Figure 4. Specificity of NPF^M neurons in regulating male courtship.

(A) Single tester males of the indicated genotypes were assayed for male-female (M–F) courtship at both permissive (23°C) and non-permissive (31°C) temperatures for Shi^ts. Newly-eclosed male flies were isolated for 5 days, after which they were housed with 5—7 w¹¹¹⁸ virgin female flies for 4 hr prior to the experiment. 7—15 day-old mature active mated w¹¹¹⁸ female flies were used as targets. The courtship index is the mean ratio of time spent by the tester male in courtship within 30 min following a 10 min incubation period. n = 8—24. Bars indicate means ± SEMs. Significance was determined using Mann-Whitney test. **p < 0.01. (B) Single tester males of the indicated genotypes were assayed for courtship at two different temperatures (23°C and 29°C). Newly-eclosed males that were isolated for 2 days were used as testers. Decapitated w¹¹¹⁸ female flies were used as the targets. Courtship index represents the mean ratio of time the male flies spent in courting within 10 min following a 5 min incubation period. n = 6—27. Bars indicate means ± SEMs. Significance was determined using Mann-Whitney test. **p < 0.01. (C—E) Immunohistochemistry showing the effect of npf RNAi knock down on NPF protein expression in male brains. Control genotypes of npf-Gal4/+ and UAS-npf-RNAi male brains and experimental genotype of npf-Gal4/+;UAS-npf-RNAi/+ male brains were immuno-stained with anti-NPF. Scale bars indicate 50 μm. (F) Effects on male-male (M–M) courtship due to RNAi knock down of npf in all neurons (elav), fru neurons, npf neurons, non-NPF^M npf neurons and NPF^M neurons. n = 7—12. The bars indicate means ± SEMs. Mann-Whitney test was used to determine significance. **p < 0.01.