The role of temperature on the development of circadian rhythms in honey bee workers.

Circadian rhythms in honey bees are involved in various processes that impact colony survival. For example, young nurses take care of the brood constantly throughout the day and lack circadian rhythms. At the same time, foragers use the circadian clock to remember and predict food availability in subsequent days. Previous studies exploring the ontogeny of circadian rhythms of workers showed that the onset of rhythms is faster in the colony environment (~2 days) than if workers were immediately isolated after eclosion (7-9 days). However, which specific environmental factors influenced the early development of worker circadian rhythms remained unknown. We hypothesized that brood nest temperature plays a key role in the development of circadian rhythmicity in young workers. Our results show that young workers kept at brood nest-like temperatures (33-35 °C) in the laboratory develop circadian rhythms faster and in greater proportion than bees kept at lower temperatures (24-26 °C). In addition, we examined if the effect of colony temperature during the first 48 h after emergence is sufficient to increase the rate and proportion of development of circadian rhythmicity. We observed that twice as many individuals exposed to 35 °C during the first 48 h developed circadian rhythms compared to individuals kept at 25 °C, suggesting a critical developmental period where brood nest temperatures are important for the development of the circadian system. Together, our findings show that temperature, which is socially regulated inside the hive, is a key factor that influences the ontogeny of circadian rhythmicity of workers.

The Role of Colony Temperature in the Entrainment of Circadian Rhythms of Honey Bee Foragers.

Honey bees utilize their circadian rhythms to accurately predict the time of day. This ability allows foragers to remember the specific timing of food availability and its location for several days. Previous studies have provided strong evidence toward light/dark cycles being the primary Zeitgeber for honey bees. Work in our laboratory described large individual variation in the endogenous period length of honey bee foragers from the same colony and differences in the endogenous rhythms under different constant temperatures. In this study, we further this work by examining the temperature inside the honey bee colony. By placing temperature and light data loggers at different locations inside the colony we measured temperature at various locations within the colony. We observed significant oscillations of the temperature inside the hive, that show seasonal patterns. We then simulated the observed temperature oscillations in the laboratory and found that using the temperature cycle as a Zeitgeber, foragers present large individual differences in the phase of locomotor rhythms for temperature. Moreover, foragers successfully synchronize their locomotor rhythms to these simulated temperature cycles. Advancing the cycle by six hours, resulting in changes in the phase of activity in some foragers in the assay. The results are shown in this study highlight the importance of temperature as a potential Zeitgeber in the field. Future studies will examine the possible functional and evolutionary role of the observed phase differences of circadian rhythms.

Neonicotinoids disrupt circadian rhythms and sleep in honey bees.

Honey bees are critical pollinators in ecosystems and agriculture, but their numbers have significantly declined. Declines in pollinator populations are thought to be due to multiple factors including habitat loss, climate change, increased vulnerability to disease and parasites, and pesticide use. Neonicotinoid pesticides are agonists of insect nicotinic cholinergic receptors, and sub-lethal exposures are linked to reduced honey bee hive survival. Honey bees are highly dependent on circadian clocks to regulate critical behaviors, such as foraging orientation and navigation, time-memory for food sources, sleep, and learning/memory processes. Because circadian clock neurons in insects receive light input through cholinergic signaling we tested for effects of neonicotinoids on honey bee circadian rhythms and sleep. Neonicotinoid ingestion by feeding over several days results in neonicotinoid accumulation in the bee brain, disrupts circadian rhythmicity in many individual bees, shifts the timing of behavioral circadian rhythms in bees that remain rhythmic, and impairs sleep. Neonicotinoids and light input act synergistically to disrupt bee circadian behavior, and neonicotinoids directly stimulate wake-promoting clock neurons in the fruit fly brain. Neonicotinoids disrupt honey bee circadian rhythms and sleep, likely by aberrant stimulation of clock neurons, to potentially impair honey bee navigation, time-memory, and social communication.

Locomotor activity patterns in three spider species suggest relaxed selection on endogenous circadian period and novel features of chronotype.

We examined the circadian rhythms of locomotor activity in three spider species in the Family Theridiidae under light-dark cycles and constant darkness. Contrary to previous findings in other organisms, we found exceptionally high variability in endogenous circadian period both within and among species. Many individuals exhibited circadian periods much lower (19-22 h) or much higher (26-30 h) than the archetypal circadian period. These results suggest relaxed selection on circadian period as well as an ability to succeed in nature despite a lack of circadian resonance with the 24-h daily cycle. Although displaying similar entrainment waveforms under light-dark cycles, there were remarkable differences among the three species with respect to levels of apparent masking and dispersion of activity under constant dark conditions. These behavioral differences suggest an aspect of chronotype adapted to the particular ecologies of the different species.

High experience levels delay recruitment but promote simultaneous time-memories in honey bee foragers.

Honey bee (Apis mellifera) foragers can remember both the location and time of day food is collected and, even in the absence of a reward, reconnoiter the food source at the appropriate time on subsequent days. This spatiotemporal memory (time-memory) is linked to the circadian clock and enables foragers to synchronize their behavior with floral nectar secretion rhythms, thus eliminating the need to rediscover productive food sources each day. Here, we asked whether the establishment of one time-memory influences the formation of another time-memory at the same time of day. In other words, can two time-place memories with the same 'time-stamp' co-exist? We simultaneously trained two groups of foragers from a single hive to two separate feeders at the same restricted time of day. After 5 days of training, one feeder was shut off. The second feeder continued being productive 4 more days. Our results showed that (1) foragers with high experience levels at the first source were significantly more likely than low-experience foragers to maintain fidelity to their original source and resist recruitment to the alternative source, (2) nearly one-third of foragers demonstrated multiple, overlapping time-memories by visiting both feeders at the correct time and (3) significantly more high-experience than low-experience foragers exhibited this multitasking behavior. The ability to maintain and act upon two different, yet contemporaneous, time-memories gives the forager bee a previously unknown level of versatility in attending to multiple food sources. These findings have major implications for understanding the formation and management of circadian spatiotemporal memories.

Effects of gender, age, and nutrition on circadian locomotor activity rhythms in the flesh fly Sarcophaga crassipalpis.

In many animal species, circadian rhythms of behavior are not constant throughout the lifetime of the individual but rather exhibit at least some degree of plasticity. In the present study, we have examined the potential influences of gender, age, and nutrition (presence or absence of liver) on the expression of circadian locomotor activity rhythms in the flesh fly Sarcophaga crassipalpis. We found no significant differences in endogenous circadian period under constant dark conditions with respect to gender, nutrition, or age for the duration of our experiments. On the other hand, both male and female flesh flies, as expected, were predominantly diurnal under light-dark cycles, but the pattern of entrainment differed between the sexes. Females also displayed higher activity levels than males. Also, in contrast with males, female activity levels increased with age. Moreover, females exhibited an extraordinary, but transient (one to three days), departure from diurnality which we characterize as "extended dark activity" (EDA). This phenomenon appeared as a continuous bout of locomotor activity that extended at least three hours into the early half of the dark phase at levels at least twice the median of the overall locomotor activity for the individual fly. EDA occurred as an age-dependent response to liver consumption, never appearing prior to day 4 post-eclosion but, thereafter, transpiring within one or two days after a 48-h exposure to liver. These results suggest a linkage between physiological events associated with egg provisioning and locomotor activity in the anautogenous flesh fly. Furthermore, our findings identify the existence of multiple influences on the expression of circadian clock-regulated behavior.

How to Train a Honey Bee.

In the early twentieth century, Karl von Frisch performed seminal work on the organization of social behavior of honey bees. Much of his work involved training individual foragers to distant artificial feeders. Similar training methods have been used in research laboratories for the better part of a century, and these methods lend themselves well to advanced undergraduate biology classes in animal behavior. In recent years, students have used these methods in group projects to study color preference and time-memory. In this Technical Paper, we describe the basic steps of training honey bees to a distant feeder. We also provide alternative methods for answering specific types of questions that students in animal behavior classes might wish to address.

Extensive reorganization of behavior accompanies ontogeny of aggression in male flesh flies.

Aggression, costly in both time and energy, is often expressed by male animals in defense of valuable resources such as food or potential mates. Here we present a new insect model system for the study of aggression, the male flesh fly Sarcophaga crassipalpis, and ask whether there is an ontogeny of aggression that coincides with reproductive maturity. After establishing that reproductive maturity occurs by day 3 of age (post-eclosion), we examined the behavior of socially isolated males from different age cohorts (days 1, 2, 3, 4, and 6) upon introduction, in a test arena, with another male of the same age. The results show a pronounced development of aggression with age. The change from relative indifference to heightened aggression involves a profound increase in the frequency of high-intensity aggressive behaviors between days 1 and 3. Also noteworthy is an abrupt increase in the number of statistically significant transitions involving these full-contact agonistic behaviors on day 2. This elevated activity is trimmed back somewhat by day 3 and appears to maintain a stable plateau thereafter. No convincing evidence was found for escalation of aggression nor the establishment of a dominance relationship over the duration of the encounters. Despite the fact that aggressive interactions are brief, lasting only a few seconds, a major reorganization in the relative proportions of four major non-aggressive behaviors (accounting for at least 96% of the total observation time for each age cohort) accompanies the switch from low to high aggression. A series of control experiments, with single flies in the test arenas, indicates that these changes occur in the absence of the performance of aggressive behaviors. This parallel ontogeny of aggressive and non-aggressive behaviors has implications for understanding how the entire behavioral repertoire may be organized and reorganized to accommodate the needs of the organism.

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

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 colony's 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.

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.

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.

Absence of consistent diel rhythmicity in mated honey bee queen behavior.

Relatively little is known about the temporal control of behavior of honey bee queens under natural conditions. To determine if mated honey bee queens possess diel rhythmicity in behavior, we observed them in glass-sided observation hives, employing two focal studies involving continuous observations of individual queens as well as a scan-sampling study of multiple queens. In all cases, all behaviors were observed at all times of the day and night. In four of the five queens examined in focal studies, there were no consistent occurrences of diel periodicity for any of the individual behaviors. A more encompassing measure for periodicity, in which the behaviors were characterized as active (walking, inspecting, egg-laying, begging for food, feeding, and grooming self) or inactive (standing), also failed to reveal consistent diel rhythmicity. Furthermore, there were no consistent diel differences in the number of workers in the queen's retinue. Behavioral arrhythmicity persisted across seasons and despite daily changes in both light and temperature levels. Both day and night levels of behavioral activity were correlated with daytime, but not with nighttime, ambient temperatures. The behavior of the one exceptional queen was not consistent: diurnal activity patterns were present during two 24-h observation sessions but arrhythmicity during another. Based on the behavior observed by all but one of the queens examined in this work, the arrhythmic behavior by the mated honey bee queen inside the colony appears to be similar to that exhibited by worker bees before they approach the age of onset of foraging behavior.

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.

Neural basis of a simple behavior: abdominal positioning in crayfish.

Crustaceans have been used extensively as models for studying the nervous system. Members of the Order Decapoda, particularly the larger species such as lobsters and crayfish, have large segmented abdomens that are positioned by tonic flexor and extensor muscles. Importantly, the innervation of these tonic muscles is known in some detail. Each abdominal segment in crayfish is innervated bilaterally by three sets of nerves. The anterior pair of nerves in each ganglion controls the swimmeret appendages and sensory supply. The middle pair of nerves innervates the tonic extensor muscles and the regional sensory supply. The superficial branch of the most posterior pair of nerves in each ganglion is exclusively motor and supplies the tonic flexor muscles of that segment. The extension and flexion motor nerves contain six motor neurons, each of which is different in axonal diameter and thus produces impulses of different amplitude. Motor programs controlling each muscle can be characterized by the identifiable motor neurons that are activated. Early work in this field discovered that specific central interneurons control the abdominal positioning motor neurons. These interneurons were first referred to as "command neurons" and later as "command elements." Stimulation of an appropriate command element causes a complex, widespread output involving dozens of motor neurons. The output can be patterned even though the stimulus to the command element is of constant interval. The command elements are identifiable cells. When a stimulus is repeated in a command element, from either the same individual or from different individuals, the output is substantially the same. This outcome depends upon several factors. First, the command elements are not only identifiable, but they make many synapses with other neurons, and the synapses are substantially invariant. There are separate flexion-producing and extension-producing command elements. Abdominal flexion-producing command elements excite other flexion elements and inhibit extensor command elements. The extension producing elements do the opposite. These interactions insure that interneurons of a particular class (flexion- or extension-producing) synaptically recruit perhaps twenty others of similar output, and that command elements promoting the opposing movements are inhibited. This strong reciprocity and the recruitment of similar command elements give a powerful motor program that appears to mimic behavior.