Functional analysis of circadian clock genes in honey bee time-place learning

• Applicant: Dr. Katharina Beer
• Subject Area: Sensory and Behavioural Biology ; General Genetics and Functional Genome Biology ; Cognitive, Systems and Behavioural Neurobiology
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 525143846

The circadian clock is ubiquitous in animals on earth. It is involved in the regulation of many behaviors and physiological processes, like sleep-wake cycle, foraging/hunting activity, metabolism and daily cycles in hormone levels. The circadian clock is also consulted in complex behaviors like time-place learning, which has so far been a behavior observed only in mammals and Hymenoptera. In the honey bee, clock genes are well described, however the link between circadian clock and structures involved in time and place dependent memory processes is largely unknown. In the described project, we investigate the function of two central circadian clock genes (namely period and insect-type 2 cryptochrome) and the neuropeptide Pigment Dispersing Factor producing gene in establishing time-place dependent memory in honey bees. For this, we mutate the genes via CRISPR-Cas9 and analyze mutant behavior: rhythmic locomotion, foraging activity, time dependent learning performance in an olfactory learning assay and time-place learning performance in a thermal-visual learning paradigm. Furthermore, we analyze clock gene expression profiles in the mutants with immunocytochemistry and qPCR. In preliminary experiments, we have already successfully mutated the Pigment Dispersing Factor gene and found that bees carrying a frame-shift mutation on both alleles are no longer able to display circadian rhythms in locomotion. Interestingly, the role of period in time-dependent learning processes is debated controversial in studies with mice and fruit flies. Our project with honey bees may help to elucidate differences among animal clock systems in respect to time memory. Additionally, we plan to characterize the neuronal clock network in the honey bee brain in more detail. Although mice and honey bees show the same extraordinary time-place learning behavior, brain structure in the honey bee is far less complex than in mice. We want to utilize this fact to find the anatomical link between the clock and structures involved in learning processes. Therefore, we plan to perform parallel immunostaining in honey bee brains of selected clock components and neuromessengers (allatropin, tachykinin, FMRF-amide, Neuropeptide F (short) and serotonin) associated to learning behavior. With this project, we aim to better understand the role of clock genes in time-place learning of the honey bee.

• DFG Programme: Research Grants
• Co-Investigator: Professorin Dr. Ricarda Scheiner-Pietsch