Effects of temperature experienced across life stages on morphology and flight behavior of painted lady butterflies (Vanessa cardui)

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Mesler, Karen E. Mabry This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4731760/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Movement Ecology → Version 1 posted 9 You are reading this latest preprint version Abstract Background: With ongoing anthropogenic climate change, there is increasing interest in how organisms are affected by higher temperatures, including how animals respond behaviorally to increasing temperatures. Movement behavior is especially relevant here, as the ability of a species to shift its range is implicitly dependent upon movement capacity and motivation. Temperature may influence movement behavior of ectotherms both directly, through an increase in body temperature, and indirectly, through temperature-dependent effects on physiological and morphological traits that can influence movement. Methods: Here, we investigate the influence of ambient temperature during two life stages, larval and adult, on body size and movement behavior of the painted lady butterfly ( Vanessa cardui ). We reared painted ladies to emergence at either a “low” (24 °C) or “high” (28 °C) temperature. At eclosion, we assessed flight behavior in an arena test, with half of the adults emerging from each rearing treatment tested at either the “low” or “high” temperature. We had a total of four treatment groups: the control (reared and tested at 24 °C), a consistently high temperature (reared and tested at 28 °C), and two treatments in which butterflies experienced flight tests at a temperature either higher or lower than the one at which they were reared. We measured adult body size, including wingspan, and determined flight speed, distance, and duration from video recordings. Results: Adult butterflies that experienced the higher temperature during development were larger. We documented an interaction effect of rearing x testing temperature on flight behavior: unexpectedly, the fastest butterflies were those who experienced a change in temperature, whether an increase or decrease, between rearing and testing. Conclusions: Individuals that experienced matching thermal environments flew more slowly, but for more time and covering more distance. Overall, the influence of body size per se on flight was minimal. We conclude that the potential role of “matching” thermal environments across life stages has been underinvestigated with regard to how organisms may respond to warming conditions. climate change temperature flight movement behavior butterfly Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background Anthropogenic climate change continues to influence life on Earth [ 1 , 2 ], with diverse responses to increasing temperatures exhibited by a range of species. Researchers often place these responses into one of three categories: species can ‘adapt’, through either adaptive evolution or acclimation to new conditions, move in either space (range shifts, typically to areas of higher latitude and/or elevation) or time (phenological shifts), or die (extinction). However, while predictions about future range shifts in relation to increasing temperatures are common in the literature, the movement behavior that would underlie such shifts by animal species is not often considered [ 3 – 6 ]. Many factors can influence an organism’s ability to move from one location to another and the search for more resources can be impacted by external conditions [ 7 ]. Ambient temperature can influence movement behavior in multiple ways, including both direct and indirect effects. For ectothermic animals in particular, the rate of vital processes is affected by the thermal conditions an individual experiences [ 8 ]. In many cases, the thermal performance curve is “hump-shaped”: performance (or the rate of a process) increases with increasing temperature up to the thermal optimum, then declines [ 9 ]. We might expect the locomotion speed of an ectothermic animal to follow such a performance curve, in which speed increases with temperature to the thermal optimum, then declines dramatically [ 6 ]. However, temperature can also have indirect effects on movement behavior. For example, thermal influences on developmental processes may produce variation in movement-related morphology [ 3 ]. Body size is a well-known predictor of movement behavior in a range of animal species [ 10 ]. Changes in body size are also a major response to warmer temperatures [ 11 , 12 ], with the “temperature-size rule” stating that organisms should be smaller at higher temperatures, leading some to predict that body sizes will decline with increasing temperatures due to anthropogenic climate change [ 13 ]. Thus, body size becomes a potential explicit link between the effect of climate warming on organisms and the ability of organisms to respond to that warming via movement and range shifts [ 3 ]: animals that develop under warmer conditions are expected to be smaller as adults, and thus potentially less able to track changing environmental conditions. Despite the importance of movement behavior as an influence on range shifts in response to climate change, relatively few researchers have attempted to link the developmental conditions that produce variation in movement-related phenotypes to movement behavior exhibited later in life. This is potentially related to a mismatch in the types of studies and systems used to investigate these questions: there has been a recent explosion in techniques for tracking the movements of large animals in the field [ 14 – 16 ], but it is almost impossible to manipulate rearing conditions for these species in ways that would be expected to result in morphological changes that are relevant to movement. Meanwhile, it is relatively straightforward to generate such morphological diversity in lab-based invertebrate systems, but devices for tracking individual movements of small ectotherms, such as insects, are more limited and our understanding of thermal effects on movement of insects remains incomplete [ 6 , 17 , 18 ]. However, it is often feasible to track the movements of individuals using automated systems [ 6 ], and this is the approach that we followed in this study. To understand the effects of temperature on both body size and movement behavior, we used the painted lady butterfly ( Vanessa cardui ) as a study organism. In general, butterflies serve as pollinators, food sources and indicators of ecosystem health [ 19 ], and butterfly range shifts have been documented in relation to climate change [ 20 ]. V. cardui is particularly suitable for the study of movement behavior due to its almost cosmopolitan distribution and high mobility [ 21 ]; transatlantic flights by V. cardui have recently been reported [ 22 ]. We manipulated rearing temperature for larval V. cardui and flight testing temperature for adults in a two-way factorial design. First, we wanted to determine how rearing temperature influenced adult morphology. Specifically, we were interested in effects of temperature on body size, due to its potential influence on movement behavior [ 3 ]. Following the “temperature size rule” [ 11 ], we predicted that at emergence, butterflies reared at a higher temperature would be smaller than those reared at a lower temperature. Second, we wanted to determine the influence of ambient temperature on flight behavior. Completion of the second objective required controlling for the potential effects of variation in the rearing environment on flight-related morphology in adults, because we predicted that larger adult butterflies (presumably those reared at a lower temperature) would fly farther and faster than would smaller individuals. Combining our predictions about the effects of rearing temperature on body size (“hotter is smaller”) and testing temperature on flight (“hotter is faster”), we expected that individuals who experienced lower rearing temperatures and higher flight testing temperatures would fly the fastest and in contrast, animals that were reared at a higher temperature and tested at a lower temperature would be the slowest. Methods Study species and culturing We obtained larval V. cardui from Carolina Biological Supply (Burlington, NC, USA). Larvae were within one developmental instar from each other upon receipt. With a paintbrush, larvae were gently transferred from travel containers to prepared rearing containers, each of which contained approximately 30 g of food mix in an even layer on the bottom of the cup (painted lady culture medium, Carolina Biological Supply, Burlington, NC, USA). Three randomly-selected larvae were placed into each container without consideration of size or stage of development. Each container was then covered with a mesh screen to allow for hanging chrysalids. A second plastic lid with air holes was placed on the cup to prevent larvae from escaping. Food was replenished as it was consumed. Manipulation of developmental temperatures Experimental temperature regimes were based on established temperature tolerances of V. cardui in laboratory studies. Symanski and Redak [ 23 ] reviewed rearing temperatures in laboratory studies of V. cardui ; they report experiments across a range from 12–33°C, with an increase in mortality at temperatures above 30°C. Symanski and Redak [ 23 ] describe 25°C as the “optimal” rearing temperature for this species. We chose two experimental temperatures, with the objective of inducing deleterious effects without increasing mortality of animals reared at the higher temperature treatment: 24°C (“low”) and 28°C (“high”). Temperatures were maintained at 24 ± 0.9°C and 28 ± 0.7°C (mean ± 1 SE) throughout the larval, chrysalis, and adult stages using electronically controlled biological chambers (BioChambers model #6194, Winnipeg, Manitoba, Canada) with lights on at 0800 and lights off at 2000. Each larval container was randomly assigned to one of the rearing temperatures. Larvae were monitored daily from 21 October 2022 to 8 December 2022, at which point all larvae had either metamorphosed or died. We determined the number of live and dead larvae, chrysalides, and newly emerged adults in each container each day. There was evidence of some cannibalism: half bodied larvae and larvae parts including decapitated heads were observed prior to the chrysalid form, when larval heads are pinched off. Once an individual reached chrysalis form, it was moved from the rearing container and placed into a 42 cm x 42 cm x 76 cm enclosure constructed of 1.27 cm diameter PVC pipe and mesh netting, with one enclosure per temperature treatment. These enclosures were kept in the environmental chambers with the remaining rearing containers. A zippered door and Velcro corner allowed for the handling of chrysalides and adults. Chrysalides were hung within the mesh containers along the walls to allow for proper development of wings after eclosion. Once an adult eclosed it was assigned a color which was painted on the dorsal side of the abdomen with non-toxic hobby paint (Apple Barrel acrylic paint, PLAID, Atlanta, GA, USA) and allowed for the simultaneous tracking of multiple individuals in filmed flight trials. Food for adults was a 1:4 mixture of sucrose and water which was placed in the adult enclosures in a tray with a sponge on top of the liquid to help prevent drowning. Measuring movement behavior under varying temperatures The movements of adult butterflies were assessed at both low (24°C) and high (28°C) temperatures. Because we expected movement of these ectothermic animals to be affected by both temperature during the flight tests [ 24 ] and the temperature at which they were reared (via an effect of rearing temperature on body size), we used a two-way factorial design in the assessment of movement behavior. Animals that were reared under the low and high temperature regimes were split evenly between testing temperatures for a total of four treatments: low-low, high-high, low-high, high-low, where “low” = 24°C and “high” = 28°C. Flight tests were conducted in an enclosed space that was warmed to the required testing temperature using a space heater (Dreo Atom One Space Heater, Dreo, New York, NY, USA). The flight arena was a 92 cm x 43 cm x 46 cm aquarium (Aqueon standard open-glass aquarium tank, 151.4 liter, Aqueon Products, Franklin, WI, USA) with a fitted lid to prevent escape of individuals during trials. The glass walls of the aquarium allowed video recording and prevented individuals from clinging to the side rather than flying. For each flight trial, five randomly selected marked adults were placed in the arena. Each trial lasted for one hour and was recorded using a small video camera (Akaso V50 X action camera, Akaso, Frederick, MD, USA). The lights were on for the entirety of the trials. After the flight trial, the butterflies were placed in another mesh enclosure of the same size (42 cm x 42 cm x 76 cm) instead of the general population enclosure so they wouldn’t be selected again. Morphological measurements After flight trials, adults were placed into envelopes with wings folded to prevent damage and secured in a -20°C freezer. Each envelope was marked with an individual’s color, flight trial number, and unique ID number. Individuals were later pinned, spread, and dried. After drying for two days, we measured total wingspan, body length, and forewing length (all in mm) using electronic calipers (15.5 cm electronic digital caliper w/ LCD readout, WEN, West Dundee, IL, USA). We did not determine sex of tested animals; sex size dimorphism is reported to be minimal in this species [ 25 ]. Scoring of movement behavior To allow butterflies to acclimate to the testing environment, the first 15 minutes of each flight trial were discarded. Flight behaviors over the next 30 minutes were scored using idTracker software [ 26 ], which allowed us to simultaneously track multiple individual flight paths. For each individual, we quantified total time spent in the air (s) and total distance traveled (cm). Speed (cm/s) was calculated from flight time and travel distance measurements. Statistical analysis All statistical analyses were conducted in R version 4.2.3 [ 27 ]. Due to expected correlations among body size measurements, we tested for statistical correlations among wingspan, body length, and forewing length before conducting further analyses. We then tested for an effect of rearing temperature treatment on body size using a t-test. The movement responses time in air (s), distance moved (cm) and speed (cm/s) were analyzed used with generalized linear mixed models (GLMMs) or general linear models (GLMs) in the package ‘lmerTest’ [ 28 ] to assess treatment effects. We tested models with and without a random effect of trial to evaluate the possibility of non-independence in behavior among individuals who were tested together. The most likely model (including or not including the random effect) was determined by comparing the AIC values of the models using the package ‘performance’ [ 29 ]. Because our first analysis found that body size was strongly affected by rearing temperature, we followed up with two sets of linear models for flight behavior: one investigating the effects of both rearing and testing temperatures on responses and another investigating the effects of wingspan and testing temperature on movement. Results A total of 398 adult butterflies emerged from the rearing treatments (N: low = 206, high = 192). As expected, all body size measurements were highly correlated with each other (wingspan to body length: r = 0.66, wingspan to forewing length: r = 0.71, body length to forewing length: r = 0.75). Thus, we chose a single measurement, wingspan, to assess the morphological response to rearing temperature. In contrast to our expectation based on the temperature-size rule, high rearing temperature individuals had larger wingspans than did low rearing temperature individuals (mean ± 1 SE (mm): low = 124.12 ± 0.69, high = 159.17 ± 1.13; Fig. 1 , t = 169.13, p < 2 x 10 − 16 ). We found statistically significant interaction effects in all models with any aspect of flight behavior as a response. The addition of a random effect of flight trial did not substantially improve the performance of models testing for a rearing x testing temperature interaction (speed: DAICc = 2.1, model weight = 0.74; time: DAICc = 2, model weight = 0.74; distance: DAICc = 1.2, model weight = 0.64; in all cases, the model with the lower AIC did not include the random effect), thus we proceeded with simpler GLMs. For the response of flight speed, butterflies in the low/low and high/high treatments flew significantly slower than did the individuals who experienced different rearing and testing treatment temperatures (GLM: rearing temperature, F = 8.57, P = 0.004, testing temperature, F = 30.15, P = 7.17 x 10 − 08 , interaction, F = 113.57, P < 2.2 x 10 − 16 ; mean ± 1 SE (cm/s): low/low: 3.61 ± 0.06, high/high: 4.08 ± 0.19, low/high: 7.01 ± 0.31, high/low: 5.27 ± 0.21; Fig. 2 ). When we decomposed speed into its two components (time flying and distance traveled), we also observed statistically significant interaction effects, and all treatment combinations were different from each other (Figs. 3 , 4 ). However, butterflies from the low/low and high/high treatments flew longer (GLM: rearing temperature, F = 0.06, P = 0.81, testing temperature, F = 25.80, P = 5.84 x 10 − 7 , interaction, F = 378.84, P < 2.2 x 10 − 16 ; mean ± 1 SE (seconds): low/low: 1,166.85 ± 19.54, high/high: 1,058.25 ± 38.41, low/high: 542.04 ± 20.35, high/low: 663.50 ± 23.50; Fig. 3 ) and for greater distances (GLM: rearing temperature, F = 155.33, P < 2.2 x 10 − 16 , testing temperature, F = 57.33, P = 2.61 x 10 − 13 , interaction, F = 1265.70, P < 2.2 x 10 − 16 ; mean ± 1 SE (cm); low/low: 4,093.00 ± 14.53, high/high: 3,689.48 ± 32.87, low/high: 3,192.82 ± 17.24, high/low: 3,060.72 ± 17.79; Fig. 4 ) than individuals in the other treatments. Thus, butterflies that experienced matching rearing and testing temperatures, whether low or high, flew further and for longer than animals from the other treatments (Figs. 3 , 4 ), but at lower speeds (Fig. 2 ). Because animals reared at the higher temperature were larger than those reared at the lower temperature, we then ran a related set of models in which the categorical variable “rearing temperature” was replaced with the continuous variable “wingspan,” which allowed us to examine the role of body size itself (wingspan) on movement behavior. For this set of models, inclusion of the random effect of flight trial did generally improve model performance (speed: DAICc = 0.1, model weight = 0.53; time: DAICc = 12.5, model weight = 0.99; distance: DAICc = 125.2, model weight > 0.99; in all cases, the model with the lower AIC included the random effect). Similar to the previous set of analyses, we found statistically significant interaction effects for all movement responses. We found a statistically significant effect of the interaction between wingspan (which results from rearing temperature, Fig. 1 ) and flight testing temperature on speed (GLMM: wingspan, F = 2.80, P = 0.10, testing temperature, F = 43.22, P = 8.18 x 10 − 10 , interaction, F = 35.12, P = 2.07 x 10 − 8 , Fig. 5 ). However, within each of the four rearing/testing temperature combinations, wingspan did not obviously influence speed (Fig. 5 ). The time spent flying was also affected by the interaction between wingspan and testing temperature (GLMM: wingspan, F = 1.87, P = 0.17, testing temperature, F = 22.83, P = 3.43 x 10 − 6 , interaction, F = 18.74, P = 2.29 x 10 − 5 , Fig. 6 ). Again, while there was variation due to temperature treatments, within those treatments, time flying was relatively consistent across wingspans (Fig. 6 ), with the exception of “high/high” animals, who showed a negative relationship between wingspan and time in flight. Finally, we observed that distance traveled was also affected by the interaction between wingspan and testing temperature (GLMM: wingspan, F = 0.19, P = 0.66, testing temperature, F = 8.83, P = 0.003, interaction, F = 5.95, P = 0.02, Fig. 7 ), but again, wingspan per se did not affect distance traveled within the treatment groups. Discussion Our results align with our general expectation that both rearing and testing temperatures would influence flight behavior by V. cardui . However, the effect of rearing temperature on body size was the opposite of our expectation: we observed smaller, rather than larger, butterflies emerging from the lower developmental temperature (Fig. 1 ). This observation leads to revised expectations for the joint effects of rearing and testing temperatures on flight: we would now expect that the lowest flight speeds would be exhibited by low/low treatment animals (smaller bodies at lower temperatures), and the highest flight speeds by high/high individuals (larger bodies at higher temperatures). However, our observations also did not align with these revised predictions. Instead, we found that higher flight speeds were exhibited by the animals who experienced a mismatch between the conditions under which they were reared and those under which they were tested (Figs. 2 , 5 ). Further, when we examined the role of wingspan itself on flight behavior, we found that there was no relationship between body size and flight behavior, aside from the effects of temperature (Figs. 5 – 7 ). Our first unexpected result was the increase in body size (quantified as wingspan) with higher rearing temperatures (Fig. 1 ). However, experimental rearing studies increasingly suggest that the temperature size rule is less consistent for arthropods than for vertebrate ectotherms [ 30 – 32 ], with some studies even describing a “reverse TSR” [ 33 ], in which size increases with temperature [ 5 , 31 ]. Further, recent meta-analyses have failed to find evidence of selection for smaller bodies at higher temperatures [ 34 , 35 ] (note that these studies focus on evolutionary rather than plastic responses to temperature). Published reports of effects of rearing temperature on body size in V. cardui are sparse, but as in the current study, the data collected by Medina-Báez and colleagues [ 26 , 36 ] are not consistent with the TSR. Although Medina-Báez et al. [ 26 ] did not directly report the effect of rearing temperature on body size, we were able to assess this using their archived data [ 36 ], which showed that V. cardui reared at 20°C and 30°C were not significantly different in body mass (mg) as adults. The range of observed responses of insect body sizes to experimental warming treatments suggests that McCauley and Mabry’s [ 3 ] concept of a positive feedback loop between temperature and body size that negatively affects movement is perhaps more broadly applicable to vertebrate than invertebrate ectotherms. Our second surprising result was the lack of effect of wingspan on movement behavior (Figs. 5 – 7 ). We did not find that larger butterflies flew faster; instead, butterflies that were flight tested at a different temperature (whether higher or lower) from their rearing temperature flew faster (Fig. 5 ). Animals that experienced “matching” rearing and testing environments flew longer and covered more distance (Figs. 6 – 7 ), at lower flight speeds (Fig. 5 ). We posit that this observation constitutes a carry-over effect, in which environmental conditions experienced early in life affect animals at a later life stage [ 37 ], potentially via developmental influences on physiology and behavior. We are not alone in observing acclimation effects to rearing temperature in V. cardui ; albeit with a different response, Medina-Báez et al. [ 25 ] found that individuals reared at 30°C had a higher critical thermal maximum (CT max ) than did butterflies reared at 20°C, and that CT max also varied across ontogeny. Our result has potential implications not only for understanding how insects may respond to climate change, but also for the design of experiments seeking to investigate movement as a response to temperature across life stages. While we deliberately set out to control for potential carry-over effects in understanding how temperature and body size influenced the flight behavior of V. cardui , many studies of temperature effects on animals across ontogeny do not conduct such controls (as reviewed by [ 38 ]). In studies of thermal effects on movement of invertebrates, it is not uncommon for researchers to rear animals at different temperatures, but conduct all movement tests at the same temperature [ 17 , 39 ]. Alternately, some researchers use a single rearing temperature and multiple testing temperatures [ 6 ]. Both of these types of experimental design can confound acclimation to a temperature and carry-over effects. This is because when such experiments use multiple temperatures for either rearing or testing (but not both), alignment of rearing and testing conditions typically occurs at just one temperature (the control), and it is not possible to evaluate whether animals reared at other temperatures would demonstrate similar responses if movement were tested at their respective rearing temperature. For example, Arambourou et al. [ 39 ] observed reduced flight time in damselflies reared under “heat wave” conditions and conclude that “carry-over effects of warming experienced during the larval stage reduce adult locomotor ability”. However, because their damselflies were reared at three different temperatures (20, 25, and 30°C) and flight trials were conducted at one of those temperatures (20°C), this finding is consistent with both carry-over effects and the degree of acclimation to the temperature under which flight was assessed. Put more simply, “heat wave” damselflies experienced elevated temperatures during development, but also experienced a dramatic decrease in temperature (of 5 or 10°C) when movement behavior was assessed. Replicating across both rearing and testing temperatures will always be logistically challenging, as required sample sizes increase dramatically with the number of treatment groups. However, we argue that given the potential influence of both thermal acclimation and carry-over effects on movement behavior of ectotherms in particular, it is imperative to use fully-replicated experimental designs. This is not to say that our experimental design (a two-way factorial in which rearing and testing temperatures were crossed) was ideal; an improvement on our study would be the inclusion of an intermediate testing temperature (26°C), which would have allowed us to further disentangle the effects of rearing environment and ambient conditions on flight. Multiple aspects of morphology have been shown to affect flight performance in butterflies: for example, Barwaerts et al. [ 40 ] found effects of body mass, thorax mass, forewing area, forewing length, wing loading, aspect ratio, and the centroid of forewing area on flight performance. Barwaerts et al. [ 40 ] found that all of the listed traits correlated positively with flight performance. Understandably, however, most researchers (including us) do not measure all of these variables when assessing the effects of flight morphology on flight performance and behavior. Differences in which variables were measured among studies complicate efforts at synthesis, as traits deemed important in one study may be unmeasured in others. For example, Reim et al. [ 17 ] found that increased reared temperature did lead to smaller body size in the tropical butterfly Bicyclus anynana , but also found that sexual dimorphism was a more important influence than rearing temperature on flight behavior. Reim and colleagues [ 17 ] reared their butterflies at three temperatures (21, 25, and 29°C) and conducted flight tests at 27°C, finding that flight distance was shorter at higher temperatures. The closest analog to the results of Reim et al. [ 17 ] in the current study is the comparison between groups of V. cardui that were reared at either 24°C or 28°C and given flight tests at 28°C (Fig. 4 ). In contrast to their findings, we documented increased flight distance by butterflies reared at the higher temperature. However, we also documented smaller body sizes for V. cardui reared at the lower temperature, the opposite of the findings for B. anynana , making it difficult to directly compare the two studies in terms of the effects of rearing temperature on movement behavior. Conclusions Drawing synthetic conclusions about how the movement behavior of insects may respond to climate change is challenging. As described above, differences in experimental designs across studies make comparisons difficult, as researchers vary in both how they manipulate rearing and testing temperatures and in the response variables measured. Further, recent studies have found unexpected results – for example, Arambourou et al. [ 39 ] found that increased rearing temperature decreased flight performance in a damselfly, but via a change in wing shape rather than body size. Thus, while insight into how thermal conditions influence development and subsequent movement behavior is needed to make realistic predictions about future changes [ 4 , 17 , 41 – 43 ], organismal responses may be complex and potentially unexpected [ 17 , 31 ]. Declarations Ethics approval and consent to participate: No permits or approvals were required for the experiments with invertebrate animals reported here. Consent for publication: Not applicable Availability of data and materials: All data generated or analysed during this study are included in this published article and its supplementary information files. Competing interests: The authors declare that they have no competing interests. Funding: This work was supported by the NMSU Biology Department. Author’s contributions: Both authors contributed to conception, study design, and data interpretation. SPM conducted all experimental work and wrote the first draft of the manuscript. KEM contributed to revisions. Both authors read and approved the final manuscript. Acknowledgements We thank Drs. Scott Bundy, Adriana Romero-Olivares, and Shannon McCauley for feedback on earlier drafts of this manuscript. References Parmesan, C. Ecological and evolutionary responses to recent climate change. Ann Rev Ecol Evol Sys. 2006;37:637-669. Brooker RW, Travis JM, Clark EJ, Dytham C. 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Cons Phys. 2023;11:coad058. Pérez-Escudero A, Vicente-Page J, Hinz R et al. idTracker: tracking individuals in a group by automatic identification of unmarked animals. Nat Meth. 2014;11 : 743-748. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. 2021. https://www.R-project.org/ Kuznetsova A, Brockhoff PB, Christensen RHB. lmerTest Package: Tests in Linear Mixed Effects Models. J Stat Soft. 2017;82:1-26. Lüdecke D, Ben-Shachar M, Patil I, Waggoner P, Makowski D. performance: An R package for assessment, comparison and testing of statistical models. J Open Source Soft . 2021;6:3139. Klok CJ, Harrison, JF. The temperature size rule in arthropods: independent of macro-environmental variables but size dependent. Integr Comp Biol. 2013;53:557-570. Davies WJ. Multiple temperature effects on phenology and body size in wild butterflies predict a complex response to climate change. Ecol. 2019;100:e02612. Wonglersak R, Fenberg PB, Langdon PG, Brooks SJ, Price BW. Temperature-body size responses in insects: a case study of British Odonata. Ecol Ent. 2020;45:795-805. Horne CR, Hirst AG, Atkinson D. Temperature-size responses match latitudinal-size clines in arthropods, revealing critical differences between aquatic and terrestrial species. Ecol Lett. 2015;18:327-335. Siepielski AM, Morrissey MB, Carlson SM, Francis CD, Kingsolver JG, Whitney KD, Kruuk LEB. No evidence that warmer temperatures are associated with selection for smaller body sizes. Proc Roy Soc B. 2019;286:20191332. Granger TN, Levine JM. Rapid evolution of life-history traits in response to warming, predation and competition: a meta-analysis. Ecol Lett. 2021;25:541-554. Diamond SE. Ontogenetic variation in physiology of the painted lady butterfly. 2023. https://doi.org/10.17605/OSF.IO/UP5BJ Benard MF, McCauley SJ. Integrating across life‐history stages: consequences of natal habitat effects on dispersal. Am Nat. 2008;171:553-567. Rebolledo AP, Sgrò CM, Monro K. Thermal performance curves are shaped by prior thermal environment in early life. Fron Phys. 2021;12:738338. Arambourou H, Sanmartín-Villar I, Stoks R. 2017. Wing shape-mediated carry-over effects of a heat wave during the larval stage on post-metamorphic locomotor ability. Glob Change Bio. 2017;184:279-291. Berwaerts K, Van Dyck H, Aerts P. Does flight morphology relate to flight performance? An experimental test with the butterfly Pararge aegeria . Funct Ecol. 2002;16:484-491. Bristow LV, Grundel R, Dzurisin JDK, Wu GC, Li Y, Hildreth A, Hellmann JJ. Warming experiments test the temperature sensitivity of an endangered butterfly across life history stages. J Insect Conserv. 2023;28:1-13. Niehaus AC, Angilletta Jr MJ, Sears MW, Franklin CE, Wilson RS. Predicting the physiological performance of ectotherms in fluctuating thermal environments. J Exp Biol. 2012;215:694-701. Van der Have TM, De Jong G. Adult size in ectotherms: temperature effects on growth and differentiation. J Theor Biol. 1996;183:329-340. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.csv File name: Additional file 1 File format: csv Title of data: Raw morphology and flight behavior data Description of data: This file contains all raw data used in the analyses. ‘rearing.trt’ is the temperature at which an individual was reared. ‘testing.trt’ is the temperature at which an individual was flight-tested. ‘ID’ is the unique identifier for an individual. ‘trial’ is the flight trial an individual participated in. ‘wingspan’ is the distance between the forewing tips of an individual in mm. ‘body.length.mm’ is an individual’s body length in mm. ‘forewing.length.mm’ is the length of the forewing in mm. ‘time.in.air.sec’ is the number of seconds an individual spent flying during the 30 minute trial. ‘distance.cm’ is the distance an individual moved during the 30 minute trial. ‘speed.cm.sec’ is ‘distance.cm’/’time.in.air.sec’. Cite Share Download PDF Status: Published Journal Publication published 03 Dec, 2024 Read the published version in Movement Ecology → Version 1 posted Editorial decision: Revision requested 30 Sep, 2024 Reviews received at journal 18 Sep, 2024 Reviews received at journal 10 Sep, 2024 Reviewers agreed at journal 28 Aug, 2024 Reviewers agreed at journal 21 Aug, 2024 Reviewers invited by journal 24 Jul, 2024 Editor assigned by journal 17 Jul, 2024 Submission checks completed at journal 17 Jul, 2024 First submitted to journal 12 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4731760","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":333126819,"identity":"038eabf2-8f58-45c1-96aa-5a3fb343cb6f","order_by":0,"name":"Sarah P. Mesler","email":"","orcid":"","institution":"New Mexico State University","correspondingAuthor":false,"prefix":"","firstName":"Sarah","middleName":"P.","lastName":"Mesler","suffix":""},{"id":333126821,"identity":"60067052-2a16-4d49-8594-c13538f84fb9","order_by":1,"name":"Karen E. Mabry","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACCQYGNiBlA+WyEa8ljXQth0nQIjnt8LPHlW3n83T7zxgwfCg7TFiLtHSaueHZttvFZjdyDBhnnCNCi5x0gplkY9vtxG03eAyYeduI0pL+DajlXOK282cMmP8So0VaOgdky4HEbQdyDJgZidEiOTun3LDhXDLQYWkFB3vOpRPWInE7fdvDhjI7oMMOb3zwo8yasBYwYIRGxwEi1YPAHxLUjoJRMApGwcgDADmDPKPn/MGTAAAAAElFTkSuQmCC","orcid":"","institution":"New Mexico State University","correspondingAuthor":true,"prefix":"","firstName":"Karen","middleName":"E.","lastName":"Mabry","suffix":""}],"badges":[],"createdAt":"2024-07-12 17:03:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4731760/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4731760/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s40462-024-00516-3","type":"published","date":"2024-12-03T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":62659100,"identity":"ddc21235-a1dc-4a43-ad5f-284bc1b82fe0","added_by":"auto","created_at":"2024-08-17 02:23:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47359,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot showing the influence of rearing temperature on adult \u003cem\u003eV. cardui\u003c/em\u003e wingspan. The open bar shows wingspan for animals reared at 24 °C and the stippled bar shows animals reared at 28 °C.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/fa14c6256fad1655abe793c7.png"},{"id":62659096,"identity":"2cd05538-5c52-4400-a037-3680cf93bacb","added_by":"auto","created_at":"2024-08-17 02:23:35","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":64524,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot showing the interactive effects of rearing and flight testing temperature on flight speed (cm/s). Rearing temperature is depicted with background color (white = 24 °C, gray = 28 °C) and testing temperature is shown with stripes (no stripes = 24 °C, stripes = 28 °C).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/d00a6cb575a767c8c90bfc0a.png"},{"id":62659093,"identity":"2d26a51e-4d9f-4e2e-b36e-0c9036b1e0e1","added_by":"auto","created_at":"2024-08-17 02:23:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":76844,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot showing the interactive effects of rearing and flight testing temperature on time spend flying (s). Rearing temperature is depicted with background color (white = 24 °C, gray = 28 °C) and testing temperature is shown with stripes (no stripes = 24 °C, stripes = 28 °C).\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/0ef61db6d40a07ffb622a56f.png"},{"id":62660152,"identity":"5c1fe7ad-360e-47cb-b2e2-1b102be93bac","added_by":"auto","created_at":"2024-08-17 02:31:35","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":67364,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot showing the interactive effects of rearing and flight testing temperature on flight distance (cm). Rearing temperature is depicted with background color (white = 24 °C, gray = 28 °C) and testing temperature is shown with stripes (no stripes = 24 °C, stripes = 28 °C).\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/fda5b4ca8f5e7f255f8a717f.png"},{"id":62659095,"identity":"fb346f00-c105-4734-8e26-1bd85c630f00","added_by":"auto","created_at":"2024-08-17 02:23:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":93478,"visible":true,"origin":"","legend":"\u003cp\u003eScatterplot showing the interactive effects of wingspan and flight testing temperature on flight speed (cm/s). Different symbols represent the rearing temperatures (triangles = 24 °C, circles = 28 °C). Gray symbols and dashed lines represent the low (24 °C) testing temperature and black symbols and solid lines represent the high (28 °C) testing temperature.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/b94565a25f3f7f81e82f612e.png"},{"id":62661249,"identity":"e5ee5ec2-0f45-46d1-a26a-29486815e6d9","added_by":"auto","created_at":"2024-08-17 02:39:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":104379,"visible":true,"origin":"","legend":"\u003cp\u003eScatterplot showing the interactive effects of wingspan and flight testing temperature on time spent flying (s). Different symbols represent the rearing temperatures (triangles = 24 °C, circles = 28 °C). Gray symbols and dashed lines represent the low (24 °C) testing temperature and black symbols and solid lines represent the high (28 °C) testing temperature.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/3eb916ad31fb80878d9dc447.png"},{"id":62660153,"identity":"fde4e09f-f12a-4eb1-9990-679745ab8d49","added_by":"auto","created_at":"2024-08-17 02:31:35","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":101985,"visible":true,"origin":"","legend":"\u003cp\u003eScatterplot showing the interactive effects of wingspan and flight testing temperature on flight distance (cm). Different symbols represent the rearing temperatures (triangles = 24 °C, circles = 28 °C). Gray symbols and dashed lines represent the low (24 °C) testing temperature and black symbols and solid lines represent the high (28 °C) testing temperature.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/771bd9c84af0a0717b47a6af.png"},{"id":70964698,"identity":"f7ae2cb9-d6c3-4943-aa11-41057359730a","added_by":"auto","created_at":"2024-12-09 16:14:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":935453,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/bf86e0df-e93b-416e-b4e1-5a05c5e19ce3.pdf"},{"id":62659099,"identity":"59d25f1d-5e5c-4e59-bf47-71f1c43b0ab2","added_by":"auto","created_at":"2024-08-17 02:23:35","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":16927,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFile name: Additional file 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFile format: csv\u003c/p\u003e\n\u003cp\u003eTitle of data: Raw morphology and flight behavior data\u003c/p\u003e\n\u003cp\u003eDescription of data:\u003c/p\u003e\n\u003cp\u003eThis file contains all raw data used in the analyses.\u003c/p\u003e\n\u003cp\u003e‘rearing.trt’ is the temperature at which an individual was reared.\u003c/p\u003e\n\u003cp\u003e‘testing.trt’ is the temperature at which an individual was flight-tested.\u003c/p\u003e\n\u003cp\u003e‘ID’ is the unique identifier for an individual.\u003c/p\u003e\n\u003cp\u003e‘trial’ is the flight trial an individual participated in.\u003c/p\u003e\n\u003cp\u003e‘wingspan’ is the distance between the forewing tips of an individual in mm.\u003c/p\u003e\n\u003cp\u003e‘body.length.mm’ is an individual’s body length in mm.\u003c/p\u003e\n\u003cp\u003e‘forewing.length.mm’ is the length of the forewing in mm.\u003c/p\u003e\n\u003cp\u003e‘time.in.air.sec’ is the number of seconds an individual spent flying during the 30 minute trial.\u003c/p\u003e\n\u003cp\u003e‘distance.cm’ is the distance an individual moved during the 30 minute trial.\u003c/p\u003e\n\u003cp\u003e‘speed.cm.sec’ is ‘distance.cm’/’time.in.air.sec’.\u003c/p\u003e","description":"","filename":"Additionalfile1.csv","url":"https://assets-eu.researchsquare.com/files/rs-4731760/v1/995f2b776f41bb0df513cfea.csv"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of temperature experienced across life stages on morphology and flight behavior of painted lady butterflies (Vanessa cardui)","fulltext":[{"header":"Background","content":"\u003cp\u003eAnthropogenic climate change continues to influence life on Earth [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], with diverse responses to increasing temperatures exhibited by a range of species. Researchers often place these responses into one of three categories: species can \u0026lsquo;adapt\u0026rsquo;, through either adaptive evolution or acclimation to new conditions, move in either space (range shifts, typically to areas of higher latitude and/or elevation) or time (phenological shifts), or die (extinction). However, while predictions about future range shifts in relation to increasing temperatures are common in the literature, the movement behavior that would underlie such shifts by animal species is not often considered [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Many factors can influence an organism\u0026rsquo;s ability to move from one location to another and the search for more resources can be impacted by external conditions [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAmbient temperature can influence movement behavior in multiple ways, including both direct and indirect effects. For ectothermic animals in particular, the rate of vital processes is affected by the thermal conditions an individual experiences [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In many cases, the thermal performance curve is \u0026ldquo;hump-shaped\u0026rdquo;: performance (or the rate of a process) increases with increasing temperature up to the thermal optimum, then declines [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. We might expect the locomotion speed of an ectothermic animal to follow such a performance curve, in which speed increases with temperature to the thermal optimum, then declines dramatically [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHowever, temperature can also have indirect effects on movement behavior. For example, thermal influences on developmental processes may produce variation in movement-related morphology [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Body size is a well-known predictor of movement behavior in a range of animal species [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Changes in body size are also a major response to warmer temperatures [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], with the \u0026ldquo;temperature-size rule\u0026rdquo; stating that organisms should be smaller at higher temperatures, leading some to predict that body sizes will decline with increasing temperatures due to anthropogenic climate change [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Thus, body size becomes a potential explicit link between the effect of climate warming on organisms and the ability of organisms to respond to that warming via movement and range shifts [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]: animals that develop under warmer conditions are expected to be smaller as adults, and thus potentially less able to track changing environmental conditions.\u003c/p\u003e \u003cp\u003eDespite the importance of movement behavior as an influence on range shifts in response to climate change, relatively few researchers have attempted to link the developmental conditions that produce variation in movement-related phenotypes to movement behavior exhibited later in life. This is potentially related to a mismatch in the types of studies and systems used to investigate these questions: there has been a recent explosion in techniques for tracking the movements of large animals in the field [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], but it is almost impossible to manipulate rearing conditions for these species in ways that would be expected to result in morphological changes that are relevant to movement. Meanwhile, it is relatively straightforward to generate such morphological diversity in lab-based invertebrate systems, but devices for tracking individual movements of small ectotherms, such as insects, are more limited and our understanding of thermal effects on movement of insects remains incomplete [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, it is often feasible to track the movements of individuals using automated systems [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and this is the approach that we followed in this study.\u003c/p\u003e \u003cp\u003eTo understand the effects of temperature on both body size and movement behavior, we used the painted lady butterfly (\u003cem\u003eVanessa cardui\u003c/em\u003e) as a study organism. In general, butterflies serve as pollinators, food sources and indicators of ecosystem health [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and butterfly range shifts have been documented in relation to climate change [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. \u003cem\u003eV. cardui\u003c/em\u003e is particularly suitable for the study of movement behavior due to its almost cosmopolitan distribution and high mobility [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]; transatlantic flights by \u003cem\u003eV. cardui\u003c/em\u003e have recently been reported [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. We manipulated rearing temperature for larval \u003cem\u003eV. cardui\u003c/em\u003e and flight testing temperature for adults in a two-way factorial design. First, we wanted to determine how rearing temperature influenced adult morphology. Specifically, we were interested in effects of temperature on body size, due to its potential influence on movement behavior [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Following the \u0026ldquo;temperature size rule\u0026rdquo; [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], we predicted that at emergence, butterflies reared at a higher temperature would be smaller than those reared at a lower temperature. Second, we wanted to determine the influence of ambient temperature on flight behavior. Completion of the second objective required controlling for the potential effects of variation in the rearing environment on flight-related morphology in adults, because we predicted that larger adult butterflies (presumably those reared at a lower temperature) would fly farther and faster than would smaller individuals. Combining our predictions about the effects of rearing temperature on body size (\u0026ldquo;hotter is smaller\u0026rdquo;) and testing temperature on flight (\u0026ldquo;hotter is faster\u0026rdquo;), we expected that individuals who experienced lower rearing temperatures and higher flight testing temperatures would fly the fastest and in contrast, animals that were reared at a higher temperature and tested at a lower temperature would be the slowest.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy species and culturing\u003c/h2\u003e \u003cp\u003eWe obtained larval \u003cem\u003eV. cardui\u003c/em\u003e from Carolina Biological Supply (Burlington, NC, USA). Larvae were within one developmental instar from each other upon receipt. With a paintbrush, larvae were gently transferred from travel containers to prepared rearing containers, each of which contained approximately 30 g of food mix in an even layer on the bottom of the cup (painted lady culture medium, Carolina Biological Supply, Burlington, NC, USA). Three randomly-selected larvae were placed into each container without consideration of size or stage of development. Each container was then covered with a mesh screen to allow for hanging chrysalids. A second plastic lid with air holes was placed on the cup to prevent larvae from escaping. Food was replenished as it was consumed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eManipulation of developmental temperatures\u003c/h2\u003e \u003cp\u003eExperimental temperature regimes were based on established temperature tolerances of \u003cem\u003eV. cardui\u003c/em\u003e in laboratory studies. Symanski and Redak [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] reviewed rearing temperatures in laboratory studies of \u003cem\u003eV. cardui\u003c/em\u003e; they report experiments across a range from 12\u0026ndash;33\u0026deg;C, with an increase in mortality at temperatures above 30\u0026deg;C. Symanski and Redak [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] describe 25\u0026deg;C as the \u0026ldquo;optimal\u0026rdquo; rearing temperature for this species. We chose two experimental temperatures, with the objective of inducing deleterious effects without increasing mortality of animals reared at the higher temperature treatment: 24\u0026deg;C (\u0026ldquo;low\u0026rdquo;) and 28\u0026deg;C (\u0026ldquo;high\u0026rdquo;). Temperatures were maintained at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u0026deg;C and 28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7\u0026deg;C (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;1 SE) throughout the larval, chrysalis, and adult stages using electronically controlled biological chambers (BioChambers model #6194, Winnipeg, Manitoba, Canada) with lights on at 0800 and lights off at 2000. Each larval container was randomly assigned to one of the rearing temperatures.\u003c/p\u003e \u003cp\u003eLarvae were monitored daily from 21 October 2022 to 8 December 2022, at which point all larvae had either metamorphosed or died. We determined the number of live and dead larvae, chrysalides, and newly emerged adults in each container each day. There was evidence of some cannibalism: half bodied larvae and larvae parts including decapitated heads were observed prior to the chrysalid form, when larval heads are pinched off.\u003c/p\u003e \u003cp\u003eOnce an individual reached chrysalis form, it was moved from the rearing container and placed into a 42 cm x 42 cm x 76 cm enclosure constructed of 1.27 cm diameter PVC pipe and mesh netting, with one enclosure per temperature treatment. These enclosures were kept in the environmental chambers with the remaining rearing containers. A zippered door and Velcro corner allowed for the handling of chrysalides and adults. Chrysalides were hung within the mesh containers along the walls to allow for proper development of wings after eclosion. Once an adult eclosed it was assigned a color which was painted on the dorsal side of the abdomen with non-toxic hobby paint (Apple Barrel acrylic paint, PLAID, Atlanta, GA, USA) and allowed for the simultaneous tracking of multiple individuals in filmed flight trials. Food for adults was a 1:4 mixture of sucrose and water which was placed in the adult enclosures in a tray with a sponge on top of the liquid to help prevent drowning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMeasuring movement behavior under varying temperatures\u003c/h2\u003e \u003cp\u003eThe movements of adult butterflies were assessed at both low (24\u0026deg;C) and high (28\u0026deg;C) temperatures. Because we expected movement of these ectothermic animals to be affected by both temperature during the flight tests [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and the temperature at which they were reared (via an effect of rearing temperature on body size), we used a two-way factorial design in the assessment of movement behavior. Animals that were reared under the low and high temperature regimes were split evenly between testing temperatures for a total of four treatments: low-low, high-high, low-high, high-low, where \u0026ldquo;low\u0026rdquo; = 24\u0026deg;C and \u0026ldquo;high\u0026rdquo; = 28\u0026deg;C. Flight tests were conducted in an enclosed space that was warmed to the required testing temperature using a space heater (Dreo Atom One Space Heater, Dreo, New York, NY, USA). The flight arena was a 92 cm x 43 cm x 46 cm aquarium (Aqueon standard open-glass aquarium tank, 151.4 liter, Aqueon Products, Franklin, WI, USA) with a fitted lid to prevent escape of individuals during trials. The glass walls of the aquarium allowed video recording and prevented individuals from clinging to the side rather than flying.\u003c/p\u003e \u003cp\u003eFor each flight trial, five randomly selected marked adults were placed in the arena. Each trial lasted for one hour and was recorded using a small video camera (Akaso V50 X action camera, Akaso, Frederick, MD, USA). The lights were on for the entirety of the trials. After the flight trial, the butterflies were placed in another mesh enclosure of the same size (42 cm x 42 cm x 76 cm) instead of the general population enclosure so they wouldn\u0026rsquo;t be selected again.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eMorphological measurements\u003c/h2\u003e \u003cp\u003eAfter flight trials, adults were placed into envelopes with wings folded to prevent damage and secured in a -20\u0026deg;C freezer. Each envelope was marked with an individual\u0026rsquo;s color, flight trial number, and unique ID number. Individuals were later pinned, spread, and dried. After drying for two days, we measured total wingspan, body length, and forewing length (all in mm) using electronic calipers (15.5 cm electronic digital caliper w/ LCD readout, WEN, West Dundee, IL, USA). We did not determine sex of tested animals; sex size dimorphism is reported to be minimal in this species [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eScoring of movement behavior\u003c/h2\u003e \u003cp\u003eTo allow butterflies to acclimate to the testing environment, the first 15 minutes of each flight trial were discarded. Flight behaviors over the next 30 minutes were scored using idTracker software [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], which allowed us to simultaneously track multiple individual flight paths. For each individual, we quantified total time spent in the air (s) and total distance traveled (cm). Speed (cm/s) was calculated from flight time and travel distance measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted in R version 4.2.3 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Due to expected correlations among body size measurements, we tested for statistical correlations among wingspan, body length, and forewing length before conducting further analyses. We then tested for an effect of rearing temperature treatment on body size using a t-test. The movement responses time in air (s), distance moved (cm) and speed (cm/s) were analyzed used with generalized linear mixed models (GLMMs) or general linear models (GLMs) in the package \u0026lsquo;lmerTest\u0026rsquo; [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] to assess treatment effects. We tested models with and without a random effect of trial to evaluate the possibility of non-independence in behavior among individuals who were tested together. The most likely model (including or not including the random effect) was determined by comparing the AIC values of the models using the package \u0026lsquo;performance\u0026rsquo; [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Because our first analysis found that body size was strongly affected by rearing temperature, we followed up with two sets of linear models for flight behavior: one investigating the effects of both rearing and testing temperatures on responses and another investigating the effects of wingspan and testing temperature on movement.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eA total of 398 adult butterflies emerged from the rearing treatments (N: low\u0026thinsp;=\u0026thinsp;206, high\u0026thinsp;=\u0026thinsp;192). As expected, all body size measurements were highly correlated with each other (wingspan to body length: r\u0026thinsp;=\u0026thinsp;0.66, wingspan to forewing length: r\u0026thinsp;=\u0026thinsp;0.71, body length to forewing length: r\u0026thinsp;=\u0026thinsp;0.75). Thus, we chose a single measurement, wingspan, to assess the morphological response to rearing temperature. In contrast to our expectation based on the temperature-size rule, high rearing temperature individuals had larger wingspans than did low rearing temperature individuals (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;1 SE (mm): low\u0026thinsp;=\u0026thinsp;124.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69, high\u0026thinsp;=\u0026thinsp;159.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.13; Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e, t\u0026thinsp;=\u0026thinsp;169.13, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e).\u003c/p\u003e\n\u003cp\u003eWe found statistically significant interaction effects in all models with any aspect of flight behavior as a response. The addition of a random effect of flight trial did not substantially improve the performance of models testing for a rearing x testing temperature interaction (speed: DAICc\u0026thinsp;=\u0026thinsp;2.1, model weight\u0026thinsp;=\u0026thinsp;0.74; time: DAICc\u0026thinsp;=\u0026thinsp;2, model weight\u0026thinsp;=\u0026thinsp;0.74; distance: DAICc\u0026thinsp;=\u0026thinsp;1.2, model weight\u0026thinsp;=\u0026thinsp;0.64; in all cases, the model with the lower AIC did not include the random effect), thus we proceeded with simpler GLMs. For the response of flight speed, butterflies in the low/low and high/high treatments flew significantly slower than did the individuals who experienced different rearing and testing treatment temperatures (GLM: rearing temperature, F\u0026thinsp;=\u0026thinsp;8.57, P\u0026thinsp;=\u0026thinsp;0.004, testing temperature, F\u0026thinsp;=\u0026thinsp;30.15, P\u0026thinsp;=\u0026thinsp;7.17 x 10\u003csup\u003e\u0026minus;\u0026thinsp;08\u003c/sup\u003e, interaction, F\u0026thinsp;=\u0026thinsp;113.57, P\u0026thinsp;\u0026lt;\u0026thinsp;2.2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;1 SE (cm/s): low/low: 3.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, high/high: 4.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19, low/high: 7.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31, high/low: 5.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). When we decomposed speed into its two components (time flying and distance traveled), we also observed statistically significant interaction effects, and all treatment combinations were different from each other (Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eHowever, butterflies from the low/low and high/high treatments flew longer (GLM: rearing temperature, F\u0026thinsp;=\u0026thinsp;0.06, P\u0026thinsp;=\u0026thinsp;0.81, testing temperature, F\u0026thinsp;=\u0026thinsp;25.80, P\u0026thinsp;=\u0026thinsp;5.84 x 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e, interaction, F\u0026thinsp;=\u0026thinsp;378.84, P\u0026thinsp;\u0026lt;\u0026thinsp;2.2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;1 SE (seconds): low/low: 1,166.85\u0026thinsp;\u0026plusmn;\u0026thinsp;19.54, high/high: 1,058.25\u0026thinsp;\u0026plusmn;\u0026thinsp;38.41, low/high: 542.04\u0026thinsp;\u0026plusmn;\u0026thinsp;20.35, high/low: 663.50\u0026thinsp;\u0026plusmn;\u0026thinsp;23.50; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e) and for greater distances (GLM: rearing temperature, F\u0026thinsp;=\u0026thinsp;155.33, P\u0026thinsp;\u0026lt;\u0026thinsp;2.2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e, testing temperature, F\u0026thinsp;=\u0026thinsp;57.33, P\u0026thinsp;=\u0026thinsp;2.61 x 10\u003csup\u003e\u0026minus;\u0026thinsp;13\u003c/sup\u003e, interaction, F\u0026thinsp;=\u0026thinsp;1265.70, P\u0026thinsp;\u0026lt;\u0026thinsp;2.2 x 10\u003csup\u003e\u0026minus;\u0026thinsp;16\u003c/sup\u003e; mean\u0026thinsp;\u0026plusmn;\u0026thinsp;1 SE (cm); low/low: 4,093.00\u0026thinsp;\u0026plusmn;\u0026thinsp;14.53, high/high: 3,689.48\u0026thinsp;\u0026plusmn;\u0026thinsp;32.87, low/high: 3,192.82\u0026thinsp;\u0026plusmn;\u0026thinsp;17.24, high/low: 3,060.72\u0026thinsp;\u0026plusmn;\u0026thinsp;17.79; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) than individuals in the other treatments. Thus, butterflies that experienced matching rearing and testing temperatures, whether low or high, flew further and for longer than animals from the other treatments (Figs. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e), but at lower speeds (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eBecause animals reared at the higher temperature were larger than those reared at the lower temperature, we then ran a related set of models in which the categorical variable \u0026ldquo;rearing temperature\u0026rdquo; was replaced with the continuous variable \u0026ldquo;wingspan,\u0026rdquo; which allowed us to examine the role of body size itself (wingspan) on movement behavior. For this set of models, inclusion of the random effect of flight trial did generally improve model performance (speed: DAICc\u0026thinsp;=\u0026thinsp;0.1, model weight\u0026thinsp;=\u0026thinsp;0.53; time: DAICc\u0026thinsp;=\u0026thinsp;12.5, model weight\u0026thinsp;=\u0026thinsp;0.99; distance: DAICc\u0026thinsp;=\u0026thinsp;125.2, model weight\u0026thinsp;\u0026gt;\u0026thinsp;0.99; in all cases, the model with the lower AIC included the random effect). Similar to the previous set of analyses, we found statistically significant interaction effects for all movement responses. We found a statistically significant effect of the interaction between wingspan (which results from rearing temperature, Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e) and flight testing temperature on speed (GLMM: wingspan, F\u0026thinsp;=\u0026thinsp;2.80, P\u0026thinsp;=\u0026thinsp;0.10, testing temperature, F\u0026thinsp;=\u0026thinsp;43.22, P\u0026thinsp;=\u0026thinsp;8.18 x 10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e, interaction, F\u0026thinsp;=\u0026thinsp;35.12, P\u0026thinsp;=\u0026thinsp;2.07 x 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eHowever, within each of the four rearing/testing temperature combinations, wingspan did not obviously influence speed (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). The time spent flying was also affected by the interaction between wingspan and testing temperature (GLMM: wingspan, F\u0026thinsp;=\u0026thinsp;1.87, P\u0026thinsp;=\u0026thinsp;0.17, testing temperature, F\u0026thinsp;=\u0026thinsp;22.83, P\u0026thinsp;=\u0026thinsp;3.43 x 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e, interaction, F\u0026thinsp;=\u0026thinsp;18.74, P\u0026thinsp;=\u0026thinsp;2.29 x 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e, Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e). Again, while there was variation due to temperature treatments, within those treatments, time flying was relatively consistent across wingspans (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), with the exception of \u0026ldquo;high/high\u0026rdquo; animals, who showed a negative relationship between wingspan and time in flight. Finally, we observed that distance traveled was also affected by the interaction between wingspan and testing temperature (GLMM: wingspan, F\u0026thinsp;=\u0026thinsp;0.19, P\u0026thinsp;=\u0026thinsp;0.66, testing temperature, F\u0026thinsp;=\u0026thinsp;8.83, P\u0026thinsp;=\u0026thinsp;0.003, interaction, F\u0026thinsp;=\u0026thinsp;5.95, P\u0026thinsp;=\u0026thinsp;0.02, Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e), but again, wingspan \u003cem\u003eper se\u003c/em\u003e did not affect distance traveled within the treatment groups.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur results align with our general expectation that both rearing and testing temperatures would influence flight behavior by \u003cem\u003eV. cardui\u003c/em\u003e. However, the effect of rearing temperature on body size was the opposite of our expectation: we observed smaller, rather than larger, butterflies emerging from the lower developmental temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This observation leads to revised expectations for the joint effects of rearing and testing temperatures on flight: we would now expect that the lowest flight speeds would be exhibited by low/low treatment animals (smaller bodies at lower temperatures), and the highest flight speeds by high/high individuals (larger bodies at higher temperatures). However, our observations also did not align with these revised predictions. Instead, we found that higher flight speeds were exhibited by the animals who experienced a mismatch between the conditions under which they were reared and those under which they were tested (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Further, when we examined the role of wingspan itself on flight behavior, we found that there was no relationship between body size and flight behavior, aside from the effects of temperature (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur first unexpected result was the increase in body size (quantified as wingspan) with higher rearing temperatures (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). However, experimental rearing studies increasingly suggest that the temperature size rule is less consistent for arthropods than for vertebrate ectotherms [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], with some studies even describing a \u0026ldquo;reverse TSR\u0026rdquo; [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], in which size increases with temperature [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Further, recent meta-analyses have failed to find evidence of selection for smaller bodies at higher temperatures [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] (note that these studies focus on evolutionary rather than plastic responses to temperature). Published reports of effects of rearing temperature on body size in \u003cem\u003eV. cardui\u003c/em\u003e are sparse, but as in the current study, the data collected by Medina-B\u0026aacute;ez and colleagues [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] are not consistent with the TSR. Although Medina-B\u0026aacute;ez et al. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] did not directly report the effect of rearing temperature on body size, we were able to assess this using their archived data [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], which showed that \u003cem\u003eV. cardui\u003c/em\u003e reared at 20\u0026deg;C and 30\u0026deg;C were not significantly different in body mass (mg) as adults. The range of observed responses of insect body sizes to experimental warming treatments suggests that McCauley and Mabry\u0026rsquo;s [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] concept of a positive feedback loop between temperature and body size that negatively affects movement is perhaps more broadly applicable to vertebrate than invertebrate ectotherms.\u003c/p\u003e \u003cp\u003eOur second surprising result was the lack of effect of wingspan on movement behavior (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). We did not find that larger butterflies flew faster; instead, butterflies that were flight tested at a different temperature (whether higher or lower) from their rearing temperature flew faster (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Animals that experienced \u0026ldquo;matching\u0026rdquo; rearing and testing environments flew longer and covered more distance (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e), at lower flight speeds (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). We posit that this observation constitutes a carry-over effect, in which environmental conditions experienced early in life affect animals at a later life stage [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], potentially via developmental influences on physiology and behavior. We are not alone in observing acclimation effects to rearing temperature in \u003cem\u003eV. cardui\u003c/em\u003e; albeit with a different response, Medina-B\u0026aacute;ez et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] found that individuals reared at 30\u0026deg;C had a higher critical thermal maximum (CT\u003csub\u003emax\u003c/sub\u003e) than did butterflies reared at 20\u0026deg;C, and that CT\u003csub\u003emax\u003c/sub\u003e also varied across ontogeny.\u003c/p\u003e \u003cp\u003eOur result has potential implications not only for understanding how insects may respond to climate change, but also for the design of experiments seeking to investigate movement as a response to temperature across life stages. While we deliberately set out to control for potential carry-over effects in understanding how temperature and body size influenced the flight behavior of \u003cem\u003eV. cardui\u003c/em\u003e, many studies of temperature effects on animals across ontogeny do not conduct such controls (as reviewed by [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]). In studies of thermal effects on movement of invertebrates, it is not uncommon for researchers to rear animals at different temperatures, but conduct all movement tests at the same temperature [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Alternately, some researchers use a single rearing temperature and multiple testing temperatures [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Both of these types of experimental design can confound acclimation to a temperature and carry-over effects. This is because when such experiments use multiple temperatures for either rearing or testing (but not both), alignment of rearing and testing conditions typically occurs at just one temperature (the control), and it is not possible to evaluate whether animals reared at other temperatures would demonstrate similar responses if movement were tested at their respective rearing temperature. For example, Arambourou et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] observed reduced flight time in damselflies reared under \u0026ldquo;heat wave\u0026rdquo; conditions and conclude that \u0026ldquo;carry-over effects of warming experienced during the larval stage reduce adult locomotor ability\u0026rdquo;. However, because their damselflies were reared at three different temperatures (20, 25, and 30\u0026deg;C) and flight trials were conducted at one of those temperatures (20\u0026deg;C), this finding is consistent with both carry-over effects and the degree of acclimation to the temperature under which flight was assessed. Put more simply, \u0026ldquo;heat wave\u0026rdquo; damselflies experienced elevated temperatures during development, but also experienced a dramatic decrease in temperature (of 5 or 10\u0026deg;C) when movement behavior was assessed. Replicating across both rearing and testing temperatures will always be logistically challenging, as required sample sizes increase dramatically with the number of treatment groups. However, we argue that given the potential influence of both thermal acclimation and carry-over effects on movement behavior of ectotherms in particular, it is imperative to use fully-replicated experimental designs. This is not to say that our experimental design (a two-way factorial in which rearing and testing temperatures were crossed) was ideal; an improvement on our study would be the inclusion of an intermediate testing temperature (26\u0026deg;C), which would have allowed us to further disentangle the effects of rearing environment and ambient conditions on flight.\u003c/p\u003e \u003cp\u003eMultiple aspects of morphology have been shown to affect flight performance in butterflies: for example, Barwaerts et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] found effects of body mass, thorax mass, forewing area, forewing length, wing loading, aspect ratio, and the centroid of forewing area on flight performance. Barwaerts et al. [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] found that all of the listed traits correlated positively with flight performance. Understandably, however, most researchers (including us) do not measure all of these variables when assessing the effects of flight morphology on flight performance and behavior. Differences in which variables were measured among studies complicate efforts at synthesis, as traits deemed important in one study may be unmeasured in others. For example, Reim et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] found that increased reared temperature did lead to smaller body size in the tropical butterfly \u003cem\u003eBicyclus anynana\u003c/em\u003e, but also found that sexual dimorphism was a more important influence than rearing temperature on flight behavior. Reim and colleagues [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] reared their butterflies at three temperatures (21, 25, and 29\u0026deg;C) and conducted flight tests at 27\u0026deg;C, finding that flight distance was shorter at higher temperatures. The closest analog to the results of Reim et al. [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] in the current study is the comparison between groups of \u003cem\u003eV. cardui\u003c/em\u003e that were reared at either 24\u0026deg;C or 28\u0026deg;C and given flight tests at 28\u0026deg;C (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). In contrast to their findings, we documented increased flight distance by butterflies reared at the higher temperature. However, we also documented smaller body sizes for \u003cem\u003eV. cardui\u003c/em\u003e reared at the lower temperature, the opposite of the findings for \u003cem\u003eB. anynana\u003c/em\u003e, making it difficult to directly compare the two studies in terms of the effects of rearing temperature on movement behavior.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eDrawing synthetic conclusions about how the movement behavior of insects may respond to climate change is challenging. As described above, differences in experimental designs across studies make comparisons difficult, as researchers vary in both how they manipulate rearing and testing temperatures and in the response variables measured. Further, recent studies have found unexpected results \u0026ndash; for example, Arambourou et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] found that increased rearing temperature decreased flight performance in a damselfly, but via a change in wing shape rather than body size. Thus, while insight into how thermal conditions influence development and subsequent movement behavior is needed to make realistic predictions about future changes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], organismal responses may be complex and potentially unexpected [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e No permits or approvals were required for the experiments with invertebrate animals reported here.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e Not applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e All data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the NMSU Biology Department.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor’s contributions:\u003c/strong\u003e Both authors contributed to conception, study design, and data interpretation. SPM conducted all experimental work and wrote the first draft of the manuscript. KEM contributed to revisions. Both authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Drs. Scott Bundy, Adriana Romero-Olivares, and Shannon McCauley for feedback on earlier drafts of this manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eParmesan, C. Ecological and evolutionary responses to recent climate change. Ann Rev Ecol Evol Sys. 2006;37:637-669.\u003c/li\u003e\n \u003cli\u003eBrooker RW, Travis JM, Clark EJ, Dytham C. Modelling species\u0026rsquo; range shifts in a changing climate: the impacts of biotic interactions, dispersal distance and the rate of climate change. J Theor Biol. 2007;245:59-65.\u003c/li\u003e\n \u003cli\u003eMcCauley SJ, Mabry KE. Climate change, body size, and phenotype dependent dispersal. 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Biol Rev. 2021;96:2113-2126.\u003c/li\u003e\n \u003cli\u003eGhazanfar M, Malik MF, Hussain, M, Iqbal R, Younas M. Butterflies and their contribution in ecosystem: A review. J Ent and Zool Stud. 2016;4:115-118.\u003c/li\u003e\n \u003cli\u003eSunde J, Franz\u0026eacute;n M, Betzholtz PE, Francioli Y, Pettersson LB, P\u0026ouml;yry J, Ryrholm N, Forsman A. Century-long butterfly range expansions in northern Europe depend on climate, land use and species traits. Comm Biol. 2023;6:601.\u003c/li\u003e\n \u003cli\u003eStefanescu C, Alarc\u0026oacute;n M, \u0026Agrave;vila A. Migration of the painted lady butterfly, \u003cem\u003eVanessa cardui\u003c/em\u003e, to north-eastern Spain is aided by African wind currents. J Anim Ecol. 2007;76:888-898.\u003c/li\u003e\n \u003cli\u003eSuchan T, Bataille CP, Reich MS, Toro-Delgado E, Vila R, Pierce NE, Talavera G. A trans-oceanic flight of over 4,200 km by painted lady butterflies. Nat Comm.2024;15:5205.\u003c/li\u003e\n \u003cli\u003eSymanski C, Redak RA. Does fluctuating asymmetry of wing traits capture relative environmental stress in a lepidopteran? Ecol Evol. 2021;11:1199-1213.\u003c/li\u003e\n \u003cli\u003eStevenson RD. The relative importance of behavioral and physiological adjustments controlling body temperature in terrestrial ectotherms. Am Nat. 1985;126:362-386.\u003c/li\u003e\n \u003cli\u003eMedina-B\u0026aacute;ez OA, Lenard A, Muzychuk RA, da Silva CRB, Diamond SE. Life cycle complexity and body mass drive erratic changes in climate vulnerability across ontogeny in a seasonally migrating butterfly. Cons Phys. 2023;11:coad058.\u003c/li\u003e\n \u003cli\u003eP\u0026eacute;rez-Escudero A, Vicente-Page J, Hinz R \u003cem\u003eet al.\u003c/em\u003e idTracker: tracking individuals in a group by automatic identification of unmarked animals. Nat Meth. 2014;11\u003cstrong\u003e:\u003c/strong\u003e743-748.\u003c/li\u003e\n \u003cli\u003eR Core Team. 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Ontogenetic variation in physiology of the painted lady butterfly. 2023. https://doi.org/10.17605/OSF.IO/UP5BJ\u003c/li\u003e\n \u003cli\u003eBenard MF, McCauley SJ. Integrating across life‐history stages: consequences of natal habitat effects on dispersal. Am Nat. 2008;171:553-567.\u003c/li\u003e\n \u003cli\u003eRebolledo AP, Sgr\u0026ograve; CM, Monro K. Thermal performance curves are shaped by prior thermal environment in early life. Fron Phys. 2021;12:738338.\u003c/li\u003e\n \u003cli\u003eArambourou H, Sanmart\u0026iacute;n-Villar I, Stoks R. 2017. Wing shape-mediated carry-over effects of a heat wave during the larval stage on post-metamorphic locomotor ability. Glob Change Bio. 2017;184:279-291.\u003c/li\u003e\n \u003cli\u003eBerwaerts K, Van Dyck H, Aerts P. Does flight morphology relate to flight performance? An experimental test with the butterfly \u003cem\u003ePararge aegeria\u003c/em\u003e. Funct Ecol. 2002;16:484-491.\u003c/li\u003e\n \u003cli\u003eBristow LV, Grundel R, Dzurisin JDK, Wu GC, Li Y, Hildreth A, Hellmann JJ. Warming experiments test the temperature sensitivity of an endangered butterfly across life history stages. J Insect Conserv. 2023;28:1-13.\u003c/li\u003e\n \u003cli\u003eNiehaus AC, Angilletta Jr MJ, Sears MW, Franklin CE, Wilson RS. Predicting the physiological performance of ectotherms in fluctuating thermal environments. J Exp Biol. 2012;215:694-701.\u003c/li\u003e\n \u003cli\u003eVan der Have TM, De Jong G. Adult size in ectotherms: temperature effects on growth and differentiation. J Theor Biol. 1996;183:329-340.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"movement-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"move","sideBox":"Learn more about [Movement Ecology](http://movementecologyjournal.biomedcentral.com/)","snPcode":"40462","submissionUrl":"https://submission.nature.com/new-submission/40462/3","title":"Movement Ecology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"climate change, temperature, flight, movement behavior, butterfly ","lastPublishedDoi":"10.21203/rs.3.rs-4731760/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4731760/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eWith ongoing anthropogenic climate change, there is increasing interest in how organisms are affected by higher temperatures, including how animals respond behaviorally to increasing temperatures. Movement behavior is especially relevant here, as the ability of a species to shift its range is implicitly dependent upon movement capacity and motivation. Temperature may influence movement behavior of ectotherms both directly, through an increase in body temperature, and indirectly, through temperature-dependent effects on physiological and morphological traits that can influence movement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eHere, we investigate the influence of ambient temperature during two life stages, larval and adult, on body size and movement behavior of the painted lady butterfly (\u003cem\u003eVanessa cardui\u003c/em\u003e). We reared painted ladies to emergence at either a “low” (24 °C) or “high” (28 °C) temperature. At eclosion, we assessed flight behavior in an arena test, with half of the adults emerging from each rearing treatment tested at either the “low” or “high” temperature. We had a total of four treatment groups: the control (reared and tested at 24 °C), a consistently high temperature (reared and tested at 28 °C), and two treatments in which butterflies experienced flight tests at a temperature either higher or lower than the one at which they were reared. We measured adult body size, including wingspan, and determined flight speed, distance, and duration from video recordings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eAdult butterflies that experienced the higher temperature during development were larger. We documented an interaction effect of rearing x testing temperature on flight behavior: unexpectedly, the fastest butterflies were those who experienced a change in temperature, whether an increase or decrease, between rearing and testing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eIndividuals that experienced matching thermal environments flew more slowly, but for more time and covering more distance. Overall, the influence of body size \u003cem\u003eper se\u003c/em\u003e on flight was minimal. We conclude that the potential role of “matching” thermal environments across life stages has been underinvestigated with regard to how organisms may respond to warming conditions.\u003c/p\u003e","manuscriptTitle":"Effects of temperature experienced across life stages on morphology and flight behavior of painted lady butterflies (Vanessa cardui)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-17 02:23:30","doi":"10.21203/rs.3.rs-4731760/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-09-30T10:06:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-18T09:54:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-10T17:13:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"42778688517142900342262097828599496540","date":"2024-08-28T11:30:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"233939522325209538221868688054347831521","date":"2024-08-21T13:16:37+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-24T09:53:24+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-17T23:04:51+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-17T23:04:42+00:00","index":"","fulltext":""},{"type":"submitted","content":"Movement Ecology","date":"2024-07-12T17:01:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"movement-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"move","sideBox":"Learn more about [Movement Ecology](http://movementecologyjournal.biomedcentral.com/)","snPcode":"40462","submissionUrl":"https://submission.nature.com/new-submission/40462/3","title":"Movement Ecology","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"069e2fb8-1256-4fd6-acf6-b60af5e42370","owner":[],"postedDate":"August 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T16:02:38+00:00","versionOfRecord":{"articleIdentity":"rs-4731760","link":"https://doi.org/10.1186/s40462-024-00516-3","journal":{"identity":"movement-ecology","isVorOnly":false,"title":"Movement Ecology"},"publishedOn":"2024-12-03 15:57:32","publishedOnDateReadable":"December 3rd, 2024"},"versionCreatedAt":"2024-08-17 02:23:30","video":"","vorDoi":"10.1186/s40462-024-00516-3","vorDoiUrl":"https://doi.org/10.1186/s40462-024-00516-3","workflowStages":[]},"version":"v1","identity":"rs-4731760","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4731760","identity":"rs-4731760","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}