Pieris Napi Research Articles

Host preference variation cannot explain microhabitat differentiation among sympatric Pieris napi and Pieris rapae butterflies

1. Often, closely related insect species feed on different host plant species, and the tremendous diversity of phytophagous insects is therefore attributed to host plant‐driven speciation. However, for most taxa, host use information comes from field observations of egg‐laying females or feeding caterpillars, which means that the underlying reason for a particular host‐affiliation is not easily determined.2. Therefore, it is often unclear whether an insect feeds on a certain host because it prefers that plant to alternative hosts, or because the host distribution overlaps with the habitat requirements of the insect.3. We ask to what extent a divergent host use in the field mirrors the host plant preferences of two closely related butterflies, Pieris napi and Pieris rapae (Pieridae). In nature, P. napi typically occurs in moister habitats than P. rapae.4. We scanned several microhabitats at a field site in Southern Sweden during multiple years, and collected Pieris eggs from three different plants, Cardamine pratensis (wet meadows), Barbarea vulgaris (drier micro‐habitats) and Alliaria petiolata (intermediate areas).5. As predicted, P. rapae eggs were more common than P. napi eggs on B. vulgaris, whereas all of the 358 individuals collected from C. pratensis were P. napi, indicating a divergence in host use between the Pieris species. However, under controlled laboratory conditions, both species had virtually identical oviposition preferences, laying eggs on all three plants, notably P. rapae also laying eggs on C. pratensis, indicating that habitat use, not plant preference, drives host plant use in nature.

Phenotypic biomarkers of climatic impacts on declining insect populations: A key role for decadal drought, thermal buffering and amplification effects and host plant dynamics.

Widespread population declines have been reported for diverse Mediterranean butterflies over the last three decades, and have been significantly associated with increased global change impacts. The specific landscape and climatic drivers of these declines remain uncertain for most declining species. Here, we analyse whether plastic phenotypic traits of a model butterfly species (Pieris napi) perform as reliable biomarkers of vulnerability to extreme temperature impacts in natural populations, showing contrasting trends in thermally exposed and thermally buffered populations. We also examine whether improved descriptions of thermal exposure of insect populations can be achieved by combining multiple information sources (i.e., integrating measurements of habitat thermal buffering, habitat thermal amplification, host plant transpiration, and experimental assessments of thermal death time (TDT), thermal avoidance behaviour (TAB) and thermally induced trait plasticity). These integrative analyses are conducted in two demographically declining and two non-declining populations of P.napi. The results show that plastic phenotypic traits (butterfly body mass and wing size) are reliable biomarkers of population vulnerability to extreme thermal conditions. Butterfly wing size is strongly reduced only in thermally exposed populations during summer drought periods. Laboratory rearing of these populations documented reduced wing size due to significant negative effects of increased temperatures affecting larval growth. We conclude that these thermal biomarkers are indicative of the population vulnerability to increasing global warming impacts, showing contrasting trends in thermally exposed and buffered populations. Thermal effects in host plant microsites significantly differ between populations, with stressful thermal conditions only effectively ameliorated in mid-elevation populations. In lowland populations, we observe a sixfold reduction in vegetation thermal buffering effects, and larval growth occurs in these populations at significantly higher temperatures. Lowland populations show reduced host plant quality (C/N ratio), reduced leaf transpiration rates and complete above-ground plant senescence during the peak of summer drought. Amplified host plant temperatures are observed in open microsites, reaching thermal thresholds that can affect larval survival. Overall, our results suggest that butterfly population vulnerability to long-term drought periods is associated with multiple co-occurring and interrelated ecological factors, including limited vegetation thermal buffering effects at lowland sites, significant drought impacts on host plant transpiration and amplified leaf surface temperature, as well as reduced leaf quality linked to the seasonal advance of plant phenology. Our results also identify multiannual summer droughts affecting larval growing periods as a key driver of the recently reported butterfly population declines in the Mediterranean biome.

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.

An automated system for CE-MALDI and on-target digestion under a fluorocarbon lid applied on spermatophore proteins from Pieris napi

A method for off-line CE-MALDI-TOF-MS and MS2, and on-target digestion under a fluorocarbon lid was developed and applied for the analysis of proteins in the spermatophore of the butterfly Pieris napi. Fractionation revealed many peptides otherwise not detected or resolved. Automated fractionation was performed with an in-lab developed robotic system, and automated on-target tryptic digestion under a fluorocarbon lid was demonstrated with the same system. Fractionation onto a pre-structured MALDI-concentration plate facilitated aligned deposition of trypsin and MALDI-matrix with the deposited sample, also under the fluorocarbon lid. Some indications of indigenous proteolysis of spermatophore proteins were seen, and searching MS2 spectra suggested three tentative sequence homologies to P. rapae. The study demonstrates the functionality of the lab-made robot. Detailed manufacturing instructions and code are provided. The feasibility of automated on-target digestion under a fluorocarbon lid, and the usefulness of a structured concentration plate in CE-MALDI fractionation was shown. Further, it constitutes a preliminary study of P. napi spermatophore proteins.

Analysis of butterfly reproductive proteins using capillary electrophoresis and mass spectrometry

A method for analysis of proteins from spermatophores transferred from male to female Pieris napi butterflies during mating has been developed. The proteins were solubilized from the dissected spermatophores using different solubilization agents (water, methanol, acetonitrile and hexafluoroisopropanol). Capillary electrophoresis (CE) analysis was performed using an acidic background electrolyte containing a fluorosurfactant to avoid protein-wall adsorption, and to increase separation performance. The samples were also analyzed with matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS), in a lower m/z range (1000–6000) and a higher m/z range (6000–12000). Solubilization with different solvents and the use of alternative matrices gave partly complementary profiles.

Sperm morph and remating frequency in the Indian meal moth, Plodia interpunctella.

All Lepidoptera produce two sperm types: normal, nucleated 'eupyrene' sperm and anucleate 'apyrene' sperm. One hypothesis for the evolution of apyrene sperm suggests that they act to reduce female remating rate. Apyrene sperm require less resources to produce than do eupyrene sperm, and could delay remating by females by acting as a 'cheap filler', packing the spermatheca and thereby reducing receptivity. This would reduce the risk of sperm competition, giving a potential adaptive advantage to the male producing these sperm. This leads to the prediction that the probability of a female remating should correlate with the number of stored apyrene sperm, which has previously been supported by experiments using the green-veined white butterfly, Pieris napi We repeated this experiment using the Indian meal moth, Plodia interpunctella We find that in this species, eupyrene, not apyrene sperm number is the best predictor of female remating probability, indicating that the 'cheap filler' hypothesis for the function of apyrene sperm is not well supported in Pl. interpunctella.

Seasonal and sexual differences in the ultraviolet reflectance and scent scale substances of the wings of Pieris napi japonica (Lepidoptera, Pieridae)

Seasonal and sexual differences in the ultraviolet reflectance and scent scale substances of the wings of Pieris napi japonica (Lepidoptera, Pieridae)

Microevolutionary selection dynamics acting on immune genes of the green-veined white butterfly, Pieris napi.

Insects rely on their innate immune system to successfully mediate complex interactions with their microbiota, as well as the microbes present in the environment. Previous work has shown that components of the canonical immune gene repertoire evolve rapidly and have evolutionary characteristics originating from interactions with fast-evolving microorganisms. Although these interactions are likely to vary among populations, there is a poor understanding of the microevolutionary dynamics of immune genes, especially in non-Dipteran insects. Here, we use the full set of canonical insect immune genes to investigate microevolutionary dynamics acting on these genes between and among populations by comparing three allopatric populations of the green-veined white butterfly, Pieris napi (Linné; Lepidoptera, Pieridae). Immune genes showed increased genetic diversity compared to genes from the rest of the genome and various functional categories exhibited different types of signatures of selection, at different evolutionary scales, presenting a complex pattern of selection dynamics. Signatures of balancing selection were identified in 10 genes, and 17 genes appear to be under positive selection. Genes involved with the cellular arm of the immune response as well as the Toll pathway appear to be enriched among our outlier loci, regardless of functional category. This suggests that the targets of selection might focus upon an entire pathway, rather than functional subsets across pathways. Our microevolutionary results are similar to previously observed macroevolutionary patterns from diverse taxa, suggesting that either the immune system is robust to dramatic differences in life history and microbial communities, or that diverse microbes exert similar selection pressures.

Description of a new subspecies of Pieris erutae Poujade, 1888 from Eastern Bhutan, with taxonomic notes on the Pieris napi group from the Himalayas (Lepidoptera, Pieridae)

Description of a new subspecies of Pieris erutae Poujade, 1888 from Eastern Bhutan, with taxonomic notes on the Pieris napi group from the Himalayas (Lepidoptera, Pieridae)

Sleeping butterflies have spirited goo

Butterfly metamorphosis is an iconic transition – an ugly caterpillar shrouds itself in a chrysalis to later emerge as a beautiful butterfly. However, in reality, it's a bit less elegant than some might imagine. ‘You have a larva in a pupal casing that turns to goo,’ says Philipp Lehmann, at the Stockholm University in Sweden. ‘And that is then used to build the adult inside the pupa.’ This transition happens directly for some species, but for others, the goo lays dormant – sleeping through winter – until it gets a cue to wake up and make an adult form. Lehmann is fascinated by what's happening in this goo throughout the phases of dormancy, called diapause. This goo, ‘an energy-rich meat broth,’ says Lehmann, contains lipids and the metabolome – a collection of small-molecule chemicals, including sugars and amino acids – that Lehmann says are the result of many, many metabolic pathways. Lehmann and other researchers had previously looked for changes in the lipids during diapause, and found that not much happens. But, ‘We had trouble believing this would be the case with the metabolome,’ Lehmann says. So he and an international team set out to see what the chemicals in the goo were doing in the sleepy pupa.Lehmann and his colleagues raised green-veined white butterflies in the lab and separated them into three groups. One group they allowed to develop directly from larva to butterfly, and in the other two they triggered diapause by subjecting the larvae to shortened day lengths. They initially held all three groups at a warm temperature, then moved one diapause group to cold, then colder temperatures, for a period of 5 months to cue them to end diapause. This period of cold is necessary in the population of green-veined whites that Lehmann studies. He says, ‘Just like seed dormancy in plants, there's a temperature that you must dip under before you kickstart the process we call diapause termination.’ Diapause termination is a not a point in time, but a process that requires months at low temperatures. The animals in diapause that were kept warm never began that process, they simply remained in diapause.The team sampled the animals’ metabolome several times throughout their development. They found that although the sugar and amino acid collection changed in all animals, it changed most in the diapause groups, and most dramatically in the diapause termination group. ‘It was so very surprising,’ Lehmann says. ‘The majority of metabolites were changing very, very much during this otherwise static phase.’‘If you would look at these pupae over this whole experiment, they're just in the fridge in a cup, in complete darkness,’ Lehmann explains. ‘Nothing happens. Metabolic rate doesn't change, weight decreases a tiny amount, lipids don't change. But if you look at their metabolites, there's so much going on. And something magic seems to happen, exactly when they terminate diapause.’ The most significant magical event: a spike in the amino acid alanine.Although they don't fully understand alanine's role, Lehmann and his collaborators believe it is key in the termination of diapause. The group is playing with experimentally adjusting the length of the alanine peak in the lab, and looking at differences in alanine peak timing and length in wild populations. Lehmann explains, ‘we have this high dynamism that warrants further explanation.’ Other studies have shown chemical peaks during diapause that are not well understood. Lehmann feels the correlation between alanine and diapause termination is a strong first step, but there's much more to do. More secrets to unlock in the spirited sleeping butterfly goo.

Sex‐linked inheritance of diapause induction in the butterfly Pieris napi

AbstractMany temperate insects survive harsh environmental conditions, such as winter, by entering a state of developmental arrest. This diapause state is predominantly induced by photoperiod. The photoperiod varies with latitude and has led to local adaptation in the photoperiodic induction of diapause in many insects. To understand the rapid evolution of the photoperiodic threshold, it is important to investigate and understand the underlying genetic mechanisms. In the present study, the genetic basis of photoperiodic diapause induction is investigated in the green‐veined white butterfly Pieris napi (Lepidoptera, Pieridae) by assaying diapause induction in a range of conditions for a Swedish and Spanish population. Furthermore, the inheritance of diapause induction is assessed in reciprocal F1 hybrids and backcrosses between the two populations. The southern population shows a clear photoperiodic threshold determining diapause or direct development, whereas the northern populations show a high incidence of diapause, regardless of photoperiod. The hybrid crosses reveal that the inheritance of diapause induction is strongly sex‐linked, and that diapause incidence in the genetic crosses is highly dependent on photoperiod. This emphasizes the importance of assaying a range of conditions in diapause inheritance studies. The results indicate a strongly heritable diapause induction with a major component on the Z‐chromosome, as well as a minor effect of the autosomal background.

Timing of diapause termination in relation to variation in winter climate

AbstractIn temperate insects, winters are typically endured by entering diapause, which comprises a deep resting stage. Correct timing of diapause termination is vital for synchronization of emergence with conspecifics and for mobilizing resources when conditions for growth and reproduction become favourable. Although critical to survival, the intrinsic and extrinsic drivers of diapause termination timing are poorly understood. In the present study, we investigate diapause development under a range of durations (10–24 weeks) spent at different temperatures (−2 to 10 °C) in the pupal diapausing butterfly Pieris napi Linnaeus (Lepidoptera:Pieridae). We determine: (i) the maximum cold temperature for diapause development; (ii) if pupae in diapause count cold days or cold sums; and (iii) whether diapause termination is distinct or gradual. The results indicate large and idiosyncratic effects of high and low nonlethal temperatures on diapause development in P. napi. Although all temperatures tested lead to diapause termination, a thermal optimum between 2 and 4 °C is observed. Lower temperatures lead to decreased eclosion propensity, whereas higher temperatures slow down development and increase emergence desynchronization. These data suggest that, rather than a simple cold‐summing process with a distinct diapause termination point, there are trade‐offs between time and temperature at the low and high end of the thermal range, resulting in a nonlinear thermal landscape showing a ridge of increasing eclosion propensity at moderate temperatures. The present study suggests that the effects of temperature on diapause development should be included in projections on post‐winter phenology models of insects, including pest species.

Female fecundity variation affects reproducibility of experiments on host plant preference and acceptance in a phytophagous insect.

Reproducibility is a scientific cornerstone. Many recent studies, however, describe a reproducibility crisis and call for assessments of reproducibility across scientific domains. Here, we explore the reproducibility of a classic ecological experiment-that of assessing female host plant preference and acceptance in phytophagous insects, a group in which host specialization is a key driver of diversification. We exposed multiple cohorts of Pieris napi butterflies from the same population to traditional host acceptance and preference tests on three Brassicaceae host species. Whereas the host plant rank order was highly reproducible, the propensity to oviposit on low-ranked hosts varied significantly even among cohorts exposed to similar conditions. Much variation could be attributed to among-cohort variation in female fecundity, a trait strongly correlated both to female size and to the size of the nuptial gift a female receives during mating. Small males provide small spermatophores, and in our experiment small females that mated with small males had a disproportionally low propensity to oviposit on low-ranked hosts. Hence, our results provide empirical support to the theoretical prediction that female host utilization is strongly affected by non-genetic, environmental variation, and that such variation can affect the reproducibility of ecological experiments even under seemingly identical conditions.

UV reflectance is associated with environmental conditions in Palaearctic Pieris napi (Lepidoptera: Pieridae).

The subject of our investigation was the visual features of wing color with special focus on the UV reflectance in the green-veined white butterfly (Pieris napi). Previous studies had concluded that UV reflectance on dorsal wing surfaces is found only in the female P. napi. Based on UV sensitive photography, we analyzed a correlation between 12 geographic and environmental factors and UV reflectance patterns on 3 patches on the forewings of 407 P. napi specimens from the Palaearctic region. Results had shown that females significantly differ from males: they exhibit a 25% higher UV reflectance. To investigate whether and how UV reflectance levels on the forewings and hindwings of both sexes are influenced by the environment, we performed a principal component analysis (PCA) with several environmental variables. For several variables (in particular, latitude and longitude, mean annual temperature and precipitation, and temperature annual range and altitude), the generalized linear model (GLM) model revealed a significant correlation in both sexes. This suggests a link between UV reflectance levels and the environment and distribution of P. napi. We found that stronger UV reflectance is associated with generally more hostile environments and concluded that large-scale environmental factors influence the UV reflectance on the forewings of both male and female green-veined white butterflies.

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.

Butterflies and plants: preference/performance studies in relation to plant size and the use of intact plants vs. cuttings

AbstractPlants have evolved a number of defences to ameliorate herbivore attacks including chemicals induced by mechanical wounding. Such changes in plant chemical composition are potential confounding factors in experiments on plant–insect interactions, which often present cuttings of potential host plants to phytophagous insects. In particular, this could affect studies of female egg‐laying preference and larval performance, because the same plant chemicals that deter certain generalist insects can elevate attacks from more specialized insects. Furthermore, plant cuttings are by definition smaller than intact plants, and any female host size preference could thus affect experiments using plant cuttings. We first assessed female preference and larval performance of a specialist herbivore, Pieris napi (L.) (Lepidoptera: Pieridae, Pierini), confronted with either intact plants or leaf‐cuttings of four Brassicaceae host plants, Alliaria petiolata (Bieb.) Cavara & Grande, Barbarea vulgaris (L.) WT Aiton, Berteroa incana (L.) DC., and Brassica napus (L.). Egg and larval survival did not differ between intact plants and leaf‐cuttings, whereas larval growth was slightly, but significantly, faster on leaf‐cuttings. Females, however, significantly preferred to lay eggs on intact plants of all four hosts, although the preference hierarchy for the intact plants was largely mirrored by that for leaf‐cuttings. We then tested the female preference for different size‐classes of intact B. napus plants. Small individuals received more eggs than larger individuals, and follow‐up experiments showed that this difference was largely generated by a strong female preference for cotyledon leaves; there was no significant difference in female preference for large and small individuals when both carried cotyledons, and females landing on cotyledons were more likely to oviposit compared to when landing on a true leaf. Our study concludes that plant cuttings can serve as adequate proxies for live plants for preference/performance studies, but that experimentalists should be aware of the variation imposed both by plant handling and plant phenology for female oviposition preference.

Black and Garlic Mustard Plants Are Highly Suitable for the Development of Two Native Pierid Butterflies.

In multivoltine insects that oviposit and develop on short-lived plants, different herbivore generations across a growing season often exploit different plant species. Here, we compare the development time, pupal mass, and survival of two closely related oligophagous herbivore species on two species of brassicaceous plants that grow in different habitats and which exhibit little overlap in temporal growth phenology. In central Europe, the green-veined white butterfly, Pieris napi L., is bivoltine, whereas the small cabbage white butterfly, Pieris rapae L., has two to three generations a year. Moreover, P. napi is primarily found in moist, open (e.g., meadow), and forest habitats, whereas P. rapae prefers drier, open habitats. Both butterflies were reared on Garlic mustard (Alliaria petiolata), which is shade-tolerant and grows early in spring in forest undergrowth, and Black mustard (Brassica nigra), which prefers open disturbed habitats and is most common in summer. Both host plant species differ in other traits such as secondary chemistry. We hypothesized that, owing to habitat preference, P. napi would develop equally well on both plants but that P. rapae would perform better on B. nigra. The results provide partial support for this hypothesis, as both herbivores performed equally well on A. petiolata and B. nigra. However, there were differences in these parameters that were species-specific: on both plants P. rapae developed faster and had larger pupae than P. napi. Our results show that specialized herbivores can exploit different species of related plants that grow at different times of the season, enabling them to have multiple generations.

It's All in the Mix: Blend-Specific Behavioral Response to a Sexual Pheromone in a Butterfly.

Among insects, sexual pheromones are typically mixtures of two to several components, all of which are generally required to elicit a behavioral response. Here we show for the first time that a complete blend of sexual pheromone components is needed to elicit a response also in a butterfly. Males of the Green-veined White, Pieris napi, emit an aphrodisiac pheromone, citral, from wing glands. This pheromone is requisite for females to accept mating with a courting male. Citral is a mixture of the two geometric isomers geranial (E-isomer) and neral (Z-isomer) in an approximate 1:1 ratio. We found that both these compounds are required to elicit acceptance behavior, which indicates synergistic interaction between processing of the isomers. Using functional Ca2+ imaging we found that geranial and neral evoke significantly different but overlapping glomerular activity patterns in the antennal lobe, which suggests receptors with different affinity for the two isomers. However, these glomeruli were intermingled with glomeruli responding to, for example, plant-related compounds, i.e., no distinct subpopulation of pheromone-responding glomeruli as in moths and other insects. In addition, these glomeruli showed lower specificity than pheromone-activated glomeruli in moths. We could, however, not detect any mixture interactions among four identified glomeruli, indicating that the synergistic effect may be generated at a higher processing level. Furthermore, correlations between glomerular activity patterns evoked by the single isomers and the blend did not change over time.

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 β-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.