Benjamin Lant, Ph.D. Biology, 2011

Expression pattern of the novel freeze-responsive genes li16, fr10 and fr47 in the wood frog, Rana sylvatica



The capacity to adapt to and survive oxygen deprivation has long been an important topic of study, in both ecological and medical fields. The freshwater crayfish, Orconectes virilis, is capable of tolerating anoxia, but the metabolic mechanisms underlying this are largely unprofiled. This thesis examines the activity and regulation of a number of stress response pathways in response to anoxia in O. virilis. The model organism Caenorhabditis elegans that shows stress-induced entry into hypometabolism (the dauer stage) was used as a template for selecting stress response pathways that could be important in crayfish anaerobiosis. The Akt signaling response showed a distinct increase in activity in crayfish tail muscle and hepatopancreas under anoxia, as assessed through phosphorylation states of the kinase and its downstream targets. This implicated a pro-survival response that functions by preventing cell cycle attenuation. Despite elevated Akt activity, residualFoxO activity remained, possibly mediating a pro-survival mechanism through transactivation of antioxidant genes (includingMnSOD) in preparation for reoxygenation. Smad and STAT transcription factors, following the pattern of pro-development Akt signaling, also showed a fairly active profile (via phosphorylation status) but upregulation was not unilateral. Hepatopancreas showed a more active profile of Smads, but this did not correlate with increased DNA binding, again hinting at a preparative mechanism for the recovery period. Apoptosis (cell death) signaling was assessed through pro-apoptosis (p53) and anti-apoptosis (Bcl) targets, whereas autophagy (a cell minimization response to stress) was assessed via expression response of multiple autophagy proteins (Atg). An anoxia-triggered, tissue-specific result arose, potentially based on the importance of individual organ integrity throughout hypometabolism. Tail muscle, which showed increased expression profiles of all three target groups (p53, Bcls, Atgs), contrasted with hepatopancreas, which appeared to be not susceptible to either apoptotic orautophagic signaling during anoxia. Finally, the cell cycle, often a target for attenuation in stress states, was analyzed. Neither tissue showed strong signs of cell cycle attenuation under anoxia, although certain inhibitor profiles were enhanced under anoxia. The data provide a comprehensive overview of the responses and integration of multiple stress-responsive signaling pathways in O. virilis that provide a novel contribution to our understanding of pro-survival mechanisms supporting invertebrate anoxia tolerance.