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Published as doi: 10.1096/fj.06-8032com.
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Natural plasticity in circadian rhythms is mediated by reorganization in the molecular clockwork in honeybees

Yair Shemesh, Mira Cohen and Guy Bloch1

Department of Evolution, Systematics, and Ecology, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel

1Correspondence: Department of Evolution, Systematics, and Ecology, The Alexander Silberman Institute of Life Sciences, Berman 114, Givat Ram Campus, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. E-mail: bloch{at}vms.huji.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Various animals naturally switch to considerable periods of around-the-clock activity with no apparent ill effects. Such plasticity in overt circadian rhythms might be observed because the clock is masked by the influence of external factors, is uncoupled from behavioral outputs, or results from genuine plasticity in the clock machinery. We studied honeybees in which plasticity in circadian rhythms is socially modulated and associated with the division of labor. We confirm that "nurse" bees care for the brood around-the-clock even when experiencing a light:dark illumination regime. However, nurses transferred from the hive to individual cages in constant conditions have robust circadian rhythms in locomotor activity with an onset of activity at the subjective morning. These data indicate that circadian rhythmicity in nurses depends on their environment, and suggest that some clockwork components were entrained even in nurses active around the clock while in the hive. Brain oscillations in transcript abundance for the putative clock genes Period, Cryptochrome-m, Cycle, and Timeout were attenuated or totally suppressed in nurses as compared to behaviorally rhythmic foragers, irrespective of the illumination regime. These findings provide the first support for the hypothesis that natural plasticity in circadian rhythms is associated with reorganization of the internal clockwork.—Shemesh, Y., Cohen, M., Bloch, G. Natural plasticity in circadian rhythms is mediated by reorganization in the molecular clockwork in honeybees.


Key Words: social behavior • clock gene • Apis mellifera


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MOST ANIMALS STUDIED SO FAR, including humans, cannot sustain long-lasting periods of activity without circadian rhythms. Recent studies, however, suggest that there are animals that adapt to the temporal organization of their environment by naturally switching to extended periods of activity around-the-clock with no apparent ill effects. For example, reindeer go through prolonged periods of activity with no circadian rhythms during the continuous light or dark days of the Arctic (1) , voles forage for food around the clock with strong ultradian rhythms (2) , and mothers and cubs of dolphins and killer whales are continuously active and have altered sleep behavior for several weeks after birth (3) . These reports suggest that some animals have evolved a circadian system capable of contending with the challenge of around-the-clock activity, but the underlying mechanism has remained elusive. Three major mechanisms may account for plasticity in overt circadian rhythms. First, motor controlling centers may be released ("uncoupled") from the influence of the internal circadian clockwork. Second, the influence of environmental (including social) factors on the motor controlling center may be stronger than that of the circadian system ("masking"). Third, there could be genuine reorganization of the internal circadian system.

We studied honeybees (Apis mellifera) in which the plasticity in circadian rhythms is modulated by the social environment. Honeybee workers naturally switch between activity with and without circadian rhythms in a way that coincides with their social role in the division of labor system that organizes their colonies. Young workers (<2 wk of age) typically specialize in maternal behavior and provide care for ("nurse") the brood around-the-clock with no circadian rhythms. Older workers (>3 wk of age) typically perform foraging activities and have strong circadian rhythms that are needed for time-compensated sun-compass navigation and timing visits to flowers (4 , 5) . This remarkable behavioral plasticity is associated with variation in the expression of the honeybee homologue for Period (Per), a gene that is necessary for circadian rhythms in flies and mice. Per is involved in a negative feedback loop in which its protein product inhibits the transcription of its mRNA. This organizational principle of the clock results in circadian oscillations in both transcript and protein abundance (6 7 8) . In the honeybee brain, Per transcript levels cycle with high levels at night in foragers and other old bees with circadian rhythms, but typically are fairly constant in around-the-clock active nurses irrespective of age (9 10 11) . Because in these analyses each time point represents the average of several bees, it is unknown whether this lack of observed cycling stems from a genuine plasticity in the molecular clockwork of each nurse bee, or whether individual nurses, each with a cycling clockwork, are not synchronized with each other because they stay inside a constantly dark and thermo-regulated hive. In the current study, we entrained nurses with a light:dark illumination (LD) regime. Light is the most potent time-giver to the circadian system and is expected to synchronize the phase of circadian functions among individual nurses. We determined the brain expression profile for four putative clock genes—Per, Cryptochrome (Cry-m), Timeout (Tim2), and Cycle (Cyc)—to arrive at improved assessment of clockwork function. This is important because the temporal expression pattern of any single clock gene (e.g., Per in earlier studies) might also be influenced by noncircadian processes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Experimental outline
We established triple-cohort colonies with 1-day-old bees (0–24 h of age), nurses, foragers (each cohort consisted of 800-1500 bees), and their mother queen. We marked all the 1-day-old bees with a paint dot on their thorax. We housed the colonies in two-frame observation hives (with transparent walls made of glass and Plexiglas); one frame contained pollen and honey and the other was empty for the queen to lay eggs. We placed the observation hive in an environmental chamber (29±1°C; relative humidity=~50%) and connected it to the outside with a clear plastic tube (length=60 cm, diameter=3 cm). We reduced genetic variation by obtaining all bees for each experiment from the same source colony in which the queen was artificially inseminated with semen from a single drone (different in each colony). Because of the haplodiploid sex determination system in Hymenoptera, full sister bees share 75% of their genome (12) .

During the first 8 days we kept the colony in constant darkness (DD, dim red light that the bees cannot see; ref. 13 ). Under these conditions, foragers experience light during the day (when they fly outside) and darkness during the night whereas nurses mostly experience the constantly dark environment of the hive. On day 9 we changed the illumination regime to 12 h light:12 h dark (see experimental outline in Fig. 1 ). This was done to guarantee exposure of nurses to a potent external cue that is known to entrain circadian clocks (14) . We made sure that light reached all parts of the hive (e.g., there were no shaded spots). The hive entrance was blocked during the dark and open during the light phase (light on=08:00, light off=20:00). This was done in order to match the illumination regimes of foragers and nurses as it prevents foragers from experiencing daylight while nurses are still in darkness. During days 8 and 9 of the experiment, we introduced additional batches of 1-day-old bees (n=400–630 bees; Fig. 1 ). We number-tagged 100–150 of the bees introduced at day 8, and paint marked (with different colors in days 8 and 9) all other 1-day-old bees. Over the following days we paint-marked a few hundred foragers (with a different color) so they could be collected at times when foragers are inactive (e.g., at night). After 7 days in LD (day 15 of the experiment), we turned off the light and kept the colonies in DD for the last day of the experiment. Nurses and foragers were identified as in previous studies (4 , 5 , 9 10 11) . Nurses are bees seen with their head in a cell containing larvae; foragers are bees returning to the hive with loads of pollen conspicuously visible on their hind legs.


Figure 1
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  		Figure 1. Experimental outline. The plot depicts a time line for the experiment. The horizontal bar in the middle shows the days of the experiment and the illumination regime: open box = light, filled box = dark, striped box = subjective light phase at times when the hive was constantly dark. The numbers below the bar show days since the experiment began. The arcs next to days 7, 13, 15, and 16 indicate that observations or sample collections during these days were performed every 3 h throughout the day.

We collected bees for mRNA analyses from 4 treatment groups: 1) nurses that were introduced on day 1, reared in DD, and collected in DD on day 7 of the experiment (when 7–8 days of age, not collected in the experiment with colony S1); 2) nurses that were introduced on day 8, reared in LD, and collected in LD on day 15 (when 8–9 days of age); 3) nurses introduced on day 9, reared in LD, and collected in DD on day 16 (when 8–9 days of age); 4) foragers of unknown age reared in LD and collected in LD on day 16 (Fig. 1) . The collection of bees under both LD and DD allowed us to uncouple the direct effect of light from that of the internal circadian clock. Bees for mRNA analyses were collected at eight different time points (every 3 h over 1 day). Bees were collected directly into liquid nitrogen to minimize RNA degradation and stored at –80°C until analyzed with a real-time PCR (see below).

We observed brood care activity of number-tagged nurses every 3 h (as in refs. 4 , 5 ) on days 4–6 in LD (days 12–14 of the experiment, in which number-tagged bees were 5–7 days of age). Each observation included six scans of 10 min each on both sides of the honeycomb. Observers (n=2) were trained before the experiment began to minimize interobserver variability. Observers used red goggles in observations conducted during the day (light phase) to minimize variation in visibility compared with night observations performed under dim red light. On day 5 or 6 in LD (day 14 or 15 of the experiment), we transferred samples (n=15–20 bees) of foragers and 6- or 7-day-old nurses for analyses of locomotor activity (see below). We repeated this experiment three times with bees from source colonies S1, S4, and S8.

RNA analysis
We measured mRNA levels with real-time RT-PCR as in ref. 15 . Briefly, we removed and freeze-dried bee heads and dissected the brains on a frozen dissecting dish in dry ice; the tissue remained frozen during the entire procedure. We removed compound eyes, ocelli, hypopharyngeal glands, and any other glandular tissues during dissection; we discarded all brains in which pieces of tissue were lost and analyzed only intact brains. We stored each brain individually at –80°C until mRNA quantification.

We measured mRNA levels with real-time quantitative RT-PCR using an ABI Prism 7000 appliance (16) . To measure Per mRNA levels we used a multiplex PCR protocol in which Per and EF-1{alpha} are amplified in the same reaction tube. Total brain RNA was isolated (Invisorb Spin Tissue RNA Mini Kit, Invitek GmbH, Berlin, Germany), treated with DNase (RQ1 RNase-Free DNase, Promega, Madison, WI, USA), and reverse-transcribed in 20–25 µl 1 x RT buffer + 2.5 u/µl reverse transcriptase (BioScript, BioLine), 4 mM deoxy NTPs mixture (Fermentas, Hanover, MD, USA), 25 ng/µl random hexamers (Invitrogen), and 1 u/µl RNase inhibitor (RiboLock ribonuclease inhibitor, Fermentas). RNA and random hexamers were incubated at 70°C for 5 min and immediately transferred to ice. Reverse transcription was carried out at 25°C for 10 min, 42°C for 60 min, 70°C for 10 min, then incubated at 4°C. Amplification reactions (25 µl) contained 1 x TaqMan Universal PCR Master Mix (ABI Applied Biosystems, Foster City, CA, USA), 0.1 µM of each primer, 0.2 µM TaqMan probe, and 20–24 ng cDNA (control samples had no reverse transcriptase). Amplification thermal profile was 50°C for 2 min and 95°C for 10 min (95°C for 15 s, 60°C for 1 min) x 40 cycles. We excluded outliers (SD among triplicates>0.3) from our analyses.

We measured levels of Cry-m, Tim2, Clk, and Cyc in singleplex reactions with the SYBR Green dye protocol. Amplification reaction (20 µl) was similar to the above but contained SYBR green master mix (ABI Applied Biosystems) instead of TaqMan Universal PCR Master Mix and did not contain oligo probes. Each cDNA sample was analyzed in triplicate. PCR reactions for all focal genes and EF-1{alpha} were loaded on the same 96-well analysis plate. To prevent amplification of genomic DNA, we designed the PCR primers to span over an exon-exon boundary. All clock genes and EF1{alpha} levels were measured from the same cDNA sample, which was obtained with the same RNA used to produce cDNA for Per measurements.

We quantified clock gene levels with the 2–{Delta}{Delta}Ct method and EF-1{alpha} as a control gene for normalization (ABI User Bulletin #2; see also ref. 16 ). Measurements with dot blots, Northern blots, and real-time RT-PCR indicated that levels of EF-1{alpha} did not vary with age, task, or time of day (9 10 11 , 15) . For statistical analyses we used {Delta}{Delta}Ct values that are normally distributed. We assessed whether brain clock gene mRNA levels differed between bees collected at different time points with a 1-way analysis of variance (ANOVA, SPSS software), with time as factor and a Fisher LSD post hoc test.

Determining circadian rhythms for individual bees
We placed each focal bee in an individual monitoring cage made of a modified Petri dish (diameter=90 mm, height=15 mm) inside an environmental chamber (29±1°C, RH=~50%). The chamber was illuminated with constant dim red light during the entire period of data acquisition. We provided each cage with sugar syrup (50%, w/w). We ensured that bees were not exposed to light or other external factors while being transferred from the hive to the laboratory. We monitored locomotor activity with the ClockLab data acquisition system (Actimetrics Inc., Evanston, IL, USA) with two light-sensitive black and white Panasonic WV-BP334, 0.08 lux CCD cameras (each camera recorded activity from 24 cages), and a high-quality monochrome image acquisition board (IMAQ 1409, National Instruments Corp., Austin, TX, USA). Data were collected continuously at a frequency of 1–2.5 Hz (17) .

We determined circadian rhythmicity for the first 5 days in the laboratory with a {chi}2 periodogram analysis with 10 min bins (ClockLab circadian analyses software, Actimetrics). For each day we determined the time at onset of activity with the aid of the ClockLab software and a visual inspection of double plotted actograms. We used the time at onset of activity on the first day and the Oriana circular statistics software package to determine the degree of synchronization among individuals and the phase of activity. We used the Rayleigh test to assess phase coherence and the Watson-Williams F test to compare the phases of nurses and foragers.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Young nurse bees cared for the brood around-the-clock with a similar level of activity during the light and dark phases of the illumination regime; activity differed between the dark and light phases in 1/37, 0/24, and 0/6 bees in colonies S1, S4, and S8, respectively (exact binomial test P<0.05; we used only data from bees that were seen to be active in ≥10 scans, Fig. 2 A, C and Supplemental Fig. S1). In an additional analysis we compared activity during the light and dark periods for a pooled sample that included all nurses that were seen active in ≥10 scans. In this more sensitive analysis, there were no differences between activity during the light and dark periods (Wilcoxon signed ranks test, P=0.86, P=0.61, P=0.16, in colonies S1, S4, S8, respectively). These results show that nurses were active around-the-clock in the LD illumination regime and suggest that they have no circadian rhythms, as is typical of nurses (4 , 5) .


Figure 2
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  		Figure 2. Nurse bees care for the brood around- the-clock in an LD illuminated hive but have circadian rhythms in locomotor activity when transferred to individual cages in constant laboratory conditions. A) Brood care activity of a nurse bee (W65) in an LD illuminated hive. Open column = brood care activity during the day (light on); filled bars = brood care activity during the night (light off). B) A double-plot actogram showing locomotor activity of the same bee immediately after transfer from the observation hive to an individual cage in the laboratory. The numbers on the Y-axis show the days after removal from the colony. The height of the small bars within each day corresponds to the locomotor activity in a 10 min bin. Horizontal bars at the top of the plot refers to the illumination regime while in the observation hive; striped bars = subjective day, filled bars = subjective night. C, D) Brood care activity (C) and actogram (D) for bee W49; details as above in panels A, B. E) Actogram showing the locomotor activity for a representative forager. Note that the onset of activity in the first few days was earlier than in the nurse bees.

We next tested whether nurses are capable of expressing circadian rhythms when removed from the hive environment. We transferred bees from the LD illuminated hive to individual cages and monitored their locomotor activity in constant conditions. We found that most nurses and all foragers exhibited circadian rhythms in locomotor activity (Table 1 ). Figure 2A, B and C, D show records of activity for two representative nurse bees (W65 and W49) that were active around the clock when observed in the LD illuminated hive (Fig. 2A , C), but switched to activity with strong circadian rhythms shortly after transfer to an individual cage in the laboratory (Fig. 2B, D , respectively). Records for additional bees are provided in Supplemental Fig. S1. For comparison, we show an actogram of a representative forager in Fig. 2E . We used the time at onset of the daily bout of activity and circular analyses to determine the degree of synchronization among individual nurses and foragers (see Materials and Methods). We found a significantly coherent phase for the nurses, similar to that observed for foragers (Rayleigh test, P<0.001, Fig. 3 , Table 1 ). The daily onset of activity in nurses was synchronized with the illumination regime in the hive in all three colonies. In colonies S4 and S8 in which we monitored locomotor activity for foragers, we found that their onset of activity occurred earlier than in nurses (Fig. 3 , Table 1 ; Watson-Williams F tests, P<0.01).


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  		Table 1. Circadian rhythms and phase coherence for the locomotor activity of nurses and foragers transferred from LD illuminated hives to individual cages under constant conditionsa


Figure 3
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  		Figure 3. Nurses and foragers have a coherent phase of circadian rhythms in locomotor activity shortly after removal from the LD illuminated hive to individual cages in a constant environment. Foragers, upper panels; nurses, lower panels. Each triangle depicts the onset of activity on the first day in the laboratory for an individual bee. The arrow dial points to the average onset of activity and its length corresponds to the degree of phase coherence (length of vector, r). Left panel, colony S1; middle panels, colony S4; right panels, colony S8. Both foragers and nurses were synchronized with each other (Rayleigh test, P<0.001). The onset of activity was significantly earlier in foragers in colonies S4 and S8 (Watson-Williams test, P<0.01, not determined for colony S1). The open and filled arrows on the perimeter point to the time of light on and light off in the observation hive, respectively. Sample size for each plot and additional details are summarized in Table 1 .

To begin elucidating the molecular bases for plasticity in circadian rhythms, we assayed brain Per mRNA abundance over time in foragers and nurses experiencing various illumination regimes. We found that brain Per RNA abundance in nurses in DD is similar throughout the day (analysis of variance, P>0.75 in all three colonies, Fig. 4 ) but was significantly higher at night in foragers (P<0.05 in all three colonies). Levels did not vary during the day for nurses synchronized for 6–7 days in LD and collected in either LD (P>0.89 in all three colonies) or DD (P>0.81 in all three colonies, Fig. 4 ).


Figure 4
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  		Figure 4. Oscillations in brain Per mRNA abundance are attenuated in nurses relative to foragers irrespective of illumination regime. Data depict mean ± SE relative Per mRNA levels. Sample size = 5–6 bees/time point. The bars at the bottom of plots represent illumination regime (as in Figs. 1 , 2 ). P values at the right bottom corner of each plot show results obtained from an analysis of variance. Brain Per mRNA levels vary during the day only in foragers. Brain mRNA levels were normalized relative to foragers collected at 9:00 AM. Representative results from colony S4; similar results were obtained with bees from colonies S1 and S8.

To determine whether the observed task-related variation in the temporal profile of Per expression is indicative of genuine plasticity in the molecular clockwork or is distinctive to Per, we measured RNA abundance over time for additional putative clock genes. We found that variation over time in Cry-m mRNA levels is significantly reduced in nurses relative to foragers in all three colonies (Fig. 5 A). The peak/trough ratio in Cyc mRNA abundance in nurses and foragers was similar (peak/trough ratio=1.4–1.5 in foragers, and 1.2–1.4 in nurses; Fig. 5B ), but the pattern differed in that in nurses there was no consistent ~12 h interval between the peak and the trough. Perhaps this is because the weak oscillations in Cyc were attenuated in nurse bees. We found no temporal variation in brain Tim2 mRNA levels in nurses from LD illuminated hives, but in foragers levels were significantly higher at night in two of the colonies (S1 and S8; P<0.05, Fig. 5C ); a similar trend was obtained for the third colony (S4) but the differences did not reach statistical significance (ANOVA, P>0.05).


Figure 5
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  		Figure 5. Oscillations in brain mRNA abundance for putative clock genes are attenuated in nurses relative to foragers in LD illumination regime. Foragers (filled diamonds, continuous line) and nurses (open circles, dashed line) were entrained for 7 days in LD and collected in LD. A) Relative brain Cry-m mRNA levels. B) Relative Cyc mRNA levels. C) Relative Tim2 mRNA levels. The bars at the bottom of plots represent illumination regime (see Fig. 1 ). Sample size = 5–6 bees/time point. P values above and below plots show the results of analyses of variance for foragers and nurses, respectively. Representative results from colony S1; similar results were obtained with bees from colonies S4 and S8.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Honeybees show plasticity in circadian rhythms that meshes with the division of labor, a socially regulated process that is crucial for social-level responses to changes in their environment. We show that their temporal pattern of activity depends on the environment; most young nurses that were active around-the- clock in the hive showed robust circadian rhythms in locomotor activity shortly after transfer to individual cages in a constant environment. We further provide the first support for the hypothesis that natural plasticity in activity rhythms is associated with plasticity in the internal circadian clock system. Brain clock gene transcript levels varied during the day in foragers but not in nurses even when both groups were entrained by a similar LD illumination regime.

Brood care is the main occupation of nurse bees and therefore provides a good estimation of their temporal pattern of activity. Our observations are consistent with earlier studies showing that nurses are active around the clock with similar levels during the light and dark periods (4) . These findings support the hypothesis that around-the-clock activity is necessary for nurses to provide improved care to the brood (5 , 17) . Nevertheless, nurse bees that were active around-the-clock in the hive switched to activity with robust circadian rhythms shortly after being isolated in individual cages in a constant laboratory environment. This suggests that the circadian system of young nurse bees can generate rhythmic behavior, but this is not expressed in the colony environment. Additional support for this hypothesis comes from studies showing that newly emerged bees isolated in individual cages typically manifest circadian rhythms in locomotor activity when 3–14 days old, ages at which most bees in the hive perform around-the-clock nursing activities (reviewed in ref. 18 ). Furthermore, nurse-age (7–10 days old) bees that grew up in small groups in the laboratory typically have oscillating levels of brain Per mRNA that is typical of bees with circadian rhythms (10 , 11 , 19) . We further show that the phase of locomotor activity rhythms in nurses removed from the observation hive was coherent and synchronized with the hive environment (day-night fluctuation outside the hive, light-dark illumination inside). Thus, although they were active around-the-clock, it appears that at least some components of their circadian system were synchronized with external time-givers ("Zeitgebers"). Together, the observations in the hive and the locomotor activity data show that the temporal activity pattern of young bees depends on the environment. We suspect that social factors that influence division of labor are important for the regulation of circadian rhythms in young bees because around-the-clock activity is linked to brood care.

Brain Per mRNA levels vary during the day in foragers but not in nurses irrespective of the illumination regime. Their synchronized phase of locomotor activity rhythm suggests that the attenuated molecular oscillations cannot be attributed to the fact that we averaged brains of several nurses, each with a cycling clock, but with a phase that is not synchronized among individuals. To establish that this task-related plasticity in Per expression is not a distinct feature of this gene, but rather represents genuine plasticity in the molecular clockwork, we measured brain levels for additional putative clock genes. The molecular clockwork in the honeybee fits better with the mouse model than with that of Drosophila (15 , 20) . Honeybees do not have true homologs to Drosophila Timeless (Tim1) and Cryptochrome (Cry-d) genes but have an ortholog to the mammalian-type Cry (Cry-m), for which transcript levels oscillate with strong amplitude and a phase similar to Per. This robust temporal variation in brain Cry-m mRNA levels in foragers was severely attenuated in nurses, similar to the task-related variation in Per expression. Brain levels of Clk do not oscillate under LD or DD illumination regimes in either foragers or nurses (ref. 15 , and data not shown), but in foragers there appears to be weak but consistent oscillations in brain Cyc mRNA levels that peak at the end of the night or early day in both LD and DD illumination regimes (15) . These low oscillations were not detected in nurses, consistent with the premise that Apis Cyc is involved in the positive loop of the clock and does not cycle in nurses. We also measured levels of Timeout (Tim2), a gene for which there is an ongoing debate concerning its involvement in the circadian clock (21 22 23 24 25) . The pattern of Tim2 expression in the honeybee brain differs from that of canonical clock genes in that there is little temporal variation in mRNA abundance and the expression profile varies with the illumination regime; the phase is similar to Cry-m and Per in LD illumination regime, and to Cyc in DD (ref. 15 , and Y. Shemesh and G. Bloch, unpublished data). Given this expression pattern in foragers, it is interesting that we found no temporal variation in brain Tim2 mRNA levels in nurses from LD illuminated hives (Fig. 5C ), reminiscent of the findings for Per and Cry. These observations suggest that Apis Tim2 is influenced both by the clock that functions differently in nurses and foragers, and by the direct effect of light on foragers. Additional studies are needed to establish that Tim2 is indeed influenced by the clock in honeybees as well as in other animals.

These molecular analyses raise the question of how nurses with clockwork that does not produce molecular cycling can manifest robust, coherent, and entrained circadian rhythms shortly after transfer to a constant environment. As mentioned above, it is possible that some of the clock cells in nurses were entrained by external Zeitgebers in the hive even though they were active around-the-clock. These entrained cells in turn could set the phase for the entire circadian system when the nurses are removed from the hive environment and exhibit overt circadian rhythms. The existence of some entrained clock cells in their brains perhaps accounts for the apparent increase in Per levels during the night seen in all plots for nurses in Fig. 4 . When the nurse bee is in the hive, these putative environmentally entrained cells are uncoupled from other components of the circadian system, as well as from downstream output pathways. A second explanation for the environmental influence on circadian rhythms in nurses is that their clock was arrested when in the hive and picked up from the same phase when transferred to a novel environment in the laboratory (26) . A crucial point is that according to both explanations the clock of nurses functions differently from that of foragers. Nevertheless, we find the latter explanation less likely because it predicts a strong correlation between the time of transfer and the phase of locomotor activity. By contrast, in nine different experiments (including the three reported here), we found that nurses that were transferred from observation hives to a constant environment were always synchronized with the subjective day irrespective of whether they were transferred during the morning or the afternoon (Y. Shemesh and G. Bloch, unpublished observations).

Our behavioral observations, locomotor activity data, and temporal expression analyses of Per, Cry-m, Tim2, and Cyc are not consistent with the masking or "uncoupling" hypotheses that predict normal molecular oscillations in the brain of light-entrained around-the-clock active nurse bees. The possibility that in nurses the circadian network produces normal oscillations that are not detected in a whole brain analysis seems unlikely because it implies that the vast majority of cells expressing clock genes are not part of the circadian system. Such an idea is not consistent with the findings that in rhythmic foragers and nurse-age bees in the laboratory, whole brain clock gene expression cycles with circadian rhythms (Figs. 4 , 5 ; refs. 9 10 11 , 15 ). The task-related variation in clock gene oscillations is consistent with the hypothesis that chronobiological plasticity in bees is mediated by reorganization in the circadian system. Such a reorganization may include the uncoupling of pacemaker cells—for example, the uncoupling of the core pacemaker from other clock cells, the uncoupling of central and peripheral clocks or "master" and "slave" pacemakers (27 28 29 30 31 32) . In addition, task-related plasticity in the transcriptional/translational feedback loops within clock cells (6 7 8 , 32) may also account for the differences in whole brain expression profile. It is notable that the pattern of brain Per expression in this study is similar in nurses collected in LD and DD illumination regimes (Fig. 4) . This suggests that the attenuated brain Per mRNA oscillations in earlier studies in which nurses were collected in DD also indicated genuine attenuation of molecular oscillations in their brains. These studies suggested profound socially modulated molecular plasticity in the clockwork. For example, bees that were induced to forage precociously showed molecular oscillations at a young age in which bees typically care for brood around-the-clock with no molecular cycling whereas foragers that reverted to brood care activity showed attenuated molecular cycling (9 10 11) .

Despite the rapid progress achieved so far in elucidating the molecular underpinnings governing circadian rhythms and photic entrainment, little is known about how this intricate molecular system functions adaptively in natural ecological contexts (7 , 32 , 33) . Some animals, including bees, have evolved remarkable plasticity in their circadian clockwork that enables them to contend with the temporal challenges of their environment. These natural forms of plasticity in circadian rhythms contrast with evidence for increased pathologies and deterioration in performance observed when around-the-clock activity is imposed on most animals, including humans. The circadian system is evolutionarily conserved in animals, both in terms of its general organizational principles and in the clock genes involved in rhythm generation. Considering this conservation, the finding that plasticity in circadian rhythms is mediated by plasticity in clock gene expression suggests that comprehensive comparative studies could reveal the inner workings of mechanisms enabling some, but not other, animals to show such profound plasticity in behavioral rhythms.


   ACKNOWLEDGMENTS
 
We thank Sharon Geva, Yfat Yair, Mordechai Applebaum, and Ada Eban for help with the bees, and Isaac Edery, Ada Eban, and two anonymous reviewers for comments on earlier versions of this manuscript. Financial support was provided by the Israel-U.S. Binational Science Foundation (BSF, grant 2003–151, to G.B.), Israel Science Foundation (ISF, grant number 606/02, to G.B.), and the Joseph H. and Belle R. Braun Senior Lectureship in Life Sciences (to G.B.).

Received for publication January 4, 2007. Accepted for publication February 15, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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