A single mutation weakens symbiont-induced reproductive manipulation through reductions in deubiquitylation efficiency


Wolbachia are maternally transmitted bacteria that infect most insects, making them the most common endosymbionts. Wolbachia achieved this status by manipulating host reproduction. For example, many Wolbachia cause cytoplasmic incompatibility (CI) that kills uninfected embryos. In females, Wolbachia can rescue CI, promoting their spread to high frequencies in host populations. CI strength varies in nature from weak to strong. Importantly, strong CI enables Wolbachia biocontrol strategies in mosquito systems, which protect millions of individuals from arboviruses. However, theory predicts that natural selection does not act to preserve genes that cause CI, suggesting mutations may disrupt it. We show that a single naturally observed mutation weakens CI by reducing deubiquitylation. These discoveries help elucidate the molecular basis of symbiont-induced reproductive manipulations.


Animals interact with microbes that affect their performance and fitness, including endosymbionts that reside inside their cells. Maternally transmitted Wolbachia bacteria are the most common known endosymbionts, in large part because of their manipulation of host reproduction. For example, many Wolbachia cause cytoplasmic incompatibility (CI) that reduces host embryonic viability when Wolbachia-modified sperm fertilize uninfected eggs. Operons termed cifs control CI, and a single factor (cifA) rescues it, providing Wolbachia-infected females a fitness advantage. Despite CI’s prevalence in nature, theory indicates that natural selection does not act to maintain CI, which varies widely in strength. Here, we investigate the genetic and functional basis of CI-strength variation observed among sister Wolbachia that infect Drosophila melanogaster subgroup hosts. We cloned, Sanger sequenced, and expressed cif repertoires from weak CI–causing wYak in Drosophila yakuba, revealing mutations suspected to weaken CI relative to model wMel in D. melanogaster. A single valine-to-leucine mutation within the deubiquitylating (DUB) domain of the wYak cifB homolog (cidB) ablates a CI-like phenotype in yeast. The same mutation reduces both DUB efficiency in vitro and transgenic CI strength in the fly, each by about twofold. Our results map hypomorphic transgenic CI to reduced DUB activity and indicate that deubiquitylation is central to CI induction in cid systems. We also characterize effects of other genetic variation distinguishing wMel-like cifs. Importantly, CI strength determines Wolbachia prevalence in natural systems and directly influences the efficacy of Wolbachia biocontrol strategies in transinfected mosquito systems. These approaches rely on strong CI to reduce human disease.


  • Author contributions: J.F.B., K.V.V., and B.S.C. designed research; J.F.B., K.V.V., D.E.A., and B.S.C. performed research; J.F.B., D.E.A., and B.S.C. contributed new reagents/analytic tools; J.F.B., K.V.V., D.E.A., and B.S.C. analyzed data; J.F.B. and B.S.C. provided funding acquisition; B.S.C. coordinated the research; and J.F.B., K.V.V., D.E.A., and B.S.C. wrote the paper.

  • The authors declare no competing interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.2113271118/-/DCSupplemental.

Data Availability

All study data are included in the article and/or supporting information.

  • Accepted August 16, 2021.

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