Habitat specialization shapes the evolution of transcriptional responses to hypoxia

Abstract Oxygen limitation is a widespread environmental constraint that shapes physiological and evolutionary responses across ecosystems. A central unresolved question is whether tolerance to hypoxia reflects generalized stress responses or coordinated regulatory strategies shaped by long-term environmental exposure. Here, we use comparative transcriptomic analyses to examine gene expression responses to low oxygen in two aquifer-dwelling stoneflies (Isocapnia sp. and Paraperla frontalis) and one benthic species (Sweltsa sp.) under controlled conditions. Time-series analysis in Isocapnia sp. revealed a multi-phase transcriptional response involving early regulatory activation, metabolic reorganization, and late-stage cellular stabilization. Across aquifer taxa, hypoxia was associated with downregulation of energy-demanding processes and upregulation of pathways related to oxidative stress mitigation, metabolite transport, and protein folding, consistent with coordinated cellular adjustment to oxygen limitation. In contrast, the river benthic species exhibited transcriptional profiles dominated by neural signaling, ion channel activity, and structural remodeling, which are patterns consistent with acute physiological stress rather than coordinated regulation. Despite these differences, all taxa showed modulation of ion transport and calcium signaling pathways, suggesting conserved mechanisms of hypoxia sensing. Together, these results indicate that transcriptional responses to hypoxia differ systematically with habitat and are consistent with the evolution of distinct regulatory strategies in chronically hypoxic environments. Significant statement Oxygen limitation is a common environmental challenge that affects organisms across aquatic and terrestrial ecosystems, yet the mechanisms by which species cope with low oxygen remain incompletely understood. A key question is whether tolerance to hypoxia reflects common stress responses or the evolution of coordinated metabolic regulatory strategies under chronic exposure. By comparing gene expression responses in closely related aquatic insects from oxygen-variable underground aquifers and oxygen-rich river habitats, we show that species that evolved under persistent hypoxia exhibit transcriptional patterns consistent with energy conservation and cellular stabilization, whereas those experiencing hypoxia as a transient stress display signature of physiological disruption. These findings highlight fundamental differences between evolutionarily adaptive and acute stress-driven responses to environmental change and provide insight into how organisms may respond to increasing hypoxia under global change. Competing Interest Statement The authors have declared no competing interest.