Levels and activity of cyclic guanosine monophosphate-dependent protein kinase in nurse and forager honeybees.

Age-dependent division of labour in honeybees was shown to be connected to sensory response thresholds. Foragers show a higher gustatory responsiveness than nurse bees. It is generally assumed that nutrition-related signalling pathways underlie this behavioural plasticity. Here, one important candidate gene is the foraging gene, which encodes a cyclic guanosine monophosphate-dependent protein kinase (PKG). Several roles of members of this enzyme family were analysed in vertebrates. They own functions in important processes such as growth, secretion and neuronal adaptation. Honeybee foraging messenger RNA expression is upregulated in the brain of foragers. In vivo activation of PKG can modulate gustatory responsiveness. We present for the first time PKG protein level and activity data in the context of social behaviour and feeding. Protein level was significantly higher in brains of foragers than in those of nurse bees, substantiating the role of PKG in behavioural plasticity. However, enzyme activity did not differ between behavioural roles. The mediation of feeding status appears independent of PKG signalling. Neither PKG content nor enzyme activity differed between starved and satiated individuals. We suggest that even though nutrition-related pathways are surely involved in controlling behavioural plasticity, which involves changes in PKG signalling, mediation of satiety itself is independent of PKG.

The reduced brood nursing by mite-infested honey bees depends on their accelerated behavioral maturation.

The parasitic mite Varroa destructor is regarded as the most important parasite of honey bees and plays a fundamental role in the decline of bee colonies observed in the last decade in the Northern hemisphere. Parasitization has a number of detrimental effects on bees, including reduced nursing, which can have important impacts on colony balance. In this work we investigated at the individual level the causes of this abnormal behavior and found that the reduced nursing activity in mite-infested workers is associated with impaired learning performance and a series of physiological traits that are typical of foragers, including reduced response to brood pheromone, limited development of hypopharyngeal glands and higher juvenile hormone titre in the haemolymph. Altogether our data confirm the premature transition to foraging already postulated based on previous genomics studies, from a physiological point of view.

Division of labour in honey bees: age- and task-related changes in the expression of octopamine receptor genes.

The honey bee (Apis mellifera L.) has developed into an important ethological model organism for social behaviour and behavioural plasticity. Bees perform a complex age-dependent division of labour with the most pronounced behavioural differences occurring between in-hive bees and foragers. Whereas nurse bees, for example, stay inside the hive and provide the larvae with food, foragers leave the hive to collect pollen and nectar for the entire colony. The biogenic amine octopamine appears to play a major role in division of labour but the molecular mechanisms involved are unknown. We here investigated the role of two characterized octopamine receptors in honey bee division of labour. AmOctαR1 codes for a Ca(2+) -linked octopamine receptor. AmOctßR3/4 codes for a cyclic adenosine monophosphate-coupled octopamine receptor. Messenger RNA expression of AmOctαR1 in different brain neuropils correlates with social task, whereas expression of AmOctßR3/4 changes with age rather than with social role per se. Our results for the first time link the regulatory role of octopamine in division of labour to specific receptors and brain regions. They are an important step forward in our understanding of complex behavioural organization in social groups.

The foraging gene of Drosophila melanogaster: spatial-expression analysis and sucrose responsiveness.

The ability to identify and respond to food is essential for survival, yet little is known about the neural substrates that regulate natural variation in food-related traits. The foraging (for) gene in Drosophila melanogaster encodes a cGMP-dependent protein kinase (PKG) and has been shown to function in food-related traits. To investigate the tissue distribution of FOR protein, we generated an antibody against a common region of the FOR isoforms. In the adult brain we localized FOR to neuronal clusters and projections including neurons that project to the central complex, a cluster within the dorsoposterior region of the brain hemispheres, a separate cluster medial to optic lobes and lateral to brain hemispheres, a broadly distributed frontal-brain cluster, axon bundles of the antennal nerve and of certain subesophageal-ganglion nerves, and the medulla optic lobe. These newly described tissue distribution patterns of FOR protein provide candidate neural clusters and brain regions for investigation of neural networks that govern foraging-related traits. To determine whether FOR has a behavioral function in neurons we expressed UAS-for in neurons using an elav-gal4 driver and measured the effect on adult sucrose responsiveness (SR), known to be higher in rovers than sitters, the two natural variants of foraging. We found that pan-neuronal expression of for caused an increase in the SR of sitters, demonstrating a neural function for PKG in this food-related behavior.

Phototactic behaviour correlates with gustatory responsiveness in honey bees (Apis mellifera L.).

The response threshold hypothesis of division of labour in honey bees assumes that individuals differ in their responsiveness to different stimulus modalities. However, previous experiments have shown that responsiveness to gustatory stimuli correlates with responsiveness to odours, pollen and tactile stimuli. Evaluation of these stimuli involves sensory receptors on the antenna. We tested whether responsiveness to gustatory stimuli correlates with responsiveness to visual stimuli in a phototaxis experiment, which is independent of antennal input. Gustatory responsiveness was measured using the proboscis extension response to antennal stimulation with water and different sucrose concentrations. Phototaxis was quantified by measuring the walking times a bee needed to reach light sources of different intensities. Walking behaviour in the darkness was measured to test for differences in locomotor behaviour. The walking time towards a light stimulus, the path length, and the walking speed depended on the intensity of the light stimulus. Responsiveness to visual stimuli correlated significantly with gustatory responsiveness. Bees displaying a high gustatory responsiveness were also very sensitive to light. Locomotor activity did not correlate with gustatory responsiveness. This shows that gustatory responsiveness is a good indicator of sensitivity for visual stimuli, which are not perceived by the antenna.

Aminergic control and modulation of honeybee behaviour.

Biogenic amines are important messenger substances in the central nervous system and in peripheral organs of vertebrates and of invertebrates. The honeybee, Apis mellifera, is excellently suited to uncover the functions of biogenic amines in behaviour, because it has an extensive behavioural repertoire, with a number of biogenic amine receptors characterised in this insect.In the honeybee, the biogenic amines dopamine, octopamine, serotonin and tyramine modulate neuronal functions in various ways. Dopamine and serotonin are present in high concentrations in the bee brain, whereas octopamine and tyramine are less abundant. Octopamine is a key molecule for the control of honeybee behaviour. It generally has an arousing effect and leads to higher sensitivity for sensory inputs, better learning performance and increased foraging behaviour. Tyramine has been suggested to act antagonistically to octopamine, but only few experimental data are available for this amine. Dopamine and serotonin often have antagonistic or inhibitory effects as compared to octopamine.Biogenic amines bind to membrane receptors that primarily belong to the large gene-family of GTP-binding (G) protein coupled receptors. Receptor activation leads to transient changes in concentrations of intracellular second messengers such as cAMP, IP(3) and/or Ca(2+). Although several biogenic amine receptors from the honeybee have been cloned and characterised more recently, many genes still remain to be identified. The availability of the completely sequenced genome of Apis mellifera will contribute substantially to closing this gap.In this review, we will discuss the present knowledge on how biogenic amines and their receptor-mediated cellular responses modulate different behaviours of honeybees including learning processes and division of labour.

Activity of protein kinase A and gustatory responsiveness in the honey bee ( Apis mellifera L.).

The cAMP-dependent protein kinase A is involved in the induction of long-term memory and habituation in the bee. Gustatory responsiveness correlates strongly with associative and non-associative learning in bees. We tested whether protein kinase A activity in the antennal lobes correlates with gustatory responsiveness. Thirty minutes after feeding, bees with high gustatory responsiveness had a significantly higher protein kinase A activity than bees with low responsiveness. Ninety minutues after feeding, when gustatory responsiveness had increased in initially unresponsive bees, no changes in protein kinase A activity were found. We also tested time-dependent effects of protein kinase A activator and protein kinase A inhibitor on gustatory responsiveness. Injection of the protein kinase A activator adenosine 3'5'-cyclic monophosphate 8-bromo-sodium salt or of the protein kinase A inhibitor KT 5720 did not affect gustatory responsiveness within the first 4 h after treatment. Feeding of adenosine 3'5'-cyclic monophosphate 8-bromo-sodium salt over 4 days increased gustatory responsiveness in newly emerged bees and adult foragers. These results enable us to distinguish between two different forms of gustatory responsiveness: basal and transient gustatory responsiveness. Basal gustatory responsiveness correlates with protein kinase A activity and can only be modulated in the range of several days. Transient gustatory responsiveness appears to be independent of protein kinase A activity and can be modulated in the range of minutes to hours.

The effects of genotype, foraging role, and sucrose responsiveness on the tactile learning performance of honey bees (Apis mellifera L.).

We analyzed sucrose responsiveness and associative tactile learning in two genetic strains of honey bees under laboratory conditions. These strains differ in their foraging behavior. Bees of the "high" strain preferentially collect pollen. "Low"-strain bees mainly forage for nectar. Responsiveness to different sucrose concentrations and tactile learning were examined using the proboscis extension reflex. Acquisition, extinction of conditioned responses, and responses to an alternative tactile stimulus were tested. High-strain bees are more responsive to sucrose than low-strain bees. Regardless of genotype, pollen foragers are more responsive to sucrose than nectar foragers. In bees of both strains we find the same relationship between responsiveness to sucrose and acquisition. Bees responding to low sucrose concentrations show more often the conditioned response during acquisition than those responding only to higher sucrose concentrations. Extinction of conditioned responses depends on the response probability during acquisition. Discrimination between the two tactile stimuli is affected by genotype but not by responsiveness to sucrose. High-strain bees discriminate better than low-strain bees. Our experiments thus establish links between division of labor, responsiveness to sucrose, and associative learning in honey bees.

Responsiveness to sucrose affects tactile and olfactory learning in preforaging honey bees of two genetic strains.

Using sucrose solution as reward, we conditioned preforaging honey bees of two genetic strains to tactile stimuli or to odours. Acquisition, extinction of conditioned responses and discrimination between the conditioned stimuli and alternative tactile or olfactory stimuli were measured. Bees of the two genetic strains were selected for their foraging behaviour. In addition, they differ in their responsiveness to sucrose. To analyse the effects of sucrose perception on learning parameters independent of foraging behaviour, we conditioned preforaging bees of the two strains and compared their performance in tactile and olfactory learning paradigms. In both strains, acquisition in tactile and olfactory conditioning is determined by responsiveness to sucrose. There is no genetic effect on the relationship between responsiveness to sucrose and acquisition. Bees responding to low sucrose concentrations perform better than ones that only respond to higher concentrations. Extinction of conditioned responses correlates with acquisition. Responses to alternative stimuli are independent of responsiveness to sucrose.

Tactile learning and the individual evaluation of the reward in honey bees (Apis mellifera L.).

Using the proboscis extension response we conditioned pollen and nectar foragers of the honey bee (Apis mellifera L.) to tactile patterns under laboratory conditions. Pollen foragers demonstrated better acquisition, extinction, and reversal learning than nectar foragers. We tested whether the known differences in response thresholds to sucrose between pollen and nectar foragers could explain the observed differences in learning and found that nectar foragers with low response thresholds performed better during acquisition and extinction than ones with higher thresholds. Conditioning pollen and nectar foragers with similar response thresholds did not yield differences in their learning performance. These results suggest that differences in the learning performance of pollen and nectar foragers are a consequence of differences in their perception of sucrose. Furthermore, we analysed the effect which the perception of sucrose reward has on associative learning. Nectar foragers with uniform low response thresholds were conditioned using varying concentrations of sucrose. We found significant positive correlations between the concentrations of the sucrose rewards and the performance during acquisition and extinction. The results are summarised in a model which describes the relationships between learning performance, response threshold to sucrose, concentration of sucrose and the number of rewards.