The Fruit Fly Drosophila melanogaster Favors Dim Light and Times Its Activity Peaks to Early Dawn and Late Dusk

Transcription

1 The Fruit Fly Drosophila melanogaster Favors Dim Light and Times Its Activity Peaks to Early Dawn and Late Dusk Dirk Rieger, Christina Fraunholz, Jochen Popp, Dominik Bichler, Rainer Dittmann, and Charlotte Helfrich-Förster 1 Institute of Zoology, University of Regensburg, Regensburg, Germany Abstract The light preferences of fruit flies were tested by 2 different means. First, flies were allowed to choose between different illuminations, and their favorite resting, grooming, and feeding places were determined with an infrared-sensitive camera. Second, the activity levels of the animals during their main daily activity period were determined photoelectrically (via infrared light beams) under different light intensities. Both methods revealed that the flies prefer dim light. They rested, groomed, and fed preferentially in places with a light intensity between 5 and 10 lux, and they showed the highest activity level when the light intensity during the day was kept at 10 lux. Furthermore, when dawn and dusk were simulated by logarithmically increasing/decreasing the light intensity during a 1.5-h interval, the flies activity maxima occurred at about 7.5 lux during early dawn and late dusk. The results suggest that fruit flies time their clocks by early dawn and late dusk and avoid bright light during the day. Key words diurnal rhythms, dawn and dusk, dim light, Drosophila melanogaster, activity, feeding The fruit fly Drosophila melanogaster is one of the preferential model organisms to study diurnal and circadian rhythms. As with many animals (Aschoff, 1966), fruit flies show bimodal activity patterns with pronounced morning (M) and evening (E) activity peaks and little activity during midday and night. Under lab conditions with artificial rectangular lightdark cycles, they restrict their activity largely to the light phase and are barely active during the night (Hamblen-Coyle, 1992; Rieger et al., 2003; Fujii et al., 2007). Therefore, fruit flies are generally regarded as diurnal animals. However, a recent study showed that flies that experience weak illumination during the night (of about quarter-moonlight intensity) shift their activity prominently into the dark phase (Bachleitner et al., 2007). The same happens when males and females are housed together (Fujii et al., 2007). These studies suggest that the flies may show a different activity pattern in nature and also indicate that the flies may prefer to be active at lower light intensities than previously assumed. Older studies applied daylight intensities ranging between 200 and 2500 lux (Hamblen-Coyle, 1992). In nature, experienced light intensities can vary between 0 and 100,000 lux, depending on the time of day, on the weather, and on the position of the fly in the environment. Little is known about the light preferences of fruit flies under natural conditions. Researchers 1. To whom all correspondence should be addressed: Charlotte Helfrich-Förster, University of Regensburg, Institute of Zoology, Regensburg, Germany; charlotte.foerster@biologie.uni-regensburg.de. JOURNAL OF BIOLOGICAL RHYTHMS, Vol. 22 No. 5, October DOI: / Sage Publications 387

2 388 JOURNAL OF BIOLOGICAL RHYTHMS / October 2007 capturing wild Drosophila find them mostly in holes of decaying fruit and partially hidden from the light (R. Costa and C. P. Kyriacou, personal communication, 2007). This underlines the result obtained with nocturnal moonlight, suggesting that fruit flies avoid bright light in nature. The present study aims to test the light preferences of D. melanogaster more objectively with 2 different methods. In a first set of experiments, individual fruit flies were confined to small photometer cuvettes that had one half shaded and the other half illuminated by green light of different intensity. The fly could freely move within the cuvette. A camera monitored its place in the cuvette, and the proportion of time the fly spent in the shaded and illuminated areas was determined. In a second set of experiments, the activity of the flies was recorded in the usual way by infrared light beams under rectangular light-dark cycles with different light intensities during the light phase. In these experiments, we determined the light intensity at which the flies were most active. Both sets of experiments showed that fruit flies favored low light intensities of 5 to 10 lux, both for rest and activity. In a last experiment, the flies were subjected to light-dark cycles with simulated dawn and dusk. Under such conditions, the flies were most active during early dawn and late dusk, when light intensity was around 7.5 lux. This suggests that the flies use a value of ~7.5 lux during dawn and dusk to time their activity. This confirms the hypothesis of Bünning (1969), who proposed that organisms time their behavior to the very low irradiances occurring during early dawn and late dusk. In summary, our experiments clearly indicate that fruit flies prefer to be active at rather low light intensities. This suggests that they are either crepuscular or that they are diurnal but prefer a shaded environment. Fly Strain MATERIALS AND METHODS The lab strain CantonS was used for the present experiments. This strain was obtained about 20 years ago from Jeffrey C. Hall (Brandeis University) and has been kept for many decades in the lab. The flies were reared under LD 12:12 cycles on Drosophila medium (0.8% agar, 2.2% sugar-beet syrup, 8.0% malt extract, 1.8% yeast, 1.0% soy flour, 8.0% corn flour, and 0.3% hydroxybenzoic acid) at either 20 C or 25 C. Only male flies at an age of 3 to 6 days were used for the experiments. Recording the Activity of Flies with a Camera The flies were briefly anesthetized with CO 2. A single male fly was confined to a photometer cuvette ( cm), the open end of which was then closed with a plain layer of Drosophila medium. Parafilm prevented the desiccation of the medium. Small holes along both long sides of the cuvette provided ventilation. One half of the cuvette had been shaded with a red Edding marker (Edding 3000 permanent marker, col. 002), which reduced light transmission to 5%. The shaded area of the cuvette was either on the side of the food or on the opposite site. Two such cuvettes with a fly each were put on a glass table, which was illuminated from below by fluorescence tubes covered by an opaque glass plate and a red foil (688 nm; ROSCO Laboratories, Ltd., London, UK) (Fig. 1A). An infrared-sensitive video camera (MTI CCD72Ex) was placed on top of the cuvettes to monitor the activity of the flies for 24 h (images were taken every 2 sec). The flies were illuminated for 12 h with green light supplied by LEDs (luxeon emitter LXHL-PE01, 505 nm; Future Electronics, München, Germany). The light was in phase with the LD 12:12 cycle during rearing, and light intensity was adjusted by a computer program to 10, 100, or 1000 lux. The corresponding light intensity under the shaded area was 0.5, 5, and 50 lux. Since the illumination by the green LEDs did not provide a clear border between the 2 areas, a gray area of 5 mm was defined between both areas that putatively received intermediate irradiances. The camera was equipped with the same red filter (688 nm) that covered the fluorescence lamps, so that the light quality entering the camera remained constant throughout the recording period (otherwise, the green light during the light phase of the LD cycle would perturb image quality). The entire setup was put into a climate box (Percival Scientific I-36VL), where the temperature was kept constant at 20 C. The camera was connected via a frame grabber (video digitizer VIDEO 1000 ST, Fricke, Berlin) with an ATARI computer (Mega ST) outside the climate box. The fly s activity was recorded using the program Oxalis and analyzed with Oxalprim (Joachim Schuster, Version 5.30x, 1994). The programs determined the position of the fly in the cuvette every 2 sec (detailed description in Schuster and Engelmann, 1990). The position was given in x and y coordinates, stored on the hard disk, and later analyzed with the program Oxalprim (Joachim Schuster, Version 5.30x, 1994). With Oxalprim, the fly s positions in the cuvette could be

3 Rieger et al. / LIGHT PREFERENCES OF FRUIT FLIES 389 Figure 1. (A) Recording system for the flies rest and activity using a video camera, digitizer, and ATARI computer. The flies were placed in photometer cuvettes that had one half shaded by red ink and the open end closed by food. Always 2 flies with the shaded halves next to and opposed to the food, respectively, were recorded simultaneously. For details, see the text. (B) Method to determine the position of the fly in the x-axis of the cuvette. On top, the cuvette is shown schematically with the divisions in illuminated and shaded areas, as well as in a gray area between both. The diagram shows the movements of the fly over a 24-h time period. High locomotor activity of the fly is indicated by horizontal white traces crossing the x-axis. Thinner vertical traces indicate that the fly was resting or only slightly moving as it would occur during grooming. During the rest and grooming phases, the x position of the fly in the cuvette could be determined easily. The gray vertical lines indicate this position at 2 different time points. depicted during a defined time interval either as isolated dots or as dots connected with lines, so that the fly could be traced in the cuvette (see Fig. 2). Alternatively, x and y diagrams of the fly s position over time could be plotted, which allowed an easy determination of the insect s position in the cuvette, especially during its rest phases (see Fig. 1B for an x diagram). To analyze the resting behavior quantitatively, we calculated the overall rest time of each individual fly during the day and night and set this value to 100%. Then, we determined the percentage of time the fly spent in the illuminated area of the cuvette, in the gray area, and in the shaded area. Later, average percentages for all flies were calculated out of the individual values. Activity was monitored during consecutive 4-min intervals. Any activity within an interval resulted in a scan value of 1, while no activity resulted in a value of 0. Since this method did not yield the absolute activity level of the fly, a new activitymonitoring device was constructed, which registered how often the fly crossed the infrared light beam within 1-min intervals. Furthermore, a LED-based lighting system was designed, which allowed a logarithmic increase in irradiance in 1-min steps within a certain interval (here 1.5 h). This allowed a rough simulation of dawn and dusk. First, the flies were recorded in our usual recording system under rectangular LD cycles with different irradiances (10 lux, 100 lux, 500 lux, 1000 lux; light source: halogen photo-optic lamps [Xenophot 12V, 120W; Osram, Berlin, Germany] equipped with a heat filter). Temperature was adjusted to 20 C and, in one experiment, to 25 C. Recording lasted from 7 to 10 days. In the second set of experiments, the flies were recorded under similar conditions (at 20 C) in the new activity-monitoring device to determine their absolute activity values (10 lux, 100 lux, 500 lux, 1000 lux; light source: white LEDs; Lumitronix LED-Technik, GmbH, Jungingen, Germany). After 1 week of recording, they were recorded under simulated dawn and dusk (again at 20 C). Within a 1.5-h interval, irradiance was increased/decreased logarithmically in 1-min steps from 0 to 10 lux, 0 to 100 lux, 0 to 500 lux, or 0 to 1000 lux. Recording the Activity of Flies with Infrared Light Beams In other experiments, locomotor activity of individual male flies was recorded photoelectrically as described previously (Helfrich-Förster, 1998). Briefly, the flies were confined to photometer cuvettes that were placed with one end in an infrared light beam. Data Analysis The raw data were displayed as actograms using the program El Temps (v.1.192, Antoni Diez-Noguera, Barcelona, 1999; ElTemps). Mean activity profiles of individual flies and of groups of flies were calculated for each chosen light intensity and light program as performed previously (Helfrich-Förster, 2000). The average days

4 390 JOURNAL OF BIOLOGICAL RHYTHMS / October 2007 shown in the diagrams are smoothed by a moving average filter. Filtering was done over 5 values for the 4-min bin data stemming from the old recording system and over 10 values for the 1-min interval data of the new system. To reveal the phase relationship of the M and E peaks to lights-on or lights-off, the maxima of both peaks were determined and plotted in phase plots as described (Helfrich-Förster, 2000; Helfrich-Förster et al., 2001; Rieger et al., 2003). Mean activity levels were calculated over the entire light-dark cycle, for the light phase, for the dark phase, and, in the case of simulated dawn, for the 1.5-h dawn and dusk phases. Statistics A 2-way analysis of variance (ANOVA) was used to analyze the effects of light and food on the resting place of the flies in the cuvettes as well as on the times they were active. The activity levels and the phases of the activity maxima found with the infrared beam recordings under the different light regimes were statistically compared with a 1-way ANOVA and Bonferroni s post hoc analysis. RESULTS Activity Patterns and Traces of Flies in the Photometer Cuvettes For the first set of experiments, we chose a light intensity of 1000 lux during the light phase. Thus, the flies could chose between 1000 lux in the illuminated area of the cuvette and 50 lux in the shaded area. Consequently, the gray zone was illuminated by values between 50 and 1000 lux. On average, a fly spent 32% of the day being active and 68% resting or grooming. As already known from the activity recordings with infrared light beams, most flies were preferentially active in the morning around lights-on and in the evening around lights-off and showed less activity during midday and midnight (Fig. 2). During these main activity times (M and E peaks), the flies ran preferentially along the borders of the cuvettes, apparently ignoring food, light, and shadows (Fig. 2A). Crosses of the cuvette centers were rare. The flies activity outside the M and E peaks was different. They neither ran along the borders of the cuvette nor completely avoided the center of the cuvette. Instead, they showed short-distance movements within Figure 2. Two typical activity traces of male wild-type flies in the photometer cuvettes. Schemes of the cuvettes indicating the position of food, light, and shadow are shown on the left; x diagrams of the flies activity over 24 h are shown in the middle; and traces or single-position dots of the flies in the cuvettes are shown on the right. The light-dark schedule is shown as white and black bars on top of the x diagram. The fly in A had the food positioned in the illuminated area of the cuvette and showed prominent M and E activity peaks, very little activity during the day, and some activity during the night. Its preferred resting position was in the shaded area of the cuvette. During the main activity period, the fly was running along the borders of the cuvettes, as can be seen in the activity trace to the right that was monitored during 1 h of its E peak. The fly in B had the food in the shaded area of the cuvette, showed less prominent M and E peaks, and had small-distance activity during the day, which was mainly directed to the food, and very little activity during the night. Its main resting place was in the shaded area of the cuvette during the day, as can be seen in the single-position dots in the cuvette shown to the right; this plot represents 6 h, beginning at lights-on. The x diagram shows that the fly always kept a certain distance to the food while resting. During the night, the fly rested in the cuvette half opposite to the food. the cuvettes that were frequently restricted to a certain cuvette half and were often directed to the food (Fig. 2B). During their rest periods, which lasted from only a few minutes up to several hours during midday through to the night, the flies appeared to clearly prefer certain positions in the cuvettes. The latter were dependent on the position of the food and, during the light phase, on the distribution of light in the cuvette (Figs. 2, 3). We determined the percentage of time the flies spent resting or grooming in the different parts of the cuvette. Influence of Food and Light on the Flies Resting Place The food was either placed in the illuminated (Fig. 2A) or the shaded side (Fig. 2B) of the cuvette.

5 Rieger et al. / LIGHT PREFERENCES OF FRUIT FLIES 391 As can already be seen in Figure 2, the flies rested preferentially in the cuvette half opposite the food, and this behavior was most pronounced during the night. When the food was placed in the illuminated half of the cuvette, the fly preferred the cuvette half opposite to the food also during the day (Fig. 2A). However, when the food was in the shaded half of the cuvette, the fly changed its resting place during the day and rested in the shaded area close to the food (Fig. 2B). Nevertheless, the flies always kept a certain distance from the food while resting (see Fig. 2B). These results indicate that the flies avoid resting close to the food and also prefer the shaded area in the cuvette during the light phase. This tendency was confirmed by a statistical analysis (Fig. 3). During the night, the flies spent about 80% of their resting time in the cuvette half opposite the food, including the defined gray zone (83.6% if food was placed in the cuvette half that was shaded during daytime and 82.8% if food was in the half illuminated during daytime), and only about 20% of their resting time in the cuvette half containing the food (16.4% if food was placed in the area that was shaded during daytime and 17.2% if food was in the area illuminated during daytime; Fig. 3). ANOVA revealed a significant effect of the food on the position of the fly in the cuvette, F (1, 40) = , p < During the day, the flies spent about 94% of their resting time in the shaded area of the cuvette, including the gray zone, and only 6% of the time in the illuminated area of the cuvette. ANOVA revealed a significant effect of light on the flies resting place, F (1, 40) = , p < 0.001, but no significant influence of the food, F (1, 40) = 0.457, p = Thus, during the day, the effect of light clearly overrides the effect of the food on the resting position of the fly. We concentrated on the effects of light on the resting position of the flies, analyzing only the light phases of the LD cycles. Although we found that the Figure 3. Mean percentages of time the flies rested in the shaded area, the illuminated area, and the gray area of the cuvette during the light phase (A, day) and the dark phase (B, night). The cuvette schemes on top of the columns indicate the position of food, light, and shadow. During the day, the flies rested preferentially in the shaded area, regardless of whether this was close to or opposite the food. During the night, the flies rested preferentially in the cuvette half that was opposite to the food. The percentage of time spent in the different areas plus the standard error of the mean areas are indicated in the columns. position of the food did not significantly influence the resting behavior of the flies during the day, we placed the food half the time in the shaded area and the other half in the illuminated area of the cuvette to avoid any hidden influences of food position. Light Preferences of the Flies for Resting The results obtained at illuminations between 50 and 1000 lux clearly indicated that the flies preferred the lower light intensity of 50 lux for resting. Our next aim was to determine the preferred light intensity of the flies. Therefore, we let them also choose between 5 and 100 lux and between 0.5 and 10 lux and determined the percentage of time they spent in the zones defined above. We found that the flies still preferred to rest in the shaded area if they could choose between 5 and 100 lux (Fig. 4). However, the percentage of time they spent in the gray area increased from 24% to 36%. When they could choose between 0.5 lux and 10 lux, they spent most of their resting time in the gray area. ANOVA revealed that the light preferences of the flies were significantly

6 392 JOURNAL OF BIOLOGICAL RHYTHMS / October 2007 Figure 4. Mean percentages of time the flies rested in the shaded area, the illuminated area, and the gray area of the cuvette during the light phase at different light intensities. The percentage of time spent in the different areas plus the standard error of the mean areas are indicated in the columns. The data indicate that the flies prefer an illumination between 5 and 10 lux for resting. different at the 3 illuminations chosen, F (2, 105) = , p < These results indicate that the flies prefer light intensities between 5 and 10 lux for resting and grooming. Influence of Food and Light on the Flies Activity The next question was whether the flies also have a light preference for being active. Our programs did not allow the calculation of absolute activity levels (e.g., distances walked during a certain interval), but we could calculate the percentage of time the flies were active during the entire day, during the light period, and during the dark period. We found that the different light intensities (10 lux, 100 lux, and 1000 lux) did not affect the overall activity times of the flies. They spent 32% of an entire light-dark cycle being active and 68% being inactive (resting and grooming) at all 3 light intensities. Nevertheless, there seemed to be differences in feeding activity between day and night, depending on the position of the food. The fly shown in Figure 2B that had the food in the shaded area was rather active during the day, and most of this activity consisted of walking to the food and back to the resting place. On the other hand, the fly with the food in the illuminated Figure 5. Effect of the position of the food on the activity during day and night. The kind of activity represented here is mainly short-distance activity from the resting place to the food and vice versa. Activity is given as the percentage of time the flies spent being active in the 12-h light phase (day) and the 12- h dark phase (night). The flies showed longer activity times during the day, if the food was in the shaded area, and increased their activity times in the night, if the food was in the illuminated area. The data suggest that the flies show foraging activity primarily in the shadow. area (Fig. 2A) showed less activity during the day but slightly more activity during the night. We calculated the percentage of time of the day and night when flies were active under the different conditions (food in the shaded or illuminated area of the cuvette under 10 lux, 100 lux, and 1000 lux). We found that the flies were more active during the day if the food was in the shaded area, whereas they were more active during the night when the food was positioned in the illuminated area (Fig. 5). These results suggest that the flies prefer to eat in a shaded area, and if this is not possible during daytime, they shift their feeding activity into the night. ANOVA revealed that this behavior was the same at all 3 tested light intensities. There was a significant effect of the position of the food on activity, F (1, 102) = 8.706, p = 0.004; no effect of light intensity, F (2, 102) = 0.305, p = 0.738); and no interaction, F (2, 102) = 0.025, p = Influence of the Light Intensity on the Activity Level The results obtained with the camera clearly indicate that the flies preferred low light intensities for resting, grooming, and feeding as well as for shortdistance movements. But did the different light intensities also affect the activity level during the main activity times in the morning and the evening? We used our conventional activity monitors, where the fly s activity was measured with an infrared light

7 Rieger et al. / LIGHT PREFERENCES OF FRUIT FLIES 393 beam. The infrared light beam was placed in one corner at the border of the cuvette, so that the fly only passed it when it was running along the borders of the cuvette, a behavior the flies showed preferentially during the morning (M) and the evening (E) (see Fig. 2). Therefore, mainly the running behavior of a fly during its activity phases was measured. Consistent with that assumption, all flies showed prominent bimodality with M and E peaks (Figs. 6, 7). In a first experiment, we recorded the activity of flies under 12:12 LD cycles with 10, 100, 500, and 1000 lux at 20 C to determine their activity profiles and activity levels. Here we used our old recording device, where we determined whether a fly was active in 4-min intervals. We found that the activity profiles of the flies altered slightly at the different irradiances (Fig. 6). The higher the irradiance, the longer the midday trough and the narrower the M and E peaks. Light intensity also had a significant effect on the mean daily activity level, F (3, 147) = 9.502, p < The higher the irradiance, the lower the activity level. The reduction of the mean activity level was due to a reduced activity during the light phase; the activity level during the dark phase did not change significantly, F (3, 147) = 0.706, p = (Fig. 6). We conclude that the flies not only prefer lower light intensities for resting, grooming, and feeding but are also more active at low light intensities than at high ones. Next, we were interested in whether the preference for lower light intensities applies also at a higher ambient temperature, and we repeated the same experiment at 25 C (again with the 4-min interval method). We found a similar tendency of the flies to increase the midday trough and to decrease the activity level at higher irradiances (Fig. 6). Nevertheless, the differences were not as pronounced as they were at 20 C: At 10 lux, the midday trough was stronger at 25 C than at 20 C, but it did not increase to the same extent at 1000 lux as it did at 20 C. Similarly, activity at 10 lux was lower at 25 C than at 20 C, but at 1000 lux, it did not decrease to the same low amount as it did at 20 C. ANOVA revealed a significant interaction between temperature and light intensity on the activity level, F (3, 318) = 2.639, p = Despite these differences at 20 C and 25 C, the flies preferred to be active at lower light intensities under both temperatures, F (3, 318) = , p < In a further experiment, we tested whether the increase in activity level under lowered irradiances could also be observed when the activity level was determined in a more stringent way (e.g., when the number of infrared light beam crosses was counted in Figure 6. Average activity profiles and mean running activity levels of flies recorded photoelectrically with infrared light beams at different light intensities and 2 temperatures. Within a 4-min bin, it was determined whether the fly was active (see Materials and Methods). The numbers in the diagrams indicate the numbers of flies tested at each light intensity. With increasing light intensity, the midday trough became large, and the activity level decreased. The latter was due to a decrease in the activity during the day (L); the activity level during the night (D) remained constant. This behavior was less pronounced at 25 C than at 20 C. 1-min intervals [= absolute activity level]). We found that the absolute activity levels were also strongly dependent on the light intensity (Fig. 7B): the higher the light intensity, the lower the activity level. Nevertheless, the relation between activity level and light intensity was not as linear as it was before; the activity levels at 10 lux and 100 lux as well as those at

8 394 JOURNAL OF BIOLOGICAL RHYTHMS / October 2007 Figure 7. Average activity profiles and mean activity levels of flies recorded photoelectrically at 20 C, whereby the absolute number of infrared light beam crosses was monitored every minute. (A) Back-calculation of the values shown in B into the 4-min interval method, (B) rectangular LD cycles, and (C) LD cycles with simulated dawn and dusk. For details, see the text. 500 lux and 1000 lux were not significantly different from each other. Furthermore, the mean activity profiles of the absolute activity levels revealed clear differences from those in which activity was judged as 1 or 0 during 4-min bins. (1) The M peak was smaller if the absolute activity was determined, (2) the activity peaks were more pointed with a clear maximum at lights-on and lights-off, and (3) a strong lights-off activity peak was present. To test whether these differences were due to the method (absolute activity in 1-min bins, instead of determining whether activity or no activity occurred in 4-min bins) or due to the different illumination (LEDs instead of halogen lamps), we converted the data into the old 4-min bin format (Fig. 7A). The conversion showed that mainly methodical differences existed. The morning peak became larger after the conversion, the activity peaks were less pointed, and the lights-off peak disappeared. Only the overall activity level did not rise to the same level as it was under the halogen illumination. Thus, we may conclude that the LED illumination had a higher subjective intensity for the flies than the halogen illumination. All other differences were due to the method. Obviously, the flies show maximal activity at lights-on and lights-off and a very high activity peak lasting for several minutes after lights-off. This lights-off peak is clearly eliminated by the 4-min bin method. Despite the methoddependent differences, we can conclude that the activity level and the broadness of the midday trough clearly depend on the light intensity. Figure 8A shows that the activity level during the M and E peaks strongly decreased with increasing light intensity. This figure depicts the absolute activity levels during 3-h intervals around lights-on and lightsoff. It also shows that the lights-off effect was stronger the higher the light intensity (see also below).

9 Rieger et al. / LIGHT PREFERENCES OF FRUIT FLIES 395 Activity under Simulated Dusk and Dawn Our next aim was to test whether the simulation of dawn and dusk influenced the activity pattern of the flies. Since the previous experiments showed that flies are most active at around 10 lux, we would expect that the flies would be most active when dawn and dusk reached a light intensity of that level. Indeed, the flies shifted the maxima of the M and E peaks into dawn and dusk (Fig. 7C). This was true at all light intensities, regardless of whether light was increased from 0 to 10 lux, 0 to 100 lux, 0 to 500 lux, or 0 to 1000 lux. Nevertheless, the light intensity still had a significant effect on the activity level. As found before, the overall activity level decreased with increasing light intensity (Fig. 7C). The same was true for the mean activity level during the light phase, including dawn and dusk (Fig. 7C), and during dawn and dusk alone (Fig. 8B). The direct comparison between the activity patterns under rectangular LD cycles and LD cycles with simulated dawn and dusk revealed several significant differences. This was especially visible when the mean activity profiles during dawn and dusk were plotted at a larger scale (Fig. 8). (1) The amount of activity before lights-on and after lights-off was significantly reduced under dawn and dusk conditions; we did not see any anticipation of lights-on or a lights-off effect (compare Fig. 8A with 8B). (2) The activity maxima of the M and E peaks occurred clearly after lights-on and before lights-off under dawn and dusk conditions (Fig. 8B), whereas these were around lights-on and at lights-off in rectangular LD cycles (Fig. 8A). (3) Under dawn and dusk conditions, the phases of the activity maxima of the M and E peaks were different at the 4 final light intensities; they occurred earlier/later depending on the steepness of the increase/decrease in irradiance (Fig. 8B). Under rectangular LD cycles, the phases of the M and E peaks remained constant at all light intensities (Fig. 8A). (4) The flies spent the highest percentage of their overall activity during the dawn and dusk period. This was 55% of their activity, as opposed to 31% of their activity during the same time interval under rectangular LD cycles (averaged over all light intensities). The percentage of activity spent during dawn and dusk increased with increasing light intensity (Fig. 8B, panels on the bottom). Simultaneously, the percentage of activity during the first half hour of the night increased slightly, whereas that during the first and last half hours of the day clearly decreased. The same light-dependent activity decrease occurred during the rest of the day (compare Fig. 7C). Under rectangular LD cycles, the most prominent lightdependent changes in activity level occurred for the lights-off peak. The percentage of activity during the latter clearly increased with increasing light intensity (Fig. 8A, panels at the bottom). In summary, these results indicate once more that the flies tend to avoid activity at higher illumination: if possible, they shift activity into dawn and dusk; if there is no dawn and dusk, they shift activity into the dark phase, showing a higher lights-off response. We then determined the peak points of M and E peaks under dawn and dusk in each single fly plus the corresponding light intensity. We found that the times of the M and E peaks clearly depended on the final light intensity (10, 100, 500, or 1000 lux; ANOVA for M peak: F (3, 97) = , p < 0.001; ANOVA for E peak: F (3, 97) = , p < 0.001), but they occurred always when light intensity had reached a value around 7.5 lux, suggesting that there is a certain threshold light intensity (below 7.5 lux) that triggered the onset of activity in all cases. The gentler the increase in light intensity (e.g., from 0 to 10 lux within the 1.5-h interval), the harder it seemed to be for the flies to determine this threshold; the distribution of activity peaks was rather broad (see wide bars of standard deviation in Fig. 8B, 10 lux). The steeper the increase in light intensity, the smaller the standard deviation from the mean of the activity peaks (Fig. 8B, 1000 lux). DISCUSSION In the present study, we show that fruit flies have a clear preference for lower light intensities: they rest, groom, and prefer to feed in the shadow. In addition, they restrict their main activity to the morning and evening hours, and they show a higher overall locomotor activity if the daytime illumination is low. Restricting activity to the morning and evening was more pronounced at higher light intensities and most extreme if the flies were offered a 1.5-h period of dawn and dusk. Then, most activity was spent during the dawn and dusk period, and the activity peaks occurred as light intensity reached 7.5 lux. Although it seems adaptive to avoid bright light because of the deleterious effects of UV light, these observations are somewhat in contrast to the view that flies are day-active species that show a positive phototactic

10 396 JOURNAL OF BIOLOGICAL RHYTHMS / October 2007 Figure 8. Average activity profiles during the morning and evening at an enlarged scale under (A) rectangular LD cycles and (B) simulated dawn and dusk. The light program with the intensity plotted logarithmically is indicated as a gray line in each activity profile. The M and E peaks (± standard deviation) are indicated on top of the activity profiles (in B). Data are smoothed by moving the average over 10 values. Therefore, the lights-off peak appears to start before lights-off under rectangular LD cycles (A); this is an artifact. The mean activity levels shown below the activity profiles are given in absolute activity values and in percentages of the whole activity in certain intervals around the M and E peaks. For the M peak, the intervals are as follows: 1 h before lights-on (ZT23-ZT0, black bars), 1.5 h after lights-on (ZT0-ZT1.5, light gray bars during rectangular LD cycles [A] and dark gray bars during dawn simulation [B]), and 1.5 to 2.5 h after lights-on (ZT1.5-ZT2.5, light gray bars). For the E peak, the intervals are as follows: 1.5 to 2.5 h before lights-off (ZT9.5- ZT10.5, light gray bars), 1.5 h before lights-off (ZT10.5-ZT12, light gray bars during rectangular LD cycles [A] and dark gray bars during dusk simulation [B]), and 1 h after lights-off (ZT12-ZT13, black bars). For details, see the text.

11 Rieger et al. / LIGHT PREFERENCES OF FRUIT FLIES 397 behavior (Benzer, 1967; Ballinger and Benzer, 1988). Our results rather indicate that fruit flies may be negatively phototactic and crepuscular, as are mosquitos (Saunders, 1982; Shinkawa et al., 1994). Alternatively, fruit flies may be day active but prefer a shaded environment under normal undisturbed conditions. The latter explanation is more likely than the former for several reasons. First of all, recordings of D. melanogaster flies in nature show that they are clearly day active (R. Costa and C. P. Kyriacou, personal communication, 2007). Second, wild Drosophila flies are often found on and in compost heaps, under leaves, and in decaying fruit inside or outside of houses, mostly hidden from the light. The latter does not exclude that the flies react with a positive phototactic response when they try to escape after a disturbance. Phototactic behavior is usually measured for several minutes after the flies have been transferred into a glass tube and forced to choose between lightened and darkened Y-wings (Hirsch and Boudreau, 1958; Hadler, 1964) or countercurrent tubes (Benzer, 1967). Under these conditions, they try to escape toward the light. It is, however, not clear what they would do over a longer period. One study indicated that phototaxis of flies depends on the experimental design (Hadler, 1964). Furthermore, not all flies prefer the high light intensities when exposed along a gradient of light intensities (Parsons, 1975). Natural populations of D. pseudoobscura are phototactically neutral but could be selected within 10 generations for positive phototaxis (Dobzhansky et al., 1974). A critical factor that could also influence the light preference of a fly is the ambient temperature. High temperatures will probably prevent a fly from moving into the light, whereas lower temperatures are less restrictive. Indeed, temperature has been shown to have a significant effect on the bimodal activity pattern of the flies (Majercak et al., 1999). At higher constant temperatures, the midday trough becomes broader, and the M and E activity peaks shift into the night, probably as an adaptation to avoid the midday heat. We found a similar effect for light in the flies recorded at 20 C; the higher the light intensity, the broader the midday trough (Fig. 6). Most interestingly, this influence of the light intensity on the behavior was dependent on the temperature, and it was less evident at 25 C. Thus, temperature and light intensity may interact in a complex manner in influencing the fly s activity level and pattern. Tomioka et al. (1998) showed that the interaction of light and temperature on the activity also works in clockless flies. per 0 mutants that were diurnal at 25 C avoided the light period at 30 C and became mainly nocturnal. Another factor that may influence the flies activity is the availability of food. Here, we show that flies preferred to feed in the shadow, and they significantly reduced their activity during the day if food was only available in the illuminated area of the cuvette. Furthermore, they avoided sitting directly on the food during rest. This may be to avoid sticking to the food or, because in nature, there may be a higher risk of predation nearer the food or in the light. Nevertheless, these observations show that the position and the availability of food may also influence activity. In temperate regions, the availability of food, irradiance, and temperatures are not constant throughout a 24-h cycle. Furthermore, the maximal and minimal values as well as the fluctuations of these parameters are quite different during the seasons. It is quite imaginable that low night and morning temperatures that are typical for spring and early summer in northern Europe will prevent a fly from becoming active in the night and early morning, whereas more favorable temperatures occurring during midday will provoke activity and perhaps completely suppress the midday trough that is observable under lab conditions. In mid- and late summer, this may be different. The favorable temperature will occur earlier and may be already beyond the optimal range during midday. Consequently, one would expect that a fly starts activity earlier and shows a more prominent midday trough than under spring conditions. That latitude can strongly influence the activity pattern of flies from the same species in the aboveindicated manner has been shown for D. ananassae captured from Sri Lanka and India (Joshi, 1999). A strain captured in the colder north had unimodal activity patterns, whereas a southern strain had bimodal activity patterns. Similar differences in the activity patterns were found in D. helvetica strains, which live at different altitudes in the Himalayas (Keny et al., 2007). A high-altitude strain showed a unimodal activity, whereas the activity pattern of the low-altitude strain was bimodal. This was true when the flies were recorded under natural conditions in the field as well as under lab conditions. Most interestingly, the activity onset and offset was strongly dependent on the ambient temperature. Flies of the high-altitude strain began activity about 4.5 h after sunrise, when temperature was ~19 C and light intensity was close to maximum levels (79,000 lux),

12 398 JOURNAL OF BIOLOGICAL RHYTHMS / October 2007 and they terminated activity 1.4 h before sunset, when temperature was ~14 C and light intensity was 17,000 lux. In contrast, the bimodal low-altitude strain started activity during early dawn (~1 h before sunrise, when temperature and light intensity were ~20 C and 1 lux, respectively) and terminated activity ~0.4 h after sunset at a temperature of ~22 C and a light intensity of 30 lux. Neither strain showed nocturnal activity. Under simulated dawn and dusk, Drosophila displayed little activity before dawn, and the onset of activity was clearly dependent on the light intensity. The so-called lights-on effect or startle response occurring after the lights have turned on was completely absent. Similarly, there was little activity after dusk, and the prominent lights-off effect that we observe under LD conditions disappeared under simulated dawn and dusk. So far, much less attention has been paid to the lights-off effect as compared to the lights-on effect. This is clearly due to the recording method. Our old recording system that measured activity in 4-min bins barely revealed the lights-off effect, but it did show the lights-on effect. The simple reasons for this difference are that the lights-on effect lasts longer than the lights-off effect and that the activity is already maximal before lights-off, whereas it is low before lights-on. Thus, the increase in activity after lights-on is easy to see, whereas a further increase of activity after lights-off is less evident and difficult to score if one only determines whether an animal is active, as we did with our old system (compare Fig. 7A and 7B, which depict the same data). For similar reasons, the lights-off effect might also have escaped the attention of the groups that recorded absolute activity in 30-min bins (e.g., Hamblen- Coyle, 1992). More important than the absence of lights-on and lights-off effects under simulated dawn and dusk conditions is the observation that the timing of M and E activity peaks strongly depends on the light intensity. This led us to speculate that a certain intensity threshold triggers the onset and end of activity and that this threshold is rather low. This leads back to Bünning s hypothesis that organisms time their behavior to the very low irradiances occurring during dawn and dusk (Bünning, 1969). The reason for this lies in the fact that the day-to-day fluctuations in light intensity are smallest during early dawn and late dusk, when the irradiances are still below 10 lux. We have recently shown that the internal molecular clock of the same strain of D. melanogaster is indeed so light sensitive that it can be shifted by irradiances of 0.03 lux (Bachleitner et al., 2007). The ability to time their behavior to the low irradiances occurring during dawn and dusk enables organisms to maintain a very stable entrainment to the environmental 24-h oscillations. That this is true has been shown for the scorpion Androctonus australis (Fleissner et al., 1996); under dawn and dusk, not only was the beginning of nocturnal activity more precise, but also the activity pattern was very compact and precisely coupled to other rhythmic parameters such as the sensitivity of the eyes. Furthermore, lower light levels were sufficient for synchronization of locomotor activity rhythms (Fleissner and Fleissner, 1998, 2002). Fleissner and Fleissner (1998) have claimed that simulating dawn and dusk is extremely important to understand the real entrainment of organisms. The present and numerous previous results gained in different species show that they have been right (Kavaliers and Ross, 1981; Boulos et al., 1996a, 1996b, 2002; Gorman and Zucker, 1998; Boulos and Macchi, 2005). In the future, it will be important to record wildtype D. melanogaster strains under the type of conditions described here and, moreover, to test whether female flies behave similarly to males. It will also be most interesting to compare wild-type strains caught at different latitudes and altitudes to see whether these have developed different activity patterns and eventually critical light thresholds for timing M and E peaks. Furthermore, studying the different photoreceptor mutants existing in D. melanogaster will help to reveal the roles of the many photorececeptors in exactly timing the M and E activity to dawn and dusk. ACKNOWLEDGEMENTS We thank Günther Stöckl for developing the new photoelectrical recording device plus the adequate software, Angelika Kühn for excellent technical assistance, and Taishi Yoshii and Christian Bergmiller for writing programs facilitating recording and data analysis. 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