Supplementary material from "Heat hardening capacity in <i>Drosophila melanogaster</i> is life stage specific and juveniles show the highest plasticity"

Variation in stress resistance and adaptive plastic responses during ontogeny have rarely been addressed, despite the possibility that differences between life stages can affect range margins and thermal tolerance of species. Here, we assessed the thermal sensitivity and hardening capacity of <i>Drosophila melanogaster</i> across developmental stages from <i>larval</i> to the adult stage. We observed strong differences between life stages in heat resistance with adults being most heat resistant followed by <i>puparia</i>, <i>pupae</i> and <i>larvae</i>. The impact of heat hardening (1 h at 35°C) on heat resistance changed during ontogeny with the highest positive effect of hardening observed in <i>puparia</i> and <i>pupae</i> and the lowest in adults. These results suggest that immobile life stages (<i>puparia</i> and <i>pupae</i>) have evolved high plasticity in upper thermal limits whereas adults and <i>larvae</i> rely more on behavioural responses to heat stress allowing them to escape from extreme high temperatures. While most studies on the plasticity of heat resistance in ectotherms have focused on the adult life stage, our findings emphasize the crucial importance of juvenile life stages of arthropods in understanding the thermal biology and life stage-specific physiological responses to variable and stressful high temperatures. Failure to acknowledge this complication might lead to biased estimates of species' ability to cope with environmental changes, such as climate change.