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.

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.

Octopamine (OA) and juvenile hormone (JH) are implicated in the regulation of age-based division of labor in the honeybee, Apis mellifera. We tested the hypothesis that these two neuroendocrine signals influence task-associated plasticity in circadian and diurnal rhythms, and in brain expression of the clock gene period (per). Treatment with OA, OA antagonist (epinastine), or both, did not affect the age at onset of circadian rhythmicity or the free running period in constant darkness (DD). Young bees orally treated with OA in light-dark (LD) illumination regime for 6 days followed by DD showed reduced alpha (the period between the daily onset and offset of activity) during the first 4 days in LD and the first 4 days in DD. Oral treatment with OA, epinastine, or both, but not manipulations of JH levels, caused increased average daily levels and aberrant patterns of brain per mRNA oscillation in young bees. These results suggest that OA and JH do not influence the development or function of the central pacemaker but rather that OA influences the brain expression of a clock gene and characteristics of locomotor behavior that are not thought to be under direct control of the circadian pacemaker.

Social factors constitute an important component of the environment of many animals and have a profound influence on their physiology and behavior. Studies of social influences on circadian rhythms have been hampered by a methodological trade-off: automatic data acquisition systems obtain high-quality data but are effective only for individually isolated animals and therefore compromise by requiring a context that may not be sociobiologically relevant. Human observers can monitor animal activity in complex social environments but are limited in the resolution and quality of data that can be gathered. The authors developed and validated a method for prolonged, automatic, high-quality monitoring of focal honey bees in a relatively complex social environment and with minimal illumination. The method can be adapted for studies on other animals. The authors show that the system provides a reliable estimation of the actual path of a focal bee, only rarely misses its location for > I min, and removes most nonspecific signals from the background. Using this system, the authors provide the first evidence of social influence on the ontogeny of activity rhythms. Young bees that were housed with old foragers show similar to 24-h rhythms in locomotor activity at a younger age and with stronger rhythms than bees housed with a similar number of young bees. By contrast, the maturation of the hypopharyngeal glands was slower in bees housed with foragers, similar to findings in previous studies. The morphology and function of the hypopharyngeal glands vary along with age-based division of labor. Therefore, these findings indicate that social inhibition of task-related maturation was effective in the experimental setup. This study suggests that although the ontogeny of circadian rhythms is typically correlated with the age-based division of labor, their social regulation is different.

Various animals naturally switch to considerable periods of around-the-clock activity with no apparent ill effects. Such plasticity in overt circadian rhythms might be observed because the clock is masked by the influence of external factors, is uncoupled from behavioral outputs, or results from genuine plasticity in the clock machinery. We studied honeybees in which plasticity in circadian rhythms is socially modulated and associated with the division of labor. We confirm that ``nurse'' bees care for the brood around-the-clock even when experiencing a light: dark illumination regime. However, nurses transferred from the hive to individual cages in constant conditions have robust circadian rhythms in locomotor activity with an onset of activity at the subjective morning. These data indicate that circadian rhythmicity in nurses depends on their environment, and suggest that some clockwork components were entrained even in nurses active around the clock while in the hive. Brain oscillations in transcript abundance for the putative clock genes Period, Cryptochrome-m, Cycle, and Timeout were attenuated or totally suppressed in nurses as compared to behaviorally rhythmic foragers, irrespective of the illumination regime. These findings provide the first support for the hypothesis that natural plasticity in circadian rhythms is associated with reorganization of the internal clock-work.

We investigated labor- related plasticity in the circadian clock of the bumblebee Bombus terrestris. Bumblebee workers vary enormously in body size, and we found that size, division of labor, and diurnal rhythms in activity are correlated in B. terrestris colonies. Large workers typically perform foraging activities with strong diurnal rhythms and low activity at night, whereas small bees typically care for ( nurse) brood around the clock with weak or no diurnal rhythms. Under constant laboratory conditions, circadian rhythms in locomotor activity were weaker, less stable, and developed at a later age in small ( nurse- size) bees compared to their larger ( forager- size) sisters. Under a light: dark illumination regime, many small bees, particularly at a young age, were active during the dark phase, fewer small bees developed rhythms, and they did so later compared to large bees. Taken together these findings reveal naturally occurring attenuation or suppression in the circadian clock of small bees that is determined during pre- adult development. This deficiency in clock function, however, does not result in pathology but rather appears to be functionally significant, because it is associated with around- the- clock brood care activity and therefore apparently improves divisions of labor and colony efficiency. This in turn suggests that variation in social biology influences traits of the circadian clock.

Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A+T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A. mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A. mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement.

The circadian clock of the honey bee is implicated in ecologically relevant complex behaviors. These include time sensing, time-compensated sun-compass navigation, and social behaviors such as coordination of activity, dance language communication, and division of labor. The molecular underpinnings of the bee circadian clock are largely unknown. We show that clock gene structure and expression pattern in the honey bee are more similar to the mouse than to Drosophila. The honey bee genome does not encode an ortholog of Drosophila Timeless (Tim1), has only the mammalian type Cryptochrome (Cry-m), and has a single ortholog for each of the other canonical ``clock genes.'' In foragers that typically have strong circadian rhythms, brain mRNA levels of amCry, but not amTim as in Drosophila, consistently oscillate with strong amplitude and a phase similar to amPeriod (amPer) under both light-dark and constant darkness illumination regimes. In contrast to Drosophila, the honey bee amCYC protein contains a transactivation domain and its brain transcript levels oscillate at virtually an anti-phase to amPer, as it does in the mouse. Phylogenetic analyses indicate that the basal insect lineage had both the mammalian and Drosophila types of Cry and Tim. Our results suggest that during evolution, Drosophila diverged from the ancestral insect clock and specialized in using a set of clock gene orthologs that was lost by both mammals and bees, which in turn converged and specialized in the other set. These findings illustrate a previously unappreciated diversity of insect clockwork and raise critical questions concerning the evolution and functional significance of species-specific variation in molecular clockwork.

We measured seasonal variation in the locomotor behavior of newly emerged adult honey bee workers in the laboratory. Analyses of bees from 12 colonies, 7 of which were tested once and 5 tested more than once, revealed seasonal changes in the free-running period (FRP) of the rhythm for locomotor behavior, with an increase from spring to summer. At the same time there was a decrease in the age at onset of circadian rhythmicity. There were no seasonal changes in overall levels of locomotor activity. Temperature and photoperiod, the only factors known to mediate plasticity in the insect clock, cannot account for the observed seasonal variation because bees were maintained under constant conditions. In a second experiment we found no differences in the FRP of nurses and foragers obtained from colonies maintained in a 12 h light: 12 h dark illumination regime. These findings suggest that exposure to unknown cues during preadult stages may affect the circadian behavior of adult bees.

To determine whether ecdysteroids are associated with reproductive division of labor in Bombus terrestris, we measured their levels in hemolymph and ovaries of queens and workers. Queens heading colonies had large active ovaries with high ecdysteroid content, whereas virgin gynes and mated queens before and after diapause had undeveloped ovaries with low ecdysteroid content. The hemolymph ecdysteroid titer was rather variable, but in a pooled analysis of mated queens before and after diapause versus colony-heading queens, ecdysteroid titers were higher in the latter group. In workers, agonistic behavior, ovarian activity, ovarian ecdysteroid content, and hemolymph ecdysteroid titers were positively correlated, and were lowest when a queen was present. In queenless workers, ecdysteroid levels were elevated in dominant workers, and were also influenced by the presence of brood and by group demography; hormone levels were higher in bees kept in larger groups. These findings are consistent with the premise that in B. terrestris the ovary is the primary site of ecdysteroid synthesis, and they show that ecdysteroids levels vary with the social environment. (c) 2005 Elsevier Ltd. All rights reserved.

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. (C) 2004 Elsevier Ltd. All rights reserved.