Fig 1.
Confocal micrographs of FISH carried out in the abdomen of an adult female D. citri.
Red (Alexa Fluor 594), green (Alexa Fluor 488), and blue (DAPI) signals indicate Carsonella, Profftella, and the host nuclei, respectively. (A) Whole-mount FISH image [maximum intensity projection (MIP)] of the ventral view of the abdomen of an adult female on the day of eclosion showing the bacteriome and immature ovaries. (B) FISH image (MIP) of a sagittal cross-section of the abdomen of an adult female at 5 days post-eclosion. Carsonella can be seen within the uninucleate bacteriocytes on the surface of the bacteriome, while Profftella is encased in syncytial cytoplasm at the center of the bacteriome. Carsonella and Profftella signals can also be seen in an oocyte at the vitellogenic stage (arrowhead), which is located in the ovariole constituting the ovary. Inset on the left is an enlarged image of the area in the yellow rectangle showing a Profftella cell in the hemocoel. (C) Enlarged image (MIP) of the bacteriome in the dotted rectangle of B, from which the Alexa Fluor 488 (Profftella) signals have been removed. Yellow dotted lines surround bacteriocytes containing large spherical Carsonella cells, while thin white dotted lines indicate bacteriocytes containing elongated thin tubular Carsonella cells. Bacteriocytes circled by thick white dotted lines contain Carsonella cells that appear to be in the process of transformation. (D) Enlarged image (optical section) of the area shown in B. Dotted lines indicate the same structures as described in C. The arrowhead indicates the syncytium harboring Profftella, which reaches the surface of the bacteriome. (E) FISH image (MIP) of another sagittal cross-section of the abdomen of the same individual shown in A–D. A mass of spherical Carsonella and Profftella cells exiting from the bacteriome is indicated within the dotted line. Abbreviations: bc, bacteriocyte; bo, bacteriome; ov, ovary; pv, previtellogenic oocyte; sy, syncytium; tg, tergite; vt, vitellogenic oocyte.
Fig 2.
Confocal micrographs (MIP) showing transovarial transmission of Carsonella and Profftella.
(A–F) FISH images showing an oocyte accepting symbionts in serial sections (5 μm thick) of an adult female D. citri at 5 days post-eclosion. Red (Alexa Fluor 594), green (Alexa Fluor 488), and blue (DAPI) signals indicate Carsonella, Profftella, and the host nuclei, respectively, unless otherwise stated. A mass of Carsonella and Profftella cells can be seen entering the posterior of a vitellogenic oocyte through follicle cells and the pedicel of the ovariole. (G) Enlarged image of the dotted rectangle shown in E. (H) Duplicate of image shown in G following the removal of Alexa Fluor 488 (Profftella) signals. Spherical or short rod-shaped Carsonella cells can be seen entering the posterior of the oocyte through follicle cells. Note that the shape of the Carsonella cells does not change after entering the oocyte. Some of the weak DAPI signals in this micrograph correspond to Profftella (see also I). (I) Duplicate of the image shown in G following the removal of Alexa Fluor 594 (Carsonella) signals. Spherical Profftella cells can be seen entering the posterior of the oocyte through follicle cells. Note that Profftella cells in the oocyte have already transformed from spherical to tubular form. Some of the weak DAPI signals in this micrograph correspond to Carsonella. Abbreviations: fc, follicle cell; pc, pedicel; pv, previtellogenic oocyte; tr, trophocyte; vt, vitellogenic oocyte. Scale bars: 50 μm in A–F, 10 μm in G–I.
Fig 3.
Confocal micrographs (optical sections) showing dynamics of Carsonella and Profftella during the embryogenesis of D. citri.
Whole-mount FISH images of embryos. Red (Alexa Fluor 594), green (Alexa Fluor 488), and blue (DAPI) signals indicate Carsonella, Profftella, and the host nuclei, respectively. (A) Blastula-stage embryo collected at 0–6 h post-oviposition. A tightly aggregated ball-like mosaic mass of Carsonella and Profftella can be observed at the posterior pole of the embryo. (B) An embryo at 6–12 h post-oviposition. The symbiont mass was loosening. (C) An embryo at 12–18 h post-oviposition. Carsonella and Profftella can be observed in the central and peripheral parts of the mass, respectively. Host nuclei are observed in the symbiont mass. (D) An embryo18–24 h post-oviposition. Carsonella and Profftella can be observed in the central syncytium and peripheral uninucleate bacteriocytes, respectively. (E) An embryo at 24–30 h post-oviposition. The head of the germ band nears the proto-bacteriome. Uninucleate bacteriocytes harboring Profftella migrate toward the posterior pole. (F) A katatrepsis-stage embryo at 30–36 h post-oviposition. The uninucleate bacteriocytes harboring Profftella assemble at the posterior pole, whereas the syncytium containing Carsonella resides on the anterior side of the mass. (G) An embryo (36–48 h post-oviposition) in the process of dorsal closure. The syncytium harboring Carsonella is divided into uninuclear bacteriocytes and begins to surround the mass of Profftella-containing bacteriocytes. (H) An embryo at 48–60 h post-oviposition. The proto-bacteriome is settled within the embryo. (I) An embryo at 60–72 h post-oviposition. The mass of bacteriocytes harboring Profftella is fused into a syncytium, which is completely surrounded by uninuclear bacteriocytes containing Carsonella. (J) An embryo at 72–84 h post-oviposition. Two wing-like protrusions start to grow from the lateral sides of the bacteriome. (K) Enlarged image of the symbiont mass with host nuclei shown in C. (L) Enlarged image of the cellularized proto-bacteriome shown in D. (M) Enlarged image of the proto-bacteriome shown in H. Profftella-containing bacteriocytes are still uninucleate. (N) Enlarged image of the proto-bacteriome shown in I. Profftella-containing bacteriocytes are fused into a syncytium. Abbreviations: a, abdomen; gb, germ band; h, head; t, thorax; y, yolk. Scale bars: 50 μm for A–J, 10 μm for K–N. Arrows indicate the presumed direction of motion of the embryo.
Fig 4.
Confocal micrographs (MIP) of whole-mount FISH assays showing the bacteriomes of the D. citri nymphs.
Red (Alexa Fluor 594), green (Alexa Fluor 488), and blue (DAPI) signals indicate Carsonella, Profftella, and the host nuclei, respectively. Bar: 100 μm. (A) 1st instar. (B) 2nd instar. (C) 3rd instar. (D) 4th instar. (E) 5th instar. The bacteriome continuously increased in size and volume during nymphal development in proportion to the increase in body size. The pair of wing-like protrusions that emerged at the late embryonic stage continued to grow throughout the nymphal stages.