Using calorespirometry to assemble a complete picture of acute hypoxic metabolic depression in fishes

人类活动使世界的水生环境越来越缺氧（低O2）。这对动物造成了威胁，因为需要O2来提供足够的细胞能量（ATP）以满足动物的高代谢需求。然而，许多动物，特别是鱼类，已经适应了缺氧的环境，我们可以看看这些物种的耐受策略，以了解什么可能促进或阻碍动物适应不断变化的世界的能力。耐受缺氧的一种非常有效的策略是代谢抑制（MD），代谢率的调节性降低-从而ATP的供应和使用-低于静息水平。不是所有的鱼类都能诱导缺氧性MD，但那些对缺氧有高度耐受性的鱼类往往能诱导缺氧性MD。一些下调的细胞ATP供应和使用的过程，有助于缺氧MD已被调查隔离，然而，上游信号，诱导他们的下调和他们的调制方式是通过时间和O2水平（PO 2）仍然未知。这项发现补助金将建立在我以前的工作，以测试总体假设，减少血红蛋白（Hb）介导的O2供应的组织作为缺氧MD在鱼类的初始信号。具体而言，当水PO 2变得太低，Hb结合和提供足够量的O2到组织，这些组织诱导MD，包括逮捕的有氧和厌氧ATP供应途径和ATP使用过程的下调。通过4个假设驱动的项目，这项工作将首先调查的作用，血红蛋白在缺氧MD诱导通过实验涉及呼吸热量和操纵Hb-O2亲和力。第二，它将确定如何细胞PO 2影响有氧ATP供应途径使用高分辨率呼吸测定。第三，它将调查如何逮捕ATP供应和使用过程中连接作为一个功能的时间和PO 2使用培养的组织细胞。第四，它将在细胞水平上研究为什么一些物种不能诱导缺氧MD。这项工作的预期结果是更好地了解什么信号缺氧MD诱导，其细胞机制是如何依次连接，以及什么细胞机制不同物种之间，不使用缺氧MD。目前，这些都是未知数。拟议的工作将培养5名研究生（4名硕士，1名博士）和6名本科生，为他们提供广泛适用的研究和分析技能。此外，它将提高我们对动物对水生缺氧反应的理解，这是一种全球范围内日益增加的现象（包括。加拿大）和威胁无数的鱼类种群。最近的研究表明，缺氧MD可能是一种快速进化的耐缺氧策略。了解其近端和最终的机制可能有利于预测模型识别潜在的脆弱物种和保护工作，以保护他们。

Human practices are rendering the world's aquatic environments increasingly hypoxic (low O2). This threatens the resident animals because O2 is required to supply sufficient quantities of cellular energy (ATP) to meet the animals' high metabolic demands. However, many animals, especially fishes, are adapted to environments that already experience hypoxia, and we can look to the tolerance strategies of these species to understand what might facilitate or hinder an animal's ability to adapt to a changing world. A highly effective strategy for tolerating hypoxia is metabolic depression (MD), a regulated reduction of metabolic rate - and thus the supply and use of ATP - below resting levels. Not all fishes can induce hypoxic MD, but those that can tend to be highly tolerant of hypoxia. Some of the downregulated cellular ATP-supply and -use processes that contribute to hypoxic MD have been investigated in isolation; however, the upstream signal that induces their downregulation and the manner by which their modulation is linked via time and O2 level (PO2) remain unknown. This Discovery Grant will build significantly upon my previous work to test the overarching hypothesis that diminished hemoglobin (Hb)-mediated O2 supply to the tissues serves as the initial signal for hypoxic MD in fish. Specifically, when water PO2 becomes too low for Hb to bind and deliver sufficient quantities of O2 to the tissues, those tissues induce MD, including the arrest of aerobic and anaerobic ATP-supply pathways and the downregulation of ATP-use processes. Through 4 hypothesis-driven projects, the work will first investigate the role of Hb in hypoxic MD induction through experiments involving calorespirometry and the manipulation of Hb-O2 affinity. Second, it will determine how cellular PO2 affects aerobic ATP supply pathways using high resolution respirometry. Third, it will investigate how the arrest of ATP-supply and -use processes are linked as a function of time and PO2 using cultured tissue cells. And fourth, it will investigate at the cellular level why some species are unable to induce hypoxic MD. The expected outcome of this work is a better understanding of what signals hypoxic MD induction, how its cellular mechanisms are linked sequentially, and what cellular mechanisms differ between species that do and do not use hypoxic MD. Currently, these are unknowns. The proposed work will train 5 graduate (4 MSc, 1 PhD) and 6 undergraduate students, providing them with widely applicable research and analytical skills. Additionally, it will improve our understanding of animal responses to aquatic hypoxia, a phenomenon that is increasing globally (incl. Canada) and threatens innumerable fish populations. Recent work indicates that hypoxic MD may be a rapidly evolved strategy for tolerating hypoxia. Understanding its proximate and ultimate mechanisms may benefit predictive models for identifying potentially vulnerable species and conservation efforts for protecting them.