Thermal stress-induced cell death pathways in Drosophila: molecular insights into apoptosis, necrosis, and autophagy

Climate change threatens ectotherms, yet the cellular mechanisms underlying thermal injury remain poorly characterized. Here, we used an integrative approach combining acclimation treatments, transcriptional profiling of cell-death-related genes, and fluorescence-based cellular markers to compare cold- and heat-induced cell death pathways in third-instar Drosophila melanogaster larvae. Individuals were exposed to cold (-3°C) or heat (38°C) stress across three acclimation phenotypes. Cold acclimation enhanced cold tolerance but reduced heat tolerance, and vice versa, revealing clear acclimation trade-offs. Melanized lesions (black spots) appeared during recovery, predominantly after cold stress, and strongly predicted mortality; cold acclimation reduced their incidence from 82% to 13%. Cold stress induced upregulation of pro-apoptotic genes (reaper, grim, sickle) and caspases (Dronc, Dcp-1, Damm), whereas heat stress predominantly induced Hsps genes (upregulation over 4000-fold) and some autophagy-related genes (Atg1, Atg6). Larvae with black spots exhibited hallmarks of necrosis, including low cell viability, severe cytoskeletal collapse, and nuclear loss. Heat-stressed larvae showed elevated cytosolic Ca²⁺, while all stressed groups exhibited increased reactive oxygen species. No autophagy-associated acidification was detected under our conditions. We propose that thermal stress induces a continuum of cellular responses: moderate stress activates protective mechanisms, greater damage triggers apoptosis, and severe injury in susceptible phenotypes leads to necrosis with secondary melanization in sensitive tissues. By distinguishing the respective signatures of apoptosis, necrosis, and autophagy under contrasting thermal stresses, our study identifies necrosis as a major and previously underappreciated driver of thermal mortality in insects. Thermal acclimation mitigates cell death but only when matching subsequent stress, highlighting physiological trade-offs.