2.5. Electroantennography (EAG) with Clerid Beetle Antennae

Electroantennography was used to test whether antennae of P. detritus of either sex responded to extracts of volatiles collected from male or female beetles. It was also used to test whether C. mutillarius males and females perceived the pheromone compounds identified in the headspace volatiles from P. detritus males. Clerus mutillarius beetles were sexed by dissection of the genitalia after the antennae had been removed. For EAG assays, an antenna from a live female (n = 6) or male (n = 7) adult C. mutillarius was excised at the base and mounted between two glass capillary electrodes containing 0.1 M KCl solution. A constant humidified airflow of ~0.7 L/min was directed over the antenna, placed ~3 mm from the exiting airflow from a Teflon™-coated steel stimulus delivery tube. One of the electrodes was grounded, and the other was connected to a high-impedance DC amplifier (IDAC-232; Ockenfels Syntech GmbH). Test compounds (100 ng) dissolved in 10 µL isopropyl alcohol were administered to a 5 × 5 mm piece of filter paper inside a Pasteur pipette. Stimuli were delivered by puffing 1 mL of air through the Pasteur pipette into the air stream flowing over the antenna. Isopropyl alcohol was used as the solvent control, while pure air was the blank control without any chemical stimulus. Response amplitudes were normalized against the means of responses to the standard (Z)-3-hexenol, which was tested before and after the test compounds.