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- Fig. 1.
Measuring and manipulating gain in fly songs and experimental setup for testing auditory responses to song amplitude structure in Drosophila. (A) We measured the amplitude gain of pulses as the relative increase in amplitude of successive pulses. For example, suppose (using arbitrary units) that a pulse has an amplitude of 1 and is followed by a pulse with an amplitude of 2. The relative increase between those pulses is 2. (B) We created playback stimuli by masking 5 min of species-specific real song with a strain's mean gain envelopes. Songs of D. melanogaster exhibit species-specific characteristics (see Table 1), and our amplitude modulation does not alter the song parameters (see Fig. 3). The notation m-m refers to D. melanogaster song with D. melanogaster mean gain. (C) Schematic of our playback setup. (D) Schematic of our copulation assay. We muted males (shown in blue; females are in pink) by removing their wings and deafened them by removing their arista. Fly mating occurs more often during song playback (right) than during silence (left).
- Fig. 2.
Amplitude envelope preference of female D. melanogaster. We present our data in two different forms: (left) violin distribution plots and (right) survivorship curves. (A) D. melanogaster CS females prefer their own strain's IPI duration (IPI = 38 ms) over shorter IPIs (3 ms), longer IPIs (73 ms), and the silence control condition. We calculated a P-value of P<0.0001 and an F statistic of F ≈ 34.51 in a one-way ANOVA test. There are nc=264 mixed-sex couples. To measure IPIs, we detected pulses in modulated playbacks automatically with FlySongSegmenter (Arthur et al., 2013) and then computed the distance between detected pulses. (B) D. melanogaster CS females preferred their own strain's song-amplitude-structure envelope (m-m) over flat gain, steeper gain (twice the D. melanogaster mean gain, which we denote by m-2m), and the silence control condition when we modulated song amplitude structure in a courtship song recording of a D. melanogaster male. We calculated P<0.0001 and F≈10.99 in a one-way ANOVA test, where nc=444 is the number of mixed-sex couples. (See Fig. 1B for an illustration of amplitude modulation in courtship-song recordings.) (C) D. melanogaster CS females preferred their own strain's amplitude envelope (m-m) over flat gain, steeper gain (m-2m), and the silence control condition when we modulated amplitude in an artificial D. melanogaster song. We calculated P<0.0001 and F≈18.33 in a one-way ANOVA with nc=228 mixed-sex couples. We show the distribution of the fraction of copulating pairs as grey kernel-density plots, which we mirror across the vertical axis and also show as box plots. A red cross indicates the mean, a horizontal line indicates the median, a box indicates the inter-quartile-range (IQR), and the whiskers indicate 1.5 × IQR. We calculated P<0.01 using two-sided Wilcoxon rank-sum (WR) tests, with Bonferroni correction for multiple testing; and we obtained P<0.025 using log-rank tests for comparing survival curves. The asterisks indicate a significant difference in the fraction of copulating pairs. The colour of the asterisks indicates which gain condition differs significantly from the others. The grey asterisks signifies that the grey condition (i.e. the m-2m envelope) differs significantly from the labelled condition (i.e. the D. melanogaster mean-gain envelope). The black asterisks indicate that the black condition (i.e. the flat gain) differs significantly from the labelled condition (i.e. the D. melanogaster mean-gain envelope). The black dots indicate that there is a significant difference in the fraction of copulating pairs between the labelled condition and the silence control; the P-value is P<0.01 (WR).
- Fig. 3.
Song parameters do not differ significantly across playback conditions. (A) Inter-pulse interval (IPI) does not differ significantly across different playback conditions. The number np of detected pulses also does not differ significantly across different playback conditions for D. melanogaster. D. melanogaster song has np=1870 for flat gain, np=1873 for m-m, and np=1869 for m-2m, where we recall that m-m refers to D. melanogaster song with D. melanogaster mean gain and m-2m refers to D. melanogaster song with twice the D. melanogaster mean gain. We show song statistics for each envelope condition (black for flat gain, orange for m-m, and grey for m-2 m) as light-grey kernel-density plots, which we mirror across the vertical axis and also show as box plots. A red cross indicates the mean, a horizontal line indicates the median, a box indicates the IQR, and the whiskers indicate 1.5 × IQR. (B) Neither pulse-song frequency nor sine-song frequency differ significantly across different playback conditions. We modulated pulse amplitude only, but we also present sine-song statistics, as sine song can influence fly behaviour (von Schilcher, 1976a). Our data suggest that sine song is similar across playback conditions, so differences in sine song are not sufficient to explain the behavioural differences that we observed. (C) Cycles per pulse (CPP) do not differ significantly across different playback conditions. (D) The mean pulse shape (computed as a pointwise mean) does not differ significantly across different playback conditions.
- Fig. 4.
Small-amplitude pulses in modulated songs arouse flies. (A) We show m-m playback as a control to illustrate the amplitude level of pulses with minimum amplitude in the playbacks. To study whether flies hear pulses with small amplitudes in modulated songs, we generated playback with pulses set to the minimum amplitude that occurred in the m-m playback. If pulses at the minimum amplitude are not heard by flies, they should behave similarly in their responses to songs with pulses at minimum amplitude as in their responses to songs with no pulses (i.e. a sine-song-only condition). (B) Flies mate significantly more in response to songs with pulses at minimum amplitude (purple curve) than to songs with no pulses (grey curve). (C) The asterisks indicate significance levels: * signifies P<0.005 and *** signifies P<0.0005. In each case, we apply Bonferroni correction for multiple testing using a two-sided Wilcoxon signed-rank test. We show song statistics for each playback condition (orange for m-m, purple for minimum-amplitude pulses, and grey for sine-song only) as light-grey kernel-density plots, which we mirror across the vertical axis and also show as box plots.
- Fig. 5.
Distance-independent variations in song amplitude are prevalent in the data of Coen et al. (2016). Because distance affects song amplitude but not all variation in song amplitude can be explained by distance (Coen et al., 2016), we search the data of Coen et al. (2016) for consecutive pulse trains at specified distances between male and female flies to investigate residual amplitude variation. (A) To analyse residual amplitude variation, we (1) measure pulse peaks for a distance category, (2) arrange consecutive pulse trains of a given distance interval around the pulse peak with maximal amplitude, and (3) plot the arranged peaks as heat maps that illustrate residual variation of pulse amplitude at a given distance interval. We observe residual amplitude variation in the spread of amplitudes along the vertical axis of the heat plots. In panel B, we consider distance intervals of 4 mm. There are N pulses at the specified distance ranges. The numbers of pulses are N=1376 for 0–4 mm (including both 0 mm and 4 mm), N=1420 for 4–8 mm (not including 4 mm), and N=985 for 8–12 mm (not including 8 mm). In panel C, we consider distance intervals of 2 mm. The numbers of pulses are N=1376 for 0–2 mm (including both 0 mm and 2 mm), N=1523 for 2–4 mm (not including 2 mm), N=969 for 4–6 mm (not including 4 mm), N=789 for 6–8 mm (not including 6 mm), N=575 for 8–10 mm (not including 8 mm), and N=500 for 10–12 mm (not including 10 mm).
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