Organisms inhabiting highly seasonal environments must cope with a wide range of environmentally induced challenges. Many seasonal challenges require extensive physiological modification to survive. In winter, to survive extreme cold and limited resources, insects commonly enter diapause, which is an endogenously derived dormant state associated with minimized cellular processes and low energetic expenditure. Due to the high degree of complexity involved in diapause, substantial cellular regulation is required, of which our understanding primarily derives from the transcriptome via messenger RNA expression dynamics. Here we aim to advance our understanding of diapause by investigating microRNA (miRNA) expression in diapausing and direct developing pupae of the butterfly Pieris napi. We identified coordinated patterns of miRNA expression throughout diapause in both head and abdomen tissues of pupae, and via miRNA target identification, found several expression patterns to be enriched for relevant diapause-related physiological processes. We also identified two candidate miRNAs, miR-14-5p and miR-2a-3p, that are likely involved in diapause progression through their activity in the ecdysone pathway, a critical regulator of diapause termination. miR-14-5p targets phantom, a gene in the ecdysone synthesis pathway, and is upregulated early in diapause. miR-2a-3p has been found to be expressed in response to ecdysone, and is upregulated during diapause termination. Together, the expression patterns of these two miRNAs match our current understanding of the timing of hormonal regulation of diapause in P. napi and provide interesting candidates to further explore the mechanistic role of microRNAs in diapause regulation.

Winter diapause in insects is commonly terminated through cold exposure, which, like vernalization in plants, prevents development before spring arrives. Currently, quantitative understanding of the temperature dependence of diapause termination is limited, likely because diapause phenotypes are generally cryptic to human eyes. We introduce a methodology to tackle this challenge. By consecutively moving butterfly pupae of the species Pieris napi from several different cold conditions to 20 °C, we show that diapause termination proceeds as a temperature-dependent rate process, with maximal rates at relatively cold temperatures and low rates at warm and extremely cold temperatures. Further, we show that the resulting thermal reaction norm can predict P. napi diapause termination timing under variable temperatures. Last, we show that once diapause is terminated in P. napi, subsequent development follows a typical thermal performance curve, with a maximal development rate at around 31 °C and a minimum at around 2 °C. The sequence of these thermally distinct processes (diapause termination and postdiapause development) facilitates synchronous spring eclosion in nature; cold microclimates where diapause progresses quickly do not promote fast postdiapause development, allowing individuals in warmer winter microclimates to catch up, and vice versa. The unveiling of diapause termination as one temperature-dependent rate process among others promotes a parsimonious, quantitative, and predictive model, wherein winter diapause functions both as an adaptation against premature development during fall and winter and for synchrony in spring.

In their simulation study, Garcia-Costoya et al. (2023) conclude that evolutionary constraints might aid populations facing climate change. However, we are concerned that this conclusion is largely a consequence of the simulated temperature variation being too small, and, most importantly, that uneven limitations to standing variation disadvantage unconstrained populations. In their simulation study, Garcia-Costoya et al. (2023) conclude that evolutionary constraints might aid populations facing climate change. However, we are concerned that this conclusion is largely a consequence of the simulated temperature variation being too small, and, most importantly, that uneven limitations to standing variation disadvantage unconstrained populations.image

The tomato leafminer, Tuta absoluta, is an invasive crop pest that has evolved resistance to many of the insecticides used for its control. To facilitate the investigation of the underpinning mechanisms of resistance in this species we generated a contiguous genome assembly using long-read sequencing data. We leveraged this genomic resource to investigate the genetic basis of resistance to the diamide insecticide chlorantraniliprole in Spanish strains of T. absoluta that exhibit high levels of resistance to this insecticide. Transcriptomic analyses revealed that, in these strains, resistance is not associated with previously reported target-site mutations in the diamide target-site, the ryanodine receptor, but rather is associated with the marked overexpression (20-to >100-fold) of a gene encoding a UDP-glycosyltransferase (UGT). Functional expression of this UGT, UGT34A23, via ectopic expression in Drosophila melanogaster demonstrated that it confers strong and significant resistance in vivo. The genomic resources generated in this study provide a powerful resource for further research on T. absoluta. Our findings on the mechanisms underpinning resistance to chlorantraniliprole will inform the development of sustainable management strategies for this important pest.

Insects have the capacity to significantly modify their metabolic rate according to environmental conditions and physiological requirement. Consequently, the 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 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. The primary goal is to explore respiratory pattern as a non-invasive method to assess whether pupae are in diapause or have transitioned to post-diapause. Respiratory pattern can also provide insight into endogenous processes taking place during diapause, and the prolonged duration of diapause allows for the detailed study of the thermal dependence of the DGE pattern. 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 rho CO2 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 temperature conditions permissive for development. Taken together, the results show that respiratory patterns are highly regulated during diapause in P. napi and change predictably as diapause progresses.

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. 

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.