but the effects of these interactions have not yet For instance, ORNs are often excited by some compounds and been systematically characterized in insect sensilla. inhibited by others (e.g. Andersson et al., 2009; Hallem and Carlson, Conifer-feeding bark (Coleoptera: Curculionidae: 2006; Said et al., 2003), and responses to compound blends cannot Scolytinae) constitute excellent models to study peripheral always be predicted based on the responses to the individual blend integration of odor mixtures. They are among the most well-studied constituents (Getz and Akers, 1997; Ochieng et al., 2002; Party et insects in terms of behavioral responses to odor blends, such as al., 2009). Furthermore, insects typically have two or more ORNs different combinations of ecologically relevant attractants and co-compartmentalized within a sensillum. The significance of such inhibitors, and much is known about the peripheral detection of these colocalization is poorly understood, but the pairing rules seem very individual compounds (Andersson et al., 2009; Tømmerås, 1985).

THE JOURNAL OF EXPERIMENTAL BIOLOGY Neurons interact in olfactory sensilla 3333

Mass-attacks on Norway spruce [Picea abies (L.) Karst.] by the bark area ca. 0.45m2) were trapped on Porapak Q (50/80 mesh; European spruce bark beetle (Ips typographus, L.) are induced by 30mg in Teflon tube: 3mmϫ35mm) for 1.5h (airflow 300mlmin–1) release of the male-produced aggregation pheromone, a mixture of and extracted with 300ml diethyl ether (Fluka >99%). (4S)-cis-verbenol (cV) and 2-methyl-3-buten-2-ol (Schlyter et al., Headspace volatiles from a non-attacked spruce log (25cm 1987). Pheromone attraction is modulated by anti-attractant non- diameter and 30cm long; freshly cut from a nearby healthy tree) host volatiles (NHVs) from leaves and bark of angiosperm plants were collected using the same aeration procedure. The film was (Zhang and Schlyter, 2004). Mechanisms to prevent overcrowding replaced by a polyacetate cooking bag (35ϫ43cm, Terinex, of host trees are thought to involve other semiochemicals, such as sampling area ca. 0.24m2). All aeration extracts were kept at –20°C verbenone, that appear in later attack phases. Verbenone synergizes before GC–EAD and GC–MS analyses. the inhibitory effect of NHVs in I. typographus (Zhang and Schlyter, 2003) and is used as a negative cue also by several other bark beetle GC–EAD and GC–MS analyses species (Lindgren and Miller, 2002; Schlyter and Birgersson, 1999). A volume of 3ml of aeration samples was injected splitless into an Very little is known about the behavioral relevance of host HP 6890 GC (Agilent, Palo Alto, CA, USA) containing a fused monoterpenes in I. typographus (Erbilgin et al., 2007) or the host silica column (HP-Innowax) with a 1:1 effluent splitter, allowing phenolics (Faccoli and Schlyter, 2007) and other less volatile host simultaneous flame ionization detection (FID) and compounds, such as sesquiterpenes. electroantennographic detection (EAD). Hydrogen was used as a It is known from single-sensillum recordings (SSRs) that several carrier gas. The column temperature was 40°C for the initial 2min, ORN classes in I. typographus are tuned to host monoterpenes, rising to 200°C at 10°Cmin–1, and held for 2min. The outlet for including two classes that are highly selective for para-cymene and the EAD was inserted into a humidified air-stream (0.5ms–1) that 1,8-cineole (Ci), respectively (Andersson et al., 2009). All Ci neurons passed over an I. typographus antennal preparation. A glass capillary (small-amplitude B cell) were co-compartmentalized with neurons indifferent electrode filled with Beadle–Ephrussi Ringer, and responding to the pheromone component cV (large-amplitude Acell). grounded by means of a silver wire, was inserted into the severed However, in other sensilla, the cV cell was paired with a different B head of a beetle. A similar recording electrode, connected to a high- cell that did not respond to the test odorants. Observations suggested impedance DC amplifier with automatic baseline drift compensation, that stimulating with Ci sometimes inhibited the cV cell, and this was placed in contact with the distal end of the antennal club. The inhibition appeared mainly in sensilla in which the cV and Ci cells antennal signal was stored and analyzed on a PC equipped with an were co-compartmentalized, but the phenomenon was not thoroughly IDAC-card and the program EAD v. 2.3 (Syntech, Kirchzarten, surveyed (Andersson et al., 2009). The organization of cV and Ci Germany). A repeatable response was defined as a depolarization neurons in the two functional types of sensilla in I. typographus is of the antennal signal at the same retention time in three out of five excellent for investigations of potential interactions between these runs. The headspace samples were also analyzed by GC–MS on an ORNs, the colocalization of which we now hypothesize has a HP 6890 GC with an HP 5973 mass-selective detector (Agilent) functional and ecological relevance. To test this hypothesis, we using the same type of GC column and conditions as described characterized, by a combination of chemistry, behavior and above. Antennally active volatiles were identified by comparison physiology, a semiochemical system involving both a behaviorally of retention times and mass spectra of standards. attractive pheromone component and inhibitory plant volatiles. In this study, we identified, by combined gas chromatographic Field trapping experiment and electroantennographic detection (GC–EAD) (Zhang et al., The behavioral effect of (±)-1,8-cineole (1,3,3-trimethyl-2- 2000), two (among others) EAD-active compounds, Ci and p- oxabicyclo[2,2,2]octane; Ci; >99%, Aldrich) and p-cymene (1- cymene, that were abundant in spruce trees heavily attacked by I. methyl-4-[1-methylethyl]benzene; >99%, Acros) on I. typographus typographus, and demonstrated that Ci significantly inhibits pheromone attraction was investigated using Lindgren multiple-funnel pheromone attraction in the field. We also detailed the inhibitory traps (12 funnel size, Pherotech International, Delta, BC, Canada). effects of Ci on the cV pheromone neuron, comparing the two types The minimum distance between traps was 50m and 15m between of sensilla by SSR using binary odor mixtures. The plant volatile traps and the nearest tree. Insects were collected when at least 30 Ci inhibited the cV neuron primarily when it was paired with a Ci beetles were caught in a pheromone-baited trap with the lowest catch. neuron, also when the cV cell was simultaneously activated by its Following collection, treatment positions were changed according to ligand. Thus, coincident responses in colocalized neurons might a Latin square experimental design (Byers, 1991). The experiment significantly influence odor perception, suggesting that the two was conducted from June to August 2004 in clear-cuts situated within neurons do not act as independent units. Such colocalization effects spruce stands (80–90 years old) in the Polana Mountains in central might sharpen adaptive responses to mixtures of semiochemicals Slovakia. Traps were placed on SW slopes at altitudes of ca. 750m with different ecological origin and significance. above sea level. The standard commercial pheromone dispenser IT- Ecolure was used as an attractant (Fytofarm, Bratislava, Slovakia). MATERIALS AND METHODS IT-Ecolure is a wick-aluminium-foil-protected dispenser (Varkonda, Headspace sampling of attacked tree and fresh log 1996) filled with 3ml of pheromone mixture. The average release Headspace volatiles from the trunk of a Norway spruce (Picea abies) rate is ca. 50mg/day under field conditions (Fytofarm). The tree heavily attacked by I. typographus for at least two weeks were monoterpenes 1,8-cineole and p-cymene were filled in membrane sampled in situ within a high-density polyacetate film (Look, dispensers (Wilhelm Biological Plant Protection, Sachsenheim, Terinex, England) enclosure by a battery-operated pump with an Germany) that consisted of a 4ml glass vial with a twisted cap that activated charcoal filter tube in the air inlet (Zhang et al., 1999; was punched (diameter 12mm) and contained four laminated Zhang et al., 2000). The plastic film around the trunk at a level of permeable membranes (0.2mm thick, diameter 17mm). Baited vials 1.1–1.6m was sealed by a portable heat sealer on the side and were positioned with the cap facing downwards, resulting in a uniform tightened to the trunk with tape at both open ends. The distance evaporation. The estimated average release rate was ca. 50mg/day between the film and bark surface was ca. 1cm. Volatiles (sampling under field conditions, as measured by weight loss over time.

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3334 M. N. Andersson and others

Single-sensillum recordings A Single-sensillum recordings with tungsten microelectrodes were β -pinene myrcene performed using standard equipment (Syntech) and well-established α -pinene experimental protocols. Details regarding insect rearing, insect preparation, stimuli preparation and the odor delivery system have been described previously (Andersson et al., 2009). Chemicals, limonene diluted in paraffin (product # 1.07162.1000, Merck, Darmstadt, 1, 8 -cineole/ β -phell a ndrene Germany), were applied in aliquots of 10ml on filter papers inside terpinolene Pasteur pipettes. Two doses (1 and 10ng) of (4S)-cis-verbenol (cis- 4,6,6-trimethylbicyclo[3.1.1]hept-3-en-2-ol; cV; 95%, Borregaard), and two doses (1 and 10mg) of (±)-1,8-cineole (>99%, Aldrich) 3 -c a rene were tested singly and as the four possible binary combinations. In the latter case, the two compounds were applied on separate filter p -cymene papers inside the same pipette to prevent any potential compound FID interactions that could have affected evaporation rates. The cV and EAD Ci cells are indifferent to p-cymene (>99%, Acros), but p-cymene (10mg) was tested in binary combinations with both doses of cV (1 1 3 57911 and 10 ng), as control stimuli. In pipettes containing single compounds, a second filter paper loaded with paraffin solvent was also present (i.e. all pipettes contained two filter papers). Stimuli B were delivered in random order and prepared before each β -pinene experimental session. Sensilla with the cV cell paired with a non- α -pinene responsive B cell (sensillum typeI) and sensilla containing the colocalized cV and Ci cells (typeII) were both tested for responses.

Attempts were made to record from both sensillum types on all 3 -c a rene limonene individuals, and we succeeded in the majority of cases. Both males myrcene 1, 8 -cineole/ β -phell a ndrene and females were used in recordings. Sex separation was based on external morphology (Schlyter and Cederholm, 1981) and confirmed * by dissection of genitalia. p -cymene

Data analysis terpinolene For the field test, absolute catch was transformed according to FID log(catch +1) and used as the variable for statistical processing. EAD Treatments were compared with one-way ANOVA, and, to compensate for unequal variances, Dunnett’s T3 was used as a post- 1 3 57911 hoc test at 0.05. Sex separation was based on dissection of  Retention time (min) genitalia and the sex ratio analyzed with 95% binomial confidence intervals (95% CI) (Newcombe, 1998). Three hundred beetles, or Fig.1. GC–EAD recording showing antennal responses (EADs) to all individuals (if <300), per treatment were dissected. monoterpenes (FID) released by a non-attacked cut spruce log (A) and a From the SSRs, response curves of high temporal resolution were spruce tree heavily attacked by I. typographus (B). The onset of the obtained by counting (in Autospike 3.0, Syntech) the number of antennal response to the combined limonene/1,8-cineole/-phellandrene spikes (action potentials) in the cV A cell in 200-ms bins, starting peak in (A), and the 1,8-cineole/-phellandrene peak in (B), corresponds to 1s before stimulus onset and ending 2s after onset. Clear excitatory the part of the FID peaks that belongs to 1,8-cineole. Because the log was cut, the total amount of monoterpenes is higher in (A), which results in odor responses were recorded during a time-period of ca. 800ms.  incomplete separation of 1,8-cineole/-phellandrene from limonene. *EAD For statistical comparisons of both excitatory and inhibitory response is delayed in comparison with the retention time of terpinolene, responses, the total number of spikes during this period was used, suggesting that the antennal response is to an unidentified trace after subtracting the number of spikes fired during stimulation with compound. control. Factorial ANOVA was used to compare the response of the cV cell in the two sensillum types. Regression analysis on log- transformed doses, log(dose+1), was used for comparisons within In the non-attacked sample, responses were elicited by myrcene, a sensillum type. t-tests were used in pair-wise comparisons. 1,8-cineole/-phellandrene, -longipinene, 1-terpineol and 4- Cohen’s d was used as a measure of standardized effect size. In allyanisole (Fig.1A; later-eluting compounds not shown). Responses addition, a d value was calculated from the adjusted R2 obtained to the two major monoterpenes, -pinene and -pinene, were not from regression analyses (Nakagawa and Cuthill, 2007). According repeatable. In the aeration sample from the heavily attacked spruce to Cohen (Cohen, 1988), a d value above 0.8 is regarded as a large tree, EAD responses were again recorded to compounds present in effect. Tests were performed in SPSS 11.0 at 0.05. the fresh sample (including Ci), but several additional compounds also elicited responses, including p-cymene, isolongifolene, the RESULTS pheromone component cis-verbenol and several unidentified GC–EAD and GC–MS analysis compounds (Fig.1B; later-eluting compounds not shown). 1,8- GC–EAD analysis of the aeration samples from both a non-attacked cineole was ca. 20–30 times more abundant than cis-verbenol in log and an attacked trunk of a spruce tree showed repeatable antennal the head-space from the attacked tree. A minute amount of the other responses (in at least three out of five runs) to several compounds. essential pheromone component, 2-methyl-3-buten-2-ol, was

THE JOURNAL OF EXPERIMENTAL BIOLOGY Neurons interact in olfactory sensilla 3335

4500 a Type I Type II 4000 3500

.e.m. .e.m. 3000 s 2500 A B A B tch ± ± tch

a 2000 a cV ? cV Ci n c a 1500 A

Me B A B 1000 cV 10 ng b 500 ccc 0 1 mV 1 s Ph Ph+Cineole Cineole cV 1 ng Ph + p-Cymene p-Cymene Blank

Fig.2. Field data showing inhibition of response to synthetic pheromone (Ph) when combined with the two EAD-active compounds (1,8-cineole or p- cymene). A blank and the two compounds alone are included as controls. Ci 10 µg N6 number of replicates (trap rotations). Treatments with the same letter are not significantly different by Dunnett’s T3 post hoc test at 0.05 of log(catch +1). cV 1 ng + Ci 10 µg detected in the monoterpene eluting range, but it did not elicit repeatable EAD responses. Again, the antennal responses to the major monoterpenes varied among the antennal preparations. Fig.3. Top: schematic drawing of ORN pairing in the two functional Field trapping experiment sensillum types. In typeI sensilla (left column), the cis-verbenol (cV) A A total of 32,363 I. typographus were caught in six replicates with neuron is paired with a non-responsive Bneuron. The typeII sensilla (right a highly significant overall variation among treatments (F5,3572.7, column) contain a cV Aneuron and a 1,8-cineole (Ci) B neuron. SSR P<0.001). The addition of 1,8-cineole to the pheromone strongly traces: the cV neuron clearly responds to the higher 10ng cis-verbenol reduced trap catch compared with pheromone alone (Fig.2; 88% dose (first row). The response threshold is close to the lower 1ng dose reduction, with a very high effect size; Cohen’s d–2.9 based on (second row). The cV response is stronger in typeII sensilla. TypeI sensilla are unaffected by Ci (10mg), whereas, in typeII sensilla, Ci elicits a log-transformed means). The reduction in trap catch by the presence powerful excitatory response in the Bneuron, whereas the cV Aneuron of p-cymene together with pheromone was weaker and not simultaneously is inhibited (third row). The cV neuron in typeI sensilla significantly different from pheromone alone (50% reduction, responds to the binary cV:Ci mixture (1ng:10mg) in a manner similar to the d–0.80). The proportion of males in the catch ranged from 19 to response to 1ng cis-verbenol alone, whereas the cV neuron in typeII 33% but did not differ significantly between treatments (data not sensilla does not respond to cis-verbenol during the Ci response in the shown). Catches in traps baited with 1,8-cineole or p-cymene alone Bneuron (fourth row). Note: in this recording, not a single spike was elicited in the cV cell in typeII sensilla by the cV:Ci mixture. However, did not attract beetles, with close to zero catches like the blank shutdown of the cV neuron by this stimulus was typically not complete. control (Fig.2). Horizontal bars indicate the 0.5s stimulation period.

Single-sensillum recordings Sensilla classified as cV sensilla are found in two types. An A neuron responding selectively to cis-verbenol is accompanied either by a demonstrated by factorial ANOVA showing a significant interaction non-responding B neuron (sensillum typeI) or with a B neuron between sensillum types and stimuli (F2,603.43, P<0.05; cV/p- responding to 1,8-cineole (typeII) (Fig.3, schematic drawing). cymene stimulus excluded from analysis; Fig.4B). In some typeII Dose–response curves for the cV and the Ci cells in typeII sensilla sensilla, the cV cell responded to the cV:Ci (1ng:10mg) stimulus have been published previously (Andersson et al., 2009). In typeI just as if the cis-verbenol was absent (Fig.3). Thus, the response of sensilla, we now show that there was no general inhibition of the the pheromone cell was in such cases shut down to a level lower cV cell by either dose of Ci. By contrast, the cV cell in typeII sensilla than or similar to the blank control. In both types of sensilla, was inhibited by both doses of Ci (Fig.4A). The difference between combining 1ng cV with 10mg p-cymene had no effect on the cV sensillum types is obvious from the recorded spike trains (Fig.3). response (Fig.4B). The B cells in both types of sensilla were, on The inhibition in typeII sensilla lasted for up to 1.4s and was average, not affected by cV stimulation (the few spikes in the B significantly stronger in typeII than in typeI sensilla during the cells in Fig.3 upon cV stimulation are due to random fluctuations 800ms time-slot (Fig.4A). in background activity). Stimulating with cis-verbenol at a lower dose (1ng) elicited a The higher dose (10ng) of cV elicited a clear response in both weak response in the cV cells (Fig.3). The response was significantly sensillum types (Fig.3). Similar to the low (1ng) cV dose, the stronger in typeII than in typeI sensilla (t21–2.12, P<0.05; Cohen’s response to the higher dose also seemed stronger in typeII sensilla d–0.94; Fig.4B). Synchronous stimulation with cV and Ci clearly than in typeI, as indicated by the relatively large effect size reduced the cV response in a dose-dependent manner. The reduction (Cohen’s d–0.75), but the difference was not significant (t21–1.81, was found only in cV cells co-compartmentalized with Ci cells, as P>0.05; Fig.4C). At the even higher screening dose (10mg) of cV,

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3336 M. N. Andersson and others

3 3 5 A Type I Type II Blank 4 Type I

Ci 1 µg .e.m. s 3 Type II Ci 10 µg ±

s 2 2 2 1 00 m 8 / / Ci 1 µg Ci 10 µg 0

pike s –1 1 1 s –2 er of –3

N u m b –4 0 0 00.40.8 1.2 1.6 2.0 2.4 2.8 00.40.8 1.2 1.6 2.0 2.4 2.8 –5 5 B 5 10 cV 1 ng Type I

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4 s Type II cV 1 ng Ci 10 µg ± s Blank 6 3 3 00 m 8 4 2 2 pike s / s 2

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N cV 1 ng cV 1 ng cV 1 ng cV 1 ng N u m b er of s pike /200 ± .e.m. 0 0 Ci 1 µg Ci 10 µg pC 10 µg 00.40.8 1.2 1.6 2.0 2.4 2.8 00.40.8 1.2 1.6 2.0 2.4 2.8 –2 15 C 15 35 cV 10 ng Type I 30 s .e.m. cV 10 ng Ci 1 µg Type II cV 10 ng Ci 10 µg s ± 25 10 Blank 10

00 m 20 8

15 pike s / s 5 5 10 er of

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N 0 cV 10 ng cV 10 ng cV 10 ng cV 10 ng 0 0 Ci 1 µg Ci 10 µg pC 10 µg 00.40.8 1.2 1.6 2.0 2.4 2.8 00.40.8 1.2 1.6 2.0 2.4 2.8 Time (s)

Fig.4. Temporal response characteristics of the cis-verbenol (cV) A cell in typeI (left column) and typeII (mid column) sensilla to single odorants and binary mixtures. Arrows indicate onset of stimulation, and blue lines indicate the 800ms integration interval. Right column: total response of both sensillum types during the integration interval, after subtracting the blank response. N10–12 for all stimuli and sensillum types. (A)Stimulating with only 1,8-cineole (Ci) inhibited the cV cell only in typeII sensilla (factorial ANOVA: F1,3915.0, P<0.001). (B)Responses to cV and binary cV–Ci mixtures at the low (1ng) cV dose. 2 Ci reduces the cV response only in typeII sensilla. Bold line (right column) indicates significant regression at P<0.01 (F1,3211.2; R 0.26, d1.27). There was no effect on the cV response by the presence of p-cymene (pC; P>>0.05 for both sensillum types). (C)Responses to cV and binary cV–Ci mixtures at 2 the high (10ng) cV dose. Bold line (right column) indicates a highly significant regression at P<0.01 (F1,337.60, R 0.19, d0.88). Narrow line indicates 2 significant regression at P<0.05 (F1,305.28, R 0.15, d0.74). A nonsignificant tendency for inhibition of the cV response by p-cymene was observed in typeI sensilla (typeI, P>0.05; typeII, P>>0.05). For clarity, the response to the cV/p-cymene stimulus is shown only in the right column in (B) and (C).

the response did not differ between the sensillum types (average p-cymene did not significantly alter the response to 10ng cV in any response typeI: 157Hz, N12; typeII: 161Hz, N7; P>>0.05). type of sensillum, although a tendency was found in sensilla without At the 10ng cV dose, the binary mixtures of cV and Ci reduced Ci neurons. the cV response in a dose-dependent manner. In contrast to the low cV dose, factorial ANOVA showed no interaction between sensillum DISCUSSION types and stimuli (F2,610.35, P>0.05; cV/p-cymene stimulus We show that the antennae of I. typographus respond to volatiles excluded from analysis) and regression analyses demonstrated from healthy host tree logs and to other volatiles released primarily significant response reductions in both types of sensilla. However, from heavily attacked trees. More EAD-active compounds were the significance levels from the regression analyses, as well as the found in the spruce that had been under attack for two weeks, in effect size measures, differed between sensillum types, indicating comparison with a non-attacked log, which indicates an important a stronger effect in typeII sensilla (Fig.4C). The addition of 10mg multi-component ‘unsuitable host signal’ that might regulate bark

THE JOURNAL OF EXPERIMENTAL BIOLOGY Neurons interact in olfactory sensilla 3337 beetle density on attacked trees. As Ci was present in both attacked 1998), but, so far, not in bark beetle pheromone neurons (Andersson and non-attacked samples, its inhibitory properties presumably et al., 2009; Mustaparta et al., 1977; Mustaparta et al., 1980). represent a quantitative rather than a qualitative effect; preliminary Consistent with behavioral observations, asynchronous arrival of results indicate that the amount of Ci increases after attack by I. pheromone constituents disrupts the spiking pattern of projection typographus, as newly attacked trees release approximately four neurons in the antennal lobe, with resulting effects on the temporal times more Ci than non-attacked trees [N6 (C. Schiebe, P. aspects of odor coding (Christensen and Hildebrand, 1997). Brodelius, G. Birgersson, J. Witzell, P. Krokene, J. Gershenzon, A. The system of cis-verbenol and 1,8-cineole in I. typographus is Hammerbacher, B. S. Hansson and F.S., personal communication)]. different from the pheromone agonist/antagonist organization in The amount of Ci released from the attacked tree was 20–30 times other insects; the compounds are of and plant origins, larger than the amount of cV. In our field test, the release ratio respectively, and unlikely to be involved in reproductive isolation. between the pheromone and Ci was 1:1, but, as the pheromone It would, rather, be a means to judge the fitness value of an integrated contains 2-methyl-3-buten-2-ol and cV in a ca. 50:1 ratio, the cV:Ci odor stimulus comprised of elements from conspecifics and potential ratio in the field test was similar to the ratio released from the hosts. It would nevertheless be of interest to investigate the effects attacked tree. In contrast to earlier reports on the kairomonal activity of spatial separation of pheromone and 1,8-cineole and compare the of mainly (–)--pinene on I. typographus (Erbilgin et al., 2007; effect of spacing with that of another anti-attractant compound (e.g. Hulcr et al., 2006; Jakus and Blazenec, 2003), a mixture of EAD- verbenone), detected by an ORN not colocalized with the cV cell. active host monoterpenes, -pinene, -pinene and p-cymene, was The colocalization of one pheromone-responding ORN with one unattractive in itself, but interrupted the pheromone response of the that responds to a plant odor is also different from that shown by closely related larch bark beetle I. subelongatus (Zhang et al., 2007). most other insects. Typically, pheromone ORNs are located in More work is surely needed to determine the behavioral roles of specific morphological sensillum types, whereas ORNs responding these antennally active volatiles from healthy and attacked hosts. to plant odors are found in other sensilla (Hansson et al., 1986; The abundance of host monoterpene-selective ORNs on the I. Hansson et al., 1999; Larsson et al., 2001) (but see Said et al., 2003). typographus antennae (Andersson et al., 2009) suggests that beetles The pairing of host odor- and pheromone-responsive ORNs in I. are able to distinguish spruces based on their odor profiles, which typographus suggests that host localization, involving both the vary among trees of different genotypes and physiological states pheromone and host-derived compounds, is an integrated system (Keeling and Bohlmann, 2006). Terpenoid compounds are involved that does not segregate between different classes of semiochemicals. in constitutive and induced defenses of both conifers and Signal modulation between receptor neurons at the peripheral angiosperms (Keeling and Bohlmann, 2006; Mumm et al., 2008) – level is another factor that could influence grouping of neurons in insects feeding on such plants would likely benefit from selecting sensilla. Indeed, our results indicate response interactions between hosts with low terpenoid content. Indeed, several monoterpenes are neighboring ORNs as the inhibition of the cV cell was much more toxic and lethal to bark beetles (Everaerts et al., 1988; Raffa and pronounced when a colocalized Ci cell responded. Neurons within Smalley, 1995) and to their symbiotic fungi (Raffa et al., 1985). contact chemosensilla of the grasshopper Schistocerca americana Although not vectored by bark beetles, inoculation of Norway spruce have also been shown to interact, in that a single spike in the small- with pathogenic fungus Heterobasidion parviporum resulted in an spiking cell resulted in an extended spike interval in the colocalized increased content of 1,8-cineole (Zamponi et al., 2007). In addition, large-spiking cell (White et al., 1990). Contacts between sensory p-cymene was the most effective of all tested terpenes in reducing cells in thermo-/hygroreceptive sensilla styloconica have been growth of coniferous decay fungi (De Groot, 1972). It is therefore found in, for example, the silkmoth Bombyx mori (Steinbrecht, 1989) likely that both Ci and p-cymene are involved in the induced defense and were hypothesized to be involved in the antagonistic response of conifers against bark beetles and/or their symbiotic fungi. characteristics of these cells. However, contacts such as electrical Because the 1,8-cineole and cis-verbenol neurons are not always or chemical synapses have not been found between insect ORNs. colocalized within the same sensilla, we have a unique model for A non-synaptic mechanism that could explain the inhibition is studying intra-sensillum interactions between a pheromone the passive electrical interaction that occurs between ORNs that are compound and a plant-derived behavioral antagonist. There is much colocalized in sensilla (Vermeulen and Rospars, 2004). According evidence suggesting that insect decision making in host choice is a to the model, excitatory responses in an ORN can hyperpolarize a matter of weighing relative ratios of different plant compounds, neighboring cell that is insensitive to the stimulus and in close possibly from different sources. Compartmentalization of neurons contact. The model also suggests that pairing of ORNs with similar in sensilla might reflect this task (Bruce et al., 2005) as it probably odor tuning might reduce the amplitude of the receptor potentials favors coincidence detection, which in turn would improve the and thus lower the sensitivity of the cells (Vermeulen and Rospars, accuracy of compound ratio discrimination of odor mixtures. In 2004). If signal modulation unavoidably occurs between receptor nature, I. typographus frequently encounters cV and Ci in neurons at the peripheral level, pairing of ORNs with differentiated combination as cV is released when a spruce tree is attacked. Along response profiles would be expected as it would circumvent an with our behavioral data, the colocalization of the neurons suggests overall reduction in olfactory sensitivity. To our knowledge, ORNs that the binary mixture constitutes an ecologically significant signal, with largely overlapping response spectra have rarely been found assuming that selective pairing of ORNs is adaptive and not due to within the same sensillum (but see Hansen, 1984), although it chance. Some male moths have a remarkable ability to distinguish theoretically could improve discrimination between chemically odor filaments from different sources with extremely high similar compounds. spatiotemporal resolution (Fadamiro et al., 1999; Witzgall and In our case, the strong excitatory response in the Ci cell might Priesner, 1991). It was suggested that such amazing feats depend change the receptor potential of the cV cell so that a higher cV dose on ORNs being located within the same sensillum (Fadamiro et al., is required to produce the same number of action potentials. If so, 1999), which is common among pheromone agonist/antagonist the inhibitory interaction would be expected to be reciprocal – that neurons in Lepidoptera (Baker et al., 1998; Larsson et al., 2002). is, a strong cV response should inhibit the Ci cell. However, we Similar arrangements are found also in Coleoptera (Wojtasek et al., were unable to investigate this owing to the fact that the small-

THE JOURNAL OF EXPERIMENTAL BIOLOGY 3338 M. N. Andersson and others amplitude spikes of the Ci cell were completely obscured by a strong central integration, which probably explains some (or most) of the response of the large-spiking cV cell. In a previous study, we found anti-attractant effect of Ci. that, when the methyl-butenol B cell responded, the colocalized A In conclusion, the host monoterpene 1,8-cineole inhibits the cell was strongly inhibited (Andersson et al., 2009). Unfortunately, attraction of I. typographus to its aggregation pheromone, and highly no excitatory responses from this A cell were recorded to any odorant specific ORNs for the compound are present on the antennae. Insects tested. In addition, it would be of great interest to identify the typically group pheromone ORNs in sensilla that are distinct from ligand(s) for the non-responsive B cell that is paired with the cV the ones that contain ORNs for plant compounds, which makes our cell in typeI sensilla, perhaps by means of GC-coupled SSRs system deviant from this general rule. In particular, the inhibitory (Stensmyr et al., 2001), to investigate whether responses in this cell interaction that occurs between the pheromone and host odor ORNs also inhibit the cV cell. Functional characterization of this ORN in is the first one described for any insect. Although the ecological conjunction with behavioral testing of the ligand would be desirable consequences of the peripheral inhibition in isolation could not be in order to consolidate the relevance of ORN interactions. In fact, established, it is clear that an inhibitory interaction occurs in the inhibition of Acells when Bcells respond seems to be a common periphery and that the cV cells differ in sensitivity depending on phenomenon (Blight et al., 1995; Hallem et al., 2004; Larsson et which type of sensilla they are housed in. Our observations thus al., 2001), but it has not been systematically addressed. question the traditional view that ORNs within a sensillum act as To our knowledge, our study is the first to investigate interactions independent response units. between ORNs in sensilla where spikes from the individual neurons could be distinguished. However, the idea of ORN interaction is LIST OF ABBREVIATIONS not novel. In honeybee sensilla placodea, neurons within a sensillum Ci (±)-1,8-cineole responded to odors in a coordinated manner, suggesting that cV (4S)-cis-verbenol responses of individual ORNs are not independent (Getz and Akers, GC–EAD gas chromatographic–electroantennographic detection GC–MS gas chromatography–mass spectrometry 1994). Unfortunately, as these sensilla contain 18–35 ORNs, spikes NHV non-host volatile from individual neurons were not distinguishable but could only be OBP odorant-binding protein grouped into three or four ‘response units’, which makes the ORN olfactory receptor neuron interpretation of the results less straightforward compared with our SSR single-sensillum recordings observations. Other studies have also shown that receptor neuron responses to binary mixtures cannot always be predicted based on ACKNOWLEDGEMENTS the responses to individual constituents, and that the mixture We thank Muhammad Binyameen and Elisabeth Marling for assistance in bark interaction can be both inhibitory and synergistic (Getz and Akers, beetle rearing. We also thank Sylvia Anton and Michel Renou (INRA, Versailles) for useful input on the manuscript. This study was funded by FORMAS, project # 1995; Getz and Akers, 1997; Ochieng et al., 2002; Party et al., 2009). 230-2005-1778, ‘Semiochemical diversity and insect dynamics’, and by the In our case, it was clear that the inhibition of cV neurons in Linnaeus-program ‘Insect Chemical Ecology, Ethology, and Evolution’ (ICE3). response to binary cV–Ci mixtures occurred exclusively in typeII M.C.L. was also funded by the Crafoord foundation and the Trygger foundation. This study was supported by the Centre of Excellence ‘Adaptive Forest sensilla at the low (1ng) cV dose. The inhibition in both sensillum Ecosystems’, ITMS: 26220120006, the Research & Development Operational types at the higher (10ng) cV dose suggests that two different Programme, funded by the ERDF. mechanisms could be operating, one being the interaction between neurons and the other possibly an effect of a second compound REFERENCES present at a high concentration. An inhibitory interaction would be Andersson, M. N., Larsson, M. C. and Schlyter, F. (2009). Specificity and redundancy in the olfactory system of the bark beetle Ips typographus: Single-cell expected if the rate of molecules entering the cuticular pores is responses to ecologically relevant odors. J. Insect Physiol. 55, 556-567. limited or if there is competition for, for example, odorant-binding Baker, T. C., Fadamiro, H. Y. and Cossé, A. A. (1998). Moth uses fine tuning for odour resolution. Nature 393, 530. proteins (OBPs) within the sensillum lymph. Possibly, the different Bengtsson, J. M., Wolde-Hawariat, Y., Khbaish, H., Negash, M., Jembere, B., degree of inhibition by Ci could also be explained by inherent Seyoum, E., Hansson, B. S., Larsson, M. C. and Hillbur, Y. (2009). Field attractants for interrupta selected by means of GC-EAD and single differences between the Aneurons or between the two types of sensillum screening. J. Chem. Ecol. 35, 1063-1076. sensilla. Interestingly, the cV neurons in typeII sensilla also Blight, M. M., Pickett, J. A., Wadhams, L. J. and Woodcock, C. M. (1995). Antennal perception of oilseed rape, Brassica napus (Brassicaceae), volatiles by the cabbage demonstrated a greater sensitivity to cV than the ones in typeI seed weevil Ceutorhynchus assimilis (Coleoptera, Curculionidae). J. Chem. Ecol. 21, sensilla. The response to 1ng cV was almost twice as strong in 1649-1664. Bruce, T. J. A., Wadhams, L. J. and Woodcock, C. M. (2005). Insect host location: a sensilla containing both the cV and Ci neuron, and a similar, albeit volatile situation. Trends Plant Sci. 10, 269-274. not significant, tendency was found at the 10ng dose. We do not Byers, J. A. (1991). Basic algorithms for random sampling and treatment randomization. Comput. Biol. Med. 21, 69-77. know why this difference exists, but it might be explained for Christensen, T. A. and Hildebrand, J. G. (1997). Coincident stimulation with instance by differences in olfactory receptor gene expression rates pheromone components improves temporal pattern resolution in central olfactory or differences in the sensillum environment, such as OBPs or other neurons. J. Neurophysiol. 77, 775-781. Cohen, J. (1988). Statistical Power Analysis for the Behavioral Sciences, 2nd edn. factors. Alternatively, cis-verbenol-sensitive neurons of typeI and Hillsdale, NJ: Erlbaum. typeII sensilla might express odorant receptors (ORs) with slightly Cossé, A. A., Todd, J. L. and Baker, T. C. (1998). Neurons discovered in male Helicoverpa zea antennae that correlate with pheromone-mediated attraction and different response profiles. Molecular characterization of I. interspecific antagonism. J. Comp. Physiol. A 182, 585-594. typographus ORs is required to distinguish between these de Bruyne, M., Foster, K. and Carlson, J. R. (2001). Odor coding in the Drosophila antenna. Neuron 30, 537-552. hypotheses. De Groot, R. C. (1972). Growth of wood-inhabiting fungi in saturated atmospheres of It is uncertain whether the recorded physiological inhibition of monoterpenoids. Mycologia 64, 863-870. Erbilgin, N., Krokene, P., Kvamme, T. and Christiansen, E. (2007). A host Ci is of ecological relevance as the Ci needs to be present at a ca. monoterpene influences Ips typographus (Coleoptera: Curculionidae, Scolytinae) 1000 times higher dose than the cV in order to elicit detectable responses to its aggregation pheromone. Agric. For. Entomol. 9, 135-140. Everaerts, C., Grégoire, J. C. and Merlin, J. (1988). The toxicity of Norway spruce inhibition. Our head-space collection from the attacked tree indicated monoterpenes to two bark beetle species and their associates. In Mechanisms of that the ratio between cV and Ci is ca. 1:20–1:30, but our sample Woody Plant Defenses Against Insects: Search for Pattern (ed. W. J. Mattson, J. Levieux and C. Bernard-Dagan), pp. 335-344. New York: Springer-Verlag. size is too small to draw any definite conclusions. In addition, the Faccoli, M. and Schlyter, F. (2007). Conifer phenolic resistance markers are bark excitatory inputs from the cV and Ci cells provide the means for beetle antifeedant semiochemicals. Agric. For. Entomol. 9, 237-245.

THE JOURNAL OF EXPERIMENTAL BIOLOGY Neurons interact in olfactory sensilla 3339

Fadamiro, H. Y., Cossé, A. A. and Baker, T. C. (1999). Fine-scale resolution of Raffa, K. F. and Smalley, E. B. (1995). Interaction of pre-attack and induced closely spaced pheromone and antagonist filaments by flying male Helicoverpa zea. monoterpene concentrations in host conifer defense against bark beetle-fungal J. Comp. Physiol. A 185, 131-141. complexes. Oecologia 102, 285-295. Getz, W. M. and Akers, R. P. (1994). Honeybee olfactory sensilla behave as Raffa, K. F., Berryman, A. A., Simasko, J., Teal, W. and Wong, B. L. (1985). Effects integrated processing units. Behav. Neural Biol. 61, 191-195. of grand fir monoterpenes on the fir engraver, Scolytus ventralis (Coleoptera: Getz, W. M. and Akers, R. P. (1995). Partitioning non-linearities in the response of Scolytidae), and its symbiotic fungus. Environ. Entomol. 14, 552-556. honey bee olfactory receptor neurons to binary odors. BioSystems 34, 27-40. Said, I., Tauban, D., Renou, M., Mori, K. and Rochat, D. (2003). Structure and Getz, W. M. and Akers, R. P. (1997). Response of American cockroach (Periplaneta function of the antennal sensilla of the palm weevil Rhynchophorus palmarum americana) olfactory receptors to selected alcohol odorants and their binary (Coleoptera, Curculionidae). J. Insect Physiol. 49, 857-872. combinations. J. Comp. Physiol. A 180, 701-709. Schlyter, F. and Birgersson, G. A. (1999). Forest beetles. In Pheromones of Non- Ghaninia, M., Ignell, R. and Hansson, B. S. (2007). Functional classification and Lepidopteran Insects Associated with Agricultural Plants (ed. J. Hardie and A. K. central nervous projections of olfactory receptor neurons housed in antennal trichoid Minks), pp. 113-148. Oxford: CAB International. sensilla of female yellow fever mosquitoes, Aedes aegypti. Eur. J. Neurosci. 26, Schlyter, F. and Cederholm, I. (1981). Separation of the sexes of living spruce bark 1611-1623. beetles Ips typographus (L), (Coleoptera, Scolytidae). Z. Angew. Entomol. 92, 42-47. Hallem, E. A. and Carlson, J. R. (2006). Coding of odors by a receptor repertoire. Schlyter, F., Birgersson, G., Byers, J. A., Löfqvist, J. and Bergström, G. (1987). Cell 125, 143-160. Field response of spruce bark beetle, Ips typographus, to aggregation pheromone Hallem, E. A., Ho, M. G. and Carlson, J. R. (2004). The molecular basis of odor candidates. J. Chem. Ecol. 13, 701-716. coding in the Drosophila antenna. Cell 117, 965-979. Shang, Y., Claridge-Chang, A., Sjulson, L., Pypaert, M. and Miesenböck, G. Hansen, K. (1984). Discrimination and production of disparlure enantiomers by the (2007). Excitatory local circuits and their implications for olfactory processing in the gypsy moth and the nun moth. Physiol. Entomol. 9, 9-18. fly antennal lobe. Cell 128, 601-612. Hansson, B. S., Löfstedt, C., Löfqvist, J. and Hallberg, E. (1986). Spatial Silbering, A. F., Okada, R., Ito, K. and Galizia, C. G. (2008). Olfactory information arrangement of different types of pheromone-sensitive sensilla in a male moth. processing in the Drosophila antennal lobe: anything goes? J. Neurosci. 28, 13075- Naturwissenschaften 73, 269-270. 13087. Hansson, B. S., Larsson, M. C. and Leal, W. S. (1999). Green leaf volatile-detecting Steinbrecht, R. A. (1989). The fine structure of thermo-/hygrosensitive sensilla in the olfactory receptor neurones display very high sensitivity and specificity in a scarab silkmoth Bombyx mori: Receptor membrane substructure and sensory cell contacts. beetle. Physiol. Entomol. 24, 121-126. Cell Tissue Res. 255, 49-57. Hulcr, J., Ubik, K. and Vrkoc, J. (2006). The role of semiochemicals in tritrophic Stensmyr, M. C., Larsson, M. C., Bice, S. and Hansson, B. S. (2001). Detection of interactions between the spruce bark beetle Ips typographus, its predators and fruit- and flower-emitted volatiles by olfactory receptor neurons in the polyphagous infested spruce. J. Appl. Entomol. 130, 275-283. fruit chafer Pachnoda marginata (Coleoptera: Cetoniinae). J. Comp. Physiol. A 187, Jakus, R. and Blazenec, M. (2003). Influence of the proportion of (-)--pinene in 509-519. pheromone bait on Ips typographus (Col., Scolytidae) catch in pheromone trap Tømmerås, B. Å. (1985). Specialization of the olfactory receptor cells in the bark barriers and in single traps. J. Appl. Entomol. 127, 91-95. beetle Ips typographus and its predator Thanasimus formicarius to bark beetle Keeling, C. I. and Bohlmann, J. (2006). Genes, enzymes and chemicals of terpenoid pheromones and host tree volatiles. J. Comp. Physiol. A 157, 335-342. diversity in the constitutive and induced defence of conifers against insects and Varkonda, S. (1996). Pheromone dispenser, Industrial pattern 5720. Office of industrial pathogens. New Phytol. 170, 657-675. ownership of The Czech Republic, Prague, Czech Republic. Larsson, M. C., Leal, W. S. and Hansson, B. S. (2001). Olfactory receptor neurons Vermeulen, A. and Rospars, J.-P. (2004). Why are insect olfactory receptor neurons detecting plant odours and male volatiles in Anomala cuprea beetles (Coleoptera: grouped into sensilla? The teachings of a model investigating the effects of the ). J. Insect Physiol. 47, 1065-1076. electrical interaction between neurons on the transepithelial potential and the Larsson, M. C., Hallberg, E., Kozlov, M. V., Francke, W., Hansson, B. S. and neuronal transmembrane potential. Eur. Biophys. J. 33, 633-643. Löfstedt, C. (2002). Specialized olfactory receptor neurons mediating intra- and White, P. R., Chapman, R. F. and Ascoli-Christensen, A. (1990). Interactions interspecific chemical communication in leafminer moths Eriocrania spp. between two neurons in contact chemosensilla of the grasshopper Schistocerca (Lepidoptera: Eriocraniidae). J. Exp. Biol. 205, 989-998. americana. J. Comp. Physiol. A. 167, 431-436. Lindgren, B. S. and Miller, D. R. (2002). Effect of verbenone on five species of bark Wibe, A. and Mustaparta, H. (1996). Encoding of plant odours by receptor neurons in beetles (Coleoptera: Scolytidae) in lodgepole pine forests. Environ. Entomol. 31, the pine weevil (Hylobius abietis) studied by linked gas chromatography- 759-765. electrophysiology. J. Comp. Physiol. A 179, 331-344. Mumm, R., Posthumus, M. A. and Dicke, M. (2008). Significance of terpenoids in Witzgall, P. and Priesner, E. (1991). Wind-tunnel study on attraction inhibitor in male induced indirect plant defence against herbivorous . Plant Cell Environ. Coleophora laricella Hbn. (Lepidoptera: Coleophoridae). J. Chem. Ecol. 17, 1355- 31, 575-585. 1362. Mustaparta, H. (1975). Responses of single olfactory cells in the pine weevil Hylobius Wojtasek, H., Hansson, B. S. and Leal, W. S. (1998). Attracted or repelled? A matter abietis L. (Col.: Curculionidae). J. Comp. Physiol. A 97, 271-290. of two neurons, one pheromone binding protein, and a chiral center. Biochem. Mustaparta, H., Angst, M. E. and Lanier, G. N. (1977). Responses of single receptor Biophys. Res. Commun. 250, 217-222. cells in the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae) to its Zamponi, L., Michelozzi, M. and Capretti, P. (2007). Terpene response of Picea aggregation pheromone, ipsdienol, and the aggregation inhibitor, ipsenol. J. Comp. abies and Abies alba to infection with Heterobasidion s. l. For. Pathol. 37, 243-250. Physiol. A 121, 343-347. Zhang, Q.-H. and Schlyter, F. (2003). Redundancy, synergism, and active inhibitory Mustaparta, H., Angst, M. E. and Lanier, G. N. (1980). Receptor discrimination of range of non-host volatiles in reducing pheromone attraction in European spruce enantiomers of the aggregation pheromone ipsdienol in two species of Ips. J. Chem. bark beetle Ips typographus. Oikos 101, 299-310. Ecol. 6, 689-702. Zhang, Q.-H. and Schlyter, F. (2004). Olfactory recognition and behavioural Nakagawa, S. and Cuthill, I. C. (2007). Effect size, confidence interval and statistical avoidance of angiosperm nonhost volatiles by conifer-inhabiting bark beetles. Agric. significance: a practical guide for biologists. Biol. Rev. 82, 591-605. For. Entomol. 6, 1-19. Newcombe, R. G. (1998). Interval estimation for the difference between independent Zhang, Q.-H., Birgersson, G., Zhu, J., Löfstedt, C., Löfqvist, J. and Schlyter, F. proportions: comparison of eleven methods. Stat. Med. 17, 873-890. (1999). Leaf volatiles from nonhost deciduous trees: Variation by tree species, Ochieng, S. A., Park, K. C. and Baker, T. C. (2002). Host plant volatiles synergize season and temperature, and electrophysiological activity in Ips typographus. J. responses of sex pheromone-specific olfactory receptor neurons in male Helicoverpa Chem. Ecol. 25, 1923-1943. zea. J. Comp. Physiol. A 188, 325-333. Zhang, Q.-H., Schlyter, F. and Birgersson, G. (2000). Bark volatiles from nonhost Olsen, S. R. and Wilson, R. I. (2008). Lateral presynaptic inhibition mediates gain angiosperm trees of spruce bark beetle, Ips typographus (L.) (Coleoptera: control in an olfactory circuit. Nature 452, 956. Scolytidae): Chemical and electrophysiological analysis. Chemoecology 10, 69-80. Party, V., Hanot, C., Said, I., Rochat, D. and Renou, M. (2009). Plant terpenes affect Zhang, Q.-H., Schlyter, F., Chen, G. and Wang, Y. (2007). Electrophysiological and intensity and temporal parameters of pheromone detection in a moth. Chem. Senses behavioral responses of Ips subelongatus to semiochemicals from its hosts, non- 34, 763-774. hosts, and conspecifics in China. J. Chem. Ecol. 33, 391-404.

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