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.     

Photic and nonphotic inputs to the diurnal circadian clock

Diurnal animals occupy a different temporal niche from nocturnal animals and are consequently exposed to different amounts of light as well as different dangers. Accordingly, some variation exists in the way that diurnal animals synchronize their internal circadian clock to match the external 24-hour daily cycle. First, though the brain mechanisms underlying photic entrainment are very similar among species with different daily activity patterns, there is evidence that diurnal animals are less sensitive to photic stimuli compared to nocturnal animals. Second, stimuli other than light that synchronize rhythms (i.e. nonphotic stimuli) can also entrain and phase shift daily rhythms. Some of the rules that govern nonphotic entrainment in nocturnal animals as well as the brain mechanisms that control nonphotic influences on rhythms do not appear to apply to diurnal animals, however. Some evidence supports the idea that arousal or activity plays an important role in entraining rhythms in diurnal animals, either during the light (active) or dark (inactive) phases, though no consistent pattern is seen. GABAergic stimulation induces phase shifts during the subjective day in both diurnal and nocturnal animals. In diurnal Arvicanthis niloticus (Nile grass rats), SCN GABA_A receptor activation at this time results in phase delays while in nocturnal animals phase advances are induced. It appears that the effect of GABA at this circadian phase results from the inhibition of period gene expression in both diurnal and nocturnal animals. Nonetheless, the resulting phase shifts are in opposite directions. It is not known what stimuli or behaviours ultimately induce changes in GABA activity in the SCN that result in alterations of circadian phase in diurnal grass rats. Taken together, studies such as these suggest that it may be problematic to apply the principles governing nocturnal nonphotic entrainment and its underlying mechanisms to diurnal species including humans.     

Insect circadian clock outputs

Insects display an impressive variety of daily rhythms, which are most evident in their behaviour. Circadian timekeeping systems that generate these daily rhythms of physiology and behaviour all involve three interacting elements: the timekeeper itself (i.e. the clock), inputs to the clock through which it entrains and otherwise responds to environmental cues such as light and temperature, and outputs from the clock through which it imposes daily rhythms on various physiological and behavioural parameters. In insects, as in other animals, cellular clocks are embodied in clock neurons capable of sustained autonomous circadian rhythmicity, and those clock neurons are organized into clock circuits. Drosophila flies spend their entire lives in small areas near the ground, and use their circadian brain clock to regulate daily rhythms of rest and activity, so as to organize their behaviour appropriately to the daily rhythms of their local environment. Migratory locusts and butterflies, on the other hand, spend substantial portions of their lives high up in the air migrating long distances (sometimes thousands of miles) and use their circadian brain clocks to provide time-compensation to their sun-compass navigational systems. Interestingly, however, there appear to be substantial similarities in the cellular and network mechanisms that underlie circadian outputs in all insects.     

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.     

Circadian rhythms from multiple oscillators: lessons from diverse organisms

The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.     

Rhythms of locomotion expressed by Limulus polyphemus, the American horseshoe crab: II. Relationship to circadian rhythms of visual sensitivity

In the laboratory, horseshoe crabs express a circadian rhythm of visual sensitivity as well as daily and circatidal rhythms of locomotion. The major goal of this investigation was to determine whether the circadian clock underlying changes in visual sensitivity also modulates locomotion. To address this question, we developed a method for simultaneously recording changes in visual sensitivity and locomotion. Although every animal (24) expressed consistent circadian rhythms of visual sensitivity, rhythms of locomotion were more variable: 44% expressed a tidal rhythm, 28% were most active at night, and the rest lacked statistically significant rhythms. When exposed to artificial tides, 8 of 16 animals expressed circatidal rhythms of locomotion that continued after tidal cycles were stopped. However, rhythms of visual sensitivity remained stable and showed no tendency to be influenced by the imposed tides or locomotor activity. These results indicate that horseshoe crabs possess at least two biological clocks: one circadian clock primarily used for modulating visual sensitivity, and one or more clocks that control patterns of locomotion. This arrangement allows horseshoe crabs to see quite well while mating during both daytime and nighttime high tides.     

Adult circadian behavior in Drosophila requires developmental expression of cycle, but not period

Circadian clocks have evolved as internal time keeping mechanisms that allow anticipation of daily environmental changes and organization of a daily program of physiological and behavioral rhythms. To better examine the mechanisms underlying circadian clocks in animals and to ask whether clock gene expression and function during development affected subsequent daily time keeping in the adult, we used the genetic tools available in Drosophila to conditionally manipulate the function of the CYCLE component of the positive regulator CLOCK/CYCLE (CLK/CYC) or its negative feedback inhibitor PERIOD (PER). Differential manipulation of clock function during development and in adulthood indicated that there is no developmental requirement for either a running clock mechanism or expression of per. However, conditional suppression of CLK/CYC activity either via per over-expression or cyc depletion during metamorphosis resulted in persistent arrhythmic behavior in the adult. Two distinct mechanisms were identified that may contribute to this developmental function of CLK/CYC and both involve the ventral lateral clock neurons (LN(v)s) that are crucial to circadian control of locomotor behavior: (1) selective depletion of cyc expression in the LN(v)s resulted in abnormal peptidergic small-LN(v) dorsal projections, and (2) PER expression rhythms in the adult LN(v)s appeared to be affected by developmental inhibition of CLK/CYC activity. Given the conservation of clock genes and circuits among animals, this study provides a rationale for investigating a possible similar developmental role of the homologous mammalian CLOCK/BMAL1 complex.     

Cryptochrome restores dampened circadian rhythms and promotes healthspan in aging Drosophila

Circadian clocks generate daily rhythms in molecular, cellular, and physiological functions providing temporal dimension to organismal homeostasis. Recent evidence suggests two‐way relationship between circadian clocks and aging. While disruption of the circadian clock leads to premature aging in animals, there is also age‐related dampening of output rhythms such as sleep/wake cycles and hormonal fluctuations. Decay in the oscillations of several clock genes was recently reported in aged fruit flies, but mechanisms underlying these age‐related changes are not understood. We report that the circadian light–sensitive protein CRYPTOCHROME (CRY) is significantly reduced at both mRNA and protein levels in heads of old Drosophila melanogaster. Restoration of CRY using the binary GAL4/UAS system in old flies significantly enhanced the mRNA oscillatory amplitude of several genes involved in the clock mechanism. Flies with CRY overexpressed in all clock cells maintained strong rest/activity rhythms in constant darkness late in life when rhythms were disrupted in most control flies. We also observed a remarkable extension of healthspan in flies with elevated CRY. Conversely, CRY‐deficient mutants showed accelerated functional decline and accumulated greater oxidative damage. Interestingly, overexpression of CRY in central clock neurons alone was not sufficient to restore rest/activity rhythms or extend healthspan. Together, these data suggest novel anti‐aging functions of CRY and indicate that peripheral clocks play an active role in delaying behavioral and physiological aging.     

Neural circuits underlying circadian behavior in Drosophila melanogaster

Circadian clocks include control systems for organizing daily behavior. Such a system consists of a time-keeping mechanism (the clock or pacemaker), input pathways for entraining the clock, and output pathways for producing overt rhythms in behavior and physiology. In Drosophila melanogaster, as in mammals, neural circuits play vital roles in all three functional subdivisions of the circadian system. Regarding the pacemaker, multiple clock neurons, each with cell-autonomous pacemaker capability, are coupled to each other in a network. The outputs of different sets of clock neurons in this network combine to produce the normal bimodal pattern of locomotor activity observed in Drosophila. Regarding input, multiple sensory modalities (including light, temperature, and pheromones) use their own circuitry to entrain the clock. Regarding output, distinct circuits are likely involved for controlling the timing of eclosion and for generating the locomotor activity rhythms. This review summarizes work on all of these circadian circuits, and discusses the broader utility of studying the fly's circadian system.     

The circadian timekeeping system of Drosophila

Daily rhythms in behavior, physiology and metabolism are controlled by endogenous circadian clocks. At the heart of these clocks is a circadian oscillator that keeps circadian time, is entrained by environmental cues such as light and activates rhythmic outputs at the appropriate time of day. Genetic and molecular analyses in Drosophila have revealed important insights into the molecules and mechanisms underlying circadian oscillator function in all organisms. In this review I will describe the intracellular feedback loops that form the core of the Drosophila circadian oscillator and consider how they are entrained by environmental light cycles, where they operate within the fly and how they are thought to control overt rhythms in physiology and behavior. I will also discuss where work remains to be done to give a comprehensive picture of the circadian clock in Drosophila and likely many other organisms.     

A fly's eye view of circadian entrainment

The Drosophila circadian clock is an ideal model system for teasing out the molecular mechanisms of circadian behavior and the means by which animals synchronize to day-night cycles. The clock that drives behavioral rhythms, located in the lateral neurons in the central brain, consists of a feedback loop of the circadian genes period (per) and timeless (tim). The molecular cycle, roughly 24 h long, is constantly reset by the environment. This review focuses on the main input pathways of the dominant circadian zeitgeber, light. Light acts directly on the clock primarily through cryptochrome (cry), a deep brain blue-light photoreceptor. CRY activation causes rapid TIM degradation, which is a predicted means of resetting the clock both on a daily basis at dawn and on an acute basis following an entraining light pulse during the night hours. In the absence of cry, the clock can still be driven by photic input through the visual system, though the mechanisms underlying this entrainment are unclear. Temperature can also entrain the clock, although the mechanisms by which this occurs are also unclear.     

Biological clocks in Arabidopsis thaliana

Biological rhythms are ubiquitous in eukaryotes, and the best understood of these occur with a period of approximately a day – circadian rhythms. Such rhythms persist even when the organism is placed under constant conditions, with a period that is close, but not exactly equal, to 24 h, and are driven by an endogenous timer – one of the many 'biological clocks'. In plants, research into circadian rhythms has been driven forward by genetic experiments using Arabidopsis . Higher plant genomes include a particularly large number of genes involved in metabolism, and circadian rhythms may well provide the necessary coordination for the control of these – for example, around the diurnal rhythm of photosynthesis – to suit changing developmental or environmental conditions. The endogenous timer must be flexible enough to support these requirements. Current research supports this notion most strongly for the input pathway, in which multiple photoreceptors have been shown to mediate light input to the clock. Both input and output components are now related to putative circadian oscillator mechanisms by sequence homology or by experimental observation. It appears that the pathways linking some domains of the basic clock model may be very short indeed, or the mechanisms of these domains may overlap. Components of the first plant circadian output pathway to be identified unequivocally will help to determine exactly how many output pathways control the various phases of overt rhythms in plants.     

Metabolism as an integral cog in the mammalian circadian clockwork

Abstract

Circadian rhythms are an integral part of life. These rhythms are apparent in virtually all biological processes studies to date, ranging from the individual cell (e.g. DNA synthesis) to the whole organism (e.g. behaviors such as physical activity). Oscillations in metabolism have been characterized extensively in various organisms, including mammals. These metabolic rhythms often parallel behaviors such as sleep/wake and fasting/feeding cycles that occur on a daily basis. What has become increasingly clear over the past several decades is that many metabolic oscillations are driven by cell-autonomous circadian clocks, which orchestrate metabolic processes in a temporally appropriate manner. During the process of identifying the mechanisms by which clocks influence metabolism, molecular-based studies have revealed that metabolism should be considered an integral circadian clock component. The implications of such an interrelationship include the establishment of a vicious cycle during cardiometabolic disease states, wherein metabolism-induced perturbations in the circadian clock exacerbate metabolic dysfunction. The purpose of this review is therefore to highlight recent insights gained regarding links between cell-autonomous circadian clocks and metabolism and the implications of clock dysfunction in the pathogenesis of cardiometabolic diseases.     

To be or not to be rhythmic? A review of studies on organisms inhabiting constant environments

Abstract

Circadian clocks are endogenous time keeping mechanisms that drive near 24-h behavioural, physiological and metabolic rhythms in organisms. It is thought that organisms possess circadian clocks to facilitate coordination of essential biological events to the external day and night (extrinsic advantage) so as to enhance Darwinian fitness. However, on Earth, there are a number of habitats that are not subject to such robust daily cycling of geo-physical factors. Do organisms living under such conditions exhibit rhythmic behaviours that are driven by endogenous circadian clocks? We attempt to critically survey studies of rhythms (or the lack of them) in organisms living in a range of constant environments. Many such organisms do show rhythms in behaviour and/or physiological variables. We suggest that such presence of rhythms may be indicative of an underlying clock that facilitates, (a) internal synchrony among rhythms, and (b) temporal partitioning of incompatible cellular processes (intrinsic advantage). We then highlight reasons that limit our interpretations about the presence (or absence) of clocks in such organisms living under constant conditions, and suggest possible methods to conclusively test whether or not rhythms in these organisms are driven by endogenous circadian clocks with the hope that it may enhance our understanding of circadian clocks in organisms under constant environments.     

Aging of the circadian system

Ageing affects all organic structures and processes, including the circadian system and its principal sign, the biological rhythms. The circadian system temporarily organizes the living organisms. It is made up of structures receiving information from the external environment (that synchronize the circadian clock), the central circadian pacemaker and the peripheral clocks that depends on it, and several outputs that are the overt rhythms. Ageing produces losses in function of all these three components: receptors (the eye in its capacity to transmit the more active light information to the circadian system), the central pacemaker (due to alterations in neuronal function) and the outputs. This leads to the alteration of overt rhythms, with losses in the phase relationship between them, a reduction in amplitude, an increase in fragmentation and an advancement of its phase.     

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day^1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).Although circadian rhythms in running-wheel activity are typically linked to the SCN clock^6-8, circadian oscillators in many other regions of the brain and body^9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN^15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators^17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.