(d) Odour stimulation

For stimulation in calcium imaging and intracellular recordings, an artificial behaviourally active pheromone blend containing (Z)-7-dodecen-1-yl acetate (Z7–12:OAc), (Z)-9-tetradecen-1-yl acetate (Z9–14:OAc) and (Z)-11-hexadecen-1-yl acetate (Z11–16:OAc) at a ratio of 4 : 1 : 4 was used [19–21] in order to compare physiological results with the previously acquired behavioural data [17]. For single-sensillum recordings, only the major pheromone component, Z7–12:OAc, was used, because ORNs are tuned to individual pheromone compounds. Responses at the peripheral and AL level are nevertheless comparable, because the vast majority of AL neurons respond to the major pheromone component [22]. Whereas ORNs are selectively only responding to a single compound, certain AL neurons also respond to other compounds in addition [23]. Responses to the major compound are generally very similar to responses to the pheromone blend, and only very few AL neurons are blend-specific in A. ipsilon [24]. For all experiments, pheromone stimuli were diluted in decadic steps in hexane and applied on a filter paper introduced in a Pasteur pipette. For single sensillum recordings, doses from 0.1 ng to 1 µg of Z7–12:Ac were used (lower doses did not elicit any responses). For calcium imaging experiments, the pheromone blend was tested at doses from 0.01 to 10 ng to limit the number of stimuli, critical for these experiments. Preliminary experiments have shown that higher stimulus doses did not further increase responses. For intracellular recordings, the pheromone blend was used at doses from 0.01 pg to 100 ng. A minimum evaporation time of 30 min was respected. To avoid mechanical stimulation at the odour onset, the antennae were constantly superfused by a humidified and charcoal-filtered air stream (70 l h⁻¹). An air pulse of 200 ms for peripheral and intracellular recordings and 1 s for imaging experiments (10 l h⁻¹) passing through a Pasteur pipette containing the stimulus on a filter paper was introduced into the constant air stream by means of a stimulation device (CS55 Syntech, Kirchzarten, Germany).