Decrease in the chaining behavior.

<p>(A) Retrieval test. Flies exposed to the continuous pulse song for 30 min were maintained in silence for 30 min (bluish-purple) or 60 min (red-purple), and returned to sound-exposure. Dotted vertical lines indicate the start and restart of the sound playback (Left panel). Blue vertical line indicates cessation of the first playback. The chain index for the initial phase (I) was significantly higher than those in later (II) and after the restart (III-a and III-b) temporal phases (Middle and Right panels). (B) GCaMP3 fluorescent change to the continuous pulse song. Left panel shows a ventral view of the AMMC. The AMMC is the primary center for JO neurons and is anatomically subdivided into zones A (magenta), B (light green), C (light blue), D (orange), and E (blue). Pseudocolor images at 0, 10, and 20 min after stimulus onset are shown (middle panels). The region of interest (ROI) was set in the AMMC zone A. 1-min averages of the ΔF/F intensity at 0, 10, and 20 min after stimulus onset was plotted (right panel). No significant difference was observed between them (N = 4, Friedman's test followed by Scheffe's multiple comparison). P, posterior; L, lateral. Scale bar  = 20 µm. (C) Dishabituation tests. Light blink (blue), vortex (red), bang (green), or a short silent period (light blue) does not restore the behavioral suppression to the continuous pulse song (Left panel). A dishabituation stimulus was applied at the start of a 40-s silent interval (arrow). Dotted vertical lines indicate when the sound playback was started (at 5 min) and restarted (at 35 min 40 s). A blue vertical line indicates cessation of the first playback. Right panels show the cumulative chain indices for four temporal phases. **p<0.01; *p<0.05; n. s., not significant.</p

Systematic counting of the chain index by ChaIN.

<p>(A) Detection of chaining behavior in fruit flies. In the top panel, green line shows the body axis of each fly. Passive recipients (the leader of the train of flies) are included in the score <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074289#pone.0074289-Eberl1" target="_blank">[11]</a>, so that the chain index in the top panel is defined as 2. Bottom panel shows a histogram for the body length of <i>D. melanogaster</i> (mean ± standard deviation 2.27 mm ±0.44 mm; n = 17,184 silhouettes of files). Inset image shows the flies forming chains. (B) Flowchart of ChaIN. Chain indices in images were exported as a time-series dataset. (C) Length and angle constants. Change in the length constant (<i>L</i>) varies the scale of the chain region (Left panel). Change in the angle constant (<i>k</i>) varies the expanse of the chain region (Right panel). Note that ChaIN_13 pixels (px) in the left panel and ChaIN_0.66 in the right panel show the same chain region (<i>L</i> = 13, <i>k</i> = 0.66). (D) Evaluation of ChaIN. Blue and red traces show the time-course of the chain index counted by ChaIN and an experimenter's manual inspection, respectively (Top panel). Sound playback starts at 5 min (dotted vertical line). Thick and thin lines represent mean ± standard error. Time windows for three temporal phases are hatched in gray. The box plot depicts the cumulative chain indices during three temporal phases of 1 min each (Bottom panel). (E) Change in the length and angle constants had less impact on the chain index. Length constant <i>L</i>, originally set as 13 pixels, was shifted to 12 or 14 pixels (Top left panel). Angle constant <i>k</i>, originally set as 0.66, was shifted to 0.5 or 0.75 (Top right panel). Box plot shows the cumulative chain indices during three temporal phases (Bottom panel). n. s., not significant.</p

Sensitivity of the chaining behavior.

<p>(A) Time traces for the artificial sound playback from a loudspeaker. Mean baseline-to-peak amplitudes of their particle velocities are: 15.2 mm/s in S1 (blue), 6.7 mm/s in S2 (red), 2.2 mm/s in S3 (green), 1.1 mm/s in S4 (light blue), and 0.4 mm/s in S5 (pink). (B) Time-course for the chain index in response to artificial sounds (Left panel; same color-code as Fig. 2A). A baseline response without sound playback is shown as the gray line. Thick and thin lines represent mean ± standard error, respectively. Sound playback begins at 5 min (dotted vertical line). Time windows for silent and sound temporal phases are hatched in gray. Box plot depicts the comparison of chain indices between the silent and sound temporal phases that span 2.5 min each (Right panel). Sound intensity 1.1 mm/s (S4) and greater evokes chaining behavior (Wilcoxon signed-rank test). Basal chaining behavior without sound declined gradually, as shown in the “no sound” group. (C) IPI oscillations have no effect in evoking the chaining behavior. Artificial songs without (blue) or with (red) an IPI oscillation induced significant increases in the chain index (Kruskal-Wallis test followed by Scheffe's multiple comparison) (Bottom panel). (D) Effect of population density on the chaining behavior. Chain indices between two (blue), four (red), and six (green) males during song playbacks are shown as time-courses (Top panel). Box plot shows the mean probability of a fly forming a chain in each second during the “Silent” and “Sound” phases (Kruskal-Wallis test followed by Scheffe's multiple comparison) (Bottom panel). Arrows indicate the increase in the probability from 2 males (24%) to 4 males (36%) or to 6 males (44%). *p<0.05; **p<0.01; ***p<0.001; n. s., not significant. n, number of behavioral chambers examined.</p

Behavioral change in wild-type and <i>rut²⁰⁸⁰</i> mutant flies.

<p>(A) Artificial songs used in this assay. (B) Chain index to prolonged pulse sounds. Top panel shows the time-course of the chain index to the continuous pulse song (red line) or intermittent pulse song (blue line). Time windows for counting the cumulative chain index in each 1-min temporal phase are marked with gray boxes (temporal phases I to V). Bottom panels show cumulative chain indices during five temporal phases, marked in the top panel. Responses to the continuous pulse song (Left panel) and intermittent pulse song (Right panel) are color-coded in red and blue, respectively. Different letters indicate significant differences between groups (Friedman's test followed by Scheffe's multiple comparison, p<0.05). (C) Time-course of chain index to acoustic stimuli that contain sequential continuous and intermittent sound clips (Top panels). Time windows of the continuous song (red underline) and intermittent song (blue underline) playbacks are underlined at the bottom of each panel. Blue vertical lines depict the time when the sound clip is switched between two songs. Bottom panels show the box plot of the cumulative chain indices during the five temporal phases. Each temporal phase spans 1 min (indicated in the top panel as temporal phases I to V). (D) Behavior in <i>rut²⁰⁸⁰</i> mutant flies. Chain indices to the continuous (red line) and intermittent (blue line) sounds are shown (Left panel). Cumulative chain indices for five temporal phases are shown in the box plot (Middle and Right panels). (E) Chain indices in wild-type and <i>rut²⁰⁸⁰</i> mutant flies. SR, suppression rate; RR, recovery rate; DR, drop rate. Magenta arrows indicate two temporal phases for Fig. 6G. (F) Behavioral changes in wild-type and <i>rut²⁰⁸⁰</i> mutant flies. *p<0.05; ***p<0.001; n. s., not significant (Student's t-test). (G) Cumulative chain indices for <i>rut²⁰⁸⁰</i> mutant flies at the onset and later of the second playback of the continuous pulse song (shown as arrows at 40 min and 60 min in Fig. 6E, respectively).</p