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.