Molecules that regulate circadian rhythmicity are also thought to influence the functioning of processes at different temporal scales. The goal of this project is to test an important prediction of this model: given that behavioral and molecular data suggest an association between behavioral maturation and behavioral circadian rhythms in the honey bee, then social and neuroendocrine factors known to influence rate of behavioral maturation should also influence per expression. The following six lines of inquiry will be pursued. 1. Characterize the bee per gene: We have generated a full-length cDNA sequence and also have partially sequenced a genomic clone and will do comparative analyses (with H. Robertson). 2. Determine whether per mRNA levels in the bee brain change during behavioral maturation. Young bees, which work in the hive, have no behavioral circadian rhythms while older bees, which forage on flowers, do; per mRNA levels in the brain change in association with this behavioral maturation. This correlative analysis provides baseline data for the treatment experiments in Aims 4-6. 3. Localize and measure levels of brain PER. Preliminary immunocytochemical results indicate that maturational changes in per mRNA are reflected at the protein level. Continued analyses will be coupled with in situ hybridization to provide additional confirmation (with S. Fahrbach and K. Siwicki). Results of this correlative study will also be used to provide a baseline for Aims 4-6. 4. Determine whether changes in social environment, known to influence behavioral maturation, also influence per expression in the brain. We will take full advantage of a uniquely powerful set of behavioral manipulations to dissociate chronological age, stage of behavioral maturation, and experience, all while working with bees that are highly related to each other. Preliminary results support this hypothesis. 5. Determine whether juvenile hormone and octopamine manipulations, known to influence behavioral maturation, cause changes in both rhythmic locomotor activity and 6. per expression. Preliminary results for locomotor activity support this hypothesis. We hypothesize that juvenile hormone analog and octopamine treatments will increase per expression while allatectomy and octopamine antagonist treatment will decrease it. The principal significance of this research is that it attempts to experimentally determine for the first time whether the expression of a clock gene is dependent upon a broader array of influences than those strictly associated with circadian rhythmicity.
