Evolution of Insect Circadian Clocks and Light Input Channels

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Bees are important pollinators of land plants, and they display robust circadian (ca. 24 hr) rhythms when foraging. Yet little is known about the circadian clock in the brains of bees and its light input channels. Cryptochromes (CRYs) and opsins are light-sensitive molecules that mediate the entrainment of circadian clocks by the external light cycle. Prior to the radiation of metazoans at least one gene duplication event occurred pergene family creating both vertebrate-like opsins (CRY2) and Drosophila-like cryptochrome (CRY1). These investigators discovered the CRY2 gene in non-drosophilid insects. While Drosophila only express CRY1, mosquitos and butterflies express both CRY1 and CRY2, and bees and beetles only express CRY2. These three different expression patterns for the two CRY genes suggest potentially three different types of insect clocks.Honeybees and bumblebees are a model system for examining the function of vertebrate-like opsin and cryptochrome in insects, because they represent one of the three ways within insects in which circadian clocks have evolved, with a vertebrate-like CRY (insect CRY2) but without a Drosophila-like CRY (insect CRY1). This project will evaluate the molecular clockwork in bees using molecular, biochemical and anatomical approaches. The molecular clock's transcriptional feedback loop will be reconstructed in cell culture using CRY2 and bee homologs of other clock proteins (such as 'period'). Anatomical methods will map the location of circadian clock and opsin proteins in the brain and eye. The temporal profiles of cry2 and period RNA and protein levels will be examined in bee heads, and the critical protein-protein interactions defined. Moreover, Drosophila transgenesis and RNA interference approaches will examine in vivo the function of bee CRY2. Finally, light input pathways to the bee clock will be evaluated, and the light sensitivity of the vertebrate-like opsin will be studied in cell culture. As bees are already famously used for behavioral investigations, the results of these studies will define a novel circadian clockwork mechanism and define specific neuroanatomical pathways that could in future studies be manipulated to determine their impact on behavioral outputs. This project will provide an excellent training environment for undergraduate and graduate students by using multi-level integrated approaches to study how circadian systems have evolved, from sensory input to integrating centers.