Axel Brockmann

Behavioral performance in adult honey bees is influenced by the temperature experienced during their pupal development

To investigate the possible consequences of brood-temperature regulation in honey bee colonies on the quality of behavioral performance of adults, we placed honey bee pupae in incubators and allowed them to develop at temperatures held constant at 32°C, 34.5°C, and 36°C. This temperature range occurs naturally within hives. On emergence, the young adult bees were marked and introduced into foster colonies housed in normal and observation hives and allowed to live out their lives. No obvious difference in within-hive behavior was noted between the temperature-treated bees and the foster-colony bees. However, when the temperature-treated bees became foragers and were trained to visit a feeder 200 m from the hive, they exhibited clear differences in dance performance that could be correlated with the temperatures at which they had been raised: bees raised at 32°C completed only ≈20% of the dance circuits when compared with bees of the higher-temperature group. Also, the variance in the duration of the waggle phase is larger in 32°C-raised bees compared with 36°C-raised bees. All other parameters compared across all groups were not significantly different. One-trial learning and memory consolidation in the bees raised at different temperatures was investigated 1 and 10 min after conditioning the proboscis-extension reflex. Bees raised at 36°C performed as expected for bees typically classified as “good learners,” whereas bees raised at 32°C and 34.5°C performed significantly less well. We propose that the temperature at which pupae are raised will influence their behavioral performance as adults and may determine the tasks they carry out best inside and outside the hive.

A honey bee odorant receptor for the queen substance 9-oxo-2-decenoic acid

By using a functional genomics approach, we have identified a honey bee [Apis mellifera (Am)] odorant receptor (Or) for the queen substance 9-oxo-2-decenoic acid (9-ODA). Honey bees live in large eusocial colonies in which a single queen is responsible for reproduction, several thousand sterile female worker bees complete a myriad of tasks to maintain the colony, and several hundred male drones exist only to mate. The “queen substance” [also termed the queen retinue pheromone (QRP)] is an eight-component pheromone that maintains the queens dominance in the colony. The main component, 9-ODA, acts as a releaser pheromone by attracting workers to the queen and as a primer pheromone by physiologically inhibiting worker ovary development; it also acts as a sex pheromone, attracting drones during mating flights. However, the extent to which social and sexual chemical messages are shared remains unresolved. By using a custom chemosensory-specific microarray and qPCR, we identified four candidate sex pheromone Ors (AmOr10, -11, -18, and -170) from the honey bee genome based on their biased expression in drone antennae. We assayed the pheromone responsiveness of these receptors by using Xenopus oocytes and electrophysiology. AmOr11 responded specifically to 9-ODA (EC50 = 280 ± 31 nM) and not to any of the other seven QRP components, other social pheromones, or floral odors. We did not observe any responses of the other three Ors to any of the eight QRP pheromone components, suggesting 9-ODA is the only QRP component that also acts as a long-distance sex pheromone.

Quantitative peptidomics reveal brain peptide signatures of behavior

The honey bee genome predicts ≈100 peptides from 36 prohormones, but the functions of many of these peptides are unknown. We used differential isotope labeling combined with mass spectrometric analysis to quantify ≈50% of known bee brain peptides in the context of foraging, with 8 showing robust and dynamic regulation. Some showed differences in brain abundance as a function of experience; specifically, nectar and pollen collection led to quick changes in abundance. These changes were related to the act of food collection, not ingestion, because foragers bring food back to the hive for storage rather than eating it themselves. Other peptide differences in brain abundance were seen in bees that either flew to a nectar feeder or a pollen feeder, but did not yet collect any food. These differences likely reflect well-known predispositions of some bees to collect either nectar or pollen, but not both. Tachykinin, PBAN, and sNPF were among the peptides with the strongest changes in association with nectar and pollen foraging. These peptides are known to be involved in regulating food intake in solitary insects, suggesting an evolutionary connection between that behavior and social foraging. These results demonstrate that it is now possible to use quantitative peptidomics to help determine which brain peptides are bioactive and to elucidate their function in the regulation of behavior.

Size determines antennal sensitivity and behavioral threshold to odors in bumblebee workers

The eusocial bumblebees exhibit pronounced size variation among workers of the same colony. Differently sized workers engage in different tasks (alloethism); large individuals are found to have a higher probability to leave the colony and search for food, whereas small workers tend to stay inside the nest and attend to nest duties. We investigated the effect of size variation on morphology and physiology of the peripheral olfactory system and the behavioral response thresholds to odors in workers of Bombus terrestris. Number and density of olfactory sensilla on the antennae correlate significantly with worker size. Consistent with these morphological changes, we found that antennal sensitivity to odors increases with body size. Antennae of large individuals show higher electroantennogram responses to a given odor concentration than those of smaller nestmates. This finding indicates that large antennae exhibit an increased capability to catch odor molecules and thus are more sensitive to odors than small antennae. We confirmed this prediction in a dual choice behavioral experiment showing that large workers indeed are able to respond correctly to much lower odor concentrations than small workers. Learning performance in these experiments did not differ between small and large bumblebees. Our results clearly show that, in the social bumblebees, variation in olfactory sensilla number due to size differences among workers strongly affects individual odor sensitivity. We speculate that superior odor sensitivity of large workers has favored size-related division of labor in bumblebee colonies.

Adult honeybees (Apis mellifera L.) abandon hemocytic, but not phenoloxidase-based immunity

Hemocytes and the (prophenol-) phenoloxidase system constitute the immediate innate immune system in insects. These components of insect immunity are present at any post-embryonic life stage without previous infection. Differences between individuals and species in these immune parameters can reflect differences in infection risk, life expectancy, and biological function. In honeybees which show an age-related division of labor within the worker caste, previous studies demonstrated that foragers show a strongly reduced number of hemoctyes compared to the younger nurse bees. This loss of immune competence has been regarded advantageous with respect to an already high mortality rate due to foraging and to redistribution of energy costs at the colony level. Based on the idea that abandoning hemocytes in all adults would be a reasonably direct regulatory mechanism, we posed the hypothesis that abandoning hemocytic immunity is not restricted to worker honeybees. We tested our hypotheses by performing a comprehensive analysis of hemocyte number and phenoloxidase (PO)-activity levels in immunologically naive workers, queens, and drones. We found that in all three adult phenotypes hemocyte number is dramatically reduced in early adult life. In contrast, we found that the dynamics of PO-activity levels have sex and caste-specific characteristics. In workers, PO activity reached a plateau within the first week of adult life, and in queens enzyme levels continuously increased with age and reached levels twice as high as those found in workers. PO-activity levels slightly declined with age in drones. These data support our hypothesis, from which we infer that the previously reported reduction of hemocyte in foragers is not worker specific but represents a general phenomenon occurring in all honeybee adult phenotypes.

Perception Space—The Final Frontier

Mapping the complex sensory behavior of animals, such as smell in bees, to relevant neural activity provides clues into how animals perceive and respond to the world through their senses

Honeybee odometry: Performance in varying natural terrain

Recent studies have shown that honeybees flying through short, narrow tunnels with visually textured walls perform waggle dances that indicate a much greater flight distance than that actually flown. These studies suggest that the bees “odometer” is driven by the optic flow (image motion) that is experienced during flight. One might therefore expect that, when bees fly to a food source through a varying outdoor landscape, their waggle dances would depend upon the nature of the terrain experienced en route. We trained honeybees to visit feeders positioned along two routes, each 580 m long. One route was exclusively over land. The other was initially over land, then over water and, finally, again over land. Flight over water resulted in a significantly flatter slope of the waggle-duration versus distance regression, compared to flight over land. The mean visual contrast of the scenes was significantly greater over land than over water. The results reveal that, in outdoor flight, the honeybees odometer does not run at a constant rate; rather, the rate depends upon the properties of the terrain. The bees perception of distance flown is therefore not absolute, but scene-dependent. These findings raise important and interesting questions about how these animals navigate reliably.

Central Projections of Sensory Systems Involved in Honey Bee Dance Language Communication

Honey bee dance language is a unique and complex form of animal communication used to inform nest mates in the colony about the specific location of food sources or new nest sites. Five different sensory systems have been implicated in acquiring and communicating the information necessary for dance language communication. We present results from neuronal tracer studies identifying the central projections from four of the five. Sensory neurons of the dorsal rim area of the compound eyes, involved in acquiring sun-compass based information, project to the dorsal-most part of the medulla. Sensory neurons of the neck hair plates, required to transpose sun-compass based information to gravity-based information in the dark hive, project to the dorsal labial neuromere of the subesophageal ganglion. Sensory neurons from the antennal joint hair sensilla and the Johnston’s organ, which perceive information on dance direction and distance from mechanostimuli generated by abdomen waggling and wing vibration, project to the deutocerebral dorsal lobe and the subesophageal ganglion, and the posterior protocerebrum, respectively. We found no ‘dance-specific’ projections relative to those previously described for drone and queen honey bees and other insect species that do not exhibit dance communication. We suggest that the evolution of dance language communication was likely based on the modification of central neural pathways associated with path integration, the capability to calculate distance, and directional information during flight.

Sex and Caste-Specific Variation in Compound Eye Morphology of Five Honeybee Species

Ranging from dwarfs to giants, the species of honeybees show remarkable differences in body size that have placed evolutionary constrains on the size of sensory organs and the brain. Colonies comprise three adult phenotypes, drones and two female castes, the reproductive queen and sterile workers. The phenotypes differ with respect to tasks and thus selection pressures which additionally constrain the shape of sensory systems. In a first step to explore the variability and interaction between species size-limitations and sex and caste-specific selection pressures in sensory and neural structures in honeybees, we compared eye size, ommatidia number and distribution of facet lens diameters in drones, queens and workers of five species (Apis andreniformis, A. florea, A. dorsata, A. mellifera, A. cerana). In these species, male and female eyes show a consistent sex-specific organization with respect to eye size and regional specialization of facet diameters. Drones possess distinctly enlarged eyes with large dorsal facets. Aside from these general patterns, we found signs of unique adaptations in eyes of A. florea and A. dorsata drones. In both species, drone eyes are disproportionately enlarged. In A. dorsata the increased eye size results from enlarged facets, a likely adaptation to crepuscular mating flights. In contrast, the relative enlargement of A. florea drone eyes results from an increase in ommatidia number, suggesting strong selection for high spatial resolution. Comparison of eye morphology and published mating flight times indicates a correlation between overall light sensitivity and species-specific mating flight times. The correlation suggests an important role of ambient light intensities in the regulation of species-specific mating flight times and the evolution of the visual system. Our study further deepens insights into visual adaptations within the genus Apis and opens up future perspectives for research to better understand the timing mechanisms and sensory physiology of mating related signals.

Wax perception in honeybees: contact is not necessary

In social insects, much progress has been made in identifying variations in the cuticular signatures of sexes, castes, kin and reproductive status. In contrast to this, we still do not know how the receivers perceive these recognition cues. This study was designed to investigate whether honeybees use contact-chemosensory or olfactory sensilla to perceive wax components. To answer this question in a behavioral assay, we combined classical conditioning of the proboscis extension reaction and a recently established method using zinc sulfate to selectively block antennal contact-chemosensory sensilla. Comparison of the responses to sucrose, wax and geraniol before and after antennal zinc sulfate treatment revealed that the sucrose response is lost after treatment but the responses to wax and geraniol are maintained. As sucrose is perceived by the contact-chemosensory sensilla, the retention of the wax response indicates that contact-chemosensory sensilla are not necessary for wax perception.