Collaborative Research: Mechanisms Underlying Circatidal Rhythms in Parhyale Hawaiensis

潮汐对海洋生物的行为和生理有着深刻的影响。 作为回应，许多物种进化出内部计时器，称为昼夜节律钟，以预测潮汐变化。例如，这些生物钟控制着生活在海岸线附近的动物何时觅食、休息和繁殖。由于缺乏适合基因水平操作的模式生物，理解昼夜节律钟的机制已被证明具有挑战性。Parhyale hawaiensis是一种沿海甲壳类动物，这种操纵是可能的。我们已经建立了协议，以同步和观察昼夜行为节律Parhyale。 因此，我们完全有能力阐明昼夜节律的基本机制。 我们的第一个目标是阐明昼夜节律钟的基本性质。 特别是，我们的目标是揭示Parhyale如何使用线索，如水位和湍流，同步其行为与潮汐。 我们的第二个目标是确定控制昼夜节律钟的基因，并确定哪些生理过程受其控制。 因此，我们的工作应该能够更好地了解动物如何应对与海岸线不断变化的水位相关的挑战。在这项工作中，我们将通过"生物发现在伍兹霍尔" REU计划（NSF DBI 1659604）涉及本科少数民族（URM）的学生。 我们还将通过创建一个“时间生物学俱乐部”，让伍斯特的高中URM学生参与生物计时研究。 最后，我们将向波多黎各环境神经科学中心的学生介绍在非标准研究生物中进行基因编辑的方法。潮间带生物使用昼夜节律钟来调整其生理和行为以适应有节奏的潮汐变化，类似于昼夜节律钟使生物体能够预测每日的环境振荡。尽管昼夜节律钟的存在早已被证实，但人们对它们的运作方式知之甚少。相比之下，昼夜节律钟是很好理解的，因此可以作为关于昼夜节律钟如何构建和功能的假设的模型。Parhyale hawaiensis是一种适合遗传操作的潮间带甲壳动物。我们已经开发出的方法，夹带Parhyale的昼夜节律人工潮汐和观察昼夜游泳行为。我们的目标是确定环境的线索，夹带昼夜行为，并确定是否Parhyale的昼夜时钟夹带不同的自然潮汐模式。我们的目标也是阐明昼夜节律的分子机制。我们将使用CRISPR/Cas9指导的基因组编辑来确定核心生物钟基因是否也会产生昼夜节律。我们将分析整个潮汐周期的基因表达，以确定核心的昼夜节律基因和调节器官特异性生理的基因。我们的工作应该决定性地推进我们对昼夜节律钟的理解，从它们与环境输入的相互作用到产生它们的分子机制。URM学生参加NSF支持的"生物发现伍兹霍尔"计划将参与这项工作。 一个"时间生物学俱乐部"将被创建，以暴露在伍斯特的高中URM学生在生物计时研究。 最后，波多黎各环境神经科学中心的学生将被介绍给非标准研究生物体的基因编辑。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

The tides profoundly impact the behavior and physiology of marine organisms. In response, many species have evolved internal timers called circatidal clocks to anticipate tidal changes. These clocks control when animals living near the coastline forage, rest and reproduce, for example. Understanding the mechanisms underlying circatidal clocks has proven challenging because of the lack of model organisms that are amenable to gene-level manipulations. Parhyale hawaiensis is a coastal crustacean in which such manipulations are possible. We have established protocols to synchronize and observe circatidal behavioral rhythms in Parhyale. We are thus well-positioned to elucidate the fundamental mechanisms underlying circatidal timing. Our first goal is to elucidate the fundamental properties of circatidal clocks. In particular, we aim to reveal how Parhyale uses cues, such as water levels and turbulence, to synchronize its behavior with tides. Our second objective is to identify the genes that control the circatidal clock, and to determine which physiological processes are under its control. Our work should thus provide a much-improved understanding of how animals cope with the challenges associated with ever-changing water levels at the coastline. In this work, we will involve undergraduate underrepresented minority (URM) students through the “Biological Discovery in Woods Hole” REU program (NSF DBI 1659604). We will also engage high school URM students in Worcester through the creation of a “Chronobiology club” that will expose them to research in biological timing. Finally, we will introduce students at the Puerto Rico Center for Environmental Neuroscience to methods of gene editing in non-standard research organisms.Intertidal organisms use circatidal clocks to adapt their physiology and behavior to rhythmic tidal changes, in a similar fashion that circadian clocks allow organisms to anticipate daily environmental oscillations. Although the existence of circatidal clocks has long been established, little is known about how they operate. By contrast, the circadian clock is well understood and can therefore serve as a model for hypotheses regarding how the circatidal clock is constructed and functions. Parhyale hawaiensis is an intertidal crustacean amenable to genetic manipulations. We have developed methods to entrain Parhyale’s circatidal rhythms to artificial tides and observe circatidal swimming behavior. We aim to identify the environmental cues that entrain circatidal behavior and determine whether the Parhyale’s circatidal clock entrains to different natural tidal patterns. We also aim to elucidate the molecular mechanisms underlying circatidal rhythms. We will use CRISPR/Cas9-guided genome editing to determine whether core circadian clock genes also generate circatidal rhythms. We will profile gene expression across the tidal cycle to identify core circatidal genes and genes regulating organ-specific physiology. Our work should decisively advance our understanding of circatidal clocks, from their interactions with environmental inputs to the molecular mechanisms that generate them. URM students participating in the NSF-supported “Biological Discovery in Woods Hole” program will be involved in this work. A “Chronobiology club” will be created to expose high school URM students in Worcester to research in biological timing. Finally, students at the Puerto Rico Center for Environmental Neuroscience will be introduced to gene editing in non-standard research organisms.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.