Fig. 3

Laser confocal microscopy (LCM) of macrogametes in situ (A–D) and harvested freshly (E–K) and fully formed oocyst (L–O) of E. necatrix (immuno-labeled with rabbit anti-rEnGAM22 pAb [visualized with FITC, green] and mouse anti-rEnGAM59 pAb [visualized with Cy3, red]). A–D Macrogametocyte genesis of Eimeria necatrix in tissue sections of chicken intestine 156 h p.i., counterstained with DAPI. A An early-stage in situ macrogametocyte showing several WFB1s (green) and WFB2s (red) dispersed in the cytoplasm. B A mature macrogametocyte in situ showing WFB1 membrane-bound in the peripheral cytoplasm and numbers of WFB2s underneath the WFB1s. C Late-stage in situ macrogametocytes showing that WFB1 formed in the outer oocyst wall, and WFB2s were underneath the outer oocyst wall and linked together. D An early oocyst in situ showing WFB2s formed in the inner wall. E A mid-stage harvested macrogametocyte exhibits spheroidal-shaped WFBs distributed throughout the cytoplasm. F, G The isolated mature macrogametocyte showing that WFB1s were arranged in necklace-like structures, while the WFB2s were dispersed throughout the cytoplasm. H, I The late-stage harvested macrogametocyte showing that WFB1s align at the parasite periphery and exocytose their contents to form the outer oocyst wall, while WFB2s lose their substructure and fuse into amorphous material that coalesces into small islands. J, K 3D confocal microscopy images of the forming oocyst revealed that WFB2s were located beneath the outer oocyst wall and fused to form an amorphous material. (L–O) The fully formed oocyst immunofluorescence co-localization of rabbit anti-rEnGAM22 [image (L)] and mouse anti-rEnGAM59 [image (M)]. N, UV autofluorescence (blue) of oocyst wall. Image (O) represents the merged image of (L) and (M), showing green fluorescence layer covering the surface of the red fluorescence layer. WFB1 wall-forming body 1, WFB2 wall-forming body 2, O outer oocyst wall, I inner oocyst wall. Bar represents 10 μm