of chromatin architecture and dynamics, pointing to the existence of a bacterial factor/s that regulate the chromatin remodeling of its eukaryotic host. Results To characterize events associated with male embryo death in eggs of Wolbachia-infected Drosophila bifasciata, strain KOS10, we analyzed microtubule distribution and chromatin organization in embryos at various stages of development in order to score zygotic spindle formation, intravitelline and syncytial mitoses, cellular blastoderm formation, and later stages of embryogenesis. To ascertain whether the primary block in development was due to sperm entry failure, we stained eggs collected from 30 minutes to 3 hours after egg deposition (AED) with an antibody directed against acetylated-a-tubulin, shown to recognize axonemal struc- tures in cilia and flagella [22]. This antibody allowed the unambiguous tracing of the position of the sperm tail within the developing Drosophila embryo. Of the 273 embryos analyzed in this study, spanning from second meiosis to cellular blastoderm stage, 81% had a distinct sperm tail. This is a relatively low percentage in comparison to Drosophila melanogaste where a large majority of eggs deposited (.95%) have been shown to contain a detectable sperm tail that ends near the elongated nucleus in the anterior region of the egg [23]. To exclude the possible influence of Wolbachia bacteria on sperm entry into the egg, we also analyzed eggs obtained by the uninfected KOS1 strain. Of the 191 embryos examined, 83% contained a distinct sperm tail. The latter suggests that the reduced fertilization rate we observed was unrelated to the presence of Wolbachia bacteria but is a characteristic of this Drosophila population. Furthermore these observations indicate that failure of sperm entrance in the oocyte is not the primary cause of the early developmental block described in eggs obtained by KOS10 females. Female meiosis and gonomeric spindle formation in the Wolbachia background To determine whether the primary lesion leading to the formation of abnormal embryos was due to aberrant female meiosis, newly laid oocytes were stained for simultaneous visualization of microtubules and DNA. Oocytes scored 20 minutes AED (n = 53) had meiotic spindles of normal shape spanning from metaphase (Fig. 1A) to telophase of the second meiosis, where two tapered spindles aligned in tandem and oriented radially with respect to the oocyte surface. These spindles are typically anastral, but a monastral array of microtubules was found between them (Fig. 1A). The central aster contained a large accumulation of centrosomin (Cnn), confirming that the microtubules of the central asters were nucleated by bona fide centrosomal material (not shown). Female meiosis ends with the formation of four haploid chromosome complements that were aligned radially to the oocyte surface: the innermost haploid complement, the presumptive female pronucleus, moved toward the center of the egg where the male pronucleus was localized, whereas the other female complements moved to the egg surface to form polar bodies. These observations indicate that meiosis proceeded normally [24–26]. The assembly of the zygotic centrosome in wild-type eggs requires the recruitment of centrosomal components from the egg cytoplasm to a site adjacent to the sperm nucleus that corresponds to the basal body region [27]. To ascertain whether, in oocytes obtained by the KOS10 strain, the centrosomal components assembled around the sperm basal body to build a functional zygotic centrosome, we double stained newly fertilized eggs with anti-tubulin and Cnn. A distinct accumulation of Cnn was found associated with the sperm aster, suggesting that the recruitment of Chromatin Remodeling and Male Killing centrosomal components is normal in these eggs (not shown). Furthermore, double immunostaining with anti-tubulin antibodies and Hoechst dye for simultaneous visualization of microtubules and chromatin showed a prominent aster of microtubules in correspondence to the sperm nucleus (not shown). Therefore, defects in sperm centrosome assembly were not a primary cause of the early developmental block found in embryos of Drosophila bifasciata. To determine whether abnormalities accounting for embryo death during early development may be due to defects occurring before or subsequent to the fusion of male and female pronuclei we analyzed the formation of the gonomeric spindle. Eggs collected 30–45 minutes AED were double stained for microtubules and DNA (n = 221; Table S1). In some eggs (n = 35; 15.8%) four haploid chromosome complements were found aligned radially to the egg surface, the innermost haploid complement being the presumptive female pronucleus together to a fifth anterior male nucleus. Other eggs (n = 25; 11.3%) displayed a large microtubule aster, the sperm aster, and two close nuclei, male and female (Fig. 1B). A gonomeric spindle composed of two closely apposed spindles holding separate paternal and maternal complements, organized during metaphase near the duplicated sperm centro- some (Fig. 1C). This spindle included the separate sets of parental chromosomes with a similar degree of condensation (Fig. 1C), as described during the fertilization of Drosophila melanogaster [28]. In 100 embryos (45.4%) the first mitosis progressed normally. At anaphase the parental complements segregated (Fig. 1D) and reached the opposite poles of the spindle during telophase (Fig. 1E). Acetylated-tubulin labeling revealed the persistence of the sperm tail at one pole of the zygotic spindle (Fig. 1E). When mitosis proceeded normally, two identical spindles were found within the inner cytoplasm (Fig. 1F). However, 10.8% (n = 24) of eggs scored during the first mitosis revealed abnormal figures spanning from disorga- nized gonomeric spindles, where the parental complements were held by widely separated spindles (Fig. 1G,H), to abnormal anaphase figures in which the chromosomes were arrested at the metaphase plate and centrosomes were detaching from the poles (Fig. 1I). We also found irregular anaphase/telophase configura- tions where chromosomes were scattered (Fig. 1J) or stretched (Fig. 1K) between the opposing poles. It was observed that 37 eggs (16.7%) scored after 30–45 minutes AED lacked a male pronucleus and a sperm tail, suggesting that fertilization failed in these eggs. These observations led to the test of whether the improper positioning of the half parental complements in the gonomeric spindle could affect the normal progression of the second division. From 270 embryos collected 60–90 minutes AED (Table S1) 46 were during the second mitotic division: 39 of these embryos had chromosomes that were properly segregated (Fig. 1F), whereas in 7 embryos the chromatids had failed to segregate (Fig. 1L). The former embryos completed the second nuclear cycle, and formed two equally shaped mitotic spindles that synchronously supported chromosome congregation at the metaphase plate and sister chromatid movements to the opposite poles. The latter embryos had evident morphological abnormalities in both spindle organi- zation and chromosome segregation and sister chromatids did not migrate properly in these spindles. The association of the sperm tail with the abnormal spindles during the first (Fig. 1I) and second (Fig. 1L) mitoses indicates that these figures were the result of problems that emerged after fertilization. Improper chromatin condensation during the syncytial mitoses is associated with defects of spindle organization The arrest phenotype in some embryos obtained from KOS10 females can be the result of defective steps in the nuclear division PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e30045