Funktionelle Charakterisierung von Adenylatzyklasen der Honigbiene Apis mellifera

Abstract

Adenylyl cyclases (ACs) are enzymes that synthesize the intracellular messenger adenosine 3',5'-cyclic monophosphate (cAMP). The physiological impact of ACs has been intensively investigated in the mammalian brain and in the fruitfly Drosophila melanogaster. Some of the AC-enzymes are involved in processes underlying learning and memory. An organism, which is well suited for learning studies, is the honeybee Apis mellifera. The bee provides a rich behavioral repertoire that can be experimentally adressed. Within the last decades, several tests to study visual, olfactory, and tactile learning skills of the honeybee have been established. However, the cellular mechanisms and molecular components controlling the bees' behavior are largely unknown. In the beginning of this study, the molecular and biochemical properties of adenylyl cyclases in the bee had not been uncovered. I have cloned three genes (Amac2, Amac3, and Amac8) that encode membrane-bound ACs from honeybee brain. The amino-acid sequences have striking similarities to ACs from mammals and Drosophila. Heterologously expressed AmAC2t and AmAC3 proteins are activated by forskolin as well as by $\alpha$-subunits of stimulatory G-proteins (Gs$\alpha$). The Amac2-gene encodes an N-terminally truncated protein (AmAC2t). Notably, AmAC2t is the first truncated AC-enzyme that could be functionally expressed. The expression profile of the Amac3 gene was analyzed by in situ hybridization of braintissue sections. The Amac3 mRNA is predominatly expressed in the mushroom bodies, the optic lobes, and the deutocerebrum. To analyze the in vivo function of AmAC3, the expression of the Amac3 gene should be suppressed in the brain. To pursue such analyses, RNA interference (RNAi) is a promising technique. When Amac3-specific, double-stranded (ds) RNA is introduced into the honeybee brain, the endogenous Amac3 mRNA-level should be reduced. Bees that were injected with Amac3-dsRNA exhibited a higher responsiveness to sugar compared to controls. These results suggest that AmAC3 modulates the gustatory sensitivity of the animal. Interestingly, the injected bees had a higher amount of Amac3 mRNA than control bees. This unexpected finding evoked an alternative interpretation of the RNAi-effect. Further experiments are necessary, however, to uncover how the RNAi mechanism in the honeybee really works and to establish this method as a reliable tool to study gene function in this insect