Figure 1.
C. lectularius Orco gene and phylogeny.
(A) Orco gene structure. Exons are boxed, introns are shown as lines. UTR – untranslated region. (B) Alignment of insect Orco proteins with transmembrane domains (TMD) indicated. (C) Phylogenetic tree.
Figure 2.
(A) The bed bug arenas are shown in the figure with clean paper on the left and fecal-stained pheromone paper on the right. White filter paper was taped to the bottom of the arenas. (B) Behavioral responses of bed bugs to a pheromone (fecal-stained paper) after the application of an ORCO agonist (VUAA1) and antagonist (VU0183254). The data were reported as the mean ratio (±SEM) of bed bugs attracted to the pheromone paper compared to bed bugs attracted to the pheromone paper in addition to bed bugs that remained wandering in the arena (N = 100). Lower case letters are for means comparisons between the dark bars while capital letters are means comparisons for the light bars.
Figure 3.
Orco protein expression in antennae and sperm - Immunofluorescence labeling with anti-Drosophila Orco antibody on sectioned specimens.
Left panels (A and E), Orco labeling (green); center (B and F), nuclear staining with DAPI (blue); right (C and G), overlay. A–C, sections of male antennae; E–G, sections of seminal vesicles. D, Illustration of antennal section made from contrast-enhanced panel C image: grey part, cuticular structure with two serrated hairs; green, anti-Orco immune reactive cells; white lines, cellular components in antenna. White arrow indicates antennal orientation to the tip. Anterior-posterior orientation is also indicated in the figure. Scale bars: A–C, 50 µm, E–G, 10 µm. Small red arrowheads in panel E indicate a portion of flagellum.
Figure 4.
Expression was assayed using q-RT-PCR. Expression values were normalized with the control expression data from the same samples. The results shown are representative of three separate repeats with similar results. RNA was isolated from organs/body parts of unfed adult female bed bugs (dark-shaded columns) and unfed adult male bed bugs (light-shaded columns). The means were separated by the Tukey-Kramer HSD (p<0.05). Means which share the same letter are not significantly different.
Figure 5.
Sperm motility modulation and effects on egg production and hatching by Orco agonist and antagonist.
(A) Phase-contrast micrograph of the bed bug sperm. Black hair-like filamentous are sperm [a linear segment is indicated by a pair of white arrowheads, see also Video S2 (control) and Video S3 (sperm treated with VUAA1). (B) Motility indices (vertical axis) of the bed bug sperm for indicated concentrations of compounds tested (horizontal axis). (C) Sperm motility from the bugs topically applied with VUAA1 (2 µL of 35 mM in acetone) or acetone alone as control. (D) Egg production and hatching success for the control and Orco agonist (35 mM VUAA1). Values are in mean ± SEM. **indicates statistical significance at p<0.01 in comparison to control by non-parametric Mann-Whitney U test for B and C. No significant difference was detected by MANOVA for D.