Activated p38 MAPK localizes to cilia in S. mansoni miracidia. (A) Scanning electron micrograph of the anterior of a freshly-hatched miracidium showing numerous cilia (c), and sensory endings (se) associated with the semi-spherical terebratorium (tb). (B - J) Immunolocalization of activated p38 MAPK (green) in S. mansoni miracidia following fixing and staining of parasites with anti-phospho p38 MAPK primary antibodies. (B) Freshly-hatched miracidium incubated without primary antibodies but with secondary antibodies alone (negative control). (C) Freshly-hatched swimming miracidium displaying weak patchy staining only localized adjacent to the tegument (tg) in areas corresponding to the cilia. (D) Serial optical z-section of a miracidium treated with 20 μM anisomycin for 30 min showing p38 MAPK activity in the regions occupied by cilia. (E and F) High power, optically zoomed, z-sections through discrete regions of cilia/tegument to capture the miracidium surface in one plane with (E) showing p38 MAPK localized to the shafts of the cilia (arrowed) and regions proximal to the tegument, and (F) showing co-localization with cilia (red) detected using anti-acetylated tubulin antibodies. (G) Miracidium treated with 20 μM anisomycin for 30 min for direct comparison with (C). (H) Autofluorescence (red) of S. mansoni egg containing (I, and J overlay) miracidium (m) with p38 MAPK activity in regions occupied by cilia. (K) Scanning electron micrograph of S. mansoni egg revealing common position of rupture during hatching, and correlating to rupture in H-J. Z-axis projections are shown in maximum pixel brightness mode. Bars: A, E and F = 2 μm; B-D, and G = 15 μm; H-K = 25 μm.