Circadian rhythms are approximately 24-h cycles in behavior and physiology generated by endogenous molecular clocks that are highly conserved across species (Creux and Harmer, 2019; Dunlap and Loros, 2017; Hardin, 2011; Johnson et al., 2017; Lane et al., 2023). These clocks function through transcriptional-translational feedback loops, where activators repress their own activation over a 24-h cycle (Allada and Chung, 2010; Patke et al., 2020). The circadian clock integrates environmental inputs to regulate various rhythmic processes. Disruptions in these rhythms are linked to sleep, metabolic, and neurodegenerative disorders, highlighting the importance of studying circadian rhythms (Leng et al., 2019; Sack et al., 2007; Shimizu et al., 2016).

Sexual dimorphism is widespread, with males and females differing in morphology, physiology, and behavior (Williams and Carroll, 2009). Sex differences in circadian patterns are also common across species (Joye and Evans, 2022). For example, women generally go to bed earlier than men, and pregnancy shortens women’s circadian period (Martin-Fairey et al., 2019; Roenneberg et al., 2007). Given the conservation of the circadian clock across species, Drosophila melanogaster is an excellent model organism for studying circadian rhythms. Previous research has shown that male and female flies exhibit distinct circadian behaviors (Helfrich-Förster, 2000). Males show more pronounced evening activity, while females display higher overall activity levels throughout the day. Additionally, male flies have shorter circadian periods than female flies. Most studies of Drosophila circadian behavior employ males partly due to anecdotal observations suggesting that males have more robust rhythms than females; however, systematic investigations into sex differences in rhythm strength are lacking.

Beyond sex differences, mating induces notable behavioral and physiological changes in female Drosophila. During copulation, males transfer Sex Peptide (SP) to females (Chen et al., 1988; Liu and Kubli, 2003), triggering various behavioral changes, including increased egg laying, heightened food intake, and sleep loss (Kubli and Bopp, 2012). Additionally, a recent study found that female mating disrupts morning anticipation, which refers to the anticipatory activity before dawn, under light:dark (LD) conditions through SP signaling (Riva et al., 2022). However, a prior study found no significant effect of female mating on circadian period length in constant darkness (DD) (Helfrich-Förster, 2000), and the impacts of female mating status and SP on circadian rhythm strength remain unexplored, as does the effect of male mating status on circadian behavior.

To investigate the roles of sex, mating status, and genetic background in Drosophila circadian behavior, we studied four wild-type and control strains, assessing circadian period length and rhythm strength in virgin males, mated males, virgin females, females mated with same-strain males, and females mated with SP-deficient males. This approach allowed us to explore the effects of sex, mating status, SP, and genetic background on circadian locomotor activity. Our data show that circadian period regulation is sexually dimorphic across strains, with males exhibiting shorter circadian periods than females regardless of mating status and genetic background. In contrast, our findings indicate that the effects of sex and mating status on rhythm strength are strongly modulated by genetic background.

We investigated how sex, mating status, and genetic background influence the period length and strength of free-running rhythms in DD in Drosophila melanogaster. Our data show that these factors impact circadian period length and rhythm strength in distinct ways. Males consistently had shorter circadian periods than females, regardless of mating status or genetic background, revealing a robust sexual dimorphism in the speed of the Drosophila circadian clock. However, rhythm strength was modulated by a complex interaction among sex, mating status, and genetic background. In three strains (Canton-S, iso31, and Oregon-R-modENCODE), females mated with SP⁺ males showed reduced rhythm strength compared to virgin females and SP⁰-mated females, with Oregon-R-P2 being the exception. Except for Oregon-R-modENCODE, males exhibited rhythm strength similar to virgin and SP⁰-mated females. These findings suggest that rhythm strength is primarily influenced by female mating status and genetic background rather than inherent sexual dimorphism. While sexual dimorphism in circadian period length is independent of mating status and genetic background, the effects of sex on rhythm strength are modulated by mating status and genetic background. This difference may arise because the circadian period is determined by the pace of the molecular clock in clock neurons, while rhythm strength is influenced by additional output mechanisms in both clock and downstream neurons, making it more susceptible to modulation by genetic background and female mating status.

Several studies have identified sexually dimorphic gene expression and function in clock neurons in Drosophila. The sex-specific neural circuitry and social behaviors in Drosophila are orchestrated by the interplay of sexually dimorphic transcription factors encoded by fruitless (fru) and doublesex (dsx) (Ellendersen and von Philipsborn, 2017; Sato and Yamamoto, 2023; Yamamoto and Koganezawa, 2013). Previous research has shown that certain clock neuron clusters contain cells expressing a male-specific isoform of fru (Fujii and Amrein, 2010), and that a greater number of clock neurons in a dorsal neuron 1 (DN1) cluster express fru in males compared to females (Hanafusa et al., 2013). A recent transcriptomic analysis of fru-expressing neurons using single-cell RNA sequencing identified a specific set of fru-expressing clock neurons (Palmateer et al., 2023). Interestingly, artificial activation of these neurons had sexually dimorphic effects on the circadian period; males exhibited a shortened period, while females displayed a lengthened period. These findings suggest that fru-expressing clock neurons contribute to the sex differences observed in the present study. Additionally, a subset of dorsal lateral neurons (LNds) shows male-specific expression of Neuropeptide F (NPF) (Hermann et al., 2012; Lee et al., 2006), which is associated with feeding, metabolism, and courtship (Cui and Zhao, 2020). Another neuropeptide, Pigment Dispensing Factor (PDF) is crucial for synchronizing rhythms in DD (Renn et al., 1999), and the absence of PDF or its receptor, PDFR, leads to arrhythmic activity-rest cycles in most male flies (Lear et al., 2005; Mertens et al., 2005; Peng et al., 2003; Yoshii et al., 2009). Intriguingly, a recent study found that mutations in Pdf and Pdfr, have a lesser impact on female circadian rhythms than on males, suggesting a sexually dimorphic role for PDF in circadian regulation (Iyer et al., 2024). The molecular mechanisms by which sex differences in neural circuits and gene expression translate into distinct circadian behaviors in Drosophila warrant further investigation.

Previous studies have documented several post-mating changes in Drosophila females, including increased egg-laying, decreased sexual receptivity, and reduced sleep (Kubli and Bopp, 2012). These changes are primarily driven by SP transferred from males during copulation (Chen et al., 1988; Liu and Kubli, 2003). Our data add reduced rhythm strength to the list of SP-dependent post-mating behavioral changes. Interestingly, the post-mating reduction in rhythm strength was absent in the Oregon-R-P2 strain, implying that unique genetic factors in this variant counteract the typical post-mating changes. Oregon-R-P2 females are known for rapid egg-laying (Lynch et al., 1989), hinting that the post-mating reduction in rhythmicity observed in the other strains may be linked to normal egg-laying. Additionally, recent studies showed that mated females exhibit significantly lower PDF levels compared to virgin females (Riva et al., 2022), and mating and SP trigger changes in the expression of core clock genes along with a loss of rhythmicity in many clock-controlled genes (Delbare et al., 2023). These molecular changes may underlie the post-mating reduction in rhythm strength.

We recently reported that female post-mating reduction in nighttime sleep depends on diet rich in nutrients, including protein, and is absent on 5% sucrose food (Duhart et al., 2023). Diet may also modulate circadian behavior. However, it is challenging to examine the effects of mating on circadian behavior under protein-rich conditions due to the rapid development of larvae, which can disturb behavioral recordings within a few days. Consequently, in our experiments, flies were raised on a nutrient-rich diet but were housed in tubes containing 5% sucrose during the circadian assays. Interestingly, a recent study investigated the effects of sex and mating status on morning anticipation using a protein-rich medium under LD conditions (Riva et al., 2022). Morning anticipation in LD can be assessed within a few days of mating before larval movement becomes problematic. The study found that mated females exhibited reduced morning anticipation compared to virgin females and males, which may be linked to a decrease in rhythm power. How mating influences the circadian period and rhythm strength in DD under nutrient-rich conditions is an interesting but technically challenging topic for future investigation.

Sex differences in circadian behaviors are common across species (Bertossa et al., 2013; Helfrich-Förster, 2000; Walton et al., 2022). For example, in rodents, some studies showed that females generally have shorter endogenous period lengths than males, although this sex difference did not always reach statistical significance (Schull et al., 1989; Sterniczuk et al., 2010; Iwahana et al., 2008; Kuljis et al., 2013). Similarly, women tend to go to bed earlier than men from childhood to menopause, at which time sex differences in sleep patterns disappear (Roenneberg et al., 2007). Moreover, a recent study reported that pregnant women had phase-advanced endogenous temperature and melatonin rhythms (Cain et al., 2010), partly because of a significantly shorter circadian period length (Duffy et al., 2011). While the effects of sex and pregnancy on circadian periods in humans and rodents are well documented, their impact on circadian rhythm strength remains an open question.

Interestingly, whereas males exhibit longer periods than females in humans and rodents, female flies display longer periods than male flies. Species-specific behaviors may underlie these distinct sexual dimorphisms across organisms. For example, a shorter circadian period in male flies may allow them to wake up earlier, making it easier for them to find and court inactive females rather than active ones (Helfrich-Förster, 2000). Investigating how sexually dimorphic tasks influence the molecular clock and circadian behavior may advance our understanding of sex-specific regulation of biological rhythms.

Neurodegenerative disorders, including Parkinson’s disease, Alzheimer’s disease, Amyotrophic Lateral Sclerosis (ALS), cardiovascular disease, and sleep disorders, are associated with circadian disruptions, which may exacerbate these disease symptoms (Fifel and Videnovic, 2021; Videnovic et al., 2014). Additionally, these diseases display sexual dimorphism (Curtis et al., 2017; Haaxma et al., 2007; McCombe and Henderson, 2010; Oveisgharan et al., 2018). Unraveling the genetic and neural factors that drive sex-specific variations in circadian rhythms may provide a broader understanding of circadian misalignment in disease contexts and suggest targeted interventions for improving health outcomes.