Class-wide sequence, structural, and docking-profile diversity of insect odorant receptors and structural specialization of Orco
- School of Life Science and Technology, Northwestern Polytechnical University, China
- College of Life Sciences, Shaanxi Normal University, China
Figures
Figure 1 with 1 supplement
Evolutionary ecology of insect OR repertoires (Figure 1—source data 1–3).
(A) Phylogeny of 115 insect species (see Materials and methodsfor details). Genome size and the number of OR genes for each species are shown as bar plots. Ecological parameters, including larval and adult diets, habitat, and circadian rhythm, are displayed as labels with color coding corresponding to the legend below. Different geological periods are separated by shaded intervals. The tree was visualized using the iTOL (Letunic and Bork, 2024). (B) Boxplot showing the number of OR genes for each insect order. (C–F) Results of two-sided pGLS tests assessing the relationship between intact OR gene numbers and various ecological parameters: adult diet (C), larval diet (D), circadian rhythm (E), and habitat (F). Dots represent OR counts of each species. N represents the counts of species. Animal silhouettes were obtained from PhyloPic.org.
© 2014, Descouens and Keesey. The Rhyacophila dorsalis silhouette is reproduced from PhyloPic, and available under a CC BY-SA 3.0 license.
© 2013, Stemonitis and Keesey. The Petrobius maritimus silhouette is reproduced from PhyloPic, and available under a CC BY-SA 3.0 license.
-
Figure 1—source data 1
Genome information.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig1-data1-v1.xlsx
-
Figure 1—source data 2
Living habits of 115 species.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig1-data2-v1.xlsx
-
Figure 1—source data 3
Insect species tree.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig1-data3-v1.zip
Figure 1—figure supplement 1
Structural screening strategy and pGLS sensitivity analysis of OR quantity.
(A) The stringent screening strategy for complete OR genes. All homologously annotated OR genes underwent structure modeling. Structural similarity was used as an indirect measure to assess sequence reliability (Dali Z-score>20). Finally, manual inspection was performed to refine the intact OR genes. For detailed procedures, refer to the Materials and methods. (B) Comparison of OR counts from this study and previous annotations between species with and without conspecific sequences in the query set. Error bars represent means ± SEM. Each dot represents one species; 4<n<5. Significance was assessed using the Mann–Whitney U test. (C) Comparison after excluding the OR annotation results for the outlier Anagrus nilaparvatae. (D–G) pGLS tests for adult diet, larval diet, habitat, and circadian rhythm after OR counts were updated to the larger of structurally intact OR counts and published OR counts.
Figure 2 with 1 supplement
Subfamily classification of ORs based on SSN (Figure 2—source data 1 and Figure 2—source data 2).
(A) Schematic representation of the ‘trunk–branch’ strategy pipeline for constructing the similarity network and classifying subfamilies. First, all ORs were subjected to greedy clustering based on sequence similarity to generate nodes. The average similarity between nodes was then calculated, and nodes were treated as units to identify core regions (trunks) using a strict similarity threshold. By gradually relaxing the threshold, new core regions and additional nodes (branches) emerged. After a final merging step based on defined criteria, the optimal community structure was determined using modularity as an index (see Materials and methods). (B) Distribution of pairwise sequence similarity among all ORs. The red line indicates the mean similarity. (C) SSN of ORs constructed using the ‘trunk–branch’ strategy. Each node represents ORs sharing >60% sequence identity. Edges represent node pairs with an average sequence alignment E-value of <1 × 10⁻²⁵. Node colors indicate SeqC classifications. (D) Heatmap depicting the number of ORs within different SeqCs across various insect orders. Due to substantial differences in OR numbers among orders, the values are ln(count +1)-transformed. The bar plot above the heatmap shows the number of SeqCs present in each order, while the bar plot on the right indicates the proportion of ORs within each SeqC generated by tandem duplication. Two ORs located within 5 kb of each other on the genome were defined as tandem duplicates. TD, tandem duplication.
-
Figure 2—source data 1
Amino acid sequences of insect ORs.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig2-data1-v1.zip
-
Figure 2—source data 2
Orthologous groups identified within SeqC26, SeqC28, and StrC23.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig2-data2-v1.zip
Figure 2—figure supplement 1
SSN intra-subfamily OR sequence similarity and evidence supporting the accuracy of SeqC.
(A) Modularity changes between SeqCs at different SSN alignment e-value thresholds. Purple represents modularity obtained using the ‘trunk–branch’ algorithm, while yellow denotes the theoretical optimal modularity based on the Louvain algorithm. (B) Pairwise sequence similarity within different SeqCs. The SeqC colors are consistent with Figure 2C, while gray indicates SeqCs with no interactions with others. Boxplots represent the first quartile minus 1.5 times the interquartile range, the interquartile range, the first quartile, the mean, the third quartile, and the third quartile + 1.5 interquartile range. Dots indicate outliers. (C) Mapping of Acromyrmex echinatior (Aech) OR subfamilies onto the SSN. Different colors represent previously defined hymenopteran subfamilies. (D) Phylogenetic relationship between previously defined hymenopteran subfamilies and our SeqC classification. Red dots indicate branches with bootstrap values greater than 80. Branches with the same subfamily designation as previous studies are collapsed. The phylogenetic tree is based on the previous study (Zhou et al., 2015). Animal silhouettes were obtained from PhyloPic.org. (E) Greyscale interaction matrix showing interactions between SeqCs under different e-value thresholds. At an e-value of 1e-25, SeqC boundaries are distinct. As the threshold increases to 1e-17, interactions between SeqCs become more frequent, with extensive interactions observed at 1e-13. The SeqC divisions remain clear within approximately 8 orders of magnitude. Different color bars represent different SeqCs, consistent with Figure 2C. (F) Proportions of Lepidopteran PRs distributed across different SeqCs. (G). Heatmap showing the number of ORs within different SeqCs across species. Due to significant variation in OR numbers among species, the OR counts are ln-transformed. The top bar plot represents the number of SeqC types in each species. Gray shading indicates boundaries between insect orders.
Figure 3 with 1 supplement
StSN and structural community diversity of ORs (Figure 3—source data 1).
(A) Sequence similarity versus structural similarity of insect ORs, assessed using pairwise comparisons with Dali. Each dot represents comparison between a pair of ORs, with color indicating point density. (B) StSN of ORs constructed using the ‘trunk–branch’ strategy. Nodes represent ORs with pairwise Dali Z-scores >50. Edges connect nodes with an average pairwise Z-score of at least 42.9. Node colors denote StrCs classifications. (C) Structural comparison of ORs and Orco. The structures were aligned using US-align, with multiple structural alignments performed for annotated Orco and OR structures (visualized in PyMol). Colors represent pLDDT scores provided by AlphaFold2, indicating the confidence of structural modeling. (D) Visualization of differences among StrCs. The heatmap shows the number of ORs within different StrC across insect orders, with OR counts ln(count +1)-transformed due to large inter-order variations. The bar plot above the heatmap indicates the number of StrC in each order. On the right, a ridge plot illustrates the flexibility rates of different structural regions across StrCs. Flexibility rate = 1 - conservation rate. Light gray represents transmembrane regions, white denotes loop regions, and dark gray marks the anchor domain of ORs. Colors correspond to StrCs, with gray indicating StrCs without interactions. (E) Two types of ORs in StrC17—long IL3 ORs and general ORs—with the IL3 regions highlighted in color. (F) Phylogenetic analysis of StrC17 ORs based on amino acid sequences. Red circles indicate bootstrap values, with most ORs collapsed based on bootstrap support. Gray branches represent the clade of long IL3 ORs, and the dN/dS values are shown in blue on the corresponding evolutionary branches.
-
Figure 3—source data 1
The likelihood ratio test results for StrC17 ORs.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
StrC classification and the phylogenetic tree of StrC17 ORs.
(A) Modularity changes between StrCs at different Dali Z-score thresholds. Purple represents modularity obtained using the ‘trunk–branch’ algorithm, while yellow denotes the theoretical optimal modularity based on the Louvain algorithm. (B) Comparison of IL2 loop amino acid lengths between all ORs (n = 8791) and Orco (n=114). A two-sided t-test was used to assess significance. (C) Grayscale interaction matrix showing interactions between StrCs under different Z-score thresholds. At a Z-score of 42.9, the boundaries between StrCs are distinct. At a threshold of 41.8, interactions between StrCs increase, with extensive interactions observed at 41.2. Different color bars represent distinct StrCs, consistent with Figure 3B. Gray indicates StrCs without interactions. (D) Binding-pocket volume of the shared structural cluster StrC23. Error bars represent means ± SEM; n = 4124, 4667, and 114 receptors for StrC23, non-StrC23, and Orco, respectively.Tukey’s post hoc test was performed after one-way ANOVA. (E) Comparison of IL3 length between the conserved fly OR cluster that recognizes 1-octen-3-ol and mosquito ORs that recognize 1-octen-3-ol. Error bars represent means ± SEM. Each dot represents one species; n = 20 and 4 for flies and mosquitoes, respectively. Welch’s t-test was used. ***p<0.001. (F) Maximum likelihood phylogenetic tree of StrC17 ORs, with Orco as the outgroup. Green indicates long IL3 ORs, and red circles mark bootstrap values. The tree was visualized using iTOL. (G) Species tree of dipteran species included in the study. Red circles represent species with long IL3 ORs, while hollow circles indicate species without long IL3 ORs. Animal silhouettes were obtained from PhyloPic.org. (H) Heatmap showing the number of ORs within different StrCs across species. Due to significant variation in OR numbers among species, the OR counts are ln-transformed. The top bar plot indicates the number of StrC types in each species. Shading marks the boundaries between insect orders. ****p<0.0001.
Figure 4 with 2 supplements
Structural features associated with Orco specialization (Figure 4—source data 1 and 2).
(A) Differences in EL2, IL2, and binding pocket properties between M. hrabei and T. domestica ORs. Only ORs with closed binding pockets were analyzed, and all scores were normalized. Circles and triangles represent the minimum and maximum scores after column normalization, respectively. The presence of an EL2 β-sheet is indicated with a check mark. (B) Differences in amino acid properties of the EL2 region between OR and Orco. Hydrophilic amino acids are shown in blue, neutral amino acids in green, and hydrophobic amino acids in black (visualized by WebLogo Crooks et al., 2004). The x-axis numbers represent amino acid positions from the multiple sequence alignment, with highly gapped positions removed. (C) MD simulation-based analysis of the movement trajectory of VOC (geranyl acetate) near Orco and OR. The red protein represents ApisOR5, and the blue protein represents ApisOrco. The green area indicates the probability of VOC remaining above the binding pocket, while the pink represents its probability of occupying other regions of the protein (see Materials and methods). Statistical significance was evaluated using a Chi-Squared test. The red pentagram indicates the initial position of geranyl acetate in the simulation. (D) Changes in relative calcium fluorescence intensity (ΔF/F0) in cells. Cells were stimulated with 100 μM of the OR47a agonist pentyl acetate (PA) at 50 s and 100 μM the Orco agonist VUAA1 at 180 s. el2.DmOR47a: OR47a mutant in which the EL2 region of OR47a was replaced with that of Orco. mut.DmOr47a: the EL2 region of wild-type DmOr47a contains an inserted short peptide. The total number of cells (n=30) was derived from three independent replicate experiments. The shading indicates the 95% confidence interval. (E) Relative calcium fluorescence intensity in cells following stimulation with PA and VUAA1, respectively. Error bars represent means ± SEM. n=30. Statistical significance was determined using Tukey’s HSD test. (F) Schematic illustration of the mechanism by which the β-sheet structure in the EL2 region of Orco influences VOC proximity to the binding pocket. (G) Comparative analysis of binding pocket parameters (volume, hydrophobicity score, and polarity score) between Orco and ORs across all studied insect species. Statistical significance was determined using a two-sided t-test. **** p<0.0001; n.s., not significant.
-
Figure 4—source data 1
Calcium fluorescence responses in cells transfected with each plasmid construct.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig4-data1-v1.xlsx
-
Figure 4—source data 2
Structural and binding-pocket parameters of MhORs and TdomORs.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig4-data2-v1.csv
Figure 4—figure supplement 1
The driving factors underlying the origin of Orco.
(A) The number of VOCs recognized by Tdom1-8 and Orco. VOCs with a DOCK score < –8 kcal/mol were considered bound. The number of bound VOCs is shown on a log2 scale. (B) Proportion of hydrophilic amino acids in the EL2 region of Orco and OR. Statistical significance was assessed using a two-sided t-test. Dots represent outliers. (C) The distance between geranyl acetate and its initial position above ApisOR5 and ApisOrco was measured after MD simulation. The results indicate that Orco exhibits a dispersive effect on small molecules. (D) Examples of calcium imaging experiments with HEK293 cells co-transfected with DmOr47a+Orco constructs, the el2.DmOr47a mutant construct (in which the EL2 region of DmOr47a was replaced with that of Orco), and the mut.DmOr47a mutant construct (in which a short peptide is inserted into the EL2 region of DmOr47a at the position corresponding to the EL2 structure of Orco, rather than forming a β-sheet structure). Schematic diagrams of different plasmid constructs are shown on the far left. CMV: cytomegalovirus promoter. c.i., chimeric intron; ori, origin of replication. Fluorescence changes are shown following stimulation with PA (middle column) and VUAA1 (right column). White dashed circles indicate the cells used for fluorescence quantification. The experiment was independently replicated three times; results from one representative trial are shown here.Scale bars, 50 μm; the same scale applies to all micrographs in panel D. (E) Box plot showing the proportion of EL2 β-sheet ORs among total ORs for different insect orders. Dots represent the proportion of β-sheet ORs in each species. (F) Average proportion of β-sheet ORs among total ORs for six major insect orders. The proportion of β-sheet ORs decreases progressively with evolutionary time. * p<0.05, **** p<0.0001.
Figure 4—video 1
Molecular dynamics trajectories of VOCs above the binding pockets of OR and Orco.
Figure 5 with 2 supplements
Classification of insect OR docking-profile communities (Figure 5—source data 1 and 2).
(A) 2D odor space based on molecule physicochemical properties. Each dot represents an odor molecule, with different colors indicating different functional groups. (B) Relationship between hit rate and docking score, inferred using Bayesian statistics. The black curve shows the distribution of hit rates and docking scores across all OR–VOC docking results. The top plateau has a hit rate of 22%, while the bottom plateau has a hit rate of 0%. Cyan dots represent the mean hit rate ± SEM for each docking score interval. n = 3.The orange line indicates the peak hit rate at a docking score of –8 kcal/mol. The gray curve represents posterior distributions from Bayesian inference, with n=500. (C) dPSN and dPC of ORs. Nodes represent ORs with pairwise PCCs >0.6 based on docking scores. Edges represent node pairs with an average PCC ≥0.48. (D) Labels for the recognition of different functional group small molecules by each dPC. Red indicates a tendency to recognize a higher number of odor molecules from a particular functional group, while blue indicates a tendency to recognize fewer. Circle size represents the degree of this tendency.
-
Figure 5—source data 1
The physicochemical properties and functional group labels of the 14,499 VOCs.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig5-data1-v1.csv
-
Figure 5—source data 2
Matrix of all OR–VOC docking scores.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig5-data2-v1.zip
Figure 5—figure supplement 1
Insect OR–VOC binding profiles, dPC labeling accuracy, and distribution of docking-profile communities across insect orders.
(A) Binding profiles of insect ORs with VOCs from different functional groups. Each point represents the interaction between an OR and a small molecule. A docking score below −8 kcal/mol was classified as predicted binding. (B) Modularity changes between docking-profile communities at different PCC thresholds. Purple represents the docking-profile community modularity obtained using the ‘trunk–branch’ algorithm, while yellow denotes the theoretical optimal community modularity based on the Louvain algorithm. (C) Distribution of terpenoids in the two-dimensional odor space. (D–H) Hit rates for ORs from single insect taxa. Species include Drosophila melanogaster, Anopheles gambiae, Helicoverpa armigera, Spodoptera littoralis, and Locusta migratoria. Cyan dots represent the mean hit rate ± SEM for each docking-score interval. The gray curve represents posterior distributions from Bayesian inference, with n=500. The black curve shows the distribution of hit rates and docking scores across all OR–VOC docking results. (I) Number of VOCs recognized by different dPCs. Colors represent different dPCs, consistent with Figure 5C. (J) Accuracy of dPC labeling using Drosophila ORs as an example. The AUC represents the relationship between the predicted small molecule recognition tendency labels and the experimentally validated number of recognized VOCs. (K) Number of ORs in different docking-profile communities across insect orders. Due to large variations in OR numbers among orders, the OR counts are ln-transformed. (L) Wilson scores for different dPCs across insect orders. Wilson scoring was applied to correct for potential errors caused by low OR numbers in certain orders, providing a more balanced evaluation of dPC representation.
Figure 5—figure supplement 2
Bayesian prior modeling to optimize VOC selection by balancing information acquisition and ligand discovery.
(A) Sigmoid functional form for the hit-rate model. (B–D) Marginal Bayesian prior (blue) and posterior (red) distributions (n=100,000) for each model parameter. (B) Top. (C) Dock50. (D) Slope.
Figure 6 with 4 supplements
Relationships between OR sequence, structure, and docking-profile communities across insect orders (Figure 6—source data 1).
The phylogenetic tree follows the structure from Figure 1. On the right, the mapping relationships between all insect OR SeqCs, StrCs, and dPCs are shown, considering only ORs classified into communities. Time nodes for different geological periods are based on the previous study (Misof et al., 2014) and are separated by gray shading. The timeline beneath the tree marks the origin of various insect orders. Animal silhouettes were obtained from PhyloPic.org.
© 2014, Descouens and Keesey. The Rhyacophila dorsalis silhouette is reproduced from PhyloPic, and available under a CC BY-SA 3.0 license.
© 2013, Stemonitis and Keesey. The Petrobius maritimus silhouette is reproduced from PhyloPic, and available under a CC BY-SA 3.0 license.
-
Figure 6—source data 1
Community annotation of insect ORs.
- https://cdn.elifesciences.org/articles/110877/elife-110877-fig6-data1-v1.csv
Figure 6—figure supplement 1
Relationships between OR sequence, structure, and docking-profile communities across insect orders (all ORs).
The phylogenetic tree follows the structure from Figure 1. On the right, the mapping relationships between all insect OR SeqCs, StrCs, and dPCs are shown. Time nodes for different geological periods are based on previous studies (Misof et al., 2014) and are separated by gray shading. The timeline beneath the tree marks the origin of various insect orders. Animal silhouettes were obtained from PhyloPic.org.
Figure 6—figure supplement 2
Sequence, structural, and docking-profile community associations of ORs in holometabolous and non-holometabolous insects.
The phylogenetic tree follows the structure from Figure 1. On the right, the mapping relationships between all insect OR SeqCs, StrCs, and dPCs are shown, considering only ORs classified into specific communities. Time nodes for different geological periods are based on previous studies (Misof et al., 2014) and are separated by gray shading. The timeline beneath the tree marks the origin of various insect orders. Animal silhouettes were obtained from PhyloPic.org.
Figure 6—figure supplement 3
Changes in insect OR SeqC types and dPC proportions across different geological periods.
(A–B) Normalized analysis of SeqC diversity in holometabolous and non-holometabolous insects at the species level (A) and OR-count level (B) Error bars represent means ± SEM; n = 97 holometabolous species and 18 non-holometabolous species. Welch’s t-test was used. ***p<0.001. (C) The number of SeqC types in insect ORs across different geological periods. (D) Changes in the number of ORs within different dPC categories before and after the EPME. (E) Proportional changes in different dPCs of insect ORs before and after the EPME. EPME, end-Permian mass extinction.
Figure 6—figure supplement 4
Comparison of OR SeqC diversity between Holometabola and non-holometabolous Neoptera.
Tables
Author response table 1
Numbers of OR genes in previously annotated species.
| Species | Annotation Method in Previous Study | Previous Reported OR Count | OR Count in This Study | Conspecific Query Available | Reference |
|---|---|---|---|---|---|
| Pieris brassicae | Genome + transcriptome | 60 | 53 | No | (Wang et al. 2023) |
| Plutella xylostella | Genome | 95 (67 intact genes + 26 truncated genes + 2 pseudogenes) | 77 | Yes | (Engsontia et al. 2014) |
| Diabrotica virgifera virgifera | Genome + transcriptome | 193 (124 intact genes) | 78 | No | (Paddock and Corcoran 2025) |
| Anagrus nilaparvatae | Genome | 88 | 330 | No | (Ma et al. 2022) |
| Solenopsis invicta | Genome | 396 | 473 | Yes | (Mier et al. 2022) |
| Gonatopus flavifemur | Genome | 54 | 38 | No | (Xiao et al. 2023) |
| Telenomus remus | Genome | 46 | 57 | No | (Xiao et al. 2023) |
| Drosophila melanogaster | Genome | 60 | 56 | Yes | (Robertson et al. 2003) |
| Bombyx mori | Genome | 66 (including 2 pseudogenes) | 64 | Yes | (Tanaka et al. 2009) |
| Anopheles gambiae | Genome | 79 | 67 | Yes | (Carey et al. 2010) |
Additional files
-
Supplementary file 1
Benchmarking of insect odorant receptor annotation.
Tables A-C assess the completeness and reliability of the odorant receptor annotation and structural-filtering workflow. Table A compares receptor counts with published repertoires. Table B summarizes correspondence between predicted receptors and curated reference annotations. Table C examines the genomic locations of selected Bombyx mori candidates to assess potential exon-merging artifacts in tandem receptor regions.
- https://cdn.elifesciences.org/articles/110877/elife-110877-supp1-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/110877/elife-110877-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Class-wide sequence, structural, and docking-profile diversity of insect odorant receptors and structural specialization of Orco
eLife 15:RP110877.
https://doi.org/10.7554/eLife.110877.4
