Electronic Thesis and Dissertation Repository

Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biology

Supervisor

Sinclair, Brent J.

Abstract

Many temperate insects enter diapause (a state of dormancy) and enhance their cold tolerance to survive the winter. During diapause, the Colorado potato beetle (CPB, Leptinotarsa decemlineata, Coleoptera: Chrysomelidae) stops developing, lowers its metabolism, and changes its physiology to avoid freezing. The extent to which diapause confers cold tolerance in CPB is currently unknown. In my thesis, I used CPB to improve our understanding of the mechanisms underlying metabolic suppression during diapause and cellular protection at sub-zero temperatures in insects. First, I used RNA-sequencing (RNA-seq) to compare gene expression in two metabolically important tissues (the fat body and flight muscle) of diapausing and non-diapausing CPB, and then generated testable hypotheses about diapause in CPB. Colorado potato beetles differentially modulate their fat body and flight muscle transcriptomes during diapause; fat body plays a larger role in driving hypoxia- and immune-related processes during diapause, whereas processes mediating proteostasis and mitochondrial metabolism are more important in the flight muscle. Next, I tested the hypothesis that flight muscle mitochondria modulate energy metabolism in diapausing CPB. Indeed, low metabolic rates coincided with a reduction in flight muscle mitochondrial function and density, increased expression of Parkin (a mitophagy-related transcript), and presence of autophagic structures inside flight muscle cells. Further, knocking down Parkin with RNA interference partially restored mitochondrial density and whole-animal metabolic rate suggesting that Parkin-mediated mitophagy drives metabolic suppression during CPB diapause. In anticipation of emergence from diapause, beetles reversed this mitophagy and increase mitochondrial biogenesis to re-grow their mitochondria. Finally, I used RNA-seq to explore the mechanisms underlying the acquisition of cold tolerance in diapausing CPB. The major transcriptomic shift associated with the acquisition of cold tolerance is related to chaperone protein-related expression, and cold-tolerant beetles activate the chaperone response to a greater extent than less cold-tolerant diapausing counterparts. Further, cold-tolerant beetles potentially have a greater capacity for chaperone-mediated protein repair. Together, these studies contribute to an updated framework for insect diapause and cold tolerance and improve our understanding of the mechanisms insects use to survive the winter.

Summary for Lay Audience

Winter is a hostile season for insects because resources are scare and freezing temperatures persist for months at a time. Many temperate insects spend most of their lives overwintering where they must survive these harsh conditions. During the winter, Colorado potato beetles (CPB) become dormant (enter diapause), where they stop developing, lower their metabolism to save energy, and enhance their ability to survive freezing temperatures. In my thesis, I used CPB to explore how overwintering insects are able to lower their metabolism and protect their tissues and cells from freezing temperatures during winter. First, I investigated patterns of gene expression during diapause in CPB in two tissues that are important for metabolism over the winter, their fat body (analogous to mammalian liver), and flight muscle. Gene expression profiles differed in each tissue; fat body expressed more genes related to surviving low oxygen environments and enhancing immunity, whereas flight muscle expressed more genes related to protein protection and mitochondrial metabolism. Next, I explored how flight muscle mitochondrial metabolism is important for diapause in overwintering CPB, and whether CPB change mitochondrial function to achieve a low metabolism over the winter. I showed that CPB do not just alter flight muscle mitochondrial metabolism to achieve a low metabolism but degrade most of their mitochondria altogether. I also showed that CPB can re-grow their flight muscle mitochondria without any cues, and in anticipation of the end of diapause. Finally, I investigated how gene expression changes in the fat body of CPB that have an enhanced ability to survive freezing temperatures (cold-tolerant CPB). Although I expected to find many genes differentially expressed in cold-tolerant CPB, I found a small number of genes responsible for cold tolerance, which were all related to the process of cellular protection by chaperone proteins. Further, cold-tolerant CPB had fewer damaged proteins in their fat body compared to less cold tolerant CPB, which suggests that they are able to better repair proteins using the cellular chaperone response. Taken together, my thesis provides novel insights into how insects regulate their metabolism over the winter and protect their cells in the cold.

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