Sucrose acceptance, discrimination and proboscis responses of honey bees (Apis mellifera L.) in the field and the laboratory.

Laboratory studies in honey bees have shown positive correlations between sucrose responsiveness, division of labour and learning. We tested the relationships between sucrose acceptance and discrimination in the field and responsiveness in the laboratory. Based on acceptance in the field three groups of bees were differentiated: (1) bees that accept sucrose concentrations >10%, (2) bees that accept some but not all of the sucrose concentrations <10% and water, and (3) bees that accept water and all offered sucrose concentrations. Sucrose acceptance can be described in a model in which sucrose- and water-dependent responses interact additively. Responsiveness to sucrose was tested in the same bees in the laboratory by measuring the proboscis extension response (PER). The experiments demonstrated that PER responsiveness is lower than acceptance in the field and that it is not possible to infer from the PER measurements in the laboratory those concentrations the respective bees accepted in the field. Discrimination between sucrose concentrations was tested in three groups of free-flying bees collecting low, intermediate or high concentrations of sucrose. The experiments demonstrated that bees can discriminate between concentrations differences down to 0.2 relative log units. There exist only partial correlations between discrimination, acceptance and PER responsiveness.

Downregulation of vitellogenin gene activity increases the gustatory responsiveness of honey bee workers (Apis mellifera).

In the honey bee (Apis mellifera), young workers usually perform tasks in the nest while older workers forage in the field. The behavioral shift from nest-task to foraging activity is accompanied by physiological and sensory changes so that foragers can be characterized by a higher juvenile hormone (JH) level, a lower vitellogenin protein titer, and an increased responsiveness to water and sucrose stimuli. JH was hypothesized to be the key mediator of behavioral development, physiology, and sensory sensitivity in honey bee workers. Recent research, however, has shown that JH is controlled by the hemolymph vitellogenin level, which implies that the fat body specific vitellogenin gene can be a key regulator of behavioral change. Here, we show that downregulation of vitellogenin activity by RNA interference (RNAi) causes an increase in the gustatory responsiveness of worker bees. Our observations suggest that vitellogenin is an important regulator of long-term changes in honey bee behavior.

Behavioural pharmacology of octopamine, tyramine and dopamine in honey bees.

In the honey bee, responsiveness to sucrose correlates with many behavioural parameters such as age of first foraging, foraging role and learning. Sucrose responsiveness can be measured using the proboscis extension response (PER) by applying sucrose solutions of increasing concentrations to the antenna of a bee. We tested whether the biogenic amines octopamine, tyramine and dopamine, and the dopamine receptor agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene (6,7-ADTN) can modulate sucrose responsiveness. The compounds were either injected into the thorax or fed in sucrose solution to compare different methods of application. Injection and feeding of tyramine or octopamine significantly increased sucrose responsiveness. Dopamine decreased sucrose responsiveness when injected into the thorax. Feeding of dopamine had no effect. Injection of 6,7-ADTN into the thorax and feeding of 6,7-ADTN reduced sucrose responsiveness significantly. These data demonstrate that sucrose responsiveness in honey bees can be modulated by biogenic amines, which has far reaching consequences for other types of behaviour in this insect.

Functional characterization of transmembrane adenylyl cyclases from the honeybee brain.

The second messenger cAMP has a pivotal role in animals' physiology and behavior. Intracellular concentrations of cAMP are balanced by cAMP-synthesizing adenylyl cyclases (ACs) and cAMP-cleaving phosphodiesterases. Knowledge about ACs in the honeybee (Apis mellifera) is rather limited and only an ortholog of the vertebrate AC3 isoform has been functionally characterized, so far. Employing bioinformatics and functional expression we characterized two additional honeybee genes encoding membrane-bound (tm)ACs. The proteins were designated AmAC2t and AmAC8. Unlike the common structure of tmACs, AmAC2t lacks the first transmembrane domain. Despite this unusual topography, AmAC2t-activity could be stimulated by norepinephrine and NKH477 with EC(50s) of 0.07 µM and 3 µM. Both ligands stimulated AmAC8 with EC(50s) of 0.24 µM and 3.1 µM. In brain cryosections, intensive staining of mushroom bodies was observed with specific antibodies against AmAC8, an expression pattern highly reminiscent of the Drosophila rutabaga AC. In a current release of the honeybee genome database we identified three additional tmAC- and one soluble AC-encoding gene. These results suggest that (1) the AC-gene family in honeybees is comparably large as in other species, and (2) based on the restricted expression of AmAC8 in mushroom bodies, this enzyme might serve important functions in honeybee behavior.

Sucrose acceptance and different forms of associative learning of the honey bee (apis mellifera L.) in the field and laboratory.

The experiments analyze different forms of learning and 24-h retention in the field and in the laboratory in bees that accept sucrose with either low (/=30% or >/=50%) concentrations. In the field we studied color learning at a food site and at the hive entrance. In the laboratory olfactory conditioning of the proboscis extension response (PER) was examined. In the color learning protocol at a feeder, bees with low sucrose acceptance thresholds (/=50%). Retention after 24 h is significantly different between the two groups of bees and the choice reactions converge. Bees with low and high acceptance thresholds in the field show no differences in the sucrose sensitivity PER tests in the laboratory. Acceptance thresholds in the field are thus a more sensitive behavioral measure than PER responsiveness in the laboratory. Bees with low acceptance thresholds show significantly better acquisition and 24-h retention in olfactory learning in the laboratory compared to bees with high thresholds. In the learning protocol at the hive entrance bees learn without sucrose reward that a color cue signals an open entrance. In this experiment, bees with high sucrose acceptance thresholds showed significantly better learning and reversal learning than bees with low thresholds. These results demonstrate that sucrose acceptance thresholds affect only those forms of learning in which sucrose serves as the reward. The results also show that foraging behavior in the field is a good predictor for learning behavior in the field and in the laboratory.

Biochemical properties of heterologously expressed and native adenylyl cyclases from the honeybee brain (Apis mellifera L.).

Cyclic AMP is an important intracellular signaling molecule participating e.g. in sensory signal transduction, cardiac myocyte regulation, learning and memory. The formation of cAMP is catalyzed by adenylyl cyclases. A variety of factors can modulate the properties of these enzymes and lead to dynamic changes of the intracellular cAMP concentration. Here we determined the tissue distribution of a recently cloned adenylyl cyclase (AmAC3) in honeybee brain. The protein is present in all neuropils. Intensive immunoreactivity was found in parts of the proto- and deutocerebrum and in the suboesophageal ganglion. Biochemical and pharmacological properties of AmAC3 and of native adenylyl cyclases in subregions of the honeybee brain were examined. Values for half-maximal activation with NKH477 were in the low micromolar range with 10.2 µM for AmAC3 and 3.6-8.1 µM for native enzymes. Biosynthesis of cAMP was specifically blocked by P-site inhibitors. Adenylyl cyclases in antennal lobes and AmAC3 share the inhibitory profile with 2',5'dd3'ATP>3'AMP>2'deoxyadenosine. In addition to P-site inhibitors AmAC3 activity was impaired by Ca(2+)/calmodulin. The results suggest that AmAC3 is a likely candidate to fulfill an integrative role in sensory, motor and higher-order information processing in the honeybee brain.

8. The development and evolution of division of labor and foraging specialization in a social insect (Apis mellifera L.).

How does complex social behavior evolve? What are the developmental building blocks of division of labor and specialization, the hallmarks of insect societies? Studies have revealed the developmental origins in the evolution of division of labor and specialization in foraging worker honeybees, the hallmarks of complex insect societies. Selective breeding for a single social trait, the amount of surplus pollen stored in the nest (pollen hoarding) revealed a phenotypic architecture of correlated traits at multiple levels of biological organization in facultatively sterile female worker honeybees. Verification of this phenotypic architecture in "wild-type" bees provided strong support for a "pollen foraging syndrome" that involves increased senso-motor responses, motor activity, associative learning, reproductive status, and rates of behavioral development, as well as foraging behavior. This set of traits guided further research into reproductive regulatory systems that were co-opted by natural selection during the evolution of social behavior. Division of labor, characterized by changes in the tasks performed by bees, as they age, is controlled by hormones linked to ovary development. Foraging specialization on nectar and pollen results also from different reproductive states of bees where nectar foragers engage in pre-reproductive behavior, foraging for nectar for self-maintenance, while pollen foragers perform foraging tasks associated with reproduction and maternal care, collecting protein.

The functions of antennal mechanoreceptors and antennal joints in tactile discrimination of the honeybee (Apis mellifera L.).

Honeybees learn and discriminate excellently between different surface structures and different forms of objects, which they scan with their antennae. The sensory plate on the antennal tip plays a key role in the perception of mechanosensory and gustatory information. It is densely covered with small tactile hairs and carries a few large taste hairs. Both types of sensilla contain a mechanoreceptor, which is involved in the antennal scanning of an object. Our experiments test the roles of the mechanoreceptors on the antennal tip in tactile antennal learning and discrimination. Joints between head capsule and scapus and between scapus and pedicellus enable the bee to perform three-dimensional movements when they scan an object. The role of these joints in tactile antennal learning and discrimination is studied in separate experiments. The mechanoreceptors on the antennal tip were decisive for surface discrimination, but not for tactile acquisition or discrimination of shapes. When the scapus-pedicellus joint or the headcapsule-scapus joint was fixed on both antennae, tactile learning was still apparent but surface discrimination was abolished. Fixing both scapi to the head capsule reduced tactile acquisition.

Sensory responsiveness and the effects of equal subjective rewards on tactile learning and memory of honeybees.

In tactile learning, sucrose is the unconditioned stimulus and reward, which is usually applied to the antenna to elicit proboscis extension and which the bee can drink when it is subsequently applied to the extended proboscis. The conditioned stimulus is a tactile object that the bee can scan with its antennae. In this paper we describe the quantitative relationships between gustatory antennal stimulation, gustatory proboscis stimulation, and tactile learning and memory. Bees are 10-fold more responsive to sucrose solutions when they are applied to the antenna compared to proboscis stimulation. During tactile conditioning, the sucrose solution applied to the proboscis determines the level of acquisition, whereas antennal input is of minor importance. Bees differing in their gustatory responsiveness measured at the antenna differ strongly in their tactile acquisition and memory. We demonstrate how these differences in tactile acquisition and memory can be greatly reduced by calculating equal subjective rewards, based on individual gustatory responsiveness.

Levels of behavioral organization and the evolution of division of labor.

The major features of insect societies that fascinate biologists are the self-sacrificing altruism expressed by colony members, the complex division of labor, and the tremendous plasticity demonstrated in the face of changing environments. The social behavior of insects is a result of complex interactions at different levels of biological organization. Genes give rise to proteins and peptides that build the nervous and muscular systems, regulate their own synthesis, interact with each other, and affect the behavior of individuals. Social behavior emerges from the complex interactions of individuals that are themselves far removed from the direct effects of the genes. In order to understand how social organization evolves, we must understand the mechanisms that link the different levels of organization. In this review, we discuss how behavior is influenced by genes and the neural system and how social behavior emerges from the behavioral activities of individuals. We show how different levels of organization share common features and are linked through common mechanisms. We focus on the behavior of the honey bee, the best studied of all social insects.

Activity of cGMP-dependent protein kinase (PKG) affects sucrose responsiveness and habituation in Drosophila melanogaster.

The cGMP-dependent protein kinase (PKG) has many cellular functions in vertebrates and insects that affect complex behaviors such as locomotion and foraging. The foraging (for) gene encodes a PKG in Drosophila melanogaster. Here, we demonstrate a function for the for gene in sensory responsiveness and nonassociative learning. Larvae of the natural variant sitter (for(s)) show less locomotor activity during feeding and have a lower PKG activity than rover (for(R)) larvae. We used rover and sitter adult flies to test whether PKG activity affects (1) responsiveness to sucrose stimuli applied to the front tarsi, and (2) habituation of proboscis extension after repeated sucrose stimulation. To determine whether the differences observed resulted from variation in the for gene, we also tested for(s2), a sitter mutant produced on a rover genetic background. We found that rovers (for(R)) were more responsive to sucrose than sitters (for(s) and for(s2)) at 1-, 2-, and 3-wk old. This was true for both sexes. Differences in sucrose responsiveness between rovers and sitters were greater after 2 h of food deprivation than after 24 h. Of flies with similar sucrose responsiveness, for(R) rovers showed less habituation and generalization of habituation than for(s) and for(s2) sitters. These results show that the PKG encoded by for independently affects sensory responsiveness and habituation in Drosophila melanogaster.

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain.

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera. The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis. Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.