Broad sensory expression and selective functional roles of Ir25a in the malaria mosquito Anopheles stephensi

Mosquitoes rely heavily on their peripheral nervous system to sense various cues during host-seeking and blood-feeding. Ionotropic receptors (IRs) are a family of chemosensory receptors that play fundamental roles in mosquito chemical and physical cue detection. Among the three IR coreceptors, Ir25a is the most evolutionarily ancestral and conserved, whereas its function remains poorly understood, particularly in Anopheles mosquitoes. In this study, we integrated transgenic fluorescent labeling, single-nucleus RNA-seq (snRNA-Seq), electrophysiology, and behavioral assays to determine the functions of Ir25a in An. stephensi, an urban malaria vector. We found that Ir25a is broadly expressed in sensory neurons of multiple appendages in both female and male mosquitoes, including the antennae, maxillary palps, labella, and tarsi. Notably, Ir25a is localized in both CO2-sensing and odor-sensing neurons in the maxillary palps. Detailed snRNA-Seq profiling further supports extensive co-expression of Ir25a with CO2-sensitive gustatory receptors (GRs) and odor-sensitive odorant receptors (ORs) in chemosensory neurons, while also delineating accessory cell types not previously characterized in mosquito snRNA-seq. Surprisingly, Ir25a mutants display minimal changes in peripheral chemical responses in both antennae and maxillary palps. In contrast, Ir25a mutants exhibit severe thermosensory deficits, reduced CO₂-evoked takeoff, complete abolishment of host-seeking and blood-feeding, along with reduced mating success. Taken together, this study systematically characterizes the broad expression of Ir25a and identifies its selective functional roles in sensory physiology, as well as the profound impact on host detection in the malaria mosquito An. stephensi, contributing to our long-term effort in Anopheles mosquito control and malaria prevention.