Single-cell genome analysis of a feminizing Wolbachia in Eurema butterflies reveals a shared origin with male-killing Wolbachia

<i>Wolbachia</i> is a ubiquitous endosymbiont in arthropods that is maternally transmitted and affects host reproduction in various ways. Among these, skewing the host sex ratio towards females, either by killing males (male killing) or producing exclusively functional females (feminization or parthenogenesis), is considered advantageous for <i>Wolbachia</i>. In the butterfly <i>Eurema mandarina</i>, individuals harbouring the <i>Wolbachia</i> strain <i>w</i>Fem exclusively produce female offspring. This occurs through a two-step mechanism in which <i>Wolbachia</i> blocks the transmission of the Z chromosome from Z0 females and feminizes the resultant Z0 offspring. Given the unique characteristics of <i>w</i>Fem, understanding its genomic features is crucial to uncover the evolution and mechanisms of <i>Wolbachia</i>-induced reproductive manipulation. However, technical challenges in isolating <i>w</i>Fem from co-infecting, closely related, non-male-killing/non-feminizing <i>w</i>CI <i>Wolbachia</i> strain have hindered genomic analyses of <i>w</i>Fem. In this study, we established a closed circular genome of <i>w</i>Fem by developing a series of <i>Wolbachia</i> purification, cell sorting and single-cell genome sequencing techniques. <i>w</i>Fem genome, ~1.3 Mb in size, specifically encodes male-killing gene homologues (<i>Em-oscar</i> and <i>wmk</i>) and other putative virulence factors that are absent in <i>w</i>CI. In addition, <i>w</i>Fem carried prophage elements that showed high similarity to previously characterized male-killing-associated prophages in <i>Wolbachia</i> strains. This study highlights the shared functional genomic features between feminizing and male-killing <i>Wolbachia</i> in Lepidoptera and suggests a mechanistic link between these two <i>Wolbachia</i>-induced reproductive phenotypes.