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.

Over the past decades, increasing environmental temperatures have been identified as one of the causes of major insect population declines and biodiversity loss. However, it is unclear how these rising temperatures affect endoheterothermic insects, like bumblebees, that have evolved thermoregulatory capacities to exploit cold and temperate habitats. To investigate this, we measured head, thoracic, and abdominal temperature of bumblebee (Bombus terrestris) workers across a range of temperatures (24 °C–32 °C) during three distinct behaviors. In resting bumblebees, the head, abdomen, and thorax conformed to the environmental temperature. In pre-flight bumblebees, the head and abdominal temperatures were elevated with respect to the environmental temperature, while the thoracic temperature was maintained, indicating a pre-flight muscle warming stage. In post-flight bumblebees, abdominal temperature increased at the same rate as environmental temperature, but the head and the thoracic temperature did not. By calculating the excess temperature ratio, we show that thermoregulation in bumblebees during flight is partially achieved by the active transfer of heat produced in the thorax to the abdomen, where it can more easily be dissipated. These results provide the first indication that the thermoregulatory abilities of bumblebees are plastic and behavior dependent. We also show that the flight speed and number of workers foraging increase with increasing temperature, suggesting that bees do not avoid flying at these temperatures despite its impact on behavioral performance.

Phenotypic plasticity is produced and maintained by processes regulating the transcriptome. While differential gene expression is among the most important of these processes, relatively little is known about other sources of transcriptional variation. Previous work suggests that alternative splicing plays an extensive and functionally unique role in transcriptional plasticity, though plastically spliced genes may be more constrained than the remainder of expressed genes. In this study, we explore the relationship between expression and splicing plasticity, along with the genetic diversity in those genes, in an ecologically consequential polyphenism: facultative diapause. Using 96 samples spread over two tissues and 10 timepoints, we compare the extent of differential splicing and expression between diapausing and direct developing pupae of the butterfly Pieris napi. Splicing differs strongly between diapausing and direct developing trajectories but alters a smaller and functionally unique set of genes compared to differential expression. We further test the hypothesis that among these expressed loci, plastically spliced genes are likely to experience the strongest purifying selection to maintain seasonally plastic phenotypes. Genes with unique transcriptional changes through diapause consistently had the lowest nucleotide diversity, and this effect was consistently stronger among genes that were differentially spliced compared to those with just differential expression through diapause. Further, the strength of negative selection was higher in the population expressing diapause every generation. Our results suggest that maintenance of the molecular mechanisms involved in diapause progression, including post-transcriptional modifications, are highly conserved and likely to experience genetic constraints, especially in northern populations of P. napi.

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

1. Numerous species shift or expand their ranges poleward in response to climate change. Even when expanding species follow their climatic niches, expanding range margin populations are likely to face unfamiliar environmental conditions and thus natural selection for local adaptation.
2. The wall brown butterfly (Lasiommata megera) has expanded northward in Sweden in the years 2000–2020, most likely as a result of climate change, and has previously been shown to have evolved local adaptations to northern daylength conditions. This evolution has occurred despite hypothesised genetic constraints to adaptation at range margins.
3. We studied local adaptation to winter conditions in four of the previously-studied L. megera populations, using a common garden laboratory experiment with a warm and short, an intermediate, and a cold and long winter treatment. We compared the winter and post-winter survival of caterpillars from two southern core range and two northern range margin populations in Sweden.
4. During the experiment, we measured metabolic rates of a subset of diapausing caterpillars to test whether populations differ in metabolic suppression during diapause. Further, we measured supercooling points, which reflect lower lethal temperature in L. megera, of the same subset of caterpillars. We also compared supercooling points between L. megera and three closely related species with more northern distributions.
5. Few individuals survived the coldest treatment all the way to successful adult emergence, so L. megera seems susceptible to cold winters. Individuals of northern descent did not survive cold winters any better than individuals from southern populations. Similarly, there were no signs of local adaptation in metabolic rates or supercooling points. The comparison among species did not reveal any clear relationship between geographical distribution and supercooling point.
6. Although northern winters probably exert strong selection on L. megera, we provide comprehensive evidence for the lack of local adaptation to winter conditions. This contrasts with the previous finding of quickly evolved local adaptation in diapause timing, highlighting the need to consider how traits associated with different seasons differ in how they may evolve and facilitate climate change-induced range expansions.

In seasonal environments, many animals, including insects, enter dormancy, where they are limited to a fixed energy budget. The inability to replenish energetic stores during these periods suggests insects should be constrained by pre-dormancy energy stores. Over the last century, the community of researchers working on survival during dormancy has operated under the strong assumption that energy limitation is a key fitness trait driving the evolution of seasonal strategies. That is, energy use has to be minimized during dormancy because insects otherwise run out of energy and die during dormancy, or are left with too little energy to complete development, reproductive maturation or other costly post-dormancy processes such as dispersal or nest building. But if energy is so strongly constrained during dormancy, how can some insects - even within the same species and population - be dormant in very warm environments or show prolonged dormancy for many successive years? In this Commentary, we discuss major assumptions regarding dormancy energetics and outline cases where insects appear to align with our assumptions and where they do not. We then highlight several research directions that could help link organismal energy use with landscape-level changes. Overall, the optimal energetic strategy during dormancy might not be to simply minimize metabolic rate, but instead to maintain a level that matches the demands of the specific life-history strategy. Given the influence of temperature on energy use rates of insects in winter, understanding dormancy energetic strategies is critical in order to determine the potential impacts of climate change on insects in seasonal environments.

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.

During winter, many organisms conserve resources by entering dormancy, suppressing metabolism and biosynthesis. The transition out of winter dormancy to summer activity requires a quick reversal of this suppression, in order to exploit now-favorable environmental conditions. To date, mechanisms by which winter climate variation affects this transition remains unelucidated. Here we experimentally manipulated snow cover for naturally overwintering montane leaf beetles (Chrysomela aeneicollis), and profiled changes in gene expression during the transition out of dormancy in spring. Upon emergence, beetles up-regulate transcripts associated with digestion and nutrient acquisition and down regulate those associated with lipid metabolism, suggesting a shift away from utilizing stored lipid and towards digestion of carbohydrate-rich host plant tissue. Development of digestive capacity is followed by up-regulation of transcripts associated with reproduction; a transition that occurs earlier in females than males. Snow manipulation strongly affected the ground thermal regime and correspondingly gene expression profiles, with beetles showing a delayed up-regulation of reproduction in the dry compared to snowy plots. This suggests that winter conditions can alter the timing and prioritization of processes during emergence from dormancy, potentially magnifying the effects of declining snow cover in the Sierra's and other snowy mountains.

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.