Insect cuticle is mainly composed by proteins that interact with chitin, an N-acetylglusamine polimer, to form the exoskeleton that gives support and protects the organism. This hard carcass is periodically renewed at each molt thus permitting insect growth and development. During the molt, the old cuticle is partially digested and subsequently discarded whereas a new cuticle is synthesized. Both, cuticle degeneration and biosynthesis require a precisely timed activation or repression of distinct set of epidermal genes, and this is coordinated by hormones, mainly ecdysteroids. Cuticle proteins have been studied in several insect species, but remain only partially characterized. The completion of the genome of A. mellifera (http://www.hgsc.bcm.tmc.edu/projects/honeybee/), brought a new dimension to the study of insect cuticle. As a result, 30 sequences encoding putative cuticle proteins have been identified, but cuticle proteome is far from being completely characterized. In addition, there are no studies on regulation and function of these proteins in the honey bee. It is our intention to explore the differential expression of cuticle protein genes, or cuticle genes, in the context of the molt and metamorphosis in this important social insect. To this end, we elected two events of metamorphosis, the pupal-adult apolysis and adult ecdysis to elucidate aspects of regulation and function of cuticle genes. Briefly, RNA will be extracted from the integument (cuticle and subjacent epidermis) of bees at the time of pupal-adult apolysis or adult ecdysis and used in microarrays aiming to identify differentially expressed cuticle genes. Since these molt events are induced by contrasting levels of ecdysteroids, high and low, respectively, we expect to characterize cuticle genes differentially regulated by these hormone. The recently sequenced genome of the honey bee, and the public data bank containing sequences encoding putative cuticle proteins in arthropods, (http://bioinformatics2.biol.uoa.gr/cuticleDB/index.jsp), will be used as tools. For microarray data validation we will make use of semiquantitative and real time RT-PCR. When necessary, cuticle gene regulation by ecdysteroids will be validated by in vitro experiments using integument pieces and hormone manipulation. With this strategy, we expect to identify new structural cuticle protein genes in the honey bee genome, and also other genes encoding non-structural cuticle proteins, in addition to study aspects of their regulation and function in specific events of molt and metamorphosis.
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