What Being small ectotherms, the body temperature of insects closely matches that of their environment; therefore physiological function is greatly affected by ambient temperature. The insect central nervous system is responsible for integrating sensory inputs and coordinating movements, and is shut down at low temperatures by a spreading depolarization, which is caused by a disruption of ion balance within the central nervous system. These events lead to a cold-induced coma. However, insects display vast intra- and interspecific variation in the temperature at which this phenomenon occurs. My project aims to investigate the physiological mechanisms that lead to such variation and how differential exposure to these temperatures affects long-term fitness. Why Insects interact with humanity in many socioeconomic aspects as disease vectors, pests and pollinators. Their latitudinal distribution is limited by their ability to tolerate thermal extremes, in particular, the ability to avoid cold-induced coma, which holds predictive power. Thus, understanding the physiological dysfunctions that limit cold tolerance is crucial in improving these predictions and is therefore likely to confer finer control and management of insect populations. However, little is known about the processes that lead to coma, and even less about how acclimation and adaptation to predicted unfavorable environments lead to variation in resistance to coma. This research project aims to uncover the physiological mechanisms leading to coma and variation in coma resistance. How Coma-inducing temperatures will be estimated in six fruit fly species, cold-acclimated flies and genetic knockout flies. Variation in this temperature will be related to differences in the ability to defend ion balance by measuring the activity and abundance of ion transporters using enzyme-linked assays, Western blots, and the resistance to ion balance disturbance by estimating extracellular volume. Additionally, the sub-lethal effects of spreading depolarization will be estimated by quantifying complex fitness-related behaviors (e.g. mating and courtship), in fruit flies and locusts before and after cold exposure. These behavioral alterations will be linked to central nervous system injury caused by the spreading depolarization by estimating cell death using LIVE/DEAD and TUNEL assays. SSR By elucidating the physiological mechanisms underlying the spreading depolarization phenomenon in insects, and variation therein, we improve our knowledge of how the distribution of potential disease vectors, pests and pollinators is shaped by temperature in a changing climate. The insight gained into the physiological processes behind a commonly used parameter of cold tolerance, the coma-inducing temperature, is also vital for ecological and evolutionary work. Additionally, the insect spreading depolarization shares several characteristics with human pathologies related to cortical spreading depression, such as migraine. Thus, understanding the mechanism in a well-established insect model can prove beneficial to human medicine.