Collaborative Research: RUI: Extraordinary circadian clocks in araneoid spiders: an integrative approach to understanding their evolutionary origins and underlying mechanisms

昼夜节律是由内部生物钟的分子周期驱动的行为、生理和细胞新陈代谢的日常节律。通过对自己的内部时钟周期进行微小的调整，内部时钟保持与地球24小时一天的同步。然而，如果时钟被迫进行较大的调整(例如，时差)，则会产生负面的生理后果，因此大多数生物体的时钟在24小时内不会有太大变化。令人惊讶的是，一群蜘蛛物种的时钟相差24小时，相差多达5小时，没有明显的后果。从理论上讲，这些物种不应该存在！然而，蜘蛛系统提供了一个独特的机会来探索生物钟的基本机制，特别是生物体如何与环境同步。这个多机构项目旨在了解：(1)蜘蛛时钟基因和昼夜节律特性的进化变化，(2)同步到24小时一天的限制和生理后果，以及(3)蜘蛛的基本分子时钟。总体而言，该项目将开发一种新的、独特强大的模型系统，以了解昼夜节律，并可能了解昼夜节律疾病。从概念上讲，学生可以接触到昼夜节律，该项目将为阿巴拉契亚地区的本科生提供丰富的机会，让他们参与所有三个机构的研究。该项目的社会影响将包括开发用户友好、开放获取的应用程序，用于严格分析昼夜节律数据；年度公共推广活动，包括针对K-12学生和成年人的自然/STEM计划，以及在当地高中进行原创实验。蜘蛛类蜘蛛的昼夜节律与地球上发现的大多数蜘蛛不同。它们在物种内部和物种之间都表现出非常广泛的内源自由运行期(FRPS)分布，包括平均FRPS特别短或长的物种(17.8-29.1小时)。生存实验表明，它们以某种方式摆脱了这些选择性的限制，而不是遭受通常与内源性昼夜节律和24小时一天之间的不和谐相关的负面后果。利用一个综合的、多层次的方法，这个项目将利用蜘蛛类和非蜘蛛类蜘蛛物种之间昼夜节律系统功能的明显进化变化来识别使这些不寻常的蜘蛛类时钟存在的时钟机制的变化。使用基因组或转录组规模的数据来估计跨越蜘蛛多样性的物种的八个不同时间生物学参数的进化变化率，将使重建祖先状态和精确确定进化转变的时间。比较蜘蛛类和非蜘蛛类物种的生存能力将确定蜘蛛类蜘蛛是否真的从夹带的成本中释放出来，进入非共振的光/暗周期。经典的相移和相响应曲线实验将探索蜘蛛类蜘蛛和非蜘蛛类蜘蛛之间夹带潜力的差异。比较蜘蛛和非蜘蛛类蜘蛛以及已建立的昆虫模型之间的典型时钟基因表达幅度，将探索潜在的时钟功能差异。比较蜘蛛类和非蜘蛛类物种之间时钟成分CRY1的功能将测试昼夜节律系统的光敏感度的差异。该项目使用分子、行为和系统发育学方法，探索野生时钟昼夜节律可塑性的范围和功能分支。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Circadian rhythms are daily rhythms of behavior, physiology, and cellular metabolism that are driven by molecular cycles of an internal biological clock. Internal clocks remain in sync with the earth’s 24-h day by making small adjustments to their own internal clock period. However, there are negative physiological consequences if the clock is forced to make large adjustments (e.g., jetlag) so most organisms’ clocks do not vary much from 24 hours. Surprisingly, a group of spider species possess clocks that differ from 24 hours by as much as 5 hours with no apparent consequences. In theory, these species should not exist! However, the spider system provides a unique opportunity to explore basic mechanisms of circadian clocks, particularly how organisms synchronize with their environment. This multi-institutional project is designed to understand: (1) the evolutionary changes in clock genes and circadian properties in spiders, (2) the limits and physiological consequences of synchronizing to the 24-h day, and (3) the fundamental molecular clockworks of spiders. Overall, this project will develop a new, and uniquely powerful, model system to understand circadian rhythms and, potentially, circadian illnesses. Circadian rhythms are conceptually accessible to students and this project will support rich opportunities for undergraduates in the Appalachian region to participate in research at all three institutions. Societal impacts of this project will include development of user-friendly, open-access applications for rigorous analyses of circadian data, annual public outreach events including nature/STEM programs for K-12 students as well as adults, and conducting original experiments in local high schools. Araneoid spider circadian rhythms are unlike most others found on Earth. They exhibit remarkably broad distributions of endogenous free-running periods (FRPs) both within and among species, including species with exceptionally short or long mean FRPs (17.8-29.1 hours). Rather than suffering negative consequences typically associated with dissonance between endogenous circadian period and the 24-hour day, survivorship experiments suggest that they are somehow released from these selective constraints. Using an integrative, multi-level approach, this project will exploit the apparent evolutionary shift in circadian clock system function between araneoid and non-araneoid spider species to identify changes in the clock mechanisms that enable these unusual araneoid clocks to exist. Using genomic or transcriptomic-scale data to estimate rates of evolutionary change in eight different chronobiological parameters for species spanning the diversity of spiders will enable reconstruction of ancestral states and pinpoint the timing of evolutionary shifts. Comparing survivorship among araneoid and non-araneoid species will determine if araneoid spiders truly have been released from the costs of entrainment to non-resonant light/dark cycles. Classic phase-shifting and phase-response curve experiments will probe differences in entrainment potential between araneoid and non-araneoid spiders. Comparing canonical clock gene expression amplitudes between araneoid and non-araneoid spiders and to established insect models will explore potential clock function differences. Comparing the functionality of the clock component CRY1 between araneoid and non-araneoid species will test for differences in light sensitivity of the circadian system. Using molecular, behavioral, and phylogenetic approaches, this project explores the extent and functional ramifications of circadian plasticity in wild clocks.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.