Fig. 3. Expression of the B-subunit of V-ATPase in the vas deferens of Spodoptera littoralis. (A) Western blot analysis of B-subunit levels (arrow) in the upper vas deferens (UVD) during a 16 h:8 h L:D cycle (LD) at the Zeitgeber times indicated above the blot. The protein levels (arrow) show daily oscillations. The lane marked 20C was treated with antigen-preabsorbed antibody. Non-specific protein (arrowhead) served as a loading control. (B) UVDs from moths kept in constant darkness (DD) maintained daily oscillations in B-subunit levels, with a lower level during former daytime at circadian time 8 (Ct8) and a higher level during former nighttime at Ct20. Western blots were repeated three times with similar results. (C–E) Immunostaining for the B-subunit of V-ATPase in longitudinal sections of UVDs dissected every 4 h in LD (C), DD (D) or constant light (LL) conditions (E). Each picture shows representative columnar epithelial cells with their apical sides on the left and blue-gray elongated nuclei. Red V-ATPase signals appear to be concentrated in small vesicle-like structures. A robust increase in the amount of V-ATPase at Zt16–20 is evident in LD and in the second cycle in DD conditions; however, the amount of protein does not show a daily rhythm in LL conditions. The experiment was performed on six males per time point in each photoregime with similar results. No staining was detected in control tissues treated with antigen-preabsorbed antibody. Scale bar, 20 µm. Horizontal bars represent day (open), night (filled) and former day (hatched) portions of the photo-regime.
