Factor four

Gland extracts and synthetic chemicals were analyzed on a Saturn 2200 GC/MS (Varian Walnut Creek, CA, USA). The ionization voltage was 70 eV (electron impact ionization) with mass scanning from m/z 30–650. The GC/MS was equipped with two different capillary columns: a non-polar 30 m × 0.25 mm ID × 0.5 µm VF5-MS capillary column (Factor four, Varian Inc., USA) and a polar 30 m × 0.25 mm ID × 0.5 µm VF23-MS capillary column (Factor four, Varian Inc.). For both columns, injection was splitless and the column oven was programmed from 80 °C (held for 1 min) to 240 °C at 10 °C/min⁻¹ and then held for 13 min. Compounds were identified by comparing retention times and mass spectra with those of synthetic compounds on the two different columns.

Coupled GC/EAD analysis of pheromone gland extracts were conducted on a Varian 3800 GC equipped with a flame ionization detector (FID), a splitless injector, a 30 m × 0.25 mm (ID) × 0.25 µm VF5-MS capillary column (Factor Four, Varian Inc.) and a Y splitter (Alltech, Deerfield, IL). The column oven temperature was programmed from 80 °C (held for 1 min) to 240 °C at 10 °C.min⁻¹. Helium was the carrier gas. The column effluent was split 1:1 between the FID and EAD apparatus. Antennal depolarization was detected using a high resistance EAD probe (Signal Interface Box, Type ID-02) and Intelligent Data Acquisition Controller (Type IDAC-02) (Syntech, Hilversum, The Netherlands). Antennae from 2–3 d-old males collected from Lincoln and Little River were excised at the base and attached to silver electrodes housed in saline-filled glass electrodes using a micromanipulator (Narishige, Tokyo, Japan). Up to five antennal preparations from each location were tested with different female extracts from the same location in GC/EAD analysis.