Defects of pre-syncytial blastoderm embryos may be amplified subsequent to the migration of nuclei to the embryo surface and following syncytial blastoderm mitoses. To investigate this possibility the embryos were scored after nuclear migration during the tenth to thirteenth mitosis (Table S2). Of 336 embryos examined 3 hours AED 103 (30.6%) had some barrel shaped spindles or abnormal microtubule clusters and represent early developmental arrests or fertilization failures. Of the 233 embryos belonging to the syncytial blastoderm mitoses, 61% (n = 142) progressed normally through the nuclear division cycles, and 39% (n = 91) showed a wide range of mitotic defects. Defects in chromosome condensation were frequently observed. This is illustrated by the embryo in Fig. 3A where prophase spindles are organized around nuclei with differently condensed chromatin. This is in contrast to normal developing embryos where all prophase nuclei had similar entangled chromatin masses (Fig. 3B). Spindles that hold abnormally condensed chromatin also look abnormal. Some embryos had small surface areas with relevant asynchrony in the nuclear division cycles and metaphase and anaphase spindles observed near each other (Fig. 3C). During normal development, slight asynchrony in the nuclear division cycle and mitotic waves were observed. At the opposite poles of the embryo nuclear division cycles usually progressed gradually. However, central surface areas do not typically show asymmetric mitotic progressions (Fig. 3D). Some embryos obtained from KOS10 females also showed surface areas devoid of nuclei (Fig. 3E). In some cases telophase figures were observed in which the spindle held only one of the two expected daughters (Fig. 3E, arrows). This contrasts with the normal developing embryo where nuclei are evenly spaced (Fig. 3F). Another commonly observed defect was in chromosome segregation during anaphase and in the formation of chromatin bridges during telophase (Fig. 3G). Also observed were a lower number of embryos sharing abnormal DNA aggregates, irregular spindles and centrosomes that were found free in the cytoplasm or appeared detaching from the spindles (Fig. 3H). Chromatin condensation defects are amplified during cellularization and later stages of development To further characterize defects associated with the develop- mental arrest of embryos obtained by KOS10 females, embryos fixed 4–5 hours AED (Table S2), when the main phenotypic class was represented by cellular blastoderm, were also examined. Of the cellularizing embryos (n = 139), 61.1% (n = 85) displayed a normal blastoderm with interphase nuclei (Fig. 4A) surrounded by microtubular baskets (Fig. 4A9). The remnant cellularizing embryos (n = 54; 38.9%) displayed picnotic nuclei arranged in clusters or rows as well as dispersed within the interphase nuclei (Fig. 4B). Picnotic nuclei were associated with smaller microtubu- lar baskets (Fig. 4B9) and were often seen to sink inward to the interior of the embryo (inset, Fig. 4B). Other embryos had large surface areas populated by irregular nuclei of various size and differeing chromatin condensation (Fig. 4C) that corresponded with abnormal arrangements of microtubular baskets (Fig. 4C9). Consequently, the distribution of surface nuclei was not uniform as seen in normal development. To ascertain whether the chromatin abnormalities observed in pre-blastoderm or cellularizing embryos may be inherited and/or amplified during further developmental stages, embryos fixed 10– 20 hours AED (n = 292) were examined (Table S2). Of these: 9.5% (n = 28) were arrested very early during development, 18.5% (n = 54) were unfertilized, 8.9% (n = 26) were observed in various stages of development spanning from cellularization to later stages of gastrulation; 63.1% (184) displayed different stages of germ Chromatin Remodeling and Male Killing band extension-retraction. In this latter group, 61% (n = 112) formed what appeared to be normal segments (Fig. 5A), whereas the remnants 39% (n = 72) had significant abnormalities ranging from defects in segment formation (Fig. 5B), to failure to differentiate whole segments and the presence of unusual large areas of mitotic divisions (Fig. 5C), or incomplete body formation (Fig. 5D). These aberrations were linked to chromatin abnormal- ities that ranged from defects in morphology and condensation of sparse nuclei (inset, Fig. 5B) to nuclear and spindle abnormalities found in large areas of the embryo surface (inset, Fig. 5C). Wolbachia activity specifically targets chromatin of male embryos Embryos with chromatin/spindle abnormalities, presumably leading to developmental arrest, represent about half of the embryos collected. KOS10 females lay eggs from which only viable females develop, therefore it is hypothesized that embryos with abnormalities in the nuclear progression cycles may represent males. To test this hypothesis we stained embryos fixed from 4 to 20 hours AED (Table S3) for DNA and the Sex-lethal protein (Sxl), a peptide required for sex determination in Drosophila and only expressed by the female line circa the beginning of nuclear cycle 12. Of the embryos collected (n = 197), 8.6% (n = 17) had an early arrested phenotype, 18.6 (n = 36) were unfertilized, and 73% (n = 144) were observed in various stages of development, spanning from last syncytial mitoses to germ-band shortening. Of the stained embryos, 81 embryos (41%) gave uniform staining with anti-Sxl antibody, whereas 63 (32%) lacked staining. If we assume that the early arrested embryos were male embryos, then the number of male is about 50% of the population of developing eggs. This observation is consistent with the expectation that Sxl is expressed only in females of Drosophila bifasciata. The Sxl gene is switched on in the syncytial blastoderm starting from the last nuclear division cycle, and embryos can be sexed unambiguously at beginning this time. Sxl expression was always found to be associated with the nuclei of embryos undergoing normal development (Fig. 6A,B), suggesting that females did not have developmental defects. By contrast, Sxl-negative embryos showed a variety of abnormalities ranging from defects in nuclear progression and chromatin condensation (Fig. 6C) to failure to form large embryonic surface areas during later developmental stages (Fig. 6D). Cytoplasmic asters are the landmark of early arrested embryos Collection of embryos made at different times showed several non-developing eggs. A common characteristic was the presence of cytoplasmic asters that appeared early in development (Fig. 7A), increased in number with time (Fig. 7B), and reached high levels after 10 hours when large asters were present in the interior of the embryos and many small asters filled the cortical region (Fig. 7C). Presence of the sperm tail in rare eggs with cytoplasmic asters (Fig. 7D) suggested that the formation of these structures may be independent of sperm entrance. Unfertilized eggs obtained by KOS 1 females after 4–5 hours AED, also showed several free cytoplasmic asters in addition to metaphase-like barrel shaped spindles (Fig. 7E). This observation suggests that microtubule based asters also formed in the absence of bacteria and that they are a landmark of developmental failure. Bacteria were found mainly in association with the astral microtubules (inset, Fig. 7D). However, some bacteria clusters were also found at the poles of the meiotic-like spindles (Fig. 7F,F9) and near the periphery of figures resulting from the fusion of two PLoS ONE | www.plosone.org 5 January 2012 | Volume 7 | Issue 1 | e30045