Electrophysiology

Antennal responses of male and female P. sanguineum to the headspace samples from both sexes were recorded using coupled gas chromatography-electroantennographic detection (GC-EAD). Two μl of the aeration samples were injected into a GC (model 7890A, Agilent Technologies, Palo Alto, CA, USA) in splitless mode with an injector temperature of 225 °C (split vent opened after 0.5 min). The GC was fitted with a DB-WAX capillary column (30 m × 0.25 mm inner ø, d.f. 0.25 μm; J&W Scientific, Folsom, CA, USA), and eluting compounds were detected with a flame ionization detector (FID). Carrier gas was hydrogen at a constant flow rate of 2.1 ml/min. The GC oven was programmed from 30 °C, with a 3 min hold, and then increased at 20 °C/min to 225 °C, and was held for 10 min. The column effluent was split with a 3D/2 low dead volume four-way-cross (Gerstel, Mülheim, Germany), 27.6 kPa of nitrogen was added through the extra arm, and the resulting effluent was split 1:1 between the FID and the EAD. The GC effluent capillary for the EAD passed through a transfer line (ODP-3, Gerstel), which tracked the GC oven temperature, into a glass tube (30 cm length × 0.8 cm inner ø), where it was diluted with a charcoal-filtered, humidified airstream (1.0 L/min).

Antennae were excised using microscissors at the first antennal segment near the head of the beetle and mounted with electrode gel (article 1330, CefarCompex, Malmö, Sweden) onto a forked electroantennographic multi-probe (Syntech, Kirchzarten, Germany). The complete beetle antenna was positioned 0.5 cm from the outlet of the effluent tube. Signals from the antenna and the GC-FID were recorded simultaneously with a Syntech IDAC-2 digital converter and Syntech GCEAD 3.1 software. Male-produced extracts were run with antennae from 7 males and 10 females, and female-produced extracts were run with antennae from 7 males and 6 females. All extracts of volatiles from males and females of P. sanguineum were analyzed with GC-EAD, using at least one antenna for each extract.