A time course analysis through diapause reveals dynamic temporal patterns of microRNAs associated with endocrine regulation in the butterfly Pieris napi.

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.

Bumblebee thermoregulation at increasing temperatures is affected by behavioral state.

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.

Seasonal energetics: are insects constrained by energy during dormancy?

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.

Intrapopulation variance in ecophysiological responses to water limitation in a butterfly metapopulation suggests adaptive resilience to environmental variability.

How organisms that are part of the same trophic network respond to environmental variability over small spatial scales has been studied in a multitude of systems. Prevailing theory suggests a large role for plasticity in key traits among interacting species that allows matching of life cycles or life-history traits across environmental gradients, for instance insects tracking host-plant phenology across variable environments (Posledovich et al. 2018). A key aspect that remains understudied is the extent of intrapopulation variability in plasticity and whether stressful conditions canalize plasticity to an optimal level, or alternatively if variation in plasticity indeed could increase fitness in itself via alternative strategies. In a From the Cover article in this issue of Molecular Ecology, Kahilainen et al. (2022) investigate this issue in a classical insect study system, the metapopulation of the Glanville fritillary butterfly (Melitea cinxia) in the Åland archipelago of Finland. The authors first establish how a key host plant responds to water limitation, then quantify among-family variation in larval growth and development across control and water-limited host plants. Finally, they use RNA sequencing to gain mechanistic insights into some of these among-family differences in larval performance in response to host-plant variation, finding results suggesting the existence of heritable, intrapopulation variability in ecologically relevant plasticity. This final step represents a critically important and often overlooked component of efforts to predict sensitivity of biological systems to changing environmental conditions, since it provides a key metric of adaptive resilience present in the system.

Extensive transcriptomic profiling of pupal diapause in a butterfly reveals a dynamic phenotype.

Diapause is a common adaptation for overwintering in insects that is characterized by arrested development and increased tolerance to stress and cold. While the expression of specific candidate genes during diapause have been investigated, there is no general understanding of the dynamics of the transcriptional landscape as a whole during the extended diapause phenotype. Such a detailed temporal insight is important as diapause is a vital aspect of life cycle timing. Here, we performed a time-course experiment using RNA-Seq on the head and abdomen in the butterfly Pieris napi. In both body parts, comparing diapausing and nondiapausing siblings, differentially expressed genes are detected from the first day of pupal development and onwards, varying dramatically across these formative stages. During diapause there are strong gene expression dynamics present, revealing a preprogrammed transcriptional landscape that is active during the winter. Different biological processes appear to be active in the two body parts. Finally, adults emerging from either the direct or diapause pathways do not show large transcriptomic differences, suggesting the adult phenotype is strongly canalized.

Thermal performance under constant temperatures can accurately predict insect development times across naturally variable microclimates.

External conditions can drive biological rates in ectotherms by directly influencing body temperatures. While estimating the temperature dependence of performance traits such as growth and development rate is feasible under controlled laboratory settings, predictions in nature are difficult. One major challenge lies in translating performance under constant conditions to fluctuating environments. Using the butterfly Pieris napi as model system, we show that development rate, an important fitness trait, can be accurately predicted in the field using models parameterized under constant laboratory temperatures. Additionally, using a factorial design, we show that accurate predictions can be made across microhabitats but critically hinge on adequate consideration of non-linearity in reaction norms, spatial heterogeneity in microclimate and temporal variation in temperature. Our empirical results are also supported by a comparison of published and simulated data. Conclusively, our combined results suggest that, discounting direct effects of temperature, insect development rates are generally unaffected by thermal fluctuations.

Developmental plasticity in metabolism but not in energy reserve accumulation in a seasonally polyphenic butterfly.

The evolution of seasonal polyphenisms (discrete phenotypes in different annual generations) associated with alternative developmental pathways of diapause (overwintering) and direct development is favoured in temperate insects. Seasonal life history polyphenisms are common and include faster growth and development under direct development than in diapause. However, the physiological underpinnings of this difference remain poorly known despite its significance for understanding the evolution of polyphenisms. We measured respiration and metabolic rates through the penultimate and final larval instars in the butterfly Pieris napi and show that directly developing larvae grew and developed faster and had a higher metabolic rate than larvae entering pupal diapause. The metabolic divergence appeared only in the final instar, that is, after induction of the developmental pathway that takes place in the penultimate instar in P. napi. The accumulation of fat reserves during the final larval instar was similar under diapause and direct development, which was unexpected as diapause is predicted to select for exaggerated reserve accumulation. This suggests that overwinter survival in diapause does not require larger energy reserves than direct development, likely because of metabolic suppression in diapause pupae. The results, nevertheless, demonstrate that physiological changes coincide with the divergence of life histories between the alternative developmental pathways, thus elucidating the proximate basis of seasonal life history polyphenisms.

Oxygen limitation is not the cause of death during lethal heat exposure in an insect.

Oxygen- and capacity-limited thermal tolerance (OCLTT) is a controversial hypothesis claiming to explain variation in, and mechanistically determine, animal thermal limits. The lack of support from Insecta is typically argued to be a consequence of their high-performance respiratory systems. However, no studies have reported internal body oxygen levels during thermal ramping so it is unclear if changes in ambient gas are partially or fully offset by a compensatory respiratory system. Here we provide such an assessment by simultaneously recording haemolymph oxygen (pO2) levels-as an approximation of tissue oxygenation-while experimentally manipulating ambient oxygen and subjecting organisms to thermal extremes in a series of thermolimit respirometry experiments using pupae of the butterfly Pieris napi. The main results are that while P. napi undergo large changes in haemolymph pO2 that are positively correlated with experimental oxygen levels, haemolymph pO2 is similar pre- and post-death during thermal assays. OCLTT predicts that reduction in body oxygen level should lead to a reduction in CTmax. Despite finding the former, there was no change in CTmax across a wide range of body oxygen levels. Thus, we argue that oxygen availability is not a functional determinant of the upper thermal limits in pupae of P. napi.

Metabolome dynamics of diapause in the butterfly Pieris napi: distinguishing maintenance, termination and post-diapause phases.

Diapause is a deep resting stage facilitating temporal avoidance of unfavourable environmental conditions, and is used by many insects to adapt their life cycle to seasonal variation. Although considerable work has been invested in trying to understand each of the major diapause stages (induction, maintenance and termination), we know very little about the transitions between stages, especially diapause termination. Understanding diapause termination is crucial for modelling and predicting spring emergence and winter physiology of insects, including many pest insects. In order to gain these insights, we investigated metabolome dynamics across diapause development in pupae of the butterfly Pieris napi, which exhibits adaptive latitudinal variation in the length of endogenous diapause that is uniquely well characterized. By employing a time-series experiment, we show that the whole-body metabolome is highly dynamic throughout diapause and differs between pupae kept at a diapause-terminating (low) temperature and those kept at a diapause-maintaining (high) temperature. We show major physiological transitions through diapause, separate temperature-dependent from temperature-independent processes and identify significant patterns of metabolite accumulation and degradation. Together, the data show that although the general diapause phenotype (suppressed metabolism, increased cold tolerance) is established in a temperature-independent fashion, diapause termination is temperature dependent and requires a cold signal. This revealed several metabolites that are only accumulated under diapause-terminating conditions and degraded in a temperature-unrelated fashion during diapause termination. In conclusion, our findings indicate that some metabolites, in addition to functioning as cryoprotectants, for example, are candidates for having regulatory roles as metabolic clocks or time-keepers during diapause.

Average rainfall and the play of colors:Colonial experience and global climate data.

This paper examines the co-construction of global and local views of the weather and climate at the turn of the twentieth century through a history of data gathering efforts in the German colonies in Africa. While both governmental officials and metropolitan practitioners aimed at producing standardized - and thus globally comparable and economically useful - data in different environments, these efforts often tended to break down in practice. Rather than being able to turn the field into a finely tuned laboratory, both European and African data gatherers were confronted with complex and challenging environmental and institutional realities. Faced with these difficulties, colonial practitioners tended to embrace alternative strategies of recording weather conditions, which placed a higher value on individual sensory perception and qualitative descriptions. Thus, in a seemingly paradoxical dynamic, the attempts to gather quantitative colonial data for global maps and models also facilitated the development of a particular colonial approach to climatology that highlighted local specificities and direct embodied experience.

The scales of experience: Introduction to the special issue Experiencing the global environment.

The Scales of Experience introduces the special issue Experiencing the Global Environment by focusing on three dimensions of the theme that are reflected to various degrees in the constitutive essays. First, the introduction highlights the links between the epistemological and political contexts of the historical constitution and development of the global environment (or global environments) in the earth and environmental sciences from the late nineteenth century to today. Second, it argues for a historical approach to the complex concept of scientific experience, whose mutable and contingent qualities are demonstrated by the contributions to the special volume. Lastly, the introduction presents one of the central issues to be tackled by the essays to follow: the development - and, at times, the failure - of strategies and technologies to bridge the seemingly incommensurate gulf between individual, localized experience and the all-encompassing scale of the global environment.

Idiosyncratic development of sensory structures in brains of diapausing butterfly pupae: implications for information processing.

Diapause is an important escape mechanism from seasonal stress in many insects. A certain minimum amount of time in diapause is generally needed in order for it to terminate. The mechanisms of time-keeping in diapause are poorly understood, but it can be hypothesized that a well-developed neural system is required. However, because neural tissue is metabolically costly to maintain, there might exist conflicting selective pressures on overall brain development during diapause, on the one hand to save energy and on the other hand to provide reliable information processing during diapause. We performed the first ever investigation of neural development during diapause and non-diapause (direct) development in pupae of the butterfly Pieris napi from a population whose diapause duration is known. The brain grew in size similarly in pupae of both pathways up to 3 days after pupation, when development in the diapause brain was arrested. While development in the brain of direct pupae continued steadily after this point, no further development occurred during diapause until temperatures increased far after diapause termination. Interestingly, sensory structures related to vision were remarkably well developed in pupae from both pathways, in contrast with neuropils related to olfaction, which only developed in direct pupae. The results suggest that a well-developed visual system might be important for normal diapause development.

Do respiratory limitations affect metabolism of insect larvae before moulting? An empirical test at the individual level.

Recent data suggest that oxygen limitation may induce moulting in larval insects. This oxygen-dependent induction of moulting (ODIM) hypothesis stems from the fact that the tracheal respiratory system of insects grows primarily at moults, whereas tissue mass increases massively between moults. This may result in a mismatch between oxygen supply and demand at the end of each larval instar because oxygen demand of growing tissues exceeds the relatively fixed supply capacity of the respiratory system. The ODIM hypothesis predicts that, within larval instars, respiration and metabolic rates of an individual larva first increase with increasing body mass but eventually level off once the supply capacity of the tracheal system starts to constrain metabolism. Here, we provide the first individual-level test of this key prediction of the ODIM hypothesis. We use a novel methodology where we repeatedly measure respiration and metabolic rates throughout the penultimate- and final-instar larvae in the butterfly Pieris napi In the penultimate instar, respiration and metabolic rates gradually decelerated along with growth, supporting the ODIM hypothesis. However, respiration and metabolic rates increased linearly during growth in the final instar, contradicting the prediction. Moreover, our data suggest considerable variation among individuals in the association between respiration rate and mass in the final instar. Overall, the results provide partial support for the ODIM hypothesis and suggest that oxygen limitation may emerge gradually within a larval instar. The results also suggest that there may be different moult induction mechanisms in larva-to-larva moults compared with the final metamorphic moult.

Energy and lipid metabolism during direct and diapause development in a pierid butterfly.

Diapause is a fundamental component of the life cycle in the majority of insects living in environments characterized by strong seasonality. The present study addresses poorly understood associations and trade-offs between endogenous diapause duration, thermal sensitivity of development, energetic cost of development and cold tolerance. Diapause intensity, metabolic rate trajectories and lipid profiles of directly developing and diapausing animals were studied using pupae and adults of Pieris napi butterflies from a population in which endogenous diapause has been well studied. Endogenous diapause was terminated after 3 months and termination required chilling. Metabolic and post-diapause development rates increased with diapause duration, while the metabolic cost of post-diapause development decreased, indicating that once diapause is terminated, development proceeds at a low rate even at low temperature. Diapausing pupae had larger lipid stores than the directly developing pupae, and lipids constituted the primary energy source during diapause. However, during diapause, lipid stores did not decrease. Thus, despite lipid catabolism meeting the low energy costs of the diapausing pupae, primary lipid store utilization did not occur until the onset of growth and metamorphosis in spring. In line with this finding, diapausing pupae contained low amounts of mitochondria-derived cardiolipins, which suggests a low capacity for fatty acid ß-oxidation. While ontogenic development had a large effect on lipid and fatty acid profiles, only small changes in these were seen during diapause. The data therefore indicate that the diapause lipidomic phenotype is developed early, when pupae are still at high temperature, and retained until post-diapause development.

Is a change in juvenile hormone sensitivity involved in range expansion in an invasive beetle?

INTRODUCTION: It has been suggested that rapid range expansion could proceed through evolution in the endocrinological machinery controlling life-history switches. Based on this we tested whether the Colorado potato beetle, Leptinotarsa decemlineata, which has rapidly expanded its range across latitudinal regions in Europe, and shows photoperiodic adaptation in overwintering initiation, has different sensitivities to juvenile hormone (JH) manipulation along a latitudinal gradient. RESULTS: A factorial experiment where beetles were reared either under a long or short day photoperiod was performed. Hormone levels were manipulated by topical applications. An allatostatin mimic, H17, was used to decrease and a juvenile hormone III analogue, pyriproxyfen, was used to increase the hormone levels. The effects of photoperiod and hormone manipulations on fecundity and overwintering related burrowing were monitored. Application of H17 decreased fecundity but did not induce overwintering related burrowing. Manipulation with pyriproxyfen increased fecundity and delayed burrowing. While small population-dependent differences in responsiveness to the topical application treatments were observed in fecundity, none were seen in overwintering related burrowing. CONCLUSIONS: The results indicate that the rapid photoperiodic adaptation manifested in several life-history and physiological traits in L. decemlineata in Europe is unlikely a result of population dependent differences in JH III sensitivity. While other endocrine factors cannot be ruled out, more likely mechanisms could be genetic changes in upstream elements, such as the photoperiodic clock or the insulin signaling pathway.

Northward range expansion requires synchronization of both overwintering behaviour and physiology with photoperiod in the invasive Colorado potato beetle (Leptinotarsa decemlineata).

Photoperiodic phenological adaptations are prevalent in many organisms living in seasonal environments. As both photoperiod and growth season length change with latitude, species undergoing latitudinal range expansion often need to synchronize their life cycle with a changing photoperiod and growth season length. Since adaptive synchronization often involves a large number of time-consuming genetic changes, behavioural plasticity might be a faster way to adjust to novel conditions. We compared behavioural and physiological traits in overwintering (diapause) preparation in three latitudinally different European Colorado potato beetle (Leptinotarsa decemlineata) populations reared under two photoperiods. Our aim was to study whether behavioural plasticity could play a role in rapid range expansion into seasonal environments. Our results show that while burrowing into the soil occurred in the southernmost studied population also under a non-diapause-inducing long photoperiod, the storage lipid content of these beetles was very low compared to the northern populations. However, similar behavioural plasticity was not found in the northern populations. Furthermore, the strongest suppression of energy metabolism was seen in pre-diapause beetles from the northernmost population. These results could indicate accelerated diapause preparation and possibly energetic adjustments due to temporal constraints imposed by a shorter, northern, growth season. Our results indicate that behavioural plasticity in burrowing may have facilitated initial range expansion of L. decemlineata in Europe. However, long-term persistence at high latitudes has required synchronization of burrowing behaviour with physiological traits. The results underline that eco-physiological life-history traits of insects, such as diapause, should be included in studies on range expansion.

Seasonality of forest insects: why diapause matters.

Insects have major impacts on forest ecosystems, from herbivory and soil-nutrient cycling to killing trees at a large scale. Forest insects from temperate, tropical, and subtropical regions have evolved strategies to respond to seasonality; for example, by entering diapause, to mitigate adversity and to synchronize lifecycles with favorable periods. Here, we show that distinct functional groups of forest insects; that is, canopy dwellers, trunk-associated species, and soil/litter-inhabiting insects, express a variety of diapause strategies, but do not show systematic differences in diapause strategy depending on functional group. Due to the overall similarities in diapause strategies, we can better estimate the impacts of anthropogenic change on forest insect populations and, consequently, on key ecosystems.

Temperature dependence of gas exchange patterns shift as diapause progresses in the butterfly Pieris napi.

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 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 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.

Seasonal specialization drives divergent population dynamics in two closely related butterflies.

Seasons impose different selection pressures on organisms through contrasting environmental conditions. How such seasonal evolutionary conflict is resolved in organisms whose lives span across seasons remains underexplored. Through field experiments, laboratory work, and citizen science data analyses, we investigate this question using two closely related butterflies (Pieris rapae and P. napi). Superficially, the two butterflies appear highly ecologically similar. Yet, the citizen science data reveal that their fitness is partitioned differently across seasons. Pieris rapae have higher population growth during the summer season but lower overwintering success than do P. napi. We show that these differences correspond to the physiology and behavior of the butterflies. Pieris rapae outperform P. napi at high temperatures in several growth season traits, reflected in microclimate choice by ovipositing wild females. Instead, P. rapae have higher winter mortality than do P. napi. We conclude that the difference in population dynamics between the two butterflies is driven by seasonal specialization, manifested as strategies that maximize gains during growth seasons and minimize harm during adverse seasons, respectively.

Time- and temperature-dependent dynamics of prothoracicotropic hormone and ecdysone sensitivity co-regulate pupal diapause in the green-veined white butterfly Pieris napi.

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.