Circadian consequence of socio-sexual interactions in fruit flies Drosophila melanogaster

Abstract

In fruit flies Drosophila melanogaster, courtship is an elaborate ritual comprising chasing, dancing and singing by males to lure females for mating. Courtship interactions peak in the night and heterosexual couples display enhanced nighttime activity. What we do not know is if such socio-sexual interactions (SSI) leave long-lasting after-effects on circadian clock(s). Here we report the results of our study aimed at examining the after-effects of SSI (as a result of co-habitation of males and females in groups) between males and females on their circadian locomotor activity rhythm. Males undergo reduction in the evening activity peak and lengthening of circadian period, while females show a decrease in overall activity. Such after-effects, at least in males, require functional circadian clocks during SSI as loss-of-function clock mutants and wild type flies interacting under continuous light (LL), do not display them. Interestingly, males with electrically silenced Pigment Dispersing Factor (PDF)-positive ventral lateral (LNv) clock neurons continue to show SSI mediated reduction in evening activity peak, suggesting that the LNv clock neurons are dispensable for SSI mediated after-effects on locomotor activity rhythm. Such after-effects in females may not be clock-dependent because clock manipulated females with prior exposure to males show decrease in overall activity, more or less similar to rhythmic wild type females. The expression of SSI mediated after-effects requires a functional olfactory system in males because males with compromised olfactory ability do not display them. These results suggest that SSI causes male-specific, long-lasting changes in the circadian clocks of Drosophila, which requires the presence of functional clocks and intact olfactory ability in males.

Figure 1. Socio-sexual interactions (SSI) reduce evening activity peak and lengthens clock period in males.

(a) Average actograms of males following 0 (control), 2, 3, 4, or 5 days of SSI. (b) Activity profiles of males following 0–5 days of SSI. Zeitgeber Time (in hours) is plotted along x-axis, while activity (in counts in one hour bins averaged across five days) is plotted along y-axis. (c) The circadian period of locomotor activity rhythm of males following 5 days of SSI is lengthened compared to control males (p<0.01). (d) Circadian period of males kept in same sex groups (under light/dark – LD cycles) following 5 days of SSI for 8 days. The circadian period of SSI males is significantly lengthened compared to control males (p<0.02). (e) Cumulative daytime and nighttime activity of males subjected to 0, 2, 3, 4, or 5 days of SSI. The number of days of SSI is plotted along x-axis and daytime or nighttime activity (in counts/12 hr average across five successive days) along y-axis. (f) Anticipation to lights-on and lights-off as measured by Anticipation Index (AI) of male flies subject to 0, 2, 3, 4, or 5 days of SSI. The shaded area indicates night and the empty area day of laboratory LD cycles. The error bars are standard error around the mean (SEM). Asterisks indicate significant differences with p values<0.05.

Figure 2. Socio-sexual interactions (SSI) cause reduction in overall activity in females.

(a) Average actograms of females following 0 (control), or 5 days of SSI. (b) Activity profiles of females following 0, or 5 days of SSI. The activity of females is significantly reduced during morning and evening activity peaks and during most nighttime phases (p<0.05) in SSI females relative to control females. (c) Cumulative daytime and nighttime activity of females subjected to 0, or 5 days of SSI. (d) Anticipation to lights-on and lights-off measured as Anticipation Index (AI) of female flies subject to 0, or 5 days of SSI. All other details same as in Fig. 1.

Figure 3. Clock mutant males do not display socio-sexual interaction (SSI) mediated after-effects.

Activity profiles of (a, left panels) per⁰ and (b, left panels) cyc⁰ males and females after 0 (controls), or 5 days of SSI. In per⁰ and cyc⁰ flies, activity profiles of SSI and control males do not differ (a, b, left panels). Activity of per⁰ females is reduced during most daytime phases (p<0.05), whereas at all other time points activity is similar to controls. In per⁰ and cyc⁰ flies, cumulative daytime and nighttime activity of SSI males (a, b, right panels) is similar to control males. In SSI females, cumulative daytime activity of per⁰ flies and nighttime activity of cyc⁰ females is reduced (p<0.05) in comparison to controls, while daytime activity of cyc⁰ and nighttime activity of per⁰ flies is similar to controls (p>0.05). (c, left panels) The figure shows that circadian phenotype of SSI males interacting under constant light (LL) is similar to controls. In females, activity is reduced at most time points tested (p<0.05) (c-left panels). (c, right panels) Cumulative daytime and nighttime activity of SSI males interacting with females in LL is similar to controls, while in SSI females it is reduced (p<0.05). (d) The circadian period of SSI males interacting with females under LL is similar to controls. All other details same as in Fig. 1.

Figure 4. Flies with electrically silenced PDF-positive LNv clock neurons display socio-sexual interaction (SSI) mediated after-effects.

(a, left panels) Activity profile of electrically silenced flies show activity of SSI males and females similar to controls except during the evening activity peak (p<0.05), when SSI males are less active than controls. (a, right panels) Cumulative daytime and nighttime activity of SSI control males does not differ (p>0.05), while in SSI females daytime activity is lower than controls (p<0.05) but nighttime activity is similar to controls (p>0.05). (b, left panels) The activity of Or83b⁰ SSI males is similar to controls (p<0.05) and in females it is reduced during the morning activity peak (p<0.05). (b, right panels) The cumulative daytime and nighttime activity of SSI males is similar to controls, while activity of SSI females is reduced during daytime (p<0.05) but is similar to controls during nighttime (p>0.05). (c, left panels) The activity of Or83b ablated SSI males is similar to controls (p>0.05), while that of females is reduced during morning and evening activity peaks (p<0.05). (c, right panels) The cumulative daytime and nighttime activity of Or83b⁻ males is similar to controls, while that of females is lower than controls (p<0.05). (d) Circadian period of Or83b⁰ SSI males does not differ from controls (p>0.05). All other details same as in Fig. 1.