Tamsin M. O. Majerus

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.

Extreme variation in the prevalence of inherited male-killing microorganisms between three populations of Harmonia axyridis (Coleoptera: Coccinellidae)

Females from three populations of Harmonia axyridis (Pallas) were assayed for two phenotypical indicators of the presence of male-killing endosymbionts: low egg hatch-rates and strongly female-biased progenic sex ratios. Samples from Sapporo City, Japan, and the Altai Mountains, Mongolia, but not from Novosibirsk, Russia, were found to contain some females displaying both of these traits. Furthermore, there was a profound difference in the prevalence of infection between the Japanese and Mongolian populations. The proportion of females infected from the Japanese sample was approximately 0.49, whereas that from Altai was only 0.02. The trait was inherited by more than 99% of the progeny of infected females. The trait was inherited maternally with the same high efficiency over five generations. Treatment with antibiotics produced a small increase in the production of males from the male-killing lines, suggesting that the male-killer is bacterial in nature. Causes of variation in prevalence are discussed, and the consequences of high levels of infection with an efficiently transmitted male-killing microorganism on host population demography are considered.

The diversity of inherited parasites of insects: the male-killing agent of the ladybird beetle Coleomegilla maculata is a member of the Flavobacteria

Many species of insects bear maternally inherited bacteria which contribute to the physiology and metabolism of their host whereas others bear inherited bacteria which are regarded as parasitic. Parasitic behaviours centre on disruption of the production, survival or fertility of male hosts, through which the microbe is not transmitted. One of the most common of these is early male-killing, where the bacteria kill male embryos they enter. The question arises as to why all inherited symbionts are not male-killers. The male-killing agents identified to date derive from two groups: the Proteobacteria and the Mollicutes. However, bacteria from other groups (Spirochaetes, Flavobacteria) are found to be inherited in insects, but contain no incidence of male-killing. We show here an association of male-killing with a bacterium from one of these groups. The bacterium causing male-killing behaviour in the ladybird Coleomegilla maculata is most closely related to Blattabacterium , a host-beneficial Flavobacteria found in cockroaches and some termites. This result indicates there is little if any taxonomic bar to the evolution of male-killing behaviour. This pattern severely contrasts with the evolution of two other parasitic symbiont behaviours, cytoplasmic incompatibility and the induction of host parthenogenesis, which have been found to be associated exclusively with bacteria from one group: Wolbachia . The result is briefly discussed in the light of the incidence of parasitic and beneficial cytoplasmic elements among insects.

Male-killing bacterium in a fifth ladybird beetle, Coleomegilla maculata (Coleoptera:Coccinellidae).

Inherited symbionts which selectively cause the death of male hosts are found widely across the Insecta. Previous studies have shown a single, but different micro-organism to be responsible for male-killing in each taxonomic group studied. We here produce evidence that within a group of insects, the Coccinellidae, there is more than one causal agent of male lethality. We report a novel observation of a male-killing trait in the species Coleomegilla maculata. Six of 26 crosses were found to produce a female-biased sex ratio associated with a low egg hatch-rate. The trait was matrilinearly inherited and was observed to be tetracycline-sensitive. However, tests which indicate the presence of a Rickettsia, previously found to cause male-killing in another member of the Coccinellidae, Adalia bipunctata, proved negative. We therefore conclude that the phenomenon of male-killing is multicausal, within, as well as between, taxonomic groups of the Insecta.

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.

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.

Male-killing in the Coccinellidae: testing the predictions

Male-killing endosymbionts have been widely reported in the invertebrates and are highly prevalent in the Coccinellidae. The presence of male-killers can lead to extreme bias in host population sex ratios and may have important and far-reaching consequences for the life-history and evolution of their hosts. Male-killers may have direct and indirect effects on host fitness and reproductive behaviour, as well as affecting the host genome, either via strong selection pressure imposed by highly female-biased population sex ratios or by selective sweeps caused as a male-killer conferring an advantage to infected individuals spreads through a population. Criteria used to predict which species are liable to male-killer invasion, based on a variety of ecological factors, have been produced. In summary male-killers are predicted to occur in aphidophageous species, that lay eggs in clutches, show sibling egg consumption and are liable to neonatal larval mortality due to starvation. We assayed 30 species of Coccinellid for the presence of such male-killers to assess the predictive accuracy of the criteria. Male-killers were identified in 8 species in which they were predicted to occur and were absent from all 10 species predicted not to harbor them. Analysis of the remaining 12 species, where male-killers were predicted by the original criteria, but where they were not found, allowed us to identify areas where the criteria can be refined and improved. We conclude that whilst the original criteria give a reasonably accurate prediction, there are refinements and improvements, concerning details of host diet and life-history, which make them more robust, especially in the light of discoveries of male-killing suppressors and when incorporated give a better fit to our findings from field samples.

Screening for common copy-number variants in cancer genes

For most cases of colorectal cancer that arise without a family history of the disease, it is proposed that an appreciable heritable component of predisposition is the result of contributions from many loci. Although progress has been made in identifying single nucleotide variants associated with colorectal cancer risk, the involvement of low-penetrance copy number variants is relatively unexplored. We have used multiplex amplifiable probe hybridization (MAPH) in a fourfold multiplex (QuadMAPH), positioned at an average resolution of one probe per 2 kb, to screen a total of 1.56 Mb of genomic DNA for copy number variants around the genes APC, AXIN1, BRCA1, BRCA2, CTNNB1, HRAS, MLH1, MSH2, and TP53. Two deletion events were detected, one upstream of MLH1 in a control individual and the other in APC in a colorectal cancer patient, but these do not seem to correspond to copy number polymorphisms with measurably high population frequencies. In summary, by means of our QuadMAPH assay, copy number measurement data were of sufficient resolution and accuracy to detect any copy number variants with high probability. However, this study has demonstrated a very low incidence of deletion and duplication variants within intronic and flanking regions of these nine genes, in both control individuals and colorectal cancer patients.

Beyond killing: intra- and interspecific nonconsumptive effects among aphidophagous competitors

1. Beyond killing, predators have nonlethal effects on their potential prey. Because aphids are highly aggregated, their predators aggregate as well, creating conditions optimal for nonconsumptive effects (NCEs) among competing larvae. Although intraguild predator (IGP) density can alter the balance between conflicting forces, little is known about its effect on competing prey.