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. Author manuscript; available in PMC: 2008 Feb 12.
Published in final edited form as: Curr Biol. 2007 Feb 6;17(3):244–251. doi: 10.1016/j.cub.2006.11.049

Nocturnal Male Sex Drive in Drosophila

Shinsuke Fujii 1, Parthasarathy Krishnan 2, Paul Hardin 2, Hubert Amrein 1,*
PMCID: PMC2239012  NIHMSID: NIHMS38366  PMID: 17276917

Summary

Many behaviors and physiological processes including locomotor activity, feeding, sleep, mating, and migration are dependent on daily or seasonally reoccurring, external stimuli [1–3]. In D. melanogaster, one of the best-studied circadian behaviors is locomotion. The fruit fly is considered a diurnal (day active/night inactive) insect, based on locomotor-activity recordings of single, socially naive flies [4, 5]. We developed a new circadian paradigm that can simultaneously monitor two flies in simple social contexts. We find that heterosexual couples exhibit a drastically different locomotor-activity pattern than individual males, females, or homosexual couples. Specifically, male-female couples exhibit a brief rest phase around dusk but are highly active throughout the night and early morning. This distinct locomotor-activity rhythm is dependent on the clock genes and synchronized with close-proximity encounters, which reflect courtship, between the male and female. The close-proximity rhythm is dependent on the male and not the female and requires circadian oscillators in the brain and the antenna. Taken together, our data show that constant exposure to stimuli emanating from the female and received by the male olfactory and other sensory systems is responsible for the significant shift in intrinsic locomotor output of socially interacting flies.

Results and Discussion

Standard activity monitors are well-suited for recording circadian activity of single flies but not that of multiple interacting flies [5]. To monitor socially interacting flies, we developed a novel assay in which two flies can be observed simultaneously in a 2D arena by using a CCD camera (see the Experimental Procedures and Figure 1). In order to compare our setup with established single-fly activity monitors, we determined the velocity of flies, entrained in 12 hr light:12 hr darkness (12:12 LD), in constant darkness (DD). Concordant with activity-monitor recordings [4, 6], single males (sM) show peak activity around anticipated dusk and dawn, slightly reduced activity during the day, and extended rest during the night (Figure 1A). The activity of single females (sF), however, is characterized by a single peak at dusk (Figure 1B), confirming a previously observed sex-specific difference in locomotor activity [7]. We verified these sex-specific differences by comparing the occurrence and amplitude of peaks in individual males and females at subjective dawn and dusk (Figures 1G and 1H). Almost 70% of males, but less than 10% of females, exhibited a distinct morning peak. Thus, single-fly recordings with “classic” event recorders [7] and our new assay produce highly similar locomotor-activity profiles, validating our experimental setup.

Figure 1. Locomotor Activity Is Different in Males and Females and Modified by Social Context.

Figure 1

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(A–F) Free-running circadian locomotor rhythm of individual flies (A, B, and F) and FM, MM, and FF combinations (C, D, and E) with a 2D arena. Locomotor-activity rhythms of single, sexually naive, and wild-type males (A) and females (B) are similar and show a minor and a major activity peak at subjective dawn and dusk, respectively, and rest during the subjective night. Note that males, but not females, exhibit reduced activity during the day. A novel activity rhythm is observed when a male and a female are present in the arena, with extended period of nightly activity (C). Cohabitation of males leads to severe disruption of the daily rhythm (D), whereas cohabitation of females has no effect on the phase of the rhythm (E). Velocity rhythm of males after prolonged sexual experience “reverts” to that of inexperienced males (F): After cohabitation of 20 males and females for 9 days in 12:12 LD, single males were transferred to dishes and monitored in DD. Error bars denote SEM. n = 8–12 for all but (C) (n = 40). Black bars indicate the subjective night, and gray bars indicate the subjective day for males (M) or females (F), respectively. Flies were combined at dusk of the last day in the 12:12 LD cycle (time point 0). The relatively rapid “dampening” of locomotor activity in FM couples over the 4 day period may be due multiple copulations and hence a reduced sex drive of the male (see also Figure 2).

(G and H) Different locomotor pattern in males and females: Peak frequency is significantly higher (chi-square test; x2 = 34.909, p < 0.0001) in males than females at dawn (CT 0.5–2.5) (G). Regardless of the presence or absence of a distinct peak, male activity during that period is approximately five times as high as that of female activity (H). n = 8 for male; n = 12 for female (4 days each). White and gray bars indicate males and females, respectively. Individual relative peak values were evaluated as follows: maximum velocity in CT 0.5–2.5/minimum velocity in CT 3.5–11.5 (morning peaks), and maximum velocity in CT 12.5–14.5/minimum velocity in CT 15.5–23.5 (evening peaks). (G) Only values greater than three were considered as individual peaks. Morning peaks are sexually dimorphic (two-tailed t test; ***p < 0.001 for h). n.s. stands for not statistically significant. Error bars denote SEM.

(I) Single M, MM couples, and FM couples exhibit distinct locomotor rhythms: Pair-wise ANOVA revealed distinct activity patterns between the three different social settings. Values for the statistical analysis were taken from Figure 1A (M; diamond), Figure 1C (MF; triangle), and Figure 1D (MM; square). For M-versus-MM and MM-versus-MF pairings, the overall effects of time, genotype, and their interaction are significant by two-way ANOVA (p < 0.0001). For an M-versus-MF pairing, the overall effects of time, genotype, and their interaction are also significant by two-way ANOVA (p < 0.01). Post-hoc analysis indicates that significant differences (p < 0.05) in velocity were found in all but the following comparisons: M versus MM flies at ZT 12.5, M versus MF flies at ZT 8.5, and MM versus MF flies at ZT 4.5 and ZT 20.5. Error bars denote SEM.

Heterosexual Couples’ Distinct Locomotor-Activity Pattern Is Coupled to Courtship

We next investigated activity patterns of flies in simple social contexts. After entrainment for several days in 12:12 LD and sexual isolation, two flies from either the same or the opposite sex were placed in the arena and monitored in DD. Differences between the locomotor-activity patterns of homosexual (FF and MM) and heterosexual (FM) couples are apparent (Figure 1): Specifically, locomotor rhythm of FF couples resembled that of single flies (compare Figure 1E to Figures 1A and 1B; see Table S1 in the Supplemental Data available with this article online). MF couples, in contrast, exhibited a distinct locomotor rhythm (compare Figure 1C to Figures 1A and 1B): First, during most of the subjective night and morning, FM couples are highly active. Second, at the approximate time of anticipated dusk (~CT 12), the phase of highest activity in sM, sF, and FF couples, FM couples are the least active. To further investigate the apparent phase shifts and differently located peaks and troughs in the three social settings of sM, MM, and FM, we performed a two-way ANOVA. This analysis shows that the overall effects of time of day, genotype, and the interaction between time and genotype are statistically significant (p < 0.01) in all three cases (Figure 1I). Lastly, we note that single males that were kept with females for several days “revert” to the basic locomotor rhythm of sexually naive males (compare Figure 1A to Figure 1F), suggesting that the FM rhythm is not entrained and requires the continuous presence of a partner of the opposite sex. Finally, MM couples exhibited an erratic rhythm, without easily identifiable peaks and troughs (Figure 1D; Table S1).

We next asked whether locomotor activity is correlated with social interactions and measured close-proximity encounters between two flies (Figure 2 and the Experimental Procedures). Whereas males and females spend only approximately 25% of time in close proximity of same sex partners, heterosexual couples do so at more than 50% of time (Figures 2A–2C). Interestingly, close-proximity encounters of MM and FF couples are evenly distributed, whereas those of FM couples exhibit a distinct 24 hr rhythm. Moreover, overlay of close-proximity and velocity graphs in FM couples revealed that these rhythms are highly synchronized, especially their troughs (Figure 2E; Table S1). We also note that the close-proximity rhythm of FM couples kept in LD show the same dynamics as couples held in DD (Figure 2D). This observation suggests that locomotor and close-proximity rhythms of FM couples are not caused by the artificial DD condition but are generated by the social context in heterosexual couples. Because the close-proximity rhythm is extremely robust and a more direct measure of male courtship than locomotor activity, it will be used in all further experiments. To test whether close-proximity encounters are indeed associated with courtship, we determined the courtship index from time-lapse recordings at three different time points (see the Experimental Procedures). At dusk, the CI was 0.03 (±0.03 SEM), whereas at midnight and dawn, it was 0.89 (±0.02 and ±0.04 SEM, respectively; Figure 2F and Movie S1). Taken together, these observations indicate that high locomotor activity and courtship are linked; indeed, a significant component of courtship involves “following/chasing” of the female by the male [8].

Figure 2. Rhythm of Close Proximity Reveals Circadian Nature of Male Sex Drive.

Figure 2

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Distinct social interactions are revealed by the frequency of close-proximity encounters (≤ 5 mm between the centers of objects) in two flies of the same or different sex. Flies of MM (A) and FF (B) couples exhibit virtually no rhythm of close-proximity encounters and show little interest in each other (see also Table S1). FM couples (C), on the other hand, are in close proximity of each other for most of the night and large parts of the day, and they are interrupted for a few hours around subjective dusk; during this time, mutual interest is reduced to the level observed in MM and FF couples. (D) shows the basic structure of the circadian pattern in 12:12 LD is identical to that in DD, with a low amount of total time in proximity at dusk and a steep increase in the early hours of the (subjective) night. Note that the curve is calibrated at “lights on” (“jump” Zeitgeber 0) because of visual stimuli, which aids the male in tracking the female and staying more effectively in close proximity. (E) shows that in FM couples, the rhythm of close proximity in DD (thick line) and 12:12 LD (thin line), as well as the rhythm of velocity in DD (white boxes), are synchronized, shown by an overlay of the graphs shown in Figures 1C, 2C, and 2D). (F) shows still pictures that are taken of FM couples at different time-points during two consecutive days. At CT 0 (time points 1 and 4 in Figure 2C) and CT 18 (3 and 6), courtship is evident in 11 of 12 pairs, whereas at CT 12 (2 and 5), it occurs only in one pair (see Movie S1). Black bars indicate the subjective night, and gray bars indicate the subjective day. Flies were combined at dusk of the last day in the 12:12 LD cycle (time point 0). Error bars denote SEM. n = 8 for each panel, except (C) (n = 40).

D. melanogaster females reject courting males after a single copulation for several days [9–11]. We therefore investigated the success rate of female rejection behavior. Initial copulation events occurred almost exclusively (38/40) within 3 hr of introducing the flies into the arena. Copulation is followed by a brief period of rest, but the male usually resumes courtship activity within 30 min without additional copulation for several hours. Over the period of an experiment (96 hr), a male copulates approximately three times, indicating that a female cannot sustain rejection behavior. This finding is somewhat in contrast with the notion that fertilized D. melanogaster females refuse to remate for several days [10, 12, 13]. Significantly, less than5% of all subsequent copulations occurred within the 4 hr window around lights-off, when the frequency of close-proximity encounters is low (Figure S1D). Importantly, copulation frequency and peaks of close proximity are highly synchronized, and conversely, the proximity troughs are negatively correlated with these peaks (Figure S1). Taken together, these data suggest that courtship and mating rhythms are directly linked.

Close-Proximity Rhythm Is Controlled by the Male Circadian Clock

Because the male is the active partner throughout courtship, we expected that he—and not the female—enforces the distinct close-proximity rhythm observed in FM couples. To test this, we entrained males or females in different “time zones” in 12:12 LD before bringing them together in the arena (Figure 3). When the female was entrained with an 11 hr difference, the phase of the rhythm was identical to that of synchronized FM couples (compare Figures 3A and 3C; Table S1). However, when the male was entrained 11 hr out of phase, the proximity rhythm was shifted by the same period (compare Figures 3A and 3B; Table S1), indicating that the close-proximity rhythm of FM couples is dependent on the circadian clock of the male.

Figure 3. The Close-Proximity Rhythm Is Dependent on the Male.

Figure 3

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Males and females were entrained separately either with time synchronized (A) or with an 11 hr difference (B and C). The subjective day and night phases for both flies are shown below the graph. Note that the last period before the flies were brought together into the arena was light-on for both flies. In (B), the male is out of phase by 11 hr, whereas in (C), the female is out of phase by 11 hr. Error bars denote SEM. For (A), n = 40, for (B), n = 12, and for (C), n = 8. Black bars indicate the subjective night, and gray bars indicate the subjective day.

Central Pacemaker and Olfactory System Are Required for Robust Courtship Rhythm

Courtship behavior is mediated by the chemosensory, auditory, visual, and possibly mechanosensory systems. Because the close-proximity rhythm is maintained in DD, vision is likely to play no major role (Figures 2C and 2D). To address the contribution of other senses, we performed a series of experiments in which the male was deprived of olfactory and auditory sensory modalities. Surgical removal of the aristae (main auditory organ) or the maxillary palps (minor olfactory organs) had no significant effect on close-proximity rhythm (Figures 4A and 4B). However, when the third antennal segments were removed, or when males were homozygous mutants for the Or83b gene required for the detection of most volatile chemicals [14], frequency of close-proximity encounters was severely reduced at night and early morning (Figures 4C and 4D). For both Or83b mutant flies and flies lacking the third antennal segment, the overall effects of time and genotype were significant by two-way ANOVA (p < 0.001), and the interaction between time and genotype was also significant for the former (p < 0.001), but not the later (p = 0.16). Taken together, these experiments indicate that olfaction is a major sensory modality required for an intense courtship rhythm.

Figure 4. Olfactory Cues Received by the Male Antenna Are Required for Maintenance of the Robust Close-Proximity Rhythm.

Figure 4

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Wild-type males (filled squares) and males in which various sensory modalities were disabled (open squares), either surgically or genetically, were subjected to close-proximity recordings with unaffected females. Removal of aristae (A) or maxillary palps (B) has little effect on close-proximity rhythm. Removal of the third antennal segments (C) or impediment of olfactory perception through a null mutation in the Or83b olfactory-receptor gene (D) in the male reduces close-proximity encounters throughout the daily cycle. For the Or83b flies versus wild-type flies, the overall effects of time, genotype, and their interaction are significant by two-way ANOVA (p < 0.001). For flies lacking the third antennal segment versus wild-type flies, the overall effects of time and genotype are significant by two-way ANOVA (p < 0.0001), but their interaction is not significant (p = 0.16). Post-hoc analysis shows significant differences (p < 0.05) in close proximity between the Or83b and wild-type flies at all times except CT 12.5. Flies were combined 12 hr before dawn of the first 24 hr period. Black bars indicate the subjective night, and gray bars indicate the subjective day. Recording of close proximity was carried out exactly as with intact males (Figure 2C), but three consecutive 24 hr periods of recording (time point 12–84) were pooled and are shown as a single 24 hr interval. Error bars denote SEM.

Drosophila has multiple circadian clocks. The main clock is located in a small number of brain neurons and controls circadian locomotor activity in flies kept in social isolation [1, 15]. Peripheral clocks reside in numerous organs, including the antenna, eyes, and testes, and appear to control intrinsic circadian rhythms within [16–18]. For example, olfactory sensitivity exhibits a daily cycle, a phenomenon that is dependent on the appropriate cycling of the clock genes in olfactory neurons but independent of the cycling of these genes in the central brain neurons [17, 19]. To address the contributions of the central and peripheral clocks to close-proximity rhythms, we exploited the per 7.2:2 transgene, which confers per expression in parts of the brain sufficient for the maintenance of intrinsic locomotor activity of single males in isolation. This transgene is not expressed in the antenna and therefore does not provide an oscillator for the olfactory sensory systems [17, 19]. FM couples in which the hemizygous per01 male did or did not contain the P7.2:2 transgene (Figures S2A and S2B; Table S1) exhibited no close-proximity rhythm, indicating that per01 males’ central clock, which mediates intrinsic locomotor rhythm in socially naive flies, is not sufficient for close-proximity rhythm. However, we cannot completely exclude the possibility that loss of courtship rhythm is in part due to a slightly altered intrinsic rhythm in the central clock of these males [20]. We also note that a functional clock restricted to the antenna and sufficient for cycling olfactory sensitivity [21] is not sufficient for close-proximity rhythm, despite the fact that cyc01 mutant males also show arrhythmic close-proximity behavior (Figure S2C and Table S1). Taken together, these findings are consistent with previous observations indicating that the mating rhythm in Drosophila is governed by clock genes [6, 22].

Resetting of the Central Pacemaker by External Sensory Input

We note a significant weakening in the close-proximity rhythm in FM couples in which the male lacks olfactory input (Figure 4; Table S1). One interpretation of these observations is that multiple sensory channels cooperate in establishing the close proximity rhythm but that the presence of at least one is sufficient for generating such a rhythm. Because a functional central pacemaker in the male is essential for the rhythm as well (Figure 3 and Figure S2) and a requirement for per and tim function in mating rhythms has been reported [6, 22], we propose that external cues perceived by the olfactory and other sensory systems feed into the central pace-maker in the male to cause a shift in circadian activity. The ability of peripheral stimuli to reset the central pacemaker may be a male-specific feature because the shift is male-induced and independent of the female’s phasing of the clock (Figure 3). It is interesting to speculate that sex-specific molecular and/or anatomical differences in the neural circuit of the central clock, also suggested by the different locomotor activities of single males and females (Figure 1) [7], are responsible for the male-induced activity shift in a heterosexual social context.

The influence of social experiences on the circadian clock has been noted [23]. Specifically, it was reported that males entrained together in 12:12 LD show an increase in robustness of circadian locomotor activity when subsequently monitored individually in DD, and conversely, males mixed with males entrained in different time zones or with per01 mutant males exhibit a decrease of robustness in the same assay. However, there are important differences between these studies and the observations presented here: First, Levine and colleagues assessed circadian activity after the social experience has occurred, whereas our recordings were done during the social experience itself; second, we observe drastically distinct circadian-activity profiles (Figure 1I), whereas Levine’s paradigm of different social contexts lead to strengthening or weakening of the one and same circadian profile.

In two previous studies [6, 22], rhythms in mating (but not courtship) were investigated in 12:12 LD-entrained socially naive flies in DD. Tauber and colleagues noted a shift in circadian timing of mating behavior between D. melanogaster and D. pseudoobscura, which they showed to be dependent on the per gene, and they proposed that this difference might contribute to mating isolation and speciation [6]. Sakai and Ishida investigated the role of clock genes in mating and observed peaks at CT 6 and 18, with a clear trough at CT 12 [22]. Interestingly, this trough corresponds to that observed in the close-proximity rhythm of FM couples (Figure 2C), establishing this period of the day as a phase of low sexual activity by two independent assays. These authors also suggested that the female and not the male clock is the determinant of the mating rhythm, whereas our time-shift experiment strongly argues for the opposite (Figures 3A–3C). One possible explanation for this discrepancy concerns the different experimental setups: We measure the locomotor and courtship activity of single pairs of flies over a 4 day period, during which social experience is acquired, whereas Sakai and Ishida monitor the occurrence of a single copulation event in socially inexperienced flies over a period of just 20 min. Thus, the two studies may measure different facets of circadian behavior: courtship drive in a simple social setting over long periods of time and copulation frequency at different zeitgeber times of virgin, socially naive, but sexually starved flies. Another explanation for the female-dependant mating rhythm suggested by Sakai and Ishida may lie in the use of clock mutants, which affect also other behaviors, including court-ship song production [5, 24]. In contrast, our time-shift experiments were performed with wild-type and w/wild-type couples, yielding identical results (Figure 3; Table S1).

To our knowledge, the only previous study that investigated courtship rhythm directly was conducted by Hardeland [25]. He investigated courtship rhythms in different species of the genus Drosophila and found that D. melanogaster exhibited the highest courtship activity during the night; this is consistent with our observation.

Our studies show that rhythms in male sex drive and intrinsic male locomotor activity are out of phase, with highest male sex drive occurring throughout the subjective night and early morning when socially isolated flies rest. Interestingly, Drosophila olfactory responses oscillate in circadian fashion in the antenna independently of the central pacemaker neurons, with the highest sensitivity during the night [17]. The authors in this study suggested that enhanced olfactory sensitivity might facilitate detection of predators, contribute to opportunistic feeding, or contribute to reproductive behaviors at times when flies are usually inactive; the third possibility is consistent with the increased, nocturnal male sex drive described here. Regardless of the biological significance, it seems likely that external chemical stimuli, which may also include allomones and food odors, as well as auditory and visual stimuli can fundamentally modify intrinsic locomotor activity.

How is the shift in locomotor activity brought about in the social context of heterosexual couples? One contributing factor may be that chemosensory detection and recognition of females that are encountered during the night (in the absence of major visual stimuli) is enhanced by a male olfactory system that is tuned to perform best during that period of the diurnal cycle [17]. Interestingly, several male-specific genes expressed in the head have also been reported to be under circadian control [26–28], and at least one of them, sex-specific enzyme1, is expressed in the chemosensory system with peak activity during the night (S.F. and H.A., unpublished data).

It is generally accepted that D. melanogaster is a diurnal insect [1, 2]. However, no studies investigating the circadian behavior of the fly in its natural environment have been reported. Regardless of whether nocturnal sex drive occurs under “real-life” conditions, our findings establish that intrinsic locomotor activity is subject to extensive modification by external social cues. Ultimately, novel assays that consider the “ecological realities” of Drosophila will have to be developed to reveal the largely unknown circadian behavior of this otherwise well-understood animal model system.

Experimental Procedures

Assays for Locomotor and Close-Proximity Rhythms

Flies were collected and separated by sex within 6 hr after eclosion, and approximately 20 animals were kept in a vial for 6–12 days under a cycle of 12 hr light and 12 hr dark. Wild-type Ore-R virgin females and males were used for establishing basic locomotor and close-proximity assays of single flies. All other locomotor and close-proximity assays with FM couples were performed with w1118 females and Ore-R males (experiments 1C, 2C, 2D, 3A–3C, and 4A–4C), Or83b/Or83b males (Figure 4D), Y/per01 males without (Figure S2A) or with (Figure 3B) the per 7.2 transgene, and p[Or83b]GAL4/UAS-CYC; cyc01 ry males (Figure S2C). Males were always kept at 12:12 LD for 6–12 days and at 25°C before use in an experiment. Virgin females were between 2 and 6 days of age when they were used for an experiment and kept under the same 12:12 LD conditions as the males, except for experiments 1B, 1E, 2B, 3B, and 3C (7–12 days of age). A male and a female were briefly anaesthetized with CO2 for loading into a 35-mm-diameter Petri dish (10 mm high) with standard fly food (7.5 ml). Movements of flies in 16 Petri dishes were recorded by a highly sensitive CCD camera and a time-lapse recorder (1 frame per 3 s) under red dim light (<1 lux) and was recorded as velocity (mm/s) for 60-min-sized bins. Experiments for measuring the velocity of sexually experienced males (Figure 1F) were performed with single Ore males, which lived together with w1118 virgin females (1:1 sex ratio) for 9 days in 12:12 LD (Figure 1F). Tracking of the same fly (male or female) in FM couples was possible because of different body size and the appropriate setting of the object-size parameter. Tracking of the same fly throughout the recording period in MM and FF is not assured, and “switching” from one to the other individual may occur. Threshold for close proximity is set to ≤5 mm between the center of the objects.

Statistical Analyses

Rhythmicity of flies or couples (column 5 in Table S1) was determined with spectral analysis, written by Levine and coworkers (for details, see the legend in Table S1) [29]. For statistical analysis of peak occurrence (Figure 1G) and the determination of rhythmicity (column 5 in Table S1), we used the chi-square test, and for statistical analysis of the phase (Table S1), we used the Watson-Williams-Stevens analysis [29]. For statistical analysis of the peak height (Figure 1H), period, rhythmic index, and rhythmic statistics (Table S1), we used the two-tailed t test. Recordings of close proximity with surgically or genetically altered males (Figure 4) were carried out exactly as those with intact males (Figure 2C), but three consecutive 24 hr periods of recording (time point 12–84) were pooled into and shown as a single 24 hr interval. Analysis of the effect of time of day, genotype, and the interaction between day and genotype were analyzed by two-way ANOVA. Post-hoc analysis was performed by Fisher LSD test with alpha values for critical ranges set at 0.05. Statistical analysis was carried out with Statistica (Statsoft).

Analysis of Recordings

The movies were analyzed by EthoVision 3.1 (Noldus) with the following settings: trial protocol: sample rate = 29.970 samples per s; detection method: method = subtraction, object intensity = only objects darker than background, minimum object size = 3, maximum object size = 50, detection thresholds = −255 <-> −25; image filtering: erosion filter = 1 × 1, dilation filter = 1 × 1, filter order = first dilate, then erode; identification: large object minimum size = 4, action if large object missing = use last position, action if small object missing = use position of large object.

Courtship

Courtship Index (CI) was measured in three different experiments at CT 12, 18, and 24 on two consecutive days with time-lapse video (condensed to ~20 s; playback speed was 0.5×). These experiments were performed in 20-mm-diameter Petri dishes (10 mm high). The CI was calculated as fraction of time in which a male was in immediate proximity of the female and engaged in following, singing (wing extension/vibration), or attempting (of copulation, i.e., bending of abdomen) as defined in Hall [8].

Surgical Procedures

Flies were anesthetized shortly at 5 days of age, and both aristae, maxillary palps, or third antennal segments, respectively, were removed with surgical forceps. The flies were allowed to recover for another 5 days at 12:12 LD before they were used in an experiment.

Supplementary Material

supp data. Supplemental Data.

Supplemental Data include additional Experimental Procedures, two figures, one table, and one movie and can be found with this article online at http://www.current-biology.com/cgi/content/full/17/3/244/DC1/.

supp movie
Download video file (1.4MB, mpeg)

Acknowledgments

Fly strains were gifts from J. Hall (per01) and J.M. Giebultowicz (per01/per01; per7.2). We thank A. Toyama for technical assistance, E. Linney for the help with the EthoVision software, and J. Levine for providing a personalized, statistics program (spectral analysis), communication prior to publication, and comments on the manuscript. This work was supported by grants from the National Institutes of Health (NIH)-National Institute on Deafness and Other Communication Disorders (RO1 DC005606-01A2) and National Science Foundation (IBN-03 49671) to H.A. and the NIH to P.H.

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Supplementary Materials

supp data. Supplemental Data.

Supplemental Data include additional Experimental Procedures, two figures, one table, and one movie and can be found with this article online at http://www.current-biology.com/cgi/content/full/17/3/244/DC1/.

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