Figure 1.
Transgenic silkmoths expressing PxOR1 in bombykol receptor neurons.
(A) EGFP expression in the antennae of male moths carrying BmOR1-GAL4 and UAS-EGFP transgenes. Magnified image shows EGFP fluorescence in ORNs innervating pheromone-sensitive long sensilla trichodea. The white and yellow arrows indicate a dendrite and an axon, respectively. The white arrowhead indicates a long sensillum trichodeum. EGFP images were acquired by confocal microscopy (LSM510, Carl Zeiss). Scale bar: 20 µm. (B) PxOR1 expression in the antennae of male moths bearing either BmOR1-GAL4 and UAS-PxOR1 transgenes or BmOR1-GAL4 or UAS-PxOR1 alone. RT-PCR was performed with RNA isolated from the male antennae of the indicated genotype using PxOR1-specific primers. RT-PCR products were separated by electrophoresis. The minus sign indicates that RT-PCR was performed without reverse transcriptase. B. mori actin1 [52] was used as a positive control in the experiments. (C) The amounts of PxOR1 and BmOR1 mRNA in BmOR1-GAL4/UAS-PxOR1 male antennae were determined using quantitative PCR. The data were normalized to the copy numbers of B. mori rp49 mRNA [54]. Data shown are the means ± SD from three different cDNA pools. (D) Two-color fluorescent in situ hybridization of BmOR1 (green) and PxOR1 (magenta). Double-labeling was performed on paraffin sections of BmOR1-GAL4/UAS-PxOR1 male antennae using fluorescein-labeled BmOR1 and DIG-labeled PxOR1 antisense RNA. Scale bar: 20 µm.
Figure 2.
Single sensillum responses of PxOR1-expressing bombykol receptor neurons to Z11-16:Ald.
(A) Structure of bombykol and P. xylostella pheromone components. (B) Typical electrophysiological recordings from bombykol receptor neurons of transgenic male moths to 10 µg of bombykol or pheromone components of P. xylostella. Spikes with large amplitude (L) and small amplitude (S) are from bombykol and bombykal receptor neurons, respectively. The stimulus was applied for 1 s, as indicated by the solid line under the traces of the recordings. (C) The response for 1 s following stimulation with 10 µg of bombykol or pheromone components of P. xylostella. Error bars represent ± SEM: BmOR1-GAL4 (n = 14), UAS-PxOR1 (n = 14), BmOR1-GAL4/UAS-PxOR1 (n = 12). Two asterisks, P<0.01 compared with responses of the corresponding line stimulated with bombykol; Scheffé's F test. (D) Dose-dependent increases in the bombykol (red) or Z11-16:Ald (blue)-induced spike frequency of BmOR1-GAL4/UAS-PxOR1 male moths. Error bars represent ± SEM (n = 10).
Figure 3.
PxOR1-expressing males exhibit pheromone-orientation behavior in response to Z11-16:Ald stimulation.
(A) Behaviorally responding percentages of male moths of the indicated genotype. The moths were exposed to 100 ng of bombykol or the pheromones of P. xylostella. The display of wing flapping was used as the criterion for a behavioral response to pheromone. The numbers of samples are as follows: BmOR1-GAL4 (n = 13), UAS-PxOR1 (n = 15), BmOR1-GAL4/UAS-PxOR1 (n = 38), BmOR3-GAL4/UAS-PxOR1 (n = 10). (B) Dose-dependent increase in the percentages of moths that responded to bombykol or Z11-16:Ald (n = 22–38). The sensitivity of PxOR1-expressing males to Z11-16:Ald was significantly lower than that to bombykol; GLM, P<0.001. (C) Walking direction orientation in a BmOR1-GAL4/UAS-PxOR1 male moth after a single pulsed stimulation (500 ms) of 40 ng of bombykol (right) or Z11-16:Ald (left) to antennae. Stimulus onset (at t = 0) is indicated by arrows, an angle of zero degrees indicates the initial forward direction.
Table 1.
Comparison of the behavioral parameters of PxOR1-expressing male moths.
Figure 4.
Ectopic expression of PxOR1 does not modify the axonal projections of pheromone receptor neurons.
The axon terminals of bombykol (A) or bombykal (B) receptor neurons in the absence (left) or presence (right) of PxOR1 expression were visualized with EGFP followed by anti-GFP immunostaining (green). Background staining was carried out with Alexa Fluor 555 (left) or an anti-synaptotagmin antibody (right) to visualize neuropil structures (magenta). Representative confocal sections are shown. C: cumulus, T: toroid, D: dorsal, L: lateral. Scale bars: 50 µm.