THE MOLECULAR MECHANISM

The Molecular Mechanism of Freez Tolerance In Insects

Acknowledgement

I would take this opportunity to thank my research supervisor, family and friends for their support and guidance without which this research would not have been possible.

Table of contents

Acknowledgement2

Abstract12

Introduction13

Materials and methods14

Chemicals14

Animals14

PKA tissue extraction and assay15

PP1 extraction and assay15

PP2 extraction and assay16

PTP extraction and assay17

Results: Winter profiles of enzyme activities18

Exposure to -4 °C and recovery20

Recovery from -20 °C exposure22

Discussion23

Conclusion27

References28

Abstract

Freeze -tolerant larvae of the goldenrod gall fly, Eurosta solidaginis Fitch, show multiple metabolic adaptations for subzero survival including the autumn synthesis of high concentrations of polyols. The induction and regulation of cold hardiness adaptations requires the intermediary action of signal transduction enzymes. The present study evaluates changes in the activities of cAMP-dependent protein kinase (PKA), protein phosphatases 1 (PP1), 2A, 2C, and protein tyrosine phosphatases (PTPs) over the course of the winter season and also in insects exposed to -4, -20 °C, or anoxic conditions in the laboratory. The increased PKA and decreased PP1 over the winter season and/or at subzero temperature support a regulatory role for these enzymes in cryoprotectant polyol synthesis. PTP activities were also strongly increased under these conditions and may act to antagonize tyrosine kinase mediated cell growth and proliferation responses and, thereby, contribute to hypometabolism and diapause over the winter.

The Molecular Mechanism of Freez Tolerance In Insects

Introduction

Larvae of the goldenrod gall fly Eurosta solidaginis Fitch (Diptera, Tephritidae) have been studied extensively as a model of previous terminsect freeze tolerancenext term (for review, see Baust and Nishino, 1991; Lee et al., 1996; [Storey and Storey, 1991] and [Storey and Storey, 1992]). previous termInsectnext term freezing survival relies on a variety of molecular adaptations that include the production of high concentrations of polyol cryoprotectants, the use of ice nucleators, metabolic rate depression (diapause) and changes in gene expression (Storey, 1997; Duman, 2001). E. solidaginis uses dual cryoprotectants, glycerol and sorbitol, accumulated with different seasonal patterns (Storey and Storey, 1986). The regulation of multiple enzymes involved in polyol production and energy metabolism in E. solidaginis has been explored (Joanisse and Storey, 1994a; Storey and Storey, 1991 K.B.

Storey and J.M. Storey, Biochemistry of cryoprotectants. In: R.E. Lee and D.L. Denlinger, Editors, previous termInsectsnext term at Low Temperature, Chapman and Hall, London (1991), pp. 64-93.[Storey and Storey, 1991] and [Storey and Storey, 1992]). A key recurring previous termmechanismnext term of regulation in this species and other cold-hardy previous terminsectsnext term is reversible protein phosphorylation. For example, low-temperature-triggered activation of glycogen breakdown for polyol synthesis results from phosphorylation-mediated activation of glycogen phosphorylase (GP) (Hayakawa, 1985), whereas the inhibition of gluconeogenesis that prevents back-flow of carbon is mediated by phosphorylation inhibition of glycogen synthetase (GS) and dephosphorylation inhibition of fructose-1,6-bisphosphatase ([Muise and Storey, 1997] and [Muise and Storey, 1999]). Changes in gene expression are also typically mediated by signal transduction cascades involving protein kinases and phosphatases. A variety of genes that are cold- or previous termfreezenext term-responsive are now known for E. solidaginis including the hypoxia-inducible transcription factor (hif-1) (Morin et ...