Collaborative Research: Mechanisms Underlying Circatidal Rhythms in Parhyale Hawaiensis

潮汐深刻地影响着海洋生物的行为和生理。作为回应，许多物种进化出了被称为圆周钟的内部计时器，以预测潮汐变化。例如，这些时钟控制着生活在海岸线附近的动物何时觅食、休息和繁殖。事实证明，由于缺乏能够在基因水平上进行操纵的模式生物，理解圆形生物钟背后的机制是具有挑战性的。夏威夷甲壳类是一种沿海甲壳类动物，在这种甲壳类动物中可以进行这种操纵。我们已经建立了同步和观察Parhyale昼夜行为节律的方案。因此，我们能够很好地阐明周转计时背后的基本机制。我们的第一个目标是阐明旋转钟的基本性质。特别是，我们的目标是揭示Parhyale如何使用诸如水位和湍流等线索来使其行为与潮汐同步。我们的第二个目标是确定控制生物钟的基因，并确定哪些生理过程受其控制。因此，我们的工作应该会更好地理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“森林洞穴中的生物发现”REU计划(美国国家科学基金会DBI 1659604)让本科生参与到未被充分代表的少数族裔(URM)学生中来。我们还将通过创建一个“时间生物学俱乐部”来吸引伍斯特的高中URM学生，让他们接触到生物计时的研究。最后，我们将向波多黎各环境神经科学中心的学生介绍非标准研究组织中的基因编辑方法。潮间带生物使用昼夜节律时钟来调整它们的生理和行为以适应有节奏的潮汐变化，类似于生物钟允许有机体预测日常环境振荡的方式。尽管圆周钟的存在早已被证实，但人们对它们是如何运作的知之甚少。相比之下，生物钟被很好地理解，因此可以作为关于昼夜钟如何构造和功能的假说的模型。夏威夷甲壳类是一种潮间带甲壳类动物，易受基因控制。我们已经开发了将Parhyale的圆周节律引入人工潮汐并观察圆周游泳行为的方法。我们的目标是找出影响浮游动物行为的环境线索，并确定浮游动物的浮游生物钟是否与不同的自然潮汐模式有关。我们还旨在阐明昼夜节律背后的分子机制。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，以确定核心旋回基因和调节器官特定生理学的基因。我们的工作应该决定性地推进我们对圆周钟的理解，从它们与环境输入的相互作用到产生它们的分子机制。参加美国国家科学基金会资助的“森林洞穴中的生物发现”项目的URM学生将参与这项工作。将成立一个“时间生物学俱乐部”，让伍斯特的高中URM学生接触到生物计时的研究。最后，波多黎各环境神经科学中心的学生将被介绍到非标准研究组织中的基因编辑。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。

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.