Competitive courtship assay

Wt Drosophila show a competitive courtship strategy. To test whether this is changed in dark-fly a competitive courtship assay was performed. The courtship performance of dark-fly is significantly improved in the group courtship assay compared to the single courtship assay. In the latter, over 50% of dark-fly were not able to perform successful copulation, while in a group setup they showed an increased courtship success even surpassing OregonR levels in both illumination conditions. This suggests a change in courtship strategy in dark-fly to tolerate other males courting the same female simultaneously.

The competitive courtship assay pairs two socially naïve males with a decapitated female. The males switch between male-male agonistic interaction and courtship behaviour towards the female.

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Figure 29Competitive courtship assay. 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) = 22, N(OregonRdark) = 10, N(dark-flylight) = 22, N(dark-flydark) = 16.

(A) Fraction of courtship behaviour. The fraction of time spent with courtship is increased from dark to light conditions in both strains. (B) Fraction of aggression behaviour. The level of aggression is elevated in light conditions compared to dark conditions. Dark-fly shows a significantly increased fraction of aggression behaviour in relation to OregonR. (C) Courtship frequency. The courtship frequency in OregonR in increased from light to dark conditions. In dark-fly illumination had no impact on the courtship frequency, which did not significantly differ from OregonR in dark conditions. (D) Aggression frequency. Aggression frequency is not influenced by illumination condition in OregonR. In dark-fly the aggression frequency is significantly increased in light conditions.

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91 The duration that each male spent with the respective behaviours was determined.

Analysing the fraction of courtship behaviour, both OregonR and dark-flies spent much more time on courtship in dark conditions. A comparison within the two strains also shows severe differences (Figure 29 A). This correlates with more aggression behaviour:

in both OregonR and dark-fly aggression behaviour is reduced by a change into dark conditions. However, dark-fly shows significantly increased aggression levels in both illumination conditions compared to OregonR (Figure 29 B).

The frequency of courtship bouts is significantly increased in OregonR with a shift from light to dark conditions (Figure 29 C). In dark-fly illumination conditions show no significant effect on courtship frequency; levels are similar to OregonR in dark conditions. In OregonR, frequency of aggression behaviour is not influenced by a change from light to darkness, while in dark-fly aggression frequency is significantly increased in light conditions compared to darkness. In both illumination conditions, dark-fly shows higher levels than OregonR (Figure 29 D)

The courtship bout duration in OregonR is significantly decreased from light to dark conditions; in dark-fly the courtship bout duration is not influenced by a changing illumination condition and similar to OregonR in darkness (Figure 29 E). The aggression bout duration in fly is significantly shorter compared to OregonR. However, dark-flies overall exhibit higher aggression levels than OregonR (Figure 29 F).

To better understand the relationship between aggression and courtship behaviour a behavioural contrast was calculated. Both tested strains have a bias towards courtship behaviour in dark conditions. On the contrary, while wt flies still prefer courtship over aggression under light, dark-fly flies completely flip their behaviour and become predominantly aggressive.

(E) Courtship bout duration. Courtship bout duration is significantly decreased from light to dark conditions in OregonR. In dark-fly levels in both illumination conditions are similar to OregonR in darkness. (F) Aggression bout duration. Dark-fly shows significantly shorter aggression bout durations compared to OregonR.

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As hypothesised above, in dark-fly a tolerance towards other males courting simultaneously might have evolved. To verify this, the synchronicity of both behaviours was analysed. In both strains the synchronicity of courtship behaviour is significantly increased by a transition from light to dark conditions. In dark-fly the level in darkness is higher and in light lower compared to OregonR. The fraction of simultaneous aggression behaviour is increased from light conditions to darkness in dark-fly and decreased in OregonR.

These findings support the hypothesis that dark-fly males indeed have evolved a higher tolerance to other males courting simultaneously.

Figure 30 Comparison of courtship and aggression behaviour in the competitive courtship assay. 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) = 22, N(OregonRdark) = 10, N(dark-flylight) = 22, N(dark-flydark) = 16. (A) Behavioural contrast. The behavioural contrast was calculated as (Dcourtship – Daggression)/(Dcourtship + Daggression). Both OregonR and dark-fly show a significantly increased inclination towards courtship in darkness. In light conditions dark-fly shows a preference towards aggression behaviour. (B) Synchronicity of courtship behaviour. The synchronicity of behaviour indicates the fraction in which both males show the same behaviour simultaneously. In both strains the fraction of simultaneous courtship is significantly increased by a switch from light to darkness. Compared to OregonR the fraction is increased in darkness but decreased in light conditions in dark-fly. (C) Synchronicity of aggression behaviour. The synchronicity of behaviour indicates the fraction in which both males show the same behaviour simultaneously. In OregonR the fraction of simultaneous aggression is decreased by a switch from light to darkness, while in dark-fly the fraction is higher in dark conditions

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