M. E. N. Majerus

Male-killing Wolbachia in two species of insect

The inherited bacterium Wolbachia spreads through the manipulation of host reproduction, and has been suggested to be an important factor in arthropod evolution, from host speciation to the evolution of sex–determination systems. Past work has shown that members of this group may produce cytoplasmic incompatibility, feminize genetically male hosts, and induce host parthenogenesis. Here, we report an expansion of the range of reproductive manipulations produced by members of this clade, recording Wolbachia strains that kill male hosts during embryogenesis in two host species, the ladybird Adalia bipunctata, and the butterfly Acraea encedon. Both male–killing bacteria belong to the B group of Wolbachia. However, phylogenetic analyses were unable to resolve whether the bacteria in the two species are monophyletic, or represent independent origins of male–killing among the B–group Wolbachia. We also found significant divergence within the wsp gene of Wolbachia strains found in different A. bipunctata individuals, suggesting this host species contains two Wolbachia strains, diverged in wsp sequence but monophyletic. Our observations reinforce the notion that Wolbachia may be an important agent driving arthropod evolution, and corroborates previous suggestions that male–killing behaviour is easily evolved by invertebrate symbionts.

The butterfly Danaus chrysippus is infected by a male-killing Spiroplasma bacterium

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.

Recombination confounds interpretations of Wolbachia evolution

Wolbachia are vertically transmitted bacteria known from arthropods and nematode worms, which are maintained in host populations because they either physiologically benefit infected individuals or parasitically manipulate their reproduction. The different manipulation phenotypes are scattered across the Wolbachia phylogeny, suggesting that there have been multiple evolutions of similar phenotypes. This conclusion relies on the assumption of an absence of recombination between bacterial strains, so that the gene used to reconstruct the phylogeny reflects the evolutionary history of the genes involved in the trait. We tested for recombination by reconstructing the phylogeny of two Wolbachia genes from seven B–subdivision strains. The two genes produced mutually incompatible topologies, indicating that these lineages are subject to genetic recombination. This means that many evolutionary patterns inferred from Wolbachia phylogenies must be re–evaluated. Furthermore, recombination may be an important feature both in the evolution of the manipulation phenotypes and avoidance of Müllers ratchet. Finally, we discuss the implications of recombination for attempts to genetically engineer Wolbachia for use in the control of crop pests and human pathogens.

Adonia variegata (Coleoptera : Coccinellidae) bears maternally inherited Flavobacteria that kill males only

Inherited bacteria that parasitically distort the pattern of sex allocation of their host, biasing allocation towards female progeny, are found in many arthropods. One such manipulation is male-killing, where male progeny of infected females die during embryogenesis. We here provide evidence for a male-killing bacterium in the coccinellid beetle, Adonia variegata. We then address 3 questions. First, is this male-killing bacterium one that is found in other hosts, or does it represent a new transition to male-killing within the eubacteria? Using the sequence of the 16S rDNA of the bacterium, we found that the male-killing bacterium is a member of the Flavobacteria--Bacteroides group, most closely related to the male-killing bacterium in another ladybird beetle, Coleomegilla maculata. Secondly, is there any evidence that this bacterium affects female host physiology? In a paired test under nutritional stress, we found no evidence for a physiological benefit to infection, and weak evidence of a physiological cost, in terms of reduced fecundity. Thirdly, is there any evidence of host involvement in the transmission of the bacterium to the germ line? We found no evidence of host involvement. Rather, bacteria migrated to the ovariole independently of host cells. We conclude that the bacterium is a parasite, and discuss how 2 different species of ladybird come to be infected with 1 lineage of bacterium, and why case studies of male-killing bacteria have generally found little evidence of any symbiont contribution to host physiological functioning.

Molecular identification of a male-killing agent in the ladybird Harmonia axyridis (Pallas) (Coleoptera: Coccinellidae).

Inherited bacteria that manipulate the reproduction of their hosts are common in invertebrates. Of four known manipulative strategies caused by bacteria in insects, three, namely cytoplasmic incompatibility, feminization and parthenogenesis induction, appear to be caused solely by members of the genus Wolbachia . In contrast, the fourth, male-killing, is associated with a variety of bacteria. Within the coccinellids, which are particularly prone to invasion by male-killing endosymbionts (Majerus & Hurst, 1997), bacteria of four groups, Rickettsia (Werren et al ., 1994), Spiroplasma (Hurst et al ., 1999a), Flavobacteria (Hurst et al ., 1997) and Wolbachia (Hurst et al ., 1999b) have been reported to be associated with the male-killing trait. That one species of coccinellid, Adalia bipunctata , harbours four different bacteria, a Rickettsia , a Spiroplasma and two Wolbachia , demonstrates the susceptibility of these beetles to male-killers. Two questions arise from these observations. First, are there constraints on the evolution of a male-killing strategy within inherited bacteria, or can all such bacteria evolve this phenotype? Second, is invasion by more than one malekiller a general feature of those insect host species which are susceptible to male-killing? The Asian ladybird Harmonia axyridis (Pallas) harbours a maternally inherited, female-biased sex ratio trait, taken to result from male-killing (Matsuka et al ., 1975; Gotoh & Niijima, 1986; Majerus et al ., 1998). This species shows all the features suggested by Majerus & Hurst (1997) to be necessary for the invasion and establishment of malekillers in a coccinellid: aphidophagy, laying eggs in tight batches, sibling egg cannibalism and significant levels of neonate larval mortality due to starvation. Previous studies have shown variation in the vertical transmission efficiencies between male-killing matrilines (Matsuka et al ., 1975; Majerus et al ., 1998), and both progressive sex ratio families (progenic sex ratio becomes progressively female biased) and revertant families (initial female bias in progenic sex ratio is lost) (Matsuka et al ., 1975). Differences in the phenotypic expression of female-biased traits might be indicative of different causative agents of male-killing, making H. axyridis a suitable candidate in which to seek diversity in male-killers over geographical distance. Furthermore, establishment of the identity of the male-killer(s) in this species may help indicate which bacteria are most prone to evolving male-killing.

A new male-killing parasitism: Spiroplasma bacteria infect the ladybird beetle Anisosticta novemdecimpunctata (Coleoptera: Coccinellidae).

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.