Widespread 'hilltopping' in Acraea butterflies and the origin of sex-role-reversed swarming in Acraea encedon and A. encedana

In some populations of the butterflies Acraea encedon and A. encedana, most females are infected with a bacterium that kills their sons. The resulting shortage of males is associated with females adopting a sex‐role‐reversed mating system, in which females swarm at landmarks such as hilltops and compete for males. We have observed the mating behaviour of Acraea species that are not known to be infected with the male‐killer. In over half of these species, males were found to aggregate on hilltops. It is likely that this behaviour was ancestral to the sex‐role‐reversed swarms of Acraea encedon and A. encedana, and we discuss how the spread of the male‐killing infection may have converted this mating system into sex‐role‐reversed swarming.     

Recent changes in phenotype and patterns of host specialization in Wolbachia bacteria

Wolbachia are a genus of bacterial symbionts that are known to manipulate the reproduction of their arthropod hosts, both by distorting the host sex ratio and by inducing cytoplasmic incompatibility. Previous work has suggested that some Wolbachia clades specialize in particular host taxa, but others are diverse. Furthermore, the frequency with which related strains change in phenotype is unknown. We have examined these issues for Wolbachia bacteria from Acraea butterflies, where different interactions are known in different host species. We found that bacteria from Acraea butterflies mostly cluster together in several different clades on the bacterial phylogeny, implying specialization of particular strains on these host taxa. We also observed that bacterial strains with different phenotypic effects on their hosts commonly shared identical gene sequences at two different loci. This suggests both that the phenotypes of the strains have changed recently between sex ratio distortion and cytoplasmic incompatibility, and that host specialization is not related to the bacterial phenotype, as suggested from previous data. We also analysed published data from other arthropod taxa, and found that the Wolbachia infections of the majority of arthropod genera tend to cluster together on the bacterial phylogeny. Therefore, we conclude that Wolbachia is most likely to move horizontally between closely related hosts, perhaps because of a combination of shared vectors for transmission and physiological specialization of the bacteria on those hosts.     

Male‐killer dynamics in the tropical butterfly,Acraea encedana (Lepidoptera: Nymphalidae)

Sex ratio distortion in the tropical butterfly Acraea encedana is caused by infection with a male‐killing bacterium of the genus Wolbachia. Previous research on this species has reported extreme female bias, high bacterial prevalences, and full sex role reversal. In this paper, we provide an assessment for the dynamics of the male‐killer, based on a survey for sex ratios and Wolbachia prevalences among wild populations of A. encedana in Uganda. The study reveals that Wolbachia infection showed considerable variation over both spatial and temporal scales.     

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.     

Wolbachia in leafcutter ants: a widespread symbiont that may induce male killing or incompatible matings

Wolbachia is a maternally inherited bacterium that manipulates host reproduction by inducing cytoplasmic incompatibility (CI), parthenogenesis or male killing (MK). Here, we report on a screening of seven leafcutter ant species of the genera Atta and Acromyrmex. Using Wolbachia‐specific polymerase chain reaction (PCR) primers we show that all species are infected, usually by double A + B strain infections. For Acromyrmex echinatior and A. octospinosus, a screening across all castes shows that gynes (prospective queens) have higher infection rates than workers and males. The low infection rate of workers suggests that workers lose their infection during development. This we interpret as adaptive, because a heritable symbiont does not benefit from being present in sterile workers. Both CI and MK could potentially account for the low infection rate of males. Formal theoretical models show greater support for the MK scenario in the free living species A. echinatior and A. octospinosus but indicate that Wolbachia in the social parasite A. insinuator may cause CI, supporting a scenario of sympatric speciation of the social parasite. We conclude that Wolbachia represents a previously unrecognized source of reproductive conflict in leafcutter ant colonies.     

Prevalence and penetrance variation of male-killing Wolbachia across Indo-Pacific populations of the butterfly Hypolimnas 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.     

Tropical Drosophila pandora carry Wolbachia infections causing cytoplasmic incompatibility or male killing

Wolbachia infections have been described in several Drosophila species, but relatively few have been assessed for phenotypic effects. Cytoplasmic incompatibility (CI) is the most common phenotypic effect that has been detected, while some infections cause male killing or feminization, and many Wolbachia infections have few host effects. Here, we describe two new infections in a recently described species, Drosophila pandora, one of which causes near‐complete CI and near‐perfect maternal transmission (the “CI” strain). The other infection is a male killer (the “MK” strain), which we confirm by observing reinitiation of male production following tetracycline treatment. No incompatibility was detected in crosses between CI strain males and MK strain females, and rare MK males do not cause CI. Molecular analyses indicate that the CI and MK infections are distantly related and the CI infection is closely related to the wRi infection of Drosophila simulans. Two population surveys indicate that all individuals are infected with Wolbachia, but the MK infection is uncommon. Given patterns of incompatibility among the strains, the infection dynamics is expected to be governed by the relative fitness of the females, suggesting that the CI infection should have a higher fitness. This was evidenced by changes in infection frequencies and sex ratios in population cages initiated at different starting frequencies of the infections.     

Positive Selection and Horizontal Gene Transfer in the Genome of a Male-Killing Wolbachia

Wolbachia are a genus of widespread bacterial endosymbionts in which some strains can hijack or manipulate arthropod host reproduction. Male killing is one such manipulation in which these maternally transmitted bacteria benefit surviving daughters in part by removing competition with the sons for scarce resources. Despite previous findings of interesting genome features of microbial sex ratio distorters, the population genomics of male-killers remain largely uncharacterized. Here, we uncover several unique features of the genome and population genomics of four Arizonan populations of a male-killing Wolbachia strain, wInn, that infects mushroom-feeding Drosophila innubila. We first compared the wInn genome with other closely related Wolbachia genomes of Drosophila hosts in terms of genome content and confirm that the wInn genome is largely similar in overall gene content to the wMel strain infecting D. melanogaster. However, it also contains many unique genes and repetitive genetic elements that indicate lateral gene transfers between wInn and non-Drosophila eukaryotes. We also find that, in line with literature precedent, genes in the Wolbachia prophage and Octomom regions are under positive selection. Of all the genes under positive selection, many also show evidence of recent horizontal transfer among Wolbachia symbiont genomes. These dynamics of selection and horizontal gene transfer across the genomes of several Wolbachia strains and diverse host species may be important underlying factors in Wolbachia’s success as a male-killer of divergent host species.     

Hidden cytoplasmic incompatibility alters the dynamics of male‐killer/host interactions

Wolbachia manipulate the reproduction of their arthropod hosts in a variety of ways. Recent work has demonstrated that these bacteria may combine phenotypes – possessing a ‘male killing’ phenotype and, where males survive, induce cytoplasmic incompatibility (CI). We here develop a mathematical model to investigate the extent to which ‘hidden’ CI may affect the evolutionary dynamics of host genes that suppress male‐killing activity. We observed that for high prevalence infections, CI drives both suppressor and bacterium to higher frequency, such that the strain appears to solely exhibit CI. In contrast, for low prevalence infections, CI impedes suppressor invasion as surviving infected males are incompatible with the majority of females in the population. Our results demonstrate that ‘hidden’ phenotypes as well as observable ones can impact on the dynamics of the interaction, and knowledge of these is therefore required to predict when suppressor genes will invade, and the consequences of their invasion.     

Mating rates and the prevalence of male‐killing Spiroplasma in Harmonia axyridis (Coleoptera: Coccinellidae)

Maternally inherited bacteria that kill male but not female hosts during embryogenesis have been widely reported in invertebrates. Harmonia axyridis is one of the species infected by male‐killing Spiroplasma. The presence of male‐killers in host populations can lead to the occurrence of extremely female‐biased sex ratios. Furthermore, infected females may have fewer chances to mate if males can discriminate between infected and uninfected females and prefer the latter. Although there have been many investigations of male‐killer infection rates in H. axyridis, little is known about the influence of host mating on male‐killer infection dynamics. We investigated copulation rates and changes in infection frequency in a wild population of H. axyridis in western Japan. Almost all infected females collected each year laid fertilized eggs and had therefore mated. Mean infection rates of females collected each year were 13% in 2003, 15% in 2012 and 23% in 2013. Statistical analysis showed that neither the copulation rate nor the infection rate differed significantly among years. These results suggest that the infection rate of H. axyridis with male‐killing Spiroplasma is kept approximately constant and that there is no difference in the chance of mating with infected and uninfected females.     

Maintenance of a male-killing Wolbachia in Drosophila innubila by male-killing dependent and male-killing independent mechanisms

Many maternally inherited endosymbionts manipulate their host's reproduction in various ways to enhance their own fitness. One such mechanism is male killing (MK), in which sons of infected mothers are killed by the endosymbiont during development. Several hypotheses have been proposed to explain the advantages of MK, including resource reallocation from sons to daughters of infected females, avoidance of inbreeding by infected females, and, if transmission is not purely maternal, the facilitation of horizontal transmission to uninfected females. We tested these hypotheses in Drosophila innubila, a mycophagous species infected with MK Wolbachia. There was no evidence of horizontal transmission in the wild and no evidence Wolbachia reduced levels of inbreeding. Resource reallocation does appear to be operative, as Wolbachia-infected females are slightly larger, on average, than uninfected females, although the selective advantage of larger size is insufficient to account for the frequency of infection in natural populations. Wolbachia-infected females from the wild-although not those from the laboratory-were more fecund than uninfected females. Experimental studies revealed that Wolbachia can boost the fecundity of nutrient-deprived flies and reduce the adverse effect of RNA virus infection. Thus, this MK endosymbiont can provide direct, MK-independent fitness benefits to infected female hosts in addition to possible benefits mediated via MK.     

Male-killing Wolbachia in the butterfly Hypolimnas bolina

Some lines of the butterfly Hypolimnas bolina L. (Lepidoptera: Nymphalidae) are characterized by their female‐biased sex ratio. In these lines, most males die before reaching the middle larval stage. However, the cause of the bias remains unclear. We detected the proteobacterium Wolbachia in all individuals in the female‐biased butterfly lines and in some of the lines with a normal sex ratio. Tetracycline treatment of adult females of a female‐biased line led to a significant increase in both the hatch rate of their eggs (F1) and the male‐to‐female ratio of F1 pupae. In addition, certain assays of tetracycline treatment on mother butterflies significantly increased the male to female ratio of F1 adults. Known bacterial sex ratio distorters other than Wolbachia were not detected by diagnostic PCR assay, nor by the sequencing of 16S rDNA amplified using general prokaryotic 16S rDNA primers. These results strongly suggest that the distortion of the sex ratio is due to the killing of males by the inherited Wolbachia. Sequences of the 16S rDNA amplified using Wolbachia‐specific primers, the cell division protein gene (ftsZ), the molecular chaperone groE genes (groE operon), and the Wolbachia surface protein gene (wsp) from Wolbachia in lines belonging to three subspecies of the butterfly (bolina, jacintha, and philippensis) revealed no variation among lines nor between female‐biased lines and a normal one.     

All-female broods and mimetic polymorphism in Acraea encedon (L.) (Lepidoptera: Acraeidae) in Tanzania

In some populations of the African butterfly, Acraea encedon, there are two kinds of females, one producing offspring in a normal 1:1 sex ratio, the other producing females only; in other populations the sex ratio is apparently normal. All-female broods had hitherto been mainly associated with populations in which field sampling revealed an excess of females. The all-female brood trait is described from a population at Dar es Salaam which field sampling suggested was normal, and this indicates that the trait may be much more widespread and common than had previously been supposed. This discovery also extends the known distribution of the trait across Africa from Sierra Leone to eastern Tanzania. The butterfly is also a polymorphic Müllerian mimic of Danaus chrysippus, which is a highly unusual phenomenon as Müllerian mimicry is almost invariably monomorphic. The relative frequencies of two corresponding colour forms of the two species of butterflies at Dar es Salaam adds support to the hypothesis that they are indeed Mullerian mimics. The results of breeding experiments suggest that the polymorphic forms in Acraea encedon are allelic with dominance.     

Influences of two coexisting endosymbionts,CI‐inducing Wolbachia and male‐killing Spiroplasma,on the performance of their host Laodelphax striatellus (Hemiptera: Delphacidae)

The small brown planthopper Laodelphax striatellus (Hemiptera: Delphacidae) is reported to have the endosymbiont Wolbachia, which shows a strong cytoplasmic incompatibility (CI) between infected males and uninfected females. In the 2000s, female‐biased L. striatellus populations were found in Taiwan, and this sex ratio distortion was the result of male‐killing induced by the infection of another endosymbiont, Spiroplasma. Spiroplasma infection is considered to negatively affect both L. striatellus and Wolbachia because the male‐killing halves the offspring of L. striatellus and hinders the spread of Wolbachia infection via CI. Spiroplasma could have traits that increase the fitness of infected L. striatellus and/or coexisting organisms because the coinfection rates of Wolbachia and Spiroplasma were rather high in some areas. In this study, we investigated the influences of the infection of these two endosymbionts on the development, reproduction, and insecticide resistance of L. striatellus in the laboratory. Our results show that the single‐infection state of Spiroplasma had a negative influence on the fertility of L. striatellus, while the double‐infection state had no significant influence. At late nymphal and adult stages, the abundance of Spiroplasma was lower in the double‐infection state than in the single‐infection state. In the double‐infection state, the reduction of Spiroplasma density may be caused by competition between the two endosymbionts, and the negative influence of Spiroplasma on the fertility of host may be relieved. The resistance of L. striatellus to four insecticides was compared among different infection states of endosymbionts, but Spiroplasma infection did not contribute to increase insecticide resistance. Because positive influences of Spiroplasma infection were not found in terms of the development, reproduction, and insecticide resistance of L. striatellus, other factors improving the fitness of Spiroplasma‐infected L. striatellus may be related to the high frequency of double infection in some L. striatellus populations.     

Sex‐linked inheritance of diapause induction in the butterfly Pieris napi

Many temperate insects survive harsh environmental conditions, such as winter, by entering a state of developmental arrest. This diapause state is predominantly induced by photoperiod. The photoperiod varies with latitude and has led to local adaptation in the photoperiodic induction of diapause in many insects. To understand the rapid evolution of the photoperiodic threshold, it is important to investigate and understand the underlying genetic mechanisms. In the present study, the genetic basis of photoperiodic diapause induction is investigated in the green‐veined white butterfly Pieris napi (Lepidoptera, Pieridae) by assaying diapause induction in a range of conditions for a Swedish and Spanish population. Furthermore, the inheritance of diapause induction is assessed in reciprocal F1 hybrids and backcrosses between the two populations. The southern population shows a clear photoperiodic threshold determining diapause or direct development, whereas the northern populations show a high incidence of diapause, regardless of photoperiod. The hybrid crosses reveal that the inheritance of diapause induction is strongly sex‐linked, and that diapause incidence in the genetic crosses is highly dependent on photoperiod. This emphasizes the importance of assaying a range of conditions in diapause inheritance studies. The results indicate a strongly heritable diapause induction with a major component on the Z‐chromosome, as well as a minor effect of the autosomal background.     

Oviposition preference and larval performance of the sweet potato butterfly Acraea acerata on Ipomoea species in Ethiopia

• 1 The sweet potato butterfly Acraea acerata is an indigenous species in Ethiopia that has become a major pest on the introduced sweet potato Ipomoea batatas. To assess the role of wild Ethiopian Ipomoea species as host plants, the presence of larvae on wild ipomoeas was studied, and female oviposition choice and larval performance were tested on five wild ipomoeas, as well as on sweet potato.
• 2 In laboratory tests, oviposition and larval development were successful on two wild ipomoeas (Ipomoea tenuirostris and Ipomoea cairica) but no oviposition occurred on the remaining three species. Of the latter, larvae did not feed on Ipomoea hochstetteri and Ipomoea indica, and survival rates were extremely low on Ipomoea purpurea.
• 3 Sweet potato was a better host plant than I. tenuirostris and I. cairica in terms of oviposition preference, larval survival and pupal size; pupae were larger, resulting in more fecund female butterflies.
• 4 In the wild butterfly populations were abundant on I. tenuirostris but absent on I. cairica. Females also tended to prefer I. tenuirostris to I. cairica in oviposition choice experiments. However, no significant differences in performance were found between larvae raised on I. tenuirostris and I. cairica in the laboratory.
• 5 Wild populations of A. acerata also existed on Ipomoea obscura, a plant not investigated in the present study.
• 6 The abundance of A. acerata on wild ipomoeas is too low to likely affect butterfly population densities on sweet potato. However, wild populations may act as reservoirs subsequent to butterfly population bottlenecks on sweet potato.

     

Wolbachia infection and an all-female trait in Ostrinia orientalis and Ostrinia zaguliaevi

Sex ratio distortion toward females (SR trait), induced by a Wolbachia bacterium, has been reported in two species of the Ostrinia furnacalis group, viz., O. furnacalis, and O. scapulalis (Lepidoptera: Crambidae). In addition, an SR trait caused by abacterial, unidentified agent(s) is known in O. scapulalis. Here we examined the SR trait in four other species of the furnacalis group, viz., O. orientalis, O. zaguliaevi, and O. zealis from Japan, and O. nubilalis from central and eastern Europe. Wolbachia infection was detected in three O. orientalis females and in one O. zaguliaevi female, and the infection was always associated with the SR trait. In terms of wsp and ftsZ gene sequences, Wolbachia strains in O. orientalis and O. zaguliaevi were indistinguishable from each other, or from those in O. furnacalis and O. scapulalis. These findings suggest that Wolbachia strains in the four Ostrinia species are probably identical. In addition, one O. zealis female, which was negative in diagnostic PCRs for Wolbachia and general bacteria, produced an all‐female brood. This trait was very similar to the SR trait caused by abacterial agent(s) in O. scapulalis.     

Population structure in relation to host-plant ecology and Wolbachia infestation in the comma butterfly

Experimental work on Polygonia c‐album, a temperate polyphagous butterfly species, has shown that Swedish, Belgian, Norwegian and Estonian females are generalists with respect to host‐plant preference, whereas females from UK and Spain are specialized on Urticaceae. Female preference is known to have a strong genetic component. We test whether the specialist and generalist populations form respective genetic clusters using data from mitochondrial sequences and 10 microsatellite loci. Results do not support this hypothesis, suggesting that the specialist and generalist traits have evolved more than once independently. Mitochondrial DNA variation suggests a rapid expansion scenario, with a single widespread haplotype occurring in high frequency, whereas microsatellite data indicate strong differentiation of the Moroccan population. Based on a comparison of polymorphism in the mitochondrial data and sequences from a nuclear gene, we show that the diversity in the former is significantly less than that expected under neutral evolution. Furthermore, we found that almost all butterfly samples were infected with a single strain of Wolbachia, a maternally inherited bacterium. We reason that indirect selection on the mitochondrial genome mediated by a recent sweep of Wolbachia infection has depleted variability in the mitochondrial sequences. We also surmise that P. c‐album could have expanded out of a single glacial refugium and colonized Morocco recently.     

Egg load is a cue for offspring sex ratio adjustment in a fig‐pollinating wasp with male‐eggs‐first sex allocation

Fig‐pollinating wasps (Agaonidae) only reproduce within fig tree inflorescences (figs). Agaonid offspring sex ratios are usually female‐biased and often concur with local mate competition theory (LMC). LMC predicts less female‐bias when several foundresses reproduce in a fig due to reduced relatedness among intra‐sexually competing male offspring. Clutch size, the offspring produced by each foundress, is a strong predictor of agaonid sex ratios and correlates negatively with foundress number. However, clutch size variation can result from several processes including egg load (eggs within a foundress), competition among foundresses and oviposition site limitation, each of which can be used as a sex allocation cue. We introduced into individual Ficus racemosa figs single Ceratosolen fusciceps foundresses and allowed each to oviposit from zero to five hours thus variably reducing their eggs‐loads and then introduced each wasp individually into a second fig. Offspring sex ratio (proportion males) in second figs correlated negatively with clutch size, with males produced even in very small clutches. Ceratosolen fusciceps lay mainly male eggs first and then female eggs. Our results demonstrate that foundresses do not generally lay or attempt to lay a ‘fixed’ number of males, but do ‘reset to zero’ their sex allocation strategy on entering a second fig. With decreasing clutch size, gall failure increased, probably due to reduced pollen. We conclude that C. fusciceps foundresses can use their own egg loads as a cue to facultatively adjust their offspring sex ratios and that foundresses may also produce more ‘insurance’ males when they can predict increasing rates of offspring mortality.