The phenotype we observed in older unfertilized eggs is reminiscent of some Drosophila mutants that display high numbers of large cytoplasmic asters at the embryo surface [59–63]. The formation of these structures is hypothesized to be a secondary effect of centrosome detachment from the spindle poles. Some Drosophila bifasciata eggs we examined did not contain a sperm tail, suggesting that they were unfertilized and, therefore, lacking paternal centriole contribution needed to assemble the first zygotic centrosome. Since the focus of the asters contains centriolar and centrosomal markers, the question remains as to how centrioles/ centrosomes can form de novo in the Drosophila bifasciata unfertilized egg. The trivial answer may be that in these eggs, as in parthenogenetic eggs, there is a mechanism leading to the de novo formation of centrosomes. This could be an intrinsic property of the Drosophila egg, since it has been shown that the cortical microtubule cytoskeleton of mature wild-type eggs was reorga- nized upon activation and expressed a transient assembly of cortical asters [64], like those observed in Drosophila bifasciata. In developing embryos, Wolbachia is known to concentrate around the astral microtubules nucleated by distinct centrosomes [65,66]. Interestingly, the symbiotic bacteria in Drosophila bifasciata were seen to associate with the free cytoplasmic asters nucleated by the maternal centrosomes, but also accumulated at the poles of the acentrosomal spindles derived by the acentriolar meiotic complements. The reason for this unexpected association is unknown. These spindles are, however, unusual in their architecture, since they present aster-like structures at the poles in the absence of recognizable centrosomes. Our results on the effects of Wolbachia-induced male-killing in Drosophila bifasciata suggest a strong correlation between the male- killer Wolbachia and chromatin remodeling in male embryos. A critical issue remaining is whether the abnormalities in nuclear divisions we observed reflect a direct action of a Wolbachia product in some aspect of chromosome structure and function or whether they arise by an indirect mechanism that is triggered by Wolbachia. Materials and Methods Drosophila stocks The lines of Drosophila bifasciata KOS-1 and 210 used in this work were established from iso-females collected in May, 2004 at Matsuhime pass (N 35u43938.50E 138u56959.40, altitude 1250 m). The Wolbachia infection was confirmed by PCR amplification of 16SrRNA, ftsZ, and wsp genes. The KOS-1 and 210 are Wolbachia uninfected and infected line, respectively. KOS-10 line has been kept by crossing males of the KOS-1 line after these lines were established and reared over circa 50 generations, thus the genetic background of these lines is nearly equal. The flies were reared on standard Drosophila medium at 17uC. No specific permissions were required for research on Drosophila bifasciata eggs/embryos as is not a protected species. Antibodies We used the following antibodies: mouse anti-b-tubulin (Boehringer, Mannheim UK; 1:200); rat YL1/2 directed against tyrosinated-a-tubulin (Harlan Sera-Lab, 1:20); mouse anti-c- tubulin (Sigma- Aldrich, 1:100); mouse anti-acetylated-a-tubulin (Sigma-Aldrich; 1:100); rabbit anti-centrosomin (Cnn, 1:400; [67]); mouse anti-Sex lethal protein (Sxl) M18 and M114 clones (Developmental Studies Hybridoma Bank, Iowa University). The secondary antibodies Alexa Fluor 488 and 555 (InVitrogen) anti- mouse, anti-rabbit and anti-rat were used at 1:800 dilutions. Egg collection Embryos were collected from 4- to 5-day-old flies placed on small agar-yeast plates. Eggs were fixed at 20 minutes to obtain meiotic Chromatin Remodeling and Male Killing stages or held for 30–45 minutes before fixation to examine gonomeric spindle formation and early mitotic divisions. Eggs that developed one to three hours after collection yielded embryos that were sufficiently young to contain intravitelline mitoses and syncytial stages mainly at nuclear cycles 8–11, and some older embryos. Embryos at cellularization and early gastrulation stages were obtained by waiting 4–5 hours, whereas later developmental stages were obtained by eggs allowed to develop 10 to 20 hours. Fixation and staining Eggs were dechorionated in a 50% bleach solution for 2– 3 minutes and rinsed in distilled water. Eggs were transferred to a heptane/methanol solution (1:1) and the vitelline membrane was removed by vigorous agitation for 3 min. Eggs were then fixed 10 min in cold methanol, washed in PBS and incubated for 1 h in PBS containing 0.1% bovine serum albumin (BSA). For simultaneous localization of microtubules and c-tubulin the eggs were incubated overnight at 4uC with the anti-c-tubulin antibody, then the YL1/2 antibody was added and the incubation was allowed to proceed for 2 hours at room temperature. For simultaneous localization of the sperm tail and a-tubulin the eggs were incubated 3 hours at room temperature in both anti- acetylated-a-tubulin and YL1/2 antibodies. For double staining of microtubules and Sxl or Cnn the eggs were incubated overnight at 4uC with the specific antisera and then with anti-a or anti-b tubulin antibodies for 3 hours at room temperature.After washing in PBS–BSA, the eggs were incubated for 1 h with the appropriate secondary antibodies. Controls of the secondary antibodies alone were done for all staining. After washing in PBS–BSA, in all cases DNA was visualized by 3–4 minute incubation in Hoechst 33258. Samples were mounted in small drops of 90% glycerol in PBS. Image acquisition and presentation The fluorescent images were taken using an Axio Imager Z1 (Carl Zeiss) microscope equipped with an HBO 50-W mercury lamp for epifluorescence and with an AxioCam HR cooled charge-coupled camera (Carl Zeiss). Image projections of embryos were done with Z-stack and Extended Focus modules (Carl Zeiss). Gray-scale digital images were collected separately and then pseudocolored and merged using Adobe Photoshop 5.5 software (Adobe Systems). Supporting Information Table S1 Developmental defects observed in early embryos obtained by Drosophila bifasciata KOS10 females. (DOC) Table S2 Developmental defects observed in late embryos obtained by Drosophila bifasciata KOS10 females. (DOC) Table S3 Anti-Sxl staining of embryos collected 4–20 hours AED by Drosophila bifasciata KOS10 females. (DOC) Acknowledgments We thank Kaarlo Nygre´n for assistance in obtaining the D. bifasciata flies from Japan and Eyal Schejter (Weizmann Institute of Science, Rehovot, Israel) for the anti-centrosomin antibody. Author Contributions Conceived and designed the experiments: MGR GC. Performed the experiments: MGR GC. Contributed reagents/materials/analysis tools: MU. Wrote the paper: MGR GC RG. PLoS ONE | www.plosone.org 12 January 2012 | Volume 7 | Issue 1 | e30045