Microinjection and imaging suite for assessing the genetic and molecular basis of physiological responses to changing environments in fishes

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Coastal ecosystems are subject to large fluctuations in salinity, temperature, and dissolved oxygen on seasonal and daily cycles, posing extreme challenges for resident fishes. Climate change forecasts predict increases in the extremes of these cycles, but it is unclear how this will impact fish populations. Critically important to predicting the effects of climate change is an understanding of physiological responses to these stressors, as these responses influence animal survival and performance. This application is for the purchase of a fluorescence stereomicroscope and a microinjection system that form a comprehensive suite that is essential to the research of Drs. Alex Zimmer and Ben Speers-Roesch. Their programs seek to understand responses used by fishes to survive in changing environments using an ecologically important fish species in Atlantic Canada, the mummichog (Fundulus heteroclitus), and will address three broad research themes: Developmental mechanisms of ion regulation. Mummichog is a model for euryhaline ion regulation and the function of ion-transporting cells (ionocytes), but little is known regarding ionoregulatory physiology of its sensitive early life stages. The stereomicroscope will be used to characterize the expression of ionocyte populations over development, and their response to changing salinity, using several fluorescent labeling techniques. The microinjection system will be used to eliminate specific ionocyte subtypes using genetic manipulations targeting transcription factors (e.g., foxi3) to understand how these ionocytes contribute to euryhaline capacity over life history. Mechanistic basis of physiological plasticity. Physiological plasticity, the ability to adjust to changing environments, is an important feature of organisms that live in dynamic environments. Euryhalinity, like other plastic traits, is dependent upon underlying mechanisms that can be fine-tuned to meet environmental demands. The importance of environmentally-regulated mechanisms, such as the salinity-dependent expression of Na+/K+-ATPase isoforms in the gills, to euryhalinity and other plastic traits will be assessed by eliminating the expression of specific gene isoforms via gene editing using the microinjection system. Linkages between temperature sensing and thermal coping responses. Changes in environmental temperature trigger behavioral and physiological coping responses needed to survive. The primary temperature sensors in vertebrates are the transient receptor potential ion channels (TRPCs). TRPCs respond to cold (TRPM8) or hot (TRPV1), but the role of TRPCs in triggering vital coping responses is unclear. The microinjection system will allow us to develop mutant lines of mummichog lacking the expression of specific TRPCs. These lines will be used to discern the roles of TRPCs in controlling responses to natural and anthropogenic thermal stress, including shifts in heat tolerance and changes in activity levels to save energy.