9. Bacteria present in other cell types than bacteriocytes can be observed (e.g. white arrow in figure part C). Green label: The Blochmannia specific probe Bfl172-FITC; red label: SYTO Orange 83. The scale bars correspond to 220 μM (A) and 35 μM (B – E), respectively. Figure 11 Schematic overview of distribution of Blochmannia in the migut epithelium during host ontogeny, summarizing

results of Fig. 1 to Fig. 10. Red coloured cells are free of Blochmannia and green coloured cells are filled with endosymbionts. In small larvae (L1) all cells of the outer layer of the midgut tissue are filled with bacteria, whereas inner layers are devoid of Blochmannia. In larger larvae (L2) and pupae directly after pupation (P1 early) the midgut-epithelium strongly expands paralleling Talazoparib in vitro the growth selleck screening library of the individual. A large number of cells in the

outer cell layer do not contain Blochmannia at this stage. During metamorphosis the larval gut epithelium is shed (P1 late to P2) and excreted, forming the meconium (dark spot) in the distal end of the pupal case. During this stage an increased number of cells in the outer layer of the midgut-epithelium harbour Blochmannia. In pupae directly before eclosion (P3) the circumference of the gut lumen is very tiny as it is empty. At this stage the whole midgut can be viewed as a bacteriome, since almost all cells forming the midgut-epithelium harbour Blochmannia. After eclosion of workers the symbiosis degrades. In old workers (W3) the majority of cells in the outer layer of the epithelium do not contain Blochmannia any longer and the inner layer even less so. The circumference of the gut lumen is larger again. MT: Malphigian tubules, HG: hingut. Males are an evolutionary dead end for the bacteria since they cannot be transmitted to the progeny

via the spermatocytes [4]. Nonetheless, just as the females, the males may require the endosymbionts for proper development during early life stages. We observed that the distribution of bacteriocytes during developmental stages of males (derived from unfertilized worker eggs) was very similar to that of workers including the fact that the midgut of Chlormezanone late pupae was nearly entirely composed of bacteria-harboring cells (data not shown). Changes in the relative bacterial population density in the midgut tissue of different developmental stages were quantified as described in the Methods section (Figure 12). Volume fractions differed significantly among groups (ANOVA: p < 0.001, F = 13.08, df = 7). The results are in perfect agreement with the optical evaluation described above showing a high proportion of bacteriocytes in L1 (40.84 ± 8.75), when a contiguous bacteriocyte layer is surrounding the midgut (Figure 1). Volume fractions were significantly reduced in comparison to all other developmental stages both in L2 (13.25 ± 4.78) and early P1 pupae (17.63 ± 10.