Sources of insects
Beetles for collection of headspace volatiles were field-collected in Hungary and reared from naturally infested host material in Sweden. The sex of the beetles was determined by antennal morphology or the banding pattern on the pronotum (males have longer antennae; yellow bands are nearly transverse in females, but directed more towards the head in males; ZI, pers. obs.). In Hungary, beetles were hand-collected in an oak forest at Mátrafüred (Online Resource 1, Table S1) during 20 May to 10 June 2014, and 5 May to 12 June 2016. Beetles were collected on or around recently cut, sunlit piles of oak logs. The sexes were held separately in ventilated clear plastic boxes (56 × 28 × 28 cm) with freshly cut oak twigs, under outdoor temperature and light conditions.
In Sweden, beetles were collected as they emerged from wood samples gathered on three occasions in Ecopark Hornsö. In the spring of 2011 and 2012, freshly cut oak branches (not infested by insects) collected from various sites in the southern Swedish counties of Skåne, Blekinge, and Kalmar were placed in the Ecopark, and collected again the following autumn after oviposition by P. arcuatus. In February 2015, ~ 1.5 m3 of oak branches and small logs containing larvae were collected from a site in Hornsö that had been logged at the end of 2013, with beetles ovipositing on the substrates in the summer of 2014 (Online Resource 1, Table S1). In the laboratory, the substrates were held in a greenhouse, in transparent plastic boxes (56 × 39 × 42 cm) with mesh-covered ventilation holes. Beetles were collected twice daily as they emerged from the infested wood, and held at 8 °C (sexes separated) with pieces of freshly cut oak wood and paper tissues soaked in a honey water solution as a food source. The majority of beetles (and extracts of volatiles, see below) were obtained from the wood substrates collected in 2015.
Collection of headspace volatiles
Headspace volatiles were collected from adult beetles of both sexes (for detailed collection methods, see Online Resource 1, Table S1). In Hungary, volatiles were trapped on activated charcoal, using a closed loop stripping system with beetles held in glass jars, whereas in Sweden, volatiles were collected on traps of Porapak™ Q, using a flow-through system. Most adults of P. arcuatus were active during midmorning through early afternoon at naturally infested sites (pers. obs.), and so headspace volatiles were collected during those hours.
Analysis of headspace extracts
At the Budapest laboratory, headspace extracts were analyzed with a 6890N gas chromatograph (Agilent Technologies Inc., Palo Alto, CA, USA), equipped with an HP-5ms column (20 m × 0.32 mm × 0.25 μm film thickness; J&W Scientific, Folsom, CA, USA). Injections were made in split mode (injector temperature 220 °C), and the oven was programmed from 50 °C for 1 min, then increased at 5 °C/min rate to 230 °C, and held for 10 min. The carrier gas was helium (initial flow rate 4.0 ml/min, initial linear velocity 56 cm/s, constant pressure 112 kPa). Samples were also analyzed on an HP-5890 gas chromatograph (Hewlett-Packard, now Agilent) equipped with a DB-WAX column (30 m × 0.32 mm × 0.25 μm film thickness; J&W Scientific, Folsom CA, USA). Injections were made in split mode (injector temperature 215 °C), with an initial oven temperature of 31 °C for 1 min, then increased at 10 °C/min to 240 °C, and held for 10 min. The carrier gas was helium (2.0 ml/min). Beetle-produced compounds were recognized by comparing the chromatograms of headspace extracts from males, females, and system controls using Agilent ChemStation software (version A.10.02). Male-specific compounds were tentatively identified by comparing their retention times with those of authentic standards on nonpolar (HP-5 ms) and polar (DB-WAX) columns. Identifications were confirmed by further analyses at UC Riverside (see below).
At the Alnarp campus, headspace samples were analyzed with a 6890N gas chromatograph (Agilent Technologies) equipped with an HP-5ms capillary column (60 m × 0.25 mm ID × 0.25 μm film thickness; Agilent), interfaced to a 5975 mass selective detector (Agilent). Extracts were injected manually (2 μl) in splitless mode with helium carrier gas (constant flow rate of 1.8 ml/min, head pressure 172 kPa), injector temperature 225 °C, and a temperature program of 30 °C for 3 min, then 8 °C/min up to 260 °C with a 10-min hold. The mass spectrometer was set with a 7-min solvent delay and scanning range of 29–400 Da. Spectra were taken in electron impact ionization mode (EI) at 70 eV. The male-specific compounds were tentatively identified by matching the mass spectra of the beetle-produced compounds to mass spectra in commercially available NIST and Wiley mass spectral databases (when possible), or to those of previously known cerambycid pheromones. Identifications were confirmed by comparing mass spectra and retention times with those of authentic standards. A subset of samples was sent to UC Riverside for additional analysis (see below).
At UC Riverside, the identifications of compounds in extracts from both the Hungarian and Swedish populations were confirmed by reanalysis of extracts. First, samples were analyzed using an HP 6890 GC equipped with a medium polarity DB-17 column (30 m × 0.25 mm ID × 0.25 mm film thickness; J&W Scientific) interfaced to an HP 5973 mass selective detector (EI, 70 eV), with a solvent delay of 3 min, and a scan range of 40–400 Da. Injections of 1 μl were made in splitless mode (split vent open at 0.5 min), with a temperature program of 40 °C for 1 min, then 10 °C/min to 280 °C, hold for 10 min, using helium carrier gas (linear flow rate, 37 cm/s). Injections were initially made with an injector temp. of 250 °C and then repeated with a temp. of 125 °C to minimize isomerization of the thermally labile hydroxyketones. Compounds were identified by matches of mass spectra with database spectra, verified by matches of mass spectra and retention times with those of authentic standards.
To determine the absolute configurations of the insect-produced compounds, aliquots of extracts were analyzed with a GC equipped with a chiral stationary phase Cyclodex B column (30 m × 0.25 mm ID × 0.25 μm film thickness). Samples were injected in split mode (split ratio ~ 20:1), with a head pressure of 172 kPa, and a temperature program of 50 °C for 1 min, then 3 °C per min to 220 °C, hold for 10 min. The injector temperature was 100 °C to minimize isomerization of the hydroxyketones. Samples were injected alone, and then spiked with authentic standards to confirm the identifications of the hydroxyketone compounds. Finally, because (R)-3-hydroxyoctan-2-one and (S)-2-hydroxyoctan-3-one were not well separated on the Cyclodex B column, samples were run on a DB-WAX column (30 m × 0.25 mm × 0.25 μm film, J&W Scientific), in split mode with an injector temp. of 150 °C and a temperature program of 50 °C for 1 min, 3 °C per min to 250 °C. Under these conditions, 2-hydroxyoctan-3-one and 3-hydroxyoctan-2-one were separated to baseline.
To determine whether the eluting solvent affected the ratios of 3-hydroxyhexan-2-one (C6), 3-hydroxyoctan-2-one (C8), and 3-hydroxydecan-2-one (C10) recovered from the headspace samples from male P. arcuatus collected on activated charcoal, replicate collectors were eluted with hexane, diethyl ether, and dichloromethane, and the ratios were calculated from the relative GC peak areas of the three compounds on the DB-WAX column (Hungary). The relative ratios of the beetle-produced compounds found in the extracts from beetles in Sweden were calculated using the chromatograms obtained from the HP-5ms column at the Alnarp campus.
Sources of chemicals, preparation of pheromone lures, and trap types
Racemic 3-hydroxyoctan-2-one and 3-hydroxydecan-2-one were synthesized from 1-octyn-3-ol and 1-decyn-3-ol, respectively, using methods described previously (Imrei et al. 2012). Racemic 3-hydroxyhexan-2-one (CAS number 54123-75-0) was purchased from Bedoukian Research, Inc. (Danbury, CT, USA).
Pheromone release devices consisted of press-sealed polyethylene bags (50 × 75 mm, 50 μm wall thickness, #018161A, Fisher Scientific, Pittsburgh, PA, USA; or 65 × 55 mm, 40 μm wall thickness, Grippie® Light Nr-02, b.n.t. Scandinavia AB, Arlöv, Sweden), attached with a metal clip (Hungary), or metal wire (Sweden) to the traps (without puncturing the bags). Traps were baited with lures containing C6, C8, and C10 3-hydroxyalkan-2-ones individually, in binary blends, and in a ternary blend, diluted in isopropanol (Table 1). Proportions in the blends approximated the average quantity of each compound found in the aeration extracts eluted with diethyl ether and hexane (see “Results”). In Hungary, a cotton dental roll (Celluron®, Paul Hartmann AG, Heidenheim, Germany) was placed in the lures to minimize leakage. In all bioassays, lures were loaded with pheromone solutions immediately before being deployed. Pheromone solutions were prepared in advance and stored at − 18 °C (Hungary) or 8 °C (Sweden) until needed.
In Hungary, field tests were carried out with funnel traps consisting of a collection bucket surmounted with a transparent funnel, with a vertical plastic sheet mounted in the funnel to intercept flying insects (modified model VARb3; Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Hungary, www.csalomontraps.com; Imrei et al. 2002; Schmera et al. 2004). This trap has been shown to be effective in catching Plagionotus floralis (Pallas), a congener of P. arcuatus (Toshova et al. 2010; Imrei et al. 2014). The funnel was coated with Teflon™ (95% polytetrafluoroethylene-based spray; B’laster Corporation, Cleveland, OH, USA) to increase trapping efficiency (Graham and Poland 2012), and a Vaportape® insecticidal strip (Hercon Environmental Inc., Emigsville, PA, USA) was placed in the collection bucket. The pheromone dispenser was suspended from the vertical plastic sheet so that it hung in the middle of the funnel opening.
In Sweden, beetles were trapped with custom-built flight-intercept traps consisting of plastic cross-vane panels mounted on a funnel with a collecting jar below, and a top cover for rain protection (for details, see Molander and Larsson 2018). The panels and the inside of the funnel were coated with Fluon® (polytetrafluoroethylene dispersion, 60 w/w% in H2O, Sigma-Aldrich, St. Louis, MO, USA, diluted 1:1 with water). Polypropylene glycol (~ 0.25 L per trap) was added to the collection bucket as a preservative and killing agent.
Field bioassays of the synthesized pheromones were carried out in similar fashion in Hungary from 26 to 31 May 2016, and Sweden from 29 May to 26 July 2015 and 5 June to 31 July 2016. The experimental sites, setups, and other details are described in Online Resource 1, Table S2. Traps were deployed in blocks, with one treatment of each type per block. Treatments were assigned randomly to traps within a replicate, and treatments were re-randomized at each inspection to control for position effects.
In Hungary, traps were spaced ~ 2 m apart, along a 150-m-long and 40-m-wide pile of oak logs (Quercus petraea [M.] Liebl.), where adults of P. arcuatus were abundant. Traps were mounted at ground level on metal posts touching the ground. All captured insects were counted, sexed, and identified using the key of Kaszab (1971). Voucher specimens have been deposited at the Hungarian laboratory.
In Sweden, traps were hung from protruding tree branches (2015) or from reinforcing bar posts (2016) at a height of 1.5–2 m, with ~ 8 m (2015) or 10 m (2016) spacing between traps within replicates. Traps were emptied by filtering the trap contents through a tea filter, and the trapped insects were saved in numbered plastic bags. Beetles were identified using the key of Ehnström and Holmer (2007) and the numbers of P. arcuatus were recorded, with the sex ratio determined in a subset of samples. After counting, the beetles were preserved in 70% ethanol. Voucher specimens will be deposited in the Lund entomological collections (Biological Museum, Lund University, Sweden).
Differences among treatment means in numbers of adult beetles captured were tested with the nonparametric Friedman’s test (PROC FREQ, option CMH; SAS Institute 2011), with replicates defined by number of traps per treatment within transects and collection date. Replicates that contained no specimens in any treatment of the beetle species in question (e.g., due to bad weather) were dropped from analyses. The two years of trapping data from Sweden were pooled. Pairs of treatments were compared using the REGWQ test (controlling experiment-wise error rates; SAS Institute 2011) and were protected (i.e., assuming a significant overall Friedman’s test).