Chromatin Remodeling and Male Killing Figure 5. Embryos at later stages of development have highly defective areas. Embryos were fixed after 15–20 hours AED and stained for microtubules (green) and DNA (red). (A) Normal developing embryos in which the shortening of germ band is completed: segments are well evident. (B,C) Defective embryos after the retraction of the germ band: (B) the integrity of some segments is affected (arrows) and (C) large areas of the embryos are incompletely formed and still showed mitotic divisions (asterisks). (D) Detail of an embryo during germ band shortening: note the lack of differentiation of the anterior region of the body (arrows). Insets are details of the surface areas. Scale bar is 25 mm in the main panels and 7 mm in insets. doi:10.1371/journal.pone.0030045.g005 mated with uninfected males [7]. However, despite the key role of these microorganisms, very little is known about the cellular mechanisms involved in this process. This study is a cytological analysis of the early development of eggs obtained by crosses of Wolbachia infected Drosophila bifasciata females with uninfected males. Previous studies [6,20,31,32] showed that the sex ratio bias distortion observed in Drosophila bifasciata crosses is a result of a reduced male hatching rate compared to control uninfected females. However, there is little precise knowledge regarding the timing of male death. Earlier observations in Drosophila willistoni and Drosophila melanogaster infected by Spiroplasma suggest that the triggering of embryo defects occur at any point of development, from intravitelline mitoses to germ band shortening, with death occurring even in later stages [10,12]. Findings using Drosophila nebulosa infected with Spiroplasma have shown that male embryos arrest development between stages 11 and 13 [11]. The cause for the discrepancy between these observations remains unclear, but may be a host dependent phenotype. Using transfection experiments, it has been demonstrated, that the expression intensity of the Spiroplasma-induced male killing varies depending on the host background within a single species [33]. Analysis of our data revealed that there is a gradual increase of embryonic death during early development. Given that the percentage of unfertilized eggs and embryos arrested during the early intravitelline mitoses was almost constant through the developmental stages, it is possible that some abnormal embryos escaped detection continue to develop and were counted at each stage. It is possible that male-killer bacteria act a specific time, but the effect of the action might be prolonged over the developmental time period. Alternatively, a threshold effect, requiring a critical concentration of bacterial product/s within the embryo, could be implicated to explain the phenotype we observed in Drosophila bifasciata females infected with Wolbachia where the arrest of male embryos was observed at any stage of development. This is supported by the finding that mitotic abnormalities occurring during blastoderm formation and gastrulation affect separate clusters of nuclei, indicating that mitotic problems occurred independently in different regions of the embryo- The effect is first seen in isolated nuclei that in turn transmit their abnormalities to progeny nuclei where the problems are amplified, forming clusters of irregularly condensed nuclei. Nuclei in these areas tend to be picnotic and sink inward suggesting the existence of a checkpoint monitoring incorrect chromatin organization. PLoS ONE | www.plosone.org 8 January 2012 | Volume 7 | Issue 1 | e30045