FIG 2.

The oocyte entry process of NcVg, Nasuia, and Sulcia in adult female N. cincticeps. (A) Confocal micrographs showed the absence of NcVg within the bacteriocytes containing Nasuia and Sulcia. Bar, 50 μm. (B) Confocal micrographs showed the colocalization of NcVg with Nasuia, rather than with Sulcia in the hemocytes. Bar, 25 μm. (C) Confocal micrographs showed that NcVg, Nasuia, and Sulcia were absent in the epithelial plug of the pre-vitellogenic-stage ovary. Bar, 100 μm. (D) Confocal micrographs showed that NcVg accompanied Nasuia, rather than Sulcia from the epithelial plug (panels I) into the oocyte to form a “symbiont ball” (panels II), wherein NcVg was released into the oocyte (panels III). Bars, 50 μm. Red arrows marked the colocalization of NcVg and Nasuia. The female bacteriocytes (A), hemocytes (B), and ovarioles (C and D) were stained with Sulcia-cy5 (blue), Nasuia-cy3 (red), and NcVg-FITC (green). (E and F) Immunoelectron micrographs showed that NcVg was absent within the bacteriocytes. Bars, 2 μm (E) and 500 nm (F). (G and H) Immunoelectron micrographs showed the presence of NcVg within the cytoplasm of Nasuia in the epithelial plug. Bars, 500 nm. (I and J) Immunoelectron micrographs showed the distribution of NcVg along the envelope invaginations of Nasuia. Bars, 500 nm. (K) Immunoelectron micrograph showed the presence of NcVg within the developing yolk granules in the oocyte cytoplasm. Bar, 500 nm. The female bacteriocytes (E and F) and ovaries (G to K) were immunolabeled with NcVg-specific IgG as the primary antibody, followed by treatment with 15-nm gold particle-conjugated goat antibodies against rabbit IgG as the secondary antibody. Panels F, H, and J are the enlargements of boxed areas in panels E, G, and I, respectively. Red arrows mark gold particles. Ep, epithelial plug; Ev, envelope; Fc, follicular cell; Sb, symbiont ball; O, oocyte; N, Nasuia; S, Sulcia. All images are representative of at least three replicates.