Abstract Neuronal activity is temperature sensitive and affects behavioral traits important for individual fitness, such as locomotion and courtship. Yet, we do not know enough about the evolutionary response of neuronal phenotypes in new temperature environments. Here, we use long-term experimental evolution of Drosophila simulans populations exposed to novel temperature regimes. Here, we demonstrate a direct relationship between thermal selective pressure and the evolution of neuronally expressed molecular and behavioral phenotypes. Several essential neuronal genes evolve lower expression at high temperatures and higher expression at low temperatures, with dopaminergic neurons standing out by displaying the most consistent expression change across independent replicates. We functionally validate the link between evolved gene expression and behavioral changes by pharmacological intervention in the experimentally evolved D. simulans populations as well as by genetically triggered expression changes of key genes in D. melanogaster. As natural temperature clines confirm our results for Drosophila and Anopheles populations, we conclude that neuronal dopamine evolution is a key factor for temperature adaptation.

How do these results from laboratory evolution relate to natural populations? There are latitudinal allele frequency clines of genes encoding neuronally expressed dopamine receptors (Dop1R1, Dop2R, DopEcR) and transporters (DAT and VMAT) in natural populations of Drosophila and Anopheles 20-24 ( Supplementary Table 11). This strongly argues that the evolution of temperature-dependent neuronal dopamine signaling is not a laboratory artefact but a common response of insects to selection at high temperatures in nature. We propose that the evolutionary response of dopamine signaling to elevated temperatures is an important step to mitigate the deleterious impact of temperature change. Assuming that the pleiotropic architecture differs between species, we anticipate an increase in phenotypic divergence across species for different traits regulated by dopamine, such as pigmentation, locomotion, courtship and other behaviors [25][26][27] . Ple Ddc K + channel E Voltagegated K + channel s Voltagegated Ca 2+ channels Presynaptic active zone SNARE complex