Removing symbiotic Wolbachia bacteria specifically inhibits oogenesis in a parasitic wasp

Abstract

Wolbachia are bacteria that live in the cells of various invertebrate species to which they cause a wide range of effects on physiology and reproduction. We investigated the effect of Wolbachia infection in the parasitic wasp, Asobara tabida Nees (Hymenoptera, Braconidae). In the 13 populations tested, all individuals proved to be infected by Wolbachia. The removal of Wolbachia by antibiotic treatment had a totally unexpected effect-aposymbiotic female wasps were completely incapable of producing mature oocytes and therefore could not reproduce. In contrast, oogenesis was not affected in treated Asobara citri, a closely related species that does not harbor Wolbachia. No difference between natural symbiotic and cured individuals was found for other adult traits including male fertility, locomotor activity, and size, indicating that the effect on oogenesis is highly specific. We argue that indirect effects of the treatments used in our study (antibiotic toxicity or production of toxic agents) are very unlikely to explain the sterility of females, and we present results showing a direct relationship between oocyte production and Wolbachia density in females. We conclude that Wolbachia is necessary for oogenesis in these A. tabida strains, and this association would seem to be the first example of a transition from facultative to obligatory symbiosis in arthropod-Wolbachia associations.

Figure 1

Genital apparatus of Asobara tabida females at emergence. (A) Apparatus from untreated control female. Note the presence of numerous mature oocytes in the basal region of ovarioles (bo). (B) Apparatus from cured female (larval treatment, rifampicin, 2 mg/g). Note the total absence of oocyte in ovarioles. ge, germarium; ov, ovipositor; vi, vitellarium. [Bar = 1 mm (for A and B).]

Figure 2

Effect of rifampicin concentration (larval treatment, from 0 to 0.1 mg/g) on oocyte load and on the rate of infection by Wolbachia (percentage of females that were PCR positive). For each value, at least 20 females were used per rifampicin concentration.

Figure 3

Comparison between ovaries and oocytes from treated females (larval treatment, rifampicin, 2.10⁻² mg/g) with ovaries and oocytes from untreated control females. (A and B) Epifluorescent images of ovaries stained with 4′,6-diamidino-2-phenylindole. Ovaries were collected from a control A. tabida female, which had 228 mature oocytes in ovaries (A), or a rifampicin-treated female, containing only 36 mature oocytes (B). The ovaries were spread to reveal the oocytes and were oriented with the germarium to the right. (C–H) Confocal images of oocytes stained with propidium iodide. Oocytes having been collected from a control female (C–E) or a rifampicin-treated female as in B (F–H). Accumulation of Wolbachia is seen at the posterior extremity of the control oocyte (C). D and E are magnified views of the nucleus and posterior regions from the same oocyte, respectively. In contrast, the oocyte in F contains very low levels of Wolbachia in the posterior region (H). [Bar in A = 500 μm (for A and B).]; [Bar in C = 50 μm (for C and F).]