Modulation of Drosophila male behavioral choice

Abstract

The reproductive and defensive behaviors that are initiated in response to specific sensory cues can provide insight into how choices are made between different social behaviors. We manipulated both the activity and sex of a subset of neurons and found significant changes in male social behavior. Results from aggression assays indicate that the neuromodulator octopamine (OCT) is necessary for Drosophila males to coordinate sensory cue information presented by a second male and respond with the appropriate behavior: aggression rather than courtship. In competitive male courtship assays, males with no OCT or with low OCT levels do not adapt to changing sensory cues and court both males and females. We identified a small subset of neurons in the suboesophageal ganglion region of the adult male brain that coexpress OCT and male forms of the neural sex determination factor, Fruitless (Fru(M)). A single Fru(M)-positive OCT neuron sends extensive bilateral arborizations to the suboesophageal ganglion, the lateral accessory lobe, and possibly the posterior antennal lobe, suggesting a mechanism for integrating multiple sensory modalities. Furthermore, eliminating the expression of Fru(M) by transformer expression in OCT/tyramine neurons changes the aggression versus courtship response behavior. These results provide insight into how complex social behaviors are coordinated in the nervous system and suggest a role for neuromodulators in the functioning of male-specific circuitry relating to behavioral choice.

Fig. 1.

Manipulating OCT levels and the sex of OCT/TYR neurons changes the usage of aggressive and courtship behaviors. (a and b) Graph depicting the percentage of aggression (black) and courtship (gray) transitions after a wing extension. Control males (Tβh^M6 and UAS-tra/+) predominantly transition to aggression (aggression/total transitions, n = 31/37 and 20/23). Experimental males [Tβh^nM18, Tβh^MF372, dTdc2-Gal4;UAS-tra, and dTdc2-Gal4 (new insertion #1);UAS-tra] transition to aggression and courtship or predominantly to courtship (courtship/total transitions: n = 24/42, 11/14, 24/31, and 30/39). The number of courtship vs. aggressive transitional patterns was compared among control and experimental genotypes with the Fisher exact probability test. Significant frequency differences were observed between Tβh^M6 and Tβh^nM18 patterns (P = 0.0002), Tβh^M6 and Tβh^MF372 patterns (P = 0.0006), and UAS-tra/+ and dTdc2-Gal4#1;UAS-tra patterns (P = 0.000001). (c–e) A still frame series of a wing extension followed by an aggressive lunge behavior. Wing vibration is not visible at this resolution or in a single clip, but see SI Movies 1 and 2. The clips show a Tβh^nM18 male approaching and starting a wing extension (c), continuing the wing extension (d), and demonstrating an aggressive lunge (e, arrow). (f–h) A still frame series of courtship behavior after a wing extension. These individual clips also are of a Tβh^nM18 male pair. One male is performing a wing extension and tapping (f), followed by abdomen bending (arrow) (g), and the courting male is rejected by the courted male (wing flick), and the wing extension ends (h) (see SI Movies 1 and 2).

Fig. 2.

OCT-deficient males court males significantly more than do controls. (a and b) We quantified the total time that a male performed wing extensions to a female or a second male during a 10-min interval in the multiple male assay. The box plot depicts the percentage of total time spent performing wing extensions first to females (red) and then males (blue). The upper and lower edges of the boxes correspond to the 25% and 75% quantiles. The median (50% quantile) is shown as a horizontal line in the box. The lines depict the 5% and 95% quantiles. Asterisk shows medians that are statistically different according to Wilcoxon rank sum for nonparametric data: Tβh^M6-Tβh^nM18 (P < 0.006), Tβh^M6-Tβh^MF372 (P < 0.001), and UAS-tra/+- dTdc2-Gal4;UAS-tra (P < 0.0001). Six assays were performed with dTdc2-Gal4;UAS-tra males and six with dTdc2-Gal4 #1;UAS-tra (new insertion) males. Results were not statistically different, and the data were pooled (n = 12 multiple male assays per line tested). (c and d) Box plot of the wing extension duration (in seconds) displayed by one male to a second fly (females are indicated in red and males are indicated in blue). The data were calculated by dividing the total wing extension time by the number of bouts for each male. The duration of male–male wing extensions by Tβh^M6 control males is significantly shorter than male–female wing extensions performed by the same males (Student's t test, P < 0.001) and male–male wing extensions performed by Tβh^nM18 and Tβh^MF372 males (asterisks; ANOVA for independent groups, P < 0.001). The duration of wing extensions to other males by UAS-tra/+ transgenic control males was significantly shorter (asterisk) than dTdc2-Gal4;UAS-tra males (Student's t test, P < 0.0001). The wing extension duration data were collected from the multiple male assays described above.

Fig. 3.

OCT and the male-specific form of Fruitless are coexpressed in VUM neurons of the SOG. (a) Confocal sections of a transgenic dTdc2-Gal4;UAS-mCD8:GFP adult male brain labeled with anti-GFP (green) and mAb nc82 (red labels neuropil regions) antibodies. The SOG region containing neurons coexpressing Fru^M and OCT is highlighted by the white box. The arrow points to the VUM 1 cluster highlighted in b, and the arrowhead points to the VUM 2 cluster in c and d. (b) Confocal sections from a dTdc2-Gal4;UAS-mCD8:GFP transgenic male labeled with anti-Fru^M (red) antiserum and anti-GFP antibody (green). Two VUM 1 cluster neurons (arrows) located in the SOG coexpress Fru^M and the GFP reporter. The GFP expression is weak in the nuclei of these VUM neurons; therefore, overlap does not appear yellow (n = 9). (c, c′, and c″) A VUM 2 cluster neuron from a dTdc2-Gal4;UAS-mCD8:GFP transgenic male coexpressing FruM (red) (c′) and GFP (green) (c″) (n = 9). (d, d′, and d″) An adult male brain section (dTdc2-Gal4;UAS-mCD8:GFP) depicting neurons of the VUM 2 cluster stained with mAb OA1 (red) (d′) and anti-GFP (green) (d″) (n = 10).

Fig. 4.

Arborizations of a single Fru^M OCT VUM 1 SOG neuron. (a) Composite of four 1-μm optical sections containing the cell body of a VUM 1 cluster Fru^M-positive hs-FLP;dTdc2-Gal4;UAS<-CD2, y⁺>CD8-GFP neuron. The arrow identifies the primary neurite. (Inset) Fru^M and GFP-reporter colocalization. (b) Composite of 18 1-μm optical sections of the VUM 1 cluster Fru^M–GFP-positive neuron identified in a. This region of dense arborization is located 12 μm ventrally to the cell body. The primary neurite branches into two symmetrical secondary neurite arbors at the arrow. Arborizations extensively ramify throughout the SOG. (c) Twenty 1-μm sections of a continued group of SOG arborizations from the VUM 1 cluster Fru^M–GFP-positive neuron. This arborization is located ventrally to the clusters observed in b.