Figure 1.
The Habitus of P. galateia and the Ultrastructure of the Trophosome Region.
(A) Squeeze preparation of a live P. galateia specimen under incident light showing the smooth, silky appearance of the trophosome and the transparent rostrum. (B) TEM trophosome region cross section. One bacteriocyte (surrounded by a dashed line), the thin epidermis, neoblasts stem cells and dorso-ventral muscles are labeled. (C) TEM section of a neoblast (nb) proximal to the epidermis (ep); the cell contains highly active chromatin in the nucleus, chromatoid bodies (cb, arrowheads) and a high nucleo-cytoplasmatic ratio. (D) TEM section of a bacteriocyte with the delicate bacteriocyte membrane (bm, arrowhead); it contains the eukaryotic nucleus (nu, arrowhead) and numerous symbiotic bacteria (ba), which in turn contain bright refractive granules. (E) Schematic drawing of the trophosome region cross section reduced to the three types of cells and tissues most important for this study. Scale bars in (A) and (B) 50 µm, in (C) and (D) 5 µm.
Figure 2.
Distribution of S-phase and Mitotic Cells.
(A) Confocal fluorescence projections of 30 min EdU pulse-labeled S-phase cells (green) and serotonergic nerves (red) superimposed with interference-contrast images of P. galateia whole-mounts. S-phase cells are restricted to the body region posterior to the brain. Longitudinal dorsal nerves extend from the brain to the posterior end of the body. (B) Superimposition of interference-contrast and confocal fluorescence projections of mitotic cells (red) in the anterior body region of a 12 h nocodazole-treated P. galateia. Mitotic cells are restricted to the body region posterior to the brain. Scale bars in (A) and (B) 100 µm.
Figure 3.
S-phase and Mitotic Cells Are Found in Subepidermal Positions.
Bright field micrographs of immuno-stained trophosome region sections. All sections were counterstained with methylene blue (blue). (A) 30 min BrdU pulse-labeled S-phase cells (brown) in subepidermal positions on the dorsal and ventral side of the trophosome region (arrows). (B) Detailed micrograph of BrdU-labeled cells in sub-epidermal positions. (C) Detailed micrograph of mitotic cells (brown) in sub-epidermal positions of trophosome region cross sections. Mitotic cells were accumulated by 12 h nocodazole treatment. One bacteriocyte is indicated with an asterisk and surrounded by a dashed line. A nucleus presumably belonging to a muscle cell is labeled by an arrowhead. Scale bars in (A) 50 µm, (B) and (C) 10 µm.
Figure 4.
Pulse-Chase Labeled Cells Migrate into the Rostrum.
Confocal projections of EdU pulse-chase labeled cells (green) and serotonergic nerves (red) superimposed with interference-contrast images of P. galateia whole-mounts. Only the anterior parts of the worms are shown. (A) Worms with EdU-pulse and 72 h-chase showing labeled cells in the moment of entering the rostrum (arrowhead). (B) After 96 h, chase-labeled cells are distributed over the whole length of the rostrum. Label is visible in the epidermis and in nerve cells of the brain (yellow double label, arrows). Scale bars in (A) and (B) 100 µm.
Figure 5:.
Pulse-Chase Labeled S-phase Cells in Trophosome Region Cross Sections.
Bright field micrographs of 30 min BrdU pulse and 120 h chase immuno-labeled P. galateia trophosome region cross sections. All sections were counterstained with methylene blue (blue). (A) BrdU-labeled cells (brown) were distributed in all regions of the section including the trophosome (arrowhead). (B) Detailed micrograph of BrdU-labeled bacteriocyte (asterisk, surrounded by a dashed line) and labeled epidermal cells (arrowhead). (C) Other BrdU-labeled cell types between the bacteriocytes. Scale bars in (A) 50 µm, (B) and (C) 10 µm.
Figure 6.
Nerve Rearrangement in a Fission Plane Area.
(A) In vivo squeeze preparation of P. galateia under incident light. The animal exhibits a fission plane (arrowhead) in the trophosome region. (B) Confocal fluorescent projection of a serotonin staining (red) superimposed with an interference-contrast image of a fission plane area. The longitudinal nerves (red) are separated into an anterior and posterior fraction. A commissure (arrowhead) in the posterior fraction indicates the reorganization of the nerves. Scale bars in (A) and (B) 100 µm.
Figure 7.
Trophosome Region Fragments Can Regenerate the Rostrum.
Squeeze preparations of live P. galateia under incident light. (A) Intact P. galateia prior to rostrum amputation. The dashed line indicates the level of cutting. (B) P. galateia directly after rostrum amputation (stage 0). Small shreds of injured bacteriocytes leak out the wound. (C) Regeneration stage 1: the wound is closed and a symbiont-free area on the anterior tip indicates ongoing rostrum regeneration. (D) Regeneration stage 2: a small outgrowing rostrum is visible on the anterior tip. (E) Regeneration stage 3: the new rostrum has its final characteristic shape and has grown almost to its full size. Scale bars in (A–E) 100 µm.
Figure 8.
Scheme of the Hypothetical Asexual Reproduction by Paratomy.
Drawing of P. galateia reproducing by paratomy (grey = trophosome). Paratomy starts when the trophosome extends to a certain length (1). Progressively, constrictions appear perpendicular to the anterior-posterior axis and zooids develop (2) which finally detach from the mother zooid (3). Then, each of the zooids grows to a complete worm (4), whereas the trophosome region continuously grows until it extends to a certain size and the process of paratomy starts again.