Female-biased sex ratios in Danaus chrysippus (Linnaeus) (family Nymphalidae, subfamily Danainae, tribe Danaini) populations are attributed to the action of an endosymbiotic ‘male-killer’ bacterium of the genus Spiroplasma. In stark contrast to the extensive geographic range of their host, the Spiroplasma appears to be restricted to East Africa, where four African D. chrysippus subspecies exist sympatrically and form a hybrid zone. In this study, specimens collected at three sample sites within the hybrid zone were screened for Spiroplasma infection. The findings demonstrate that, within the hybrid zone, the frequency of Spiroplasma infection varies both spatially and temporally. Host genotype at three biallelic wing pattern loci, representative of subspecific genetic divergence, is correlated to Spiroplasma infection. Linkage between the frequency of the recessive a allele and Spiroplasma is established, suggesting the presence of a linked allele controlling infection susceptibility. In addition, a negative correlation between D. chrysippus forewing length and Spiroplasma infection is identified, suggesting that infection has a deleterious effect on body size.

Whilst most animals invest equally in males and females when they reproduce, a variety of vertically transmitted parasites has evolved the ability to distort the offspring sex ratios of their hosts. One such group of parasites are male-killing bacteria. Here we report the discovery of females of the ladybird Anisosticta novemdecimpunctata that produced highly female-biased offspring sex ratios associated with a 50% reduction in egg hatch rate. This trait was maternally transmitted with high efficiency, was antibiotic sensitive and was infectious following experimental haemolymph injection. We identified the cause as a male-killing Spiroplasma bacterium and phylogenetic analysis of rDNA revealed that it belongs to the Spiroplasma ixodetis clade in which other sex ratio distorters lie. We tested the potential for interspecific horizontal transfer by injection from an infected A. novemdecimpunctata line into uninfected individuals of the two-spot ladybird Adalia bipunctata. In this novel host, the bacterium was able to establish infection, transmit vertically and kill male embryos.

Many insects carry maternally inherited bacteria which kill male offspring. Such bacteria will spread if male death benefits the female siblings who transmit the bacterium, and they are therefore expected in insects with antagonistic sibling interactions. We report that the butterfly Danaus chrysippus is host to a maternally inherited male-killing bacterium. Using diagnostic PCR and rDNA sequence, the bacterium was identified as a Spiroplasma closely related to 2 ladybird beetle male-killers and the tick symbiont Spiroplasma ixodetis. The male-killer was found to have a geographically restricted distribution, with up to 40% of females being infected in East Africa, but no detectable infection in small samples from other populations. Danaus chrysippus is a surprising host for a male-killer as its eggs are laid singly. This suggests that the ecological conditions permitting male-killers to invade may be more widespread than previously realized.