Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer‐feeding bark beetles Ips typographus L. (“Ityp”) and Dendroctonus ponderosae Hopkins (“Dpon”) (Scolytinae), and the pine weevil Hylobius abietis L. (“Habi”; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. The results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2‐phenylethanol (2‐PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2‐PE, suggesting a shared evolutionary history of 2‐PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer‐feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm nonhost plants, and 2‐PE being important for intraspecific communication and/or playing a putative role in beetle–microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs.

Insects are able to detect a plethora of olfactory cues in the environment using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. We addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer-feeding bark beetles Ips typographus ('Ityp') and Dendroctonus ponderosae ('Dpon') (both in subfamily Scolytinae), and the pine weevil Hylobius abietis ('Habi'; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. Results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2-phenylethanol (2-PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatile (GLV) alcohols. Both groups of orthologues belong to the major coleopteran OR group 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2-PE, suggesting a shared evolutionary history of 2-PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer-feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm non-host plants, and 2-PE being important for intraspecific communication and/or playing a putative role in beetle-microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of beetle ORs.

Introduction: The striped ambrosia beetle Trypodendron lineatum (Coleoptera, Curculionidae, Scolytinae) is a major forest pest in the Holarctic region. It uses an aggregation pheromone and host and non-host volatiles to locate suitable host trees, primarily stressed or dying conifer trees. The beetles bore into the xylem and inoculate spores of their obligate fungal mutualist Phialophoropsis ferruginea inside their excavated egg galleries, with the fungus serving as the main food source for the developing larvae. Olfactory sensory neuron (OSN) responses to pheromones and host volatiles are poorly understood in T. lineatum and other ambrosia beetles, and nothing is known about potential responses to fungal volatiles.Methods: We screened responses of OSNs present in 170 antennal olfactory sensilla using single sensillum recordings (SSR) and 57 odor stimuli, including pheromones, host and non-host compounds, as well as volatiles produced by P. ferruginea and fungal symbionts of other scolytine beetles.Results and Discussion: Thirteen OSN classes were characterized based on their characteristic response profiles. An OSN class responding to the aggregation pheromone lineatin was clearly the most abundant on the antennae. In addition, four OSN classes responded specifically to volatile compounds originating from the obligate fungal mutualist and three responded to non-host plant volatiles. Our data also show that T. lineatum has OSN classes tuned to pheromones of other bark beetles. Several OSN classes showed similar response profiles to those previously described in the sympatric bark beetle Ips typographus, which may reflect their shared ancestry.