No Oxygen, No Water: the Unique Physiology of Annual Killifish Embryos Across Development

For most vertebrates, an abundance of oxygen is necessary for the production of ATP and the maintenance of cellular homeostasis. The absence of oxygen, even for brief periods, quickly leads to ATP depletion which can lead to irreparable damages to sensitive organs, such as the brain and heart. However, certain vertebrates demonstrate an extraordinary ability to thrive and recover fully from periods of no oxygen (anoxia). The annual killifish (Austrofundulus limnaeus) lives in ephemeral ponds in the Maracaibo basin of Venezuela and their embryos have the remarkable ability to not only survive anoxic periods for months, but also dehydrating conditions. Survival of this species is dependent on the ability for embryos to enter profound metabolically dormancy termed diapause, as part of their normal life cycle. Survival of dehydrating conditions in A. limnaeus embryos is believed to be achieved through reduction of evaporative water loss and thus is likely to highly limit gas exchange. Thus, embryos exposed to dehydration stress may self-impose severe oxygen stress in order to survive without water. There have been many advances in understanding the extreme anoxia tolerance of this system, but little research has been done on dehydration tolerance and how it relates to anoxia tolerance or stress tolerance in general. There is even less known about the evolutionary route that led to increased stress tolerance associated with diapause in A. limnaeus. I hypothesize that anoxia tolerance is a pre-adaptation that allowed for the evolution of dehydration tolerance. Thus, I predict that A. limnaeus embryos exposed to dehydrating conditions will show similar responses at the molecular level to embryos exposed to anoxia. A quintessential feature of anoxia-tolerance is the accumulation of neurotransmitter γ-aminobutyric acid (GABA). Despite knowledge of the accumulation of GABA in A. limnaeus, little is known about its role in embryos during anoxia or if it plays a role during dehydration stress. The overall goals of this project were to: (1) determine how GABA accumulation relates to stress tolerance of A. limnaeus embryos, and (2) explore the physiological mechanisms that allow A. limnaeus embryos to survive dehydration stress by monitoring survival, measuring oxygen consumption, and performing a metabolomics analysis. This project was the first to show that inhibition of GABA production or degradation leads to a reduction in anoxia tolerance. Only embryos that can produce GABA are able to successfully recover from long-term anoxia which may be suggestive of its role as a neurotransmitter, an energy source, and/or an antioxidant during anoxia and aerobic recovery. This project uncovered many new aspects of the biology of A. limnaeus embryos during dehydration stress. Diapausing embryos are able to survive extremely dehydrating conditions for over 1.5 years, while developing embryos are able to survive for over 100 days. Embryos in diapause respond to dehydration stress by increasing rates of oxygen consumption while post-diapause II embryos either exhibit the same or reduced rates of oxygen consumption when compared to aqueous embryos. Dormant and actively developing embryos respond to dehydration stress in an active manner by significantly altering their metabolic profile. A number of metabolites accumulate during exposure to dehydration stress that may play an important role in survival, including the identification of known antioxidants and neuroprotectants. In addition, a number of unique metabolites not yet discussed in the dehydration literature are identified. Despite high oxygen availability, embryos accumulate the anaerobic glycolytic end-product lactate and neurotransmitter GABA. We show that there is undoubtedly overlap in the molecular responses to anoxia and dehydration stress. However, the response to dehydration stress is complex and we have only just begun to scratch the surface into understanding survival of A. limnaeus embryos during this crucial time in their life cycle