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. 2022 Apr 20;25(5):104272.
doi: 10.1016/j.isci.2022.104272. eCollection 2022 May 20.

Silencing the odorant receptor co-receptor impairs olfactory reception in a sensillum-specific manner in the cockroach

Kosuke Tateishi  1 , Takayuki Watanabe  2   3 , Hiroshi Nishino  4 , Makoto Mizunami  3 , Hidehiro Watanabe  1
Affiliations

Silencing the odorant receptor co-receptor impairs olfactory reception in a sensillum-specific manner in the cockroach

Kosuke Tateishi et al. iScience. .

Abstract

Insects detect odors via a large variety of odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). The insect OR is a heteromeric complex composed of a ligand-specific receptor and the co-receptor (ORco). In this study, we identified the ORco gene of the cockroach, Periplaneta americana (PameORco), and performed RNAi-based functional analysis of PameORco. All OSNs in the basiconic sensilla expressed PameORco and received a large variety of odors including sex pheromones. In trichoid sensilla, a PameORco-positive OSN was consistently paired with a PameORco-negative OSN tuned to acids. In adult cockroaches injected with PameORco dsRNA at the nymphal stage, the expression of PameORco, odor receptions via ORs, and its central processing were strongly suppressed. These results provide new insights into the molecular basis of olfactory reception in the cockroach. The long-lasting and irreversible effects of PameORco RNAi would be an effective method for controlling the household pest.

Keywords: Entomology; Molecular neuroscience; Sensory neuroscience.

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Conflict of interest statement

The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Identification of Periplaneta americana ORco (PameORco) (A) Organization of PameORco. The positions of primers used for RT-qPCR analysis, and the target region of dsRNA for PameORco RNAi are illustrated. (B) Amino acid sequence alignment of PameORco isoform 2 and its homologs in five different insect species. The letters are shaded by alignment strength. The positions of seven transmembrane helixes, putative phosphorylation sites (yellow shaded), putative calmodulin-binding site (orange shaded), and putative N-linked glycosylation site (purple shaded) are illustrated. The polyclonal anti-PameORco antiserum was generated against the 14-a.a. synthetic antigenic peptide located within the second intracellular loop (red shaded). GenBank IDs: PameORco isoform 1, LC657818; and 2, LC657819; Drosophila melanogaster ORco (DmelORco), Q9VNB5; Bombyx mori ORco (BmorORco), NP_001,037,060; Apis mellifera ORco (AmelORco), NP_001,128,415; Blattella germanica ORco (BgerORco), PSN39983; and Locusta migratoria ORco (LmigORco), ALD51504. See also Figure S1.
Figure 2
Figure 2
Sensillar type-specific distribution patterns of PameORco-expressing OSNs (A–D) Anti-PameORco-immunopositive and immunonegative OSNs in all morphological types of antennal olfactory sensilla. Six morphological types of antennal olfactory sensilla are unambiguously identified via differential interference contrast (DIC) observations and indicated by different colored arrowheads (A1–D1). All antennal sensory neurons labeled by anti-HRP antibody are green-colored (A2–D2 and A4–B4), whereas anti-PameORco-immunopositive OSNs are magenta-colored (A3–D3 and A4–B4). Sensilla and corresponding OSNs are denoted as arrowheads and arrows of the same color, respectively. Two OSNs in single trichoid sensilla (sw-C1 and sw-C2 sensilla) are indicated by thin arrows (B2–B4 and C2–C4). In single trichoid sensilla, a PameORco-positive OSN was consistently paired with a PameORco-negative OSN (thin arrows in B3,4 and C3,4). Scale bar = 20 μm. See also Figure S2 and Videos S1, S2, S3, and S4. (E) A schematic overview of sensillar type-specific distribution patterns of PameORco-expressing OSNs.
Figure 3
Figure 3
Experimental conditions for PameORco RNAi and PameORco expressions (A) Experimental conditions for PameORco RNAi. Experimental conditions are summarized in the scheme and abbreviations. At arbitrary selected days after dsRNA injection, RNAi effects were examined by single sensillum recordings and RT-qPCRs (yellow arrowheads). Abbreviations denoted in the scheme are used in the following figures. Developmental processes of OSNs and sexual maturation are indicated by green bars (Silverman, 1977; Watanabe et al., 2018a). (B) PameORco expressions in Naive, β-lac, and PameORco dsRNA-injected cockroaches. In dsRNA-injected cockroaches, RT-qPCRs were performed at 7 days after dsRNA injection. The expression levels of PameORco mRNA were normalized with that of PameEf1α mRNA. (C) PameORco expressions across the different post-injection periods of dsRNA. The expression levels of PameORco were normalized to the average of those of Naive cockroaches (baseline expression level). In (B and C), the sample number is noted in parentheses and black bars indicate means. The different letters above each plot indicate significant differences (ANOVA post-hoc Tukey-Kramer test; p < 0.05). Samples from eight Naive cockroaches (black dots in B and C) were specifically prepared for the RT-qPCR analysis, but those obtained from RNAi cockroaches were also used in the following electrophysiological experiments. Therefore, throughout following figures, results obtained from the same individuals are represented as symbols of the same color and shape.
Figure 4
Figure 4
Effects of PameORco RNAi on the reception of sex pheromones in sw-B sensilla (A) Response properties of four OSNs in sw-B sensilla based on Boeckh and Ernst (1987). (B) Spontaneous activities of four OSNs in a sw-B sensillum of a β-lac dsRNA-injected cockroach. Spikes were sorted based on spike shapes (See STAR Methods and Figure S3). Two sex pheromone-responsive SNs (PA-SN and PB-SN) hardly exhibited spontaneous activities. (C) Typical responses of single sw-B sensilla from three different RNAi cockroaches. Electrophysiological traces show spontaneous activities of OSN1 and OSN2 (left), PB-responses of PB-SN (middle), and PA-responses of PA-SN (right) in single sw-B sensilla from three different RNAi cockroaches. (D–F) Effects of PameORco RNAi on activities of sw-B sensilla. Spontaneous spike frequencies (D), responses of PB-SNs to 2 ng of PB (E), and responses of PA-SNs to 2 ng of PA (F) in each of the RNAi conditions are summarized. Spontaneous activities and responses to each of the two sex pheromones are compared across different RNAi conditions; different letters above each column indicate significant differences (ANOVA post-hoc Tukey-Kramer test; p < 0.05). Black bars indicate means. Throughout figures, symbols of the same color and shape represent recordings from the same individuals. The numbers of recorded sensilla are noted in parentheses. (G and H) Dose-response curves of PB-SNs (G) and PA-SNs (H). The averaged response intensities of PB-SNs (G) and PA-SNs (H) to a given concentration of PB and PA are plotted with standard error (vertical bars), respectively. In each RNAi condition, response intensities to different concentrations of pheromones are statistically compared (lower tables). In the tables, the numbers of recorded sensilla are noted in parentheses, and different letters in each RNAi condition indicate significant differences (ANOVA post-hoc Tukey-Kramer test; p < 0.05).
Figure 5
Figure 5
PameORco RNAi impairs general odor receptions in sw-A sensilla but not in dw-A2 sensilla (A) Response intensities of single sw-A sensilla to ten tested odorants in both Control and PameORco RNAi cockroaches. Responses to a given odorant are color-coded according to the response intensity. Olfactory responses of 9 sensilla from 6 Control cockroaches and of 30 sensilla from 14 PameORco RNAi cockroaches are summarized. (B and C) Effects of PameORco RNAi on activities of sw-A sensilla. Both spontaneous activities (B) and responses to the best ligand (C: nonanol; C9-ol) of sw-A sensilla were impaired in PameORco RNAi cockroaches. (D) Response intensities of single dw-A2 sensilla to the three selected odors in both Control and PameORco RNAi cockroaches. Among ten tested odors, OSNs in the dw-A2 sensilla are selectively activated by three acids (See also Figure S4A). Responses to a given odorant are color-coded according to the response intensity. Olfactory responses of 6 sensilla from 5 Control cockroaches and of 13 sensilla from 10 PameORco RNAi cockroaches are summarized. (E and F) Effects of PameORco RNAi on activities of dw-A2 sensilla. Both spontaneous activities (E) and responses to the best ligand (F: heptanoic acid; C7-acid) of dw-A2 sensilla were not affected by PameORco RNAi. Black bars in each plot indicate means. Throughout figures, symbols of the same color and shape correspond to recordings from the same individual. Significant differences are denoted above each plot (ANOVA post-hoc Tukey-Kramer test; ∗∗∗ = p < 0.001, n.s. = p > 0.05).
Figure 6
Figure 6
Heteromeric combination of a PameORco-positive OSN and a PameORco-negative OSN in single sw-C2 sensilla (A) Response intensities of single sw-C2 sensilla to the three selected odors in both Control and PameORco RNAi cockroaches. Among ten tested odors, OSNs in the sw-C2 sensilla were selectively activated by three acids (See also Figure S4B). Responses to a given odorant were color-coded according to the response intensity. Olfactory responses of 8 sensilla from 6 Control cockroaches and of 20 sensilla from 6 PameORco RNAi cockroaches are summarized. (B) Effects of PameORco RNAi on the acid-responses of sw-C2 sensilla. Responses to the best ligand (heptanoic acid; C7-acid) of sw-C2 sensilla were not affected by PameORco RNAi. (C) Typical spontaneous activities of single sw-C2 sensilla in both Control and PameORco RNAi cockroaches. In Control cockroaches, single sw-C2 sensilla exhibited large (red) and small (blue) amplitude spontaneous spikes. (D) Effect of PameORco RNAi on the spontaneous spike activities of single sw-C2 sensilla. PameORco RNAi selectively impaired the spontaneous activities of large amplitude spikes but not those of small amplitude spikes. Black bars in each plot indicate means. Throughout figures, symbols of the same color and shape represent recordings from the same individual. Significant differences are denoted above each plot (ANOVA post-hoc Tukey-Kramer test; ∗∗ = p < 0.01, ∗ = p < 0.05, n.s. = p > 0.05).
Figure 7
Figure 7
Effects of PameORco RNAi on sex pheromones processing in the antennal lobe (A) PB-responsive L1-PN. Uniglomerular projection neurons (PNs) with dendrites throughout the B-glomerulus (B-glo) were intracellularly stained (Green) after recordings of olfactory responses. Arrow shows the insertion site of the glass electrode. AN; antennal nerve, AL; antennal lobe, m-ALT; medial AL tract, MB; mushroom body, LH; lateral horn. (B) Arborization patterns of the recorded L1-PNs (upper panel) and their typical responses to PB (lower panels). To visualize the B-glomerulus, antennal afferents were anterogradely stained (magenta) after staining the recorded L1-PN (green). Although the responses to 1 ng of PB in Naive and PameORco RNAi cockroaches differed, arborization patterns of L1-PN dendrites (primary dendrite, arrowheads; major collateral, double arrowheads in upper panel) and volumes of B-glomeruli (see Figure S5) were conserved. (C) PB-responses of L1-PNs in both Naive and PameORco RNAi cockroaches. In cockroaches injected with PameORco dsRNA at the last instar stage (LI injection), the PB-responses of L1-PNs were significantly impaired. Black bars indicate means. Because PB stimulus was presented to antennae 5–8 times in each of recordings, symbols of the same color and shape represent stimulus trials from the same L1-PN. Significant differences are denoted above the plot (ANOVA post-hoc Tukey-Kramer test; ∗∗∗ = p < 0.001, n.s. = p > 0.05). (D) Temporal activity patterns of recorded L1-PNs in both Naive and PameORco RNAi cockroaches. The temporal activity pattern of each recorded L1-PN form a single individual is displayed as peri-stimulus time histogram with 20 ms bins. Solid lines and shaded area represent averaged responses and +/− SEM of repeated trials, respectively. Behavioral responses to PB in Naive and PameORco RNAi (Adult injection) cockroaches are shown in Figure S6, and Videos S5 and S6. And Scale bar = 200 μm in (A); 50 μm in (B).
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References

    1. Ai M., Min S., Grosjean Y., Leblanc C., Bell R., Benton R., Suh G.S.B. Acid sensing by the Drosophila olfactory system. Nature. 2010;468:691–695. doi: 10.1038/nature09537. - DOI - PMC - PubMed
    1. Altner H., Sass H., Altner I. Relationship between structure and function of antennal chemo-hygro-and thermoreceptive sensilla in Periplaneta americana. Cell Tissue Res. 1977;176:389–405. doi: 10.1007/bf00221796. - DOI - PubMed
    1. Batra S., Corcoran J., Zhang D.D., Pal P., K P.U., Kulkarni R., Lofstedt C., Sowdhamini R., Olsson S.B. A functional agonist of insect olfactory receptors: behavior, physiology and structure. Front. Cell. Neurosci. 2019;13:134. doi: 10.3389/fncel.2019.00134. - DOI - PMC - PubMed
    1. Benton R., Sachse S., Michnick S.W., Vosshall L.B. Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol. 2006;4:e20. doi: 10.1371/journal.pbio.0040020. - DOI - PMC - PubMed
    1. Benton R., Vannice K.S., Gomez-Diaz C., Vosshall L.B. Variant ionotropic glutamate receptors as chemosensory receptors in Drosophila. Cell. 2009;136:149–162. doi: 10.1016/j.cell.2008.12.001. - DOI - PMC - PubMed

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