Male-killing Bacteria Research Articles

Discovery and identification of a male-killing agent in the Japanese ladybird Propylea japonica (Coleoptera: Coccinellidae)

BackgroundEndosymbionts that manipulate the reproduction of their hosts have been reported widely in invertebrates. One such group of endosymbionts is the male-killers. To date all male-killers reported are bacterial in nature, but comprise a diverse group. Ladybirds have been described as a model system for the study of male-killing, which has been reported in multiple species from widespread geographic locations. Whilst criteria of low egg hatch-rate and female-biased progenic sex ratio have been used to identify female hosts of male-killers, variation in vertical transmission efficiency and host genetic factors may result in variation in these phenotypic indicators of male-killer presence. Molecular identification of bacteria and screening for bacterial presence provide us with a more accurate method than breeding data alone to link the presence of the bacteria to the male-killing phenotype. In addition, by identifying the bacteria responsible we may find evidence for horizontal transfer between endosymbiont hosts and can gain insight into the evolutionary origins of male-killing. Phylogenetic placement of male-killing bacteria will allow us to address the question of whether male-killing is a potential strategy for only some, or all, maternally inherited bacteria. Together, phenotypic and molecular characterisation of male-killers will allow a deeper insight into the interactions between host and endosymbiont, which ultimately may lead to an understanding of how male-killers identify and kill male-hosts.ResultsA male-killer was detected in the Japanese coccinellid, Propylea japonica (Thunberg) a species not previously known to harbour male-killers. Families produced by female P. japonica showed significantly female-biased sex ratios. One female produced only daughters. This male-killer trait was maternally inherited and antibiotic treatment produced a full, heritable cure. Molecular analysis identified Rickettsia to be associated with the trait in this species of ladybird.ConclusionWe conclude that P. japonica is host to a bacterial male-killer that is vertically inherited with variable transmission efficiency. Rickettsia presence correlates with the male-killing trait, but there is some variation in the phenotypic expression of the trait due to interaction with host factors. Phylogenetic analysis using the 16S rRNA and 17 kDa antigen genes suggests there may have been horizontal transfer of Rickettsial male-killers between different ladybird hosts.

Rapid spread of male‐killing Wolbachia in the butterfly Hypolimnas bolina

Reproductive parasites such as Wolbachia can spread through uninfected host populations by increasing the relative fitness of the infected maternal lineage. However, empirical estimates of how fast this process occurs are limited. Here we use nucleotide sequences of male-killing Wolbachia bacteria and co-inherited mitochondria to address this issue in the island butterfly Hypolimnas bolina. We show that infected specimens scattered throughout the species range harbour the same Wolbachia and mitochondrial DNA as inferred from 6337 bp of the bacterial genome and 2985 bp of the mitochondrial genome, suggesting this strain of Wolbachia has spread across the South Pacific Islands at most 3000 years ago, and probably much more recently.

Rapidly Shifting Sex Ratio across a Species Range

Sex ratios are subject to distortion by a range of inherited parasites. Although it has been predicted that the presence of these elements will result in spatial and temporal variation in host sex ratio, testing of this hypothesis has been constrained by availability of historical data. We here determine spatial and temporal variation in sex ratio in a interaction between a butterfly and male-killing Wolbachia bacteria by assaying infection presence in museum specimens, and from this inferring infection prevalence and phenotype in historical populations. Comparison of contemporary and museum samples revealed profound change in four of five populations examined. Two populations become extremely female biased, associated with spread of the male-killer bacterium. One evolved from extremely female biased to a sex ratio near parity, resulting from the infection losing male-killing activity. The final population fluctuated widely in sex ratio, associated with varying frequency of the male killer. We conclude that asynchronous invasion and decline of sex-ratio distorters combines with the evolution of host suppressors to produce a rapidly changing mosaic of sex ratio. As a consequence, the reproductive ecology of the host species is likely to be fundamentally altered over short time scales. Further, the study demonstrates the utility of museum specimens as "silent witnesses" of evolutionary change.

THE EFFECT OF SIBMATING ON THE INFECTION DYNAMICS OF MALE-KILLING BACTERIA

Male-killing (MK) bacteria are vertically transmitted endosymbionts that selectively kill the male offspring of their hosts. Simple mathematical models describe the infection dynamics using two parameters, the bacterial transmission rate and a fitness compensation for siblings of killed males. These models cannot explain two phenomena that have been observed in nature: the persistence of extreme MK causing all-female broods, and the coexistence of two different strains of MK bacteria in the same host population. In the present study, we extend the simple MK models and investigate theoretically the effects of sibmating on the infection dynamics. We demonstrate analytically that, in general, sibmating reduces MK prevalence, and can even cause its extinction. As a special case of this finding, we show that sibmating allows a stable coexistence between no infection and extreme MK. Furthermore, we performed computer simulations and showed that, depending on male mating capacity, a stable coexistence of two strains is possible if sibmating occurs but is below a threshold. The results suggest that sibmating might be an important factor for understanding the infection dynamics of MK bacteria.

The pathology of embryo death caused by the male-killing Spiroplasma bacterium in Drosophila nebulosa

BackgroundInherited bacteria that kill male offspring, male-killers, are known to be common in insects, but little is understood about the mechanisms used by male-killing bacteria to kill males. In this paper we describe the tempo and changes that occur during male-killing by Spiroplasma bacteria in the host Drosophila nebulosa.ResultsSpiroplasma infected D. nebulosa males were developmentally retarded from 6–8 h into embryonic development at 25°C, and arrested at between stages 12 and 13 of embryogenesis (10–12 h). Dying males were characterized by a failure to form segments, and ultimately disintegration of the normal oval embryonic shape. Prior to death, dying males exhibited widespread apoptosis, as testified by TUNEL staining.ConclusionThe Spiroplasma kills male Drosophila in a narrow developmental period, shortly after the formation of the host dosage compensation complex that is required for male-killing. Male death is preceded by widespread apoptosis, but it is uncertain if this is primary or secondary apoptosis.

Male-Killing Bacteria Trigger a Cycle of Increasing Male Fatigue and Female Promiscuity

Sex-ratio distorters are found in numerous species and can reach high frequencies within populations. Here, we address the compelling, but poorly tested, hypothesis that the sex ratio bias caused by such elements profoundly alters their host's mating system. We compare aspects of female and male reproductive biology between island populations of the butterfly Hypolimnas bolina that show varying degrees of female bias, because of a male-killing Wolbachia infection. Contrary to expectation, female bias leads to an increase in female mating frequency, up to a point where male mating capacity becomes limiting. We show that increased female mating frequency can be explained as a facultative response to the depleted male mating resources in female biased populations. In other words, this system is one where male-killing bacteria trigger a vicious circle of increasing male fatigue and female promiscuity.

The Impact of Male-Killing Bacteria on Host Evolutionary Processes

Male-killing bacteria are maternally inherited endosymbionts that selectively kill male offspring of their arthropod hosts. Using both analytical techniques and computer simulations, we studied the impact of these bacteria on the population genetics of their hosts. In particular, we derived and corroborated formulas for the fixation probability of mutant alleles, mean times to fixation and fixation or extinction, and heterozygosity for varying male-killer prevalence. Our results demonstrate that infections with male-killing bacteria impede the spread of beneficial alleles, facilitate the spread of deleterious alleles, and reduce genetic variation. The reason for this lies in the strongly reduced fitness of infected females combined with no or very limited gene flow from infected females to uninfected individuals. These two properties of male-killer-infected populations reduce the population size relevant for the initial emergence and spread of mutations. In contrast, use of Wright's equation relating sex ratio to effective population size produces misleading predictions. We discuss the relationship to the similar effect of background selection, the impact of other sex-ratio-distorting endosymbionts, and how our results affect the interpretation of empirical data on genetic variation in male-killer-infected populations.

Conflict within the Genome: Evolving Defenses to Suppress the Male Killers

In the game of survival, anything goes even the selective extermination of males. Male killing is the preferred strategy for a diverse group of bacteria that infect insects and other arthropods. Aside from its tabloid appeal, male killing offers biologists a platform for investigating genetic conflict evolutionary battles between competing elements within the same genome. Male-killing bacteria are passed from mother to offspring, but only males die from infection, suggesting that males harbor genetic elements that allow them to succumb to infection. In keeping with evolutionary theory, these selfish genetic elements, which spread at the expense of the organism, should engender counteracting elements that promote male survival. Yet scant evidence has linked the evolution of host suppressors to selfish elements that mediate male killing. But now, Emily Hornett, Gregory Hurst, and colleagues report the first case of total suppression of male killing in a butterfly, Hypolimnas bolina, infected with the wBol1 strain of the male-killing bacterium Wolbachia. They attribute survival to genetic elements expressed in the male embryo, an effect called zygotic suppression. Because this mechanism of suppression can inactivate male killers which lie dormant until presented with a novel, vulnerable host it's possible that insects that don't succumb to male killing today may have in fact evolved the means to counteract lethal infection. H. bolina is found throughout the Indo-Pacific. Because wBol1 infection kills males in Polynesia but not in Southeast Asia, breeding individuals from each region could reveal genetic elements in the different populations that favor life over death. And because infected females transmit infection directly to offspring, breeding could also introduce wBol1 genes (and infection) onto the butterfly genetic background (a technique called introgression). The breeding experiments tested two questions: would male-killing wBol1 taken from Moorea in Polynesia lose that ability against Southeast Asian males with a Thai or Philippine genetic background, and would benign wBol1 from Thailand or the Philippines turn lethal against males with a Moorean genetic background? To find out, Hornett et al. mated infected Moorean females with Thai and Philippine males, and mated infected Thai and Philippine females with Moorean males. As a control, wBol1-infected females from both regions were also crossed with males from their native populations. Crossing the Moorean and Southeast Asian populations suppressed the male-killing effects of wBol1 from Moorea in just a single generation in stark contrast to the control crosses (Moorean females mated with Moorean males), which yielded no males at all. But when Moorean wBol1 infection was reintroduced to its native host background by backcrossing first-generation hybrid Moorean/Southeast Asian females with wild Moorean males it became male-lethal again. Egg hatch rates decreased dramatically and just a fraction of males survived. In contrast, continued introduction of Moorean wBol1 infection onto the Southeast Asian male genetic background produced high hatch rates and a normal sex ratio. Infected Thai and Philippine females were serially mated with Moorean males, thus progressively increasing the proportion of Moorean genetic material. By the second generation, some male killing occurred, and by the third generation, males were killed in five out of 13 crosses. By the fifth generation, no males survived. From these results, the researchers concluded that suppression occurs in the embryo, because male offspring of Moorean females crossed with the Southeast Asian males survived even though the mother's genetic profile allows killing. The fact that first-generation hybrids survived at nearly the same ratio as seen in wild Southeast Asian males, they explain, suggests the effect is dominant (requires just one copy of the gene) and is at high frequency in the population. A dominant effect also explains why male killing didn't occur in first-generation crosses between Southeast Asian females and Moorean males the suppressor elements had not been segregated out of the population yet. Through simulations, the researchers show that the suppressor could spread through the population in just 100 generations, suggesting that male killing could disappear relatively quickly after a suppressor mutation occurs. Thus, genetic conflict between killing abettors and suppressors may be far more widespread than once thought, but has simply eluded detection. Given the diversity of species afflicted by male-killing bacteria, researchers will have plenty of options for testing this possibility.

Evolution of Male-Killer Suppression in a Natural Population

Male-killing bacteria are widespread in arthropods, and can profoundly alter the reproductive biology of their host species. Here we detail the first case of complete suppression of a male killer. The nymphalid butterfly Hypolimnas bolina is infected with a strain of the bacterium Wolbachia, wBol1, which kills male host embryos in Polynesian populations, but does not do so in many areas of Southeast Asia, where both males and female adults are naturally infected, and wBol1-infected females produce a 1:1 sex ratio. We demonstrate that absence of male killing by wBol1 is associated with dominant zygotic suppression of the action of the male killer. Simulations demonstrate host suppressors of male-killer action can spread very rapidly, and historical data indicating the presence of male killing in Southeast Asia in the very recent past suggests suppressor spread has been a very recent occurrence. Thus, male killer/host interactions are much more dynamic than previously recognised, with rapid and dramatic loss of the phenotype. Our results also indicate that suppression can render male killers completely quiescent, leading to the conclusion that some species that do not currently express a male killer may have done so in the past, and thus that more species have had their biology affected by these parasites than previously believed.

A new male-killing parasitism:Spiroplasmabacteria infect the ladybird beetleAnisosticta 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.

The impact of male-killing bacteria on the evolution of aphidophagous coccinellids

Many aphidophagous coccinellids harbour male-killing bacteria. These maternally inherited bacteria kill males early in embryogenesis, female offspring of infected mothers gaining a large resource advantage from the consumption of their dead brothers. In this paper, the diversity of male-killing bacteria and their coccinellid hosts will be briefly reviewed. Thereafter, the impact that invasion by male-killers has on coccinellid hosts will be addressed in two ways. First, the selective effects due to the loss of male progeny, and the intra-genomic conflict resulting from the cytoplasmic inheritance of the bacteria will be considered. Reduc- tions in mitochondrial diversity, effects on clutch sizes and the evolution of male-killer suppresser systems will be discussed. Second, the impact of female biased population sex ratios on the evolution of reproductive strategies and the primary sex ratio will be investigated. Preliminary results suggesting changes in male investment per copulation, reductions in female testing behaviour of males, and male biases in the progenic sex ratios of some uninfected male lines will be presented. Avenues for future work will be outlined.

Prevalence and penetrance variation of male‐killingWolbachiaacross Indo‐Pacific populations of the butterflyHypolimnas bolina

Male-killing bacteria are generally thought to attain low to intermediate prevalence in natural populations, with only mild effects on the host population sex ratio. This view was recently challenged by reports of extremely high infection frequencies in three butterfly species, raising the prospect that male killers, by making males rare, might drive many features of host ecology and evolution. To assess this hypothesis, it is necessary to evaluate how often male killers actually produce a highly female-biased population sex ratio in nature, which requires both high prevalence of infection and high penetrance of action. To this end, we surveyed South Pacific and Southeast Asian populations of Hypolimnas bolina, a butterfly in which extreme prevalence of male-killing Wolbachia bacteria has recently been recorded. Our results indicate that highly female-biased populations are common in Polynesia, with 6 out of 12 populations studied having in excess of 70% of females infected with a fully efficient male killer. However, heterogeneity is extreme in Polynesia, with the male-killing Wolbachia absent from three populations. In contrast to the Polynesian situation, Wolbachia does not kill males in any of the three Southeast Asian populations studied, despite its very high prevalence there. We conclude that male killers are likely to have significant ongoing ecological and evolutionary impact in 6 of the 15 populations surveyed. The causes and consequences of the observed spatial variation are discussed with respect to host resistance evolution, host ecology and interference with additional symbionts.

Male‐killingSpiroplasmanaturally infectingDrosophila melanogaster

Elucidation of the mechanism of action of selfish genetic elements is difficult outside species with well-defined genetics. Male-killing, the phenomenon whereby inherited bacteria kill male hosts during embryogenesis, is thus uncharacterized in mechanistic terms despite being common and important in insects. We characterized the prevalence, identity and source of the male-killing infection recently discovered in Drosophila melanogaster in Brazil. Male-killing was found to be present in 2.3% of flies from Recife, Brazil, and was uniquely associated with the presence of Spiroplasma infection. The identity of sequences across part of the 16S and across the 16S-23S ITS region indicated that the male-killing infection of D. melanogaster was very closely related to S. poulsonii, the source of the male-killing infection in willistoni group flies also found in South America. The sequences of two further protein-coding genes indicated the D. melanogaster infection to be most closely related to that found in D. nebulosa, from the willistoni group. Our data suggest that the establishment of D. melanogaster in South America was associated with the movement of male-killing bacteria between species.

Prevalence of Inherited Male-Killing Microorganisms in Japanese Population of Ladybird Beetle <I>Harmonia axyridis</I> (Coleoptera: Coccinellidae)

Abstract Maternally inherited bacteria that kill male but not female hosts during embryogenesis occur in Harmonia axyridis (Pallas). In two populations in Japan, Fukuyama, Hiroshima, and Muikamachi, Niigata, H. axyridis were infected with male-killing bacteria. According to the sequence analysis of the gene fragment for 16S rDNA, these bacteria belong to the genus Spiroplasma. Tetracycline-treated infected females produced female and male progeny, although untreated females produced only female progeny, demonstrating that H. axyridis is male-killed by the Spiroplasma. The proportion of females infected in Muikamachi and Fukuyama was 0.039 and 0.135, respectively, indicating that prevalence of male-killer in H. axyridis is low in Japan.

HAPLODIPLOIDY AS AN OUTCOME OF COEVOLUTION BETWEEN MALE-KILLING CYTOPLASMIC ELEMENTS AND THEIR HOSTS

Haplodiploidy (encompassing both arrhenotoky and paternal genome elimination) could have originated from coevolution between male-killing endosymbiotic bacteria and their hosts. In insects, haplodiploidy tends to arise in lineages that rely on maternally transmitted bacteria for nutrition and that have gregarious broods in which competition between siblings may occur. When siblings compete, there is strong selection on maternally transmitted elements to kill males. I consider a hypothetical bacterial phenotype that renders male zygotes effectively haploid by preventing chromosome decondensation in male-determining sperm nuclei. By causing high male mortality, such a phenotype can be advantageous to the bacterial lineage. By eliminating paternal genes, it can also be advantageous to the host female. A simple model shows that the host female will benefit under a wide range of values for the efficiency of resource re-allocation, the efficiency of transmission, and the viability of haploid males. This hypothesis helps to explain the ecological correlates of the origins of haplodiploidy, as well as such otherwise puzzling phenomena as obligate cannibalism by male Micromalthus beetles, reversion to diploidy by aposymbiotic male stictococcid scale insects, and the bizarre genomic constitution of scale insect bacteriomes.

Host resistance does not explain variation in incidence of male-killing bacteria in Drosophila bifasciata

BackgroundSelfish genetic elements that distort the sex ratio are found widely. Notwithstanding the number of records of sex ratio distorters, their incidence is poorly understood. Two factors can prevent a sex ratio distorter from invading: inability of the sex ratio distorter to function (failure of mechanism or transmission), and lack of drive if they do function (inappropriate ecology for invasion). There has been no test to date on factors causing variation in the incidence of sex ratio distorting cytoplasmic bacteria. We therefore examined whether absence of the male-killing Wolbachia infection in D. bifasciata in Hokkaido island of Japan, in contrast to the presence of infection on the proximal island of Honshu, was associated with failure of the infection to function properly on the Hokkaido genetic background.ResultsThe male-killer both transmitted and functioned well following introgression to each of 24 independent isofemale inbred lines carrying Hokkaido genetic backgrounds. This was maintained even under stringent conditions of temperature. We therefore reject the hypothesis that absence of infection is due to its inability to kill males and transmit on the Hokkaido genetic background. Further trap data indicates that D. bifasciata may occur at different densities in Hokkaido and Honshu populations, giving some credence to the idea that ecological differentiation could be important.ConclusionsThe absence of the infection from the Hokkaido population is not caused by failure of the male-killer to function on the Hokkaido genetic background.

Male-Killing Wolbachia and Mitochondrial DNA: Selective Sweeps, Hybrid Introgression and Parasite Population Dynamics

Mitochondrial DNA (mtDNA) sequences are widely used as neutral genetic markers in insects. However, patterns of mtDNA variability are confounded by the spread of maternally transmitted parasites, which are genetically linked to the mitochondria. We have investigated these effects in the butterflies Acraea encedon (which is host to two strains of male-killing Wolbachia bacteria) and A. encedana (which is host to one strain). Within a population, the mitochondria are in linkage disequilibrium with the different male-killers. Furthermore, there has been a recent selective sweep of the mtDNA, which has led to the loss of mitochondrial variation within populations and erased any geographical structure. We also found that one of the male-killers, together with the associated mtDNA, has introgressed from A. encedana into A. encedon within the last 16,000 years. Interestingly, because butterflies are female heterogametic, this will presumably have also led to the introgression of genes on the W sex chromosome. Finally, in A. encedon the mitochondria in uninfected females are unaltered by the spread of the male-killer and have diverse, geographically structured mtDNA. This means we can reject the hypothesis that the male-killer is at a stable equilibrium maintained by imperfect transmission of the bacterium. Instead, some other form of balancing selection may be maintaining uninfected females in the population and preventing the species from going extinct due to a shortage of males.

Space and the persistence of male-killing endosymbionts in insect populations.

Male-killing bacteria are bacteria that are transmitted vertically through the females of their insect hosts. They can distort the sex ratio of their hosts by killing infected male offspring. In nature, male-killing endosymbionts (male killers) often have a 100% efficient vertical transmission, and multiple male-killing bacteria infecting a single population are observed. We use different model formalisms to study these observations. In mean-field models a male killer with perfect transmission drives the host population to extinction, and coexistence between multiple male killers within one population is impossible; however, in spatially explicit models, both phenomena are readily observed. We show how the spatial pattern formation underlies these results. In the case of high transmission efficiencies, waves with a high density of male killers alternate with waves of mainly wild-type hosts. The male killers cause local extinction, but this creates an opportunity for uninfected hosts to re-invade these areas. Spatial pattern formation also creates an opportunity for two male killers to coexist within one population: different strains create spatial regions that are qualitatively different; these areas then serve as different niches, making coexistence possible.

History of infection with different male-killing bacteria in the two-spot ladybird beetle Adalia bipunctata revealed through mitochondrial DNA sequence analysis.

The two-spot ladybird beetle Adalia bipunctata (Coleoptera: Coccinellidae) is host to four different intracellular maternally inherited bacteria that kill male hosts during embryogenesis: one each of the genus Rickettsia (alpha-Proteobacteria) and Spiroplasma (Mollicutes) and two distinct strains of Wolbachia (alpha-Proteobacteria). The history of infection with these male-killers was explored using host mitochondrial DNA, which is linked with the bacteria due to joint maternal inheritance. Two variable regions, 610 bp of cytochrome oxidase subunit I and 563 bp of NADH dehydrogenase subunit 5, were isolated from 52 A. bipunctata with known infection status and different geographic origin from across Eurasia. Two outgroup taxa were also considered. DNA sequence analysis revealed that the distribution of mitochondrial haplotypes is not associated with geography. Rather, it correlates with infection status, confirming linkage disequilibrium between mitochondria and bacteria. The data strongly suggest that the Rickettsia male-killer invaded the host earlier than the other taxa. Further, the male-killing Spiroplasma is indicated to have undergone a recent and extensive spread through host populations. In general, male-killing in A. bipunctata seems to represent a highly dynamic system, which should prove useful in future studies on the evolutionary dynamics of this peculiar type of symbiont-host association.

Incidence of male-killing Rickettsia spp. (alpha-proteobacteria) in the ten-spot ladybird beetle Adalia decempunctata L. (Coleoptera: Coccinellidae).

The diversity of endosymbiotic bacteria that kill male host offspring during embryogenesis and their frequencies in certain groups of host taxa suggest that the evolution of male killing and the subsequent spread of male-killing symbionts are primarily determined by host life history characteristics. We studied the 10-spot ladybird beetle, Adalia decempunctata L. (Coleoptera: Coccinellidae), in which male killing has not been recorded previously, to test this hypothesis, and we also assessed the evolution of the male killer identified by DNA sequence analysis. Our results show that A. decempunctata harbors male-killing Rickettsia (alpha-proteobacteria). Male-killing bacteria belonging to the genus Rickettsia have previously been reported only for the congeneric two-spot ladybird beetle, Adalia bipunctata L. Phylogenetic analysis of Rickettsia DNA sequences isolated from different populations of the two host species revealed a single origin of male killing in the genus Rickettsia. The data also indicated possible horizontal transfer of symbionts between host species. In addition, A. bipunctata is known to bear at least four different male-killing symbionts in its geographic range two of which coexist in the two locations from which A. decempunctata specimens were obtained for the present study. Since only a single male-killing taxon was found in A. decempunctata, we assume that the two closely related ladybird beetle species must differ in the number and/or geographic distribution of male killers. We discuss the importance of these findings to our understanding of the evolution and dynamics of symbiotic associations between male-killing bacteria and their insect hosts.