Evolution of Insect Circadian Clocks and Light Input Channels

蜜蜂是陆地植物的重要传粉者，它们表现出强大的昼夜节律（约。24小时）的节奏时觅食。然而，人们对蜜蜂大脑中的生物钟及其光输入通道知之甚少。隐花色素和视蛋白是光敏感分子，通过外部光周期介导生物钟的夹带。 在多细胞动物受辐射之前，每个基因家族至少发生了一次基因复制事件，产生了脊椎动物样视蛋白（pepsin 2）和果蝇样隐色素（pepsin 1）。这些研究人员在非果蝇的昆虫中发现了EST 2基因。果蝇只表达α 1，蚊子和蝴蝶同时表达α 1和α 2，蜜蜂和甲虫只表达α 2。蜜蜂和大黄蜂是研究昆虫类脊椎动物视蛋白和隐色素功能的模型系统，因为它们代表了昆虫生物钟进化的三种方式之一，具有脊椎动物样的CRY（昆虫CRY 2），但没有果蝇样的CRY（昆虫CRY 1）。这个项目将使用分子、生物化学和解剖学方法来评估蜜蜂的分子时钟机制。 分子钟的转录反馈环将在细胞培养中使用clock 2和其他时钟蛋白（如“周期”）的蜜蜂同源物重建。解剖学方法将绘制昼夜节律钟和视蛋白在大脑和眼睛中的位置。cry 2和周期RNA和蛋白质水平的时间分布将在蜂头中进行检查，并定义关键的蛋白质-蛋白质相互作用。 此外，果蝇转基因和RNA干扰的方法将在体内检查蜜蜂的功能。最后，将评估蜜蜂生物钟的光输入途径，并在细胞培养中研究脊椎动物样视蛋白的光敏性。 由于蜜蜂已经被用于行为研究，这些研究的结果将定义一种新的昼夜节律时钟机制，并定义特定的神经解剖学通路，在未来的研究中可以操纵这些通路，以确定它们对行为输出的影响。该项目将为本科生和研究生提供一个良好的培训环境，通过使用多层次的综合方法来研究昼夜节律系统是如何演变的，从感觉输入到整合中心。

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.