DOI: 10.5061/dryad.5mkkwh752
title: The female pheromone (Z)-4-undecenal mediates flight attraction and courtship in Drosophila melanogaster

description: 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.
description: 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). <br> 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.<br> 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)<br>
 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.<br>  
description: 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).