Wolbachia-Mediated Male Killing Is Associated with Defective Chromatin Remodeling Maria Giovanna Riparbelli1, Rosanna Giordano 2 , Morio Ueyama 3 , Giuliano Callaini1* 1 Department of Evolutionary Biology, University of Siena, Siena, Italy, 2 Illinois Natural History Survey, Institute of Natural Resource Sustainability, University of Illinois at Urbana-Champaign, Champaign, Illinois, United States of America, 3 Laboratory of Cell Biology, Department of Bioinformatics, Soka University, Hachioji, Tokyo, Japan Abstract Male killing, induced by different bacterial taxa of maternally inherited microorganisms, resulting in highly distorted female- biased sex-ratios, is a common phenomenon among arthropods. Some strains of the endosymbiont bacteria Wolbachia have been shown to induce this phenotype in particular insect hosts. High altitude populations of Drosophila bifasciata infected with Wolbachia show selective male killing during embryonic development. However, since this was first reported, circa 60 years ago, the interaction between Wolbachia and its host has remained unclear. Herein we show that D. bifasciata male embryos display defective chromatin remodeling, improper chromatid segregation and chromosome bridging, as well as abnormal mitotic spindles and gradual loss of their centrosomes. These defects occur at different times in the early development of male embryos leading to death during early nuclear division cycles or large defective areas of the cellular blastoderm, culminating in abnormal embryos that die before eclosion. We propose that Wolbachia affects the development of male embryos by specifically targeting male chromatin remodeling and thus disturbing mitotic spindle assembly and chromosome behavior. These are the first observations that demonstrate fundamental aspects of the cytological mechanism of male killing and represent a solid base for further molecular studies of this phenomenon. Citation: Riparbelli MG, Giordano R, Ueyama M, Callaini G (2012) Wolbachia-Mediated Male Killing Is Associated with Defective Chromatin Remodeling. PLoS ONE 7(1): e30045. doi:10.1371/journal.pone.0030045 Editor: John R. Battista, Louisiana State University and A & M College, United States of America Received August 2, 2011; Accepted December 8, 2011; Published January 23, 2012 Copyright: 2012 Riparbelli et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Callaini@unisi.it Introduction Several maternally inherited symbiotic bacteria are known to affect the reproductive biology of their host species by favouring female over male offspring. Mechanisms of sex-ratio distortion include the induction of parthenogenesis, feminization and male- killing of their arthropod host species [1–4]. The most dramatic form of sex-ratio distortion is male-killing in which bacteria pass from infected females to their progeny and selectively kill males they infect during embryogenesis, resulting in female-biased sex- ratios in their insect host. Male killing bacteria belong to diverse taxa and are widespread among arthropods and common within insects [5–8]. in the genus Drosophila, male killing has been associated with the presence of maternally transmitted Spiroplasma and Wolbachia [9]. While the biology of male killing bacteria has been extensively studied at the ecological level, little is known about how bacteria and their hosts interact at the cellular and molecular level. Past works have examined male killing phenotypes induced by Spiroplasma poulsonii in Drosophila willistoni [10], Drosophila nebulosa [11], and Drosophila melanogaster [12,13]. However, the underlying mechanisms that produce male-specific lethality remain unknown [4]. Cytological observations in Drosophila willistoni have suggested that male killing Spiroplasma interfere with the early development of embryos by affecting normal mitotic progression [10]. Whereas genetic evidence suggests that Spiroplasma can target some components of the male-specific sex-determination pathway [14]. Another male-killing organism, the c-proteobacterium Arsenophonus nasoniae has been shown to induce male killing in the wasp Nasonia vitripennis by targeting maternally inherited centro- somes [15]. These findings suggest that male killing bacteria have evolved different modes of interaction with their insect hosts resulting in diverse pathways to embryo male death. Wolbachia has been implicated in female biased sex-ratios in diverse arthropod host orders: from the arachnid Pseudoscor- piones [16], to the insect Coleoptera [17], Lepidoptera [18] and Diptera [19]. In Drosophila bifasciata, molecular systematic analysis and susceptibility to antibiotics have demonstrated that the male killing phenotype is not of viral origin [20], but is associated with a Wolbachia infection [21]. Moreover, it has also been shown that the male killing phenotype in D. bifasciata has high penetrance at low temperatures (18uC) and is reduced at high temperatures (26uC). This difference may be the result of reduced bacterial density at elevated temperatures [21]. This study aimed to examine the interaction between Wolbachia bacteria and Drosophila bifasciata by analysing early developmental stages of embryos obtained from crosses of infected females and uninfected males using fluorescent staining of both chromatin and microtubules. Results from this study demonstrate that male embryos derived from Wolbachia infected Drosophila bifasciata females mated with uninfected males show severe defects of chromatin remodeling and spindle organization that result in abnormal mitoses and development failure. Our work leads us to conclude that this male-killing strain of Wolbachia plays a crucial role as a modulator PLoS ONE | www.plosone.org 1 January 2012 | Volume 7 | Issue 1 | e30045