Chromatin Remodeling and Male Killing Figure 6. Sxl staining reveals that only male embryos have improper chromatin condensation. Embryos were stained for DNA (larger panels) and Sxl (insets, green). Sxl positive staining is associated with embryos showing normal blastoderm (A) and proper segmentation (B). Sxl antibody fails to stain embryos that have abnormal blastoderm (C) and improper segmentation (D). Scale bar is 25 mm in the main panels and 70 mm in insets. doi:10.1371/journal.pone.0030045.g006 It has been demonstrated that reducing bacterial density within eggs following exposure to elevated temperature leads to the decrease of male killing effects [6]. Consistently, mothers with a higher density of bacteria have more female-biased offspring sex- ratio [34]. Moreover, a Spiroplasma strain that normally causes early male killing also induces late male mortality in larvae and pupae in the offspring of young females [35], suggesting a correlation with maternal host age. The stage 13 male embryos we observed with no apparent defects or only small abnormalities could account for a class of late male-killing phenotypes, likely obtained by young females. Consistent with this hypothesis these embryos were found in batches in which there were large numbers of unfertilized eggs produced more frequently by young females. Intracellular density of the endosymbionts also represents a limiting factor for the intensity of cytoplasmic incompatibility and the strength of the phenotype induced [36–41]. Although male killing almost invariably results in death of the male embryos, past studies have suggested that the mechanisms potentially involved in this process do not require the Y chromosome as a specific target, since XXY females survive, and transmit the infection to their progeny [42]. Rather, it appears that flies with a single X chromosome are killed regardless of their phenotypic sex [43]. This indicates that elements of the dosage compensation system may represent the target of male-killing bacteria. This system compensates the unbalance between the unique X chromosome of males and the two chromosomes of females by augmenting X-chromosome linked transcription of the X-linked genes in males. Consistently, mutants of the tra gene that bear two X chromosomes are not killed by Spiroplasma and develop as somatic males [42]. Genetic analysis showed, that Spiroplasma failed to kill males of Drosophila melanogaster lacking any of the components of the dosage compensation complex [14], pointing to the requirement of a functional dosage compensation complex for the expression of bacteria mediated male-killing. Consistently, the expression of a doublesex homologue is altered in sexual mosaics of Ostrinia scapulalis moths infected with Wolbachia [44]. However, this does not appear to be the sole mechanism of male-killing activity. Male-killing Wolbachia have been described in both male and female heterogametic species suggesting that the dosage compensation pathway may not be the single focus of male-killing activity [5]. Moreover, the observation that the action of Wolbachia in the lepidopteran Hypolimnas can also contribute to male death in larval stages indicates that male-killing is not solely dependent on a process confined to embryos but may encompass the sex determination system that occurs early in embryogenesis [45]. Our finding of an earlier embryonic lethal phase, at the time of parental chromosome segregation during the first and second mitoses not related to the downstream male-specific developmen- tal pathways that are activated at later developmental stages [46], support the possibility of multiple targets for male-killing bacteria PLoS ONE | www.plosone.org 9 January 2012 | Volume 7 | Issue 1 | e30045