Electrophysiological analysis: GC-EAD
This protocol is extracted from research article:
An ancient push-pull pollination mechanism in cycads
Sci Adv, Jun 12, 2020; DOI: 10.1126/sciadv.aay6169

The physiological response of R. furfuracea weevils to host plant Z. furfuracea volatile compounds was determined using GC-EAD. The coupled GC-EAD system used was as previously described by Crook et al. (36), with a few modifications. Samples of aerations or standards were injected (2 μl), splitless, onto a Hewlett Packard (Agilent Technologies, Santa Clara, CA) 6890 GC with a DB-5MS-DG column (J&W Scientific Inc., Folsom, CA; 0 m by 0.25 mm ID, 0.25-μm film thickness) and a 1:1 effluent splitter that allowed simultaneous flame ionization detector (FID) and EAD detection of the separated volatile compounds. Helium was the carrier gas (2.5 ml/min). Oven temperature was held at 50°C for 1 min, programmed to 280°C at 10°C/min, and held for 15 min. Injector temperature was 280°C. The GC outlets for the EAD and FID were 300°C. The column outlet for the EAD was held in a water-cooled humidified air stream (20°C) flowing at 20 ml/min over the antennal preparation of adult R. furfuracea attached to an EAG probe (Syntech, Hilversum, the Netherlands). Whole heads of an adult beetle were removed so that both antennae could be used for recording. Tips of both antennae were cut off to make a clean opening for conducting gel (Spectra 360, Parker Laboratories, Fairfield, NJ) to form an uninterrupted connection to the EAG probe. The EAG probe was connected to an IDAC-232 serial data acquisition controller (Syntech). Signals were stored and analyzed on a PC equipped with the program EAD (version 2.6, Syntech). GC-EADs using whole Z. furfuracea plant volatile collections were performed on six males and four females, and GC-EADs of the 1,3-octadiene standard were performed on three males and three females.

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