Darrell Moore

Honey bee circadian clocks: behavioral control from individual workers to whole-colony rhythms

Abstract In the field of insect circadian rhythms, the honey bee is best known for its foraging time-sense, or Zeitgedachtnis, which permits the forager bee to make precise associations between the presence of food and the time of day. A number of studies, now considered classics, established that bees could be trained to collect food at virtually any time of the circadian cycle and that this timekeeping ability was controlled by an endogenous circadian clock. Recently, behavioral rhythms in bees have been examined using a variety of approaches, in both laboratory and field studies. The following areas of new research are reviewed: (a) the ontogeny of behavioral rhythmicity in newly emerged worker bees; (b) the integration of behavioral rhythmicity with the colonys division of labor; (c) the evidence for social entrainment of behavioral rhythms and for a ‘clock of the colony’; (d) the potential linkage between circadian rhythms of general locomotor activity and the foraging time-sense; (e) learning and entrainment hypotheses proposed to explain the mechanism underlying the time-sense; (f) the interplay between extinction and persistence of the time-memory as revealed from the differential behavior of individuals within the foraging group; and (g) comparisons of the Zeitgedachtnis with food-anticipatory rhythms in other animals.

Timekeeping in the honey bee colony: integration of circadian rhythms and division of labor

Abstract The daily patterns of task performance in honey bee colonies during behavioral development were studied to determine the role of circadian rhythmicity in age-related division of labor. Although it is well known that foragers exhibit robust circadian patterns of activity in both field and laboratory settings, we report that many in-hive tasks are not allocated according to a daily rhythm but rather are performed 24 h per day. Around-the-clock activity at the colony level is accomplished through the performance of some tasks by individual workers randomly with respect to time of day. Bees are initially arrhythmic with respect to task performance but develop diel rhythmicity, by increasing the occurrence of inactivity at night, prior to becoming foragers. There are genotypic differences for age at onset of rhythmicity and our results suggest that these differences are correlated with genotypic variation in rate of behavioral development: genotypes of bees that progressed through the age polyethism schedule faster also acquired behavioral rhythmicity at an earlier age. The ontogeny of circadian rhythmicity in honey bee workers ensures that essential in-hive behaviors are performed around the clock but also allows the circadian clock to be engaged before the onset of foraging.

Light and temperature entrainment of a locomotor rhythm in honeybees

Abstract. The circadian locomotor (walking) rhythms of forager honeybees (Apis mellifera ligustica L.) were entrained to eight different 24 h light‐dark cycles. The phases of activity onset, peak activity, and offset were correlated with the lights‐off transition, suggesting lights‐off as the primary zeitgeber for the rhythm. Further support for this hypothesis was provided by LD 1:23 experiments, in which entrainment occurred when the light pulse was situated at the end, but not at the beginning, of the subjective photophase. Steady‐state entrainment of the locomotor rhythm was achieved with square‐wave temperature cycles of 10oC amplitude under constant dark: most of the activity occurred within the early thermophase. Smaller amplitude temperature cycles yielded relative coordination of the rhythm. Interactions of temperature and light‐dark cycles resulted in entrainment patterns different from those elicited in response to either cycle alone or those formed by a simple combination of the two separate responses. Furthermore, temperature cycles having amplitudes insufficient for entrainment of the rhythm nevertheless modified the pattern of entrainment to light ‐ dark cycles, suggesting a synergism of light and temperature effects on the underlying circadian clock system.

DIURNAL CHANGES IN THE ACCURACY OF THE HONEYBEE FORAGING RHYTHM

The ability of honeybees to time foraging visits to an artificial nectar source was analyzed with respect to the time of day of food source presentation. A consistent regimen of orientation and training to the food source, which was available only during a specific one-hour period of the day for each particular experiment, allowed quantitative comparisons to be made among groups of bees trained at different feeding times. Bees trained early in the day showed the most precise time-keeping ability whereas those trained to midday or late afternoon food sources were signif icantly less accurate. In all of the experiments, the bees anticipated the onset of the training period, but the duration ofthe anticipatory component ofthe response was dependent upon the training time. Similarly, other parameters (coefficients of skew ness and kurtosis and the shut-down in the number of arrivals immediately after the end of the training time) describing the distribution of foraging flights in time varied according to times in the diurnal cycle when the food source was offered. These results suggest that honeybees more efficiently exploit a daily floral nectar source if it is available early in the morning than if it is offered late in the day. Possible mechanisms underlying the observed differences in temporal orientation are discussed.

Neurogenomic signatures of spatiotemporal memories in time-trained forager honey bees

Honey bees can form distinct spatiotemporal memories that allow them to return repeatedly to different food sources at different times of day. Although it is becoming increasingly clear that different behavioral states are associated with different profiles of brain gene expression, it is not known whether this relationship extends to states that are as dynamic and specific as those associated with foraging-related spatiotemporal memories. We tested this hypothesis by training different groups of foragers from the same colony to collect sucrose solution from one of two artificial feeders; each feeder was in a different location and had sucrose available at a different time, either in the morning or afternoon. Bees from both training groups were collected at both the morning and afternoon training times to result in one set of bees that was undergoing stereotypical food anticipatory behavior and another that was inactive for each time of day. Between the two groups with the different spatiotemporal memories, microarray analysis revealed that 1329 genes were differentially expressed in the brains of honey bees. Many of these genes also varied with time of day, time of training or state of food anticipation. Some of these genes are known to be involved in a variety of biological processes, including metabolism and behavior. These results indicate that distinct spatiotemporal foraging memories in honey bees are associated with distinct neurogenomic signatures, and the decomposition of these signatures into sets of genes that are also influenced by time or activity state hints at the modular composition of this complex neurogenomic phenotype.

Acquisition of a time-memory in forager honey bees.

Forager honey bees can associate the time of day with the presence of food at locations outside the hive. It is thought that this time-memory enables the bee to make a spatio-temporal match between its behavior and floral nectar secretion rhythms. Despite a long tradition of research, the mechanisms by which the time-memory becomes established are unknown. We investigated the influences of two experiential factors on the acquisition of time-memory: (1) the number of collecting visits made by the forager within a feeding bout during a restricted time of day and (2) the number of days of exposure to the restricted feeding time. Our results indicate that these two factors control different processes. The number of days of experience influences the temporal accuracy of reconnaissance behavior to the food source. The cumulative number of collecting visits within the feeding bouts has no apparent effect on time-accuracy but, instead, determines the probability of exhibiting food-anticipatory behavior and, if that overt behavior is performed, the intensity of its expression.

Diminishing returns: the influence of experience and environment on time-memory extinction in honey bee foragers

Classical experiments demonstrated that honey bee foragers trained to collect food at virtually any time of day will return to that food source on subsequent days with a remarkable degree of temporal accuracy. This versatile time-memory, based on an endogenous circadian clock, presumably enables foragers to schedule their reconnaissance flights to best take advantage of the daily rhythms of nectar and pollen availability in different species of flowers. It is commonly believed that the time-memory rapidly extinguishes if not reinforced daily, thus enabling foragers to switch quickly from relatively poor sources to more productive ones. On the other hand, it is also commonly thought that extinction of the time-memory is slow enough to permit foragers to ‘remember’ the food source over a day or two of bad weather. What exactly is the time-course of time-memory extinction? In a series of field experiments, we determined that the level of food-anticipatory activity (FAA) directed at a food source is not rapidly extinguished and, furthermore, the time-course of extinction is dependent upon the amount of experience accumulated by the forager at that source. We also found that FAA is prolonged in response to inclement weather, indicating that time-memory extinction is not a simple decay function but is responsive to environmental changes. These results provide insights into the adaptability of FAA under natural conditions.

Effects of environmental factors on circadian activity in the flesh fly,Sarcophaga crassipalpis

Abstract.The diel locomotor activity patterns of wandering larvae in the flesh fly, Sarcophaga crassipalpis Macquart (Diptera: Sarcophagidae), were examined using a novel apparatus and shown to be primarily diurnal, but with a minority (37%) showing nocturnal activity. In response to the environmental stress of heat shock, a significantly larger proportion (72%) of the larvae became nocturnal. In comparison, adult circadian activity also was predominantly diurnal, but not correlated with the larval activity patterns. In addition, adult patterns showed age‐related changes in entrainment and free running period. Finally, the phase of circadian‐gated adult eclosion was shown to be entrained by a 3‐day exposure to light–dark cycles delivered prior to pupariation, with the phase maintained throughout pupal–adult metamorphosis under constant dark conditions. These results demonstrate that environmental changes may have profound effects on the expression of 24‐h activity patterns and circadian rhythms during different life stages throughout development.

Persistence, reticence and the management of multiple time memories by forager honey bees

SUMMARY Honey bee foragers form time memories that enable them to match their foraging activity to the time of day when a particular food source is most productive. Persistent foragers show food-anticipatory activity by making reconnaissance flights to the previously productive food source and may continue to inspect it for several days. In contrast, reticent foragers do not investigate the source but wait for confirmation from returning persistent foragers. To determine how persistent and reticent foragers might contribute to the colonys ability to rapidly reallocate foragers among sources, we trained foragers to collect sucrose from a feeder at a restricted time of day for several days and then observed their behavior for three consecutive days during which the feeder was empty. In two separate trials, video monitoring of the hive entrance during unrewarded test days in parallel with observing reconnaissance visits to the feeder revealed a high level of activity, in both persistent and reticent foragers, thought to be directed at other food sources. This ‘extracurricular’ activity showed a high degree of temporal overlap with reconnaissance visits to the feeder. In some cases, inspection flights to the unrewarded feeder were made within the same trip to an extracurricular source, indicating that honey bees have the ability to manage at least two different time memories despite coincidence with respect to time of day. The results have major implications for understanding flower fidelity throughout the day, flower constancy within individual foraging excursions, and the sophisticated cognitive management of spatiotemporal memories in honey bees.

Diel nectar secretion rhythm in squash (Cucurbita pepo) and its relation with pollinator activity

Most studies of foraging behavior in bees have been performed under artificial conditions. One highly neglected area is the daily nectar secretion rhythm in flowers including how nectar properties may vary with time of day. As a first step in understanding the connections between forager behavior and nectar presentation under more natural conditions, we examined nectar secretion patterns in flowers of the squash Cucurbita pepo. Under greenhouse conditions, squash flowers exhibit consistent diel changes in nectar volume and concentration through anthesis. These temporal patterns are robust, persisting under field conditions as well as simulated drought conditions in the greenhouse. In the presence of active pollinators, diel patterns are evident but with highly variable, severely reduced volumes. The potential consequences of these factors for pollinator behavior are discussed.