Differential interactions of sex pheromone and plant odour in the olfactory pathway of a male moth.

Insects Adult males and females of the noctuid moth, A. ipsilon Hufnagel, were reared in the laboratory, and behavioural and physiological experiments were performed as described previously [21] , [25] . Briefly, 5-day old sexually mature virgin and mated males were used for experiments during the 8-hour scotophase. Newly-mated males were obtained by pairing virgin 5-day-old males and 3-day-old sexually mature females before the onset of the scotophase. Newly–mated males were prepared for calcium imaging or electrophysiological recordings within one to two hours after the end of copulation, and females were dissected to confirm the presence of the male spermatophore.

Odour Stimulation

For electrophysiological experiments, odour stimulations with sex pheromone blend and heptanal were performed as described previously [21] , [40] . Briefly, the behaviourally active pheromone blend consisting of ( Z )7-dodecen-1-yl acetate, ( Z )9-tetradecen-1-yl acetate and ( Z ) 11-hexadecen-1-yl acetate at a ratio 4∶1∶4 [41] , and the behaviourally attractive plant odour, heptanal [27] , were used in all experiments. Ten ng of the pheromone blend diluted in hexane were used in ORN recordings and imaging experiments because they elicit a clear response [25] . For AL intracellular recordings 1 ng and 10 ng doses of the pheromone were used for virgin and mated males respectively, because of a higher sensitivity of central neurons in virgin males [21] . Four doses of heptanal (1, 10, 100 and 1000 µg each diluted in 10 µl of mineral oil, resulting in concentrations of 1/10.000 to 1/10 volume/volume) were used for electrophysiological recordings and imaging experiments. All compounds were purchased from Sigma Aldrich (Saint-Quentin Fallavier, France) and 10 µl of stimulus solution were applied on a piece of filter paper (0.5×2 cm, Fisherbrand, Fisher Bioblock, Illkirch, France) introduced in a Pasteur pipette. The solvents hexane and mineral oil applied on a filter paper were used as control stimuli. When stimulating with mixtures, two filter papers were inserted into a glass pipette; then the pheromone (diluted in hexane) and heptanal (diluted in mineral oil) were added separately on the filter papers. This procedure avoided interactions between the two odour solutions, but allowed simultaneous application of the two stimuli in the same air puff. To exclude potential absorption of the pheromone in mineral oil or changes in the airflow due to a second filter paper in the pipette, we carried out control experiments under exactly the same experimental conditions as in the main experiments, in which we stimulated Phe-ORNs with: 1- a single filter paper with pheromone (phe), 2- one filter paper with pheromone and a second clean filter paper (phe+cfp), 3- one filter paper with pheromone and a second filter paper with mineral oil (phe+oil), 4- a single filter paper with mineral oil (oil). Phe-ORN spike response frequencies did not change significantly when a second clean filter paper or a filter paper with mineral oil was inserted in the pipette with respect to the pheromone alone (Tukey test p>0.05), but as expected, responses were significantly lower when only mineral oil was used (Tukey test, oil vs. phe, phe+cfp, phe+oil, p