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.     

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.     

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.     

Rickettsial relative associated with male killing in the ladybird beetle (Adalia bipunctata).

A cytoplasmically inherited microorganism associated with male killing in the two-spot ladybird beetle, Adalia bipunctata, is shown to be closely related to bacteria in the genus Rickettsia. Sequencing of a PCR-amplified product of the 16S genes coding for rRNA (16S rDNA) shows the organism associated with male killing in ladybirds to share a common ancestry with the Rickettsias relative to other genera (e.g., Anaplasma, Ehrlichia, and Cowdria). The rickettsial 16S rDNA product is found in four strains of ladybird beetle showing male embryo lethality and is absent from two uninfected strains and an antibiotic-cured strain. In addition, a revertant strain that had naturally lost the male-killing trait failed to amplify the rickettsial 16S rDNA product. Use of PCR primers for a 17-kDa protein antigen which is found only in rickettsias also resulted in an amplified product from infected strains. Uninfected, cured, and revertant strains and insect species infected with related bacteria (cytoplasmic-incompatibility bacteria from Nasonia wasps) failed to amplify the product. Discovery of a close relative of rickettsias associated with sex ratio distortion in insects has implications for the evolution and population dynamics of this bacterial genus.     

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.     

Habitat or prey specialization in predatory ladybird beetles: a case study of two closely related species

This study examines the niche and diet breadth of two closely related sympatric aphidophagous ladybirds: Adalia decempunctata and A. bipunctata. The degree of habitat specialization of these species is investigated, and its effect on life history traits of females is explored. The importance of prey quality in determining the diet breadth is also examined. The niches occupied by these species in three countries, the UK, Belgium and southern France, are similar: A. decempunctata is an arboreal habitat specialist with a narrower set of prey than A. bipunctata, which is commonly found on several types of vegetation. The niches of the two species overlap on trees. Experiments indicate that habitat specialization has resulted in A. decempunctata investing more in each of its offspring than A. bipunctata. A. decempunctata females lay, relative to their body size, heavier eggs than those of the more generalist A. bipunctata, which results in A. decempunctata having bigger larvae. In addition, A. decempunctata larvae are better at surviving starvation than A. bipunctata larvae. In contrast to the expected pattern in food specialization, our study failed to demonstrate a better efficiency of the specialist when fed its usual prey and a detrimental effect when fed on prey that it is unlikely to encounter in the field. The reproductive performance of the specialist ladybird was better when fed an aphid that it was unlikely to regularly feed on in the field. Therefore, the narrow diet of the specialist ladybird is most likely a consequence of it occupying a narrow habitat rather than the quality of the prey. Although further studies on specialization in predatory insects are needed, the results indicate that unlike the role of plant quality in host specialization in herbivorous insects, prey quality has not been the main determinant of ecological specialization in these predatory insects.     

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.     

Genetic linkage between melanism and winglessness in the ladybird beetle Adalia bipunctata

We report a case of genetic linkage between the two major loci underlying different wing traits in the two-spot ladybird beetle, Adalia bipunctata (L.) (Coleoptera: Coccinellidae): melanism and winglessness. The loci are estimated to be 38.8?cM apart on one of the nine autosomes. This linkage is likely to facilitate the unravelling of the genetics of these traits. These traits are of interest in the context of the evolution of intraspecific morphological diversity, and for the application of ladybird beetles in biological control programs.     

Egg dumping by predatory insects

Synovigenic insects resorb oocytes when food is scarce and mature oocytes when food is plentiful. These two antagonistic processes allow an optimal allocation of resources to reproduction and somatic functions. Unlike hymenopteran parasitoids, ladybirds cannot resorb mature oocytes present in the oviducts. Is the energy contained in these oocytes lost or is there a mechanism for recovering it when needed? Females of two species of ladybird beetles Adalia bipunctata (L.) and Adalia decempunctata (L.) that are starved for >24 h lay single infertile eggs, which they immediately eat, and these eggs comprise the mature oocytes in the oviducts at the onset of starvation. This behaviour has some similarities to egg dumping reported in herbivorous insects and is part, in ladybird beetles, of a process to retrieve energy invested in reproduction. Such behaviour may exist in other predatory synovigenic insects species that do not invest in maternal care.     

Assessment of patch quality by ladybirds: role of larval tracks

Gravid females of the two-spot ladybird, Adalia bipunctata (L.), were deterred from ovipositing when kept in petri dishes that had previously contained conspecific larvae but not conspecific adults, or the larvae of another two species of ladybird, Adalia decempunctata (L.) and Coccinella septempunctata L. The deterrent effect was density dependent and mediated via a chloroform-soluble contact pheromone present in the larval tracks. Similarly, gravid females of C. septempunctata were deterred from ovipositing by conspecific larval tracks and chloroform extracts of these tracks, but not by the tracks or extracts of tracks of A. bipunctata larvae. That is, in ladybirds the larvae produce a species-specific oviposition-deterring pheromone. In the field, the incidence of egg cannibalism in ladybirds increases very rapidly with the density of conspecific eggs or larvae per unit area. Thus, in responding to the species specific oviposition deterring pheromone female ladybirds reduce the risk of their eggs being eaten and spread their offspring more equally between patches. Received: 14 March 1997 / Accepted: 26 August 1997     

The value of an egg: resource reallocation in ladybirds (Coleoptera: Coccinellidae) infected with male-killing bacteria

Male-killing bacteria are thought to persist in host populations by vertical transmission and conferring direct and/or indirect fitness benefits to their hosts. Here, we test the role of indirect fitness benefits accrued from resource reallocation in species that engage in sibling egg cannibalism. We found that a single-egg meal significantly increased larval survival in 12 ladybird species, but the value of an egg (to survival) differed substantially between species. Next, we tested the impact of three male-killing bacteria on larval survival in one ladybird species, Adalia bipunctata. Spiroplasma reduced larval survival, whereas Wolbachia and Rickettsia had no effect. However, Spiroplasma-infected larvae showed the greatest response to a single-egg meal. The indirect fitness benefit obtained from a single egg is thus so large that even male-killing bacteria with direct fitness costs can persist in host populations. This study supports the hypothesis that fitness compensation via resource reallocation can explain male-killing bacteria persistence.     

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.     

Divergent preferences of Rhagoletis pomonella host races for olfactory and visual fruit cues

The quality of different species of aphids as food for aphidiphagous ladybird beetles varies greatly. The optimal oviposition theory predicts that a female should lay eggs preferentially in patches of suitable prey and should be reluctant to oviposit in patches of less suitable prey. A no‐choice experiment was used to test whether aphids (Homoptera: Aphididae) of different suitability influence the oviposition behaviour of the two‐spot ladybird beetle, Adalia bipunctata (L.) (Coleoptera: Coccinellidae). The results indicate that A. bipunctata females are not more reluctant to lay eggs in the presence of moderately suitable compared to highly suitable aphids. However, females laid fewer eggs in the presence of toxic aphids.     

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.     

Releases of a natural flightless strain of the ladybird beetle Adalia bipunctata reduce aphid-born honeydew beneath urban lime trees

Aphids can cause major environmental problems in urban areas. One important problem is the annual outbreaks of lime aphid, Eucallipterus tiliae (L.) (Hemiptera: Aphididae), which spoil the surroundings of lime trees by depositing honeydew. To date no environmentally friendly method has been demonstrated to yield effective control of lime aphids. Attempts are made in some cities to control lime aphids by releasing larvae of the native two-spot ladybird beetle, Adalia bipunctata (L.) (Coleoptera: Coccinellidae). However, it is known that adult ladybird beetles disperse soon after release, and there is little indication they provide control of the aphids. Here, we demonstrate experimentally that releases of a flightless strain of A. bipunctata, obtained from natural variation in wing length, can reduce the impact of honeydew from lime aphid outbreaks on two species of lime in an urban environment. Both larvae and adult beetles were released, and we discuss the contribution of the flightless adults to the decline in honeydew.     

Rickettsia Symbionts Cause Parthenogenetic Reproduction in the Parasitoid Wasp Pnigalio soemius (Hymenoptera: Eulophidae)

Bacteria in the genus Rickettsia are intracellular symbionts of disparate groups of organisms. Some Rickettsia strains infect vertebrate animals and plants, where they cause diseases, but most strains are vertically inherited symbionts of invertebrates. In insects Rickettsia symbionts are known to have diverse effects on hosts ranging from influencing host fitness to manipulating reproduction. Here we provide evidence that a Rickettsia symbiont causes thelytokous parthenogenesis (in which mothers produce only daughters from unfertilized eggs) in a parasitoid wasp, Pnigalio soemius (Hymenoptera: Eulophidae). Feeding antibiotics to thelytokous female wasps resulted in production of progeny that were almost all males. Cloning and sequencing of a fragment of the 16S rRNA gene amplified with universal primers, diagnostic PCR screening of symbiont lineages associated with manipulation of reproduction, and fluorescence in situ hybridization (FISH) revealed that Rickettsia is always associated with thelytokous P. soemius and that no other bacteria that manipulate reproduction are present. Molecular analyses and FISH showed that Rickettsia is distributed in the reproductive tissues and is transovarially transmitted from mothers to offspring. Comparison of antibiotic-treated females and untreated females showed that infection had no cost. Phylogenetic analyses of 16S rRNA and gltA gene sequences placed the symbiont of P. soemius in the bellii group and indicated that there have been two separate origins of the parthenogenesis-inducing phenotype in the genus Rickettsia. A possible route for evolution of induction of parthenogenesis in the two distantly related Rickettsia lineages is discussed.The genus Rickettsia contains a group of obligate intracellular symbionts of eukaryotic cells and belongs to the family Rickettsiaceae in the order Rickettsiales of the Alphaproteobacteria (58, 90). Many species have medical importance as they are pathogens of humans and other vertebrates; pathogenic Rickettsia species infect their hosts through blood-feeding arthropods, including lice, fleas, ticks, and mites (51, 80). In addition to Rickettsia species that cause infectious diseases in vertebrates, symbiotic species have been found in disparate groups of organisms, including arthropods, annelids, amoebae, hydrozoa, and plants (53). Rickettsia appears to be especially common in arthropods, having been found in a wide range of taxa in the classes Entognatha (springtails), Insecta (booklice, lice, bugs, leafhoppers, aphids, whiteflies, fleas, flies, lacewings, moths, beetles, and wasps), and Acarina (ticks and mites) (86). However, in most cases, the effect of Rickettsia on the invertebrate host has not been established yet. In general, Rickettsia bacteria are facultative symbionts, but in the booklouse Liposcelis bostrychophila the association is strictly obligate and Rickettsia has an essential role in oocyte development (54, 92). Facultative symbiotic Rickettsia strains have been reported to negatively affect some aspects of host fitness, causing reductions in body weight, fecundity, and longevity in the pea aphid (16, 60, 64), reductions in viability in some blood-feeding arthropod vectors (5, 46), and increased susceptibility to insecticides in the sweet potato whitefly (41). There is also evidence that Rickettsia has positive effects on host fitness, such as a larger body size in infected leeches (40) and a possible role in the oogenesis of a bark beetle (93). Finally, facultative symbiotic rickettsiae can be reproductive parasites of insects. Rickettsia strains are the causal agents of male killing (infected male embryos die) in some ladybird (79, 88) and buprestid leaf-mining (42) beetles. They are also the cause of thelytokous parthenogenesis (in which mothers produce only daughters from unfertilized eggs) in a parasitoid wasp (32). Both kinds of reproductive manipulation bias the host sex ratio toward females and favor the spread of the transovarially inherited Rickettsia strains in the infected populations. In general, Rickettsia is transmitted primarily vertically to host progeny, but in pathogenic species there is concomitant horizontal transmission via intermediate vertebrate hosts, which plays an important role in maintaining the infection in populations of blood-feeding arthropods (53, 57). An exception is Rickettsia prowazekii, the epidemic typhus agent, which spreads only via horizontal transmission in louse host populations (5). Only one Rickettsia is known to be a plant pathogen, and leafhoppers transfer this pathogen horizontally between plants (20). The fact that Rickettsia can be transmitted horizontally and then perpetuated vertically in host descendants has probably been one of the most important factors determining the enormous diversity of Rickettsia symbiotic associations. This point has been emphasized by analyses that have revealed considerable incongruence between Rickettsia and host phylogenies, indicating that horizontal transfer has occurred multiple times over evolutionary timescales (53, 54, 86).In addition to Rickettsia, diverse heritable bacteria are known to manipulate host reproduction to enhance their transmission in arthropods (12, 23). Wolbachia (order Rickettsiales, family Anaplasmataceae), a close relative of Rickettsia (90), is able to induce all known forms of manipulation of reproduction, including cytoplasmic incompatibility, feminization of genetic males, male killing, and parthenogenesis (68). Previously, only Cardinium (Sphingobacteria) has been shown to cause a similar range of reproductive phenotypes, except for male killing (35). The emerging diversity of Rickettsia associated with arthropods (53, 86), combined with evidence that it can manipulate host reproduction in more than one way, suggests that this symbiont may also be a master manipulator.In the Hymenoptera, the dominant mode of reproduction is arrhenotoky; that is, diploid females develop from fertilized eggs, and haploid males develop from unfertilized eggs (76). However, thelytokous parthenogenesis is common, and in some lineages, like the superfamilies Chalcidoidea and Cynipoidea, it is strongly associated with Wolbachia or Cardinium infection (33, 35). Parthenogenesis-inducing (PI) bacteria cause restoration of diploidy in unfertilized haploid eggs, which results in female offspring (28, 50, 69). PI Wolbachia and PI Cardinium also occur in other groups of haplodiploid arthropods, such as mites (82), scale insects (56), and thrips (4). Previously, the only example of PI caused by Rickettsia was found in the eulophid parasitoid wasp Neochrysocharis formosa (1, 32). Besides PI bacteria, uniparental (thelytokous) reproduction in haplodiploid arthropods can also be caused by feminizing bacteria that are able to interact with the host sex determination system and force the development of genotypic males toward functional phenotypic females. To date, only Cardinium has been reported to be a causal agent of feminization in haplodiploid arthropods, and only two examples are known: a mite in which Cardinium causes haploid male embryos to develop as haploid females (18, 83) and a parasitoid wasp in which diploid males are converted to females (27).In this paper, thelytokous reproduction in a parasitoid wasp, Pnigalio soemius (Hymenoptera: Eulophidae), was studied. This wasp, which is probably a complex of cryptic species (8), is a solitary ectoparasitoid that attacks larvae of many leafminer insect species in the orders Coleoptera, Diptera, Hymenoptera, and Lepidoptera (48), some of which are pests of agricultural crops (37, 61). Female P. soemius wasps paralyze host larvae by injection of venom and subsequently lay a single egg next to the host inside a leaf mine; then the parasitoid larva eats the killed host (7). Commonly, P. soemius reproduces biparentally, and the occurrence of thelytoky has not been reported previously. The aims of this study were to determine whether symbiotic bacteria are involved in manipulating the reproduction of P. soemius and then to determine the taxonomic affiliation and phenotype of the manipulators of reproduction discovered. By using antibiotic treatments and karyological analysis of the insect studied, molecular and phylogenetic characterization of the symbiotic bacteria, and detection of intracellular symbionts by means of fluorescence in situ hybridization, it was demonstrated that a PI Rickettsia causes thelytokous reproduction in P. soemius.     

Expansion of the geographical distribution of an exotic ladybird beetle, Adalia bipunctata (Coleoptera: Coccinellidae), and its interspecific relationships with native ladybird beetles in Japan

The first record of the exotic ladybird beetle, Adalia bipunctata (Coleoptera; Coccinellidae), in Japan was in 1993 at Osaka Nanko Central Park. Since that time, studies on the life history and geographical distribution of A. bipunctata have been ongoing, and its establishment in the Osaka Nanko area has been confirmed. A. bipunctata is a predacious ladybird beetle and a member of a guild that overlaps in habitat and prey with that of native ladybird beetles such as Harmonia axyridis and Menochilas sexmaculatus. We investigated the distribution of A. bipunctata and its interspecific relationships with native predacious ladybird beetles. In some areas, A. bipunctata was dominant in interspecific relationships with native ladybird beetles. For the first 10 years after A. bipunctata was discovered, it occurred only in the Osaka Nanko area, but the present geographical distribution indicates that it has expanded its range. Though the population density of this species was highest at the area recorded first, and tended to decrease in inverse proportion to the distance from Osaka Nanko Central Park, a satellite occurrence was observed in a remote area. The numbers of aphid and tree species (leaf shelter for aestivation and over-wintering) utilized by A. bipunctata have recently increased. Such increases will cause the rate of distribution of A. bipunctata to accelerate. Interspecific competition between H. axyridis and A. bipunctata, which occurs earlier than H. axyridis, may be avoided by desynchronization of the occurrence seasons, and another common predacious ladybird beetle, Coccinella septempunctata, may escape interspecific competition by habitat segregation.     

Extreme Costs of Mating for Male Two-Spot Ladybird Beetles

Male costs of mating are now thought to be widespread. The two-spot ladybird beetle (Adalia bipunctata) has been the focus of many studies of mating and sexual selection, yet the costs of mating for males are unknown. The mating system of A. bipunctata involves a spermatophore nuptial gift ingested by females after copulation. In this study, we investigate the cost to males of mating and of transferring spermatophores in terms of lifespan, ejaculate production and depletion of nutritional reserves. We found that males faced a strong trade-off between mating and survival, with males that were randomly assigned to mate a single time experiencing a 53% reduction in post-mating lifespan compared to non-mating males. This is among the most severe survival costs of a single mating yet reported. However, spermatophore transfer did not impact male survival. Instead, the costs associated with spermatophores appeared as a reduced ability to transfer spermatophores in successive matings. Furthermore, males ingested more food following spermatophore transfer than after matings without spermatophores, suggesting that spermatophore transfer depletes male nutritional reserves. This is to our knowledge the first report of an effect of variation in copulatory behaviour on male foraging behaviour. Overall, our study highlights the advantages of assessing mating costs using multiple currencies, and suggests that male A. bipunctata should exhibit mate choice.     

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.     

The inheritance of a wingless character in the 2spot ladybird (Adalia bipunctata)

A viable wingless 2spot ladybirdAdalia bipunctata (L.) was found in the wild. Breeding through four generations revealed that the wingless trait was controlled by a recessive allele which displays variable levels of expression. The wingless ladybird is discussed in relation to its potential as a biocontrol agent. One ladybird also occurred in this stock which is suggestive of a supergene controlling the colour polymorphism in this species.