The objective of this project is to contribute to an understanding of the molecular genetic basis of behavior. To achieve this objective, the following hypothesis will be tested: specific patterns of gene expression are associated with specific behavioral states in the honey bee (Apis mellifera). This will be addressed by studying genetic expression in the bee brain during post-embryonic behavioral development. Animals will collected at three discrete stages of normal post-embryonic behavioral development, day-olds, brood care (nursing), and foraging. Comparisons of gene expression will be accomplished using the """"""""differential display"""""""" technique (see proposal). RNA will isolated from the brain and cDNA copies will be made using specific primers. These will be amplified by PCR and compared on a sequencing gel. {Differentially expressed putative cDNAs will be used to generate digoxygenin-labeled probes. These will be used screen a cDNA bee brain library to recover a larger and higher quality fragment representing the expressed gene. To confirm that the gene is differentially expressed during different behavioral states, RNA dot blots of brain tissue from bees exhibiting the relevant behaviors will be performed using a probe made from the library insert cDNA. In addition, by using RNA on the dot blots from brain tissue of animals behaviorally accelerated, retarded, or reverted at similar states, it can determined whether differences in gene expression are related to differences in behavior rather than age (nurses are typically younger than foragers).} If differences are reliably associated with behavior, the gene will then be sequenced and I will attempt to determine function by looking for homologies to genes already in GenBank, in situ whole mounts for region specific expression, and developmental northerns for timing of expression. In the event that behaviorally relevant genes cannot be found with the above method, a cDNA library of bee brains will be probed with Drosophila genes that have been implicated in regulating behavior. Genes that can be used are per genes, dunce, biogenic amine receptor genes, and certain immediate early genes, such as c-fos. If found, these genes will be used to probe dot blots of behaviorally staged bees, outlined above, to see if they are involved in post-embryonic behavioral development. It is hoped that results from these experiments will provide insight into the neural and genetic mechanisms of an organism's ability to cope with environmentally influenced changes.
