Unraveling the regulatory mechanisms of pupal diapause termination

Sammanfattning: Diapause is an essential part of many insect’s life cycle and is a developmental halt induced by environmental cues in advance of deteriorating conditions. Insects typically enter diapause to avoid unfavorable environmental conditions like low temperatures, poor food quality and the absence of conspecifics. The aim of my PhD thesis was to describe how diapause is regulated and what determines its termination timing in the green-veined white butterfly Pieris napi. Answers to these questions deepens the knowledge about species distribution and population dynamics in times of a rapidly changing climate. In ectothermic insects, developmental rate generally follows a thermal performance curve (TPC) with higher temperatures leading to a faster development. However, in the diapause of P. napi low temperatures result in a higher proportion of terminations. In Chapter I, I assessed the thermal performance of diapause termination rate in P. napi by exposing them to several different temperature treatments. The diapause termination rate follows a left-shifted TPC with an optimum termination rate at 0°C and slower termination rates toward higher temperatures. This left-shifted TPC prevents the precocious termination of diapause in autumn while at the same time enabling populations to remain synchronized over a long period. Development in insects is regulated by the major developmental hormones, insulins, juvenile hormones, ecdysone, and the prothoracicotropic hormone (PTTH). The pupal diapause is regulated by the neuropeptide PTTH produced in the brain and ecdysone produced in the prothoracic glands. In Chapter II I studied this hormonal axis by assessing PTTH levels and injections with 20-hydroxyecdysone (20E), the active form of the ecdysone pathway. The PTTH neuropeptide shows diapause stage dependent changes to haemolymph levels and intracellular structure and the sensitivity to 20E returns in a time- and temperature dependent manner correlating with diapause termination progression. This indicates that diapause termination timing is regulated by the PTTH ecdysone axis and that the ecdysteroid receptor has a central part in the regulation.  Diapause is a prolonged period without the ability to replenish energy resources, insects therefore accumulate resources before diapause and reduce the metabolic rate to a minimum. In Chapter III, I investigated how the diapause physiology as well as the reduced metabolic rate affect the mode of respiration. Respiratory patterns change with diapause stages and in diapause, discontinuous gas exchange is defended even at high temperatures, supporting the finding that the diapause physiology affects the metabolic rate and with it the respiratory patterns. In Chapter IV I followed up the lead from Chapter II and tested a hypothesis on the hormonal regulation of diapause termination in which the transcription factor Foxo, the ecdysteroid receptor as well as insulin interact to regulate ecdysone sensitivity. While many findings in the transcriptome and the protein levels support the hypothesis we need further investigations into this mechanism. Furthermore, the protein levels of most proteins studied do not correlate with the mRNA levels, and while this has been described under direct developing conditions too it would be interesting to study where those different levels stem from. These findings show that the three levels, temperature, metabolic rate and the hormonal pathways are tightly linked to the diapause physiology and are most likely involved in the regulation of diapause termination timing.