Not All Bark Beetles Smell the Same: Population-Level Functional Olfactory Polymorphisms in Ips typographus Pheromone Receptor ItypOR33

Eurasian spruce bark beetle Ips typographus, a natural part of forest ecosystems, is a major threat to Norway spruce forests during outbreaks. Olfaction plays a crucial role in the survival and range expansion of these beetles, amid forest disturbances and climate change. As the current management strategies are suboptimal for controlling outbreaks, the reverse chemical ecology approaches based on pheromone receptors offer promising alternatives. While the search for pheromone receptors is in progress, recently found chromosomal inversions indicates signs of adaptation in this species. Our attempts to characterise one of the highly expressed odorant receptors, ItypOR33, located in an inversion, led to the discovery of polymorphic variants distributed with similar frequency across 18 European populations. Deorphanizing ItypOR33 and its variant ItypOR33a using the Drosophila empty-neuron system (DeNS) revealed ItypOR33 tuned to amitinol, a heterospecific pheromone component in Ips spp., whereas its variant tuned to (S)-(-)-ipsenol, a conspecific pheromone component of I. typographus. The in silico approaches revealed the structural basis of variations by predicting putative ligand-binding sites, tunnels and ligand-receptor interactions. However, no sex-specific differences were found in the ItypOR33 expression, and its ligand amitinol elicited behavioural and electrophysiological responses. Reporting population-level functional olfactory polymorphisms for the first time in a non-model organism-bark beetles, provides key evidence for further exploring their survival and adaptation in forests. Additionally, these findings indicate potential long-term complexities of managing bark beetles in forests.