Octopamine and tyramine influence the behavioral profile of locomotor activity in the honey bee (Apis mellifera)

The biogenic amines octopamine and tyramine are believed to play a number of important roles in the behavior of invertebrates including the regulation of motor function. To investigate the role of octopamine and tyramine in locomotor behavior in honey bees, subjects were injected with a range of concentrations of octopamine, tyramine, mianserin or yohimbine. Continuous observation of freely moving worker bees was used to examine the effects of these treatments on the amount of time honey bees spent engaged in different locomotor behaviors such as walking, grooming, fanning and flying. All treatments produced significant shifts in behavior. Decreases in time spent walking and increases in grooming or stopped behavior were observed for every drug. However, the pattern of the shift depended on drug, time after injection and concentration. Flying behavior was differentially affected with increases in flying seen in octopamine treated bees, whereas those receiving tyramine showed a decrease in flying. Taken together, these data provide evidence that octopamine and tyramine modulate motor function in the honey bee perhaps via interaction with central pattern generators or through effects on sensory perception.     

Biogenic amines and division of labor in honey bee colonies: behaviorally related changes in the antennal lobes and age-related changes in the mushroom bodies

Levels of the biogenic amines dopamine, serotonin, and octopamine were measured in different brain regions of adult worker honey bees as a function of age-related division of labor, using social manipulations to unlink age and behavioral state. In the antennal lobes, foragers had higher levels of all three amines than nurses, regardless of age. Differences were larger for octopamine than serotonin or dopamine. In the mushroom bodies, older bees had higher levels of all three amines than younger bees, regardless of behavioral state. These correlative results suggest that increases in octopamine in the antennal lobes may be particularly important in the control of age-related division of labor in honey bees. Accepted: 10 February 1999     

Aversive learning in honeybees revealed by the olfactory conditioning of the sting extension reflex

Invertebrates have contributed greatly to our understanding of associative learning because they allow learning protocols to be combined with experimental access to the nervous system. The honeybee Apis mellifera constitutes a standard model for the study of appetitive learning and memory since it was shown, almost a century ago, that bees learn to associate different sensory cues with a reward of sugar solution. However, up to now, no study has explored aversive learning in bees in such a way that simultaneous access to its neural bases is granted. Using odorants paired with electric shocks, we conditioned the sting extension reflex, which is exhibited by harnessed bees when subjected to a noxious stimulation. We show that this response can be conditioned so that bees learn to extend their sting in response to the odorant previously punished. Bees also learn to extend the proboscis to one odorant paired with sugar solution and the sting to a different odorant paired with electric shock, thus showing that they can master both appetitive and aversive associations simultaneously. Responding to the appropriate odorant with the appropriate response is possible because two different biogenic amines, octopamine and dopamine subserve appetitive and aversive reinforcement, respectively. While octopamine has been previously shown to substitute for appetitive reinforcement, we demonstrate that blocking of dopaminergic, but not octopaminergic, receptors suppresses aversive learning. Therefore, aversive learning in honeybees can now be accessed both at the behavioral and neural levels, thus opening new research avenues for understanding basic mechanisms of learning and memory.     

Cognitive components of color vision in honey bees: how conditioning variables modulate color learning and discrimination

Since the demonstration of color vision in honey bees 100 years ago by Karl von Frisch, appetitive conditioning to color targets has been used as the principal way to access behavioral aspects of bee color vision. Yet, analyses on how conditioning parameters affect color perception remained scarce. Conclusions on bee color vision have often been made without referring them to the experimental context in which they were obtained, and thus presented as absolute facts instead of realizing that subtle variations in conditioning procedures might yield different results. Here, we review evidence showing that color learning and discrimination in bees are not governed by immutable properties of their visual system, but depend on how the insects are trained and thus learn a task. The use of absolute or differential conditioning protocols, the presence of aversive reinforcement in differential conditioning and the degrees of freedom of motor components determine dramatic variations in color discrimination. We, thus, suggest top-down attentional modulation of color vision to explain the changes in color learning and discrimination reviewed here. We discuss the possible neural mechanisms of this modulation and conclude that color vision experiments require a careful consideration of how training parameters shape behavioral responses.     

Octopamine modulates responsiveness to foraging-related stimuli in honey bees (Apis mellifera)

The biogenic amine neurochemical octopamine is involved in the onset of foraging behaviour in honey bees. We tested the hypothesis that octopamine influences honey bee behavioural development by modulating responsiveness to task-related stimuli. We examined the effect of octopamine treatment on responsiveness to brood pheromone (an activator of foraging) and to the presence of older bees in the colony (an inhibitor of foraging in young bees). Octopamine treatment increased responsiveness to brood pheromone and decreased responsiveness to social inhibition. These results identify octopamine both as an important source of variation in response thresholds and as a modulator of pheromonal communication in insect societies. We speculate that octopamine plays more than one role in the organisation of behavioural development indicating a very high level of integration between the neurochemical system and the generation of complex behaviour.     

Hygienic behavior of the honey bee (Apis mellifera) is independent of sucrose responsiveness and foraging ontogeny

Hygienic behavior in honey bees is a behavioral mechanism of disease resistance. Bees bred for hygienic behavior exhibit an increased olfactory sensitivity to odors of diseased brood, which is most likely differentially enhanced in the hygienic line by the modulatory effects of octopamine (OA), a noradrenaline-like neuromodulator. Here, we addressed whether the hygienic behavioral state is linked to other behavioral activities known to be modulated by OA. We specifically asked if, during learning trials, bees from hygienic colonies discriminate better between odors of diseased and healthy brood because of differences in sucrose (reward) response thresholds. This determination had to be tested because sucrose response thresholds are susceptible to OA modulation and may have influenced the honey bee's association of the conditioned stimulus (odor) with the unconditioned stimulus (i.e., the sucrose reward). Because the onset of first foraging is also modulated by OA, we also examined whether bees from hygienic colonies differentially forage at an earlier age compared to bees from non-hygienic colonies. Our study revealed that 1-day- and 15- to 20-day-old bees from the hygienic line do not have lower sucrose response thresholds compared to bees from the non-hygienic lines. In addition, hygienic bees did not forage at an earlier age or forage preferentially for pollen as compared to non-hygienic bees. These results support the idea that OA does not function in honey bees simply to enhance the detection of all chemical cues non-selectively or control related behaviors regardless of their environmental milieu. Our results indicate that the behavioral profile of the hygienic bee is sculpted by multiple factors including genetic, neural, social and environmental systems.     

Effect of various insecticides on the larval growth and biogenic amine levels of Tribolium castaneum Herbst

Various insecticides reduced larval growth of the red flour beetle (tribolium castaneum Herbst) and various biogenic amines, including octopamine (OA), dopamine (DA), serotonin (5-HT), epinephrine (E), norepinephrine (NE), their precursors and metabolites in the insects were measured by high-performance liquid chromatography coupled with electrochemical detection. Tyrosine occurred in the highest concentration followed by OA, tryptophan and 3,4-dihydroxymandelic acid (DOMA). Tyramine (a precursor of OA in the biosynthetic pathway), synephrine (N-methyl OA), DA, 5-HT, E, NE and their related substances occurred in extremely low quantities compared with OA. The insects were stressed by various insecticides, which resulted in a dramatic change of biogenic amine levels: the OA levels increased, whereas the levels of other biogenic amines and related substances decreased.     

Biogenic amine levels change in the brains of stressed honeybees

Three different stress treatments, CO2 anesthesia, chilling anesthesia, and vertical spin, were applied to test whether honeybee (Apis mellifera) workers express stress responses in rewarding behaviors. In the present work, we defined the rewarding behaviors as the bees flying between the hive and feeder. The results from behavioral observation show that the flight time interval of the rewarding behavior of bee workers, flying between hive and feeder, was longer when they were stressed, suggesting that the stress treatments affected the workers' rewarding behavior. The biogenic amine levels in the workers' brains were measured to examine the rapid biochemical brain response to the stressors. After the chilling anesthesia, the dopamine (DA) and octopamine (OA) levels were significantly decreased; with the CO2 anesthesia for durations of both 2 min and 4 min, only DA showed a significant decrease. In the non-anesthesia treatments, the vertical spin with a velocity of 50 and 60 rpm for 90 s, the DA and OA levels were significantly decreased. Our results suggest that when the bees were under stress, the brain levels of OA and DA were depressed, and this may have caused latency in the rewarding behavior. The serotonin (5-HT) levels under these stress treatments were not changed.     

Octopamine influences division of labor in honey bee colonies

Forager honey bees have higher brain levels of octopamine than do bees tending larvae in the hive. To test the hypothesis that octopamine influences honey bee division of labor we treated bees orally with octopamine or its immediate precursor tyramine and determined whether these treatments increased the probability of initiating foraging. Octopamine treatment significantly elevated levels of octopamine in the brain and caused a significant dose-dependent increase in the number of new foragers. This effect was seen for precocious foragers in single-cohort colonies and foragers in larger colonies with more typical age demographies. Tyramine treatment did not increase the number of new foragers, suggesting that octopamine was exerting a specific effect. Octopamine treatment was effective only when given to bees old enough to forage, i.e., older than 4 days of age. Treatment when bees were 1-3 days of age did not cause a significant increase in the number of new foragers when the bees reached the minimal foraging age. These results demonstrate that octopamine influences division of labor in honey bee colonies. We speculate that octopamine is acting in this context as a neuromodulator.     

The role of biogenic amines in the control of blood glucose level in the decapod crustacean,Carcinus maenas L.

1. Administration of biogenic amines into intact Carcinus maenas induces dose- and timedependent elevation of hemolymph glucose level.2. Removal of the neurosecretory centre containing the crustacean hyperglycemic hormone (CHH) by ablation of the eyestalks did not induce hypoglycemia.3. Injection of dopamine (DA) into eyestalkless crabs showed no hyperglycemic effect, while serotonin (5-HT), epinephrine (E), norepinephrine (NE), and octopamine (OA) elevated glucose levels.4. The dopaminergic effect was significantly reduced by administration of trifluoperazine (TFP).5. 5-HT and OA were found to be strong elevators of glucose levels, while the other biogenic amines had moderate effects only.6. The results indicate, that DA exerts its hyperglycemic effect by stimulating the release of CHH from the eyestalk neurosecretory centre. Elevation of hemolymph glucose level by 5-HT, OA, E, and NE, occurs independently of CHH.     

Biogenic amines and division of labor in honey bee colonies

Brain levels of dopamine, serotonin, and octopamine were measured in relation to both age-related division of labor and inter-individual differences in task specialization independent of age in honey bee colonies. The only differences among similarly aged bees performing different tasks were significantly lower levels of dopamine in food storers than comb builders and significantly lower levels of octopamine in soldiers than foragers, but soldiers also were slightly younger than foragers. Differences associated with age-related division of labor were stronger. Older bees, notably foragers, had significantly higher levels of all three amines than did younger bees working in the hive. Using social manipulations to unlink chronological age and behavioral status, octopamine was found to exhibit the most robust association between behavior and amine level, independent of age. Octopamine levels were significantly lower in normal-age nurses versus precocious foragers and overage nurses versus normal-age foragers, but not different in reverted nurses versus reversion colony foragers. Dopamine levels were significantly lower in normal-age nurses versus precocious foragers, but higher in reverted nurses versus reversion colony foragers. Serotonin levels did not differ in any of these comparisons. These correlative results suggest that octopamine is involved in the regulation of age-related division of labor in honey bees. Accepted: 10 February 1999     

Brain biogenic amines and reproductive dominance in bumble bees (Bombus terrestris)

To begin to explore the role of biogenic amines in reproductive division of labor in social insects, brain levels of dopamine, serotonin, and octopamine were measured in bumble bee (Bombus terrestris) workers and queens that differ in behavioral and reproductive state. Levels of all three amines were similar for mated and virgin queens. Young workers that developed with or without a queen had similar amine levels, but in queenright colonies differences in biogenic amine levels were associated with differences in behavior and reproductive physiology. Dominant workers had significantly higher octopamine levels compared with workers of lower dominance status but of similar size, age, and ovary state. High dopamine levels were associated with the last stages of oocyte development irrespective of worker social status and behavior. These results suggest that biogenic amines are involved in behavioral and physiological aspects of regulation of reproduction in bumble bees. Accepted: 10 December 1999     

Genotypic Influence on Aversive Conditioning in Honeybees,Using a Novel Thermal Reinforcement Procedure

In Pavlovian conditioning, animals learn to associate initially neutral stimuli with positive or negative outcomes, leading to appetitive and aversive learning respectively. The honeybee (Apis mellifera) is a prominent invertebrate model for studying both versions of olfactory learning and for unraveling the influence of genotype. As a queen bee mates with about 15 males, her worker offspring belong to as many, genetically-different patrilines. While the genetic dependency of appetitive learning is well established in bees, it is not the case for aversive learning, as a robust protocol was only developed recently. In the original conditioning of the sting extension response (SER), bees learn to associate an odor (conditioned stimulus - CS) with an electric shock (unconditioned stimulus - US). This US is however not a natural stimulus for bees, which may represent a potential caveat for dissecting the genetics underlying aversive learning. We thus first tested heat as a potential new US for SER conditioning. We show that thermal stimulation of several sensory structures on the bee’s body triggers the SER, in a temperature-dependent manner. Moreover, heat applied to the antennae, mouthparts or legs is an efficient US for SER conditioning. Then, using microsatellite analysis, we analyzed heat sensitivity and aversive learning performances in ten worker patrilines issued from a naturally inseminated queen. We demonstrate a strong influence of genotype on aversive learning, possibly indicating the existence of a genetic determinism of this capacity. Such determinism could be instrumental for efficient task partitioning within the hive.     

Flight and fight: a comparative view of the neurophysiology and genetics of honey bee defensive behavior

Honey bee nest defense involves guard bees that specialize in olfaction-based nestmate recognition and alarm-pheromone-mediated recruitment of nestmates to sting. Stinging is influenced by visual, tactile and olfactory stimuli. Both quantitative trait locus (QTL) mapping and behavioral studies point to guarding behavior as a key factor in colony stinging response. Results of reciprocal F1 crosses show that paternally inherited genes have a greater influence on colony stinging response than maternally inherited genes. The most active alarm pheromone component, isoamyl acetate (IAA) causes increased respiration and may induce stress analgesia in bees. IAA primes worker bees for 'fight or flight', possibly through actions of neuropeptides and/or biogenic amines. Studies of aggression in other species lead to an expectation that octopamine or 5-HT might play a role in honey bee defensive response. Genome sequence and QTL mapping identified 128 candidate genes for three regions known to influence defensive behavior. Comparative bioinformatics suggest possible roles of genes involved in neurogenesis and central nervous system (CNS) activity, and genes involved in sensory tuning through G-protein coupled receptors (GPCRs), such as an arrestin (AmArr4) and the metabotropic GABA(B) receptor (GABA-B-R1).     

Biogenic amines and DOPA in the central nervous system of decapod crustaceans

The biogenic amines serotonin (5-HT), dopamine (DA), noradrenaline (NA), octopamine (OA) and the amino acid dihydroxyphenylalanine (DOPA) were identified and measured in the brain and the eyestalks of five decapod crustacean species using high pressure liquid chromatography (HPLC) with electrochemical detection. The amounts fall within 0.01-1.1 micrograms/g or 0.17-60 pmoles, and OA is the dominating amine in most species. THe DOPA levels in many of the species varied considerably between different measurements. It is concluded that the biogenic amines and DOPA are ubiquitous in the central nervous system of decapod crustaceans and the presence of NA and DOPA increases the number of presumed neurotransmitter/modulator candidates in the crustacean nervous system.     

Selection on a haploid genotype for discrimination learning performance: Correlation between drone honey bees (Apis mellifera) and their worker progeny (Hymenoptera: Apidae)

Successful bidirectional selection for discriminative olfactory learning is reported for drone honey bees (Apis mellifera). Learning performance was evaluated using a discrimination conditioning procedure that required drones to discriminate between an appetitively reinforced odorant and one that was followed by punishment. Selective breeding produced high- and low-learning-performance lines of worker progeny that diverged from performance of workers whose fathers were selected at random. Furthermore, we show that levels of sucrose-induced sensitization are not correlated to learning performance. These results corroborate earlier findings and further demonstrate the power of selection on a haploid (drone) genotype. In addition, this study now shows that the demonstrated differences in learning performance cannot be completely accounted for by alteration of sucrose-induced sensitization. Thus, using this technique, it may be possible to select for associative conditioning without a pleiotropic increase in sensitization. The honey bee will be ideally suited to these types of correlation analyses in future studies.     

Ethanol-Induced Effects on Sting Extension Response and Punishment Learning in the Western Honey Bee (Apis mellifera)

Acute ethanol administration is associated with sedation and analgesia as well as behavioral disinhibition and memory loss but the mechanisms underlying these effects remain to be elucidated. During the past decade, insects have emerged as important model systems to understand the neural and genetic bases of alcohol effects. However, novel assays to assess ethanol''s effects on complex behaviors in social or isolated contexts are necessary. Here we used the honey bee as an especially relevant model system since bees are typically exposed to ethanol in nature when collecting standing nectar crop of flowers, and there is recent evidence for independent biological significance of this exposure for social behavior. Bee''s inhibitory control of the sting extension response (SER) and a conditioned-place aversion assay were used to study ethanol effects on analgesia, behavioral disinhibition, and associative learning. Our findings indicate that although ethanol, in a dose-dependent manner, increases SER thresholds (analgesic effects), it disrupts the ability of honey bees to inhibit SER and to associate aversive stimuli with their environment. These results suggest that ethanol''s effects on analgesia, behavioral disinhibition and associative learning are common across vertebrates and invertebrates. These results add to the use of honey bees as an ethanol model to understand ethanol''s effects on complex, socially relevant behaviors.     

Effect of pheromones,hormones, and handling on sucrose response thresholds of honey bees (Apis mellifera L.)

The responsiveness of bees to sucrose is an important indicator of honey bee foraging decisions. Correlated with sucrose responsiveness is forage choice behavior, age of first foraging, and conditioned learning response. Pheromones and hormones are significant components in social insect systems associated with the regulation of colony-level and individual foraging behavior. Bees were treated to different exposure regimes of queen and brood pheromones and their sucrose responsiveness measured. Bees reared with queen or brood pheromone were less responsive than controls. Our results suggest responsiveness to sucrose is a physiologically, neuronally mediated response. Orally administered octopamine significantly reduced sucrose response thresholds. Change in response to octopamine was on a time scale of minutes. The greatest separation between octopamine treated and control bees occurred 30 min after feeding. There was no significant sucrose response difference to doses ranging from 0.2 g to 20 g of octopamine. Topically applied methoprene significantly increased sucrose responsiveness. Handling method significantly affected sucrose responsiveness. Bees that were anesthetized by chilling or CO₂ treatment were significantly more responsive than control bees 30 min after handling. Sixty minutes after handling there were no significant treatment differences. We concluded that putative stress effects of handling were blocked by anesthetic.Abbreviations BP brood pheromone - JH juvenile hormone - OA octopamine - PER proboscis extension response - PER-RT proboscis extension response threshold - QMP queen mandibular pheromone     

The Potential Influence of Bumble Bee Visitation on Foraging Behaviors and Assemblages of Honey Bees on Squash Flowers in Highland Agricultural Ecosystems

Bee species interactions can benefit plant pollination through synergistic effects and complementary effects, or can be of detriment to plant pollination through competition effects by reducing visitation by effective pollinators. Since specific bee interactions influence the foraging performance of bees on flowers, they also act as drivers to regulate the assemblage of flower visitors. We selected squash (Cucurbita pepo L.) and its pollinators as a model system to study the foraging response of honey bees to the occurrence of bumble bees at two types of sites surrounded by a high amount of natural habitats (≥ 58% of land cover) and a low amount of natural habitats (≤ 12% of land cover) in a highland agricultural ecosystem in China. At the individual level, we measured the elapsed time from the departure of prior pollinator(s) to the arrival of another pollinator, the selection of honey bees for flowers occupied by bumble bees, and the length of time used by honey bees to explore floral resources at the two types of sites. At the community level, we explored the effect of bumble bee visitation on the distribution patterns of honey bees on squash flowers. Conclusively, bumble bee visitation caused an increase in elapsed time before flowers were visited again by a honey bee, a behavioral avoidance by a newly-arriving honey bee to select flowers occupied by bumble bees, and a shortened length of time the honey bee takes to examine and collect floral resources. The number of overall bumble bees on squash flowers was the most important factor explaining the difference in the distribution patterns of honey bees at the community level. Furthermore, decline in the number of overall bumble bees on the squash flowers resulted in an increase in the number of overall honey bees. Therefore, our study suggests that bee interactions provide an opportunity to enhance the resilience of ecosystem pollination services against the decline in pollinator diversity.