Odorant receptor orthologues in conifer-feeding beetles display conserved responses to ecologically relevant odours

Abstract

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.

Keywords: Coleoptera; Curculionidae; HEK293 cells; de-orphanization; evolutionary conservation; functional characterization.

FIGURE 1

Maximum‐likelihood phylogeny of odorant receptors (ORs) from beetles of Curculionidae and Cerambycidae. Included are OR amino acid sequences from the curculionids Hylobius abietis (“Habi”; red), Ips typographus (“Ityp”; blue), Dendroctonus ponderosae (“Dpon”; orange) and the cerambycid Megacyllene caryae (“Mcar”; black). The tree is based on a mafft alignment, constructed using fasttree, and rooted with the conserved Orco lineage. The major coleopteran OR groups are indicated by the black arcs (Mitchell et al., 2020). The 12 groups of simple (1:1:1) OR orthologues across the three curculionids are highlighted in yellow; two clades housing putative simple orthologues shared by the cerambycid M. caryae and two of the curculionids are highlighted in purple. Receptors in OR group 2B that were functionally characterized in the present study and the previously characterized McarOR5 (Mitchell et al., 2012) are labelled by the compounds that activate them (GLV‐OHs = green leaf volatile alcohols; 2‐PE =2‐phenylethanol). Local node support values were calculated using the Shimodaira–Hasegawa (SH) test implemented in fasttree, and are indicated on branch nodes by the shaded circles; support increases with the brightness of the circles. The scale bar indicates the number of amino acid substitutions per site. The sources of sequence data and explanation of receptor suffixes are detailed in the Materials and Methods section

FIGURE 2

Conserved responses to green leaf volatile (GLV) alcohols in curculionid odorant receptor (OR) orthologues. (a) Response of Hylobius abietis OR4 (Homo sapiens codon optimized; HabiOR4^HsCO) to select compounds in the screening experiments (30 µm stimulus concentration; n = 3 biological replicates, n _total =9). (b) Dose‐dependent response of HabiOR4^HsCO to the five active GLVs, indicating similar sensitivities to four of the compounds (see main text for EC₅₀ values; n = 3–5 biological replicates, n _total =9–15). (c) Screening responses of Dendroctonus ponderosae OR9 (H. sapiens codon‐optimized; DponOR9^HsCO; n = 3 biological replicates, n _total =9), and (d) dose‐dependent responses of DponOR9^HsCO to the two most active ligands (n = 5 biological replicates, n _total =15). (e) Screening response of Ips typographus OR5 (ItypOR5; n = 3–5 biological replicates, n _total =9–15), and (f) dose‐dependent response of ItypOR5 to the most active ligand (n = 6 biological replicates, n _total =18). Ligand‐induced activation was recorded from cells induced (+) to express the exogenous Orco and OR genes and from noninduced (−) control cells. VUAA1 was tested at 50 µm as a control for functional Orco expression. Asterisks indicate significantly stronger responses in induced compared to noninduced cells (at p <.001; see main text for details on statistics). Ligands eliciting <3% increased fluorescence in screening assays were excluded from dose–response trials. Error bars show SEM. All data from induced and noninduced cells to all 62 test compounds are reported in Data S1

FIGURE 3

Conserved responses to 2‐phenylethanol in curculionid odorant receptor (OR) orthologues. (a) Response of Hylobius abietis OR3 (HabiOR3) to select compounds in the screening experiments (30 µm stimulus concentration; n = 3–4 biological replicates, n _total =9–12). (b) Dose‐dependent responses of HabiOR3 (n = 3 biological replicates, n _total =9; see main text for EC₅₀ value). (c) Screening responses of Dendroctonus ponderosae OR8 (H. sapiens codon‐optimized; DponOR8^HsCO; n = 3 biological replicates, n _total =9), and (d) dose‐dependent responses of DponOR8^HsCO (n = 9 biological replicates, n _total =27; see main text for EC₅₀ value). (e) Screening responses of Ips typographus OR6 (ItypOR6; n = 3 biological replicates, n _total =9), and (f) dose‐dependent responses of ItypOR6 (n = 3 biological replicates, n _total =9; see main text for EC₅₀ value). Ligand‐induced activation was recorded from cells induced (+) to express the exogenous beetle Orco and OR genes and from noninduced (−) control cells. VUAA1 was tested at 50 µm as a control for functional Orco expression. Asterisks indicate significantly stronger responses in induced compared to noninduced cells (at p <.001; see main text for details on statistics). Error bars show SEM. All data from induced and noninduced cells to all 62 test compounds are reported in Data S1