Table 1.
Primers for experiments.
Figure 1.
Sequence analysis of six candidate pheromone receptors.
Seven putative trans-membrane domains are indicated with red bar and “TMH ‘n’” designation, where ‘n’ designates sequential order of the putative transmembrane domain. Trans-membrane domains were predicted by TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).
Figure 2.
Phylogenetic analysis of six candidate pheromone receptors.
The branch colored in purple represents the sub-grouping of pheromone receptors. GenBank accession numbers are listed in Material S1.
Figure 3.
Tissue specificity and expression pattern of six candidate pheromone receptors amplified in male and female moth.
(A) tissue specificity of PxylOR1 and 3–7; M is D2000 marker (Tiangen), 1 is antennae, 2 is heads (without antennae), 3 is proboscis, 4 is labial palps, 5 is genitals, 6 is throats, 7 is abdomens, 8 is legs and 9 is wings, 10 is negative control. (B) Relative expression quantity of six candidate pheromone receptors amplified in male and female antennae. The x-axis shows the candidate pheromone receptors in male and female moths, the Y-axis indicates relative expression quantity (mean+standard error of mean). The expression of female PxylOR3 is taken as the reference standard.
Figure 4.
Responses of Xenopus oocytes with co-expressed PxylOR1/PxylOrco or PxylOR4/PxylOrco to stimulation with pheromone compounds.
(A) (Upper left) Inward current responses of PxylOR1/PxylOrco Xenopus oocytes in response to 10−4 M of pheromone compounds and analogs. (Upper right) Response profile of PxylOR1/PxylOrco Xenopus oocytes. Error bars indicate SEM (n = 6). (Lower left) PxylOR1/PxylOrco Xenopus oocytes stimulated with a range of Z11-16: Ald concentrations. (Lower right) Dose–response curve of PxylOR1/PxylOrco Xenopus oocytes to Z11-16:Ald. Responses are normalized by defining the maximal response as 100%. Z11-16: Ald EC50 = 2.39×10−5 (n = 5). Error bar indicates SEM. (B) (Upper left) Inward current responses of PxylOR4/PxylOrco Xenopus oocytes in response to 10−4 M of pheromone compounds and analogs. (Upper right) Response profile of PxylOR4/PxylOrco Xenopus oocytes. Error bars indicate SEM (n = 7). (Lower left) PxylOR4/PxylOrco Xenopus oocytes stimulated with a range of pheromone Z9-14: Ac and analog Z9, E12-14: Ac concentrations, respectively. (Lower right) Dose–response curve of PxylOR4/PxylOrco Xenopus oocytes to Z9-14: Ac and Z9, E12-14: Ac. Responses are normalized by defining the maximal response as 100%. Z9-14: Ac EC50 = 2.43×10−7 (n = 7) and Z9, E12-14: Ac EC50 = 1.94×10−6 (n = 5). Error bar indicates SEM.
Figure 5.
Expression of PxylOR1 and PxylOR4 genes in male antenna of P. xylostella.
In situ hybridizations were performed with digoxigenin-labelled antisense RNA probes on longitudinal tissue sections of male antennae. Signals were visualized using an anti-DIG antibody. (A) Hybridization signals in one segment of the P. xylostella antenna are shown. (B) Negative control with a DIG-labeled sense probe. (C) Higher magnification of long trichoid sensilla with hybridization signals. (D) to (G) No hybridization signal was detected under short trichoid sensilla, basiconi sensilla, coeloconic sensilla and chaetica sensilla. Scale bars: 5 µm in A–B and 2 µm in C–G.
Figure 6.
PBP-mediated responses of PxylOR1/PxylOrco Xenopus oocytes.
(A) Inward current responses of PxylOR1/PxylOrco Xenopus oocytes in response to 10−5 M of Z11-16: Ald solubilized by DMSO, 1XRinger, or each of three PxylPBPs, respectively. (B) Response profile of PxylOR1/PxylOrco Xenopus oocytes. Error bars indicate SEM (n = 5). Statistical comparison of responses of oocytes was assessed using one-way analysis of variance (ANOVA). (C) Dose–response profile of PxylOR1/PxylOR2Xenopus oocytes upon stimulation with different Z11-16: Ald concentrations solubilized by DMSO (n = 5), 1 µM PxylPBP1 (n = 4) and 1 µM PxylPBP3 (n = 4), respectively. Responses are normalized by defining the maximal response as 100%. Error bar indicates SEM.
Figure 7.
PBP-mediated responses of PxylOR4/PxylOrco Xenopus oocytes to Z9-14: Ac.
(A) Inward current responses of PxylOR4/PxylOrco Xenopus oocytes in response to 10−5 M of Z9-14: Ac solubilized by 1XRinger, or each of three PxylPBPs, respectively. (B) Response profile of PxylOR4/PxylOrco Xenopus oocytes. Error bars indicate SEM (n = 5). Statistical comparison of responses of oocytes was assessed using one-way analysis of variance (ANOVA). (C) Dose–response profile of PxylOR4/PxylOrco Xenopus oocytes upon stimulation with different Z9-14: Ac concentrations solubilized by 1XRinger (n = 7), 1 µM PxylPBP1 (n = 4), 1 µM PxylPBP2 (n = 4), 1 µM PxylPBP3 (n = 4), respectively. Responses are normalized by defining the maximal response as 100%. Error bar indicates SEM.
Figure 8.
PxylOR4/PxylOrco Xenopus oocytes to Z9, E12-14: Ac of PxylPBP1-mediated (A), PxylPBP2-mediated (B), PxylPBP3-mediated (C)response, respectively.
(Left)Inward current responses of PxylOR4/PxylOrco Xenopus oocytes in response to 10−5 M of Z9, E12-14: Ac solubilized by 1XRinger, PxylPBP and 1XRinger successively. (Right) Response profile of PxylOR4/PxylOrco Xenopus oocytes. Error bars indicate SEM (n = 3). Statistical comparison of responses of oocytes was assessed using one-way analysis of variance (ANOVA).