Intergenomic Arms Races: Detection of a Nuclear Rescue Gene of Male-Killing in a Ladybird

Many species of arthropod are infected by deleterious inherited micro-organisms. Typically these micro-organisms are inherited maternally. Consequently, some, particularly bacteria of the genus Wolbachia, employ a variety of strategies that favour female over male hosts. These strategies include feminisation, induction of parthenogenesis and male-killing. These strategies result in female biased sex ratios in host populations, which lead to selection for host factors that promote male production. In addition, the intra-genomic conflict produced by the difference in transmission of these cytoplasmic endosymbionts and nuclear factors will impose a pressure favouring nuclear factors that suppress the effects of the symbiont. During investigations of the diversity of male-killing bacteria in ladybirds (Coccinellidae), unexpected patterns of vertical transmission of a newly discovered male-killing taxon were observed in the ladybird Cheilomenes sexmaculata. Initial analysis suggested that the expression of the bacterial male-killing trait varies according to the male(s) a female has mated with. By swapping males between females, a male influence on the expression of the male-killing trait was confirmed. Experiments were then performed to determine the nature of the interaction. These studies showed that a single dominant allele, which rescues male progeny of infected females from the pathological effect of the male-killer, exists in this species. The gene shows typical Mendelian autosomal inheritance and is expressed irrespective of the parent from which it is inherited. Presence of the rescue gene in either parent does not significantly affect the inheritance of the symbiont. We conclude that C. sexmaculata is host to a male-killing γ-proteobacterium. Further, this beetle is polymorphic for a nuclear gene, the dominant allele of which rescues infected males from the pathogenic effects of the male-killing agent. These findings represent the first reported case of a nuclear suppressor of male-killing in a ladybird. They are considered in regard to sex ratio and intra-genomic conflict theories, and models of the evolutionary dynamics and distribution of inherited symbionts.     

Silence of the killers: discovery of male-killing suppression in a rearing strain of the small brown planthopper,Laodelphax striatellus

According to evolutionary theory, sex ratio distortions caused by reproductive parasites such as Wolbachia and Spiroplasma are predicted to be rapidly normalized by the emergence of host nuclear suppressors. However, such processes in the evolutionary arms race are difficult to observe because sex ratio biases will be promptly hidden and become superficially unrecognizable. The evolution of genetic suppressors has been reported in just two insect species so far. In the small brown planthopper, Laodelphax striatellus, female-biases caused by Spiroplasma, which is a ‘late’ male-killer, have been found in some populations. During the continuous rearing of L. striatellus, we noted that a rearing strain had a 1 : 1 sex ratio even though it harboured Spiroplasma. Through introgression crossing experiments with a strain lacking suppressors, we revealed that the L. striatellus strain had the zygotic male-killing suppressor acting as a dominant trait. The male-killing phenotype was hidden by the suppressor even though Spiroplasma retained its male-killing ability. This is the first study to demonstrate the existence of a late male-killing suppressor and its mode of inheritance. Our results, together with those of previous studies, suggest that the inheritance modes of male-killing suppressors are similar regardless of insect order or early or late male killing.     

Prevalence of a non-male-killing spiroplasma in natural populations of Drosophila hydei

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.     

Prevalence of a Non-Male-Killing Spiroplasma in Natural Populations of Drosophila hydei

Male-killing phenotypes are found in a variety of insects and are often associated with maternally inherited endosymbiotic bacteria. In several species of Drosophila, male-killing endosymbionts of the genus Spiroplasma have been found at low frequencies (0.1 to 3%). In this study, spiroplasma infection without causing male-killing was shown to be prevalent (23 to 66%) in Japanese populations of Drosophila hydei. Molecular phylogenetic analyses showed that D. hydei was infected with a single strain of spiroplasma, which was closely related to male-killing spiroplasmas from other Drosophila species. Artificial-transfer experiments suggested that the spiroplasma genotype rather than the host genotype was responsible for the absence of the male-killing phenotype. Infection densities of the spiroplasma in the natural host, D. hydei, and in the artificial host, Drosophila melanogaster, were significantly lower than those of the male-killing spiroplasma NSRO, which was in accordance with the hypothesis that a threshold infection density is needed for the spiroplasma-induced male-killing expression.     

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.     

Phenotype and transmission efficiency of artificial and natural male-killing Spiroplasma infections in Drosophila melanogaster

Many insect species carry inherited Spiroplasma bacteria which act as important partners and antagonists. The nature of symbioses between Spiroplasma and insects has been most extensively studied in the interaction between male-killing Spiroplasma infection and Drosophila melanogaster. For historical reasons, these studies have largely focussed on the Spiroplasma strain known as NSRO, derived from Drosophila nebulosa and transinfected into D. melanogaster. More recently, D. melanogaster naturally infected with Spiroplasma were discovered. Whilst the well studied strain NSRO is closely related to that found natively in D. melanogaster, it is unclear whether strains from D. nebulosa reflect a natural interaction when placed in D. melanogaster. In this paper, we determine if NSRO has similar or different properties from strains of Spiroplasma naturally infecting D. melanogaster in terms of transmission efficiency and the strength and timing of male-killing. Native infections were observed to have higher transmission efficiency than introduced NSRO infections during the early phases of host reproduction, but not during late reproduction. The timing and intensity of male-killing did not differ between infection classes. As a precautionary measure, it is proposed that future work seeking to reveal the nature of coevolved Spiroplasma-Drosophila interactions use the native strain.     

Ladybirds as a model system for the study of male-killing symbionts

Maternally inherited bacteria that kill male but not female hosts during embryogenesis occur in a number of aphidophagous coccinellids. Work on EnglishAdalia bipunctata (L.), has shown the causative agent of male-killing to be a member of the bacterial genusRickettsia. In coccinellids, the primary advantage of male-killing behaviour to the bacterium has been identified. Following male death, resource reallocation occurs through sibling egg cannibalism: female neonate larvae of infected mothers gain a significant survival advantage by eating the soma of their dead male siblings. In addition, daughters of infected females suffer a reduced risk of cannibalism as a result of the lower egg hatch rate in infected clutches. Predictions as to which species of coccinellid are liable to harbour male-killers may be made on the basis of the selective advantages of male-killing identified inA. bipunctata. Species which may harbour male-killers are likely to lay eggs in clutches, show sibling egg cannibalism, and exhibit high neonate mortality. Recent work has shown male-killing to occur in a number of other aphidophagous coccinellids with the predicted characteristics. Molecular genetic analysis has putatively identified three bacterial symbionts associated with male-killing, coming from three phylogenetically distant bacterial taxa. We therefore suggest that within coccinellids that possess these features, male-killing may evolve in a taxonomically diverse range of inherited bacteria. The implications of the presence of male-killing bacteria on the population demography of host coccinellids, and on host mitochondrial DNA variability are discussed. The aphidophagous coccinellids are proposed as a model system for studying the evolution and consequences of infection with male-killers.     

Low Temperature Reveals Genetic Variability Against Male-Killing Spiroplasma in Drosophila melanogaster Natural Populations

Spiroplasma endosymbionts are maternally inherited microorganisms which infect many arthropod species. In some Drosophila species, it acts as a reproductive manipulator, spreading in populations by killing the sons of infected mothers. Distinct Drosophila melanogaster populations from Brazil exhibit variable male-killing Spiroplasma prevalences. In this study, we investigated the presence of variability for the male-killing phenotype among Drosophila and/or Spiroplasma strains and verified if it correlates with the endosymbiont prevalence in natural populations. For that, we analyzed the male-killing expression when Spiroplasma strains from different populations were transferred to a standard D. melanogaster line (Canton-S) and when a common Spiroplasma strain was transferred to different wild-caught D. melanogaster lines, both at optimal and challenging temperatures for the bacteria. No variation was observed in the male-killing phenotype induced by different Spiroplasma strains. No phenotypic variability among fly lines was detected at optimal temperature (23 °C), as well. Conversely, significant variation in the male-killing expression was revealed among D. melanogaster lines at 18.5 °C, probably caused by imperfect transmission of the endosymbiont. Distinct lines differed in their average sex ratios as well as in the pattern of male-killing expression as the infected females aged. Greater variation occurred among lines from one locality, although there was no clear correlation between the male-killing intensity and the endosymbiont prevalence in each population. Imperfect transmission or male killing may also occur in the field, thus helping to explain the low or intermediate prevalences reported in nature. We discuss the implications of our results for the dynamics of male-killing Spiroplasma in natural populations.     

Incidence of Male-Killing Rickettsia spp. (α-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 (α-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.     

Two Strains of Male-Killing Wolbachia in a Ladybird,Coccinella undecimpunctata,from a Hot Climate

Ladybirds are a hot-spot for the invasion of male-killing bacteria. These maternally inherited endosymbionts cause the death of male host embryos, to the benefit of female sibling hosts and the bacteria that they contain. Previous studies have shown that high temperatures can eradicate male-killers from ladybirds, leaving the host free from infection. Here we report the discovery of two maternally inherited sex ratio distorters in populations of a coccinellid, Coccinella undecimpunctata, from a hot lowland region of the Middle East. DNA sequence analysis indicates that the male killing is the result of infection by Wolbachia, that the trait is tetracycline sensitive, and that two distinct strains of Wolbachia co-occur within one beetle population. We discuss the implications of these findings for theories of male-killing and suggest avenues for future field-work on this system.     

Negative Effects of Low Temperatures on the Vertical Transmission and Infection Density of a Spiroplasma Endosymbiont in <Emphasis Type="Italic">Drosophila hydei</Emphasis>

Maternally transmitted endosymbionts of the genus Spiroplasma infecting several species of Drosophila are known to cause selective death of male offspring (male killing). The male-killing trait is considered to be advantageous for maternally transmitted endosymbionts. However, a non-male-killing spiroplasma is present in Japanese populations of Drosophila hydei at high frequencies (23-66%). This spiroplasma is phylogenetically closely related to the male-killing spiroplasma infecting other Drosophila species. It is unknown why this spiroplasma is maintained in its host populations despite its inability to cause male killing. We examined the susceptibilities of the spiroplasma in D. hydei to four different temperatures (28, 25, 18, and 15 degrees C). Diagnostic PCR revealed that vertical transmission of the spiroplasma was nearly perfect at 28 and 25 degrees C, partially suppressed at 18 degrees C, and completely blocked at 15 degrees C. Furthermore, quantitative PCR demonstrated that offspring treated at 18 degrees C exhibited dramatically lower densities of spiroplasma (i.e., approximately one-tenth) compared to offspring treated at 28 and 25 degrees C. Considering the low temperatures during winter in Japan, some unknown advantageous effects of the spiroplasma that compensate for the failure of vertical transmission are suggested to act in natural populations of D. hydei.     

Infection densities of three Spiroplasma strains in the host Drosophila melanogaster

Maternally transmitted endosymbiotic bacteria of the genus Spiroplasma associate with numerous insect species, including the genus Drosophila. Among the Spiroplasma strains associated with Drosophila, several manipulate their host??s reproduction by killing the male offspring of the infected females. Although the male-killing mechanism is not well understood, previous studies of non-native strains transferred to D. melanogaster (strain Oregon-R) indicate that the male-killing strain achieves higher densities than two non-male-killing strains. Whether this pattern of higher male-killing strain densities occurs in other host-symbiont strain combinations is not known. Herein, we used quantitative PCR to examine infection densities of one non-male-killing strain native to D. hydei (Hyd1), and two male-killing strains; one native to D. nebulosa (NSRO), and one native to D. melanogaster (MSRO; recently discovered), upon artificial transfer to D. melanogaster (strain Canton-S). Infection densities were examined at four weekly intervals in adult flies, across three consecutive generations following artificial transfer. Infection densities of the non-male-killing strain were significantly lower than those of the two male killers immediately after adult emergence. At later time points, however, the non-male-killing strain (Hyd1) is capable of proliferating to densities similar to those of the two male-killing strains (NSRO and MSRO) in D. melanogaster (Canton-S). We also examined the effect of co-infection by the heritable bacterium Wolbachia, on Spiroplasma densities and male-killing ability. Wolbachia had little to no effect of Spiroplasma densities, but the male-killing ability of MSRO was lower in the presence of Wolbachia. Generation post-infection had little effect on Spiroplasma densities, but affected the male-killing ability.     

Male-killing <Emphasis Type="Italic">Wolbachia</Emphasis> do not protect <Emphasis Type="Italic">Drosophila bifasciata</Emphasis>against viral infection

BACKGROUND: Insect symbionts employ multiple strategies to enhance their spread through populations, and some play a dual role as both a mutualist and a reproductive manipulator. It has recently been found that this is the case for some strains of Wolbachia, which both cause cytoplasmic incompatibility and protect their hosts against viruses. Here, we carry out the first test as to whether a male-killing strain of Wolbachia also provides a direct benefit to its host by providing antiviral protection to its host Drosophila bifasciata. We infected flies with two positive sense RNA viruses known to replicate in a range of Drosophila species (Drosophila C virus and Flock House virus) and measure the rate of death in Wolbachia positive and negative host lines with the same genetic background. RESULTS: Both viruses caused considerable mortality to D. bifasciata flies, with Drosophila C virus killing 43% more flies than the uninfected controls and Flock House virus killing 78% more flies than the uninfected controls. However, viral induced mortality was unaffected by the presence of Wolbachia. CONCLUSION: In the first male-killing Wolbachia strain tested for antiviral effects, we found no evidence that it conferred protection against two RNA viruses. We show that although antiviral resistance is widespread across the Wolbachia phylogeny, the trait seems to have been lost or gained along some lineages. We discuss the potential mechanisms of this, and can seemingly discount protection against these viruses as a reason why this symbiont has spread through Drosophila populations.     

Expression and modulation of embryonic male-killing in Drosophila innubila: opportunities for multilevel selection

Organisms and the symbionts they harbor may experience opposing forces of selection. In particular, the contrasting inheritance patterns of maternally transmitted symbionts and their host's nuclear genes can engender conflict among organizational levels over the optimal host offspring sex ratio. This study uses a male-killing Wolbachia endosymbiont and its host Drosophila innubila to experimentally address the potential for multilevel selection in a host-symbiont system. We show that bacterial density can vary among infected females, and that females with a higher density have a more female-biased offspring sex ratio. Furthermore, bacterial density is an epigenetic and heritable trait: females with a low bacterial load have daughters with a lower-than-average bacterial density, whose offspring then experience less severe male-killing. For infected sons, the probability of embryonic mortality increases with the bacterial density in their mothers. The frequency distribution of Wolbachia density among individual D. innubila females, and therefore the dynamics of infection within populations of these flies, results both from processes affecting the growth and regulation of bacterial populations within cytoplasmic lineages and from selection among cytoplasmic lineages that vary in bacterial density. Estimates of effective population size of Wolbachia within cytoplasmic lineages and of D. innubila at the host population level suggest that selection among cytoplasmic lineages is likely to overwhelm the results of selection within lineages.     

Male killing in three species of the tripunctata radiation of Drosophila (Diptera: Drosophilidae)

An excess of females in progenies is commonly referred to as sex-ratio (SR). In this report, we describe three new occurrences of SR in species of the tripunctata radiation, Drosophila neocardini , Drosophila ornatifrons and Drosophila paraguayensis. Repeated backcrosses with males from normal strains were used to maintain the SR strains in the laboratory, always with all-female broods. The egg–larva viability of the SR strains was approximately half that of a normal strain, and antibiotic treatment restored the production of males in SR strains. PCR screening with Spiroplasma -specific primers showed that only SR strains tested positive for Spiroplasma . The results showed that in the three species, SR was maternally inherited and involved early male-killing bacteria, most likely spiroplasmas. These new occurrences increase the number of cytoplasmic male killers described in Drosophila from 11 to 14. In this particular collection of flies, one quarter (3 out of 12) of the species from the tripunctata radiation of Drosophila was infected with a male killer, suggesting that this group may be a hot spot for the presence of male-killing organisms.     

High and low temperatures differently affect infection density and vertical transmission of male-killing Spiroplasma symbionts in Drosophila hosts

We investigated the vertical transmission, reproductive phenotype, and infection density of a male-killing Spiroplasma symbiont in two Drosophila species under physiological high and low temperatures through successive host generations. In both the native host Drosophila nebulosa and the nonnative host Drosophila melanogaster, the symbiont infection and the male-killing phenotype were stably maintained at 25 degrees C, rapidly lost at 18 degrees C, and gradually lost at 28 degrees C. In the nonnative host, both the high and low temperatures significantly suppressed the infection density of the spiroplasma. In the native host, by contrast, the low temperature suppressed the infection density of the spiroplasma whereas the high temperature had little effect on the infection density. These results suggested that the low temperature suppresses both the infection density and the vertical transmission of the spiroplasma whereas the high temperature suppresses the vertical transmission preferentially. The spiroplasma density was consistently higher in the native host than in the nonnative host, suggesting that the host genotype may affect the infection density of the symbiont. The temperature- and genotype-dependent instability of the symbiont infection highlights a complex genotype-by-genotype-by-environment interaction and may be relevant to the low infection frequencies of the male-killing spiroplasmas in natural Drosophila populations.     

Wolbachia--a new bacteria causing sex ratio bias in the two-spot lady-bird Adalia bipunctata L

Some of the male-killing lines of the two-spot ladybird Adalia bipunctata L. isolated from the populations of Moscow and Tomsk and having a female-biased sex ratio were found to be infected with a bacterium of the genus Wolbachia. This fact is the first demonstration of the ability of Wolbachia to kill males of a host insect. The coexistence of females infected with different male-killing bacteria was recorded in the population of Moscow.     

Molecular evolution and phylogenetic utility of Wolbachia ftsZ and wsp gene sequences with special reference to the origin of male-killing

A detailed assessment of the evolution and phylogenetic utility of two genes, ftsZ and wsp, was used to investigate the origin of male-killing Wolbachia, previously isolated from the ladybird Adalia bipunctata and the butterfly Acraea encedon. The analysis included almost all available sequences of B-group Wolbachia and two outgroup taxa and showed that (1) the two gene regions differ in phylogenetic utility, (2) sequence variation is here correlated with phylogenetic information content, (3) both genes show significant rate heterogeneity between lineages, (4) increased substitution rates are associated with homoplasy in the data, (5) wsp sequences of some taxa appear to be subject to positive selection, and (6) only a limited number of clades can be inferred with confidence due to either lack of phylogenetic information or the presence of homoplasy. With respect to the evolution of male-killing, the two genes nevertheless seemed to provide unbiased information. However, they consistently produce contradictory results. Current data therefore do not permit clarification of the origin of this behavior. In addition, A. bipunctata was found to be a host to two recently diverged strains of male-killing Wolbachia that showed increased substitution rates for both genes. Moreover, the wsp gene, which codes for an outer membrane protein, was found to be subject to positive selection in these taxa. These findings were postulated to be the product of high selection pressures due to antagonistic host-symbiont interactions in this ladybird species. In conclusion, our study demonstrates that the results of a detailed phylogenetic analysis, including characterization of the limitations of such an approach, can serve as a valuable basis for an understanding of the evolution of Wolbachia bacteria. Moreover, particular features of gene evolution, such as elevated substitution rates or the presence of positive selection, may provide information about the dynamics of Wolbachia-host associations.     

Discovery and identification of a male-killing agent in the Japanese ladybird <Emphasis Type="Italic">Propylea japonica</Emphasis> (Coleoptera: Coccinellidae)

Background  

Endosymbionts 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.     

Fighting back against male-killers

Male-killing endosymbionts create a genetic black hole into which host nuclear genes vanish. In a recent paper, Hornett et al. transferred male-killing Wolbachia between different strains of the butterfly Hypolimnas bolina through hybridization and backcrossing. Their results provide unambiguous evidence of genetic variation for resistance to male-killers. A possible consequence of such variation is that male-killing might appear and disappear quickly on an evolutionary timescale.