Specific mate communication and recognition underlies reproduction and hence speciation. Mate communication evolves during adaptation to ecological niches and makes use of social signals and habitat cues. Our study provides new insights in Drosophila melanogaster premating olfactory communication, showing that female pheromone (Z)-4-undecenal (Z4-11Al) and male pheromone cVA interact with food odour in a sex-specific manner. Furthermore, Z4-11Al, which mediates upwind flight attraction in both sexes, also elicits courtship in experienced males. Twin variants of the olfactory receptor Or69a are co-expressed in the same olfactory sensory neurons, and feed into the same glomerulus in the antennal lobe. Z4-11Al is perceived via Or69aB, while the food odorant (R)-linalool is a main ligand for the other variant, Or69aA. That Z4-11Al mediates courtship in experienced males, not (R)-linalool, is probably due to courtship learning. Behavioural discrimination is reflected by calcium imaging of the antennal lobe, showing distinct glomerular activation patterns by these two compounds. Male sex pheromone cVA is known to affect male and female courtship at close range, but does not elicit upwind flight attraction as a single compound, in to contrast to Z4-11Al. A blend of cVA and the food odour vinegar attracted females, while a blend of female pheromone Z4-11Al and vinegar attracted males instead. Sex-specific upwind flight attraction to blends of food volatiles and male and female pheromone, respectively, adds a new element to Drosophila olfactory premating communication and is an unambiguous paradigm for identifying the behaviourally active components, towards a more complete concept of food-pheromone odour objects.

Wind tunnel data

Upwind flight attraction was observed in a glass wind tunnel (30x30x100 cm). The wind tunnel was lit diffusely from above, at 13 lux, temperature ranged from 20°C to 24°C, relative humidity from 38% to 48% and charcoal filtered air (Camfil AB, Malmö, Sweden), at a velocity of 0.25 m/s, was produced by a fan (Fischbach GmbH, Neunkirchen, Germany). Yeast headspace and odour blends were delivered from the centre of the upwind end of the wind tunnel via a piezo-electric microsprayer (Becher et al. 2010). 
Forty flies were flown individually to each treatment. Flies were scored when flying upwind over 80 cm in the wind tunnel centre, from the release cage towards the odour source. Unmated, fed, 3-d-old males and females were flown towards yeast and vinegar headspace alone and in blends with Z4-11Al sprayed at 10 ng/min (Lebreton et al. 2017) and 300 ng/min cis-vaccenyl acetate (cVA) (Lebreton et al. 2015). Isomeric purity of the synthetic compounds, according to gas chromatography, was 98.6%. and 97.3%, respectively.
Lebreton S, Trona S, Borrero-Echeverry F, Bilz F, Grabe V, Becher PG, Carlsson MA, Nässel DR, Hansson BS, Sachse S, Witzgall P. 2015. Feeding regulates sex pheromone attraction and courtship in Drosophila females. Sci Rep 5:13132 (doi:10.1038/srep13132)
Lebreton, S., Borrero-Echeverry, F., Gonzalez, F., Solum, M., Wallin, E.A., Hedenström, E., Hansson, B.S., Gustavsson, A.-L., Bengtsson, M., Birgersson, G., et al. 2017 A Drosophila female pheromone elicits species-specific long-range attraction via an olfactory channel with dual specificity for sex and food. BMC Biol. 15, 88. (doi:10.1186/s12915-017-0427-x).

Courtship data

Courtship experiments were done on a light box (37x28x2.5 cm; color temperature 5.000 ± 5% Kelvin; Kaiser Slimlite 2422, Kaiser Fototechnik GmbH & Co. KG, Buchen, Germany). The courtship arena consisted of three glass plates (17x13x0.5 cm) placed on top of each other. Twelve circular holes (ø 3 cm) were cut in the middle plate to form 12 circular single-pair mating chambers. All glassware was heated to 350°C for 8 h before use. Tests were done between 2 and 5 h after onset of scotophase (16:8 L:D photoperiod). Target flies, unmated males or females, were anesthetized on ice and decapitated before experiments. Test flies were either mated or unmated males. A single target fly was added to each mating chamber, and 1 μl heptane (control) or 1 μl heptane containing 1 ng Z4-11Al was applied onto its abdomen. After solvent evaporation (2 min), a single live test fly was introduced into each mating chamber and observed during 20 min. Males were scored when showing vigorous courtship behaviour, including wing vibration, licking, and attempted copulation. Treatments (n = 80) and controls (n = 80) were conducted simultaneously, and each fly was tested once.
 

Data files should be explicit, especially in combination with figures and legends.

Wind tunnel (Figure 1): Odour-mediated upwind flight attraction of fruit fly Drosophila melanogaster males (blue bars) and females (ochre bars) to (a) vinegar and yeast headspace, (b) 10 ng/min male sex pheromone cVA, (c) 10 ng/min female sex pheromone Z4-11Al, alone and in blends with vinegar or yeast headspace, respectively. Letters of the corresponding colours show differences between treatments using Wald pairwise comparisons. Asterisks show treatments with significant differences between sexes (n=40). 

Courtship (Figure 2): Effect of Z4-11Al on courtship in D. melanogaster males. Decapitated target flies were painted with 1 ng test compound or heptane (solvent control). The number of courting and non-courting males in each test was compared using a chi-square test, asterisks show significant differences. (a) Proportion of unmated or mated D. melanogaster (Dalby) test males courting unmated decapitated female or male target flies treated with 1 ng Z4-11Al or heptane (solvent control) (n=80, P=0.006 and P=0.007). (b) Proportion of mated Canton-S and Dalby strain D. melanogaster males and D. simulans males, courting decapitated Dalby or D. simulans females, treated with 1 ng Z4-11Al or heptane (n=80, P=0.009 and P=0.039). (c) Effect of RNA interference, in Orco-Gal4/uas-Or69aRNAi mated males, courting Dalby target females, treated with 1 ng Z4-11Al or heptane. Parental lines, Orco-Gal4 and uas-Or69aRNAi, show significant courtship behaviour (n=80, P=0.036 and P=0.032). (d) Proportion of mated Dalby males courting decapitated Dalby females, treated with E4-11Al, (R)-linalool or ethyl butyrate (n=80).