Figure 2.

Parallel cVA pathways signal male distance and sustained presence differently

(A–C) GCaMP6f responses to a male at ten distances (shown in B) in ORN (A, n = 10), lPN (B, n = 9), and lvPN (C, n = 8) axons, 3 trials/fly/distance. Shaded area is SEM of biological replicates. Black line: male presentation, right y axis: distance from starting position. In (A), inset: experimental setup for in vivo two-photon imaging and male presentation. The distance is measured between the male’s abdomen and the receiver fly’s antennae.

(D–F) Distance response curves in ORN (D), lPN (E), and lvPN (F), based on (A), (B), and (C), respectively. y axis: peak values of normalized traces at the ten distances from all measured flies, 3 trials; error bars are SEM of biological replicates. Dashed line shows the best sigmoidal fit: residual standard error (RSE) and half-maximal distance (ED₅₀) were: RSE = 0.0101 and ED₅₀ = 2.2 mm in (F), RSE = 0.0184 and ED₅₀ = 2.4 mm in (G), and RSE = 0.0177 and ED₅₀ = 1.5 mm in (H).

(G) GCaMP6f responses in ORN axons to 10-s male presentation (0.75 mm), female fly imaged. Average response from 10 flies, 6 trials, and gray area is SEM of biological replicates. Bottom black trace: male position.

(H and I) GCaMP6f responses in females to a male stimulus (solid line, n = 10), in males to a male stimulus (dashed line, n = 7), and in females to a virgin female stimulus (dotted line, n = 6) in lPN (H) and lvPN (I) axons. Presentation same as in (G), 6 trials. Quantification in Figure S2C.

(J) Top: a representative video frame with a stationary fly (“stimulus”) and a free fly (“receiver”). The circle shows 5 mm around the male. Bottom: receiver trajectory, body centroid tracked, 20 min.

(K) Time spent by a receiver female (magenta) or male (green) within 5 mm from a stimulus fly (female or male), during 20 min.

(L) Opposite-sex preference (OSP) score: wild-type males and females spend more time within 5-mm radius of an opposite-sex stimulus fly compared with their own sex. OSP = (time spent at a given distance to opposite sex − time spent at a given distance to same sextotal time recorded) ^∗ 100. OSP(females) = 7.95 ± 4, OSP(males) = 13.29 ± 5.6.

(M) OSP at increasing distances from stimulus: for both wild-type males and females, OSP increased until 8 mm from stimulus then started decreasing. Lines represent mean OSP within cumulative 1-mm bins. Shaded area is SEM.

(N) Two Orco null strains, Orco¹ and Orco² females, have impaired OSP within 5 mm from a male stimulus. OSP(white) = 7.44 ± 4.8; OSP(Orco¹) = 2.61 ± 4.1; OSP(Orco²) = 0.72 ± 7.4.

(O) OSP at increasing distances from stimulus: Orco¹ and Orco² females shifted their OSP to greater distances from a male stimulus. Lines represent mean OSP within cumulative 1-mm bins. Shaded area is SEM.

See also Figure S2.