Wolbachia is an endosymbiont of diverse arthropod lineages that can induce various alterations of host reproduction for its own benefice. Cytoplasmic incompatibility (CI) is the most common phenomenon, which results in embryonic lethality when males that bear Wolbachia are mated with females that do not. In the cherry fruit fly, Rhagoletis cerasi, Wolbachia seems to be responsible for previously reported patterns of incompatibility between populations. Here we report on the artificial transfer of two Wolbachia variants (wCer1 and wCer2) from R. cerasi into Drosophila simulans, which was performed with two major goals in mind: first, to isolate wCer1 from wCer2 in order to individually test their respective abilities to induce CI in the new host; and, second, to test the theoretical prediction that recent Wolbachia-host associations should be characterized by high levels of CI, fitness costs to the new host, and inefficient transmission from mothers to offspring. wCer1 was unable to develop in the new host, resulting in its rapid loss after successful injection, while wCer2 was established in the new host. Transmission rates of wCer2 were low, and the infection showed negative fitness effects, consistent with our prediction, but CI levels were unexpectedly lower in the new host. Based on these parameter estimates, neither wCer1 nor wCer2 could be naturally maintained in D. simulans. The experiment thus suggests that natural Wolbachia transfer between species might be restricted by many factors, should the ecological barriers be bypassed.Wolbachia is a maternally inherited ␣-proteobacterium and symbiont of arthropods (4,26,34,42). This bacterium has an intracellular lifestyle, and infections occur throughout host somatic and germ line tissues of insect species (15). As a reproductive parasite, it manipulates host reproduction and favors in this way its own dispersal in host populations. The most common Wolbachia effect described so far is cytoplasmic incompatibility (CI) (8,21). CI arises when infected males mate with uninfected females and results in embryonic lethality. Reciprocal crosses between infected females and uninfected males do not express CI. This pattern can be interpreted through a two-function model (29, 42): Wolbachia would somehow modify the sperm of infected males during spermatogenesis (modification, or mod function), leading to embryo death unless Wolbachia is present in the egg and restores viability (rescue, or resc function). The mod and resc functions seem to interact in a specific manner, because CI can also be observed in crosses between males and females that are both infected, if the two partners bear different Wolbachia variants.CI allows Wolbachia to invade host populations because it increases the fitness of infected females relative to that of uninfected ones. Both theoretical and empirical studies (6,16,19,36) have highlighted the key role of three parameters in the invasion dynamics: (i) CI level (the percentage of embryos killed by CI in incompatible crosses), (ii) the fitness ef...