Distribution and Evolutionary Impact of Wolbachia on Butterfly Hosts

Wolbachia are maternally inherited endosymbiotic alpha-proteobacteria found in terrestrial arthropods and filarial nematodes. They are transmitted vertically through host cytoplasm and alter host biology by inducing various reproductive alterations, like feminization, parthenogenesis, male killing (MK) and cytoplasmic incompatibility. In butterflies, some effects especially MK and sperm-egg incompatibility are well established. All these effects skew the sex ratio towards female and subsequently favor the vertical transmission of Wolbachia. Some of the insects are also infected with multiple Wolbachia strains which may results in some complex phenomenon. In the present review the potential of Wolbachia for promoting evolutionary changes in its hosts with emphasis on recent advances in interactions of butterfly–Wolbachia is discussed. In addition to this, strain diversity of Wolbachia and its effects on various butterfly hosts are also highlighted.     

Male-killing Wolbachia in a live-bearing arthropod: brood abortion as a constraint on the spread of a selfish microbe

Maternally inherited, cellular endosymbionts can enhance their fitness by biasing host sex ratio in favor of females. Male killing (MK), an extreme form of sex-ratio manipulation, is selectively advantageous, if the death of males results in increased microbe transmission through female siblings. In live-bearing hosts, females typically produce more embryos than are brought to term, and reproductive compensation through maternal resource reallocation from dead male embryos to female siblings provides a direct, physiological mechanism that could increase the number of daughters born to infected females, thereby promoting MK endosymbiont spread. In this study, a Wolbachia-infected line and an uninfected line of the viviparous pseudoscorpion, Cordylochernes scorpioides were genetically homogenized for nuclear DNA by repeated backcrossing of the infected line with the uninfected, laboratory population. Photomicroscopy of early-stage embryos demonstrated that female C. scorpioides invariably produced an excess of embryos, with Wolbachia-infected females producing as many early-stage embryos as uninfected female controls. However, Wolbachia-infected females that successfully carried broods to term gave birth to significantly fewer offspring, indicating that the extreme female bias characteristic of their broods results from the killing rather than the feminization of male embryos. Infected females that carried broods to term gave birth to significantly larger nymphs and did produce 10% more female offspring than uninfected females. However, the slight transmission advantage that the MK Wolbachia accrued from this reproductive compensation appears to be heavily outweighed by the high rate of spontaneous brood abortion suffered by infected females.     

Loss of reproductive parasitism following transfer of male-killing Wolbachia to Drosophila melanogaster and Drosophila simulans

Wolbachia manipulate insect host biology through a variety of means that result in increased production of infected females, enhancing its own transmission. A Wolbachia strain (wInn) naturally infecting Drosophila innubila induces male killing, while native strains of D. melanogaster and D. simulans usually induce cytoplasmic incompatibility (CI). In this study, we transferred wInn to D. melanogaster and D. simulans by embryonic microinjection, expecting conservation of the male-killing phenotype to the novel hosts, which are more suitable for genetic analysis. In contrast to our expectations, there was no effect on offspring sex ratio. Furthermore, no CI was observed in the transinfected flies. Overall, transinfected D. melanogaster lines displayed lower transmission rate and lower densities of Wolbachia than transinfected D. simulans lines, in which established infections were transmitted with near-perfect fidelity. In D. simulans, strain wInn had no effect on fecundity and egg-to-adult development. Surprisingly, one of the two transinfected lines tested showed increased longevity. We discuss our results in the context of host-symbiont co-evolution and the potential of symbionts to invade novel host species.     

Effects of crowding and temperature on Wolbachia infection density among life cycle stages of Aedes albopictus

Species of the genus Wolbachia are a group of Rickettsia-like, maternally-inherited bacteria (gram negative), which cause various reproductive alterations in their arthropod and nematode hosts including cytoplasmic incompatibility (CI), male-killing, parthenogenesis and feminization. They can be divided into supergroups such as A and B based on phylogenetic analysis of 16S rDNA sequences. In this study, we examined the relative infection densities of Wolbachia strains among life cycle stages in the mosquito, Aedes albopictus in terms of crowding effect and temperature effect. A. albopictus is known to be superinfected with both A- and B-supergroup Wolbachia which cause CI. The relative Wolbachia densities within each individual mosquito were determined and quantified by using real-time quantitative PCR assay based on the wsp gene. We found that B-supergroup Wolbachia strain densities in this host species were consistently and significantly higher than in the A-supergroup. Larval crowding also reduced adult size of mosquitoes. Our results show clearly that the higher densities of mosquito larvae cause lower densities of Wolbachia strains. Examination of the effect of temperature on Wolbachia density in each stage of the mosquito clearly revealed a significant decrease in bacterial density following exposure to elevated temperature (37 °C) in both males and females.     

Double trouble: combined action of meiotic drive and Wolbachia feminization in Eurema butterflies

Arthropod sex ratios can be manipulated by a diverse range of selfish genetic elements, including maternally inherited Wolbachia bacteria. Feminization by Wolbachia is rare but has been described for Eurema mandarina butterflies. In this species, some phenotypic and functional females, thought to be ZZ genetic males, are infected with a feminizing Wolbachia strain, wFem. Meanwhile, heterogametic WZ females are not infected with wFem. Here, we establish a quantitative PCR assay allowing reliable sexing in three Eurema species. Against expectation, all E. mandarina females, including wFem females, had only one Z chromosome that was paternally inherited. Observation of somatic interphase nuclei confirmed that W chromatin was absent in wFem females, but present in females without wFem. We conclude that the sex bias in wFem lines is due to meiotic drive (MD) that excludes the maternal Z and thus prevents formation of ZZ males. Furthermore, wFem lines may have lost the W chromosome or harbour a dysfunctional version, yet rely on wFem for female development; removal of wFem results in all-male offspring. This is the first study that demonstrates an interaction between MD and Wolbachia feminization, and it highlights endosymbionts as potentially confounding factors in MD of sex chromosomes.     

Monitoring long-term evolutionary changes following Wolbachia introduction into a novel host: the Wolbachia popcorn infection in Drosophila simulans

Wolbachia may act as a biological control agent for pest management; in particular, the Wolbachia variant wMelPop (popcorn) shortens host longevity and may be useful for dengue suppression. However, long-term changes in the host and Wolbachia genomes can alter Wolbachia spread and/or host effects that suppress disease. Here, we investigate the phenotypic effects of wMelPop in a non-native host, Drosophila simulans, following artificial transinfection approximately 200 generations ago. Long-term rearing and maintenance of the bacteria were at 19°C in the original I-102 genetic background that was transinfected with the popcorn strain. The bacteria were then introgressed into three massbred backgrounds, and tetracycline was used to create uninfected sublines. The effect of wMelPop on longevity in this species appears to have changed; longevity was no longer reduced at 25°C in some nuclear backgrounds, reflecting different geographical origin, selection or drift, although the reduction was still evident for flies held at 30°C. Wolbachia influenced productivity and viability, and development time in some host backgrounds. These findings suggest that long-term attenuation of Wolbachia effects may compromise the effectiveness of this bacterium in pest control. They also emphasize the importance of host nuclear background on Wolbachia phenotypic effects.     

High‐prevalence male‐killing Wolbachia in the butterfly Acraea encedana

Inherited bacteria which kill males during early development are widely distributed throughout the insects, but have been little studied outside of a single family of beetles, the Coccinellidae. We have investigated a male‐killing bacterium discovered in the butterfly Acraea encedana. This bacterium belongs to the genus Wolbachia and is identical in wsp gene sequence to a male‐killer in the closely related butterfly A. encedon, suggesting that it has either recently moved between host species or was inherited from a common ancestor of the butterflies. The prevalence of Wolbachia is remarkably high, 95% of females are infected and only 6% of wild caught butterflies are male. Measurements of the vertical transmission efficiency were used to calculate that this high prevalence is the result of infected females producing at least 1.79 times the number of surviving daughters as uninfected females (lower confidence limit is 1.25).     

Rapid evolution of Wolbachia incompatibility types

In most insects, the endosymbiont Wolbachia induces cytoplasmic incompatibility (CI), an embryonic mortality observed when infected males mate either with uninfected females or with females infected by an incompatible Wolbachia strain. Although the molecular mechanism of CI remains elusive, it is classically viewed as a modification–rescue model, in which a Wolbachia mod function disables the reproductive success of the sperm of infected males, unless eggs are infected and express a compatible resc function. The extent to which the modification–rescue model can predict highly complex CI pattern remains a challenging issue. Here, we show the rapid evolution of the mod–resc system in the Culex pipiens mosquito. We have surveyed four incompatible laboratory isofemale lines over 50 generations and observed in two of them that CI has evolved from complete to partial incompatibility (i.e. the production of a mixture of compatible and incompatible clutches). Emergence of the new CI types depends only on Wolbachia determinants and can be simply explained by the gain of new resc functions. Evolution of CI types in Cx. pipiens thus appears as a gradual process, in which one or several resc functions can coexist in the same individual host in addition to the ones involved in the self-compatibility. Our data identified CI as a very dynamic process. We suggest that ancestral and mutant Wolbachia expressing distinct resc functions can co-infect individual hosts, opening the possibility for the mod functions to evolve subsequently. This gives a first clue towards the understanding of how Wolbachia reached highly complex CI pattern in host populations.     

Detection and characterization of Wolbachia infection in silkworm

Wolbachia naturally infects a wide variety of arthropods, where it plays important roles in host reproduction. It was previously reported that Wolbachia did not infect silkworm. By means of PCR and sequencing we found in this study that Wolbachia is indeed present in silkworm. Phylogenetic analysis indicates that Wolbachia infection in silkworm may have occurred via transfer from parasitic wasps. Furthermore, Southern blotting results suggest a lateral transfer of the wsp gene into the genomes of some wild silkworms. By antibiotic treatments, we found that tetracycline and ciprofloxacin can eliminate Wolbachia in the silkworm and Wolbachia is important to ovary development of silkworm. These results provide clues towards a more comprehensive understanding of the interaction between Wolbachia and silkworm and possibly other lepidopteran insects.     

Superinfection of cytoplasmic incompatibility-inducing Wolbachia is not additive in Orius strigicollis (Hemiptera: Anthocoridae)

Cytoplasmic incompatibility (CI) allows the intracellular, maternally inherited bacterial symbiont Wolbachia to invade arthropod host populations by inducing infertility in crosses between infected males and uninfected females. The general pattern is consistent with a model of sperm modification, rescued only by egg cytoplasm infected with the same strain of symbiont. The predacious flower bug Orius strigicollis is superinfected with two strains of Wolbachia, wOus1 and wOus2. Typically, superinfections of CI Wolbachia are additive in their effects; superinfected males are incompatible with uninfected and singly infected females. In this study, we created an uninfected line, and lines singly infected with wOus1 and wOus2 by antibiotic treatment. Then, all possible crosses were conducted among the four lines. The results indicated that while wOus2 induces high levels of CI, wOus1 induces very weak or no CI, but can rescue CI caused by wOus2 to a limited extent. Levels of incompatibility in crosses with superinfected males did not show the expected pattern. In particular, superinfected males caused extremely weak CI when mated with either singly infected or uninfected females. An analysis of symbiont densities showed that wOus1 densities were significantly higher than wOus2 densities in superinfected males, and wOus2 densities were lower, but not significantly, in superinfected relative to singly infected males. These data lend qualified support for the hypothesis that wOus1 interferes with the ability of wOus2 to cause CI by suppressing wOus2 densities. To our knowledge, this is the first clear case of non-additive CI in a natural superinfection.     

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.     

The genetics and evolution of obligate reproductive parasitism in Trichogramma pretiosum infected with parthenogenesis-inducing Wolbachia

Parthenogenesis-inducing (PI) Wolbachia belong to a class of intracellular symbionts that distort the offspring sex ratio of their hosts toward a female bias. In many PI Wolbachia-infected species sex ratio distortion has reached its ultimate expression-fixation of infection and all-female populations. This is only possible with thelytokous PI symbionts as they provide an alternative form of reproduction and remove the requirement for males and sexual reproduction. Many populations fixed for PI Wolbachia infection have lost the ability to reproduce sexually, even when cured of the infection. We examine one such population in the species Trichogramma pretiosum. Through a series of backcrossing experiments with an uninfected Trichogramma pretiosum population we were able to show that the genetic basis for the loss of female sexual function could be explained by a dominant nuclear effect. Male sexual function had not been completely lost, though some deterioration of male sexual function was also evident when males from the infected population (created through antibiotic curing of infected females) were mated to uninfected females. We discuss the dynamics of sex ratio selection in PI Wolbachia-infected populations and the evolution of non-fertilizing mutations.     

Phylogenetically distinct Wolbachia gene and pseudogene sequences obtained from the African onchocerciasis vector Simulium squamosum

Wolbachia are intracellular bacteria mostly found in a diverse range of arthropods and filarial nematodes. They have been classified into seven distinct ‘supergroups’ and other lineages on the basis of molecular phylogenetics. The arthropod-infecting Wolbachia are usually regarded as reproductive parasites because they manipulate their host species’ sexing system to enhance their own spread, and this has led to their investigation as potential agents of genetic control in medical entomology. We report 12 partial Wolbachia gene sequences from: aspC, aspS, dnaA, fbpA, ftsZ, GroEL, hcpA, IDA, rpoB, rpe, TopI and wsp as well as a single ftsZ pseudogene sequence, which have all been PCR-amplified from Simulium squamosum (Diptera: Simuliidae). To our knowledge this is the first such report from Simuliidae. Uninterrupted open-reading frame sequences were obtained from all 12 genes, covering ∼6.2 kb of unique DNA sequence. Phylogenetic analyses with the different coding genes gave consistent results suggesting that the Wolbachia sequences obtained here do not derive from any of the known Wolbachia supergroups or lineages. Consistent with a unique genetic status for the S. squamosumWolbachia, the hypervariable regions of the Wolbachia-specific wsp gene were distinct from all previous records in both sequence and length. As well as potential implications for newly emerging Wolbachia-based disease control methods, the results may be relevant to some problems experienced in the laboratory colonisation of Simulium damnosum sensu lato and why it is such a diverse species complex.     

Identification of Wolbachia-responsive microRNAs in the two-spotted spider mite,Tetranychus urticae

Background

The two-spotted spider mite, Tetranychus urticae, is infected with Wolbachia, which have the ability to manipulate host reproduction and fitness. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes such as development, reproduction and host-pathogen interactions. Although miRNA was observed to involve in Wolbachia-host interactions in the other insect systems, its roles have not been fully deciphered in the two-spotted spider mite.

Results

Small RNA libraries of infected and uninfected T. urticae for both sexes (in total four libraries) were constructed. By integrating the mRNA data originated from the same samples, the target genes of the differentially expressed miRNAs were predicted. Then, GO and pathway analyses were performed for the target genes. Comparison of libraries showed that Wolbachia infection significantly regulated 91 miRNAs in females and 20 miRNAs in males, with an overall suppression of miRNAs in Wolbachia-infected libraries. A comparison of the miRNA and mRNA data predicted that the differentially expressed miRNAs negatively regulated 90 mRNAs in females and 9 mRNAs in males. An analysis of target genes showed that Wolbachia-responsive miRNAs regulated genes with function in sphingolipid metabolism, lysosome function, apoptosis and lipid transporting in both sexes, as well as reproduction in females.

Conclusion

Comparisons of the miRNA and mRNA data can help to identify miRNAs and miRNA target genes involving in Wolbachia-host interactions. The molecular targets identified in this study should be useful in further functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1122) contains supplementary material, which is available to authorized users.     

Rapid Sequential Spread of Two Wolbachia Variants in Drosophila simulans

The maternally inherited intracellular bacteria Wolbachia can manipulate host reproduction in various ways that foster frequency increases within and among host populations. Manipulations involving cytoplasmic incompatibility (CI), where matings between infected males and uninfected females produce non-viable embryos, are common in arthropods and produce a reproductive advantage for infected females. CI was associated with the spread of Wolbachia variant wRi in Californian populations of Drosophila simulans, which was interpreted as a bistable wave, in which local infection frequencies tend to increase only once the infection becomes sufficiently common to offset imperfect maternal transmission and infection costs. However, maternally inherited Wolbachia are expected to evolve towards mutualism, and they are known to increase host fitness by protecting against infectious microbes or increasing fecundity. We describe the sequential spread over approximately 20 years in natural populations of D. simulans on the east coast of Australia of two Wolbachia variants (wAu and wRi), only one of which causes significant CI, with wRi displacing wAu since 2004. Wolbachia and mtDNA frequency data and analyses suggest that these dynamics, as well as the earlier spread in California, are best understood as Fisherian waves of favourable variants, in which local spread tends to occur from arbitrarily low frequencies. We discuss implications for Wolbachia-host dynamics and coevolution and for applications of Wolbachia to disease control.     

Multi-Infections of Feminizing Wolbachia Strains in Natural Populations of the Terrestrial Isopod Armadillidium Vulgare

Maternally inherited Wolbachia (α-Proteobacteria) are widespread parasitic reproductive manipulators. A growing number of studies have described the presence of different Wolbachia strains within a same host. To date, no naturally occurring multiple infections have been recorded in terrestrial isopods. This is true for Armadillidium vulgare which is known to harbor non simultaneously three Wolbachia strains. Traditionally, such Wolbachia are detected by PCR amplification of the wsp gene and strains are characterized by sequencing. The presence of nucleotide deletions or insertions within the wsp gene, among these three different strains, provides the opportunity to test a novel genotyping method. Herein, we designed a new primer pair able to amplify products whose lengths are specific to each Wolbachia strain so as to detect the presence of multi-infections in A. vulgare. Experimental injections of Wolbachia strains in Wolbachia-free females were used to validate the methodology. We re-investigated, using this novel method, the infection status of 40 females sampled in 2003 and previously described as mono-infected based on the classical sequencing method. Among these females, 29 were identified as bi-infected. It is the first time that naturally occuring multiple infections of Wolbachia are detected within an individual A. vulgare host. Additionally, we resampled 6 of these populations in 2010 to check the infection status of females.     

Evolution of early male-killing in horizontally transmitted parasites

Early male-killing (MK) bacteria are vertically transmitted reproductive parasites which kill male offspring that inherit them. Whereas their incidence is well documented, characteristics allowing originally non-MK bacteria to gradually evolve MK ability remain unclear. We show that horizontal transmission is a mechanism enabling vertically transmitted bacteria to evolve fully efficient MK under a wide range of host and parasite characteristics, especially when the efficacy of vertical transmission is high. We also show that an almost 100% vertically transmitted and 100% effective male-killer may evolve from a purely horizontally transmitted non-MK ancestor, and that a 100% efficient male-killer can form a stable coexistence only with a non-MK bacterial strain. Our findings are in line with the empirical evidence on current MK bacteria, explain their high efficacy in killing infected male embryos and their variability within and across insect taxa, and suggest that they may have evolved independently in phylogenetically distinct species.     

Macronutrients mediate the functional relationship between Drosophila and Wolbachia

Wolbachia are maternally inherited bacterial endosymbionts that naturally infect a diverse array of arthropods. They are primarily known for their manipulation of host reproductive biology, and recently, infections with Wolbachia have been proposed as a new strategy for controlling insect vectors and subsequent human-transmissible diseases. Yet, Wolbachia abundance has been shown to vary greatly between individuals and the magnitude of the effects of infection on host life-history traits and protection against infection is correlated to within-host Wolbachia abundance. It is therefore essential to better understand the factors that modulate Wolbachia abundance and effects on host fitness. Nutrition is known to be one of the most important mediators of host–symbiont interactions. Here, we used nutritional geometry to quantify the role of macronutrients on insect–Wolbachia relationships in Drosophila melanogaster. Our results show fundamental interactions between diet composition, host diet selection, Wolbachia abundance and effects on host lifespan and fecundity. The results and methods described here open a new avenue in the study of insect–Wolbachia relationships and are of general interest to numerous research disciplines, ranging from nutrition and life-history theory to public health.     

Cloning and Characterization of a Gene Encoding the Major Surface Protein of the Bacterial Endosymbiont Wolbachia pipientis

The maternally inherited intracellular symbiont Wolbachia pipientis is well known for inducing a variety of reproductive abnormalities in the diverse arthropod hosts it infects. It has been implicated in causing cytoplasmic incompatibility, parthenogenesis, and the feminization of genetic males in different hosts. The molecular mechanisms by which this fastidious intracellular bacterium causes these reproductive and developmental abnormalities have not yet been determined. In this paper, we report on (i) the purification of one of the most abundantly expressed Wolbachia proteins from infected Drosophila eggs and (ii) the subsequent cloning and characterization of the gene (wsp) that encodes it. The functionality of the wsp promoter region was also successfully tested in Escherichia coli. Comparison of sequences of this gene from different strains of Wolbachia revealed a high level of variability. This sequence variation correlated with the ability of certain Wolbachia strains to induce or rescue the cytoplasmic incompatibility phenotype in infected insects. As such, this gene will be a very useful tool for Wolbachia strain typing and phylogenetic analysis, as well as understanding the molecular basis of the interaction of Wolbachia with its host.     

Wolbachia in the spittlebug Prosapia ignipectus: Variable infection frequencies,but no apparent effect on host reproductive isolation

Animals serve as hosts for complex communities of microorganisms, including endosymbionts that live inside their cells. Wolbachia bacteria are perhaps the most common endosymbionts, manipulating host reproduction to propagate. Many Wolbachia cause cytoplasmic incompatibility (CI), which results in reduced egg hatch when uninfected females mate with infected males. Wolbachia that cause intense CI spread to high and relatively stable frequencies, while strains that cause weak or no CI tend to persist at intermediate, often variable, frequencies. Wolbachia could also contribute to host reproductive isolation (RI), although current support for such contributions is limited to a few systems. To test for Wolbachia frequency variation and effects on host RI, we sampled several local Prosapia ignipectus (Fitch) (Hemiptera: Cercopidae) spittlebug populations in the northeastern United States over two years, including closely juxtaposed Maine populations with different monomorphic color forms, “black” and “lined.” We discovered a group‐B Wolbachia (wPig) infecting P. ignipectus that diverged from group‐A Wolbachia—like model wMel and wRi strains in Drosophila—6 to 46 MYA. Populations of the sister species Prosapia bicincta (Say) from Hawaii and Florida are uninfected, suggesting that P. ignipectus acquired wPig after their initial divergence. wPig frequencies were generally high and variable among sites and between years. While phenotyping wPig effects on host reproduction is not currently feasible, the wPig genome contains three divergent sets of CI loci, consistent with high wPig frequencies. Finally, Maine monomorphic black and monomorphic lined populations of P. ignipectus share both wPig and mtDNA haplotypes, implying no apparent effect of wPig on the maintenance of this morphological contact zone. We hypothesize P. ignipectus acquired wPig horizontally as observed for many Drosophila species, and that significant CI and variable transmission produce high but variable wPig frequencies.