The ability to tolerate environmental stress is a key adaptation for insects across the world. As ectotherms, insects are unable to regulate internal temperature, and their small body size makes them particularly susceptible to extremes in temperature and water availability. Insects rely on numerous physiological adaptations to cope with environmental stress, and recent advances in molecular biology and “omics” technologies have made it possible to study these mechanisms in detail. In this dissertation, I explore molecular mechanisms of stress tolerance in both temperate and polar insects.
In a process called rapid cold-hardening (RCH), brief exposure (i.e. minutes to hours) to nonlethal low temperatures significantly enhances tolerance to cold-shock conditions. While the ecological context of RCH is well-established, the underlying mechanisms are poorly understood. Using cDNA microarrays, we measured changes in gene expression accompanying RCH (2 h at 0°C) in the flesh fly, Sarcophaga bullata. To our surprise, no transcripts were differentially expressed during RCH, suggesting RCH occurs without the need for new gene products. Rather, cold-sensing and RCH appear to be primarily governed by second messenger systems, including calcium signaling. In Chapter 3, we show that chilling evoked an increase in intracellular calcium and activated calcium/calmodulin-dependent protein kinase II. Blocking calcium signaling pharmacologically prevented RCH, indicating calcium signaling is required during cold-sensing and RCH.
In the latter 4 chapters of this dissertation, I investigated physiological and molecular mechanisms of stress tolerance in the Antarctic midge, Belgica antarctica, the world’s southernmost insect and the only insect endemic to the continent. In the unpredictable climate of Antarctica, larvae are likely exposed to multiple bouts of both cold and desiccation stress, thus I quantified the survival and energetic consequences of repeated cold and dehydration exposure in B. antarctica. Larvae exposed to five diurnal freeze-thaw cycles experienced significant mortality and energy depletion. However, this was only true if larvae were frozen during repeated cold exposure; supercooled larvae exposed to the same temperatures experienced no significant mortality or energy depletion. Repeated bouts of dehydration and rehydration were also energetically costly, as 5 cycles of dehydration and rehydration caused larvae to consume 67% of their carbohydrate energy reserves.
In the final two chapters, I explored transcriptional mechanisms of extreme stress tolerance in B. antarctica. Targeted qPCR experiments revealed significant restructuring of metabolic gene expression during periods of stress. Cold stress caused upregulation of genes involved in glucose mobilization, while dehydration stress increased expression of genes required for glucose, trehalose, and proline synthesis. Finally, using RNA-seq, I measured changes in gene expression accompanying extreme dehydration in larvae of B. antarctica. Expression results identified upregulation of pathways involved in cellular recycling and energy conservation, such as ubiquitin-mediated proteasome and autophagy, with concurrent downregulation of numerous genes involved in central metabolism.