<i>In vivo</i> phagocytosis of primary symbionts and injected secondary symbionts in LL01 and YR2-Amp aphids (devoid of SS).

<p>(A–B) Fluorescent images of AHP from LL01 (A) and YR2-Amp (B) unchallenged lines. Hemocytes have taken up <i>B. aphidicola</i> (<i>Ba</i>) cells (arrowheads); F-actin in green, DNA in blue, arrows indicate cell nuclei. (C) Fluorescent images of AHP from the LL01 line, 24 h after injection of <i>R. insecticola</i> (<i>Ri</i>) secondary symbionts. <i>Ri</i> are observed in the cytoplasm of a granulocyte (arrowhead); F-actin in green, DNA in blue, arrows indicate cell nuclei. (D) Overlay of DIC and fluorescent images from AHP of unchallenged LL01 aphids, showing FISH detection of <i>Ba</i> cells (red fluorescence) in contact or within adherent hemocytes. Nuclei are counterstained with DAPI (blue fluorescence). (E) Overlay of DIC and confocal images showing FISH detection of <i>Ri</i> secondary symbionts (yellow fluorescence, false color) in a granulocyte of an LL01 aphid, 24 h post-injection. (F–G) TEM showing secondary symbionts <i>Ri</i> within LL01 hemocytes 24 h post-injection. Notice the high vacuolization (<i>V</i>: Vacuoles), the presence of membrane ruffles (<i>Mb</i>) and the phagosomes containing ingested <i>Ri</i>. (F) LL01 Plasmatocyte showing two zipper-like phagosomes containing individual bacteria (<i>P1</i>) and a macropinocytosis-like phagosome (<i>P3</i>) containing several symbionts, some in advanced state of degradation (high darkening). (G) LL01 granulocytes showing zipper-like phagosome (<i>P1</i>) and phagolysosomes containing symbionts (asterisk and box magnified). Scale bar: 10 µm (A–E), 1 µm (F) and 2 µm (G). <i>N</i>: Nucleus; <i>M</i>: Mitochondria; <i>RER</i>: Rough endoplasmic reticulum; <i>G</i>: Granule.