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Carried over: Heat stress in the egg stage reduces subsequent performance in a butterfly

Carried over: Heat stress in the egg stage reduces subsequent performance in a butterfly

  • Michael Klockmann, 
  • Friederike Kleinschmidt, 
  • Klaus Fischer


Increasing heat stress caused by anthropogenic climate change may pose a substantial challenge to biodiversity due to associated detrimental effects on survival and reproduction. Therefore, heat tolerance has recently received substantial attention, but its variation throughout ontogeny and effects carried over from one developmental stage to another remained largely neglected. To explore to what extent stress experienced early in life affects later life stages, we here investigate effects of heat stress experienced in the egg stage throughout ontogeny in the tropical butterfly Bicyclus anynana. We found that detrimental effects of heat stress in the egg stage were detectable in hatchlings, larvae and even resulting adults, as evidenced by decreased survival, growth, and body mass. This study shows that even in holometabalous insects with discrete life stages effects of stress experienced early in life are carried over to later stages, substantially reducing subsequent fitness. We argue that such effects need to be considered when trying to forecast species responses to climate change.


Temperature is one of the most important ecological factors for ectothermic organisms, and the ability to cope with different temperatures is of key importance for species survival and distributions [1,2]. Exposure to high temperatures typically decreases individual fitness and does ultimately cause death [3,4]. Due to ongoing anthropogenic climate change, stressfully high temperatures will be more frequently encountered in the future, which may strongly affect biodiversity [5,6]. Increasing temperature extremes might be of particular importance here, as they have stronger effects on species distributions than mean temperatures [7,8]. Therefore, upper critical thermal limits have recently received substantial attention [911]. Here, tropical ectotherms may be particularly sensitive, as they live already close to their upper thermal limits [8,1214].

When trying to assess the thermal tolerance of a given species, different aspects need to be considered. First, thermal tolerance may differ substantially throughout ontogeny, because developmental stages vary in size, morphology, physiology, and behaviour, which may easily affect thermal tolerance [11,15,16]. Second, life stages may not be entirely independent of one another, such that thermal stress experienced in a specific developmental stage may affect later life (“carry-over effects”; [1721]). Third, even different generations may not be independent, being prone to transgenerational effects [2224]. Nevertheless, the majority of studies on heat tolerance has focussed exclusively on a single stage, typically the adult one [13,25,26]. The concomitant neglect of other stages as well as potential carry over and transgenerational effects may lead to spurious results and misinterpretations [2629], and it is therefore surprising that carry-over effects have not received more attention in the given context to date [20,21,30,31].

To add further complexity, negative effects of high temperatures on growth and development can also be important [14,32,33]. For instance, heat stress during development may reduce adult body size, which may in turn reduce subsequent heat tolerance [16,32,3436]. Besides stress tolerance, body size may furthermore affect longevity, reproductive success as well as competitiveness [32,3739], thereby potentially affecting even subsequent generations. Thus, enhancing our abilities to predict responses to climate change obviously requires the consideration of heat stress on survival and other fitness components throughout development and perhaps even across generations [19,25,40].

Against this background, we here investigate the effects of different temperatures experienced in the egg stage on the heat tolerance of later developmental stages in the tropical butterfly Bicyclus anynana (Butler 1897). Therefore, we test for heat survival (and growth) of hatchlings (i.e. shortly after temperature exposure), larvae and adult butterflies to test for the occurrence and extent of carry-over effects across developmental stages. We predicted that increasing heat stress perceived in the egg stage would reduce subsequent performance, but that detrimental effects are largely restricted to subsequent (i.e. larval) stages. We indeed found that heat stress experienced in the egg stage decreased subsequent survival, growth, and body mass, and that effects were detectable up to the adult stage. Given the holometabolic life cycle of butterflies, these results are surprising and exemplify the non-independence of developmental stages and the important role carry over effects may play in determining tolerance to environmental stressors.

Materials and methods

Study organism and egg sampling

Bicyclus anynana is a tropical, fruit-feeding butterfly ranging from southern Africa to Ethiopia [41]. The species inhabits a highly seasonal environment with alternating wet-warm and dry-cool seasons, such that it relies heavily on phenotypic plasticity to master associated challenges [42,43]. Temperature variation induces, for instance, plastic responses in wing coloration, growth and development, reproduction, and survival [14,16,4447]. Reproduction is confined to the favorable wet season during which oviposition plants are abundantly available [48,49]. A laboratory stock population was established at Greifswald University, Germany, in 2008, from several hundred eggs derived from a well-established stock population at Leiden University, the Netherlands. Several hundred adults are used per generation to produce the subsequent generation, maintaining high levels of heterozygosity at neutral loci [50]. All animals were reared at 27°C, 70% relative humidity, and a photoperiod of L12:D12 within a single temperature-, light- and humidity-controlled climate chamber. To initiate the experiments, we sampled eggs from several hundred females. We did not use a family design here as previous work showed that family as compared with temperature effects are negligible [16].

Experimental design

About 500 B. anynana females were allowed to oviposit on small maize plants, from which eggs were collected one day after oviposition. Eggs were placed into petri dishes in groups of 10 eggs per dish. Dishes were randomly divided among six groups, and afterwards exposed for 24 hours to 27 (control), 29, 31, 33, 35 or 37°C in climate cabinets (Sanyo MLR-351H; Bad Nenndorf, Germany). The climate cabinets were heated up to the target temperature before the transfer of petri dishes, thus, no ramping assay was used. All eggs were exposed to temperature treatments on day 2 after oviposition, and were kept at 27°C throughout except for the 24 h exposure time. The temperatures used are based on previous results, promoting strong differences in survival rates [16]. These temperatures are clearly within the range of temperatures experienced by B. anynana in its natural habitat [14], although exposure times are typically shorter than 24 h. Egg hatching success (mean per petri dish) was subsequently scored under control conditions (27°C, 70% relative humidity, and L12:D12 photoperiod). We used the percentage of dead individuals per dish for further analyses.

Resulting hatchlings were randomly divided into two cohorts (Fig 1). In the first part of the experiment, hatchlings were randomly divided and exposed for 24 h to either control (27°C) or heat (37°C) conditions (climate cabinets Sanyo MLR-351H; Bad Nenndorf, Germany). Therefore, hatchlings were transferred one day after hatching to petri dishes lined with moist tissue and fresh cuttings of their larval host plant (maize) in groups of 10 per dish. We used 23 to 55 replicates per egg temperature and stress treatment. Survival rate (in %) per petri dish was scored under control conditions 24 hours after exposure. Additionally we measured head capsule width of dead and alive hatchlings, using one individual per petri dish.

Fig 1. Schematic figure of the experimental design used.

Eggs collected from stock population females were randomly divided and exposed to six temperatures for 24 hours each. Thereafter, one day-old hatchlings were exposed to either 27°C or 37°C, after which survival and head capsule width were measured. Another cohort of resulting hatchlings was reared under control conditions until adult eclosion and then exposed for 24 h to 37°C, after which heat survival and other traits were scored.

In the second part of the experiment, hatchlings were randomly divided among five cages per egg temperature with 30 individuals each, and were reared under control conditions until adult eclosion. Survival rates during larval and pupal development were scored per cage (%). One day after adult eclosion, all butterflies were individually transferred to plastic cups (125 ml) being provided with water, and exposed for 24 h to 37°C. Afterwards, individuals were back-transferred to control conditions. Note that all individuals were exposed to stressful conditions (37°C), as we were interested in the long-term effects of egg temperature on stress resistance. Survival rate was scored 24 h later (dead or alive). Then, all butterflies were frozen at -80°C. Thus, we here scored heat tolerance in adults having experienced different treatments exclusively in the egg stage. We measured adult body mass, thorax-abdomen ratio, and abdomen fat content for all butterflies after heat exposure. Therefore, frozen butterflies were first weighed to the nearest 0.01 mg (Sartorius LE225D). Afterwards legs, head, and wings were removed on dry ice, and the thorax and abdomen were separated and weighed. We calculated thorax-abdomen ratio as an indicator for the allocation trade-off between mobility (thorax) and reproduction (abdomen) [51]. Abdomen fat content, as an important indicator of condition, was measured after Fischer et al. (2003) but using the less poisonous acetone instead of dichloromethane [45]. In short, abdomens were weighed and subsequently dried for 48 h at 60°C. Abdomen dry masses were scored. Then, fat was extracted using acetone for 48 h, after which abdomens were once again dried and then weighed. Total fat content was calculated by subtracting the fat-free dry mass from the initial dry mass and is given as a percentage.

Statistical analyses

We analysed (1) survival rates of eggs and hatchlings as the percentage of alive individuals per dish, (2) survival rates of larvae and pupae as the percentage of alive individuals per cage, and (3) variation in head capsule width, adult body mass, thorax-abdomen ratio, and abdomen fat content using general / generalized linear mixed models (GLMMs) with egg temperature, heat stress, and sex as fixed effects and cage as random effect (if applicable). Adult survival after heat stress was analysed using a nominal logistic regression on binary data (dead or alive) with egg temperature and sex as fixed effects, cage as a random effect (nested within egg temperature) and adult body mass, thorax-abdomen ratio, and fat content as covariates. Pair-wise comparisons after GLMs were performed employing Tukey’s HSD for unequal sample size. Throughout the text, means are given ± 1 SE. Data were analysed using STATISTICA 8.0 (StatSoft, Tulsa, OK, USA) or JMP 7.0.1 (SAS institute, Cary, NC, USA).


Egg hatching success decreased significantly with increasing temperature from 52.8 ± 1.7% at 27°C to 30.2 ± 1.4% at 37°C (Table 1A; Fig 2A). Hatchling survival was significantly affected by both egg temperature and heat stress, being reduced at higher temperatures and following heat stress (control: 91.1 ± 1.7% > heat: 70.5 ± 1.2%; Table 1B; Fig 2B). Furthermore, a larger difference in hatchling survival between control and stress conditions was found at an egg temperature of 37°C (40.8%) compared with the other temperatures (8.6–20.4%; significant temperature x heat stress interaction). Hatchling head capsule width was significantly negatively affected by egg temperature and heat stress (control: 0.66 ± 0.007 mm > heat: 0.60 ± 0.004 mm; Table 1C; Fig 2C). Moreover, surviving hatchlings had significantly larger head capsule widths than dead individuals (alive: 0.69 ± 0.004 mm > dead: 0.57 ± 0.006 mm). However, all three main factors were involved in significant two-way interactions. First, effects of egg temperature on head capsule width were larger in control (reduction by 17.1% between 27 and 37°C) than in heat-stressed individuals (reduction by 10.0%; significant egg temperature x heat interaction). Second, effects of egg temperature were smaller in dead (reduction by 10.7% between 27 and 37°C) compared with surviving hatchlings (reduction by 16.2%; significant egg temperature x survival interaction). Third, effects of heat stress were restricted to surviving individuals (17.2% versus 0.3% difference between control and stressed individuals; significant heat x survival interaction). Data of the second cohort showed that egg temperature significantly affected survival rate during larval development while pupal development remained unaffected (Table 1D and 1E; Fig 3A).

Fig 2.

Egg survival rates in relation to temperature (a; 24 h at 27, 29, 31, 33, 35 or 37°C), hatchling survival rates in relation to egg temperature and heat stress (b; exposure of hatchlings for 24 h to 27°C or 37°C), and head capsule (HC) width in relation to egg temperature and heat stress for dead and alive individuals (c) in Bicyclus anynana. Given are means ± 1 SE. Sample sizes range between 132 and 199 groups (a), 23 and 55 groups (b), and 6 and 55 groups (c) with 10 individuals each. Different lower case letters above bars indicate significant differences among egg temperatures (Tukey’s HSD for unequal sample size).

Fig 3.

Survival rates until pupation and adult eclosion (a) and male and female adult body mass (b) in relation to egg temperature (24 h at 27, 29, 31, 33, 35 or 37°C) in Bicyclus anynana. Given are means ± 1 SE. Sample size were 5 cages with 30 individuals each (a) and 29 to 73 individuals each (b). Different lower case letters above bars indicate significant differences among temperatures (Tukey’s HSD for equal sample size).

Table 1. Results of general linear models (GLMs) for the effects of (a) egg temperature on egg survival rate, (b) egg temperature and larval heat stress on hatchling survival rate, (c) egg temperature, larval heat stress and survival (dead versus alive individuals) on head capsule width of hatchlings, and for the effects of egg temperature on the survival rate during (d) the larval and (e) the pupal stage in Bicyclus anynana.

Significant P-values are given in bold.

Adult survival after heat stress was only affected by adult body mass, being higher in surviving than in dead butterflies (49.8 ± 0.7 mg > 45.3 ± 1.1 mg). Adult survival did not differ among egg temperatures (Table 2). Butterflies differed significantly in adult body mass, which was lowest at 35°C and 37°C (Table 3A; Fig 3B). Additionally, adult mass was significantly higher in females than in males (females: 60.0 ± 0.6 mg > males: 36.5 ± 0.5 mg) and also differed between rearing cages. Thorax-abdomen ratio, in contrast, was significantly affected by sex only, being higher in males (57.3 ± 0.2%) than in females (43.0 ± 0.2%; Table 3B). Relative fat content, finally, differed significantly between males (16.3 ± 0.4%) and females (5.2 ± 0.2%) and among rearing cages (Table 3C).

Table 2. Results of a nominal logistic regression for the effects of egg temperature (fixed), cage (nested within temperature; random), sex (fixed), adult body mass, thorax-abdomen ratio and abdomen fat content (covariates) on adult heat survival in Bicyclus anynana.

Significant P-values are given in bold.

Table 3. Results of general linear mixed models (GLMMs) for the effects of egg temperature (fixed), cage (nested within temperature; random), and sex (fixed) on (a) adult body mass, (b) thorax-abdomen ratio, and (c) abdomen fat content in Bicyclus anynana.

Significant P-values are given in bold.


In line with earlier studies, we found strong negative effects of increasing temperatures on egg hatching success [16,5257]. Such detrimental effects may arise, for instance, from denaturation of proteins, disruption of membrane structure, interactions with oxygen supply, or dehydration [5557]. Similar considerations may apply to the increased mortality found in hatchlings exposed to heat stress (37°C). More interestingly, our results additionally demonstrate severe and long-lasting effects of thermal stress experienced early in ontogeny on later life. Specifically, we found that the temperatures exclusively experienced in the egg stage yielded effects on hatchling and larval survival resembling those found for egg hatching rate, and a similar tendency even for pupal survival. Thus, survival probability was clearly reduced in later life stages when having experienced temperature stress early in life. Especially in individuals that were exposed to heat stress twice (egg and hatchling stage) survival was compromised. Even in the adult stage, negative effects of higher egg temperatures were still visible, as adult mass was reduced in individuals having experienced higher egg temperatures. Reduced body mass may have detrimental effects on other fitness components such as stress tolerance and reproduction [32,34,5860]. Thus, while egg temperature in our study had no direct effect on adult heat survival, our data suggest an indirect effect through reduced body mass.

The negative effects reported above suggest the existence of (energetic) costs associated with high temperatures. For instance, the heat shock response is considered to be costly which may reduce subsequent body size [56,61,62]. Small body size in turn often compromises other aspects of life such as stress tolerance, reproduction or competition [32,60]. Note here that higher temperatures experienced during development generally result in smaller bopdy size in ectotherms [16,32].

We measured hatchling head capsule width to also test for changes in body mass early in development. Note that head capsule width is closely related to hatchling mass in B. anynana [63]. Indeed, we found smaller head capsule widths in hatchlings that resulted from eggs exposed to higher temperatures [18,64,65]. This suggests increased metabolic losses at higher temperatures, which results in reduced body mass in turn contributing to the diminished overall performance. Alternatively, reduced feeding rate may have caused smaller head capsule widths [14]. Effects of egg temperature were larger in control than in heat-stressed hatchlings, likely reflecting the overall strongly reduced head capsule width in heat-stressed individuals (Fig 2C). Note that negative effects of heat stress were restricted to surviving individuals, indicating detrimental effects of heat on hatchling growth. Additionally, surviving hatchlings were larger than dead ones, meaning that larger individuals had a higher heat resistance [32,60] or that surviving individuals had more time for feeding and thus growth. The latter may also explain why the effects of egg temperature were more pronounced in surviving than in dead hatchlings. These results show that, as expected, acute heat stress has detrimental effects on hatchling size likely through negative effects on feeding and metabolism.

Taken together, the stress imposed on the egg stage was clearly visible throughout all subsequent life stages including resulting adults, being measurable as reduced body mass and survival. In holometabolous insects, the life cycle is clearly divided into distinct developmental stages (egg, larva, pupa, adult) separated by major developmental transitions. Potter et al. (2011), for instance, showed that negative effects of different egg temperatures were strong in early life but disappeared rapidly in subsequent life stages ([18]; see also [33]). Our results are somewhat similar in that effect size also declined with increasing time since the original stress. However, in our study effects were still visible even in the adult stage, indicating substantial carry-over effects throughout life and that developmental stages are not independent [1821,31,64,66]. Furthermore, our results suggest that the stress and the concomitant deficiencies experienced early in life cannot be fully compensated for during development, although compensatory growth is a common feature in insects [21,67,68].

The females’ higher adult body mass compared with males is likely driven by a positive relation between body size and fecundity, while male insects are typically selected for fast development increasing mating opportunities [14,69]. Likewise, the males’ higher thorax-abdomen ratio and fat content seem to reflect sex-specific selection pressures, favouring flight ability and duration in males in order to succeed in competition for females [14,33,70].

Ongoing climate change, increasing ambient temperature and the frequency of extreme weather events like heat waves and drought periods, will probably have important consequences for life on earth [71,72]. Especially heat waves may have dramatic effects on population dynamics [7274]. Our data show that even relatively short heat waves may have severe impacts based on (1) the direct mortality induced but (2) also through fitness reductions throughout the entire life cycle even if surviving the acute stress. Here, we demonstrated that heat stress during the egg stage reduced subsequent survival and body mass up to the adult stage. One caveat of our study is that we used an arbitrarily chosen time period of 24 hours to simulate heat weaves, which does obviously not resemble natural conditions particularly closely. However, based on earlier results we do not expect that more natural settings would change our conclusions substantially [75]. The shown detrimental effects even on adult body mass seem highly relevant for two reasons. First, body mass seems to be a crucial constraint on heat stress tolerance in B. anynana in general [16]. Second, this may even cause transgenerational effects [76], because smaller females typically lay fewer and/or smaller eggs potentially giving rise to offspring with reduced fitness [32,3739,45]. In summary, our findings may have important implications for enhancing our abilities to predict the fate of particular species under ongoing climate change, indicating that carry-over effects throughout the life cycle as well as transgenerational effects need to be considered when trying to forecast species responses to climate change [19].


  1. 1. Sunday JM, Bates AE, Dulvy NK. Global analysis of thermal tolerance and latitude in ectotherms. Proc R Soc London B Biol Sci. 2011;278: 1823–1830. pmid:21106582
  2. 2. Araújo MB, Ferri-Yáñez F, Bozinovic F, Marquet PA, Valladares F, Chown SL. Heat freezes niche evolution. Ecol Lett. 2013;16: 1206–1219. pmid:23869696
  3. 3. Le Moullac G, Haffner P. Environmental factors affecting immune responses in Crustacea. Aquaculture. 2000;191: 121–131.
  4. 4. Overgaard J, Sørensen JG. Rapid thermal adaptation during field temperature variations in Drosophila melanogaster. Cryobiology. 2008;56: 159–162. pmid:18295194
  5. 5. Clusella-Trullas S, Blackburn TM, Chown SL. Climatic predictors of temperature performance curve parameters in ectotherms imply complex responses to climate change. Am Nat. 2011;177: 738–751. pmid:21597251
  6. 6. Hoffmann AA, Chown SL, Clusella-Trullas S. Upper thermal limits in terrestrial ectotherms: how constrained are they? Funct Ecol. 2013;27: 934–949.
  7. 7. Zimmermann NE, Yoccoz NG, Edwards TC, Meier ES, Thuiller W, Guisan A, et al. Climatic extremes improve predictions of spatial patterns of tree species. Proc Natl Acad Sci U S A. 2009;106: 19723–19728. pmid:19897732
  8. 8. Kellermann V, Overgaard J, Hoffmann AA, Flojgaard C, Svenning J-C, Loeschcke V. Upper thermal limits of Drosophila are linked to species distributions and strongly constrained phylogenetically. Proc Natl Acad Sci U S A. 2012;109: 16228–16233. pmid:22988106
  9. 9. Sunday JM, Bates AE, Dulvy NK. Thermal tolerance and the global redistribution of animals. Nat Clim Chang. 2012;2: 686–690.
  10. 10. Kaspari M, Clay NA, Lucas J, Yanoviak SP, Kay A. Thermal adaptation generates a diversity of thermal limits in a rainforest ant community. Glob Chang Biol. 2015;21: 1092–102. pmid:25242246
  11. 11. Pincebourde S, Casas J. Warming tolerance across insect ontogeny: influence of joint shifts in microclimates and thermal limits. Ecology. 2015;96: 986–997. pmid:26230019
  12. 12. Deutsch CA, Tewksbury JJ, Huey RB, Sheldon KS, Ghalambor CK, Haak DC, et al. Impacts of climate warming on terrestrial ectotherms across latitude. Proc Natl Acad Sci U S A. 2008;105: 6668–6672. pmid:18458348
  13. 13. Kingsolver JG. The well-temperatured biologist. Am Nat. 2009;174: 755–768. pmid:19857158
  14. 14. Fischer K, Klockmann M, Reim E. Strong negative effects of simulated heat waves in a tropical butterfly. J Exp Biol. 2014;217: 2892–2898. pmid:24902752
  15. 15. Krebs RA, Loeschcke V. Resistance to thermal stress in preadult Drosophila buzzatii: Variation among populations and changes in relative resistance across life stages. Biol J Linn Soc. 1995;56: 517–531.
  16. 16. Klockmann M, Günter F, Fischer K. Heat resistance throughout ontogeny: body size constrains thermal tolerance. Glob Chang Biol. 2017;23: 686–696. pmid:27371939
  17. 17. Hernández Moresino RD, Gonçalves RJ, Helbling EW. Sublethal effects of ultraviolet radiation on crab larvae of Cyrtograpsus altimanus. J Exp Mar Bio Ecol. 2011;407: 363–369.
  18. 18. Potter KA, Davidowitz G, Arthur Woods H. Cross-stage consequences of egg temperature in the insect Manduca sexta. Funct Ecol. 2011;25: 548–556.
  19. 19. Hettinger A, Sanford E, Hill TM, Russell AD, Sato KNS, Hoey J, et al. Persistent carry-over effects of planktonic exposure to ocean acidification in the Olympia oyster. Ecology. 2012;93: 2758–2768. pmid:23431605
  20. 20. Zhang W, Rudolf VHW, Ma C-S. Stage-specific heat effects: timing and duration of heat waves alter demographic rates of a global insect pest. Oecologia. 2015;179: 947–957. pmid:26255274
  21. 21. Zhang W, Chang X-Q, Hoffmann A, Zhang S, Ma C-S. Impact of hot events at different developmental stages of a moth: the closer to adult stage, the less reproductive output. Sci Rep. 2015;5: 10436. pmid:26000790
  22. 22. Marshall DJ. Transgenerational plasticity in the sea: context-dependent maternal effects across the life history. Ecology. 2008;89: 418–427. pmid:18409431
  23. 23. Donelson JM, Wong M, Booth DJ, Munday PL. Transgenerational plasticity of reproduction depends on rate of warming across generations. Evol Appl. 2016;9: 1072–1081. pmid:27695516
  24. 24. Fox CW. The Ecology of Body Size in a Seed Beetle, Stator limbatus: Persistence of Environmental Variation Across Generations? Evolution. 1997;51: 1005. pmid:28568589
  25. 25. Kingsolver JG, Arthur Woods H, Buckley LB, Potter KA, MacLean HJ, Higgins JK. Complex life cycles and the responses of insects to climate change. Integr Comp Biol. 2011;51: 719–732. pmid:21724617
  26. 26. Radchuk V, Turlure C, Schtickzelle N. Each life stage matters: the importance of assessing the response to climate change over the complete life cycle in butterflies. J Anim Ecol. 2013;82: 275–285. pmid:22924795
  27. 27. Pahkala M, Laurila A, Merilä J. Carry–over effects of ultraviolet–B radiation on larval fitness in Rana temporaria. Proc R Soc London B Biol Sci. 2001;268: 1699–1706.
  28. 28. Fischer J, Phillips NE. Carry-over effects of multiple stressors on benthic embryos are mediated by larval exposure to elevated UVB and temperature. Glob Chang Biol. 2014;20: 2108–2116. pmid:24259382
  29. 29. Levy O, Buckley LB, Keitt TH, Smith CD, Boateng KO, Kumar DS, et al. Resolving the life cycle alters expected impacts of climate change. Proc R Soc London B Biol Sci. 2015;282: 20150837. pmid:26290072
  30. 30. Vonesh JR. Sequential predator effects across three life stages of the African tree frog, Hyperolius spinigularis. Oecologia. 2005;143: 280–290. pmid:15657758
  31. 31. Weinig C, Delph LF. Phenotypic plasticity early in life constrains developmental responses later. 2001;55: 930–936. pmid:11430653
  32. 32. Kingsolver JGJG Huey RB. Size, temperature, and fitness: three rules. Evol Ecol Res. 2008;10: 251.
  33. 33. Klockmann M, Karajoli F, Reimer S, Kuczyk J, Fischer K. Fitness implications of simulated climate change in three species of Copper butterflies (Lepidoptera: Lycaenidae). Biol J Linn Soc. 2016.
  34. 34. Sibly RM, Atkinson D. How rearing temperature affects optimal adult size in ectotherms. Funct Ecol. 1994;8: 486–493.
  35. 35. Terblanche JS, Hoffmann AA, Mitchell KA, Rako L, le Roux PC, Chown SL. Ecologically relevant measures of tolerance to potentially lethal temperatures. J Exp Biol. 2011;214: 3713–3725. pmid:22031735
  36. 36. Nielsen ME, Papaj DR. Effects of developmental change in body size on ectotherm body temperature and behavioral thermoregulation: caterpillars in a heat-stressed environment. Oecologia. 2015;177: 171–179. pmid:25367578
  37. 37. Fischer K, Bot ANM, Zwaan BJ, Brakefield PM, Central P. Genetic and environmental sources of egg size variation in the butterfly Bicyclus anynana. Heredity. 2004;92: 163–9. pmid:14722579
  38. 38. Hopwood PE, Moore AJ, Tregenza T, Royle NJ. Niche variation and the maintenance of variation in body size in a burying beetle. Ecol Entomol. 2016;41: 96–104.
  39. 39. English S, Cowen H, Garnett E, Hargrove JW. Maternal effects on offspring size in a natural population of the viviparous tsetse fly. Ecol Entomol. 2016;41: 618–626.
  40. 40. Bowler K, Terblanche JS. Insect thermal tolerance: what is the role of ontogeny, ageing and senescence? Biol Rev. 2008;83: 339–355. pmid:18979595
  41. 41. Larsen TB. The butterflies of Kenya and their natural history. Oxford: Oxford University Press; 1991.
  42. 42. Lyytinen A, Brakefield PM, Lindström L, Mappes J. Does predation maintain eyespot plasticity in Bicyclus anynana? Proc R Soc London B Biol Sci. 2004;271: 279–283. pmid:15058439
  43. 43. Pijpe J, Brakefield PM, Zwaan BJ. Phenotypic plasticity of starvation resistance in the butterfly Bicyclus anynana. Evol Ecol. 2007;21: 589–600.
  44. 44. Wijngaarden PJ, Koch PB, Brakefield PM. Artificial selection on the shape of reaction norms for eyespot size in the butterfly Bicyclus anynana: direct and correlated responses. J Evol Biol. 2002;15: 290–300.
  45. 45. Fischer K, Brakefield PM, Zwaan BJ. Plasticity in butterfly egg size: why larger offspring at lower temperatures? Ecology. 2003;84: 3138–3147.
  46. 46. Fischer K, Dierks A, Franke K, Geister TL, Liszka M, Winter S, et al. Environmental effects on temperature stress resistance in the tropical butterfly Bicyclus anynana. PLoS One. 2010;5: e15284. pmid:21187968
  47. 47. Franke K, Heitmann N, Tobner A, Fischer K. Fitness costs associated with different frequencies and magnitudes of temperature change in the butterfly Bicyclus anynana. J Therm Biol. 2014;41: 88–94. pmid:24679977
  48. 48. Brakefield PM, Reitsma N. Phenotypic plasticity, seasonal climate and the population biology of Bicyclus butterflies (Satyridae) in Malawi. Ecol Entomol. 1991;16: 291–303.
  49. 49. Brakefield PM. Phenotypic plasticity and fluctuating asymmetry as response to environmental stress in the butterfly Bicyclus anynana. In: Bijlsma R, Loeschke V, editors. Environmental Stress: Adaptation and Evolution. Birkhäuser, Basel; 1997. pp. 65–78.
  50. 50. Van’t Hof AE, Zwaan BJ, Saccheri IJ, Daly D, Bot ANM, Brakefield PM. Characterization of 28 microsatellite loci for the butterfly Bicyclus anynana. Mol Ecol Notes. 2005;5: 169–172.
  51. 51. Hughes CL, Hill JK, Dytham C. Evolutionary trade-offs between reproduction and dispersal in populations at expanding range boundaries. Proc R Soc London B Biol Sci. 2003;270: 147–150.
  52. 52. Tewksbury JJ, Huey RB, Deutsch CA. Putting the heat on tropical animals. Science. 2008;320: 1296–1297. pmid:18535231
  53. 53. Andrew NR, Hart RA, Jung M-P, Hemmings Z, Terblanche JS. Can temperate insects take the heat? A case study of the physiological and behavioural responses in a common ant, Iridomyrmex purpureus (Formicidae), with potential climate change. J Insect Physiol. 2013;59: 870–880. pmid:23806604
  54. 54. Rukke BA, Aak A, Edgar KS. Mortality, temporary sterilization, and maternal effects of sublethal heat in bed bugs. PLoS One. 2015;10: e0127555. pmid:25996999
  55. 55. Klose MK, Robertson RM. Stress-induced thermoprotection of neuromuscular transmission. Integr Comp Biol. 2004;44: 14–20. pmid:21680481
  56. 56. Chown SL, Terblanche JS. Physiological diversity in insects: ecological and evolutionary contexts. Adv In Insect Phys. 2006;33: 50–152. pmid:19212462
  57. 57. Potter K, Davidowitz G, Woods HA. Insect eggs protected from high temperatures by limited homeothermy of plant leaves. J Exp Biol. 2009;212: 3448–3454. pmid:19837886
  58. 58. Blanckenhorn WU. The evolution of body size: What keeps organisms small? Q Rev Biol. 2000;75: 385–407. pmid:11125698
  59. 59. Gibbs AG. Water balance in desert Drosophila: lessons from non-charismatic microfauna. Comp Biochem Physiol Part A Mol Integr Physiol. 2002;133: 781–789.
  60. 60. Chidawanyika F, Terblanche JS. Rapid thermal responses and thermal tolerance in adult codling moth Cydia pomonella (Lepidoptera: Tortricidae). J Insect Physiol. 2011;57: 108–117. pmid:20933517
  61. 61. Krebs RA, Loeschcke V. Costs and benefits of activation of the heat-shock response in Drosophila melanogaster. Funct Ecol. 1994;8: 730–737.
  62. 62. Sørensen JG, Kristensen TN, Loeschcke V. The evolutionary and ecological role of heat shock proteins. Ecol Lett. 2003;6: 1025–1037.
  63. 63. Fischer K, Zwaan B, Brakefield P. How does egg size relate to body size in butterflies? Oecologia. 2002;131: 375–379. pmid:28547709
  64. 64. Pechenik JA. Larval experience and latent effects—metamorphosis is not a new beginning. Integr Comp Biol. 2006;46: 323–33. pmid:21672745
  65. 65. Russell J, Phillips NE. Synergistic effects of ultraviolet radiation and conditions at low tide on egg masses of limpets (Benhamina obliquata and Siphonaria australis) in New Zealand. Mar Biol. 2009;156: 579–587.
  66. 66. Marshall DJ, Morgan SG. Ecological and Evolutionary Consequences of Linked Life-History Stages in the Sea. Curr Biol. 2011;21: 718–725. pmid:21959162
  67. 67. Metcalfe NB, Monaghan P. Compensation for a bad start: grow now, pay later? Trends Ecol Evol. 2001;16: 254–260. pmid:11301155
  68. 68. Dmitriew C, Rowe L. Resource limitation, predation risk and compensatory growth in a damselfly. Oecologia. 2005;142: 150–154. pmid:15372227
  69. 69. Karl I, Fischer K. Why get big in the cold? Towards a solution to a life-history puzzle. Oecologia. 2008;155: 215–225. pmid:18000685
  70. 70. Fischer K, Fiedler K. Sex-related differences in reaction norms in the butterfly Lycaena tityrus (Lepidoptera: Lycaenidae). Oikos. 2000;90: 372–380.
  71. 71. Battisti DS, Naylor RL. Historical warnings of future food insecurity with unprecedented seasonal heat. Science. 2009;323: 240–244. pmid:19131626
  72. 72. Coumou D, Rahmstorf S. A decade of weather extremes. Nat Clim Chang. 2012;2: 1–6.
  73. 73. McKechnie AE, Wolf BO. Climate change increases the likelihood of catastrophic avian mortality events during extreme heat waves. Biol Lett. 2010;6: 253–256. pmid:19793742
  74. 74. Sentis A, Hemptinne J-L, Brodeur J. Effects of simulated heat waves on an experimental plant-herbivore-predator food chain. Glob Chang Biol. 2013;19: 833–842. pmid:23504840
  75. 75. Fischer K, Kölzow N, Höltje H, Karl I. Assay conditions in laboratory experiments: is the use of constant rather than fluctuating temperatures justified when investigating temperature-induced plasticity? Oecologia. 2011;166: 23–33. pmid:21286923
  76. 76. Sgrò CM, Terblanche JS, Hoffmann AA. What Can Plasticity Contribute to Insect Responses to Climate Change? Annu Rev Entomol. 2016;61: 433–451. pmid:26667379