Phylogenetic reconstruction of termite ORs

The tree is based on 182 protein sequences from five species of termites and the bristletail Lepisma saccharina as a basal insect outgroup, and also includes the sequences of OR co-receptor (OrCO). The topology and branching supports were inferred using the IQ-TREE maximum likelihood algorithm with the JTT+F+R8 model and supported by 10,000 iterations of ultrafast bootstrap approximation. Protein sequences of termite ORs can be found under the same labeling in Johny et al. (2023). Lepisma saccharina sequences are listed in Thoma et al. (2019). Arrowheads highlight the four ORs from Prorhinotermes simplex selected for functional characterization. Fully annotated version of the tree is provided as Supplementary Fig. S1. Heatmap shows the abundances of 50 OR transcripts identified in the RNAseq data from P. simplex antennae available in NCBI SRA archive under accession SRX17749141.

SSR responses of transgenic D. melanogaster ab3 sensillum expressing PsimOR9, 14, 30 and 31 to the panel of 17 volatiles with biological relevance for termites

A Heatmap showing the average responses of the four ORs as Δ spikes/s from 3–6 independent replicates. B. Comparison of SSR responses of transgenic Drosophila melanogaster ab3A neurons expressing PsimOR14 (ab3A:PsimOR14) and W1118 D. melanogaster. The bars show the average Δ spikes/s values from five independent replicates ± SEM. C. Characteristic SSR traces of ab3A:PsimOR14 and W1118 flies for 1 µg dose of neocembrene and geranylgeraniol. D. Dose response curve of ab3A:PsimOR14 SSR responses to neocembrene. The graph shows average Δ spikes/s values ± SEM based on nine replicates (8 in case of 100 ng and 4 in case of 500 ng stimulations). The curve fit and ED50 value were calculated using log(agonist) vs. response non-linear algorithm with least square fit method and the constraint of minimal response > 0. The raw data for all graphs is provided in Supplementary Tables S1–S6.

SSR responses of transgenic D. melanogaster ab3 sensillum expressing PsimOR14 to the complete set of 73 compounds (Panels 1–4)

A. Heatmap showing the average responses as Δ spikes/s from 3–6 independent replicates. C. Tuning curve of PsimOR14 for the 73 compounds contained in panels 1–4. The raw data is provided in Supplementary Tables S7–S9.

Distribution of olfactory sensilla on the last antennal segment of P. simplex workers

A. Arrows show the localization of selected representatives of the five distinguished multiporous grooved sensilla types (S I–V) on a SEM photograph of the last flagellomere. Scale bar represents 50 µm. B. Detailed view on the five identified types of multiporous grooved sensilla using HR-SEM. Scale bar represents 500 nm. C. Detailed view on SSR traces of spontaneous spikes from the neocembrene-detecting sensillum of S I type, showing spikes of three different amplitudes. D. SSR traces recorded on neocembrene-detecting sensillum for neocembrene and geranylgeraniol.

SSR responses of the neocembrene-detecting SB I sensillum on the last flagellomere of P. simplex worker

A SSR responses to the main panel of volatiles with biological relevance for termites. The bars show the average Δ spikes/s values from 8–12 replicates ± SEM. The raw data is provided in Supplementary Tables S10. B. Characteristic SSR traces of the neocembrene-detecting SB I sensillum for neocembrene and geranylgeraniol. C. Dose response curve of the SSR responses to neocembrene by the SB I sensillum. The graph shows average Δ spikes/s values ± SEM based on 9–11 replicates. The curve fit and ED50 value were calculated using log(agonist) vs. response non-linear algorithm with least square fit method and the constraint of minimal response > 0. The raw data is provided in Supplementary Table S11.

PsimOR14 gene, transcript and protein structures, docking and MD simulations

A Genomic locus containing PsimOR14 and PsimOR15. PsimOR14 gene consists of 1 non-coding and 5 protein coding exons. B. PsimOR14 transcript with 6 exons represented as blue boxes with variable height according to protein-coding (higher) or untranslated (smaller) regions. ORF is marked by blue arrow. C. Transmembrane architecture of PsimOR14. In red are shown seven residues interacting with each of the three studied ligands, the green ellipse shows the intracellular flap the most impacted by ligand binding. D. Modelled apoform of PsimOR14. Red region denotes the binding site identified via docking, green region represents the intracellular flap region. E. Holoforms of PsimOR14 with sticks representing ligands without hydrogens (cyan = neocembrene, red = geranylgeraniol, orange = squalene). F. Relative mean volumes explored by atoms of PsimOR14 per simulation step when liganded with neocembrene, geranylgeraniol and squalene. PsimOR14 apoform represents the baseline zero. Gray lines show the binding site region, the green line delimits the intracellular flap the most affected by ligation. The whole PsimOR14 protein shown on the left, detail on the binding region in the middle and the intracellular flap on the right. Nucleotide and protein sequences of PsimOR14 are provided in Supplementary Table S12 and as NCBI entry under accession OR921181.

Docking scores, contributions of van der Walls and electrostatic forces and relative dynamicity values of PsimOR14 upon binding of neocembrene, geranylgeraniol and squalene

Caste comparison of PsimOR14 expression and EAG responses between P. simplex workers and soldiers

A Volcano plot representing edgeR differential gene expression analysis of all 50 P. simplex ORs in RNAseq data from soldier and workers heads. Colored dots mark ORs reaching absolute value of log2 fold change ≥ 1, horizontal lines represent p-value thresholds of 0.05 and 0.01. Numeric values of the edgeR and DESeq2 differential expression analysis are provided in Supplementary Table S15. B. EAG responses of whole antenna preparations of workers and soldiers to neocembrene at a dose of 10 ng (mean ± SD shown on log2 scale). Inter-caste differences were compared using t-test on log2-transformed data. Raw data is shown in Supplementary Table S16.