Experimental insects
Adults of P. aubei were collected during the end of their overwintering period (March) by taking twigs of Thuja occidentalis ‘Smaragd’ with overwintering tunnels, occupied by a hibernating adult beetle. The beetles were inactive when the collections were performed. Collections took place in tree nurseries in Central-Hungary (Tahi Tree Nursery, Tahi, Pest-county, GPS 47.7653704536209 N, 19.06798750162125 E) and in West-Hungary (Prenor Tree Nursery, Szombathely, Vas-county, GPS 47.26419949232103 N, 16.59957200288773 E). Each twig with a P. aubei adult inside was kept separately during transport to the Experimental Station of the Plant Protection Institute (Budapest, Hungary, GPS 47.54803661427715 N, 18.93472731113434 E) where the tunnels were carefully opened, and the beetles inside were sexed, without taking them out from the tunnel. Twigs containing male beetles were kept separately from those containing females in an open wall greenhouse under natural conditions in 4 L glass jars.
Observation Of Courtship Behavior
As courtship takes place right after the females have produced nuptial chambers in the trunks (Bozsik and Szőcs 2017), a piece of cut T. occidentalis ‘Smaragd’ trunk was offered to females before they leave their overwintering tunnels in spring:
The females were let freely to leave their hibernating tunnels in the twigs and find a piece of trunk (ca. 5–6 cm diameter, and 10 cm long) placed next to it inside a 4 L glass jar. Altogether 10 trunks with a single female were tested. When females completed their nuptial chamber, the trunk was placed under an Alpha STO-4T zoom stereo microscope (Elektro-Optika Kft., Érd, Hungary), focusing on the opening. A single male was placed on the surface of the trunk, and the behavior of the male and the appearance of the female were monitored. The courtship behavior was video recorded by an Euromex HD-ULTRA 6Mp HDMI + USB camera through the microscope and, if possible, also visually observed.
Preparations of mid- and hindgut extracts
For extraction, pre-conditioned females were used. Briefly for their maturation feeding fresh twigs with leaves of Thuja were placed into the jar of the beetles. At the beginning of their mating period (middle of April) a semi-dried ca. 20 cm long T. occidentalis ‘Smaragd’ trunk piece was added. To synchronize the preparation of nuptial chambers and the pheromone production of all females, they were removed from their first nuptial chambers when producing boring dust (middle of May), and were offered a new, semi-dried, intact (no tunnels) piece of trunk to make a new nuptial chamber. After three days, the female beetles were removed from their new chambers.
The JHIII topical treatment was prepared by dissolving 10 mg of synthetic JHIII (CAS: 24198-95-6, SC-252931, Santa Cruz Biotechnology, 10410 Finnell Street, Dallas, TX 75220, US) in 250 µL of acetone (analytical reagent grade, Reanal, Budapest, Hungary) to make a 40 µg/µL stock solution. To treat female beetles, 0.5 µL of the solution containing 20 µg JHIII were administrated by a bevel-tip Hamilton syringe to the ventral abdominal surface of females, as described by Shepherd et al. (2010). Beetles were incubated in a Petri-dish for one day under ambient conditions. Bark of T. occidentalis was placed into the Petri-dish to feed the beetles. After the incubation period, the mid- and hindguts of seven JHIII treated female beetles were excised by sharp forceps and combined, followed by extraction with 19 µL n-hexane (reagent plus grade, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). Extraction duration was 15–20 minutes on a ca. -20°C iron plate during preparation, followed by 5 minutes extraction at room temperature.
Mid- and hindgut extracts were also prepared from beetles of the control group, without juvenile hormone treatment. In this case preparations were made instantly after the beetles were removed from their second nuptial chambers, without incubation time. Fourteen female P. aubei mid- and hindguts were combined and extracted with 45 µL n-hexane.
In order to corroborate data, the experiments were repeated in the subsequent year. The gut extracts from JHIII treated and untreated female P. aubei were obtained at the same day: Mid- and hindguts of twenty-three untreated female P. aubei were combined and extracted with 40 µL n-hexane as control. Mid- and hindguts of sixteen JHIII treated females were combined and extracted with 30 µL n-hexane. All extracts were kept in a -40 ° C until GC-EAD tests.
Volatile collection from T. occidentalis ‘Smaragd’ leafy twigs for identification the enantiomeric ratio of α-pinene
For this purpose, headspace volatiles were collected from living T. occidenatlis ‘Smaragd’ leaves and twigs from a healthy and undamaged plant cultivated in a flowerpot under natural condition (ambient temperature was 24°C) in an open-wall greenhouse. A ca. 20 cm long tip of twig was isolated by chemically inert baking bag (Alufix GmbH, Wiener Neudorf, Austria). The compounds of the gas phase were adsorbed on a charcoal CLSA filter (1.5 mg load, Brechbühler AG, Schlieren, Switzerland) by pumping air through the bag with a flow of 2.6 L min− 1 for 2 h under closed loop conditions, using a DC12 rotary vane pump (Fürgut GmbH, Tannheim, Germany). Volatiles were eluted from the filter with three portions of 20 µL n-pentane (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). The extract (ca. 50 µL) was kept in a deep freezer at -40°C.
Analyses of gut 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 a DB-WAX column (30 m x 0.32 mm x 0.25 µm film thickness; J&W Scientific, Folsom, CA, USA), was connected to an electroantennographic detector (Ockenfels SYNTECH GmbH, Buchenbach, Germany). The inlet temperature was set to 220 °C. The oven temperature was held at 60°C for 1 min, then programmed at 10°C/min to 220°C and held for 20 min. The carrier gas was helium (flow: 4.0 mL/min), and helium was also used as the makeup gas. The injected amount of the extracts was 3 µL in splitless mode.
The set of extracts, prepared in the second year was analyzed on the same GC-EAD setup, however, using a HP-5 column (J&W, 30 m x 0.32 mm, 0.25 µm film thickness; Agilent Technologies Inc.). The temperature program was set to an initial temperature of 50 °C for 1 min with a heating rate of 10 °C/min to 230 °C. The final temperature was held for 10 min. Helium was used as the carrier gas (flow: 4.0 mL/min) and helium was also used as the makeup gas. The injected amount of the extracts was 3 µL in splitless mode. The control of the GC equipment and the data acquisition were performed by the Agilent ChemStation software (version Rev. A. 10.02).
Antennae-head preparations of P. aubei males were placed between glass capillary electrodes (1.17 mm i.d.), filled with Ringer solution (Beadle and Ephrussi 1936), and equipped with silver wires for the signal transmission to the pre-amplifier. MP15 micromanipulators established the connections. The EAD outlet was introduced to a glass tube through which moistened air delivered the compounds to the antenna. The signals were analyzed by an IDAC2 amplifier and SYNTECH Syntech GC-EAD 2014 v. 1.2.5. software (Ockenfels SYNTECH GmbH, Buchenbach, Germany).
Identification of antennally active compounds and determination of their absolute configurations by gas chromatography coupled to mass spectrometry (GC/MS)
The first series of extracts was analyzed in the Institute of Organic Chemistry, Hamburg University, Germany. For identification of the antennally active components in the extracts, a GC 7890A gas chromatograph, linked to a 5975C inert XL MSD mass spectrometer (Agilent Technologies – MSD ChemStation E.02.02.1431), ran at 70 eV, was used. The gas chromatograph was equipped with a fused silica capillary column (VF-WAXms, 60 m x 0.25 mm, 0.25 µm film thickness; Agilent). Samples were injected in splitless mode (30 s). The temperature program was set to 50°C for 3 min, then increased to 80°C at a rate of 3°C/min, then increased to 150°C with 5°C/min, and then finally to 250°C with 8°C/min. The carrier gas was Helium. The MS conditions were as follows: auxiliary heater was set to 280°C, the MS source to 230°C, and the MS quad to 150°C. The scanning mass range was set to m/z 33–433 with a scan speed of 3.58 scan/s. Compounds were identified by their mass spectra using the Wiley 9th Edition/NIST 2008 MS Library.
In order to corroborate the chemical identifications, the extracts of the second year were compared with synthetic reference compounds from the laboratory of PPI, Budapest, Hungary. Injections were performed in splitless mode into a GC 6890 gas chromatograph (Agilent Technologies Inc.) coupled to an Agilent 5973 mass selective detector, operating in scan mode. Helium was used as the carrier gas (flow rate: 1 mL/min in constant flow mode). The purge flow was 20 mL/min after 1 min. For GC separation, a HP-5ms UI (30 m x 0.32 mm x 0.25 µm, J&W, Agilent) column was used. The gas chromatograph was programmed to hold 40°C for 1 min, increased with 10 °C/min to 270 °C and held for 10 minutes. For MS detection, EI ionization was used with standard 70 eV energy. The auxiliary heater was set to 290°C, the MS source to 250 °C, and the MS quad to 150 °C. The scanning mass range was set to m/z 35–400 with a scan speed of 2 scan/s.
The enantiomeric ratios of the antennally active compounds from P. aubei females and T. occidentalis leaves were determined using a GC 7890A gas chromatograph, coupled to a 5975C inert XL MSD mass spectrometer (Agilent Technologies) operating in EI mode at 70 eV. The separation of the enantiomers of both α-pinene and myrtenol was achieved using a CP-Chirasil-Dex CB (25 m x 0.25 mm, 0.25 µm film) chiral capillary GC column (Varian, Lake Forest, USA). Helium was used as the carrier gas, and the oven was programmed from 50°C to 180°C at a rate of 2°C/min. The mass spectrometer was set to single ion monitoring (SIM) mode monitoring the following ions: m/z 79, 91, 93 and 107. A solution of the synthetic enantiomers was used as reference: (–)- and (+)-α-pinene and (–)- and (+)-myrtenol with concentrations of 30, 10, 30 and 10 ng/µL, respectively.
Synthetic samples of (–)-myrtenol, (–)- and (+)-α-pinene
Synthetic samples of (–)-myrtenol (purity: 95%), (–)- and (+)-α-pinene (purity: 99%) were purchased from Sigma Aldrich (Merck KGaA, Darmstadt, Germany).
Synthesis of (+)-Myrtenol
(+)-Myrtenol was synthesized in two steps, via the resolution of the diastereomeric ester of myrtenol (Research Centre for Natural Sciences, Budapest, Hungary).
Step 1: Synthesis of myrtenyl 2-methoxy-2-phenylacetate
A solution of (+)-enantioenriched myrtenol (7.3 g, 35 mmol, 1 equiv), synthesized from commercially available (R)-(+)-enantioriched α-pinene (40% ee) using the method of Dvornikova et al. (2007), and (R)-(–)-2-methoxy-2-phenylacetic acid (5.8 g, 35 mmol, 1 equiv) (Kozel et al. 2017) was cooled to 0–5°C in dichloromethane (100 mL, 0.35 M). Then N,N′-dicyclohexylcarbodiimide (DCC, 5.4 g, 35 mmol, 1 equiv) was added followed by catalytic 4-dimethylaminopyridine (DMAP) (43 mg, 0.35 mmol, 0.01 equiv). The solution was stirred in an ice bath for 30 min and for further 3h at room temperature. The precipitated dicyclohexylurea was filtered off with neutral alumina and was washed twice with 30 mL dichloromethane. The combined filtrates were concentrated under reduced pressure. The residue (12 g) was purified by flash column chromatography on silica gel (0% ethyl acetate in hexanes increasing to 5% ethyl acetate in hexanes) to separate the diastereomers of the ester. The dominant isomer (–)-(1S,5R)-myrtenyl (R)-2-methoxy-2-phenylacetate (4.0 g, 13.3 mmol, 38%) was obtained as an oil (Fig. 1).
\({\left[{\alpha }\right]}_{D}^{23}\) = -5.6 (c = 8.0; CHCl3)
1H NMR (500 MHz, CDCl3) δ 7.44 (dd, J = 7.9, 1.3 Hz, 2H), 7.39–7.29 (m, 3H), 5.50–5.44 (m, 1H), 4.76 (s, 1H), 4.51 (d, J = 1.4 Hz, 2H), 3.42 (s, 3H), 2.32–2.14 (m, 3H), 2.07–2.02 (m, 1H), 1.94 (td, J = 5.7, 1.2 Hz, 1H), 1.21 (s, 3H), 1.06 (d, J = 8.7 Hz, 1H), 0.71 (s, 3H).
13C NMR (126 MHz, CDCl3) δ 170.7, 142.8, 136.6, 128.8, 128.7, 127.3, 122.3, 82.9, 77.2, 67.7, 57.6, 43.6, 40.8, 38.1, 31.6, 31.4, 26.2, 21.1.
HRMS (ESI): calcd. for [C19H24NaO3]+ 323.1618 found 323.1620.
TLC: Rf = 0.62 (12% ethyl acetate in hexanes).
Step 2: Synthesis of (+)-myrtenol
Dried potassium carbonate (19.3 g, 140 mmol, 7 equiv) was added to a solution of (–)-(1S,5R)-myrtenyl (R)-2-methoxy-2-phenylacetate (6.0 g, 20 mmol, 1 equiv) in methanol (300 mL, 0.06 M) was added. The suspension was stirred at room temperature for 3h until analysis by thin layer chromatography indicated full conversion. The reaction mixture was concentrated under reduced pressure. Then, diethyl ether (100 mL) was added and the suspension was filtered through a Celite pad. The filtered salts were washed with additional 100 mL diethyl ether and the combined filtrates were concentrated under reduced pressure. The residue (4.0 g) was purified by flash column chromatography on silica gel (0% ethyl acetate in hexanes increasing to 15% ethyl acetate in hexanes) to yield (+)-(1S,5R)-myrtenol (2.7 g, 18 mmol, 90%) as an oil (Fig. 1).
\({\left[{\alpha }\right]}_{D}^{23}\) = +57.3 (c = 3.22; CHCl3)
1H NMR (500 MHz, CDCl3) δ 5.48 (s, 1H), 3.99 (s, 2H), 2.41 (dt, J = 8.6, 5.6 Hz, 1H), 2.28 (q, J = 17.8 Hz, 2H), 2.14 (dd, J = 14.2, 8.6 Hz, 2H), 1.30 (s, 3H), 1.18 (d, J = 8.7 Hz, 1H), 0.84 (s, 3H).
TLC: Rf = 0.3 (12% ethyl acetate in hexanes).
Electroantennographic bioassays (EAG)
The electroantennography set-up and antennae-head preparation technique were the same as described above for GC-EAD. Stimuli were delivered from flushing ca. 1 mL air through a Pasteur pipette with a 1 x 1 cm piece of filter paper inside to which 10 µL n-pentane solution of the synthetic compounds had been applied. The testing method followed the original concept of Roelofs (1977, 1984), also recommended by SYNTECH (Ockenfels SYNTECH GmbH, Buchenbach, Germany) (Ockenfels 2015) and established by Olsson and Hansson (2013). The flush was generated by a SYNTECH stimulus controller (CS-55 v2.8, Ockenfels SYNTECH GmbH, Buchenbach, Germany). A humidified airstream (flow velocity 50 cm/s) delivered the compounds in the air flush to the antenna. Synthetic samples were dissolved in n-pentane in four concentrations: 10 ng/µL, 100 ng/µL, 1 µg/µL, 10 µg/µL.
Stimulations were implemented in series, starting with a blank stimulus (clean air), followed by n-pentane (10 µL) as the solvent control, then by beetle specific (–)-myrtenol (10 µg dose), which was used as the standard stimulus. This sequence was followed by the four doses of the synthetics, in randomized order. At the end of the series, the three stimuli, blank, solvent control, and standard were tested again. Stimulations followed each other in ca. 40 s intervals. Six female and six male antennae were tested. To avoid pseudo-replicates, antennae of an individual were tested only once.
Behavioral studies
Laboratory bioassays with a four-arm arena olfactometer
The experiments were carried out during the mating season of P. aubei (middle of April and May), in the early afternoon when mating occurs in the field, as described by Bozsik and Szőcs (2017).
A four-arm arena olfactometer was used to investigate the behavioral effects of the identified components. The method was based on the concept of Petterson (1970), described also by other authors (Karunaratne et al. 2008; Kerchev and Pousheva 2016; Mwando et al. 2018; Vuts et al. 2021), and adapted to our study with modifications as follows.
P. aubei adults were tested in a 30 x 30 x 2.5 cm size four-arm arena (Sigma Scientific LLC, Micanopy, FL, United States). The main body of the arena as well as all inlet and outlet ports were made from solid King Star Board (no phthalates), with a clear plexiglass removable lid. The system included an insect inlet adapter and an all-glass, two-piece combination of tube adapter and insect-isolation traps that collected insects responding to the odor source.
A continuous air flow was ensured by a Clean Air Delivery System (CADS, Sigma Scientific LLC) connected to a Stanley DST100/8/6 silent compressor. The CADS was equipped with a four push and one pull vacuum system, while the incoming and outgoing air was filtered through activated carbon filters (ICAF 2.5 x 8, Sigma Scientific LLC). The CADS and the arms of the olfactometer were connected by Teflon tubes.
The experimental settings of the system were the following: the compressor pressure was set to 30 psi, the olfactometer pressure was 16 psi, the source air pressure was 20 psi. The four flowmeters that regulate the air flow to the four arms were set to 1.43 L/min. The venturi pressure for the outgoing air was set to 20 psi, the olfactometer vacuum was − 2.45 psi. The flowmeter of the outgoing air was set to 4.6 L/min.
Female and male P. aubei adults were tested separately and one-by-one in the different assays shown in Table 1. Forty beetles per sex were tested in each test, thus altogether n = 120 beetles/sex were tested.
Table 1
4-Arm olfactometer assays to test the attractiveness of α-pinene and myrtenol for P. aubei.
Assay No.
|
arm1
|
arm2
|
arm3
|
arm4
|
1)
|
control
(n-hexane)
|
(+)-α-pinene 2 µg;
(–)-α-pinene 2 µg
|
(–)-myrtenol 1 µg
|
(–)-myrtenol 1 µg;
(+)-α-pinene 2 µg;
(–)-α-pinene 2 µg
|
2)
|
control
(n-hexane)
|
(+)-α-pinene 2 µg;
(–)-α-pinene 2 µg
|
(+)-myrtenol 1 µg
|
(+)-myrtenol 1 µg;
(+)-α-pinene 2 µg;
(–)-α-pinene 2 µg
|
3)
|
control
(n-hexane)
|
(+)-α-pinene 2 µg;
(–)-α-pinene 2 µg
|
(+)-myrtenol
0.5 µg;
(–)-myrtenol
0.5 µg
|
(+)-myrtenol 0.5 µg;
(–)-myrtenol 0.5 µg;
(+)-α-pinene 2 µg;
(–)-α-pinene 2 µg
|
α-Pinene was tested as the racemate, since we could show that the host plant produces both enantiomers (see results and discussion, and also Bozsik et al. 2016; 2022; Ložienė and Labokas 2012; Wang et al. 2022). In contrast, we found myrtenol only in extracts of the beetles. Therefore we tested the pure enantiomers and the racemate.
The stimuli components were dissolved in n-hexane. Ten µL of the test solution and of hexane as the control were applied directly before the test to previously purified filter papers. Then the papers were placed inside of the different glass arms. The combinations of test stimuli used to equip the four arms of the olfactometer, and the applied quantities are shown in Table 1 (see also Fig. 5).
Prior to each assay, a cleaning procedure was performed. The glass parts of the olfactometer system were washed with distilled water and acetone, and then kept overnight at 160°C. The body of the olfactometer was washed with 96% ethanol, and then dried as room temperature, as suggested by the producer.
Prior to each assay, the respective unmated beetles were kept under laboratory conditions for 2 hours, in glass jars in the laboratory of the olfactometer.
Then the beetles were tested one by one. Each beetle was placed in the center of the arena, its moving activity was checked during 12 min and the time spent in the chosen arms was recorded visually. Beetles which did not choose any arm within 12 min were excluded. To avoid possible positioning effects, the body of the olfactometer was randomly rotated by 90° between the tests. A circular lamp placed 50 cm above the arena ensured a consistent light intensity (1200 lux).
Field trapping tests
Based on the GC-EAD experiments, the female produced compounds (–)-α-pinene and (–)-myrtenol were selected for field studies. Field trapping tests were carried out in a T. occidentalis ‘Smaragd’ plantation, in Szombathely (Prenor Tree Nursery, Vas-county, Hungary, GPS 47.26419949232103 N, 16.59957200288773 E). The area of the planation was ca. 0.2 ha, where the traps were placed along the edge of the field with a distance of ca. 5–9 meters from each other.
For trapping, four-unit, black Lindgren funnel traps (Contech Enterprises Inc., 19 Dallas Rd Unit 115, Victoria, BC V8V 5A6, Canada) were used. Traps were spaced randomly in the rows of ca. 10-year-old, 3 m tall T. occidentalis ‘Smaragd’ trees, at the height of 180 cm and at ca. 60–70 cm distance from the trees. The traps were baited with a blend of 250 µL (–)-α-pinene and 50 µL (–)-myrtenol, dissolved in 1 mL mineral oil (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), mimicking the ratio found in the extracts of females. A 2 cm piece of dental cotton wool (Celluron®, Paul Hartmann AG, Heidenheim, Germany) inserted into a polyethylene bag (wall thickness: 0.02 mm) was used as a dispenser. Lures were replaced every second week. Three LFT traps were equipped with the baits, while two unbaited LFT traps were used as the control. Traps were emptied and trap catches were recorded once per week. The experiment was conducted from the beginning of the mating season till the date when flying P. aubei adults still occurred (from middle of April till middle of June). Seven recordings were made during the trapping period. The containers of the traps were filled with 20% propylene glycol (Reanal, Budapest, Hungary) in water to preserve the captured the insects.
Statistical Analyses
Analyses of ratios of α-pinene and myrtenol in extracts of JHIII treated and untreated females
To compare the α-pinene : myrtenol ratios of the gut extracts of JHIII treated and untreated females, a two-tailed z-score test was used at 0.05 significance level (Social Science Statistics 2022, https://www.socscistatistics.com/tests/ztest/default2.aspx).
Evaluation of electroantennographic responses
The intensities (mV) of the EAG responses given by the P. aubei antennae were normalized to the responses to the standard, 10 µg of (–)-myrtenol. Log10 transferred datasets were used for statistical analyses. For evaluation of homogeneity of variance, the Levene-test was used comparing the data pairwise, solvent control versus each tested compound (Social Science Statistics 2022, https://www.socscistatistics.com/tests/levene/default.aspx). The criteria of homogeneity were met in most cases. A t-test with a log10 transferred dataset was used to establish which compound elicited a significantly higher antennal response with respect to the solvent control (MS Excel). To compare all responses to each other, means of log10 transferred values, analyses of variance (ANOVA) followed by Tukey’s HSD tests were used (The jamovi project, 2021, jamovi, Version 2.2, MAC OS, retrieved from https://www.jamovi.org). Alpha was set to 5% in both cases.
Statistical evaluation of the results of four-arm arena olfactometry
For the evaluation of differences between the assays, non-parametric mean time-spent datasets, measured during the experiments, were analyzed by Kruskall-Wallis ANOVA (The jamovi project, 2021, jamovi, Version 2.2, MAC OS, retrieved from https://www.jamovi.org). Alpha was set to 5%.
Evaluation of trapping data
To compare the number of catches of baited traps to those of unbaited ones, a two tailed Welch’s t-test was used (unequal sample size). Alpha was set to 5% (Statology 2021, https://www.statology.org/welchs-t-test-calculator/).