Tactile conditioning and movement analysis of antennal sampling strategies in honey bees (Apis mellifera L.).

Honey bees (Apis mellifera L.) are eusocial insects and well known for their complex division of labor and associative learning capability(1, 2). The worker bees spend the first half of their life inside the dark hive, where they are nursing the larvae or building the regular hexagonal combs for food (e.g. pollen or nectar) and brood(3). The antennae are extraordinary multisensory feelers and play a pivotal role in various tactile mediated tasks(4), including hive building(5) and pattern recognition(6). Later in life, each single bee leaves the hive to forage for food. Then a bee has to learn to discriminate profitable food sources, memorize their location, and communicate it to its nest mates(7). Bees use different floral signals like colors or odors(7, 8), but also tactile cues from the petal surface(9) to form multisensory memories of the food source. Under laboratory conditions, bees can be trained in an appetitive learning paradigm to discriminate tactile object features, such as edges or grooves with their antennae(10, 11, 12, 13). This learning paradigm is closely related to the classical olfactory conditioning of the proboscis extension response (PER) in harnessed bees(14). The advantage of the tactile learning paradigm in the laboratory is the possibility of combining behavioral experiments on learning with various physiological measurements, including the analysis of the antennal movement pattern.

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.

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.

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.

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.

Slanted joint axes of the stick insect antenna: an adaptation to tactile acuity.

Like many flightless, obligatory walking insects, the stick insect Carausius morosus makes intensive use of active antennal movements for tactile near range exploration and orientation. The antennal joints of C. morosus have a peculiar oblique and non-orthogonal joint axis arrangement. Moreover, this arrangement is known to differ from that in crickets (Ensifera), locusts (Caelifera) and cockroaches (Blattodea), all of which have an orthogonal joint axis arrangement. Our hypothesis was that the situation found in C. morosus represents an important evolutionary trait of the order of stick and leaf insects (Phasmatodea). If this was true, it should be common to other species of the Phasmatodea. The objective of this comparative study was to resolve this question. We have measured the joint axis orientation of the head-scape and scape-pedicel joints along with other parameters that affect the tactile efficiency of the antenna. The obtained result was a complete kinematic description of the antenna. This was used to determine the size and location of kinematic out-of-reach zones, which are indicators of tactile acuity. We show that the oblique and non-orthogonal arrangement is common to eight species from six sub-families indicating that it is a synapomorphic character of the Euphasmatodea. This character can improve tactile acuity compared to the situation in crickets, locusts and cockroaches. Finally, because molecular data of a recent study indicate that the Phasmatodea may have evolved as flightless, obligatory walkers, we argue that the antennal joint axis arrangement of the Euphasmatodea reflects an evolutionary adaptation to tactile near range exploration during terrestrial locomotion.