The virulent Wolbachia strain wMelPop increases the frequency of apoptosis in the female germline cells of Drosophila melanogaster

Abstract

Background: Wolbachia are bacterial endosymbionts of many arthropod species in which they manipulate reproductive functions. The distribution of these bacteria in the Drosophila ovarian cells at different stages of oogenesis has been amply described. The pathways along which Wolbachia influences Drosophila oogenesis have been, so far, little studied. It is known that Wolbachia are abundant in the somatic stem cell niche of the Drosophila germarium. A checkpoint, where programmed cell death, or apoptosis, can occur, is located in region 2a/2b of the germarium, which comprises niche cells. Here we address the question whether or not the presence of Wolbachia in germarium cells can affect the frequency of cyst apoptosis in the checkpoint.

Results: Our current fluorescent microscopic observations showed that the wMel and wMelPop strains had different effects on female germline cells of D. melanogaster. The Wolbachia strain wMel did not affect the frequency of apoptosis in cells of the germarium. The presence of the Wolbachia strain wMelPop in the D. melanogasterw1118 ovaries increased the number of germaria where cells underwent apoptosis in the checkpoint. Based on the appearance in the electron microscope, there was no difference in morphological features of apoptotic cystocytes between Wolbachia-infected and uninfected flies. Bacteria with normal ultrastructure and large numbers of degenerating bacteria were found in the dying cyst cells.

Conclusions: Our current study demonstrated that the Wolbachia strain wMelPop affects the egg chamber formation in the D. melanogaster ovaries. This led to an increase in the number of germaria containing apoptotic cells. It is suggested that Wolbachia can adversely interfere either with the cystocyte differentiation into the oocyte or with the division of somatic stem cells giving rise to follicle cells and, as a consequence, to improper ratio of germline cells to follicle cells and, ultimately, to apoptosis of cysts. There was no similar adverse effect in D. melanogaster Canton S infected with the Wolbachia strain wMel. This was taken to mean that the observed increase in frequency of apoptosis was not the general effect of Wolbachia on germline cells of D. melanogaster, it was rather induced by the virulent Wolbachia strain wMelPop.

Figure 1

A schematic representation of an ovariole of D. melanogaster. A, an ovariole of D. melanogaster consisting of the germarium (g) and the vitellarium. B, a detailed scheme of the germarium structure composed of regions 1, 2a, 2b, 3. The checkpoint is framed (red). C, a 16-cell cyst; SSCN, a somatic stem cell niche; SSC, a somatic stem cell; FC, a follicle cell.

Figure 2

Visualisation of acridine orange (AO)- and TUNEL-stained germarium cells of D. melanogaster. A, C, E, G, germaria containing apoptotic cells in region 2a/2b from 5 day-old uninfected (A, E) and Wolbachia-infected (C, G) females (AO staining). B, D, F, H, germaria not containing apoptotic cells from the same fly stocks (AO staining). Arrows indicate small punctate AO-staining in regions 1 and 2a/2b (C, D, G, H). I, relative proportion of germaria containing apoptotic cells from ovaries of the uninfected (w1118T, Canton ST) and Wolbachia-infected (w1118, Canton S) flies. The total number of examined germaria is indicated by blue number; bars show the average percentage per experiment ± s. e. m. J, L, germaria containing apoptotic cells in region 2a/2b in the wMelPop- and wMel-infected fly stocks, respectively (TUNEL). K, M, germaria not containing apoptotic cells from the same fly stocks. Region 2a/2b of the germarium is indicated by red brackets. Scale bars: 20 μm.

Figure 3

Visualisation of germarium cells in semi-thin and ultra-thin sections. A, B, longitudinal semi-thin sections of germaria stained with methylene blue. C, D, ultrastructure of cyst cells from the uninfected and the wMelPop-infected flies. Arrows point to bacteria; arrowheads denote ring canals between neighboring cells. Scale bars correspond to 10 μm (A, B) and 2 μm (C, D), respectively.

Figure 4

Morphology of apoptotic cystocytes in region 2a/2b of the germarium from the wMelPop-infected D. melanogaster^w1118. A, swollen mitochondria (black arrows) in the cytoplasm of cyst cells. White arrows indicate bacteria. B, a fragment of a cyst cell with two mitochondria: one is normal, the other is swollen with the matrix of low electron density and the disintegrated cristae. C, a cyst cell, the cytoplasm appears dense, the nucleus is pyknotic. D, apoptotic bodies (ab) containing intracellular organelles. Scale bars: 1 μm.

Figure 5

Ultrastructure of the Wolbachia strain wMelPop in apoptotic cystocytes in region 2a/2b of the germarium. A, B, Wolbachia accumulations in apoptotic cyst cells, low magnification view. C,D, bacteria framed in panels A, B depicted at higher magnification. Bacteria showing normal morphology (arrows), bacteria with matrix of low electron density (white arrowheads), bacteria with matrix of low electron density and disrupted cell wall (black arrowheads) in the cytoplasm of dying cysts. Scale bars: 2 μm.

Figure 6

Ultrastructure of the germarium cells at the periphery of region 1 in wMelPop-infected D. melanogaster^w1118. A, a fragment of region 1 of the germarium, low magnification view. Normal cells and two fragments of cells (brackets), whose cytoplasm is filled with autophagosomes, bacteria and multilayered membranes. B, multilayered membranes and fragments of a disintegrated nucleus (white arrowhead). C, a fragment of a cell with electron-dense cytoplasm containing Wolbachia of two types: one normal (black arrows), the other with matrix of low density (white arrows). D, electron-dense bacteria-like structures engulfed by autophagosome. E, higher magnification of the bacteria-like structure framed in panel D. F, an autophagosome containing electron-dense structures and vesicles . G, autophagosomes enclosed individual bacteria. Arrowheads indicate autophagosome membranes. Scale bars: 1 μm.

Figure 7

Pathways along which Wolbachia may affect egg chamber formation in region 2a/2b of the germarium. A, localization of regions in the germarium (framed) where the bacteria may interfere with normal function of cells. B, the bacteria disturb the differentiation of cystocytes (white) into the oocyte (light orange) and the nurse cells (light violet). C, the bacteria skew the proper ratio of germline cells to follicle cells. Crescent shape, SSCN; green circle, SSC; green ovals, follicle cells. Red points represent the bacteria.