Figure 5

Transmission electron microscopy image showing immunogold labeling of SIFamide and SIFamide receptor (SIFa_R) in type III acinus of unfed Ixodes ricinus female salivary glands. (A) The SIFa-axons (yellow asterisks) were found in close proximity to the acinar duct (AD) and its valve (V). Myoepithelial cell (MC, pink arrows) and basal epithelial cells (ECs, blue arrows) surround the acinar valve (see also Fig. 2A,B,G). The inset in A is magnified in (B). (B) Basal SIFa-axon containing electron-dense neurosecretory vesicles (yellow dots indicate the reaction with SIFamide antibody). Four small axons (white arrows) containing electron-lucent vesicles are associated with the large SIFa-axon. Note that the axons (green) are separated from the acinar duct (AD) and are enclosed by highly convoluted ECs (gold) forming septate junctions (black arrowheads in C). Mitochondria (red) within and near the axons, and apparent microtubules (aqua-blue) are shown. For 3D animation of panel (B) see Video 2. (D,E) SIFa_R staining (white empty arrows in E show 6 nm nanoparticles) is observed on the EC canaliculi in the AD apex region. Black arrowheads in E show EC membranes. (F–M) Double labeling of SIFamide (black arrows indicate 15 nm nanoparticles) and SIFa_R (white empty arrows or white arrowheads show 6 nm nanoparticles) in the region above the acinar valve. Insets in F and J are magnified in (G) and (K) respectively. Insets in K are magnified in (L,M). Note that two different SIFa_R reactions were recognized in close proximity to the prominent SIFa-axon (H,I,K–M). First, SIFa_R-positive vesicle-like structures (white arrowheads in H,I) and the MC plasma membrane (white arrowheads in H). Second, the small axons containing electron-lucent vesicles accompanying the SIFa-axon were also positive for SIFa_R (white empty arrowheads in H,L,M). Numerous mitochondria (red) and microtubules (aqua-blue) were recognized in the MC (gold in K) surrounding the axons (green). (B,D,K) Overlay of one tomographic slice and 3D rendered model, single-axis electron tomography (ET). (C,E,H,L,M) Slices of ET. Scale bars: 2 μm (A,J), 500 nm (B,F,G), 200 nm (D,E,H,K,L), 100 nm (C,M).