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Figure 1. Single sensillum recording on different types of olfactory sensilla in the common bed bug, C. lectularius.(A) Schematic image of single sensillum recording in the olfactory sensilla on bed bug antennae. (B) SEM photo (modified from Liu et al., 2014) showing the different types of olfactory sensilla on bed bug antennae. The scale bar indicates 20 μM. (C) The highest neural responses for each type of olfactory sensillum to different human odorants.
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Figure 2. Summary of the responses of olfactory sensilla in the common bed bug, C. lectularius, to human odorants.(A) Distribution of firing frequencies for different strengths of responses to different odorant/sensillum combinations; (B) Response biases to different odorant categories with firing frequencies higher than 50 spikes/s. Sensilla that failed to show a response ≥15 spikes/s were considered non-responders. The excitatory response of 50 spikes/s was selected as the criterion which represents a 20% increase of the largest firing frequency recorded (248.5 spikes/s, for nonanal in Dα sensilla). (C) Different tuning curves of olfactory sensilla for human odorants. The 104 odorants are distributed along the x axis according to the strengths of the responses they elicited from each sensillum. The odors that elicited the strongest responses are near the center of the distribution; those that elicited the weakest responses are near the edges. The order of the odorants therefore differs for different sensilla. Negative values indicate inhibitory responses. The kurtosis value, K value, as a statistical measure of ‘peakedness’, is shown on the right side for each plot.
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Figure 3. A heatmap presentation of the responses of olfactory sensilla of the common bed bug, Cimex lectularius, to human odorants.Distinctive response profiles (spikes/s) of Dα, Dβ, Dγ, C, E1 and E2 sensilla to different chemical groups of human odorants were tested through single sensillum recording, with at least six replicates for each odorant on different individual sensilla at a dose of 1:100 v/v. The solvent, DMSO, produced no stimulation in any of the sensilla types.
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Figure 4. Dose-dependent responses of bed bug olfactory sensilla to human odorants.The dose-dependent response curve is presented as a mean value ± SEM, n ≥ 6. (A) Dose-dependent response of Dα sensilla to two stereoisomers of 2-hexen-1-ol; (B) Dose-dependent response of Dβ sensilla to ketones; (C) Dose-dependent response of Dβ sensilla to halides; (D) Dose-dependent response of Dγ sensilla to aromatics; (E) Dose-dependent responses of Dγ sensilla to aldehydes; and (F) Dose-dependent response of grooved peg C sensilla to two amines, propylamine and butylamine. The X axis describes the logarithm dilution series from 1:10 to 1:105 v/v in (A–F) and from 1:102 to 1:106 v/v in (E).
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Figure 5. Temporal dynamics of olfactory sensilla in response to human odorants.(A) Temporal structures of neuronal responses of Dα sensilla in response to aldehyde, ketone and aromatic odorants at a dose of 1:100 v/v. The left side of the figure shows a trace representing the mean value of spikes (n = 8, error bars are not shown) recorded during each 100 ms sampling period. The right side of the figure shows the hierarchical cluster analysis for the odorants, with the corresponding categories based on the action potential number in each single 100 ms sampling period. (B) Temporal structures of dose-dependent responses of Dα sensilla in response to hexanal, nonanal and sucaltone at doses ranging from 1:102 (10 µg/µL) to 1:106 (0.001 µg/µL) v/v. Horizontal bars indicate the duration of the stimulation (500 ms).
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Figure 6. Primary presentations of odorant space among the olfactory sensilla.(A) Hierarchical cluster analysis for human odorants based on the Euclidean distances between them. Odorants are color coded by chemical class. (B) Typical odorants with close chemical structure are clustered together in the Hierarchical cluster analysis. (C) Relationships among human odorants of the indicated chemical classes at a dose of 1:102 v/v revealed by PCA. Odorants are color coded by chemical class as in Fig. 6A. In PCA, vectors quantifying the responses of the 6 antennal sensilla to each tested odor are projected onto a three-dimensional region. Each axis represents the normalized neuronal responses of the olfactory sensilla in a new coordinate system determined by PCA. This three-dimensional representation captures 87.67% of the variation in the original 6-dimensional data set.
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Figure 7. Phylogenetic analysis of ClOrco and ClOrs and their tissue-specific expression in the common bed bug.(A) Phylogenetic analysis of ClOrco with Orco orthologs from other insect species. The tree was constructed with MEGA6 based on a Clustal alignment of the amino acid sequences and selected Orco sequences from eight other insects. Numbers above individual branches indicate the percentage of 1,000 bootstrap replication trees in that branch. The scale bar indicates 10% divergence. The accession numbers for each Orco of insect species are: KC881255.1 for Apolygus lucorum; JQ639214.1 for Lygus lineolaris; RPRC000476; Rhodnius proxilus; CLEC006196 for Cimex lectularius; AM689918 for Tribolium castaneum; XM_001359327 for Drosophila pseudoobscura; XM_001651376 for Aedes aegypti; AY843205 for Anopheles gambiae; DQ231246.1 for Culex quinquefaciatus. (B) Phylogenetic analysis of ClOrs. The phylogenetic tree shows the relationships of two ClORs and ClOrco to their equivalents in Rhodnius proxilus. The tree was rooted with 76 odorant receptors of R. proxilus from the Vectorbase. Numbers above branches represent the percentage of 1,000 bootstrap replication trees in that branch, with only those above 50% shown. The ClOrco is clearly clustered with RpOrco, with 99% bootstrap support; ClOr2 is clustered with RpOr105, with 94% bootstrap support; and ClOr1 is clustered with RpOr10, although with only 38% bootstrap support. There is no support for the backbone of the relationships within the branches. (C) Tissue-specific expression profiles for ClOr1, ClOr2 and ClOrco. The house-keeping gene, Clrpl8, was selected as the control in the semi-quantitative PCR of different tissue of bed bug. The symbols above the gel picture represent antennae (An), head (He), thorax (Th), legs (L) and abdomen (Ab), respectively.
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Figure 8. Current responses of ClOr1/Orco and ClOr2/Orco expressed in the Xenopus oocyte with two-electrode voltage clamp recording.(A) Oocytes expressing ClOr1/Orco were perfused with a panel of odorant compounds, eliciting a firing rate ≥50 spikes/s in single or multiple sensilla on the bed bug antennae. Each odorant was applied at a concentration of 10−4 M for 10 sec with immediate washes until the residue effect of the odorant was totally eradicated. All responses are normalized to the response of the same oocyte to 10−4 M nonanal (mean ± SEM, N = 4–5). The strongest responses are in the center and the weaker responses near the edges of the column graph. Representative current response traces elicited by C4–C10 aldehydes are on the right. (B) Oocytes expressing ClOr2/Orco were perfused with the same panel of odorant compounds as applied for ClOr1/Orco. All responses are normalized to the response of the same oocyte to 10−4 M decanal (mean ± SEM, N = 4–5). The strongest responses are again in the center and the weaker responses near the edges of the column graph. The representative current response traces elicited by C4–C10 aldehydes are on the left.
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Figure 9. Dose-dependent responses of bed bug ClOr1/Orco and ClOr2/Orco to nonanal/octanal and decanal, respectively.(A) Current response traces of oocytes expressed ClOr1/Orco when challenged with nonanal at doses from 10−9 to 10−4 M; (B) Fitted dose-response curve from the current responses presented in Fig. 9A (Mean ± SEM, N = 4–6); (C) Current response traces of oocytes expressed ClOr1/Orco when challenged with octanal at doses from 10−9 to 10−4 M; (D) Fitted dose-response curve from the current responses presented in Fig. 9C (Mean ± SE/M, N = 4–6); (E) Current response traces of oocytes expressed ClOr2/Orco when challenged with decanal at doses from 10−9 to 10−4 M; (F) Fitted dose-response curve from the current responses presented in Fig. 9E (Mean ± SEM, N = 4–6). Each concentration of odorants was applied for 10 sec with immediate washes until the residue effect of the odorant was totally eradicated. Responses were normalized by defining the maximal response as 100.
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