Following general anesthesia, people are often confused about the time of day and experience sleep disruption and fatigue. It has been hypothesized that these symptoms may be caused by general anesthesia affecting the circadian clock. The circadian clock is fundamental to our well-being because it regulates almost all aspects of our daily biochemistry, physiology, and behavior. Here, we investigated the effects of the most common general anesthetic, isoflurane, on time perception and the circadian clock using the honeybee (Apis mellifera) as a model. A 6-h daytime anesthetic systematically altered the time-compensated sun compass orientation of the bees, with a mean anticlockwise shift in vanishing bearing of 87 in the Southern Hemisphere and a clockwise shift in flight direction of 58 in the Northern Hemisphere. Using the same 6-h anesthetic treatment, time-trained bees showed a delay in the start of foraging of 3.3 h, and whole-hive locomotor-activity rhythms were delayed by an average of 4.3 h. We show that these effects are all attributable to a phase delay in the core molecular clockwork. mRNA oscillations of the central clock genes cryptochrome-m and period were delayed by 4.9 and 4.3 h, respectively. However, this effect is dependent on the time of day of administration, as is common for clock effects, and nighttime anesthesia did not shift the clock. Taken together, our results suggest that general anesthesia during the day causes a persistent and marked shift of the clock effectively inducing ``jet lag'' and causing impaired time perception. Managing this effect in humans is likely to help expedite postoperative recovery.
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. (C) 2012 Elsevier Ltd. All rights reserved.
Honey bee workers care for (''nurse'') the brood around the clock without circadian rhythmicity, but then they forage outside with strong circadian rhythms and a consolidated nightly rest. This chronobiological plasticity is associated with variation in the expression of the canonical ``clock genes'' that regulate the circadian clock: nurse bees show no brain rhythms of expression, while foragers do. These results suggest that the circadian system is organized differently in nurses and foragers. Nurses switch to activity with circadian rhythms shortly after being removed from the hive, suggesting that at least some clock cells in their brain continue to measure time while in the hive. We performed a microarray genome-wide survey to determine general patterns of brain gene expression in nurses and foragers sampled around the clock. We found 160 and 541 transcripts that exhibited significant sinusoidal oscillations in nurses and foragers, respectively, with peaks of expression distributed throughout the day in both task groups. Consistent with earlier studies, transcripts of genes involved in circadian rhythms, including Clockwork Orange that has not been studied before in bees, oscillated in foragers but not in nurses. The oscillating transcripts also were enriched for genes involved in the visual system, ``development'' and ``response to stimuli'' (foragers), ``muscle contraction'' and ``microfilament motor gene expression'' (nurses), and ``generation of precursor metabolites'' and ``energy'' (both). Transcripts of genes encoding P450 enzymes oscillated in both nurses and foragers but with a different phase. This study identified new putative clock-controlled genes in the honey bee and suggests that some brain functions show circadian rhythmicity even in nurse bees that are active around the clock.
The circadian clock is a core molecular mechanism that allows organisms to anticipate daily environmental changes and adapt the timing of behaviors to maximize efficiency. In social insects, the ability to maintain the appropriate temporal order is thought to improve colony efficiency and fitness. We used the newly sequenced fire ant (Solenopsis invicta) genome to characterize the first ant circadian clock. Our results reveal that the fire ant clock is similar to the clock of the honeybee, a social insect with an independent evolutionary origin of sociality. Gene trees for the eight core clock genes, period, cycle, clock, cryptochrome-m, timeout, vrille, par domain protein 1 & clockwork orange, show ant species grouping closely with honeybees and Nasonia wasps as an outgroup to the social Hymenoptera. Expression patterns for these genes suggest that the ant clock functions similar to the honeybee clock, with period and cry-m mRNA levels increasing during the night and cycle and clockwork orange mRNAs cycling approximately anti-phase to period. Gene models for five of these genes also parallel honeybee models. In particular, the single ant cryptochrome is an ortholog of the mammalian-type (cry-m), rather than Drosophila-like protein (cry-d). Additionally, we find a conserved VPIFAL C-tail region in clockwork orange shared by insects but absent in vertebrates. Overall, our characterization of the ant clock demonstrates that two social insect lineages, ants and bees, share a similar, mammalian-like circadian clock. This study represents the first characterization of clock genes in an ant and is a key step towards understanding socially-regulated plasticity in circadian rhythms by facilitating comparative studies on the organization of circadian clockwork.
We identified a predicted compact cysteine-rich sequence in the honey bee genome that we called Raalin. Raalin transcripts are enriched in the brain of adult honey bee workers and drones, with only minimum expression in other tissues or in pre-adult stages. Open-reading frame (ORF) homologues of Raalin were identified in the transcriptomes of fruit flies, mosquitoes and moths. The Raalin-like gene from Drosophila melanogaster encodes for a short secreted protein that is maximally expressed in the adult brain with negligible expression in other tissues or pre-imaginal stages. Raalin-like sequences have also been found in the recently sequenced genomes of six ant species, but not in the jewel wasp Nasonia vitripennis. As in the honey bee, the Raalin-like sequences of ants do not have an ORF. A comparison of the genome region containing Raalin in the genomes of bees, ants and the wasp provides evolutionary support for an extensive genome rearrangement in this sequence. Our analyses identify a new family of ancient cysteine-rich short sequences in insects in which insertions and genome rearrangements may have disrupted this locus in the branch leading to the Hymenoptera. The regulated expression of this transcript suggests that it has a brain-specific function.
Female bumble bee workers of the same species often show a profound body size variation that is linked to a division of labour. Large individuals are more likely to forage whereas small individuals are more likely to perform in-nest activities. A higher sensory sensitivity, stronger circadian rhythms as well as better learning and memory performances appear to better equip large individuals for outdoor activities compared to their smaller siblings. The molecular mechanisms underlying worker functional polymorphism remain unclear. Proteins are major determinants of an individual's morphology and behaviour. We hypothesized that the abundance of proteins such as metabolic enzymes as well as proteins involved in neuronal functions would differ with body size and provide insights into the mechanisms underlying size-dependent physiological specialization in bumble bee workers. We conducted protein quantification measurements based on liquid chromatography coupled with tandem mass spectrometry on tissue samples derived from small and large Bombus impatiens and Bombus terrestris workers. Proteins found to differ significantly in abundance between small and large workers belong to the categories of structure, energy metabolism and stress response. These findings provide the first proteomic insight into mechanisms associated with size-based division of labour in social insects.
The diverse social lifestyle and the small and accessible nervous system of insects make them valuable for research on the adaptive value and the organization principles of circadian rhythms and sleep. We focus on two complementary model insects, the fruit fly Drosophila melanogaster, which is amenable to extensive transgenic manipulations, and the honey bee Apis mellifera, which has rich and well-studied social behaviors. Social entrainment of activity rhythms (social synchronization) has been studied in many animals. Social time givers appear to be specifically important in dark cavity-dwelling social animals, but here there are no other clear relationships between the degree of sociality and the effectiveness of social entrainment. The olfactory system is important for social entrainment in insects. Little is known, however, about the molecular and neuronal pathways linking olfactory neurons to the central clock. In the honey bee, the expression, phase, and development of circadian rhythms are socially regulated, apparently by different signals. Peripheral clocks regulating pheromone synthesis and the olfactory system have been implicated in social influences on circadian rhythms in the fruit fly. An enriched social environment increases the total amount of sleep in both fruit flies and honey bees. In fruit flies, these changes have been linked to molecular and neuronal processes involved in learning, memory, and synaptic plasticity. The studies on insects suggest that social influences on the clock are richer than previously appreciated and have led to important breakthroughs in our understanding of the mechanisms underlying social influences on sleep and circadian rhythms. (C) 2012, Elsevier Inc.
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.
Bees provide an excellent model with which to study the neuronal and molecular modifications associated with the evolution of sociality because relatively closely related species differ profoundly in social behaviour, from solitary to highly social. The recent development of powerful genomic tools and resources has set the stage for studying the social behaviour of bees in molecular terms. We review `ground plan' and `genetic toolkit' models which hypothesize that discrete pathways or sets of genes that regulate fundamental behavioural and physiological processes in solitary species have been co-opted to regulate complex social behaviours in social species. We further develop these models and propose that these conserved pathways and genes may be incorporated into `social pathways', which consist of relatively independent modules involved in social signal detection, integration and processing within the nervous and endocrine systems, and subsequent behavioural outputs. Modifications within modules or in their connections result in the evolution of novel behavioural patterns. We describe how the evolution of pheromonal regulation of social pathways may lead to the expression of behaviour under new social contexts, and review plasticity in circadian rhythms as an example for a social pathway with a modular structure.
Although texts and wall paintings suggest that bees were kept in the Ancient Near East for the production of precious wax and honey, archaeological evidence for beekeeping has never been found. The Biblical term ``honey'' commonly was interpreted as the sweet product of fruits, such as dates and figs. The recent discovery of unfired clay cylinders similar to traditional hives still used in the Near East at the site of Tel Rehov in the Jordan valley in northern Israel suggests that a large-scale apiary was located inside the town, dating to the 10th-early 9th centuries B.C.E. This paper reports the discovery of remains of honeybee workers, drones, pupae, and larvae inside these hives. The exceptional preservation of these remains provides unequivocal identification of the clay cylinders as the most ancient beehives yet found. Morphometric analyses indicate that these bees differ from the local subspecies Apis mellifera syriaca and from all subspecies other than A. m. anatoliaca, which presently resides in parts of Turkey. This finding suggests either that the Western honeybee subspecies distribution has undergone rapid change during the last 3,000 years or that the ancient inhabitants of Tel Rehov imported bees superior to the local bees in terms of their milder temper and improved honey yield.
The social environment influences the circadian clock of diverse animals, but little is known about the functional significance, the specifics of the social signals, or the dynamics of socially mediated changes in the clock. Honey bees switch between activities with and without circadian rhythms according to their social task. Forager bees have strong circadian rhythms, whereas ``nurse'' bees typically care for the brood around-the-clock with no circadian rhythms in behavior or clock gene expression. Here we show that nurse-age bees that were restricted to a broodless comb inside or outside the hive showed robust behavioral and molecular circadian rhythms. By contrast, young nurses tended brood with no circadian rhythms in behavior or clock gene expression, even under a light-dark illumination regime or when placed with brood-but no queen-in a small cage outside the hive. This behavior is context-dependent because nurses showed circadian rhythms in locomotor activity shortly after removal from the hive, and in clock gene expression after similar to 16 h. These findings suggest that direct interaction with the brood modulates the circadian system of honey bees. The dynamics of rhythm development best fit models positing that at least some pacemakers continue to oscillate and be entrained by the environment in nurses that are active around the clock. These cells set the phase to the clock network when the nurse is removed from the hive. These findings suggest that despite its robustness, the circadian system exhibits profound plasticity, enabling adjustment to rapid changes in the social environment.
The honeybee has long been an important model for studying the interplay between the circadian clock and complex behaviors. This article reviews studies further implicating the circadian clock in complex social behaviors in bees. The article starts by introducing honeybee social behavior and sociality and then briefly summarizes current findings on the molecular biology and neuroanatomy of the circadian system of honeybees that point to molecular similarities to the mammalian clockwork rather than to that of Drosophila. Foraging is a social behavior in honeybees that relies on the circadian clock for timing visits to flowers, time-compensated sun-compass navigation, and dance communication used by foragers to recruit nestmates to rewarding flower patches. The circadian clock is also important for the social organization of honeybee societies. Social factors influence the ontogeny of circadian rhythms and are important for social synchronization of worker activities. Both queen and worker bees switch between activities with and without circadian rhythms. In workers this remarkable plasticity is associated with the division of labor; nurse bees care for the brood around the clock with similar levels of clock gene expression throughout the day, whereas foragers have strong behavioral circadian rhythms with oscillating brain clock gene levels. This plasticity in circadian rhythms is regulated by direct contact with the brood and is context-specific in that nurse bees that are removed from the hive exhibit activity with strong behavioral and molecular rhythms. These studies on the sociochronobiology of honeybees and comparative studies with other social insects suggest that the evolution of sociality has influenced the characteristics of the circadian system in honeybees.
Background: Regulation of worker behavior by dominant queens or workers is a hallmark of insect societies, but the underlying molecular mechanisms and their evolutionary conservation are not well understood. Honey bee and bumble bee colonies consist of a single reproductive queen and facultatively sterile workers. The queens' influences on the workers are mediated largely via inhibition of juvenile hormone titers, which affect division of labor in honey bees and worker reproduction in bumble bees. Studies in honey bees identified a transcription factor, Kruppel-homolog 1 (Kr-h1), whose expression in worker brains is significantly downregulated in the presence of a queen or queen pheromone and higher in forager bees, making this gene an ideal candidate for examining the evolutionary conservation of socially regulated pathways in Hymenoptera. Results: In contrast to honey bees, bumble bees foragers do not have higher Kr-h1 levels relative to nurses: in one of three colonies levels were similar in nurses and foragers, and in two colonies levels were higher in nurses. Similarly to honey bees, brain Kr-h1 levels were significantly downregulated in the presence versus absence of a queen. Furthermore, in small queenless groups, Kr-h1 levels were downregulated in subordinate workers with undeveloped ovaries relative to dominant individuals with active ovaries. Brain Kr-h1 levels were upregulated by juvenile hormone treatment relative to a vehicle control. Finally, phylogenetic analysis indicates that KR-H1 orthologs are presence across insect orders. Though this protein is highly conserved between honey bees and bumble bees, there are significant differences between orthologs of insects from different orders. Conclusions: Our results suggest that Kr-h1 is associated with juvenile hormone mediated regulation of reproduction in bumble bees. The expression of this transcription factor is inhibited by the queen and associated with endocrine mediated regulation of social organization in two species of bees. Thus, KR-H1 may transcriptionally regulate a conserved genetic module that is part of a pathway that has been co-opted to function in social behavior, and adjusts the behavior of workers to their social environmental context.
Chapter Outline 30.1 Introduction 1028 30.2 Overview of Division of Labor in Insect Societies 1028 30.2.1 Division of Labor for Reproduction 1029 30.2.2 Division of Labor among Workers 1029 30.2.3 Primitive and Advanced Eusociality 1030 30.3 Insect Hormones That Influence Division of Labor 1031 30.4 Division of Labor for Reproduction: Endocrine-Mediated Social Interactions among Adult Colony Members 1032 30.4.1 JH and Primitively Eusocial Insects 1033 30.4.2 JH and Advanced Eusocial Insects 1035 30.4.3 Ecdysteroids and Reproductive Division of Labor 1038 30.4.4 Biogenic Amines and Reproductive Division of Labor 1040 30.5 Division of Labor for Reproduction: Endocrine-Mediated Queen/Worker Determination 1041 30.5.1 Physical Factors 1041 30.5.2 Pheromones 1041 30.5.3 Nutrition 1042 30.5.4 Hormonal Integration 1042 30.5.5 Tissue Responses to Caste-Determining Endocrine Factors 1044 30.6 Division of Labor for Colony Growth and Development: Endocrine Influences on Age-Related Division of Labor among Workers 1044 30.6.1 Other Neuroendocrine and Neuromodulatory Factors That Influence Age-Related Division of Labor in Honeybee Colonies 1047 30.7 Division of Labor for Colony Growth and Development: Endocrine Influences on Worker Size and Subcaste 1049 30.7.1 Physical Factors 1049 30.7.2 Nutrition and Pheromones 1049 30.7.3 Hormonal Integration 1050 30.7.4 Tissue Responses to Worker Caste-Determining Endocrine Factors 1050 30.8 Summary 1051 30.9 Speculation on the Evolution of Division of Labor: A Neuroendocrine Perspective 1051 30.9.1 Level One: Incipient Societies and Endocrine-Mediated Social Inhibition among Adults 1053 30.9.2 Level Two: Pre-Adult, Endocrine-Mediated Social Inhibition 1054 30.9.3 Level Three: Pre-Adult, Endocrine-Mediated Social Inhibition Enhanced by Disruptive Selection 1054 30.9.4 Level Four: Division of Labor among Adult Workers and Its Regulation by Endocrine-Mediated Social Inhibition 1055 30.9.5 Level Five: Division of Labor among Morphologically Distinct Adult Workers and Its Regulation by Pre-Adult, Endocrine-Mediated Social Inhibition 1056 30.9.6 Concluding Remarks 1056 References 1057 Further Reading 1067
Large bumblebee (Bombus terrestris) workers typically visit flowers to collect pollen and nectar during the day and rest in the nest at night. Small workers are less likely to forage, but instead stay in the nest and tend brood around the clock. Because Pigment Dispersing Factor (PDF) has been identified as a neuromodulator in the circadian network of insects, we used an antiserum that recognizes this peptide to compare patterns of PDF-immunoreactivity (PDF-ir) in the brains of large and small workers. Our study provides the first description of PDF distribution in the bumblebee brain, and shows a pattern that is overall similar to that of the honey bee,Apis mellifera. The brains of large bumblebee workers contained a slightly but significantly higher number of PDF-ir neurons than did the brains of small sister bees. Body size was positively correlated with area of the PDF-ir somata and negatively correlated with the maximal staining intensity. These results provide a neuronal correlate to the previously reported body size-associated variation in behavioral circadian rhythmicity. These differences in PDF-ir are consistent with the hypothesis that body size-based division of labor in bumblebees is associated with adaptations of the morphology and function of the brain circadian system. (C) 2009 Published by Elsevier Ltd.
Honeybee (Apis mellifera) foragers are among the first invertebrates for which sleep behavior has been described. Foragers (typically older than 21 days) have strong circadian rhythms; they are active during the day, and sleep during the night. We explored whether young bees (similar to 3 days of age), which are typically active around-the-clock with no circadian rhythms, also exhibit sleep behavior. We combined 24-hour video recordings, detailed behavioral observations, and analyses of response thresholds to a light pulse for individually housed bees in various arousal states. We characterized three sleep stages in foragers on the basis of differences in body posture, bout duration, antennae movements and response threshold. Young bees exhibited sleep behavior consisting of the same three stages as observed in foragers. Sleep was interrupted by brief awakenings, which were as frequent in young bees as in foragers. Beyond these similarities, we found differences in the sleep architecture of young bees and foragers. Young bees passed more frequently between the three sleep stages, and stayed longer in the lightest sleep stage than foragers. These differences in sleep architecture may represent developmental and/or environmentally induced variations in the neuronal network underlying sleep in honeybees. To the best of our knowledge, this is the first evidence for plasticity in sleep behavior in insects.
Little is known about the temporal organization of defensive behavior in honeybees. We studied ``guards'', the best-characterized class of colony defenders. We synchronized small groups under a light-dark illumination regime (LD), and video recorded their aggression toward an intruder bumblebee worker. In 1 out of 3 trials (each trial with a different source colony), the latency before the first attack was longer during the night in LD, or subjective night in constant conditions (DD); a similar trend was observed in DD in the two other trials. In 2 out of 3 trials, the number of stinging attempts varied with highest levels during the day in DD, but not in LD. There was a similar trend for the number of biting events. These findings reveal temporal variation in aggression under constant conditions, consistent with the hypothesis that the circadian clock influences guard aggressiveness. Nevertheless, the variability between LD and DD and across colonies calls for additional studies before reaching a definitive conclusion.
We identified and characterized eight genes encoding putative Takeout/juvenile hormone binding proteins (To/JHBP) in the honeybee genome. Phylogenetic analyses revealed nine distinct lineages within this gene family, including those containing Takeout (To) and JHBP for which there are no honeybee homologs. Their diversity and ubiquitous expression suggest that To/JHBP proteins are involved in diverse and important processes in insects. We further characterized the expression of one of these genes, GB19811 that is ubiquitously expressed. GB19811 transcript levels in the abdomen increased, and decreased in the head with worker age. There was no influence of colony environment or brood care behavior on GB19811 expression in young bees. Young bees treated with juvenile hormone (JH) showed a decrease in head GB19811 mRNA levels. This finding is consistent with the premise that JH, for which titers typically increase with age, is involved in age-related modulation of GB19811 expression. In contrast to Drosophila Takeout, the expression of GB19811 did not vary with diurnal or circadian rhythms. Taken together, these findings suggest that GB19811 is not an ortholog of Takeout, and is involved in JH-mediated regulation of adult honeybee worker development. (c) 2007 Elsevier Ltd. All rights reserved.