cis-Verbenol and verbenone as male-produced pheromone components of the cypress bark beetle, Phloeosinus aubei: Identification and behavioral tests in combination with the female-produced pheromone components

cis-Verbenol and verbenone as male-produced pheromone components of the cypress bark beetle, Phloeosinus aubei: Identification and behavioral tests in combination with the female-produced pheromone components | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article cis -Verbenol and verbenone as male-produced pheromone components of the cypress bark beetle, Phloeosinus aubei : Identification and behavioral tests in combination with the female-produced pheromone components Gábor Bozsik, Armin Tröger, Stefan Schulz, Gábor Szőcs This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6305155/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Jun, 2025 Read the published version in Agricultural and Forest Entomology → Version 1 posted You are reading this latest preprint version Abstract 1. The need for monitoring the spread of the invasive cypress bark beetle, Phloeosinus aubei , a pest of scale-leaved conifers across Europe is urging. The aggregation pheromone of females was reported earlier; however, an effective trapping method has not yet been developed. 2. Therefore, this study aimed to unravel the males’ pheromonal communication system. 3. Volatiles produced by P. aubei males, as well as sex- and mating status-specific extracts from mid- and hindguts of unmated and mated males and females were analyzed by GC-EAD and compounds eliciting antennal responses identified by GC-MS. 4. Two male-specific pheromone components, cis -verbenol and verbenone, were identified in the extracts from mated males. α-Pinene, known to be a component of the host plant kairomone, as well as the female-produced pheromone, was also found in extracts of mated males. None of these components were found in unmated males. 5. Four-arm olfactometer tests showed that females were attracted to the combination of (–)-myrtenol, (–)-α-pinene and (–)- cis -verbenol, rather than to either (–)-myrtenol, or (–)-α-pinene alone. When this ternary blend was combined with (–)-verbenone, the attractiveness for both males and females was reduced. 6. Female and male P. aubei spent significantly more time in the olfactometer arm containing (–)-α-pinene and (–)- cis -verbenol, than in the control arms. 7. The ternary mixture of (–)-myrtenol, (–)-α-pinene and (–)- cis -verbenol proved to be the most effective trap bait in the field, which could serve as a potent attractant for monitoring of P. aubei . male-produced pheromone cypress bark beetle GC-EAD behavioral test cis-verbenol verbenone Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Chemical communication in bark beetles consists of inter- (host finding) and intra-specific (aggregation and mate finding) channels, which are often intertwined, not only in function, but also in the set of compounds involved. For example, in many species, host finding is initiated by volatiles from the host tree, while pioneer beetles emit aggregation pheromones to attract conspecifics, which is an effective way to collectively defeat the defense mechanisms of the attacked tree (Byers, 1989 ; Keeling et al., 2021 ). When males and females aggregate on the colonized tree, this greatly facilitates finding mating partners. This is where aggregation pheromones come into play. In Ips species, the male is the pioneer sex and pheromone producer, while in Dendroctonus species it is the female, but both sexes release sex-specific pheromones (Blomquist et al., 2010 ; Tittiger & Blomquist, 2016 ). Often, these aggregation pheromone bouquets, together with host volatiles, control complex behavioral sequences of mate finding and mating. In contrast to the bark beetles associated with needle-leaved conifers, little is known about the chemical communication of bark beetles that live exclusively on scale-leaved conifers (Cupressaceae). The most prominent representative of the latter group is the genus Phloeosinus . In the West-Palearctic region, the cypress bark beetle, Phloeosinus aubei (Perris, 1855) (Coleoptera, Curculionidae, Scolytinae) extended its range from the Mediterranean areas towards Western and Middle-Europe, threatening Thuja , Chamaecyparis and Juniperus ornamental trees in urban areas and in tree nurseries. Followed by detailed description of life cycle and mating habit of settled populations under continental climate (Bozsik & Szőcs, 2017 ), we identified key compounds in the host plant volatiles as a kairomone for the cypress bark beetle, P. aubei (Bozsik et al., 2016 ; 2023 ) and identified the predominant components of the female-produced aggregation pheromone (Bozsik et al., 2024 ). Here, we report on the identification of a male-produced pheromone of P. aubei and the interaction of its compounds with those emitted by females in the attraction of both sexes. MATERIALS AND METHODS Experimental Insects Female and male adults of P. aubei were collected in West-Hungary (Prenor Tree Nursery, Szombathely, Vas-county, GPS 47.26419949232103 N, 16.59957200288773 E) at the end of their overwintering period. They build individual overwintering tunnels in the tips of twigs of Thuja occidentalis ‘Smaragd’. Infected twigs were cut ca. 15 cm from the tip with beetles inside the tunnels and were transported to the experimental station of the Plant Protection Institute (Budapest, Hungary, GPS 47.547946 N, 18.934632 E). Tunnels were carefully opened and the sex of P. aubei was determined using a binocular microscope without removing the beetles from the tunnels. Subsequently, males and females were kept separately in a 4L glass jars, with T . occidentalis ‘Smaragd’ twigs under ambient conditions. Wind-broken branches of Juniperus chinenis ‘Spartan’ with freshly carved mating chambers of P. aubei , were collected at the Tahi Tree Nursery (Tahi, Pest-county, Central-Hungary, GPS 47.7661127 N, 19.0684354E) and transported to the experimental station of the Plant Protection Institute at the beginning of the mating season (April 2023). By opening the galleries for collecting the beetles just before preparing the extracts, we found that the nuptial chambers were occupied by a male and a female P. aubei . Typically, a mating pair was found, although usually no longer in copula. A second male was never found and, in line with our earlier observations (Bozsik et. al, 2024), they were most probably all mated. This assumption was based on our previous finding that when a male arrives at a nuptial chamber with a female inside, they mate immediately. After opening the nuptial chambers, males and females were separated from each other. These specimens were taken as mated. Mid- and hindgut extracts were made immediately afterwards. Preparations of Pheromone Extracts Mid- and hindguts of 50 mated males were excised with a sharp forceps and extracted with 55 μL of n -hexane (reagent plus grade, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). During the preparation procedure (approximately 1 h), the extract was stored in a vial placed on a -20°C iron plate. When all guts were put into the cooled n -hexane the vial was removed from the cooling plate and the mixture was kept for 10 min at room temperature (dissolving period). Similarly, mid- and hindguts of 60 mated females freshly removed from their nuptial chambers, were extracted in 60 μL of n -hexane. As a control, extracts were also made from unmated males and females, respectively. We developed a strict protocol to ensure that the beetles in this control group were in as similar a physiological state as possible to those in the mated group. Beetles, collected individually from their overwintering tunnels before the mating season, were taken as unmated, kept separately and allowed to complete the maturation feeding. An approximately 20 cm long branch of a semi-dried trunk of T. occidentalis ‘Smaragd’ (previously not infested by P. aubei ) was placed in the jar of unmated females. When all females had prepared their nuptial chambers, mid- and hindgut extracts were made using the same method as above. Guts from 20 females were dissolved in 40 μL of n -hexane. Unmated males were also collected from their overwintering tunnels, and as males do not build nuptial chambers (Bozsik & Szőcs, 2017), they were allowed to feed on T. occidentalis ‘Smaragd’ twigs before the preparation of extracts. Mid- and hindguts of 9 unmated males were dissolved in 20 μL of n -hexane using the same method previously described. The extracts were stored at -40°C, until GC-EAD analysis. Analyses of Extracts by Gas Chromatography Coupled to an Electroantennographic Detector (GC-EAD) A gas chromatograph (6890 N, Agilent Technologies Inc., Santa Clara, CA, USA), equipped with an HP-5 column (J&W, 30 m x 0.32 mm, 0.25 μm film thickness; Agilent Technologies Inc.) was connected to an electroantennographic detector (Ockenfels SYNTECH GmbH, Buchenbach, Germany) and operated with a beetle preparation (antenna-head) to analyze the extracts. The initial temperature was 50 °C for 1 min, then it was heated to 230 °C at a heating rate of 10 °C/min, and the final temperature was held for 10 min. Helium was used as the carrier gas (flow: 2.0 mL/min) and also as the make-up gas. Extracts were injected in splitless mode. Agilent ChemStation (version Rev. A. 10.02) was used as the control software. Mounted head-antennae of male P. aubei , placed between glass capillary electrodes (1.17 mm i.d.) filled with Ephrussi-Beadle Ringer solution and equipped with silver wires, were used for EAD detection. Connections were established by MP15 micromanipulators. Humidified air flow through a glass tube, connected to the outlet of the EAD, ensured the transport of compounds to the antenna. Signals were analyzed using an IDAC2 amplifier and SYNTECH Syntech GC-EAD 2014 v. 1.2.5. software (Ockenfels SYNTECH GmbH, Buchenbach, Germany). Identification of Antennally Active Compounds by Gas Chromatography Coupled to Mass Spectrometry (GC/MS) The structure elucidation of the antennally active compounds in the natural samples was carried out using a GC 7890A GC coupled to a 5975C inert XL MSD (Agilent Technologies - MSD ChemStation E.02.02.1431). The identification of the compounds was based on their mass spectra and retention indices using fused silica capillary columns of two different polarities. The assignments were verified by GC/MS comparison with synthetic reference compounds. The gas chromatograph was equipped with a DB-heavyWAX GC column (60 m x 0.25 mm, 0.25 μm film thickness, Agilent J&W) and an HP-5ms column (60 m x 0.25 mm x 0.25 μm, Agilent J&W), respectively. Samples were injected in splitless mode (30 s). The temperature program was set at 50 °C for 3 min, then increased to 80 °C at a rate of 3 °C/min, then increased to 150 °C at 5 °C/min, and then finally to 250 °C at 8 °C/min for the DB-heavyWAX and at 50 °C for 3 min, then increased to 80 °C at a rate of 3 °C/min, to 150 °C at 5 °C/min, and to 300 °C at 7.5 °C/min for the HP-5ms. Helium was used as the carrier gas. The MS was operated in the EI 70 eV mode. The auxiliary heater was set at 280 °C, the MS source at 230 °C, and the MS quad at 150 °C. The scan mass range was m/z 33-433 with a scan speed of 3.58 scans/s. Chemicals Synthetic samples of (1 R ,5 S )-(–)-myrtenol (purity: 95%), (1 S ,5 S )-(–)-α-pinene, (purity: 99%), (1 S ,2 S ,5 S )-(–)- cis -verbenol (purity: 95%) and (1 S ,5 S )-(–)-verbenone (purity: 93%) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). (1 R ,2 R ,5 R )-(+)- cis -Verbenol (purity: 99%) was synthesized by the late Prof. Wittko Francke (University of Hamburg, Germany). Synthetic samples were dissolved in n -pentane (for analysis; Merck KGaA) at three concentrations: 100 ng/μL, 1 μg/μL, 10 μg/μL. Electroantennographic Bioassays (EAG) The antenna head preparation technique was the same as described above for GC-EAD. Stimuli were passed through a Pasteur pipette containing a 12.7 mm Rotilabo filter paper (Carl Roth GmbH, Karlsruhe, Germany). 10 μL of n -pentane solution of the synthetic compounds was applied to a filter paper. The method is based on the concept of Roelofs (1977, 1984) and the recommendation of SYNTECH (Ockenfels SYNTECH GmbH, Buchenbach, Germany) (Ockenfels, 2015), which is also supported by Olsson & Hansson (2013). The flush was generated by a SYNTECH stimulus controller (CS-55 v2.8, Ockenfels SYNTECH GmbH, Buchenbach, Germany). The flow velocity of the humidified air stream to the antenna was 50 cm/s. Stimuli were started with a blank stimulus (clean air), followed by n -pentane (10 μL) as the solvent control, then by the standard stimulus, i.e. the known female-produced aggregation component (–)-myrtenol, at a dose of 10 μg (Bozsik et al ., 2024). This was followed by the three doses of the synthetic candidate compounds, in a randomized order. At the end of the series, the three control stimuli, blank, solvent control, and standard were tested again. The stimulation intervals were 30 s. Five male, and five female antennae were tested, and each antenna was tested only once. Laboratory Behavior Tests with a Four-arm Arena Olfactometer The experiments were conducted during the natural mating season of P. aubei (mid-April and May), in the early afternoon hours, when P. aubei mating occurs (Bozsik & Szőcs, 2017). The method used was based on the concept of Petterson (1970), in line with some more recent developments (Karunaratne et al ., 2008; Kerchev & Pousheva, 2016; Mwando et al ., 2018; Vuts et al ., 2021). The main body of the arena was a 30 x 30 x 2.5 cm size, the connecting four-arm equipment was made of solid King StarBoard ® (no phthalates) with a removable clear Plexiglas lid (Sigma Scientific LLC, Micanopy, FL, United States). An insect inlet adapter and a two-piece all-glass combination tube adapter and insect isolation traps were attached to the arena body. Continuous airflow was provided by a Clean Air Delivery System (CADS, Sigma Scientific LLC) which was connected to a Stanley DST100/8/6 silent compressor. The CADS was equipped with a four-push and one-pull vacuum system. The supply and exhaust air was filtered by activated carbon filters (ICAF 2.5 x 8, Sigma Scientific LLC). The CADS and the olfactometer arms were connected by Teflon tubing. The compressor pressure was set at 30 psi, the olfactometer pressure was set at 16 psi, and the source air pressure was set at 20 psi. The four flowmeters controlling air flow to the four arms were set at 1.43 L/min. The exhaust venturi pressure was set at 20 psi, and the olfactometer vacuum was set at -2.45 psi. The outlet air flowmeter was set at 4.6 L/min. These settings provided approximately laminar airflow within the olfactometer. The synthetic samples tested were basically the same as those used in the EAG assays, with the only exception of using only the (1 S ,2 S ,5 S )-(–)- cis -verbenol enantiomer for the behavior tests. Synthetic compounds were dissolved in n -pentane, and 10 μL each of the test solution and pentane as a control were applied to 12.7 mm Rotilabo filter paper and then it was placed in the different glass arms. The combinations used in the different arms and assays are shown in the Table 1. Before each test, a cleaning procedure was performed: the glass parts were washed with distilled water and acetone and then baked out at 160 °C overnight. The olfactometer body was washed with 96% ethanol and then dried at room temperature as recommended by the manufacturer. The beetles were tested individually. Twenty-five males and females were tested in each assay, for a total of n=75 beetles were tested per sex. The beetles were acclimated in the olfactometer laboratory for 2 hours prior to testing. Each test-beetle was placed in the center of the olfactometer, and its locomotor behavior was continuously monitored for 12 min. The time spent in selected arms was recorded during this time. The body of the olfactometer was randomly rotated by 90° between the tests. A circular lamp was placed 50 cm above the arena to provide a uniform, omnidirectional, constant light intensity of 1200 lux to each arm. Behavior Tests on the Field The trapping tests were carried out during the breeding season of P. aubei (from mid-April to mid-June), in 2024. The experiments were conducted in a T. occidentalis ‘Smaragd’ plantation in Szombathely (Prenor Tree Nursery, Vas-county, Hungary, GPS 47.26419949232103 N, 16.59957200288773 E). The size of the plantation was about 0.2 ha, where the traps were placed at the edge of the field at a distance of about 6-7 m from each other. The traps were randomly placed in the rows of T. occidentalis ‘Smaragd’ trees, at a height of 180 cm and about 60-70 cm from the trees, hanging from bamboo poles. Eight-unit, black Lindgren funnel traps (SEDQ Healthy Crops S.L., Barcelona, Spain) were used for trapping. Synthetic stock solutions contained the same compounds in the same ratios as used in the olfactometer bioassays, but at much higher doses. In addition, a separate set of tests was performed: To mimic the most volatile cues of decaying trees (Bozsik et al ., 2023), five traps were also baited with (–)-α-pinene (purity: 96%, 85% ee, technical; abcr GmbH, Karlsruhe, Germany) in separate dispensers. We included this set of combined “plant-odour – pheromone” lures to make these baits more similar to the natural situation. The concept was based on our studies, which showed that the most abundant host odour component of the decaying T. occidentalis was α-pinene (Bozsik et al ., 2023), which attracted P. aubei in four-arm olfactometer tests (Bozsik et al ., 2024). The composition of the baits was the same as that tested in the four-arm olfactometry assays, but at two million times higher doses. The synthetic compounds were first dissolved in n -pentane and then further diluted in silicone oil (20 cSt, 25 °C, polydimethylsiloxane; VWR International, Radnor, PA, USA) (see the arrangement in the Table 2). Two 4 cm pieces of dental cotton wool (Celluron®, Paul Hartmann AG, Heidenheim, Germany) were placed in a resealable polyethylene bag (6x9cm, 0.2 mm wall thickness). When preparing the baits, the required amounts of solutions were administrated on the dental roll, then the polyethylene bag was carefully heat-sealed. Baits were transferred to the field under cooling to approximately -4 C° and attached to the edge of the funnel of the traps using a plastic flip. Baits were changed every other week. Each mixture was used in two replicates (two traps) and two unbaited traps were used as controls. Traps were emptied and recorded once a week, and the beetles were removed from the traps and transported to the experimental station of the Institute for sex determination. Ten records were made for each trap. The collecting containers of the traps were filled with 20% propylene glycol (Reanal, Budapest, Hungary) in water to preserve the captured insects. Statistical Analyses. The values of the EAG responses (mV) were normalized to the responses of the standard, 10 μg of (–)-myrtenol. LOG10 transformation was performed on each data and statistics were performed on these data sets. The Levene test was used to analyze the homogeneity of variance (Social Science Statistics 2024, https://www.socscistatistics.com/tests/levene/default.aspx) as each compound was compared pairwise with the solvent control. The homogeneity criteria were met in the most cases. A T-test was used to determine which compound elicited a significantly higher response than the solvent control (MS Excel). Analyses of variance (ANOVA), normality tests (Shapiro-Wilk) followed by Tukey's HSD tests were used to compare all responses (The jamovi project, 2021, jamovi, version 2.2, MAC OS, retrieved from https://www.jamovi.org). In both cases the significance level was set at 5%. The behavioral test data sets, as four-arm olfactometry and field trapping, were analyzed by Kruskal-Wallis ANOVA, and followed by a Wilcoxon rank sum test to analyze the differences between the means (p=5%; R version 4.4.0). RESULTS Analyses of Extracts by GC-EAD and Identification of Signalling Compounds by GC/MS Three compounds from the gut extracts of mated males with retention times of 5.25 min, 8.30 min, and 9.30 min and retention indices (RI) of 937, 1147, and 1217, on an HP-5 GC phase elicited antennal responses in males. The GC/MS analysis of the extracts showed that these compounds were α-pinene, cis -verbenol and verbenone (Figure 1). None of these compounds were detected in the extract from unmated males (Table 3/B). The ratio and the relative content of the identified compounds are also shown in Table 3/B. We confirmed the presence of the female-produced aggregation pheromone compounds of P. aubei , α-pinene and myrtenol, in the gut extracts of females, as we described it in our previous study (Bozsik et. al 2024). Both unmated and mated females produced these compounds, but the ratio and the relative levels differed depending on the mating status. In mated females, the level of α-pinene was decreased, while the level of myrtenol was increased, compared to unmated females (Table 3/A). Electroantennographic Bioassays (EAG) Electroantennographic analyses of synthetic compounds using male and female antennae showed that the highest antennal responses were elicited by (–)-myrtenol, (+)- cis -verbenol and (–)- cis -verbenol, at the doses of 10 μg and 100 μg. (–)-Myrtenol at 100 μg elicited numerically the highest antennal responses in both males and females, but these were not significantly higher than those evoked by the other compounds tested at this dose. At the 1 μg dose level, the responses were generally low, only slightly higher than the solvent control. However, many stimuli evoked statistically higher responses than the solvent. Only (–)-α-pinene at the lowest dose level did not elicit significantly greater responses than the solvent in males, while (–)-myrtenol at 1 μg was the only compound with a nonsignificant response in females (Figure 2). Behavioral Tests Four-arm Arena Olfactometer Bioassays P. aubei males and females spent significantly more time in the arms containing the mixtures of [1 μg (–)-myrtenol and 5 μg (–)-α-pinene], [1 μg (–)-myrtenol, 5 μg (–)-α-pinene and 1 μg (–)- cis -verbenol], [1 μg (–)-myrtenol, 5 μg (–)-α-pinene, 1 μg (–)- cis -verbenol and 1 μg (–)-verbenone], [5 μg (–)-α-pinene and 1 μg (–)- cis -verbenol], and 5 μg (–)-α-pinene alone, compared to the solvent control. Females spent significantly more time in the arms containing the combination of (–)- cis -verbenol and the two female-produced compounds, (–)-myrtenol and (–)-α-pinene, (mean time spent: 4.54 min±0.78) than in the arm containing only (–)-myrtenol and (–)-α-pinene (mean time spent: 2.46±0.73). The addition of (–)-verbenone to the three-component mixture, significantly reduced the time spent by both males and females (Figure 3). Field Trapping The results of the laboratory olfactometry bioassays were reflected by the trap catches, as by far the most P. aubei were caught in traps baited with the ternary combination of 200 mg (–)-myrtenol, 1 g (–)-α-pinene and 200 mg (–)- cis -verbenol dissolved in 1.5 mL of pentane and 1 g of silicone oil. Traps baited with this mixture plus 200 mg (–)-verbenone caught significantly fewer P. aubei . The combination of 200 mg (–)-myrtenol and 1 g (–)-α-pinene (the female produced pheromone) was used as a positive control according to Bozsik et al . (2024). Both males and females were recorded in the traps baited with the synthetic compounds (Figure 4). Traps with 4g of technical grade (–)-α-pinene caught significantly more P. aubei than the control traps. Traps in assays marked with an asterisk in Figure 4 also contained 4g of technical grade (–)-α-pinene in separate dispensers. In these assays, the combination of (–)-myrtenol, (–)-α-pinene, and (–)- cis -verbenol attracted significantly more P. aubei , than technical (–)-α-pinene alone. Traps with (–)- cis -verbenol alone caught slightly but significantly more P. aubei adults than the unbaited control traps. The addition of (–)-verbenone significantly reduced its attractiveness (Figure 4). The numbers of P. aubei captured in the traps are shown in the Table 4. A few individuals of a species closely related to P. aubei , P. thujae (Perris, 1855), were caught in some of the traps. One female was caught in a trap baited with (–)-myrtenol and (–)-α-pinene, while one male was caught in a trap with the mixture of (–)-myrtenol, (–)-α-pinene, and (–)- cis -verbenol and one in a trap baited with (–)-α-pinene and (–)- cis -verbenol. Most P. thujae were recorded in the traps baited with the mixture of (–)-myrtenol and (–)-α-pinene, (–)- cis -verbenol, and (–)-verbenone. Their total number was 7 (5 females, 2 males) and their mean number was 1.75. DISCUSSION In this study, three electrophysiologically active components were detected in mid- and hindgut extracts of mated male cypress bark beetles P. aubei by GC-EAD and their structures were determined by GC-MS as α-pinene, cis -verbenol and verbenone, respectively. None of these compounds were found in unmated males. α-Pinene has been previously reported as a major component in the volatiles of arborvitae, Thuja occidentalis ‘Smaragd’, a common host tree of P. aubei and as a component of the aggregation pheromone, produced by unmated P. aubei females (Bozsik et al., 2024 ). In this study, we also detected α-pinene in the mid- and hindgut extracts of mated P. aubei females but at much lower levels than in unmated females. Identification of a pheromone of P. aubei males, produced only after mating, provides a deeper insight into the chemical ecology of host and mate finding of this invasive bark beetle species, associated with scale-leaved conifers: The male-produced pheromone, it is emitted right after mating during the colonization period of the attacked tree, when the couple is in the nuptial chamber for some time (for details on the mating sequence, see Bozsik & Szőcs, 2017 ; Bozsik at al., 2024). Newly approaching beetles therefore encounter a mixture of host volatiles and female- and male-produced pheromones. To mimic this complex of chemical signals in this phase of colonization, we developed different synthetic multicomponent mixtures. For this, we used (–)-α-pinene, a key component of the host kairomone and a part of the pheromones of both females and males, female-specific (–)-myrtenol, and (+)- and (–)- cis -verbenol and (–)-verbenone, representing male volatiles. Each of these compounds elicited antennal responses in both sexes. We compared their behavioral effects under laboratory and field conditions. The results gained in the laboratory with a four-arm olfactometer and in the field in trapping tests were in very good agreement. A ternary mixture of (–)-α-pinene, (–)-myrtenol and (–)- cis -verbenol in a ratio of 5:1:1 attracted the highest number of P. aubei to the traps. This mixture was also most attractive to the beetles in the olfactometer. Both sexes were attracted in both types of tests, however the sex ratio was somewhat biased towards females. In contrast, the addition of (–)-verbenone to the ternary mixture greatly reduced its attractiveness in both the field and the laboratory tests. Single compounds and two-component blends were less attractive. The attractiveness enhancing effect of (–)- cis -verbenol while also influencing the sex ratio and the anti-aggregation effect of (–)-verbenone indicate the importance of a male pheromone in the process of host colonization of P. aubei . In the field test a few adults of the closely related P. thujae were also captured. This indicates similarities in chemical communication between these two co-occurring Phloeosinus species. However, these sparse data do not allow any more precise conclusions to be drawn. Nevertheless, the communication channels must be specific, as we never found P. thujae males in mating chambers of P. aubei . Currently, P. aubei is the economically most important pest, while P. thujae is much less abundant. Should this dominance change in the future, elucidating the pheromonal system of P. thujae would become more important. cis -Verbenol is known to be a component of the aggregation pheromones of Dendroctonus species. In this context, it is produced by female D. ponderosae Hopkins, 1902 (Miller & Lafontaine, 1991 ), by female D. frontalis Zimmermann, 1868 (Sullivan, 2005 ), and by both sexes of D. valens LeConte, 1860 (Xu et al., 2015 ). Biosynthetically, α-pinene is the precursor for cis -verbenol (Renwick et al., 1976 ). Furthermore, the biosynthetic oxidation of verbenol provides verbenone, which is used by D. frontalis males as an anti-aggregation pheromone (Byers, 1983 ; Frühbrodt et al., 2024 ). Its effect correlates with its concentration: it has no inhibitory effect at low levels, but it does at high doses (Audley et al., 2020 ; Byers et al., 2021 ). This phenomenon was also observed in our recent studies on P. aubei and may be reflected in the finding that the verbenone production increases after the mating. In other bark beetles, e.g. Hylurgus ligniperda (Fabricius, 1787) or Hylastes attenuatus Erichson, 1836 it has dose-dependent synergistic effect with pheromonal or kairomonal attractants (Romón et al., 2007 ; Frühbrodt et al., 2024 ). (–)-α-Pinene, (–)-verbenol and (–)-verbenone not only share a common carbon skeleton but also the same configuration of the stereocenters. Further investigations of the absolute configuration of the natural products will show whether our assumption derived from the behavioral tests of a common configuration of the male-produced compounds is confirmed. The results of our research are an important step in establishing an effective method of alternative control for P. aubei . A mixture of the female and male aggregation pheromone compounds, (–)-myrtenol and (–)- cis -verbenol, respectively, and female and host-produced (–)-α-pinene in a ratio of 1:1:5 is the most effective bait to date and could enable mass trapping for the first time. Declarations Author Contributions Gábor Bozsik developed the concept, made the experimental design, conducted the laboratory and field experiments, evaluation of datasets and wrote the main text of the manuscript. Armin Tröger carried out the mass spectrometry analyses to identify the aggregation pheromone compounds and wrote the corresponding sections of the manuscript. Stefan Schulz was the consultant for the spectral analyses and made the proofreading of the manuscript. Gábor Szőcs supervised the project and wrote the main text of the manuscript. All authors have reviewed the manuscript. Acknowledgements We are grateful to Mr. G. Izer, Director, Mr. Gy. Both and Mr. M. Bokor, Phytosanitary Advisors (Prenor Nursery Ltd, Szombathely, Hungary), for allowing us to conduct trapping assays and collect P. aubei beetles, and to Mr. Balázs Kulin, Director (Tahi Tree Nursery Ltd, Tahi, Hungary), for allowing us to collect P. aubei and tree samples, at the tree nursery. Conflict of Interest Statement The authors declare that they have no conflict of interest to declare. Data Availability Data supporting the findings of this study are available from the corresponding authors on reasonable request. Funding The study was financed by the National Research, Development and Innovation Office (NKFIH), Budapest, Hungary, project number PD 138089 to Gábor Bozsik. References Audley, J.P., Bostock, R.M. & Seybold, S.J. (2020) Trap assays of the walnut twig beetle, Pityophthorus juglandis Blackman (Coleoptera: Curculionidae: Scolytinae), reveal an effective semiochemical repellent combination. Journal of Chemical Ecology , 46, 1047–1058. https://doi.org/10.1007/s10886-020-01228-9 Blomquist, G.J., Figueroa-Teran, R., Aw, M., Song, M., Gorzalski, A., Abbott, N.L., Chang, E. & Tittiger, C. (2010) Pheromone production in bark beetles. Insect Biochemistry and Molecular Biology , 40, 699–712. https://doi.org/10.1016/j.ibmb.2010.07.013 Bozsik, G., Tröger, A., Francke, W. & Szőcs, G. (2016) Thuja occidentalis : identification of volatiles and electroantennographic response by the invasive cedar bark beetle, Phloeosinus aubei . Journal of Applied Entomology , 140, 434–443. https://doi.org/10.1111/jen.12272 Bozsik, G. & Szőcs, G. (2017) Phenology, behavior and infestation levels of the invasive small cypress bark beetle, Phloeosinus aubei , on some cultivars of Thuja and Juniper spp, in Hungary. Phytoparasitica , 45, 201–210. http://doi.org/10.1007/s12600-017-0585-y Bozsik, G., Molnár, B.P., Domingue, M.J. & Szőcs, G. (2023) Changes to volatile profiles of arborvitae, Thuja occidentalis , from drought and insect infestation: olfactory cues for the cypress bark beetle, Phloeosinus aubei . Chemoecology , 33, 113–124. https://doi.org/10.1007/s00049-023-00389-9 Bozsik, G., Molnár, B.P., Hegedüs, K., Soós, T., Schulz, S., Tröger, A., Francke, W., & Szőcs, G. (2024) (−)-myrtenol and (−)-α-pinene: Aggregation pheromone components of the cypress bark beetle Phloeosinus aubei . Journal of Applied Entomology , 148, 351–363. https://doi.org/10.1111/jen.13231 Byers, J.A. (1983) Bark beetle conversion of a plant compound to a sex specific inhibitor of pheromone attraction. Science , 220, 624–626. https://doi.org/10.1126/science.220.4597.624 Byers, J.A. (1989) Chemical ecology of bark beetles. Experientia , 45, 271–283. https://doi.org/10.1007/BF01951813 Byers, J.A., Maoz, Y., Cohen, B., Golani, M., Fefer, D. & Levi-Zada, A. (2021) Protecting avocado trees from ambrosia beetles by repellents and mass trapping (push–pull): Experiments and simulations. Journal of Pest Science , 94, 1–12. https://doi.org/10.1007/s10340-020-01310-x Frühbrodt, T., Schebeck, M., Andersson, M.N., Holighaus, G., Kreuzwieser, J., Burzlaff, T., Delb, H. & Biedermann, P.H.W. (2024) Verbenone—the universal bark beetle repellent? Its origin, effects, and ecological roles. Journal of Pest Science 97, 35–71. https://doi.org/10.1007/s10340-023-01635-3 Karunaratne, W.S., Kumar, V., Pettersson, J., Savitri, N. & Kumar, N. (2008) Response of the shot-hole borer of tea, Xyleborus fornicatus (Coleoptera: Scolytidae) to conspecifics and plant semiochemicals. Acta Agriculturae Scandinavica, Section B — Soil & Plant Science , 58, 345–351. https://doi.org/10.1080/09064710701788802 Keeling, C.I., Tittiger, C., MacLean, M., & Blomquist, G.J. (2021) Pheromone production in bark beetles. In: Blomquist, G.J. & Vogt R.G. (Eds.) Insect Pheromone Biochemistry and Molecular Biology (Second Edition) . New York, New York: Academic Press, pp. 123–162. https://doi.org/10.1016/B978-0-12-819628-1.00004-3 , Kerchev, I. A. & Pousheva, M.S. (2016) Olfactometric evidence for aggregation pheromone production by females of the four-eyed fir bark beetle Polygraphus proximus Blandf. (Coleoptera, Curculionidae: Scolytinae). Entomological Review , 96, 821–825. https://doi.org/10.1134/S0013873816070010 Miller, D.R. & Lafontaine, J.P. (1991) cis-Verbenol: An aggregation pheromone for the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Scolytidae). Journal of the Entomological Society of British Columbia , 88, 34–38. Mwando, N.L., Tamiru, A., Nyasani, J.O., Obonyo, M.A.O., Caulfield, J.C., Bruce, T.J.A. & Subramanian, S. (2018) Maize chlorotic mottle virus induces changes in host plant volatiles that attract vector thrips species. Journal of Chemical Ecology , 44, 681–689. https://doi.org/10.1007/s10886-018-0973-x Ockenfels, P. (2015) Electroantennography a practical introduction. Buchenbach, Germany: Syntech GmbH. Rerieved from http://www.ockenfels-syntech.com/wp-content/uploads/EAGpract_man_fin [Accessed 5th November 2024] Olsson, S.B. & Hansson, B.S. (2013) Electroantennogram and single sensillum recording in insect antennae. In: Touhara, K. (Ed.) Pheromone signalling. Methods in molecular biology (methods and protocols) . Totowa, New Jersey: Humana Press, pp. 157–177. Pettersson, J. (1970) An aphid sex attractant. I. Biological studies. Entomologica Scandinavica , 1, 63–73. Renwick, J.A., Hughes, P.R. & Krull, I.S. (1976) Selective production of cis- and trans-verbenol from (-)-and (+)-alpha by a bark beetle. Science , 191, 199–201. https://doi.org/10.1126/science.1246609 Roelofs, W.L. (1977) The scope and limitations of the electroantennogram technique in identifying pheromone components. In: McFarlane, N.R. (Ed.) Crop protection agents – their biological evaluation . New York, New York: Academic Press, pp. 147–165. Roelofs, W.L. (1984) Electroantennogram assay: rapid and convenient screening procedures for pheromones. In: Hummel, H.E. & Miller, T.A. (Eds.) Techniques in pheromone research . New York, New York: Springer Verlag, pp. 131–160. Romón, P., Iturrondobeitia, J.C., Gibson, K., Lindgren, B.S. & Goldarazena, A. (2007) Quantitative association of bark beetles with pitch canker fungus and effects of verbenone on their semiochemical communication in Monterey pine forests in Northern Spain. Environmental entomology , 36, 743–750. https://doi.org/10.1603/0046-225x(2007)36[743:qaobbw]2.0.co;2 Sullivan, B.T. (2005) Electrophysiological and behavioral responses of Dendroctonus frontalis (Coleoptera: Curculionidae) to volatiles isolated from conspecifics. Journal of Economic Entomology , 98, 2067–2078. https://doi.org/10.1093/jee/98.6.2067 Tittiger, C. & Blomquist, G.J. (2016) Pheromone Production in Pine Bark Beetles, In: Tittiger, C. & Blomquist G.J. (Eds.) Advances in Insect Physiology . New York, New York: Academic Press, pp. 235–263. https://doi.org/10.1016/bs.aiip.2016.02.002 Vuts, J., Szanyi, Sz., Szanyi, K., König, L., Nagy, A., Imrei, Z., Birkett, M.A. & Tóth, M. (2021) Development of a phytochemical-based lure for the dried bean beetle Acanthoscelides obtectus Say (Coleoptera: Chrysomelidae). Journal of Chemical Ecology , 47, 987–997. https://doi.org/10.1007/s10886-021-01305-7 Xu, L., Lou, Q., Cheng, C., Lu. M. & Sun, J. (2015) Gut-associated bacteria of Dendroctonus valens and their involvement in verbenone production. Microbial Ecology , 70, 1012–1023. https://doi.org/10.1007/s00248-015-0625-4 Tables Table 1 Arrangement of the four-arm olfactometer bioassays: Concentrations and combinations of the solutions. Assay No. arm1 arm2 arm3 arm4 control ( n -pentane 10 μL) (–)-myrtenol 1 μg (–)-α-pinene 5 μg (–)-myrtenol 1 μg (–)-α-pinene 5 μg (–) -cis -verbenol 1 μg (–)- cis -verbenol 1 μg control ( n -pentane 10 μL) (–)-myrtenol 1 μg (–)-α-pinene 5 μg (–)-myrtenol 1 μg (–)-α-pinene 5 μg (–)- cis -verbenol 1 μg (–)-verbenone 1 μg (–) -cis -verbenol 1 μg (–)-verbenone 1 μg control ( n -pentane 10 μL) (–)-α-pinene 5 μg (–)- cis -verbenol 1 μg (–)-α-pinene 5 μg (–)- cis -verbenol 1 μg (–)-verbenone 1 μg (–)-α-pinene 5 μg Table 2 Mixtures applied in the trapping trials. Lures/traps No. 1. 2. 3. 4. 5. 6. 7. (–)-α-pinene 1 g (–)-α-pinene 1 g (–)-α-pinene 1 g – – (–)-α-pinene 1 g (–)-α-pinene techn. 4 g (–)-myrtenol 200 mg (–)-myrtenol 200 mg (–)-myrtenol 200 mg – – – – (–)- cis -verbenol 200 mg (–) -cis -verbenol 200 mg (–) -cis -verbenol 200 mg (–) -cis -verbenol 200 mg (–)- cis -verbenol 200 mg – – (–)-verbenone 200 mg – (–)-verbenone 200 mg – silicone oil 1 g silicone oil 1 g silicone oil 1 g silicone oil 1 g silicone oil 1 g silicone oil 1 g pentane 1.5 mL pentane 1.5 mL pentane 1.5 mL pentane 1.5 mL pentane 1.5 mL pentane 1.5 mL and and and and in a separate dispenser: in a separate dispenser: in a separate dispenser: in a separate dispenser: (–)-α-pinene techn. 4 g (–)-α-pinene techn. 4 g (–)-α-pinene techn. 4 g – – (–)-α-pinene techn. 4 g Table 3 Ratios and amounts of the identified aggregation pheromone compounds in unmated and mated female (A.) and male (B.) P. aubei . A. ratio female equivalent (ng/FE) α-pinene myrtenol α-pinene myrtenol unmated female 1.35 1 0.81 0.59 mated female 0.16 1 0.5 3.2 B. ratio male equivalent (ng/ME) α-pinene cis -verbenol verbenone α-pinene cis -verbenol verbenone unmated male NO NO NO NO NO NO mated male 0.083 2.67 1 0.08 2.72 1.02 Table 4 Total and number of P. aubei in traps. Significant differences between the experiment means were defined as P values less than 0.05. (For the detailed composition of mixtures/lures, see Table 2) Additional Declarations The authors declare no competing interests. Cite Share Download PDF Status: Published Journal Publication published 19 Jun, 2025 Read the published version in Agricultural and Forest Entomology → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6305155","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":433856492,"identity":"8e0a833c-becb-4370-980a-f4096ea3e9a9","order_by":0,"name":"Gábor Bozsik","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABCklEQVRIiWNgGAWjYBACAxCRcABISADxB5CABExOgggtjDMYDIjUwgDVwsxDjBZz9vbHHx6cYbDrn9187LNNzR9jBukesweMe2zygAwDbFosew6kSSTcYEiecedY8uycYwZmDDJnzA0YnqUVAxlYtRjcSDjGkPCBIZnhRo4xcw6bgQ2DRI6Z9J8DhxMbJHJwaEls/gDSIg/SYvEPokWC4cB/PFqSGUAOszMAaWFsAzoMouUATi2WPcfYJBLOSCQYAv3C2NtnbMwmkVYG1JKc2CaRVoArxD7+OGZjL3e7+TDDj29yhv0SyduAWuwSgYwN2IMZDCQSG2BMNgwGDmBPQH4UjIJRMApGMgAA8nNcavzKNRYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3832-4890","institution":"Department of Chemical Ecology, Plant Protection Institute, Centre for Agricultural Research, HUN-REN, Brunszvik str. 2., Martonvásár, Hungary","correspondingAuthor":true,"prefix":"","firstName":"Gábor","middleName":"","lastName":"Bozsik","suffix":""},{"id":433856493,"identity":"454a92d1-e169-453d-8f06-04d5c6e7bbac","order_by":1,"name":"Armin Tröger","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABHUlEQVRIiWNgGAWjYJCCAwhmBQMDGwMPAwNjA5hrgEcLTO4MkVoQcoxtIJKAFv7ZpxMPFzD8yednb7/4uHLe4Tw+Bt6Dn27uOJwv78C88QEWLRLncjccnsFgYDmz50yx4dlth4vZGPiSpXPPHLbceICtGKs1Z3g3HOZhMDAwuJGTJtm47XBim/wbA+nctsMGhg08ZhJYdMjDtdx/k/6zcQ5QCwOP8W+oFvMf2PyNsIX9GGNjA1iLGdgWeSADm7sMwVoMjA0ke3KYJRuOpYO1WOeeSTcwYGYrxuYwuTO8mz/zVMgZ8LMff/ixocY6cX4Dj/Ht3B3WBvLtzRs/YLMG4jwQwYMWPAaHcaqHAfYHqHz5BoJaRsEoGAWjYGQAALEeYneJGx7/AAAAAElFTkSuQmCC","orcid":"","institution":"Institute of Organic Chemistry, University of Hamburg, Germany","correspondingAuthor":true,"prefix":"","firstName":"Armin","middleName":"","lastName":"Tröger","suffix":""},{"id":433856494,"identity":"54653160-9ccb-45b9-b330-96a41cdb06c6","order_by":2,"name":"Stefan Schulz","email":"","orcid":"https://orcid.org/0000-0002-4810-324X","institution":"Institute of Organic Chemistry, TU Braunschweig, Braunschweig, Germany","correspondingAuthor":false,"prefix":"","firstName":"Stefan","middleName":"","lastName":"Schulz","suffix":""},{"id":433856495,"identity":"ad094321-7973-4604-ad0f-58805a2c77f1","order_by":3,"name":"Gábor Szőcs","email":"","orcid":"https://orcid.org/0000-0001-8714-6405","institution":"Department of Chemical Ecology, Plant Protection Institute, Centre for Agricultural Research, HUN-REN, Brunszvik str. 2., Martonvásár, Hungary","correspondingAuthor":false,"prefix":"","firstName":"Gábor","middleName":"","lastName":"Szőcs","suffix":""}],"badges":[],"createdAt":"2025-03-25 15:21:19","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-6305155/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6305155/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1111/afe.12695","type":"published","date":"2025-06-20T00:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79239886,"identity":"1dc80a49-b71f-438f-b845-c9171aced0a7","added_by":"auto","created_at":"2025-03-26 05:28:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":145966,"visible":true,"origin":"","legend":"\u003cp\u003eGC-EAD analyses of a gut extract of mated male \u003cem\u003eP. aubei\u003c/em\u003e using an HP-5 column.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6305155/v1/40edbc822b2d7d3b02bd846e.png"},{"id":79239885,"identity":"0dceacbe-5dc6-4071-bfba-6f7288831f44","added_by":"auto","created_at":"2025-03-26 05:28:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":266988,"visible":true,"origin":"","legend":"\u003cp\u003eRelative antennal responses of male and female \u003cem\u003eP. aubei\u003c/em\u003e to (–)-α-pinene, (–)-myrtenol, (+)- and (–)-\u003cem\u003ecis\u003c/em\u003e-verbenol and (–)-verbenone at different dose levels. Responses to (–)-myrtenol (10 μg) were used as standard. Means labelled with the same letter are not significantly different from each other at the alpha=5% level (ANOVA, followed by \u003cem\u003eTukey’s HSD\u003c/em\u003e, df=15). Means marked with an asterisk are significantly different from the solvent control (\u003cem\u003et-test\u003c/em\u003e at alpha=5%, df=15).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6305155/v1/706174536f65e9d13953e2bc.png"},{"id":79239889,"identity":"48671792-e335-449e-bc31-63b07346c133","added_by":"auto","created_at":"2025-03-26 05:28:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":413507,"visible":true,"origin":"","legend":"\u003cp\u003eTime spent by \u003cem\u003eP. aubei\u003c/em\u003e males and females in the olfactometer arms containing the different mixtures of \u003cem\u003eP. aubei\u003c/em\u003e pheromone and host compounds. Means marked with the same letter within a graph are not significantly different at alpha=5% level (Kruskall-Wallis ANOVA, Wilcoxon test, df=3).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6305155/v1/316828d3683b26d185ab545b.png"},{"id":79239892,"identity":"b6aef192-984f-4ae0-9774-f0c69bcaf7d9","added_by":"auto","created_at":"2025-03-26 05:28:28","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":230620,"visible":true,"origin":"","legend":"\u003cp\u003eMean number of \u003cem\u003eP. aubei\u003c/em\u003e males and females captured in traps baited with the identified male and female \u003cem\u003eP. aubei\u003c/em\u003epheromone and host compounds and in unbaited traps. Means marked with the same letter within a graph are not significantly different at the alpha=5% level (Kruskall-Wallis ANOVA, Wilcoxon test, df=7). Experiments with technical grade (–)-α-pinene in an additional separate dispenser are marked with an asterisk below its label.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6305155/v1/98e34e1d41868d66d02e8ae5.png"},{"id":85126906,"identity":"36b64fbb-9be4-4021-aa69-57db29af78bb","added_by":"auto","created_at":"2025-06-21 23:31:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1837075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6305155/v1/662950a3-ea65-4fcc-a0cd-89c5cb20eebc.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ecis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-Verbenol and verbenone as male-produced pheromone components of the cypress bark beetle, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePhloeosinus aubei\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e: Identification and behavioral tests in combination with the female-produced pheromone components\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eChemical communication in bark beetles consists of inter- (host finding) and intra-specific (aggregation and mate finding) channels, which are often intertwined, not only in function, but also in the set of compounds involved. For example, in many species, host finding is initiated by volatiles from the host tree, while pioneer beetles emit aggregation pheromones to attract conspecifics, which is an effective way to collectively defeat the defense mechanisms of the attacked tree (Byers, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Keeling et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). When males and females aggregate on the colonized tree, this greatly facilitates finding mating partners. This is where aggregation pheromones come into play. In \u003cem\u003eIps\u003c/em\u003e species, the male is the pioneer sex and pheromone producer, while in \u003cem\u003eDendroctonus\u003c/em\u003e species it is the female, but both sexes release sex-specific pheromones (Blomquist et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tittiger \u0026amp; Blomquist, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Often, these aggregation pheromone bouquets, together with host volatiles, control complex behavioral sequences of mate finding and mating.\u003c/p\u003e \u003cp\u003eIn contrast to the bark beetles associated with needle-leaved conifers, little is known about the chemical communication of bark beetles that live exclusively on scale-leaved conifers (Cupressaceae). The most prominent representative of the latter group is the genus \u003cem\u003ePhloeosinus\u003c/em\u003e. In the West-Palearctic region, the cypress bark beetle, \u003cem\u003ePhloeosinus aubei\u003c/em\u003e (Perris, 1855) (Coleoptera, Curculionidae, Scolytinae) extended its range from the Mediterranean areas towards Western and Middle-Europe, threatening \u003cem\u003eThuja\u003c/em\u003e, \u003cem\u003eChamaecyparis\u003c/em\u003e and \u003cem\u003eJuniperus\u003c/em\u003e ornamental trees in urban areas and in tree nurseries. Followed by detailed description of life cycle and mating habit of settled populations under continental climate (Bozsik \u0026amp; Szőcs, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), we identified key compounds in the host plant volatiles as a kairomone for the cypress bark beetle, \u003cem\u003eP. aubei\u003c/em\u003e (Bozsik et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) and identified the predominant components of the female-produced aggregation pheromone (Bozsik et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Here, we report on the identification of a male-produced pheromone of \u003cem\u003eP. aubei\u003c/em\u003e and the interaction of its compounds with those emitted by females in the attraction of both sexes.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cem\u003eExperimental Insects\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFemale and male adults of \u003cem\u003eP. aubei\u003c/em\u003e were collected in West-Hungary (Prenor Tree Nursery, Szombathely, Vas-county, GPS 47.26419949232103 N, 16.59957200288773 E) at the end of their overwintering period. They build individual overwintering tunnels in the tips of twigs of \u003cem\u003eThuja occidentalis\u003c/em\u003e \u0026lsquo;Smaragd\u0026rsquo;. Infected twigs were cut ca. 15 cm from the tip with beetles inside the tunnels and were transported to the experimental station of the Plant Protection Institute (Budapest, Hungary, GPS 47.547946 N, 18.934632 E). Tunnels were carefully opened and the sex of \u003cem\u003eP. aubei\u003c/em\u003e was determined using a binocular microscope without removing the beetles from the tunnels. Subsequently, males and females were kept separately in a 4L glass jars, with \u003cem\u003eT\u003c/em\u003e. \u003cem\u003eoccidentalis\u003c/em\u003e \u0026lsquo;Smaragd\u0026rsquo; twigs under ambient conditions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWind-broken branches of \u003cem\u003eJuniperus chinenis\u003c/em\u003e \u0026lsquo;Spartan\u0026rsquo; with freshly carved mating chambers of \u003cem\u003eP. aubei\u003c/em\u003e, were collected at the Tahi Tree Nursery (Tahi, Pest-county, Central-Hungary, GPS 47.7661127 N, 19.0684354E) and transported to the experimental station of the Plant Protection Institute at the beginning of the mating season (April 2023). By opening the galleries for collecting the beetles just before preparing the extracts, we found that the nuptial chambers were occupied by a male and a female \u003cem\u003eP. aubei\u003c/em\u003e. Typically, a mating pair was found, although usually no longer in copula. A second male was never found and, in line with our earlier observations (Bozsik et. al, 2024), they were most probably all mated. This assumption was based on our previous finding that when a male arrives at a nuptial chamber with a female inside, they mate immediately. After opening the nuptial chambers, males and females were separated from each other. These specimens were taken as mated. Mid- and hindgut extracts were made immediately afterwards.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePreparations of Pheromone Extracts\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMid- and hindguts of 50 mated males were excised with a sharp forceps and extracted with 55 \u0026mu;L of \u003cem\u003en\u003c/em\u003e-hexane (reagent plus grade, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). During the preparation procedure (approximately 1 h), the extract was stored in a vial placed on a -20\u0026deg;C iron plate. When all guts were put into the cooled \u003cem\u003en\u003c/em\u003e-hexane the vial was removed from the cooling plate and the mixture was kept for 10 min at room temperature (dissolving period). Similarly, mid- and hindguts of 60 mated females freshly removed from their nuptial chambers, were extracted in 60 \u0026mu;L of \u003cem\u003en\u003c/em\u003e-hexane.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAs a control, extracts were also made from unmated males and females, respectively. We developed a strict protocol to ensure that the beetles in this control group were in as similar a physiological state as possible to those in the mated group. Beetles, collected individually from their overwintering tunnels before the mating season, were taken as unmated, kept separately and allowed to complete the maturation feeding. An approximately 20 cm long branch of a semi-dried trunk of \u003cem\u003eT. occidentalis\u003c/em\u003e \u0026lsquo;Smaragd\u0026rsquo; (previously not infested by \u003cem\u003eP. aubei\u003c/em\u003e) was placed in the jar of unmated females. When all females had prepared their nuptial chambers, mid- and hindgut extracts were made using the same method as above. Guts from 20 females were dissolved in 40 \u0026mu;L of \u003cem\u003en\u003c/em\u003e-hexane. Unmated males were also collected from their overwintering tunnels, and as males do not build nuptial chambers (Bozsik \u0026amp; Szőcs, 2017), they were allowed to feed on \u003cem\u003eT. occidentalis\u003c/em\u003e \u0026lsquo;Smaragd\u0026rsquo; twigs before the preparation of extracts. Mid- and hindguts of 9 unmated males were dissolved in 20 \u0026mu;L of \u003cem\u003en\u003c/em\u003e-hexane using the same method previously described.\u003c/p\u003e\n\u003cp\u003eThe extracts were stored at -40\u0026deg;C, until GC-EAD analysis. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAnalyses of Extracts by Gas Chromatography Coupled to an Electroantennographic Detector (GC-EAD)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eA gas chromatograph (6890 N, Agilent Technologies Inc., Santa Clara, CA, USA), equipped with an HP-5 column (J\u0026amp;W, 30 m x 0.32 mm, 0.25 \u0026mu;m film thickness; Agilent Technologies Inc.) was connected to an electroantennographic detector (Ockenfels SYNTECH GmbH, Buchenbach, Germany) and operated with a beetle preparation (antenna-head) to analyze the extracts. The initial temperature was 50 \u0026deg;C for 1 min, then it was heated to 230 \u0026deg;C at a heating rate of 10 \u0026deg;C/min, and the final temperature was held for 10 min. Helium was used as the carrier gas (flow: 2.0 mL/min) and also as the make-up gas. Extracts were injected in splitless mode. Agilent ChemStation (version Rev. A. 10.02) was used as the control software.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMounted head-antennae of male \u003cem\u003eP. aubei\u003c/em\u003e, placed between glass capillary electrodes (1.17 mm i.d.) filled with Ephrussi-Beadle Ringer solution and equipped with silver wires, were used for EAD detection. Connections were established by MP15 micromanipulators. Humidified air flow through a glass tube, connected to the outlet of the EAD, ensured the transport of compounds to the antenna. Signals were analyzed using an IDAC2 amplifier and SYNTECH Syntech GC-EAD 2014 v. 1.2.5. software (Ockenfels SYNTECH GmbH, Buchenbach, Germany). \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eIdentification of Antennally Active Compounds by Gas Chromatography Coupled to Mass Spectrometry (GC/MS)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe structure elucidation of the antennally active compounds in the natural samples was carried out using a GC 7890A GC coupled to a 5975C inert XL MSD (Agilent Technologies - MSD ChemStation E.02.02.1431). The identification of the compounds was based on their mass spectra and retention indices using fused silica capillary columns of two different polarities. The assignments were verified by GC/MS comparison with synthetic reference compounds.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe gas chromatograph was equipped with a DB-heavyWAX GC column (60 m x 0.25 mm, 0.25 \u0026mu;m film thickness, Agilent J\u0026amp;W) and an HP-5ms column (60 m x 0.25 mm x 0.25 \u0026mu;m, Agilent J\u0026amp;W), respectively. Samples were injected in splitless mode (30 s). The temperature program was set at 50 \u0026deg;C for 3 min, then increased to 80 \u0026deg;C at a rate of 3 \u0026deg;C/min, then increased to 150 \u0026deg;C at 5 \u0026deg;C/min, and then finally to 250 \u0026deg;C at 8 \u0026deg;C/min for the DB-heavyWAX and at 50 \u0026deg;C for 3 min, then increased to 80 \u0026deg;C at a rate of 3 \u0026deg;C/min, to 150 \u0026deg;C at 5 \u0026deg;C/min, and to 300 \u0026deg;C at 7.5 \u0026deg;C/min for the HP-5ms. Helium was used as the carrier gas. The MS was operated in the EI 70 eV mode. The auxiliary heater was set at 280 \u0026deg;C, the MS source at 230 \u0026deg;C, and the MS quad at 150 \u0026deg;C. The scan mass range was \u003cem\u003em/z\u0026nbsp;\u003c/em\u003e33-433 with a scan speed of 3.58 scans/s. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eChemicals\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSynthetic samples of\u0026nbsp;(1\u003cem\u003eR\u003c/em\u003e,5\u003cem\u003eS\u003c/em\u003e)-(\u0026ndash;)-myrtenol (purity: 95%), (1\u003cem\u003eS\u003c/em\u003e,5\u003cem\u003eS\u003c/em\u003e)-(\u0026ndash;)-\u0026alpha;-pinene, (purity: 99%), (1\u003cem\u003eS\u003c/em\u003e,2\u003cem\u003eS\u003c/em\u003e,5\u003cem\u003eS\u003c/em\u003e)-(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol (purity: 95%) and (1\u003cem\u003eS\u003c/em\u003e,5\u003cem\u003eS\u003c/em\u003e)-(\u0026ndash;)-verbenone (purity: 93%) were purchased from Sigma-Aldrich (Merck\u0026nbsp;KGaA, Darmstadt, Germany). (1\u003cem\u003eR\u003c/em\u003e,2\u003cem\u003eR\u003c/em\u003e,5\u003cem\u003eR\u003c/em\u003e)-(+)-\u003cem\u003ecis\u003c/em\u003e-Verbenol (purity: 99%) was synthesized by the late Prof. Wittko Francke (University of Hamburg, Germany). Synthetic samples were dissolved in \u003cem\u003en\u003c/em\u003e-pentane (for analysis; Merck KGaA) at three concentrations: 100 ng/\u0026mu;L, 1 \u0026mu;g/\u0026mu;L, 10 \u0026mu;g/\u0026mu;L. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eElectroantennographic Bioassays (EAG)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe antenna head preparation technique was the same as described above for GC-EAD. Stimuli were passed through a Pasteur pipette containing a 12.7 mm Rotilabo filter paper (Carl Roth GmbH, Karlsruhe, Germany). 10 \u0026mu;L of \u003cem\u003en\u003c/em\u003e-pentane solution of the synthetic compounds was applied to a filter paper. The method is based on the concept of Roelofs (1977, 1984) and the recommendation of SYNTECH (Ockenfels SYNTECH GmbH, Buchenbach, Germany) (Ockenfels, 2015), which is also supported by Olsson \u0026amp; Hansson (2013). The flush was generated by a SYNTECH stimulus controller (CS-55 v2.8, Ockenfels SYNTECH GmbH, Buchenbach, Germany). The flow velocity of the humidified air stream to the antenna was 50 cm/s.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStimuli were started with a blank stimulus (clean air), followed by \u003cem\u003en\u003c/em\u003e-pentane (10 \u0026mu;L) as the solvent control, then by the standard stimulus, i.e. the known female-produced aggregation component (\u0026ndash;)-myrtenol, at a dose of 10 \u0026mu;g (Bozsik \u003cem\u003eet al\u003c/em\u003e., 2024). This was followed by the three doses of the synthetic candidate compounds, in a randomized order. At the end of the series, the three control stimuli, blank, solvent control, and standard were tested again. The stimulation intervals were 30 s. Five male, and five female antennae were tested, and each antenna was tested only once. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLaboratory Behavior Tests with a Four-arm Arena Olfactometer\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments were conducted during the natural mating season of \u003cem\u003eP. aubei\u0026nbsp;\u003c/em\u003e(mid-April and May), in the early afternoon hours, when \u003cem\u003eP. aubei\u003c/em\u003e mating occurs (Bozsik \u0026amp; Szőcs, 2017).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe method used was based on the concept of Petterson (1970), in line with some more recent developments (Karunaratne \u003cem\u003eet al\u003c/em\u003e., 2008; Kerchev \u0026amp; Pousheva, 2016; Mwando \u003cem\u003eet al\u003c/em\u003e., 2018; Vuts \u003cem\u003eet al\u003c/em\u003e., 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe main body of the arena was a 30 x 30 x 2.5 cm size, the connecting four-arm equipment was made of solid King StarBoard\u003csup\u003e\u0026reg;\u003c/sup\u003e (no phthalates) with a removable clear Plexiglas lid (Sigma Scientific LLC, Micanopy, FL, United States). An insect inlet adapter and a two-piece all-glass combination tube adapter and insect isolation traps were attached to the arena body. Continuous airflow was provided by a Clean Air Delivery System (CADS, Sigma Scientific LLC) which was connected to a Stanley DST100/8/6 silent compressor. The CADS was equipped with a four-push and one-pull vacuum system. The supply and exhaust air was filtered by activated carbon filters (ICAF 2.5 x 8, Sigma Scientific LLC). The CADS and the olfactometer arms were connected by Teflon tubing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe compressor pressure was set at 30 psi, the olfactometer pressure was set at 16 psi, and the source air pressure was set at 20 psi. The four flowmeters controlling air flow to the four arms were set at 1.43 L/min. The exhaust venturi pressure was set at 20 psi, and the olfactometer vacuum was set at -2.45 psi. The outlet air flowmeter was set at 4.6 L/min. These settings provided approximately laminar airflow within the olfactometer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe synthetic samples tested were basically the same as those used in the EAG assays, with the only exception of using only the\u0026nbsp;(1\u003cem\u003eS\u003c/em\u003e,2\u003cem\u003eS\u003c/em\u003e,5\u003cem\u003eS\u003c/em\u003e)-(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol enantiomer for the behavior tests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSynthetic compounds were dissolved in \u003cem\u003en\u003c/em\u003e-pentane, and 10 \u0026mu;L each of the test solution and pentane as a control were applied to 12.7 mm Rotilabo filter paper and then it was placed in the different glass arms. The combinations used in the different arms and assays are shown in the Table 1.\u003c/p\u003e\n\u003cp\u003eBefore each test, a cleaning procedure was performed: the glass parts were washed with distilled water and acetone and then baked out at 160 \u0026deg;C overnight. The olfactometer body was washed with 96% ethanol and then dried at room temperature as recommended by the manufacturer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe beetles were tested individually. Twenty-five males and females were tested in each assay, for a total of n=75 beetles were tested per sex. The beetles were acclimated in the olfactometer laboratory for 2 hours prior to testing. Each test-beetle was placed in the center of the olfactometer, and its locomotor behavior was continuously monitored for 12 min. The time spent in selected arms was recorded during this time. The body of the olfactometer was randomly rotated by 90\u0026deg; between the tests. A circular lamp was placed 50 cm above the arena to provide a uniform, omnidirectional, constant light intensity of 1200 lux to each arm. \u0026nbsp;\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBehavior Tests on the Field\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe trapping tests were carried out during the breeding season of \u003cem\u003eP. aubei\u003c/em\u003e (from mid-April to mid-June), in 2024. The experiments were conducted in a \u003cem\u003eT. occidentalis\u003c/em\u003e \u0026lsquo;Smaragd\u0026rsquo; plantation in Szombathely (Prenor Tree Nursery, Vas-county, Hungary, GPS 47.26419949232103 N, 16.59957200288773 E). The size of the plantation was about 0.2 ha, where the traps were placed at the edge of the field at a distance of about 6-7 m from each other. The traps were randomly placed in the rows of \u003cem\u003eT. occidentalis\u003c/em\u003e \u0026lsquo;Smaragd\u0026rsquo; trees, at a height of 180 cm and about 60-70 cm from the trees, hanging from bamboo poles. Eight-unit, black Lindgren funnel traps (SEDQ Healthy Crops S.L., Barcelona, Spain) were used for trapping.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSynthetic stock solutions contained the same compounds in the same ratios as used in the olfactometer bioassays, but at much higher doses. In addition, a separate set of tests was performed: To mimic the most volatile cues of decaying trees (Bozsik \u003cem\u003eet al\u003c/em\u003e., 2023), five traps were also baited with (\u0026ndash;)-\u0026alpha;-pinene (purity: 96%, 85% ee, technical; abcr GmbH, Karlsruhe, Germany) in separate dispensers. We included this set of combined \u0026ldquo;plant-odour \u0026ndash; pheromone\u0026rdquo; lures to make these baits more similar to the natural situation. The concept was based on our studies, which showed that the most abundant host odour component of the decaying \u003cem\u003eT. occidentalis\u003c/em\u003e was \u0026alpha;-pinene (Bozsik \u003cem\u003eet al\u003c/em\u003e., 2023), which attracted \u003cem\u003eP. aubei\u003c/em\u003e in four-arm olfactometer tests (Bozsik \u003cem\u003eet al\u003c/em\u003e., 2024).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe composition of the baits was the same as that tested in the four-arm olfactometry assays, but at two million times higher doses. The synthetic compounds were first dissolved in \u003cem\u003en\u003c/em\u003e-pentane and then further diluted in silicone oil (20 cSt, 25 \u0026deg;C, polydimethylsiloxane; VWR International, Radnor, PA, USA) (see the arrangement in the Table 2). Two 4 cm pieces of dental cotton wool (Celluron\u0026reg;, Paul Hartmann AG, Heidenheim, Germany) were placed in a resealable polyethylene bag (6x9cm, 0.2 mm wall thickness). When preparing the baits, the required amounts of solutions were administrated on the dental roll, then the polyethylene bag was carefully heat-sealed. Baits were transferred to the field under cooling to approximately -4 C\u0026deg; and attached to the edge of the funnel of the traps using a plastic flip. Baits were changed every other week. Each mixture was used in two replicates (two traps) and two unbaited traps were used as controls. Traps were emptied and recorded once a week, and the beetles were removed from the traps and transported to the experimental station of the Institute for sex determination. Ten records were made for each trap. The collecting containers of the traps were filled with 20% propylene glycol (Reanal, Budapest, Hungary) in water to preserve the captured insects. \u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical Analyses.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe values of the EAG responses (mV) were normalized to the responses of the standard, 10 \u0026mu;g of (\u0026ndash;)-myrtenol. LOG10 transformation was performed on each data and statistics were performed on these data sets. The \u003cem\u003eLevene test\u003c/em\u003e was used to analyze the homogeneity of variance (Social Science Statistics 2024, https://www.socscistatistics.com/tests/levene/default.aspx) as each compound was compared pairwise with the solvent control. The homogeneity criteria were met in the most cases. A \u003cem\u003eT-test\u003c/em\u003e was used to determine which compound elicited a significantly higher response than the solvent control (MS Excel). Analyses of variance (ANOVA), normality tests (Shapiro-Wilk) followed by Tukey\u0026apos;s HSD tests were used to compare all responses (The jamovi project, 2021, jamovi, version 2.2, MAC OS, retrieved from https://www.jamovi.org). In both cases the significance level was set at 5%.\u003c/p\u003e\n\u003cp\u003eThe behavioral test data sets, as four-arm olfactometry and field trapping, were analyzed by Kruskal-Wallis ANOVA, and followed by a Wilcoxon rank sum test to analyze the differences between the means (p=5%; R version 4.4.0).\u0026nbsp;\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cem\u003eAnalyses of Extracts by GC-EAD and Identification of Signalling Compounds by GC/MS\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThree compounds from the gut extracts of mated males with retention times of 5.25 min, 8.30 min, and 9.30 min and retention indices (RI) of 937, 1147, and 1217, on an HP-5 GC phase elicited antennal responses in males. The GC/MS analysis of the extracts showed that these compounds were \u0026alpha;-pinene, \u003cem\u003ecis\u003c/em\u003e-verbenol and verbenone (Figure 1). None of these compounds were detected in the extract from unmated males (Table 3/B). The ratio and the relative content of the identified compounds are also shown in Table 3/B.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe confirmed the presence of the female-produced aggregation pheromone compounds of \u003cem\u003eP. aubei\u003c/em\u003e, \u0026alpha;-pinene and myrtenol, in the gut extracts of females, as we described it in our previous study (Bozsik et. al 2024). Both unmated and mated females produced these compounds, but the ratio and the relative levels differed depending on the mating status. In mated females, the level of \u0026alpha;-pinene was decreased, while the level of myrtenol was increased, compared to unmated females (Table 3/A).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eElectroantennographic Bioassays (EAG)\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eElectroantennographic analyses of synthetic compounds using male and female antennae showed that the highest antennal responses were elicited by (\u0026ndash;)-myrtenol, (+)-\u003cem\u003ecis\u003c/em\u003e-verbenol and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol, at the doses of 10 \u0026mu;g and 100 \u0026mu;g. (\u0026ndash;)-Myrtenol at 100 \u0026mu;g elicited numerically the highest antennal responses in both males and females, but these were not significantly higher than those evoked by the other compounds tested at this dose. At the 1 \u0026mu;g dose level, the responses were generally low, only slightly higher than the solvent control. However, many stimuli evoked statistically higher responses than the solvent. Only (\u0026ndash;)-\u0026alpha;-pinene at the lowest dose level did not elicit significantly greater responses than the solvent in males, while (\u0026ndash;)-myrtenol at 1 \u0026mu;g was the only compound with a nonsignificant response in females (Figure 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBehavioral Tests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFour-arm Arena Olfactometer Bioassays\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eP. aubei\u003c/em\u003e males and females spent significantly more time in the arms containing the mixtures of [1 \u0026mu;g (\u0026ndash;)-myrtenol and 5 \u0026mu;g (\u0026ndash;)-\u0026alpha;-pinene], [1 \u0026mu;g (\u0026ndash;)-myrtenol, 5 \u0026mu;g (\u0026ndash;)-\u0026alpha;-pinene and 1 \u0026mu;g (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol], [1 \u0026mu;g (\u0026ndash;)-myrtenol, 5 \u0026mu;g (\u0026ndash;)-\u0026alpha;-pinene, 1 \u0026mu;g (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol and 1 \u0026mu;g (\u0026ndash;)-verbenone], [5 \u0026mu;g (\u0026ndash;)-\u0026alpha;-pinene and 1 \u0026mu;g\u003cem\u003e\u0026nbsp;\u003c/em\u003e(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol], and 5 \u0026mu;g (\u0026ndash;)-\u0026alpha;-pinene alone, compared to the solvent control. Females spent significantly more time in the arms containing the combination of (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol and the two female-produced compounds, (\u0026ndash;)-myrtenol and (\u0026ndash;)-\u0026alpha;-pinene, (mean time spent: 4.54 min\u0026plusmn;0.78) than in the arm containing only (\u0026ndash;)-myrtenol and (\u0026ndash;)-\u0026alpha;-pinene (mean time spent: 2.46\u0026plusmn;0.73). The addition of (\u0026ndash;)-verbenone to the three-component mixture, significantly reduced the time spent by both males and females (Figure 3). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eField Trapping\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe results of the laboratory olfactometry bioassays were reflected by the trap catches, as by far the most \u003cem\u003eP. aubei\u003c/em\u003e were caught in traps baited with the ternary combination of 200 mg (\u0026ndash;)-myrtenol, 1 g (\u0026ndash;)-\u0026alpha;-pinene and 200 mg (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol dissolved in 1.5 mL of pentane and 1 g of silicone oil. Traps baited with this mixture plus 200 mg (\u0026ndash;)-verbenone caught significantly fewer \u003cem\u003eP. aubei\u003c/em\u003e. The combination of 200 mg (\u0026ndash;)-myrtenol and 1 g (\u0026ndash;)-\u0026alpha;-pinene (the female produced pheromone) was used as a positive control according to Bozsik \u003cem\u003eet al\u003c/em\u003e. (2024). Both males and females were recorded in the traps baited with the synthetic compounds (Figure 4).\u003c/p\u003e\n\u003cp\u003eTraps with 4g of technical grade (\u0026ndash;)-\u0026alpha;-pinene caught significantly more \u003cem\u003eP. aubei\u003c/em\u003e than the control traps. Traps in assays marked with an asterisk in Figure 4 also contained 4g of technical grade (\u0026ndash;)-\u0026alpha;-pinene in separate dispensers. In these assays, the combination of (\u0026ndash;)-myrtenol, (\u0026ndash;)-\u0026alpha;-pinene, and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol attracted significantly more \u003cem\u003eP. aubei\u003c/em\u003e, than technical (\u0026ndash;)-\u0026alpha;-pinene alone.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTraps with (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol alone caught slightly but significantly more \u003cem\u003eP. aubei\u003c/em\u003e adults than the unbaited control traps. The addition of (\u0026ndash;)-verbenone significantly reduced its attractiveness (Figure 4).\u003c/p\u003e\n\u003cp\u003eThe numbers of \u003cem\u003eP. aubei\u003c/em\u003e captured in the traps are shown in the Table 4.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eA few individuals of a species closely related to \u003cem\u003eP. aubei\u003c/em\u003e, \u003cem\u003eP. thujae\u003c/em\u003e (Perris, 1855), were caught in some of the traps. One female was caught in a trap baited with (\u0026ndash;)-myrtenol and (\u0026ndash;)-\u0026alpha;-pinene, while one male was caught in a trap with the mixture of (\u0026ndash;)-myrtenol, (\u0026ndash;)-\u0026alpha;-pinene, and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol and one in a trap baited with (\u0026ndash;)-\u0026alpha;-pinene and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol. Most \u003cem\u003eP. thujae\u003c/em\u003e were recorded in the traps baited with the mixture of (\u0026ndash;)-myrtenol and (\u0026ndash;)-\u0026alpha;-pinene, (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol, and (\u0026ndash;)-verbenone. Their total number was 7 (5 females, 2 males) and their mean number was 1.75.\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, three electrophysiologically active components were detected in mid- and hindgut extracts of mated male cypress bark beetles \u003cem\u003eP. aubei\u003c/em\u003e by GC-EAD and their structures were determined by GC-MS as α-pinene, \u003cem\u003ecis\u003c/em\u003e-verbenol and verbenone, respectively. None of these compounds were found in unmated males. α-Pinene has been previously reported as a major component in the volatiles of arborvitae, \u003cem\u003eThuja occidentalis\u003c/em\u003e \u0026lsquo;Smaragd\u0026rsquo;, a common host tree of \u003cem\u003eP. aubei\u003c/em\u003e and as a component of the aggregation pheromone, produced by unmated \u003cem\u003eP. aubei\u003c/em\u003e females (Bozsik et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In this study, we also detected α-pinene in the mid- and hindgut extracts of mated \u003cem\u003eP. aubei\u003c/em\u003e females but at much lower levels than in unmated females.\u003c/p\u003e \u003cp\u003eIdentification of a pheromone of \u003cem\u003eP. aubei\u003c/em\u003e males, produced only after mating, provides a deeper insight into the chemical ecology of host and mate finding of this invasive bark beetle species, associated with scale-leaved conifers: The male-produced pheromone, it is emitted right after mating during the colonization period of the attacked tree, when the couple is in the nuptial chamber for some time (for details on the mating sequence, see Bozsik \u0026amp; Szőcs, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Bozsik at al., 2024). Newly approaching beetles therefore encounter a mixture of host volatiles and female- and male-produced pheromones.\u003c/p\u003e \u003cp\u003eTo mimic this complex of chemical signals in this phase of colonization, we developed different synthetic multicomponent mixtures. For this, we used (\u0026ndash;)-α-pinene, a key component of the host kairomone and a part of the pheromones of both females and males, female-specific (\u0026ndash;)-myrtenol, and (+)- and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol and (\u0026ndash;)-verbenone, representing male volatiles. Each of these compounds elicited antennal responses in both sexes. We compared their behavioral effects under laboratory and field conditions. The results gained in the laboratory with a four-arm olfactometer and in the field in trapping tests were in very good agreement. A ternary mixture of (\u0026ndash;)-α-pinene, (\u0026ndash;)-myrtenol and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol in a ratio of 5:1:1 attracted the highest number of \u003cem\u003eP. aubei\u003c/em\u003e to the traps. This mixture was also most attractive to the beetles in the olfactometer. Both sexes were attracted in both types of tests, however the sex ratio was somewhat biased towards females. In contrast, the addition of (\u0026ndash;)-verbenone to the ternary mixture greatly reduced its attractiveness in both the field and the laboratory tests. Single compounds and two-component blends were less attractive. The attractiveness enhancing effect of (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol while also influencing the sex ratio and the anti-aggregation effect of (\u0026ndash;)-verbenone indicate the importance of a male pheromone in the process of host colonization of \u003cem\u003eP. aubei\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eIn the field test a few adults of the closely related \u003cem\u003eP. thujae\u003c/em\u003e were also captured. This indicates similarities in chemical communication between these two co-occurring \u003cem\u003ePhloeosinus\u003c/em\u003e species. However, these sparse data do not allow any more precise conclusions to be drawn. Nevertheless, the communication channels must be specific, as we never found \u003cem\u003eP. thujae\u003c/em\u003e males in mating chambers of \u003cem\u003eP. aubei\u003c/em\u003e. Currently, \u003cem\u003eP. aubei\u003c/em\u003e is the economically most important pest, while \u003cem\u003eP. thujae\u003c/em\u003e is much less abundant. Should this dominance change in the future, elucidating the pheromonal system of \u003cem\u003eP. thujae\u003c/em\u003e would become more important.\u003c/p\u003e \u003cp\u003e \u003cem\u003ecis\u003c/em\u003e-Verbenol is known to be a component of the aggregation pheromones of \u003cem\u003eDendroctonus\u003c/em\u003e species. In this context, it is produced by female \u003cem\u003eD. ponderosae\u003c/em\u003e Hopkins, 1902 (Miller \u0026amp; Lafontaine, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1991\u003c/span\u003e), by female \u003cem\u003eD. frontalis\u003c/em\u003e Zimmermann, 1868 (Sullivan, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), and by both sexes of \u003cem\u003eD. valens\u003c/em\u003e LeConte, 1860 (Xu et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Biosynthetically, α-pinene is the precursor for \u003cem\u003ecis\u003c/em\u003e-verbenol (Renwick et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e1976\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the biosynthetic oxidation of verbenol provides verbenone, which is used by \u003cem\u003eD. frontalis\u003c/em\u003e males as an anti-aggregation pheromone (Byers, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e1983\u003c/span\u003e; Fr\u0026uuml;hbrodt et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Its effect correlates with its concentration: it has no inhibitory effect at low levels, but it does at high doses (Audley et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Byers et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). This phenomenon was also observed in our recent studies on \u003cem\u003eP. aubei\u003c/em\u003e and may be reflected in the finding that the verbenone production increases after the mating. In other bark beetles, e.g. \u003cem\u003eHylurgus ligniperda\u003c/em\u003e (Fabricius, 1787) or \u003cem\u003eHylastes attenuatus\u003c/em\u003e Erichson, 1836 it has dose-dependent synergistic effect with pheromonal or kairomonal attractants (Rom\u0026oacute;n et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Fr\u0026uuml;hbrodt et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e(\u0026ndash;)-α-Pinene, (\u0026ndash;)-verbenol and (\u0026ndash;)-verbenone not only share a common carbon skeleton but also the same configuration of the stereocenters. Further investigations of the absolute configuration of the natural products will show whether our assumption derived from the behavioral tests of a common configuration of the male-produced compounds is confirmed.\u003c/p\u003e \u003cp\u003eThe results of our research are an important step in establishing an effective method of alternative control for \u003cem\u003eP. aubei\u003c/em\u003e. A mixture of the female and male aggregation pheromone compounds, (\u0026ndash;)-myrtenol and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol, respectively, and female and host-produced (\u0026ndash;)-α-pinene in a ratio of 1:1:5 is the most effective bait to date and could enable mass trapping for the first time.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG\u0026aacute;bor Bozsik developed the concept, made the experimental design, conducted the laboratory and field experiments, evaluation of datasets and wrote the main text of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eArmin Tr\u0026ouml;ger carried out the mass spectrometry analyses to identify the aggregation pheromone compounds and wrote the corresponding sections of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStefan Schulz was the consultant for the spectral analyses and made the proofreading of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eG\u0026aacute;bor Szőcs supervised the project and wrote the main text of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll authors have reviewed the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to Mr. G. Izer, Director, Mr. Gy. Both and Mr. M. Bokor, Phytosanitary Advisors (Prenor Nursery Ltd, Szombathely, Hungary), for allowing us to conduct trapping assays and collect \u003cem\u003eP. aubei\u003c/em\u003e beetles, and to Mr. Balázs Kulin, Director (Tahi Tree Nursery Ltd, Tahi, Hungary), for allowing us to collect \u003cem\u003eP. aubei\u003c/em\u003e and tree samples, at the tree nursery.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflict of interest to declare.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eData supporting the findings of this study are available from the corresponding authors on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was financed by the National Research, Development and Innovation Office (NKFIH), Budapest, Hungary, project number PD 138089 to Gábor Bozsik.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAudley, J.P., Bostock, R.M. \u0026amp; Seybold, S.J. (2020) Trap assays of the walnut twig beetle, \u003cem\u003ePityophthorus juglandis\u003c/em\u003e Blackman (Coleoptera: Curculionidae: Scolytinae), reveal an effective semiochemical repellent combination. \u003cem\u003eJournal of Chemical Ecology\u003c/em\u003e, 46, 1047\u0026ndash;1058. https://doi.org/10.1007/s10886-020-01228-9 \u003c/li\u003e\n\u003cli\u003eBlomquist, G.J., Figueroa-Teran, R., Aw, M., Song, M., Gorzalski, A., Abbott, N.L., Chang, E. \u0026amp; Tittiger, C. (2010) Pheromone production in bark beetles. \u003cem\u003eInsect Biochemistry and Molecular Biology\u003c/em\u003e, 40, 699\u0026ndash;712. https://doi.org/10.1016/j.ibmb.2010.07.013 \u003c/li\u003e\n\u003cli\u003eBozsik, G., Tr\u0026ouml;ger, A., Francke, W. \u0026amp; Szőcs, G. (2016) \u003cem\u003eThuja occidentalis\u003c/em\u003e: identification of volatiles and electroantennographic response by the invasive cedar bark beetle, \u003cem\u003ePhloeosinus aubei\u003c/em\u003e. \u003cem\u003eJournal of Applied Entomology\u003c/em\u003e, 140, 434\u0026ndash;443. https://doi.org/10.1111/jen.12272\u003c/li\u003e\n\u003cli\u003eBozsik, G. \u0026amp; Szőcs, G. (2017) Phenology, behavior and infestation levels of the invasive small cypress bark beetle, \u003cem\u003ePhloeosinus aubei\u003c/em\u003e, on some cultivars of \u003cem\u003eThuja\u003c/em\u003e and \u003cem\u003eJuniper\u003c/em\u003e spp, in Hungary. \u003cem\u003ePhytoparasitica\u003c/em\u003e, 45, 201\u0026ndash;210. http://doi.org/10.1007/s12600-017-0585-y\u003c/li\u003e\n\u003cli\u003eBozsik, G., Moln\u0026aacute;r, B.P., Domingue, M.J. \u0026amp; Szőcs, G. (2023) Changes to volatile profiles of arborvitae, \u003cem\u003eThuja occidentalis\u003c/em\u003e, from drought and insect infestation: olfactory cues for the cypress bark beetle, \u003cem\u003ePhloeosinus aubei\u003c/em\u003e. \u003cem\u003eChemoecology\u003c/em\u003e, 33, 113\u0026ndash;124. https://doi.org/10.1007/s00049-023-00389-9\u003c/li\u003e\n\u003cli\u003eBozsik, G., Moln\u0026aacute;r, B.P., Heged\u0026uuml;s, K., So\u0026oacute;s, T., Schulz, S., Tr\u0026ouml;ger, A., Francke, W., \u0026amp; Szőcs, G. (2024) (\u0026minus;)-myrtenol and (\u0026minus;)-\u0026alpha;-pinene: Aggregation pheromone components of the cypress bark beetle \u003cem\u003ePhloeosinus aubei\u003c/em\u003e. \u003cem\u003eJournal of Applied Entomology\u003c/em\u003e, 148, 351\u0026ndash;363. https://doi.org/10.1111/jen.13231\u003c/li\u003e\n\u003cli\u003eByers, J.A. 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(Eds.) \u003cem\u003eInsect Pheromone Biochemistry and Molecular Biology (Second Edition)\u003c/em\u003e. New York, New York: Academic Press, pp. 123\u0026ndash;162. https://doi.org/10.1016/B978-0-12-819628-1.00004-3 ,\u003c/li\u003e\n\u003cli\u003eKerchev, I. A. \u0026amp; Pousheva, M.S. (2016) Olfactometric evidence for aggregation pheromone production by females of the four-eyed fir bark beetle \u003cem\u003ePolygraphus proximus\u003c/em\u003e Blandf. (Coleoptera, Curculionidae: Scolytinae). \u003cem\u003eEntomological Review\u003c/em\u003e, 96, 821\u0026ndash;825. https://doi.org/10.1134/S0013873816070010 \u003c/li\u003e\n\u003cli\u003eMiller, D.R. \u0026amp; Lafontaine, J.P. (1991) cis-Verbenol: An aggregation pheromone for the mountain pine beetle, \u003cem\u003eDendroctonus ponderosae\u003c/em\u003e Hopkins (Coleoptera: Scolytidae). \u003cem\u003eJournal of the Entomological Society of British Columbia\u003c/em\u003e, 88, 34\u0026ndash;38.\u003c/li\u003e\n\u003cli\u003eMwando, N.L., Tamiru, A., Nyasani, J.O., Obonyo, M.A.O., Caulfield, J.C., Bruce, T.J.A. \u0026amp; Subramanian, S. (2018) Maize chlorotic mottle virus induces changes in host plant volatiles that attract vector thrips species. \u003cem\u003eJournal of Chemical Ecology\u003c/em\u003e, 44, 681\u0026ndash;689. https://doi.org/10.1007/s10886-018-0973-x\u003c/li\u003e\n\u003cli\u003eOckenfels, P. (2015) Electroantennography a practical introduction. Buchenbach, Germany: Syntech GmbH. Rerieved from http://www.ockenfels-syntech.com/wp-content/uploads/EAGpract_man_fin [Accessed 5th November 2024]\u003c/li\u003e\n\u003cli\u003eOlsson, S.B. \u0026amp; Hansson, B.S. (2013) Electroantennogram and single sensillum recording in insect antennae. In: Touhara, K. (Ed.) \u003cem\u003ePheromone signalling. Methods in molecular biology (methods and protocols)\u003c/em\u003e. Totowa, New Jersey: Humana Press, pp. 157\u0026ndash;177. \u003c/li\u003e\n\u003cli\u003ePettersson, J. (1970) An aphid sex attractant. I. Biological studies. \u003cem\u003eEntomologica Scandinavica\u003c/em\u003e, 1, 63\u0026ndash;73.\u003c/li\u003e\n\u003cli\u003eRenwick, J.A., Hughes, P.R. \u0026amp; Krull, I.S. (1976) Selective production of cis- and trans-verbenol from (-)-and (+)-alpha by a bark beetle. \u003cem\u003eScience\u003c/em\u003e, 191, 199\u0026ndash;201. https://doi.org/10.1126/science.1246609 \u003c/li\u003e\n\u003cli\u003eRoelofs, W.L. (1977) The scope and limitations of the electroantennogram technique in identifying pheromone components. In: McFarlane, N.R. (Ed.) \u003cem\u003eCrop protection agents \u0026ndash; their biological evaluation\u003c/em\u003e. New York, New York: Academic Press, pp. 147\u0026ndash;165. \u003c/li\u003e\n\u003cli\u003eRoelofs, W.L. (1984) Electroantennogram assay: rapid and convenient screening procedures for pheromones. In: Hummel, H.E. \u0026amp; Miller, T.A. (Eds.) \u003cem\u003eTechniques in pheromone research\u003c/em\u003e.\u003cem\u003e \u003c/em\u003eNew York, New York: Springer Verlag, pp. 131\u0026ndash;160.\u003c/li\u003e\n\u003cli\u003eRom\u0026oacute;n, P., Iturrondobeitia, J.C., Gibson, K., Lindgren, B.S. \u0026amp; Goldarazena, A. (2007) Quantitative association of bark beetles with pitch canker fungus and effects of verbenone on their semiochemical communication in Monterey pine forests in Northern Spain. \u003cem\u003eEnvironmental entomology\u003c/em\u003e, 36, 743\u0026ndash;750. https://doi.org/10.1603/0046-225x(2007)36[743:qaobbw]2.0.co;2 \u003c/li\u003e\n\u003cli\u003eSullivan, B.T. (2005) Electrophysiological and behavioral responses of \u003cem\u003eDendroctonus frontalis\u003c/em\u003e (Coleoptera: Curculionidae) to volatiles isolated from conspecifics. \u003cem\u003eJournal of Economic Entomology\u003c/em\u003e, 98, 2067\u0026ndash;2078. https://doi.org/10.1093/jee/98.6.2067 \u003c/li\u003e\n\u003cli\u003eTittiger, C. \u0026amp; Blomquist, G.J. (2016) Pheromone Production in Pine Bark Beetles, In: Tittiger, C. \u0026amp; Blomquist G.J. (Eds.) \u003cem\u003eAdvances in Insect Physiology\u003c/em\u003e. New York, New York: Academic Press, pp. 235\u0026ndash;263. https://doi.org/10.1016/bs.aiip.2016.02.002 \u003c/li\u003e\n\u003cli\u003eVuts, J., Szanyi, Sz., Szanyi, K., K\u0026ouml;nig, L., Nagy, A., Imrei, Z., Birkett, M.A. \u0026amp; T\u0026oacute;th, M. (2021) Development of a phytochemical-based lure for the dried bean beetle \u003cem\u003eAcanthoscelides obtectus\u003c/em\u003e Say (Coleoptera: Chrysomelidae). \u003cem\u003eJournal of Chemical Ecology\u003c/em\u003e, 47, 987\u0026ndash;997. https://doi.org/10.1007/s10886-021-01305-7\u003c/li\u003e\n\u003cli\u003eXu, L., Lou, Q., Cheng, C., Lu. M. \u0026amp; Sun, J. (2015) Gut-associated bacteria of \u003cem\u003eDendroctonus valens \u003c/em\u003eand their involvement in verbenone production. \u003cem\u003eMicrobial Ecology\u003c/em\u003e, 70, 1012\u0026ndash;1023. https://doi.org/10.1007/s00248-015-0625-4 \u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 Arrangement of the four-arm olfactometer bioassays: Concentrations and combinations of the solutions.\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"604\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003cp\u003eAssay No.\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003earm1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003earm2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003earm3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003earm4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003col\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003econtrol\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003en\u003c/em\u003e-pentane\u003c/p\u003e\n \u003cp\u003e10 \u0026mu;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-myrtenol 1 \u0026mu;g\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene 5 \u0026mu;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-myrtenol 1 \u0026mu;g\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene 5 \u0026mu;g\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)\u003cem\u003e-cis\u003c/em\u003e-verbenol 1 \u0026mu;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol 1 \u0026mu;g\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003col start=\"2\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003econtrol\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003en\u003c/em\u003e-pentane\u003c/p\u003e\n \u003cp\u003e10 \u0026mu;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-myrtenol 1 \u0026mu;g\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene 5 \u0026mu;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-myrtenol 1 \u0026mu;g\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene 5 \u0026mu;g\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol 1 \u0026mu;g\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-verbenone 1 \u0026mu;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)\u003cem\u003e-cis\u003c/em\u003e-verbenol 1 \u0026mu;g\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-verbenone 1 \u0026mu;g\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 66px;\"\u003e\n \u003col start=\"3\"\u003e\n \u003cli\u003e\u0026nbsp;\u003c/li\u003e\n \u003c/ol\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 85px;\"\u003e\n \u003cp\u003econtrol\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(\u003cem\u003en\u003c/em\u003e-pentane\u003c/p\u003e\n \u003cp\u003e10 \u0026mu;L)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene 5 \u0026mu;g\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol 1 \u0026mu;g\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene 5 \u0026mu;g\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol 1 \u0026mu;g\u003c/p\u003e\n \u003cp\u003e(\u0026ndash;)-verbenone 1 \u0026mu;g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 151px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene 5 \u0026mu;g\u003c/p\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 2 Mixtures applied in the trapping trials.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"624\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 100%;\" colspan=\"8\"\u003e\u003cstrong\u003eLures/traps\u003c/strong\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eNo.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e1.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e2.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e3.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e5.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e6.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e7.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene\u0026nbsp;\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene\u0026nbsp;\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene\u0026nbsp;\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene\u0026nbsp;\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene techn.\u003cbr\u003e\u0026nbsp;4 g\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-myrtenol\u0026nbsp;\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-myrtenol\u0026nbsp;\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-myrtenol\u0026nbsp;\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)\u003cem\u003e-cis\u003c/em\u003e-verbenol\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e(\u0026ndash;)\u003cem\u003e-cis\u003c/em\u003e-verbenol\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e(\u0026ndash;)\u003cem\u003e-cis\u003c/em\u003e-verbenol\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-verbenone\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-verbenone\u003cbr\u003e\u0026nbsp;200 mg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003esilicone oil\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003esilicone oil\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003esilicone oil\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003esilicone oil\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003esilicone oil\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003esilicone oil\u003cbr\u003e\u0026nbsp;1 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003epentane\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003epentane\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003epentane\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003epentane\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003epentane\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003epentane\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e1.5 mL\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 90px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cem\u003eand\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eand\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003eand\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cem\u003eand\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cem\u003ein a\u003cbr\u003e\u0026nbsp; separate dispenser:\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003ein a\u0026nbsp;\u003cbr\u003e\u0026nbsp;separate dispenser:\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003ein a\u0026nbsp;\u003cbr\u003e\u0026nbsp;separate dispenser:\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 80px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 90px;\"\u003e\n \u003cp\u003e\u003cem\u003ein a\u0026nbsp;\u003cbr\u003e\u0026nbsp;separate dispenser:\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 35px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd style=\"width: 88px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene techn.\u003cbr\u003e\u0026nbsp;4 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene techn.\u003cbr\u003e\u0026nbsp;4 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 85px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene techn.\u003cbr\u003e\u0026nbsp;4 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 76px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 80px;\"\u003e\n \u003cp\u003e\u0026ndash;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 90px;\"\u003e\n \u003cp\u003e(\u0026ndash;)-\u0026alpha;-pinene techn.\u003cbr\u003e\u0026nbsp;4 g\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 3 Ratios and amounts of the identified aggregation pheromone compounds in unmated and mated female (A.) and male (B.) \u003cem\u003eP. aubei\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"618\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 170px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"bottom\" style=\"width: 180px;\"\u003e\n \u003cp\u003e\u003cstrong\u003efemale equivalent (ng/FE)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026alpha;-pinene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003emyrtenol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026alpha;-pinene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\n \u003cp\u003emyrtenol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eunmated female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.81\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003emated female\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\n \u003cp\u003e3.2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eB.\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eratio\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"bottom\" style=\"width: 255px;\"\u003e\n \u003cp\u003e\u003cstrong\u003emale equivalent (ng/ME)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u0026alpha;-pinene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003ecis\u003c/em\u003e-verbenol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003everbenone\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\n \u003cp\u003e\u0026alpha;-pinene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e\u003cem\u003ecis\u003c/em\u003e-verbenol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003everbenone\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003eunmated male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNO\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 107px;\"\u003e\n \u003cp\u003emated male\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e0.083\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.67\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 95px;\"\u003e\n \u003cp\u003e0.08\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 85px;\"\u003e\n \u003cp\u003e2.72\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003e1.02\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTable 4 Total and number of \u003cem\u003eP. aubei\u003c/em\u003e in traps. Significant differences between the experiment means were defined as P values less than 0.05. (For the detailed composition of mixtures/lures, see Table 2)\u003c/p\u003e\n\u003cp\u003e\u003cimg 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\" style=\"width: 1019px;\" width=\"1019\" height=\"545\"\u003e\u003cbr\u003e\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"Department of Chemical Ecology, Plant Protection Institute, Centre for Agricultural Research, HUN-REN, Brunszvik str. 2., Martonvásár, Hungary","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"male-produced pheromone, cypress bark beetle, GC-EAD, behavioral test, cis-verbenol, verbenone","lastPublishedDoi":"10.21203/rs.3.rs-6305155/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6305155/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e1. The need for monitoring the spread of the invasive cypress bark beetle, \u003cem\u003ePhloeosinus aubei\u003c/em\u003e, a pest of scale-leaved conifers across Europe is urging. The aggregation pheromone of females was reported earlier; however, an effective trapping method has not yet been developed.\u003c/p\u003e \u003cp\u003e2. Therefore, this study aimed to unravel the males\u0026rsquo; pheromonal communication system.\u003c/p\u003e \u003cp\u003e3. Volatiles produced by \u003cem\u003eP. aubei\u003c/em\u003e males, as well as sex- and mating status-specific extracts from mid- and hindguts of unmated and mated males and females were analyzed by GC-EAD and compounds eliciting antennal responses identified by GC-MS.\u003c/p\u003e \u003cp\u003e4. Two male-specific pheromone components, \u003cem\u003ecis\u003c/em\u003e-verbenol and verbenone, were identified in the extracts from mated males. α-Pinene, known to be a component of the host plant kairomone, as well as the female-produced pheromone, was also found in extracts of mated males. None of these components were found in unmated males.\u003c/p\u003e \u003cp\u003e5. Four-arm olfactometer tests showed that females were attracted to the combination of (\u0026ndash;)-myrtenol, (\u0026ndash;)-α-pinene and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol, rather than to either (\u0026ndash;)-myrtenol, or (\u0026ndash;)-α-pinene alone. When this ternary blend was combined with (\u0026ndash;)-verbenone, the attractiveness for both males and females was reduced.\u003c/p\u003e \u003cp\u003e6. Female and male \u003cem\u003eP. aubei\u003c/em\u003e spent significantly more time in the olfactometer arm containing (\u0026ndash;)-α-pinene and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol, than in the control arms.\u003c/p\u003e \u003cp\u003e7. The ternary mixture of (\u0026ndash;)-myrtenol, (\u0026ndash;)-α-pinene and (\u0026ndash;)-\u003cem\u003ecis\u003c/em\u003e-verbenol proved to be the most effective trap bait in the field, which could serve as a potent attractant for monitoring of \u003cem\u003eP. aubei\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"cis-Verbenol and verbenone as male-produced pheromone components of the cypress bark beetle, Phloeosinus aubei: Identification and behavioral tests in combination with the female-produced pheromone components","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 05:28:24","doi":"10.21203/rs.3.rs-6305155/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0c03e646-d6e6-4564-bc62-fcdf8a5ca407","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-21T23:30:41+00:00","versionOfRecord":{"articleIdentity":"rs-6305155","link":"https://doi.org/10.1111/afe.12695","journal":{"identity":"agricultural-and-forest-entomology","isVorOnly":true,"title":"Agricultural and Forest Entomology"},"publishedOn":"2025-06-20 00:00:00","publishedOnDateReadable":"June 20th, 2025"},"versionCreatedAt":"2025-03-26 05:28:24","video":"","vorDoi":"10.1111/afe.12695","vorDoiUrl":"https://doi.org/10.1111/afe.12695","workflowStages":[]},"version":"v1","identity":"rs-6305155","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6305155","identity":"rs-6305155","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}