Male-killing Wolbachia in the butterfly Hypolimnas bolina

Some lines of the butterfly Hypolimnas bolina L. (Lepidoptera: Nymphalidae) are characterized by their female-biased sex ratio. In these lines, most males die before reaching the middle larval stage. However, the cause of the bias remains unclear. We detected the proteobacterium Wolbachia in all individuals in the female-biased butterfly lines and in some of the lines with a normal sex ratio. Tetracycline treatment of adult females of a female-biased line led to a significant increase in both the hatch rate of their eggs (F1) and the male-to-female ratio of F1 pupae. In addition, certain assays of tetracycline treatment on mother butterflies significantly increased the male to female ratio of F1 adults. Known bacterial sex ratio distorters other than Wolbachia were not detected by diagnostic PCR assay, nor by the sequencing of 16S rDNA amplified using general prokaryotic 16S rDNA primers. These results strongly suggest that the distortion of the sex ratio is due to the killing of males by the inherited Wolbachia. Sequences of the 16S rDNA amplified using Wolbachia-specific primers, the cell division protein gene (ftsZ), the molecular chaperone groE genes (groE operon), and the Wolbachia surface protein gene (wsp) from Wolbachia in lines belonging to three subspecies of the butterfly (bolina, jacintha, and philippensis) revealed no variation among lines nor between female-biased lines and a normal one.