2.10 Electrophysiological responses of male Ostrinia nubilalis
Antennal responses of male ECB adults to biologically-derived pheromone blend (BioPhe) were evaluated by electroantennography (EAG) using a commercially available electroantennographic system (Ockenfels Syntech GmbH, Buchenbach, Germany). The antenna of a virgin two-to-three-day old male adult was excised from the head using micro-scissors and attached between two stainless-steel wire electrodes with the assistance of electrically conductive gel (G008, Fiab, Italy). The base of the antenna was connected to the indifferent electrode, whilst the distal end was connected to the recording electrode after cutting off a few segments of the tip. The signal was amplified 10X by a Universal AC/DC pre-amplifier probe connected to the recording electrode, and the analog signal was amplified and detected with a data acquisition controller (IDAC-4, Ockenfels Syntech GmbH).
Test stimulus was the biologically-derived pheromone blend BioPhe containing 7.2% Z 11-14:OAc, according to GC-MS analysis. Commercially available Z 11-14:OAc, the major sex pheromone of theO. nubilalis Z-race, was used as a reference compound. The BioPhe blend, as well as commercially available pheromone compounds, were diluted in pentane. For each stimulus, a 10 µL aliquot of the solution was pipetted to a piece of filter paper (7 x 30 mm, Whatman no. 1), and the solvent was allowed to evaporate before being inserted into a Pasteur pipette for odor delivery. Control stimulus consisted of a clean Pasteur pipette with a filter paper impregnated with solvent (pentane). Stimuli were provided as 0.3 s air puffs into a continuous flow of filtered and humidified air. The airflow, at 25  cm3/s rate, tube diameter 1 cm, was generated by an air stimulus controller (CS-55, Syntech, The Netherlands).
The reference stimulus, consisting of a filter paper carrying 100 ng ofZ 11-14:OAc, was provided at regular intervals during each recording session. The EAG response to each reference stimulus was defined as 100%, and all responses to test stimuli between adjacent references were normalized in % relative to the references. At least 1 min for weak and moderate stimuli and 5 min for strong stimuli were allowed between successive stimulations to let the antenna recover. The BioPhe blend was tested at a 100 ng dose (containing ~7 ng Z 11-14:OAc), as well as at a 1 μg dose (i.e ., 70 ngZ 11-14:OAc), in order to reach an amount of major sex pheromone comparable to the 100 ng dose of the Z 11-14:OAc stimulus.
The minor sex pheromone, E 11-14:OAc, was also tested, as well as both isomers at a 97:3 Z:E ratio, based on the ratio released from the female moth gland as reported in the literature for ECB Z-race [7,21] . In addition, considering the high amount of tetradecyl acetate (14:OAc) in the BioPhe blend (~50%), the EAG response to 50 ng of this compound was also evaluated (for consistency reasons 50 ng is approx. the amount of 14:OAc contained in the 100 ng BioPhe test dose). All aforementioned stimuli were tested on a total of 20 antennal preparations.