Evidence for an one-step mechanism of endosymbiont-induced thelytoky in the parasitoid wasp, Muscidifurax uniraptor

Wolbachia manipulates host reproduction in various haplodiploid insect species, in which fertilized eggs normally develop into diploid females while unfertilized eggs develop into haploid males. Females infected with a thelytoky-inducing Wolbachia produce diploid daughters from unfertilized eggs (thelytoky), but in some infected species diploid males spontaneously occur in low numbers. This suggests that diploidization and feminization are induced separately. In the Wolbachia- infected thelytokous parasitoid wasp, Muscidifurax uniraptor , occasional males have been found but with unknown ploidy. Therefore, we studied the mechanism of Wolbachia -induced thelytoky in M. uniraptor in the context of sex determination. We started by feeding different concentrations of tetracycline (antibiotic) to M. uniraptor females to gradually reduce the Wolbachia titre. We found that a decreased Wolbachia titre leads to an increased proportion of haploid male offspring, but we found no diploid males. Next, we studied the effect of Wolbachia infection on the expression and splicing of the sex determination genes transformer ( Mutra ) and transformer-2 ( Mutra2 ) in female ovaries and conclude that Wolbachia does neither affect the expression nor the splicing of Mutra and Mutra2 . We then measured Mutra and Mutra2 expression levels in developing zygotes at different time points and found a two-fold Mutra expression increase in Wol + compared to Wol -. Finally, we used the sexually reproducing sister species, Muscidifurax raptorellus and artificially created triploid females to determine whether diploidization is sufficient for feminization. These triploid females, when virgin, produced haploid sons and diploid daughters, showing that in Muscidifurax feminization solely depends on ploidy level. This strongly suggests that Wolbachia only needs to induce diploidization and that bi-allelic Mutra expression is sufficient for female development. ### Competing Interest Statement The authors have declared no competing interest.