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

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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.