Date Published: September 10, 2019
Publisher: Public Library of Science
Author(s): Jessamyn I. Perlmutter, Sarah R. Bordenstein, Robert L. Unckless, Daniel P. LePage, Jason A. Metcalf, Tom Hill, Julien Martinez, Francis M. Jiggins, Seth R. Bordenstein, David S. Schneider.
Wolbachia are the most widespread maternally-transmitted bacteria in the animal kingdom. Their global spread in arthropods and varied impacts on animal physiology, evolution, and vector control are in part due to parasitic drive systems that enhance the fitness of infected females, the transmitting sex of Wolbachia. Male killing is one common drive mechanism wherein the sons of infected females are selectively killed. Despite decades of research, the gene(s) underlying Wolbachia-induced male killing remain unknown. Here using comparative genomic, transgenic, and cytological approaches in fruit flies, we identify a candidate gene in the eukaryotic association module of Wolbachia prophage WO, termed WO-mediated killing (wmk), which transgenically causes male-specific lethality during early embryogenesis and cytological defects typical of the pathology of male killing. The discovery of wmk establishes new hypotheses for the potential role of phage genes in sex-specific lethality, including the control of arthropod pests and vectors.
Wolbachia (order Rickettsiales) infect an estimated 40–52% of all arthropod species [1, 2] and 47% of filarial nematode species , making them the most widespread intracellular bacterial symbiont in animals. Concentrated in host testes and ovaries, Wolbachia primarily transmit cytoplasmically from mother to offspring [4, 5]. In arthropod reproductive tissues and embryos, Wolbachia deploy cunning manipulations to achieve a greater proportion of transmitting females in the host population. Collectively, these strategies are categorized as reproductive parasitism.
This study reports twelve key results supporting wmk as a male-killing gene candidate: (i) wmk recurrently associates with genomic screens for reproductive parasitism; it is on the shortlists of candidate phage WO genes in Wolbachia male-killers and CI-inducers . (ii) The wmk gene is found in all sequenced male-killers including the reduced phage WO genome of wRec (which retains ~25% of the full phage WO genome) and the divergent phage WO genome of wBif. (iii) wmk is common, divergent in sequence, and located in the eukaryotic association module of phage WO that is enriched with sequences predicted or known to contain eukaryotic function and homology . In this region, wmk is a few genes away from the two causative cytoplasmic incompatibility genes, cifA and cifB, that modify arthropod gametes . (iv) Transgenic expression of wmk consistently induces a sex-ratio bias, but the phenotype does not recapitulate other forms of reproductive parasitism. (v) No sex ratio bias results from expression of other transgenes tested thus far under the same expression system, making the phenotype specific to wmk. (vi) Canonical DNA defects are recapitulated under transgenic expression at the same time in development as natural systems. (vii) wmk is naturally expressed in wMel and wBif embryos at the time the defects are known to occur in D. bifasciata. (viii) The Wmk protein is predicted to interact with DNA when DNA defects are a hallmark of Wolbachia male killing. (ix) wmk is unique to Wolbachia, and the Wolbachia male-killing mechanism has some unique phenotypic features compared to other male-killers. For example, the dosage compensation complex is not mislocalized in Wolbachia infection, but it is in Spiroplasma infection [13, 49]. (x) The phenotype can be induced with drivers that yield approximately ten-fold variation in expression levels, indicating the highest Act5c levels of expression are not necessary for the phenotype. (xi) DNA damage is more common in wmk males than in controls and it is associated with H4K16ac, which parallels data in natural infections. (xii) Wmk’s predicted structure is conserved across arthropod hosts despite sequence divergence, indicating it likely has conserved function.
Statistical analyses were done using GraphPad Prism software (version 5 or 8) or GraphPad online tools, unless otherwise noted. For comparisons among only two data categories, we used the two-tailed, non-parametric Mann-Whitney U test. For comparisons with more groups, a non-parametric Kruskal-Wallis one-way analysis of variance was used, followed by Dunn’s test for multiple comparisons, if significant. In cases of comparisons among groups where only a single measurement was taken per group (such as cytology experiments), a Chi-square test was used. Exact tests used and other important information are listed in the figure legends of each experiment.