Ectothermic animals like insects have long been studied for their dependence on environmental temperatures on life-history trait performance. However, most research in temperate regions has focused on summer growth seasons, when insects develop and reproduce. Yet, periods between growth seasons, such as winters, constitute the major portion of insects’ lives in the temperate zone. During these unfavorable conditions, insects generally enter a dormant physiological state called diapause. Here, we quantify the temperature dependence of diapause termination and couple it with spring development. Using a novel methodology, we estimate diapause termination rates at four constant temperatures and show that diapause termination in the butterfly Pieris napi follows a unimodal thermal reaction norm, with optimal rates at winter temperatures. We model this reaction norm as a mirrored version of a typical thermal performance curve (TPC) and use it to predict diapause termination successfully in multiple fluctuating laboratory temperatures, though predictions in field settings were unsuccessful. Moreover, we convincingly show that P. napi post-diapause development resumes directly after diapause termination in a sequential manner and that the temperature-dependence of this development follows a typical development rate TPC. As diapause termination typically takes place in winter, development is halted until spring since winter temperatures usually reside around or below the minimum temperature for development. We propose that this sequence of two thermally separated TPCs is crucial for synchronizing the spring emergence of adult butterflies. In conclusion, we present a new and unified temperature-based framework linking winter and spring biology in diapausing insects, with great applications for future predictive phenology modeling.

Diapause, a general shutdown of developmental pathways, is a vital adaptation allowing insects to adjust their life cycle to adverse environmental conditions such as winter. Diapause in the pupal stage is regulated by the major developmental hormones prothoracicotropic hormone (PTTH) and ecdysone. Termination of pupal diapause in the butterfly Pieris napi depends on low temperatures; therefore, we study the temperature-dependence of PTTH secretion and ecdysone sensitivity dynamics throughout diapause, with a focus on diapause termination. While PTTH is present throughout diapause in the cell bodies of two pairs of neurosecretory cells in the brain, it is absent in the axons, and the PTTH concentration in the haemolymph is significantly lower during diapause than during post diapause development, indicating that the PTTH signaling is reduced during diapause. The sensitivity of pupae to ecdysone injections is dependent on diapause stage. While pupae are sensitive to ecdysone during early diapause initiation, they gradually lose this sensitivity and become insensitive to non-lethal concentrations of ecdysone about 30 days into diapause. At low temperatures, reflecting natural overwintering conditions, diapause termination propensity after ecdysone injection is precocious compared to controls. In stark contrast, at high temperatures reflecting late summer and early autumn conditions, sensitivity to ecdysone does not return. Thus, here we show that PTTH secretion is reduced during diapause, and additionally, that the low ecdysone sensitivity of early diapause maintenance is lost during termination in a temperature dependent manner. The link between ecdysone sensitivity and low-temperature dependence reveals a putative mechanism of how diapause termination operates in insects that is in line with adaptive expectations for diapause.

Respiration in insects takes place in the tracheal system. Tracheae are tubes of decreasing diameter that run from the exocuticle to target tissues. Airflow in the tracheal system is regulated by spiracles that in some insect species can be opened and closed independently. In these species, respiratory patterns can range from continuous gas exchange (CGE) to discontinuous gas exchange (DGE). In the latter, spiracles are kept closed during much of the time, and gas exchange occurs only during short periods when spiracles are opened. While ultimate causes and benefits of DGE remain debated, it is often seen during insect diapause, a deep resting stage that insects induce in order to survive unfavourable environmental conditions, such as winter. The present study explores the shifts between CGE and DGE during diapause by performing long continuous respirometry measurements at multiple temperatures during key diapause stages in the green-veined white butterfly Pieris napi. Pupae change from CGE to DGE a few days after pupation, and this shift coincides with metabolic rate suppression during diapause initiation. Once in diapause, pupae maintain DGE even at elevated temperatures that significantly increase CO2 production. Instead of shifting respiratory pattern to CGE, pupae increase the frequency of DGE cycles. Since total CO2 released during a single open phase remains unchanged, our results suggest that P. napi pupae defend a maximum internal ρCO2 set point, even in their heavily suppressed diapause state. During post-diapause development, CO2 production increases as a function of development and changes to CGE during high temperature conditions. Taken together, the results show that respiratory patterns are highly regulated during diapause in P. napi and change predictably as diapause progresses. 

Insect diapause is an alternative developmental pathway that is induced in advance of adverse environmental conditions and constitutes an absence of development. The hormonal regulation of diapause has been studied extensively, however concise hypotheses on how the timing of diapause termination is regulated is lacking. Several studies have implicated the Prothoracicotropic hormone (PTTH) ecdysone axis as being central to diapause regulation, since application of either hormone can terminate diapause and initiate development. This is of further interest as diapause in many insect species is terminated by accumulating time in cold conditions, indicating a link between temperature and the major developmental hormones. For this study, we integrate the transcriptional profile of genes in the major developmental hormone pathways with protein levels in the butterfly Pieris napi to test the hypothesis that the insulin pathway is co-regulating the timing of diapause termination with FoxO and the ecdysone receptor Ultraspiracle. Furthermore, we want to put P. napi diapause regulation in context with the hormonal regulation of diapause in other insects that diapause as pupa.