Figure 4

The binucleation process was observed in the late 5th instar nymph of Cimex. (A–C) Growth of internal reproductive organ primordia during the 5th instar nymph stage. At the (A) 0–2nd, (B) 2nd-3rd, and (C) 4th-6th days after molting to the 5th instar nymph. SV: seminal vesicle. MR: mesadenial reservoir. MG: mesadenial gland. Green: phalloidin staining. Magenta: DAPI staining. Scale bars in A-C are 100 μm. (D,E) Cells in the last mitotic phase were detected by staining with the anti-pH3 antibody (white) at the 5th day after molting to the 5th instar nymph. (D) Low magnification surface view of the MG and MR. (E) High magnification views of the MR. (Square photo in E) Surface view. (Rectangular photos in E) Longitudinal and transverse sectional views that were reconstituted from the Z-stacks of the confocal images on the yellow dashed lines in the square photo in E. Green: phalloidin staining. White: pH3 staining. Magenta: DAPI staining. Scale bars in (D,E) are 100 and 10 μm, respectively. Note the spherical shapes of the mitotic phase cells on the apical surface of the MR epithelium and the polarities of nuclear division horizontal to the epithelial plane. (F) MG cells after their last cytokinesis at the 6th day after molting to the 5th instar nymph. (G–H’) Cells during binucleation at the 7th day after molting to the 5th instar nymph. In the MR (G,G’), arrows indicate the phalloidin-positive puncta between the two nuclei. Dashed lines denote the outlines of the two epithelial cells. In the MG (H,H’), most of the newly binucleated cells display linear arrays of phalloidin-positive puncta between the two nuclei. Green: phalloidin staining. Magenta: DAPI staining. Scale bars in G and H are 10 and 25 μm, respectively. (I) Double staining by phalloidin (green) and LysoTracker (magenta) in the MG during binucleation. The LysoTracker-positive puncta are rarely observed and are not associated with phalloidin-positive puncta, unlike those of the starvation-induced autophagy (Fig. 3). The scale bar in I is 10 μm.