Figure 5

Immunofluorescence and confocal analysis. a) Western blot analysis of the AGAP009370-like protein (i.e. AgAcp34A-2 and/or AgAcp34A-3 gene products) using a polyclonal antibody raised against the C-terminal peptide fragment of the protein. AGAP009370-like is detected in the male accessory glands (MAG) and mating plug, but not in the carcass (rest of the body) of adult males. Arrows indicate the AGAP009370-like immunoreactive bands. Actin was used as a loading control. b) Confocal analysis of AGAP009370-like localization in the plug. The protein (green) is specifically localized in the posterior part. Scale bar: 44 μm. Fluorescent microscope analysis of MAG of virgin 3-day-old A. gambiae, A. arabiensis and A. merus males: c) AGAP009370-like (green) is primarily present in the posterior part of MAG (near the aedeagus) in all three species. Scale bar: 50 μm. d) The apotome analysis reveals that AGAP009370-like (green) is either present in channels formed by phalloidin-Alexa 546 (red)-labelled actin-rich muscle cells or in the lumen of the gland. The three species do not differ in this pattern. Here, channel localized protein is shown for A. gambiae and A. arabiensis and lumen presence for A. merus. Nuclei are labeled with DAPI (blue). Scale bar: 20 μm.