The colony environment, but not direct contact with conspecifics, influences the development of circadian rhythms in honey bees

Honey bee (Apis mellifera) workers emerge from the pupae with no circadian rhythms in behavior or brain clock gene expression but show strong rhythms later in life. This postembryonic development of circadian rhythms is reminiscent of that of infants of humans and other primates but contrasts with most insects, which typically emerge from the pupae with strong circadian rhythms. Very little is known about the internal and external factors regulating the ontogeny of circadian rhythms in bees or in other animals. We tested the hypothesis that the environment during early life influences the later expression of circadian rhythms in locomotor activity in young honey bees. We reared newly emerged bees in various social environments, transferred them to individual cages in constant laboratory conditions, and monitored their locomotor activity. We found that the percentage of rhythmic individuals among bees that experienced the colony environment for their first 48 h of adult life was similar to that of older sister foragers, but their rhythms were weaker. Sister bees isolated individually in the laboratory for the same period were significantly less likely to show circadian rhythms in locomotor activity. Bees experiencing the colony environment for only 24 h, or staying for 48 h with 30 same-age sister bees in the laboratory, were similar to bees individually isolated in the laboratory. By contrast, bees that were caged individually or in groups in single- or double-mesh enclosures inside a field colony were as likely to exhibit circadian rhythms as their sisters that were freely moving in the same colony. These findings suggest that the development of the circadian system in young adult honey bees is faster in the colony than in isolation. Direct contact with the queen, workers, or the brood, contact pheromones, and trophallaxis, which are all important means of communication in honey bees, cannot account for the influence of the colony environment, since they were all withheld from the bees in the double-mesh enclosures. Our results suggest that volatile pheromones, the colony microenvironment, or both influence the ontogeny of circadian rhythms in honey bees.     

Juvenile hormone and circadian locomotor activity in the honey bee Apis mellifera

Age-related division of labor in honeybees is associated with plasticity in circadian rhythms. Young nest bees care for brood around the clock with no circadian rhythms while older foragers have strong circadian rhythms that are used for sun compass navigation and for timing visits to flowers. Since juvenile hormone (JH) is involved in the coordination of physiological and behavioral processes underlying age-related division of labor in honey bees, we tested the hypothesis that JH influences the ontogeny of circadian rhythms and other clock parameters in young worker bees. Treatments with the JH analog methoprene or allatectomy did not influence the onset of rhythmicity, overall locomotor activity, or the free-running period of rhythmic locomotor behavior. There were, however, significant differences in the onset of rhythmicity, overall locomotor activity, and longevity between bees from different source colonies, suggesting that there is significant genetic variation for these traits. Our results suggest that JH does not coordinate all aspects of division of labor in bees and that coordination of task performance with circadian rhythms is probably mediated by other regulatory systems.     

period expression in the honey bee brain is developmentally regulated and not affected by light, flight experience, or colony type

Changes in circadian rhythms of behavior are related to age-based division of labor in honey bee colonies. The expression of the clock gene period (per) in the bee brain is associated with age-related changes in circadian rhythms of behavior, but previous efforts to firmly associate per brain expression with division of labor or age have produced variable results. We explored whether this variability was due to differences in light and flight experience, which vary with division of labor, or differences in colony environment, which are known to affect honey bee behavioral development. Our results support the hypothesis that per mRNA expression in the bee brain is developmentally regulated. One-day-old bees had the lowest levels of expression and rarely showed evidence of diurnal fluctuation, while foragers and forager-age bees (> 21 days of age) always had high levels of brain per and strong and consistent diurnal patterns. Results from laboratory and field experiments do not support the hypothesis that light, flight experience, and colony type influence per expression. Our results suggest that the rate of developmental elevation in per expression is influenced by factors other than the ones studied in our experiments, and that young bees are more sensitive to these factors than foragers.     

Behavioral rhythmicity, age, division of labor and period expression in the honey bee brain.

Young adult honey bees work inside the beehive "nursing" brood around the clock with no circadian rhythms; older bees forage for nectar and pollen outside with strong circadian rhythms. Previous research has shown that the development of an endogenous rhythm of activity is also seen in the laboratory in a constant environment. Newly emerging bees maintained in isolation are typically arrhythmic during the first few days of adult life and develop strong circadian rhythms by about a few days of age. In addition, average daily levels of period (per) mRNA in the brain are higher in foragers or forager-age bees (> 21 days of age) relative to young nest bees (approximately 7 days of age). The authors used social manipulations to uncouple behavioral rhythmicity, age, and task to determine the relationship between these factors and per. There was no obligate link between average daily levels of per brain mRNA and either behavioral rhythmicity or age. There also were no differences in per brain mRNA levels between nurse bees and foragers in social environments that promote precocious or reversed behavioral development. Nurses and other hive-age bees can have high or low levels of per mRNA levels in the brain, depending on the social environment, while foragers and foraging-age bees always have high levels. These findings suggest a link between honey bee foraging behavior and per up-regulation. Results also suggest task-related differences in the amplitude of per mRNA oscillation in the brain, with foragers having larger diurnal fluctuation in per than nurses, regardless of age. Taken together, these results suggest that social factors may exert potent influences on the regulation of clock genes.     

Physiological correlates of division of labor among similarly aged honey bees

Hormone analyses and exocrine gland measurements were made to probe for physiological correlates of division of labor among similarly aged adult worker honey bees (Apis mellifera L.). Middle-age bees (ca. 2 weeks old) performing different tasks showed significant differences in both juvenile hormone (JH) biosynthesis rates and hemolymph titers; guards and undertakers had high JH, and wax producers and food storers, low JH. Guards and undertakers had similar hormone levels to foragers, even though they were 10 days younger than foragers. No differences in JH were detected among young bees (1-week-old queen attendants and nurses) or older bees (3–4 week-old pollen foragers, non-pollen foragers, and soldiers). Hypopharyngeal gland size was inversely correlated with worker age and rate of JH biosynthesis, but soldiers had significantly larger hypopharyngeal glands than did foragers, despite their similar age and JH level. Results from soldiers indicate that exocrine gland development is not always linked with age-related behavior and endocrine development; they also support the recent claim that soldiers constitute a group of older bees that are distinct from foragers. Hormonal analyses indicate that the current model of JH's role in honey bee division of labor needs to be expanded because high levels of JH are associated with several other tasks besides foraging. JH may be involved in the regulation of division of labor among similarly aged workers in addition to its role in age-related division of labor.Abbreviations JH Juvenile hormone - RIA radioimmunoassay - CA corpora allata - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Maternity-related plasticity in circadian rhythms of bumble-bee queens

Unlike most animals studied so far in which the activity with no circadian rhythms is pathological or linked to deteriorating performance, worker bees and ants naturally care for their sibling brood around the clock with no apparent ill effects. Here, we tested whether bumble-bee queens that care alone for their first batch of offspring are also capable of a similar chronobiological plasticity. We monitored locomotor activity of Bombus terrestris queens at various life cycle stages, and queens for which we manipulated the presence of brood or removed the ovaries. We found that gynes typically emerged from the pupae with no circadian rhythms, but after several days showed robust rhythms that were not affected by mating or diapauses. Colony-founding queens with brood showed attenuated circadian rhythms, irrespective of the presence of ovaries. By contrast, queens that lost their brood switched again to activity with strong circadian rhythms. The discovery that circadian rhythms in bumble-bee queens are regulated by the life cycle and the presence of brood suggests that plasticity in the circadian clock of bees is ancient and related to maternal behaviour or physiology, and is not a derived trait that evolved with the evolution of the worker caste.     

The involvement of the antennae in mediating the brood influence on circadian rhythms in "nurse" honey bee (Apis mellifera) workers

Age-related division of labor in honey bees is associated with plasticity in circadian rhythms. Forager bees that are typically older than 3 weeks of age show strong behavioral and molecular circadian rhythms with higher activity during the day. Younger bees that typically care for ("nurse") the brood are active around the clock with similar brain clock gene levels throughout the day. However, nurses that are caged on brood-less combs inside or outside the hive show robust circadian rhythms with higher activity during the day, suggesting that direct contact with the brood mediates the plasticity in the circadian system. The nature of the brood signals affecting the workers' circadian system and the modalities by which they are detected are unknown. Given that the antennae are pivotal sensory organs in bees, we hypothesized that they are involved in mediating the brood influence on the plasticity in circadian rhythms. The flagella of the antennae are densely covered with diverse sensory structures able to detect a wide range of signals. To test our hypothesis, we removed the flagella of nurses and observed their behavior in isolation and in free-foraging colonies. We found that individually-isolated flagella-less bees under constant laboratory conditions show robust circadian rhythms in locomotor activity. In observation hives, flagella-less bees cared for the brood, but were more active during the day. By contrast, sham-treated bees were active around the clock as typical of nurses. Detailed video recordings showed that the brood-tending behavior of flagella-less and sham-treated bees is similar. These observations suggest that the difference in the patterns of brood care activity is not because the flagella-less bees did not contact the brood. Our results suggest that nurses are able to find the brood in the dark environment of the hive without their flagella, perhaps by using other sensory organs. The higher activity of flagella-less bees during the day further suggests that the flagella are involved in mediating the brood signals modulating plasticity in the circadian system.     

Daily rhythms related to distinct social tasks inside an eusocial bee colony

A bee colony is often compared to a multicellular organism, mainly because of its spatial organization. We propose that a temporal organization of equal importance is also present. To support this view, we studied the reproductive processes of two closely related species of stingless bees. Stingless bees enable observations of daily rhythms that are performed by distinct social classes. The emergent process, POP, is cyclic and consists of the building and provisioning of brood cells by the worker bees and egg-laying by the queen. Colonies were kept in the laboratory under constant conditions with the exit tube opening to the environment; thus, foragers had direct access to environmental cycles. At a later stage of the experiment, the exit tube was closed by a sieve; in this case, bees had their own stock of food, but the environmental LD cycle could still be detected when they were inside the exit tube. Daily POP rhythms were present and showed distinct temporal patterns in each species. A third condition was imposed on one of the species only: the exit tube was closed by a sieve and maintained inside a box that was provided with constant illumination. In this colony, the POP rhythm was perturbed by the destruction of the brood cells. Restoration of POP consisted of a rapid reconstruction of cells followed by a late oviposition in the same day. As different rhythmic patterns were detected, but showed regular timings with respect to one another, an interpretation based upon the concept of an internal temporal order is suggested.     

Circadian rhythms of locomotor activity in solitary and social species of African mole-rats (family: Bathyergidae)

Mole-rats are strictly subterranean and hardly, if ever, come into contact with external light. As a result, their classical visual system is severely regressed and the circadian system proportionally expanded. The family Bathyergidae presents a unique opportunity to study the circadian system in the absence of the classical visual system in a range of species. Daily patterns of activity were studied in the laboratory under constant temperature but variable lighting regimes in individually housed animals from 3 species of mole-rat exhibiting markedly different degrees of sociality. All 3 species possessed individuals that exhibited endogenous circadian rhythms under constant darkness that entrained to a light-dark cycle. In the solitary species, Georychus capensis, 9 animals exhibited greater activity during the dark phase of the light cycle, while 2 individuals expressed more activity in the light phase of the light cycle. In the social, Cryptomys hottentotus pretoriae, 5 animals displayed the majority of their activity during the dark phase of the light cycle and the remaining 2 exhibited more activity during the light phase of the light cycle. Finally in the eusocial Cryptomys damarensis, 6 animals displayed more activity during the light phase of the light cycle, and the other 2 animals displayed more activity during the dark phase of the light cycle. Since all three mole-rat species are able to entrain their locomotor activity to an external light source, light must reach the SCN, suggesting a functional circadian clock. In comparison to the solitary species, the 2 social species display a markedly poorer response to light in all aspects. Thus, in parallel with the sociality continuum, there exists a continuum of sensitivity of the circadian clock to light.     

Nurse bees support the physiological development of young bees (Apis mellifera L.)

Newly emerged worker honeybees (focal bees) were caged individually for 8 days either isolated or together with one companion bee of known age (2–30 days) taken from a colony. The companion bee was replaced every 2nd day. After 8 days, various parameters were investigated in the focal bees as indicators of the level of development. Focal bees which had been caged with 6-day-old companion bees were better developed than isolated focal bees, newly emerged bees, or focal bees caged with almost all other ages of companion bees. They had hypopharyngeal glands that were larger and contained more protein, their thoraces had a higher protein content, and they had a higher rate of proteolytic activity in the midgut. Although the focal bees were supplied with pollen as well as honey, they consumed only small amounts of pollen. We attribute their better development to their having been fed worker jelly by the accompanying companion bees. The 6-day-old companion bees consumed high quantities of pollen and spent more time (18.7 ± 11.85 s/h) feeding focal bees than 12-day-old bees (6.5 ± 4.09 s/h) or foragers (no feeding of focal bees). The results show that even under such artificial conditions, the exchange of food (trophallaxis) promotes the development of young honeybee workers. Accepted: 26 February 1999     

Mechanisms of social synchrony between circadian activity rhythms in cohabiting marmosets

In marmosets, social synchrony between circadian profiles of activity is stronger in animals that cohabit in a family. The activity of three breeding pairs was recorded by actiwatches to investigate the mechanisms involved in the synchrony between the circadian activity profiles during cohabitation in marmoset reproductive pairs. The dyads were submitted to LD 12:12 (21 days) and LL: 1) cohabitation (24 days), 2) removal of the cage mate (20 days), 3) reintroduction of the mate into the cage of the 1^st situation (30 days) and 4) removal of the cage mate (7 days). Next, they were rejoined and maintained in LD 12:12 (11 days). In conditions involving cohabitation of pair, the general and maximum correlation indexes between circadian profiles were higher in cage mates compared to animals of the same or different sex with which they maintain only acoustic and olfactive contact. This strong synchrony between rhythms was accompanied by a stable phase relationship at the activity onset and offset, with identical circadian periods between mates. When the pairs were separated, there was a break in stability in the phase relationships between activity profiles with different circadian periods and a greater phase angle difference between rhythms of cage mates. During separation, two females and one male progressively anticipated the activity onset and offset in a phase similar to that in previous conditions, expressing entrainment to the mate. During the first reintroduction, two pairs exhibited signs of masking in rhythm. Although modulation in the rhythm of some animals has been observed through acoustic cues from animals outside the colony, we suggest that cohabitation favors strong synchrony between the circadian activity profiles of marmoset reproductive pairs involving synchronization by entrainment and masking. Further studies in the absence of external social cues are necessary to clarify the role of these mechanisms on social synchronization in marmosets.     

Ultradian and circadian rhythms of sleep-wake and food-intake behavior during early infancy

The early development of sleep-wake and food-intake rhythms in human infants is reviewed. The development of a 24h day-night rhythm contains two observable developmental processes: the alterations in the periodic structure of behavior (decreased ultradian, increased circadian components) and the process of synchronization to external time (entrainment). The authors present the results of their studies involving 26 German children and compare them with previous investigations. In their research, it became evident that, during the first weeks of life, the time pattern of sleep-wake and food-intake behavior is characterized by different ultradian periodicities, ranging from 2h to 8h. In the course of further ontogenesis, the share of ultradian rhythms in the sleep-wake behavior decreases, while it remains dominant for food-intake behavior. The circadian component is established as early as the first weeks of life and increases in the months that follow. Besides, the authors' study supports the notion of broad interindividual variation in ultradian rhythms and in the development of a day-night rhythm. Examples of free-running rhythms of sleep-wake and food-intake behavior by various authors are strong indicators of the endogenous nature of the circadian rhythms in infants and show that the internal clock is already functioning at birth. It is still uncertain when the process of synchronization to external and social time cues begins and how differences in the maturation of perceptive organs affect the importance of time cues for the entrainment. Prepartally, the physiological maternal entrainment factors and mother-fetus interactions may be most important; during the first weeks of life, the social time cues gain importance, while light acts as a dominant “zeitgeber” at a later time only.     

The flow of jelly within a honeybee colony

Summary The flow of jelly from 100 nurse bees to the members of two normal-sized colonies was measured during one night. To follow the flow, nurses were injected with ¹⁴C-phenylalanine. They incorporated this label into the protein of their hypopharyngeal (brood food) glands and their own body protein. When they were allowed trophallactic contacts during the investigation period a loss of label and a shift away from the abdomen was observed, indicating protein synthesis in the hypopharyngeal glands from previously stored protein. Very young larvae were fed less frequently than older ones. Younger workers received larger amounts of jelly than older ones, but considerable amounts were given to foragers. Drones behaved similarly. Between one-third and one-half of the distributed jelly was given to imagines; 10% and 16% of all workers received radioactive jelly from 100 nurses in the two colonies during one night. Thus, jelly is a very important food for adult honey bees. There was a remarkable exchange of label within the class of nurses themselves that is interpreted as communication within the social system.Abbreviation dpm decays per minute     

Circadian Activity Rhythm of the Foragers of a Eusocial Bee (Scaptotrigona aff depilis,Hymenoptera, Apidae,Meliponinae) outside the Nest

In all bee colonies of the Meliponinae subfamily, activity inside the nest is temporally organized around the oviposition by the queen, assisted by nurse bees. This class is constituted by young bees that remain inside the nest. In a colony of Scaptotrigona aff depilis, the oviposition cycle occurs in a 3-hour period. The foragers are older bees that collect food for the colony in the field. Other tasks in the nest are performed by workers of ages intermediate between nurses and foragers. With the aim of studying activity rhythms, foragers were kept under constant light, with food constantly available and no flight restriction. The results showed that, although inside the nest the prevailing period is 3 hours, the activity of the foragers is a circadian rhythm, synchronized by the light/dark cycle and probably influenced by other environmental cycles as temperature and the availability of food sources.     

Testicular hormones modulate circadian rhythms of the diurnal rodent, Octodon degus

Sex differences have been identified in a variety of circadian rhythms, including free-running rhythms, light-induced phase shifts, sleep patterns, hormonal fluctuations, and rates of reentrainment. In the precocial, diurnal rodent Octodon degus, sex differences have been found in length of free-running rhythm (tau), phase response curves, rates of reentrainment, and in the use of social cues to facilitate reentrainment. Although gonadal hormones primarily organize circadian rhythms during early development, adult gonadal hormones have activational properties on various aspects of circadian rhythms in a number of species examined. Gonadectomy of adult female O. degus did not influence tau, phase angle of entrainment, or activity patterns in previous experiments. The present experiment examined the role of gonadal hormones in adult male degus' circadian wheel-running rhythms. We predicted that male gonadal hormones would have an activational effect on some aspects of circadian rhythms, particularly those in which we see sex differences. Phase angles of entrainment, tau, length of the active period (alpha), maximum and mean activity levels, and activity amplitude were examined for intact and castrated males housed in LD 12:12. Responses to light pulses while housed in constant darkness (DD) were also compared. Castration had no significant effect on tau or light-induced phase shifts. However, castration significantly increased phase angle of entrainment and decreased activity levels. The data indicate that adult gonadal steroids are not responsible for the sex differences in endogenous circadian mechanisms of O. degus (tau, PRC), although they influence activity level and phase angle of entrainment. This is most likely due to masking properties of testosterone, similar to the activity-increasing effects of estrogen during estrus in O. degus females.     

Circadian rhythms of the sex steroids of female Papio hamadryas in long-term hypokinesia

Radioimmunoassay was used to study the effect of restricted motor activity on circadian rhythms of blood plasma sexual steroids and cortisol in female Papio hamadryas during different phases of the menstrual cycle. Instact animals manifested clear-cut circadian rhythms of the testosterone and cortisol content during both the phases and those of estradiol and progesterone during the follicular phase of the cycle. Two-week immobilization did not produce any changes in the general pattern of circadian rhythms of cortisol and testosterone but led to a decrease in their mean daily concentrations, with that decrease being more pronounced for testosterone of females immobilized during the luteal phase. The authors established unbalance of sexual steroids with a dramatic fall of the mean daily concentrations of estradiol and progesterone and desynchronism of their circadian rhythms regardless of the cycle phase at the moment of immobilization. The mechanisms responsible for alterations in circadian rhythms of sexual steroids in hypokinetic females are discussed.     

Social influences on mammalian circadian rhythms: animal and human studies

While light is considered the dominant stimulus for entraining (synchronizing) mammalian circadian rhythms to local environmental time, social stimuli are also widely cited as 'zeitgebers' (time-cues). This review critically assesses the evidence for social influences on mammalian circadian rhythms, and possible mechanisms of action. Social stimuli may affect circadian behavioural programmes by regulating the phase and period of circadian clocks (i.e. a zeitgeber action, either direct or by conditioning to photic zeitgebers), by influencing daily patterns of light exposure or modulating light input to the clock, or by associative learning processes that utilize circadian time as a discriminative or conditioned stimulus. There is good evidence that social stimuli can act as zeitgebers. In several species maternal signals are the primary zeitgeber in utero and prior to weaning. Adults of some species can also be phase shifted or entrained by single or periodic social interactions, but these effects are often weak, and appear to be mediated by social stimulation of arousal. There is no strong evidence yet for sensory-specific nonphotic inputs to the clock. The circadian phase-dependence of clock resetting to social stimuli or arousal (the 'nonphotic' phase response curve, PRC), where known, is distinct from that to light and similar in diurnal and nocturnal animals. There is some evidence that induction of arousal can modulate light input to the clock, but no studies yet of whether social stimuli can shift the clock by conditioning to photic cues, or be incorporated into the circadian programme by associative learning. In humans, social zeitgebers appear weak by comparison with light. In temporal isolation or under weak light-dark cycles, humans may ignore social cues and free-run independently, although cases of mutual synchrony among two or more group-housed individuals have been reported. Social cues may affect circadian timing by controlling sleep-wake states, but the phase of entrainment observed to fixed sleep-wake schedules in dim light is consistent with photic mediation (scheduled variations in behavioural state necessarily create daily light-dark cycles unless subjects are housed in constant dark or have no eyes). By contrast, discrete exercise sessions can induce phase shifts consistent with the nonphotic PRC observed in animal studies. The best evidence for social entrainment in humans is from a few totally blind subjects who synchronize to the 24 h day, or to near-24 h sleep-wake schedules under laboratory conditions. However, the critical entraining stimuli have not yet been identified, and there are no reported cases yet of social entrainment in bilaterally enucleated blind subjects. The role of social zeitgebers in mammalian behavioural ecology, their mechanisms of action, and their utility for manipulating circadian rhythms in humans, remains to be more fully elaborated.     

Division of Labor Associated with Brood Rearing in the Honey Bee: How Does It Translate to Colony Fitness?

Division of labor is a striking feature observed in honey bees and many other social insects. Division of labor has been claimed to benefit fitness. In honey bees, the adult work force may be viewed as divided between non-foraging hive bees that rear brood and maintain the nest, and foragers that collect food outside the nest. Honey bee brood pheromone is a larval pheromone that serves as an excellent empirical tool to manipulate foraging behaviors and thus division of labor in the honey bee. Here we use two different doses of brood pheromone to alter the foraging stimulus environment, thus changing demographics of colony division of labor, to demonstrate how division of labor associated with brood rearing affects colony growth rate. We examine the effects of these different doses of brood pheromone on individual foraging ontogeny and specialization, colony level foraging behavior, and individual glandular protein synthesis. Low brood pheromone treatment colonies exhibited significantly higher foraging population, decreased age of first foraging and greater foraging effort, resulting in greater colony growth compared to other treatments. This study demonstrates how division of labor associated with brood rearing affects honey bee colony growth rate, a token of fitness.     

Birds of a feather clock together--sometimes: social synchronization of circadian rhythms

Biological systems use internal circadian clocks to efficiently organize physiological and behavioral activity within the 24-hour time domain. In the absence of time cues, circadian periods vary slightly from 24 hours, but in nature, ambient light serves as the most salient synchronizer for these rhythms, fine-tuning them to exactly 24 hours each day. For some species, social cues can serve to synchronize circadian rhythms in the absence of other time cues or to amplify ambiguous light cues. This has been demonstrated to various degrees in fruit flies, degus, birds, fish, bats, beavers and humans; however, studies in rats and hamsters have shown that social cues are less salient time cues for these species. Social influences on circadian timing might function to tightly organize the social group, thereby decreasing the chances of predation and increasing the likelihood of mating.