Flying on empty: reduced mitochondrial function and flight capacity in food-deprived monarch butterflies.

Mitochondrial function is fundamental to organismal performance, health and fitness - especially during energetically challenging events, such as migration. With this investigation, we evaluated mitochondrial sensitivity to ecologically relevant stressors. We focused on an iconic migrant, the North American monarch butterfly (Danaus plexippus), and examined the effects of two stressors: 7 days of food deprivation and infection by the protozoan parasite Ophryocystis elektroscirrha (known to reduce survival and flight performance). We measured whole-animal resting metabolic rate (RMR) and peak flight metabolic rate, and mitochondrial respiration of isolated mitochondria from the flight muscles. Food deprivation reduced mass-independent RMR and peak flight metabolic rate, whereas infection did not. Fed monarchs used mainly lipids in flight (respiratory quotient 0.73), but the respiratory quotient dropped in food-deprived individuals, possibly indicating switching to alternative energy sources, such as ketone bodies. Food deprivation decreased mitochondrial maximum oxygen consumption but not basal respiration, resulting in lower respiratory control ratio (RCR). Furthermore, food deprivation decreased mitochondrial complex III activity, but increased complex IV activity. Infection did not result in any changes in these mitochondrial variables. Mitochondrial maximum respiration rate correlated positively with mass-independent RMR and flight metabolic rate, suggesting a link between mitochondria and whole-animal performance. In conclusion, low food availability negatively affects mitochondrial function and flight performance, with potential implications for migration, fitness and population dynamics. Although previous studies have reported poor flight performance in infected monarchs, we found no differences in physiological performance, suggesting that reduced flight capacity may be due to structural differences or low energy stores.

The genetics of monarch butterfly migration and warning colouration.

The monarch butterfly, Danaus plexippus, is famous for its spectacular annual migration across North America, recent worldwide dispersal, and orange warning colouration. Despite decades of study and broad public interest, we know little about the genetic basis of these hallmark traits. Here we uncover the history of the monarch's evolutionary origin and global dispersal, characterize the genes and pathways associated with migratory behaviour, and identify the discrete genetic basis of warning colouration by sequencing 101 Danaus genomes from around the globe. The results rewrite our understanding of this classic system, showing that D. plexippus was ancestrally migratory and dispersed out of North America to occupy its broad distribution. We find the strongest signatures of selection associated with migration centre on flight muscle function, resulting in greater flight efficiency among migratory monarchs, and that variation in monarch warning colouration is controlled by a single myosin gene not previously implicated in insect pigmentation.

Effects of flight and food stress on energetics, reproduction, and lifespan in the butterfly Melitaea cinxia.

Environmental change can have drastic effects on natural populations. To successfully predict such effects, we need to understand how species that follow different life-history strategies respond to stressful conditions. Here I focus on two stressors, increased flight and dietary restriction, and their effects on bioenergetics and life-history. Using the Glanville fritillary butterfly (Melitaea cinxia), I subjected mated females to three treatments: (1) control conditions, (2) repeated forced flight with unlimited food, and (3) repeated forced flight coupled with food restriction. Interestingly, flight increased fecundity: females in both flight treatments initiated oviposition earlier, laid more egg clutches, and had higher total fecundity than control females. However, food-restriction by 50% reduced clutch size and resulted in an approximately 25% decrease in total fecundity compared to flown females with unlimited food. There were no differences in egg wet mass, water content or hatching success. Flown females with unlimited food appeared to exhibit a trade-off between reproduction and lifespan: they had higher mass-independent resting metabolic rate and shorter lifespan than females in the other treatments. Mass-independent flight metabolic rate, reflecting flight capacity, did not differ among the treatments. There were no differences in the rate of metabolic senescence across the treatments. The current findings suggest a mechanistic link between flight and reproduction, potentially mediated by juvenile hormone signalling. It appears that this wing-monomorphic butterfly does not show an oogenesis-flight trade-off often found in wing-dimorphic insects. Nevertheless, nectar-feeding is needed for achieving maximum reproductive output, suggesting that diminishing nectar resources may negatively impact natural populations.

Resting Metabolic Rate Does Not Predict Peak Metabolic Rate in the Glanville Fritillary Butterfly.

Peak metabolic rate reflects maximal performance and may have direct fitness consequences, whereas resting metabolic rate (RMR) represents the maintenance cost of the whole animal. These traits may be linked, which has significant implications for the evolution of both traits. In vertebrates, a positive correlation between RMR and aerobic capacity has been proposed to explain the origin of endothermy. However, as studies on the relationship between RMR and aerobic capacity have focused on vertebrates, we know much less about these traits in ectothermic insects. I measured RMR in the Glanville fritillary butterfly (Melitaea cinxia) using two configurations: one optimized for measuring flight metabolic rate and the other optimized for RMR. The relationship between RMR and body mass was similar for the two configurations. Body mass explained 82% of the variation in RMR when it was measured using the "flight" configuration at 32°C, and 91% when using the "rest" configuration at 23°C. The Q10 coefficient calculated based on the two RMR measurements was 2.8. Mass-independent RMR was positively correlated between measurements obtained using the two instrument configurations. However, neither measure of RMR was correlated with peak metabolic rate, which indicates that RMR cannot be used as a surrogate measure for aerobic capacity in the Glanville fritillary. Ectothermic insects may be able to combine high metabolic capacity with no apparent increase in maintenance cost. Even though RMR is among the most frequently measured physiological variables, it may have limited predictive power when it comes to questions related to activity or aerobic capacity, or in the case of butterflies, flight performance.

Insect Flight: State of the Field and Future Directions.

The evolution of flight in an early winged insect ancestral lineage is recognized as a key adaptation explaining the unparalleled success and diversification of insects. Subsequent transitions and modifications to flight machinery, including secondary reductions and losses, also play a central role in shaping the impacts of insects on broadscale geographic and ecological processes and patterns in the present and future. Given the importance of insect flight, there has been a centuries-long history of research and debate on the evolutionary origins and biological mechanisms of flight. Here, we revisit this history from an interdisciplinary perspective, discussing recent discoveries regarding the developmental origins, physiology, biomechanics, and neurobiology and sensory control of flight in a diverse set of insect models. We also identify major outstanding questions yet to be addressed and provide recommendations for overcoming current methodological challenges faced when studying insect flight, which will allow the field to continue to move forward in new and exciting directions. By integrating mechanistic work into ecological and evolutionary contexts, we hope that this synthesis promotes and stimulates new interdisciplinary research efforts necessary to close the many existing gaps about the causes and consequences of insect flight evolution.

A long life in the fast lane: positive association between peak metabolic rate and lifespan in a butterfly.

High peak metabolic rates may provide a performance advantage, but it may also entail a physiological cost. A long-held assumption is that high mass-specific energy expenditure is associated with short lifespan. To examine the relationship between energy expenditure and lifespan we asked two questions. First, do individuals have a consistent rate of metabolism throughout their life? Second, is metabolic rate correlated with lifespan? We analysed the repeatability of measurements of resting (RMR) and peak flight metabolic rate (MR(peak)) throughout the life of the Glanville fritillary butterfly (Melitaea cinxia). Measurements of MR(peak) showed significant repeatability. Senescence occurred only shortly before death. RMR showed a U-shaped relationship with age and very low repeatability. Intraspecific association between metabolic rates and lifespan was tested under three conditions: in the laboratory, under field conditions and in a laboratory experiment with repeated flight treatments. There was a significant correlation between MR(peak) and lifespan in all three experiments, but the correlation was positive, not negative. RMR was not correlated with lifespan. Both MR(peak) and lifespan may reflect physiological condition and therefore be positively correlated. Individuals with a large resource pool may be able to invest in mechanisms that slow down ageing. Individuals with high metabolic capacity may also possess adaptations against ageing. Molecular polymorphism in the gene phosphoglucose isomerase (Pgi) was significantly associated with both MR(peak) and lifespan, and may have coevolved with defence mechanisms against senescence. Generalisations such as 'live fast, die young' may be too simple to explain the complex processes affecting ageing and lifespan.

Flight metabolic rate has contrasting effects on dispersal in the two sexes of the Glanville fritillary butterfly.

Evolution of dispersal is affected by context-specific costs and benefits. One example is sex-biased dispersal in mammals and birds. While many such patterns have been described, the underlying mechanisms are poorly understood. Here, we study genetic and phenotypic traits that affect butterfly flight capacity and examine how these traits are related to dispersal in male and female Glanville fritillary butterflies (Melitaea cinxia). We performed two mark-recapture experiments to examine the associations of individuals' peak flight metabolic rate (MR(peak)) and Pgi genotype with their dispersal in the field. In a third experiment, we studied tethered flight in the laboratory. MR(peak) was negatively correlated with dispersal distance in males but the trend was positive in females, and the interaction between MR(peak) and sex was significant for long-distance dispersal. A similar but nonsignificant trend was found in relation to molecular variation at Pgi, which encodes a glycolytic enzyme: the genotype associated with high MR(peak) tended to be less dispersive in males but more dispersive in females. The same pattern was repeated in the tethered flight experiment: the relationship between MR(peak) and flight duration was positive in females but negative in males. These results suggest that females with high flight capacity are superior in among-population dispersal, which facilitates the spatial spreading of their reproductive effort. In contrast, males with high flight capacity may express territorial behaviour, and thereby increase the number of matings, whereas inferior males may be forced to disperse. Thus, flight capacity has opposite associations with dispersal rate in the two sexes.

Tracking butterfly movements with harmonic radar reveals an effect of population age on movement distance.

We used harmonic radar to track freely flying Glanville fritillary butterfly (Melitaea cinxia) females within an area of 30 ha. Butterflies originated from large and continuous populations in China and Estonia, and from newly established or old (> 5 years) small local populations in a highly fragmented landscape in Finland. Caterpillars were raised under common garden conditions and unmated females were tested soon after eclosion. The reconstructed flight paths for 66 individuals comprised a total distance of 51 km with high spatial resolution. Butterflies originating from large continuous populations and from old local populations in Finland exhibited similar movement behaviors, whereas butterflies originating from newly established local populations in the fragmented landscape in Finland moved significantly more than the others. There was no difference in the lengths of individual flight bouts, but the new-population females flew more frequently, resulting in longer daily movement tracks. The flight activity of all individuals was affected by environmental conditions, peaking at 19-23 degrees C (depending on population type), in the early afternoon, and during calm weather. Butterflies from all population types showed a strong tendency to follow habitat edges between the open study area and the neighboring woodlands.

Genotype by temperature interactions in the metabolic rate of the Glanville fritillary butterfly.

Metabolic rate is a highly plastic trait. Here I examine factors that influence the metabolic rate of the Glanville fritillary butterfly (Melitaea cinxia) in pupae and resting and flying adults. Body mass and temperature had consistent positive effects on metabolic rate in pupae and resting adults but not in flying adults. There was also a consistent nonlinear effect of the time of the day, which was strongest in pupae and weakest in flying adults. Flight metabolic rate was strongly affected by an interaction between the phosphoglucose isomerase (Pgi) genotype and temperature. Over a broad range of measurement temperatures, heterozygous individuals at a single nucleotide polymorphism (SNP) in Pgi had higher peak metabolic rate in flight, but at high temperatures homozygous individuals performed better. The two genotypes did not differ in resting metabolic rate, suggesting that the heterozygotes do not pay an additional energetic cost for their higher flight capacity. Mass-independent resting and flight metabolic rates were at best weakly correlated at the individual level, and therefore, unlike in many vertebrates, resting metabolic rate does not serve as a useful surrogate of the metabolic capacity of this butterfly.

Mitochondrial physiology varies with parity and body mass in the laboratory mouse (Mus musculus).

The life-history patterns that animals display are a product of their ability to maximize reproductive performance while concurrently balancing numerous metabolic demands. For example, the energetic costs of reproduction may reduce an animal's ability to support self-maintenance and longevity. In this work, we evaluated the impact of parity on mitochondrial physiology in laboratory mice. The theory of mitohormesis suggests that modest exposure to reactive oxygen species can improve performance, while high levels of exposure are damaging. Following this theory, we hypothesized that females that experienced one bout of reproduction (primiparous) would display improved mitochondrial capacity and reduced oxidative damage relative to non-reproductive (nulliparous) mice, while females that had four reproductive events (multiparous) would have lower mitochondrial performance and greater oxidative damage than both nulliparous and primiparous females. We observed that multiple reproductive events enhanced the mitochondrial respiratory capacity of liver mitochondria in females with high body mass. Four-bout females showed a positive relationship between body mass and mitochondrial capacity. In contrast, non-reproductive females showed a negative relationship between body mass and mitochondrial capacity and primiparous females had a slope that did not differ from zero. Other measured variables, too, were highly dependent on body mass, suggesting that a female's body condition has strong impacts on mitochondrial physiology. We also evaluated the relationship between how much females allocated to reproduction (cumulative mass of all young weaned) and mitochondrial function and oxidative stress in the multiparous females. We found that females that allocated more to reproduction had lower basal respiration (state 4), lower mitochondrial density, and higher protein oxidation in liver mitochondria than females that allocated less. These results suggest that, at least through their first four reproductive events, female laboratory mice may experience bioenergetic benefits from reproduction but only those females that allocated the most to reproduction appear to experience a potential cost of reproduction.

Flight metabolic rate and Pgi genotype influence butterfly dispersal rate in the field.

Dispersal is a key life-history trait, especially in species inhabiting fragmented landscapes. The process of dispersal is affected by a suite of morphological, physiological, and behavioral traits, all of which have a more or less complex genetic basis and are affected by the prevailing environmental conditions. To be able to identify genetic and phenotypic effects on dispersal, movements have to be recorded over relevant spatial and temporal scales. We used harmonic radar to track free-flying Glanville fritillary butterflies (Melitaea cinxia) released in the field and reconstructed their flight tracks for several hours. Flight track lengths for individual butterflies ranged from tens of meters to several kilometers. Butterflies were most mobile at midday and in intermediate temperatures. Flight metabolic rate (MR), measured prior to the tracking, explained variation in mobility at all scales studied. One-third of the variation in the distance moved in one hour could be attributed to variation in flight MR. Heterozygous individuals at a single nucleotide polymorphism in the phosphoglucose isomerase (Pgi) gene moved longer distances in the morning and at lower ambient temperatures than homozygous individuals. A similar genotype x temperature interaction was found to affect the metabolic rate. Our results establish connections from molecular variation in a single gene to flight physiology and movement behavior at the landscape level. These results indicate a fitness advantage to the heterozygous genotype in low temperatures and suggest a mechanism by which varying environmental conditions maintain genetic polymorphism in populations.

Genetics of dispersal.

Dispersal is a process of central importance for the ecological and evolutionary dynamics of populations and communities, because of its diverse consequences for gene flow and demography. It is subject to evolutionary change, which begs the question, what is the genetic basis of this potentially complex trait? To address this question, we (i) review the empirical literature on the genetic basis of dispersal, (ii) explore how theoretical investigations of the evolution of dispersal have represented the genetics of dispersal, and (iii) discuss how the genetic basis of dispersal influences theoretical predictions of the evolution of dispersal and potential consequences. Dispersal has a detectable genetic basis in many organisms, from bacteria to plants and animals. Generally, there is evidence for significant genetic variation for dispersal or dispersal-related phenotypes or evidence for the micro-evolution of dispersal in natural populations. Dispersal is typically the outcome of several interacting traits, and this complexity is reflected in its genetic architecture: while some genes of moderate to large effect can influence certain aspects of dispersal, dispersal traits are typically polygenic. Correlations among dispersal traits as well as between dispersal traits and other traits under selection are common, and the genetic basis of dispersal can be highly environment-dependent. By contrast, models have historically considered a highly simplified genetic architecture of dispersal. It is only recently that models have started to consider multiple loci influencing dispersal, as well as non-additive effects such as dominance and epistasis, showing that the genetic basis of dispersal can influence evolutionary rates and outcomes, especially under non-equilibrium conditions. For example, the number of loci controlling dispersal can influence projected rates of dispersal evolution during range shifts and corresponding demographic impacts. Incorporating more realism in the genetic architecture of dispersal is thus necessary to enable models to move beyond the purely theoretical towards making more useful predictions of evolutionary and ecological dynamics under current and future environmental conditions. To inform these advances, empirical studies need to answer outstanding questions concerning whether specific genes underlie dispersal variation, the genetic architecture of context-dependent dispersal phenotypes and behaviours, and correlations among dispersal and other traits.

Effects of ambient and preceding temperatures and metabolic genes on flight metabolism in the Glanville fritillary butterfly.

Flight is essential for foraging, mate searching and dispersal in many insects, but flight metabolism in ectotherms is strongly constrained by temperature. Thermal conditions vary greatly in natural populations and may hence restrict fitness-related activities. Working on the Glanville fritillary butterfly (Melitaea cinxia), we studied the effects of temperature experienced during the first 2 days of adult life on flight metabolism, genetic associations between flight metabolic rate and variation in candidate metabolic genes, and genotype-temperature interactions. The maximal flight performance was reduced by 17% by 2 days of low ambient temperature (15 °C) prior to the flight trial, mimicking conditions that butterflies commonly encounter in nature. A SNP in phosphoglucose isomerase (Pgi) had a significant association on flight metabolic rate in males and a SNP in triosephosphate isomerase (Tpi) was significantly associated with flight metabolic rate in females. In the Pgi SNP, AC heterozygotes had higher flight metabolic rate than AA homozygotes following low preceding temperature, but the trend was reversed following high preceding temperature, consistent with previous results on genotype-temperature interaction for this SNP. We suggest that these results on 2-day old butterflies reflect thermal effect on the maturation of flight muscles. These results highlight the consequences of variation in thermal conditions on the time scale of days, and they contribute to a better understanding of the complex dynamics of flight metabolism and flight-related activities under conditions that are relevant for natural populations living under variable thermal conditions.

Effects of Increased Flight on the Energetics and Life History of the Butterfly Speyeria mormonia.

Movement uses resources that may otherwise be allocated to somatic maintenance or reproduction. How does increased energy expenditure affect resource allocation? Using the butterfly Speyeria mormonia, we tested whether experimentally increased flight affects fecundity, lifespan or flight capacity. We measured body mass (storage), resting metabolic rate and lifespan (repair and maintenance), flight metabolic rate (flight capacity), egg number and composition (reproduction), and food intake across the adult lifespan. The flight treatment did not affect body mass or lifespan. Food intake increased sufficiently to offset the increased energy expenditure. Total egg number did not change, but flown females had higher early-life fecundity and higher egg dry mass than control females. Egg dry mass decreased with age in both treatments. Egg protein, triglyceride or glycogen content did not change with flight or age, but some components tracked egg dry mass. Flight elevated resting metabolic rate, indicating increased maintenance costs. Flight metabolism decreased with age, with a steeper slope for flown females. This may reflect accelerated metabolic senescence from detrimental effects of flight. These effects of a drawdown of nutrients via flight contrast with studies restricting adult nutrient input. There, fecundity was reduced, but flight capacity and lifespan were unchanged. The current study showed that when food resources were abundant, wing-monomorphic butterflies living in a continuous meadow landscape resisted flight-induced stress, exhibiting no evidence of a flight-fecundity or flight-longevity trade-off. Instead, flight changed the dynamics of energy use and reproduction as butterflies adopted a faster lifestyle in early life. High investment in early reproduction may have positive fitness effects in the wild, as long as food is available. Our results help to predict the effect of stressful conditions on the life history of insects living in a changing world.

Aging, life span, and energetics under adult dietary restriction in lepidoptera.

Stressful conditions can affect resource allocation among different life-history traits. The effect of dietary restriction (DR) on longevity and reproduction has been studied in many species, but we know little about its effects on energetics, especially in flying animals that have high energy demand. We assessed the effects of DR on metabolic rate throughout the entire adult life span in two butterfly species, Colias eurytheme and Speyeria mormonia. We cut the food intake of adult females in half and measured resting metabolic rate (RMR) and flight metabolic rate (FMR) together with body mass repeatedly throughout life. In both species, DR reduced body mass, but mass-corrected FMR was not affected, indicating that flight capacity was retained. DR lowered RMR and reduced fecundity but had no effect on life span. FMR declined with age, but the rate of senescence was not affected by DR. In contrast, aging had a strong negative effect on RMR only in control females, whereas food-restricted females had more stable RMR throughout their lives. The results suggest that flight capacity is conserved during nutritional stress but that investment in flight and survival may negatively affect other important physiological processes when resources are limited.