4. Results
4.7 Courtship songs are influenced by light-deprivation
The analysis of dark-fly courtship behaviour showed a rather surprising decrease in courtship success under dark conditions. Restored success in a group courtship assay suggests that this effect can be mended by simultaneous song production of the male flies. The obvious next step was to analyse the male courtship songs, which were recorded during the single couple courtship assay.
In Drosophila, the courtship song produced by male wing vibration is one of the critical features of courtship behaviour. Upon perceiving the courtship songs, female Drosophila pauses, indicating receptivity (Schilcher, 1976). The song can be divided in three distinct modes: two pulse songs, Pfast and Pslow, the use of which correlates with the distance to mating partner. Pfast is used in longer distance to the female, whereas Pslow is used in close proximity (Clemens et al, 2018). Furthermore, one type of sine song can be identified, which together with the interpulse interval (IPI) communicates species identification (R J Greenspan and Ferveur, 2000).
To extract possible differences in the shape of Pfast and Pslow, the pulses were z-scored scored (normalised to mean = 0 and standard deviation = 1) and superimposed. This rendered no difference between the four groups (Figure 33 A). However, a change in amplitude on both pulse forms within the two strains and two illumination conditions can be observed, indicating a change in volume.
Analysing the amplitude of pulse songs shows an increase in both strains when changing the illumination condition from light to dark. In both conditions the dark-fly pulse songs have a higher amplitude than OregonR (Figure 33 B). This demonstrates that the courtship song of dark-fly is indeed increased in volume compared to OregonR.
Subdividing the amplitudes into the two pulse forms Pslow and Pfast indicates a trend towards higher amplitudes in darkness in both strains but does not reach significance.
Dark-fly produced significantly louder courtship song in both illumination conditions compared to OregonR flies, corresponding to the observation that the overall pulse amplitude is increased in dark-fly (Figure 33 C & D).
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Figure 33 Pulse songs and courtship songs amplitude. Dots represent individual values; black line indicates median. Boxes include 50% of the data set around the medians; whiskers indicate 1.5*
interquartile distance; outliers are marked by black circles. N(OregonRlight) = 21, N(OregonRdark) = 21, N(dark-flylight) = 21, N(dark-flydark) = 21. To test for significance, Kruskal-Wallis test was used.
Significance is indicated as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 (A) Shape of pulse songs. Z-scored superposition of pulse shapes. The shape of the two forms of pulse songs Pfast and Pslow does not differ between dark-fly and OregonR in both illumination conditions. (B) Overall amplitude of pulse songs. In OregonR and dark-fly the amplitude of pulse songs is significantly increased from light to dark conditions. The overall amplitude is significantly increased if comparing dark-fly and OregonR.
[p-values: ORL vs ORD 0.27; DFL vs DFD 0.04 ; ORL vs DFL 2 x 10-3; ORD vs DFD 1 x 10-4]
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(C)Amplitude of Pslow. Pslow shows a trend to higher amplitudes in dark conditions compared to light conditions in both dark-fly and OregonR. The amplitude in dark-fly is significantly increased compared to OregonR. [p-values: ORL vs ORD 0.74; DFL vs DFD 0.72 ; ORL vs DFL 1 x 10-4; ORD vs DFD 1 x 10-4] (D) Amplitude of Pfast. Pfast shows a trend to higher amplitudes in dark conditions compared to light conditions in both dark-fly and OregonR. The amplitude in dark-fly is significantly increased compared to OregonR. [p-values: ORL vs ORD 0.14; DFL vs DFD 0.15; ORL vs DFL 0.02; ORD vs DFD 0.03]
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The latency to performance of first courtship song was not influenced by illumination condition in OregonR flies and similar to dark-fly in light conditions. Interestingly, in dark conditions the latency was significantly prolonged in dark-fly compared to all other groups (Figure 34 A).
As mentioned above, the IPI has an important role in species identification and was therefore compared between the four groups. Both dark-fly (40.6 ms) and OregonR (41.3 ms) show a similar IPI in light conditions. Interestingly, with a change to dark conditions, the interval is significantly reduced in OregonR but significantly increased in dark-fly (OregonRdark 39.0 ms; dark-flydark 45.9 ms) (Figure 33 B).
Comparing the pulse to sine ratio, a significantly higher proportion of pulse song is found in dark-fly for both illumination conditions. Within the groups a trend to an increase in proportion of pulse song can be observed but does not reach significance (Figure 33 C).
Correspondingly, the fraction of Pfast is significantly increased in dark-fly for both illumination conditions compared to OregonR (Figure 33 D). In dark-fly the proportion is also significantly higher in dark conditions than in light conditions, whereas in OregonR
Figure 34 Male courtship song characteristics. Dots represent individual values; black line indicates median. Boxes include 50% of the data set around the medians; whiskers indicate 1.5* interquartile distance; outliers are marked by black circles. N(OregonRlight) = 21, N(OregonRdark) = 21, N(dark-flylight) = 21, N(dark-flydark) = 21. To test for significance, Kruskal-Wallis test was used. Significance is indicated as follows: * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001 (A) Latency to first courtship song. Latency to first courtship song is similar for OregonR in both illumination conditions and dark-fly in light conditions. In dark conditions, dark-fly shows a significant increase in latency compared to the other three groups. [p-values:
ORL vs ORD 0.97; DFL vs DFD 2 x 10-4 ; ORL vs DFL 0.88; ORD vs DFD 1 x 10-4] (B) Median interpulse interval (IPI). In OregongR the IPI is significantly reduced from light to dark conditions. In dark-fly the IPI is significantly increased from light to dark conditions. Median IPI is similar for both strains in light conditions but significantly different in dark conditions. [p-values: ORL vs ORD 43 x 10-4 ; DFL vs DFD 99 x 10-4 ; ORL vs DFL 0.85; ORD vs DFD 1 x 10-4] (C) Pulse to sine ratio. Pulse to sine ratio shows a trend to be increased in darkness in both strains. Compared to OregonR, the ratio is significantly increased in dark-fly. [p-values:
ORL vs ORD 0.09; DFL vs DFD 0.12; ORL vs DFL 4 x 10-3; ORD vs DFD 7 x 10-3] (D) Fraction of Pfast. The fraction of Pfast is increased in darkness for both strains. Overall, dark-fly shows a higher fraction of Pfast
compared to OregonR. [p-values: ORL vs ORD 0.27; DFL vs DFD 0.04; ORL vs DFL 15 x 10-3; ORD vs DFD 1 x 10-3]
4. Results
101 a trend towards an increased fraction of Pfast in dark conditions can be observed but does not reach significance (Figure 33 D).
In dark conditions, courtship song volume is increased compared to light conditions.
Since male are not able to reliably locate the female in the absence of visual cues and distance to the female mediates courtship song volume, this result was to be expected.
However, dark-fly males increase their courtship song volume significantly compared to OregonR males, suggesting an underlying mechanism that favours louder courtship songs.