Heat stress reduced survival but sped up development in Heliconius erato butterflies

Anthropogenic climate change is thought to present a significant threat to biodiversity, in particular to tropical ectotherms, and the effects of long‐term developmental heat stress on this group have received relatively little research attention. Here, we studied the effects of experimentally raising developmental temperatures on a tropical butterfly. We measured survival, development time, adult body mass and wing size of Heliconius erato demophoon (Linnaeus) (Lepidoptera: Nymphalidae) across three temperature treatments. Egg survival was lower in the hotter treatments, with 84%, 73% and 49% of eggs hatching in the 20–30°C (fluctuating temperature with 12 h at 20°C followed by 12 h at 30°C), 23–33°C and 26–36°C treatments, respectively. Larval survival was three times lower in the 26–36°C treatment (8%) compared with the 20–30°C treatment (26%), and we did not detect differences in pupal survival across treatments due to high mortality in earlier stages. Under a moderately increased temperature at 23–33°C, larvae developed faster and adults had a higher body mass and wing loading, but this was not seen in the hottest treatment (26–36°C). Females were also heavier than males in the 23–33°C treatment, but there was no associated increase in wing size. This may suggest a different developmental response to moderately elevated temperatures between the sexes. In summary, high developmental temperatures are particularly lethal for eggs and less so for larvae and also affect adult morphology. This highlights the importance of understanding the effects of temperature variation across ontogeny in tropical ectotherms.


INTRODUCTION
Environmental temperature is one of the most critical ecological parameters for ectothermic species, as they have limited ability to adjust their body temperature physiologically (Sunday et al., 2011).Thus, both prevailing weather and long-term climate change directly impact insects, and their capacity to cope with varying temperatures is critical for species survival and dispersal.Exposure to a high temperature typically reduces individual fitness and ultimately causes death through protein denaturation, disruption of membrane structure and desiccation (Klockmann, Kleinschmidt, & Fischer, 2017).Furthermore, heat stress may lead to long-term life history trade-offs in those survivors, restricting future reproductive success (Jourdan et al., 2019).
Under current global warming projections, high temperatures and extreme climate events will be more frequently encountered in the future, which may strongly impact biodiversity (IPCC, 2021).
Although half of the known animal species are tropical ectotherms, our knowledge about how well they can withstand high temperatures remains limited (García-Robledo et al., 2016;Sheldon, 2019).There are many studies on the effects of heat stress on the development of animals (Piyaphongkul et al., 2012), but fewer have focused on tropical insects.Furthermore, the majority of these studies have focused on adults, thus, much less is known about the impact of thermal stress throughout development (Klockmann, Kleinschmidt, & Fischer, 2017).Tropical insects are predicted to be especially vulnerable to elevated temperatures (Janzen, 1967).Since environmental temperatures in the tropics are largely stable throughout the year, this could potentially lead to narrower thermal tolerance in tropical compared with temperate species (Polato et al., 2018;Sheldon, 2019).As such, tropical ectotherms are likely to be living near their upper-temperature limits already and may not be able to cope with a large increase in developmental temperatures (Deutsch et al., 2008;Fischer et al., 2014).Thus, it is important to determine the ability of tropical ectotherms to overcome the physiological constraints posed by increasing temperatures (Piyaphongkul et al., 2012).
Thermal stress experienced by juveniles may further affect adult fitness, and there may also be differences between responses of males and females to this earlier heat stress.Under elevated temperatures, there may be a trade-off in phenology and morphology, which may differ between males and females.Males may respond with shorter development time and smaller body sizes to facilitate earlier mating opportunities, whereas females may be selected for maintaining larger body sizes for egg production (Fischer et al., 2014;Fischer & Fiedler, 2000;Kingsolver & Huey, 2008).Interestingly, however, a previous study with the tropical Bicyclus anynana (Butler) (Lepidoptera: Nymphalidae) butterflies found a decrease in female body mass and no difference in larval development time within sexes under heat stress (Fischer et al., 2014).Thus, further research is needed with other tropical butterfly species and across a wider range of long-term developmental heat stress to improve our understanding of their response to global warming.
This study investigates the effects of increased temperatures on survival and development in a Neotropical butterfly Heliconius erato demophoon (Linnaeus).H. erato is a pollen-feeding butterfly ranging from southern Texas to northern Paraguay and often inhabits fringes of tropical rainforests (Turner, 1971).Previous studies have shown plasticity in heat tolerance when the offspring of butterflies from different elevations were reared in a common-garden environment and they showed similar heat tolerances despite strong differences in the wild populations (Montejo-Kovacevich et al., 2020).Here, we test the vulnerability of juvenile stages to sustained heat stress by testing the survival of eggs, larvae and pupae, as well as the growth rate in larvae.We then investigated the effects of elevated temperatures on other traits in the surviving adults, including adult mass and wing development.

Study system
We used an established laboratory stock population at the Madingley rearing facility, University of Cambridge, UK, that was started in 2017 with 25 mated female and 35 male H. erato demophoon butterflies from Panama.The larvae of the stock population were reared at 25 C, 75% relative humidity and an LD 12:12 h photocycle within a single temperature-, light-and humidity-controlled climate room similar to the average conditions experienced in the source population (World Bank Group, 2021).The adults were kept in humidity-and temperaturecontrolled insectaries that experienced more variable diurnal temperature changes.The larvae were fed with tips of Passiflora biflora (Lamarck) (Malpighiales: Passifloraceae) and adults were fed with pollen from Lantana camara and supplemented with protein-incorporated sugar solution.The generation time is around 21 days from eggs to adult eclosion, and the survival rate is around 15% in the lab.

Experimental design
Eggs were collected from dozens of females in the stock population in the summer of 2021.Since the stock populations had been reared and bred together for multiple generations, effects due to genetic differences between parents were considered negligible, and thus, we did not track pedigrees in this experiment.However, each egg was tracked individually throughout development.
The host plants P. biflora were removed from the cages with adults 24-48 h before collecting eggs, to encourage females to lay when the host plants were reintroduced.Approximately 20 H. erato demophoon females were allowed to oviposit on a small P. biflora plant for 3 h at a similar temperature on three consecutive days, from which the eggs were collected hourly, and time was noted.Three eggs were placed per small plastic pot.Pots were randomly placed among three treatment groups within 2 h of laying.
Climate chambers were heated up to the target temperature before the transfer of pots, and the L:D photocycle and temperature cycle were 12:12 h across cabinets.The 20-30 C temperature (i.e., 12 h of 20 C in dark conditions followed by 12 h of 30 C in light conditions, also referred to as the cold treatment) was used as a control for the comparison, with the same mean temperature as the normal rearing temperature of the stock population, that is, 25 C.The 23-33 C temperature (also referred to as the intermediate treatment) used is within the range of temperature experienced by H. erato demophoon in some locations of its natural habitat, and 26-36 C (also referred to as the hot treatment) represents temperatures only experienced occasionally in forest canopies in the Equator (Montejo-Kovacevich et al., 2020) but that could become common in degraded lowland habitats in the tropics (Luber & McGeehin, 2008).
PHC MLR-352H-PE climate chambers were used for the experiment, and based on the Panasonic MLR-352H series performance curve, the rate of acclimation from the minimum to maximum rearing temperature was within 0.5 h (Panasonic, 2012).The annual mean temperature in Panama is around 25 C, with the highest temperature occurring in April at 30 C and the lowest in January at 21.5 C (Figure 1, World Bank Group, 2021).The daily air temperature range in the Panama rain forest where the lab colony founders were caught varies between 8 and 18 C (Lubchenco et al., 1984;Zotz et al., 1997).
Thus, a daily fluctuating temperature of 10 C was selected for this experiment (World Bank Group, 2021).Nevertheless, these rapid experimental fluctuations are unlikely to be found in nature today but allow us to better test the effects of heat stress and environmental extremes on development, which may become commonplace in a warmer planet.
Survival rates and development times for eggs, larvae and pupae were measured.Egg hatchings were checked three times a day at 10 AM, 1 PM and 4 PM for 7 days after laying, and time was noted.
Eggs were considered dead if no larvae had emerged after 7 days.The larvae from each temperature group were transferred into plastic boxes lined with a moist tissue and P. biflora with three to five larvae per box and survival was checked every 2 days.Pupation and emergence dates were recorded to obtain larval and pupal development time.Pupae were weighed between 2 and 4 days after pupation.Each pupa was hung in an individual, labelled pot once weighed.After emergence, butterfly wings were carefully removed from the thorax with forceps to best maintain the whole structure.Adult body mass was measured within 2 days after emergence with wings removed, but several hours after emergence, as butterflies were allowed to expand their wings and dry out.

Wing image analysis
Wing size was obtained from images of the detached wings of the individuals that reached adulthood (N = 78).Detached wings were photographed dorsally and ventrally with a DSLR camera with a 100-mm macro lens in standardised conditions.Any damaged or folded specimens were excluded from the analysis.A custom script for Fiji (Schindelin et al., 2012), which automatically crops, extracts the forewings and performs particle size analysis, was used to obtain wing measurements from the images (Montejo-Kovacevich et al., 2019).We obtained an average wing area between the forewings (where possible, in square millimetres, hereafter 'size') since butterflies predominantly use their forewings for flight (Le Roy et al., 2019).

Statistical analysis
We analysed (1) the survival rates of eggs/larvae/pupae as the per-

Survival and development time
Heat stress decreased survival rates across several life stages (Figure 2).Egg and larval survival were both affected by heat stress (χ 2 [2, N = 928] = 74.95,p < 0.0001, and χ 2 [2, N = 633] = 16.01,p < 0.001, respectively).Bonferroni post hoc pairwise comparisons of survival revealed that egg survival was lower in the two hotter treatments (egg survival was 83.8%, 73.3% and 49.2% in the cold, intermediate and hot treatments, respectively, Figure 2), whereas larval survival was only significantly lower in the 26-36 C treatment compared with the 20-30 C treatment (larval survival was 26.5%, 19.0% and 7.8% in the cold, intermediate and hot treatments, respectively).We did not detect differences in pupal survival across treatments, but sample sizes were lower due to mortality in earlier stages Overall development time (from egg laying to adult emerging) was fastest in the intermediate treatment (19.0 ± 1.4 days) and similar in the cold and hot treatments (22.6 ± 1.9 days and 23.1 ± 0.6 days, respectively, Figure 3a).There was no significant difference in development times between sex in each treatment group (analysis of variance [ANOVA], p = 0.970, Table 1).
Larval development time, however, was not significantly different between each sex and treatment group.Those that died as pupae (classed as unknown in Figure 3b) took around 30 h longer to develop as larvae compared with those that successfully emerged as adults (p = 0.002, Table 1).

Mass and growth rate
Pupal mass was similar between the cold and the intermediate groups (ANOVA, p = 0.41, Table 1) but decreased significantly at 26-36 C ( p = 0.04; Figure 3c).
Adult body mass increased with a mild increase in temperature in the 23-33 C treatment (ANOVA, p < 0.001, Table 1 and Figure 3d).This change was not observed for the 26-36 C treatment (Figure 3d), although this is a preliminary conclusion based on pairwise t-test comparison of mean adult body mass with 20-30 C treatment.This was because the hot treatment was omitted from the main ANOVA models due to low sample sizes.Furthermore, females in the 23-33 C treatment were heavier than males in the same treatment (Tukey HSD, p = 0.003).Thus, the difference in mean body mass between cold and intermediate treatment was mainly driven by heavier females in the intermediate treatment (Figure 3d).It is interesting to note that adult female bodies are heavier than male bodies in the 23-33 C treatment but pupae were not.This could be because the pupal weight includes wings, whereas adult weight only included the body mass in our study.Since wing size varied considerably across individuals, pupal mass does not correlate perfectly with adult mass.
Growth rate, which is the ratio between adult body mass and larval development time (as growth only occurs at the larval stage), was highest in the 23-33 C (ANOVA, p < 0.001, Table 1).Females in the 23-33 C treatment had a higher growth rate than males in the same treatment (Tukey HSD, p = 0.003).Since there was no difference between development times, females with higher temperatures grew both faster and heavier.In summary, a small increase of 3 C in temperature led to a higher pupal mass and adult body mass, but they dropped when the temperature was too high (+6 C).

Wing size and loading
The three emerging adults from the 26-36 C group did not have fully developed wings, and thus, this group was omitted from wing analysis.
Wing size increased with adult body mass (ANOVA, p < 0.001, Table 1), but there was no difference in wing size between temperature treatments or sexes (p = 0.12 and p = 0.77 respectively, Table 1) despite the differences in adult body mass (Figure 3d).4).As a result, both males and females had a higher wing loading, which is the ratio between wing area and adult body mass, in the 23-33 C compared with 20-30 C (ANOVA, p < 0.001), but males, in general, had a lower wing loading than females (ANOVA, p = 0.02, Table 1).These differences in wing loading were likely driven by the higher adult body mass in the warmer treatment since there was no difference in wing size between the treatments alone.

DISCUSSION
Insects have developed a variety of adaptations to endure temperature variation (Overgaard et al., 2008), yet there are temperature thresholds beyond which species cannot live.This study revealed that heat stress lowers fitness by increasing mortality in the early developmental stages, with a 3 C temperature increase leading to higher egg mortality and a 6 C temperature increase leading to both higher egg and larval mortality compared with developing at 20-30 C. A moderately elevated temperature reduced developmental time in both sexes and boosted female adult body mass and wing loading.These effects were reversed at highly elevated temperatures (26-36 C).

Higher temperature lowers survival and affects development
In line with earlier studies, we found strong adverse effects of high temperature on both egg-hatching success and larval survival (Kingsolver et al., 2015;Klockmann, Günter, & Fischer, 2017;Piyaphongkul et al., 2012).These detrimental effects could be explained by, for instance, denaturation of proteins, disruption of membrane structure or desiccation (Chown & Terblanche, 2006;Klose & Robertson, 2004;Potter et al., 2009).The H. erato demophoon larvae studied here seem to be well equipped to bear temperatures Many studies have looked at the maximum temperature organisms can endure (critical thermal maximum) or perform short exposures to high temperatures and measured the time until they are knocked down (Bowler & Terblanche, 2008;Ju et al., 2014;Nandi & Chakraborty, 2015).Our findings suggest that responses to short exposures may not correlate so well to long exposures.In the wild, short exposures may be relevant to complex habitats such as tropical forests where microclimates are buffering against heat stress, acting as refugia (Scheffers et al., 2014).But for other more open or degraded habitats, temperature increases may be more homogenous throughout space and time of the day (De Frenne et al., 2019;Montejo-Kovacevich et al., 2020).Therefore, testing different exposure times and temperatures for different habitat types would be interesting for future studies.
The ability to survive in hot conditions changes throughout development, with eggs having the lowest survival, followed by larvae.
T A B L E 1 Results of linear models for the effects of various explanatory variables on the effect of larval development time, overall development time, pupal mass, adult body mass, growth rate, wing area and wing loading.Note: Larvae and pupae that died before reaching adulthood were classified as 'Sex (unknown)' in the models.Significant p-values are given in bold.
Different hypotheses as to why heat tolerance varies throughout development have been proposed.First, individuals with a larger size may have higher survival under heat stress (Kingsolver & Huey, 2008;Klockmann, Günter, & Fischer, 2017).Klockmann, Kleinschmidt, and Fischer (2017), for example, found that survival was higher for larger caterpillars of B. anynana.Nonetheless, we did not find size dependency in heat tolerance within larval development as the time of larval death did not differ between the treatment groups.Second, the enhanced survival of larvae compared to eggs may be attributed to behavioural responsiveness.Since desiccation is an important factor contributing to the high mortality under heat stress, the consumption of food by the larvae may allow them to acquire water and energy, which are essential for evaporative cooling to prevent desiccation (Klockmann, Günter, & Fischer, 2017).Finally, habitat use by wild butterflies may further explain why eggs are sensitive to high temperatures.Eggs may not be under strong selection pressure to evolve heat tolerance in the wild if they are laid in the understory and near the moist leaves, which can provide microclimate buffering against heating (Madigosky, 2004).The understory of tropical forests inhabited by Heliconius in Ecuador has, on average, 1.75 C cooler daily maximum temperature than the sub-canopy (Montejo-Kovacevich et al., 2020).
The absence of such refugia in our experiment, with only a few leaves and/or stems of the plant in each pot, may have exacerbated the heat stress suffered by the eggs.Overall, our findings are broadly in line with similar experiments on B. anynana butterflies (Kingsolver & Huey, 2008;Klockmann, Günter, & Fischer, 2017) and highlight the importance of preserving habitat complexity for buffering against heat stress in vulnerable and immobile early life stages of tropical ectotherms.
Our results showed that adult body mass was higher at 23-33 C relative to the 20-30 C treatment, but lower at 26-36 C. Since temperature is known to have a major influence on various developmental processes in ectotherms (Lailvaux & Irschick, 2007), higher temperatures may lead to butterflies reaching adulthood faster.Faster development for both sexes at moderately increased temperatures likely decreases mortality due to predation since the larvae will be exposed to predators for a shorter time.In line with previous studies, the overall trend of growth rate is similar to a typical temperatureperformance curve, with higher growth rates in intermediate to low temperatures (Huey & Stevenson, 1979;Lee & Roh, 2010).
Female adult body mass responds more plastically to temperature Our results also found differential responses to temperature between sexes.Under mildly elevated temperatures (23-33 C), females showed an increase in body mass and growth rate, whereas males did not (Figure 3d).Females often have a higher body mass due to a positive correlation between body size and fecundity, whereas males are typically selected for larger wings, which increases their mating opportunities (Blanckenhorn et al., 2007;Deinert et al., 1994;Gotthard et al., 1994;Honěk, 1993;Montejo-Kovacevich et al., 2019).This sexual difference in adult body mass has been seen in other butterfly species, including the copper butterfly (Fischer & Fiedler, 2000) and the B. anynana butterflies (Reim et al., 2019).Although this can be explained by fecundity selection, where female reproductive success is determined by adult body mass (Blanckenhorn et al., 2007;Gotthard, 2008), this effect might be more subtle in Heliconius butterflies as they feed on pollen to boost both lifespan and egg production.
An increase in female body mass may give them a head start in the competition for egg-laying sites with other females, or allow them to save time on feeding for egg laying in early life in environments where predation risk is high.Males clearly would not benefit as much from these responses, and thus, the optimal adult body mass may differ between the sexes.This is further supported by differences in the Monarch butterflies (Soule et al., 2020), potentially leading to lower fitness and reproductive success despite the enhanced growth rate under warmer temperatures.This has been found in the tropical B. anynana butterflies as well, where under increased developmental temperature, male butterflies tend to show better flight performance and higher mobility and stronger intrinsic motivation for movement and exploratory behaviour than females (Reim et al., 2019).Similar results to the temperature-wing loading relationship are also found in Drosophila (Fallen) (Diptera: Drosophilidae) (Fraimout et al., 2018).
While adult body mass is correlated with higher egg production and fitness for females, it may not benefit males if their flight ability is hindered by higher wing loading (Almbro & Kullberg, 2012).Intense sexual competition in pupal-mating species like H. erato, where they copulate with females as they emerge from their pupae, may further select for lower wing loadings in males (Thurman et al., 2018).As locating female pupae and competing with other males for them requires extensive flight ability, male H. erato may be under stronger selection to maintain their flight ability even when the temperature is no longer a limiting factor for body mass.Thus, higher wing loading due to greater body mass under mildly elevated temperatures may be disadvantageous to male butterflies in the wild.
Overall, increased adult body mass benefits females but hinders males, so the two sexes have evolved contrasting developmental responses to moderately elevated temperatures, to which the early stages of the butterflies are regularly exposed in nature.Females potentially exploited the opportunity for enhanced fecundity by increasing their adult body mass, whereas males maintained their mass with lower wing loading for better flight performance.On the contrary, they rarely experience highly elevated temperatures of +6 C in nature, so the morphological and developmental effects detected are likely due to physiological stress rather than potentially adaptive responses.

CONCLUSIONS
This study investigated the effect of temperature on the development of butterflies, which has been rarely studied in tropical insects.Our findings reveal that long-term heat stress has deleterious effects on both survival and developmental traits in a common neotropical butterfly.Eggs were found to be most vulnerable to heat stress, followed by larvae.Furthermore, the growth rate followed a typical thermalperformance curve, which showed an optimal growth temperature of 23-33 C. The observed decline in adult development rate and body mass at 26-36 C, which is known to be correlated with lower fitness (Klockmann, Kleinschmidt, & Fischer, 2017;Piyaphongkul et al., 2012), is of particular concern for two reasons.First, body mass may be a critical restriction on H. erato demophoon heat stress tolerance in general (Klockmann, Günter, & Fischer, 2017;Klockmann, Kleinschmidt, & Fischer, 2017).Second, because smaller females often produce fewer and/or smaller eggs, this may have a transgenerational effect, resulting in offspring with lower fitness (Kingsolver & Huey, 2008;Klockmann, Kleinschmidt, & Fischer, 2017).Thus, it is clear that highly elevated temperatures will be detrimental to the butterflies.
The effect of slightly raised temperatures on population dynamics is, however, difficult to predict.Under moderately increased temperatures, males and females have shown different developmental responses, potentially to enhance either fecundity or manoeuvrability.If females gain fitness through increased adult mass at moderately elevated temperatures, and the early stages of both sexes benefit from shorter developmental time in warmer environments, this will contrast with the increased mortality of both sexes due to heat stress in early life stages.In nature, early life mortality caused by heat stress may be reduced by careful selection of shaded egg-laying sites by the females and also by mobile larvae avoiding exposure to direct sunlight.If this is true, then a small temperature rise would be more likely to increase than decrease population numbers (Klockmann, Kleinschmidt, & Fischer, 2017;Theng et al., 2020).
Thus, this study highlights the importance of taking plastic responses of phenotypic traits into account when predicting popula- centage of hatching/pupation/emergence per treatment group, respectively; (2) development time across treatments; (3) pupal mass, adult body mass and growth rate per treatment group and sexes and (4) wing size and wing loading between treatment groups and sexes.We define wing loading as the ratio between adult body mass (excluding the wings) and wing area.Chi-squared tests and Bonferroni post hoc pairwise comparisons were performed for the survival rates.For all other analyses (i.e., larval development time, pupal mass, adult body mass, growth rate, wing area and wing loading), we used linear regression models ('lm' function in the R Stats package) followed by Tukey post hoc tests for all significant explanatory variables.For the adult wing size and body mass regression analysis, a regression line is plotted for each sex of each treatment.All statistical analyses were run in R V1.3.1093(RDevelopment Core Team, 2011), and graphics were generated with the package 'ggplot2'(Ginestet, 2011).

F
I G U R E 1 Mean monthly temperature variation from daily recordings in Panama between 1991 and 2020 (data from World Bank Group, 2021).Dots represent monthly maximum temperatures, triangles represent monthly mean temperatures and squares represent monthly minimum temperatures.
Females in the 23-33 C treatment had a lower increase in wing size per mass gained in adult body weight compared with males in both treatments (pairwise comparison of the regression gradient of 23-33 C females with 20-30 C males and 23-33 C males: p = 0.045 and p = 0.011, respectively, Figure

F
I G U R E 2 Percentage of individuals surviving during egg, larval and pupal stages in three temperature treatments (blue/circle = 20-30 C, yellow/triangle = 23-33 C and red/square = 26-36 C).Sample sizes for each treatment at the beginning of each stage are shown in corresponding colours.Chi-squared test was performed on the survival rate for each developmental stage followed by post hoc Bonferroni-adjusted pairwise comparisons.The statistically significant comparisons were shown (**p < 0.01, ***p < 0.001).slightly above the normal rearing temperature (25 C) of the stock, as there were no differences in larval and pupal mortality between the 20-30 C and 23-33 C treatments.Furthermore, it is remarkable that some individuals survived cycles of 12 h at 36 C in the 26-36 C treatment, as individuals of the same species have been shown to lose locomotor performance (i.e., get knocked down) after only 16 min when exposed to temperatures between 39 C and 41 C (Montejo-Kovacevich et al., 2020).

F
I G U R E 3 Growth and development across treatments and sexes: (a) overall development time, (b) larval development time, (c) pupal weight and (d) adult body mass.Pink, blue and green bars represent females, males and unsexed larvae that died as pupal, respectively.The bottom and the top of the boxes represent the first and third quartiles, respectively, and the middle line represents the median, the points represent the outliers and the vertical line delimits maximum and minimum non-outlier observations.Sample sizes for each sex in the treatment are indicated above their label.Data with the same lowercase letters are not significantly different (Tukey's honest significance test, p > 0.05).

F
I G U R E 4 Adult wing size and body mass allometry.Regression lines and 95% confidence intervals (shading) are shown for wing analyses between treatment groups (left panel = 20-30 C, right panel = 23-33 C) and sex (pink dots/solid line = female, blue triangles/dashed line = male).adultwing size and body mass allometry for the males and females in the 23-33 C, where the females showed a significant increase in adult body mass for the same wing size compared with males from the same temperature treatment (Figure4).In contrast, males may benefit from maintaining their manoeuvrability and flight duration with lower wing loading under moderately elevated temperatures.Wing loading, which is the mass carried per unit area of wing size, was higher in both sexes under elevated temperatures (23-33 C).Greater wing loading may translate into reduced flight performance and dispersal ability as shown in experiments with tion viability in response to recent global warming.It calls for incorporating the effects of temperature on development and life history traits across ontogeny into models forecasting population dynamics (Jourdan et al., 2019).AUTHOR CONTRIBUTIONS Yuqian Huang: Writingoriginal draft; conceptualization; data curation; writingreview and editing.Josie McPherson: Conceptualization; data curation; writingreview and editing.Chris D. Jiggins: Conceptualization; writingreview and editing.Gabriela Montejo-Kovacevich: Conceptualization; supervision; writingreview and editing.