Evolution of Wolbachia cytoplasmic incompatibility types

The success of obligate endosymbiotic Wolbachia infections in insects is due in part to cytoplasmic incompatibility (CI), whereby Wolbachia bacteria manipulate host reproduction to promote their invasion and persistence within insect populations. The observed diversity of CI types raises the question of what the evolutionary pathways are by which a new CI type can evolve from an ancestral type. Prior evolutionary models assume that Wolbachia exists within a host individual as a clonal infection. While endosymbiotic theory predicts a general trend toward clonality, Wolbachia provides an exception in which there is selection to maintain diversity. Here, evolutionary trajectories are discussed that assume that a novel Wolbachia variant will co-exist with the original infection type within a host individual as a superinfection. Relative to prior models, this assumption relaxes requirements and allows additional pathways for the evolution of novel CI types. In addition to describing changes in the Wolbachia infection frequency associated with the hypothesized evolutionary events, the predicted impact of novel CI variants on the host population is also described. This impact, resulting from discordant evolutionary interests of symbiont and host, is discussed as a possible cause of Wolbachia loss from the host population or host population extinction. The latter is also discussed as the basis for an applied strategy for the suppression of insect pest populations. Model predictions are discussed relative to a recently published Wolbachia genome sequence and prior characterization of CI in naturally and artificially infected insects.     

SPECIALIZATION AND GEOGRAPHIC ISOLATION AMONG WOLBACHIA SYMBIONTS FROM ANTS AND LYCAENID BUTTERFLIES

Wolbachia are the most prevalent and influential bacteria described among the insects to date. But despite their significance, we lack an understanding of their evolutionary histories. To describe the evolution of symbioses between Wolbachia and their hosts, we surveyed global collections of two diverse families of insects, the ants and lycaenid butterflies. In total, 54 Wolbachia isolates were typed using a Multi Locus Sequence Typing (MLST) approach, in which five unlinked loci were sequenced and analyzed to decipher evolutionary patterns. AMOVA and phylogenetic analyses demonstrated that related Wolbachia commonly infect related hosts, revealing a pattern of host association that was strongest among strains from the ants. A review of the literature indicated that horizontal transfer is most successful when Wolbachia move between related hosts, suggesting that patterns of host association are driven by specialization on a common physiological background. Aside from providing the broadest and strongest evidence to date for Wolbachia specialization, our findings also reveal that strains from New World ants differ markedly from those in ants from other locations. We, therefore, conclude that both geographic and phylogenetic barriers have promoted evolutionary divergence among these influential symbionts.     

Intense transpositional activity of insertion sequences in an ancient obligate endosymbiont

The streamlined genomes of ancient obligate endosymbionts generally lack transposable elements, such as insertion sequences (IS). Yet, the genome of Wolbachia, one of the most abundant bacterial endosymbionts on Earth, is littered with IS. Such a paradox raises the question as to why there are so many ISs in the genome of this ancient endosymbiont. To address this question, we investigated IS transpositional activity in the unculturable Wolbachia by tracking the evolutionary dynamics and history of ISWpi1 elements. We show that 1) ISWpi1 is widespread in Wolbachia, being present in at least 55% of the 40 sampled strains, 2) ISWpi1 copies exhibit virtually identical nucleotide sequences both within and among Wolbachia genomes and possess an intact transposase gene, 3) individual ISWpi1 copies are differentially inserted among Wolbachia genomes, and 4) ISWpi1 occurs at variable copy numbers among Wolbachia genomes. Collectively, our results provide compelling evidence for intense ISWpi1 transpositional activity and frequent ISWpi1 horizontal transmission among strains during recent Wolbachia evolution. Thus, the genomes of ancient obligate endosymbionts can carry high loads of functional and transpositionally active transposable elements. Our results also indicate that Wolbachia genomes have experienced multiple and temporally distinct ISWpi1 invasions during their evolutionary history. Such recurrent exposition to new IS invasions may explain, at least partly, the unusually high density of transposable elements found in the genomes of Wolbachia endosymbionts.     

Genotypic diversity of Wolbachia pipientis in native and invasive Harmonia axyridis Pall., 1773 (Coleoptera,Coccinellidae) populations

The distribution and variability of reproductive symbiotic Wolbachia pipientis bacteria were studied in seven native and six invasive H. axyridis populations. Wolbachia-infected individuals were found in two invasive and two native populations. We demonstrated for the first time an infection of invasive H. axyridis populations with Wolbachia. Two new molecular forms of Wolbachia were detected by a system of multilocus typing. The supergroup A Wolbachia was found for the first time in H. axyridis. The detected genotypic diversity of Wolbachia indicates repeated and independent infection events in the evolutionary past of H. axyridis.     

Recombination confounds interpretations of Wolbachia evolution.

Wolbachia are vertically transmitted bacteria known from arthropods and nematode worms, which are maintained in host populations because they either physiologically benefit infected individuals or parasitically manipulate their reproduction. The different manipulation phenotypes are scattered across the Wolbachia phylogeny, suggesting that there have been multiple evolutions of similar phenotypes. This conclusion relies on the assumption of an absence of recombination between bacterial strains, so that the gene used to reconstruct the phylogeny reflects the evolutionary history of the genes involved in the trait. We tested for recombination by reconstructing the phylogeny of two Wolbachia genes from seven B-subdivision strains. The two genes produced mutually incompatible topologies, indicating that these lineages are subject to genetic recombination. This means that many evolutionary patterns inferred from Wolbachia phylogenies must be re-evaluated. Furthermore, recombination may be an important feature both in the evolution of the manipulation phenotypes and avoidance of Müller's ratchet. Finally, we discuss the implications of recombination for attempts to genetically engineer Wolbachia for use in the control of crop pests and human pathogens.     

Survival of Wolbachia pipientis in cell-free medium

Wolbachia pipientis is an obligate intracellular bacterium found in a wide range of invertebrate taxa. While over ecological timescales Wolbachia infections are maintained by strict maternal inheritance, horizontal transfer events are common over evolutionary time. To be horizontally transferred between organisms, Wolbachia bacteria must pass through and survive an extracellular phase. We used BacLight live-dead staining, PCR, and fluorescence in situ hybridization to assess the ability for purified Wolbachia bacteria to survive in cell-free media. We found that purified Wolbachia bacteria were able to survive extracellularly for up to 1 week with no decrease in viability. While no replication was observed in the extracellular phase, purified Wolbachia bacteria were able to reinvade cells and establish stable infections at all time points. The ability of Wolbachia bacteria to survive outside host cells may increase the probability of successful horizontal transfer and the exploitation of new ecological niches. Our development of methods to purify and maintain viable Wolbachia bacteria from cultured cells will be useful for other researchers studying Wolbachia biology.     

Analysis of Sarcoptes scabiei finds no evidence of infection with Wolbachia

The endosymbiont Wolbachia has been detected in a range of filarial nematodes and parasitic mites and is known to affect host reproductive compatibility and potentially evolutionary processes. PCR of Wolbachia surface protein (wsp), ftsZ and 16SrRNA genes from individual Sarcoptes scabiei mites obtained from a series of individual hosts, and database searches of an S. scabiei var. hominis EST library failed to detect Wolbachia genes. Therefore, Wolbachia appears not to be involved in the genetic subdivision observed between varieties of host-associated S. scabiei or, involved in the inflammatory disease pathogenesis of scabies unlike its activity in filarial infection.     

Influence of two Wolbachia strains on population structure of East African Drosophila simulans

Drosophila simulans is hypothesized to have originated in continental East Africa or Madagascar. In this study, we investigated evolutionary forces operating on mitochondrial DNA (mtDNA) in populations of D. simulans from Zimbabwe, Malawi, Tanzania, and Kenya. Variation in mtDNA may be affected by positive selection, background selection, demographic history, and/or any maternally inherited factor such as the bacterial symbiont Wolbachia. In East Africa, the wRi and wMa Wolbachia strains associate with the siII or siIII mitochondrial haplogroups, respectively. To ask how polymorphism relates to Wolbachia infection status, we sequenced 1776 bp of mitochondrial DNA and 1029 bp of the X-linked per locus from 79 lines. The two southern populations were infected with wRi and exhibited significantly reduced mtDNA variation, while Wolbachia-uninfected siII flies from Tanzania and Kenya showed high levels of mtDNA polymorphism. These are the first known populations of D. simulans that do not exhibit reduced mtDNA variation. We observed no mitochondrial variation in the siIII haplogroup regardless of Wolbachia infection status, suggesting positive or background selection. These populations offer a unique opportunity to monitor evolutionary dynamics in ancestral populations that harbor multiple strains of Wolbachia.     

Diversification of Wolbachia endosymbiont in the Culex pipiens mosquito

The α-proteobacteria Wolbachia are among the most common intracellular bacteria and have recently emerged as important drivers of arthropod biology. Wolbachia commonly act as reproductive parasites in arthropods by inducing cytoplasmic incompatibility (CI), a type of conditional sterility between hosts harboring incompatible infections. In this study, we examined the evolutionary histories of Wolbachia infections, known as wPip, in the common house mosquito Culex pipiens, which exhibits the greatest variation in CI crossing patterns observed in any insect. We first investigated a panel of 20 wPip strains for their genetic diversity through a multilocus scheme combining 13 Wolbachia genes. Because Wolbachia depend primarily on maternal transmission for spreading within arthropod populations, we also studied the variability in the coinherited Cx. pipiens mitochondria. In total, we identified 14 wPip haplotypes, which all share a monophyletic origin and clearly cluster into five distinct wPip groups. The diversity of Cx. pipiens mitochondria was extremely reduced, which is likely a consequence of cytoplasmic hitchhiking driven by a unique and recent Wolbachia invasion. Phylogenetic evidence indicates that wPip infections and mitochondrial DNA have codiverged through stable cotransmission within the cytoplasm and shows that a rapid diversification of wPip has occurred. The observed pattern demonstrates that a considerable degree of Wolbachia diversity can evolve within a single host species over short evolutionary periods. In addition, multiple signatures of recombination were found in most wPip genomic regions, leading us to conclude that the mosaic nature of wPip genomes may play a key role in their evolution.     

Diversity of Wolbachia endosymbionts in heteropteran bugs

An extensive survey of Wolbachia endosymbionts in Japanese terrestrial heteropteran bugs was performed by PCR detection with universal primers for wsp and ftsZ genes of Wolbachia, cloning of the PCR products, restriction fragment length polymorphism analysis of infecting Wolbachia types, and molecular phylogenetic characterization of all the detected Wolbachia strains. Of 134 heteropteran species from 19 families examined, Wolbachia infection was detected in 47 species from 13 families. From the 47 species, 59 Wolbachia strains were identified. Of the 59 strains, 16 and 43 were assigned to A group and B group in the Wolbachia phylogeny, respectively. The 47 species of Wolbachia-infected bugs were classified into 8 species with A infection, 28 species with B infection, 2 species with AA infection, 3 species with AB infection, 5 species with BB infection, and 1 species with ABB infection. Molecular phylogenetic analysis showed little congruence between Wolbachia phylogeny and host systematics, suggesting frequent horizontal transfers of Wolbachia in the evolutionary course of the Heteroptera. The phylogenetic analysis also revealed several novel lineages of Wolbachia. Based on statistical analyses of the multiple infections, we propose a hypothetical view that, in the heteropteran bugs, interactions between coinfecting Wolbachia strains are generally not intense and that Wolbachia coinfections have been established through a stochastic process probably depending on occasional horizontal transfers.     

The evolution of obligate mutualism: if you can't beat 'em, join 'em

Wolbachia is best known as a facultative endosymbiotic parasite, manipulating host reproduction. However, it has also evolved as an obligate mutualist at least twice. In a recent paper, Pannebakker et al. identify a possible mechanism for such a transition from facultative parasitism to obligate mutualism in a parasitic wasp in which Wolbachia are required for producing eggs (oogenesis). Their proposed mechanism suggests that compensatory evolution in the host to counter the harmful effects of Wolbachia is the basis of this evolutionary transition.     

Evidence for widespread Wolbachia infection in isopod crustaceans: molecular identification and host feminization.

Wolbachia are maternally inherited, intracellular, alpha proteobacteria that infect a wide range of arthropods. They cause three kinds of reproductive alterations in their hosts: cytoplasmic incompatibility, parthenogenesis and feminization. There have been many studies of the distribution of Wolbachia in arthropods, but very few crustacean species are known to be infected. We investigated the prevalence of Wolbachia in 85 species from five crustacean orders. Twenty-two isopod species were found to carry these bacteria. The bacteria were found mainly in terrestrial species, suggesting that Wolbachia came from a continental environment. The evolutionary relationships between these Wolbachia strains were determined by sequencing bacterial genes and by interspecific transfers. All the bacteria associated with isopods belonged to the Wolbachia B group, based on 16S rDNA sequence data. All the terrestrial isopod symbionts in this group except one formed an independent clade. The results of interspecific transfers show evidence of specialization of Wolbachia symbionts to their isopod hosts. They also suggest that host species plays a more important role than bacterial phylogeny in determining the phenotype induced by Wolbachia infection.     

Infection with Wolbachia protects mosquitoes against Plasmodium-induced mortality in a natural system

In recent years, there has been a shift in the one host-one parasite paradigm with the realization that, in the field, most hosts are coinfected with multiple parasites. Coinfections are particularly relevant when the host is a vector of diseases, because multiple infections can have drastic consequences for parasite transmission at both the ecological and evolutionary timescales. Wolbachia pipientis is the most common parasitic microorganism in insects, and as such, it is of special interest for understanding the role of coinfections in the outcome of parasite infections. Here, we investigate whether Wolbachia can modulate the effect of Plasmodium on what is, arguably, the most important component of the vectorial capacity of mosquitoes: their longevity. For this purpose, and in contrast to recent studies that have focused on mosquito-Plasmodium and/or mosquito-Wolbachia combinations not found in nature, we work on a Wolbachia-mosquito-Plasmodium triad with a common evolutionary history. Our results show that Wolbachia protects mosquitoes from Plasmodium-induced mortality. The results are consistent across two different strains of Wolbachia and repeatable across two different experimental blocks. To our knowledge, this is the first time that such an effect has been shown for Plasmodium-infected mosquitoes and, in particular, in a natural Wolbachia-host combination. We discuss different mechanistic and evolutionary explanations for these results as well as their consequences for Plasmodium transmission.     

Evolution of Wolbachia pipientis transmission dynamics in insects.

Wolbachia pipientis is an intracellular bacterial parasite of arthropods that enhances its transmission by manipulating host reproduction, most commonly by inducing cytoplasmic incompatibility. The discovery of isolates with modified cytoplasmic incompatibility phenotypes and others with novel virulence properties is an indication of the potential breadth of evolutionary strategies employed by Wolbachia.     

Phylogeny and PCR-based classification of Wolbachia strains using wsp gene sequences.

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a number of different reproductive phenotypes in their hosts, such as cytoplasmic incompatibility, parthenogenesis and feminization. While it is known that the bacterial strains responsible for these different host phenotypes form a single clade within the alpha-Proteobacteria, until now it has not been possible to resolve the evolutionary relationships between different Wolbachia strains. To address this issue we have cloned and sequenced a gene encoding a surface protein of Wolbachia (wsp) from a representative sample of 28 Wolbachia strains. The sequences from this gene were highly variable and could be used to resolve the phylogenetic relationships of different Wolbachia strains. Based on the sequence of the wsp gene from different Wolbachia isolates we propose that the Wolbachia pipientis clade be initially divided into 12 groups. As more sequence information becomes available we expect the number of such groups to increase. In addition, we present a method of Wolbachia classification based on the use of group-specific wsp polymerase chain reaction (PGR) primers which will allow Wolbachia isolates to be typed without the need to clone and sequence individual Wolbachia genes. This system should facilitate future studies investigating the distribution and biology of Wolbachia strains from large samples of different host species.     

THE POPCORN WOLBACHIA INFECTION OF DROSOPHILA MELANOGASTER: CAN SELECTION ALTER WOLBACHIA LONGEVITY EFFECTS?

Wolbachia popcorn ( w MelPop), a life-shortening strain of Wolbachia, has been proposed as an agent for suppressing transmission of dengue fever following infection of the vectoring mosquito Aedes aegypti . However, evolutionary changes in the host and Wolbachia genomes might attenuate any life span effects mediated by w MelPop. Here we test for attenuation by selecting strains of Drosophila melanogaster infected with w MelPop for early and late reproduction in three independent outcrossed populations. Selection caused divergence among the lines in longevity. This divergence was mostly associated with the host genetic background rather than the Wolbachia infection, although there were also interactions between the host and Wolbachia genomes. Development time, viability, and productivity were not altered by selection. The implications of these results are discussed in light of the intended use of w MelPop for suppressing disease transmission.     

The distribution of Wolbachia in fig wasps: correlations with host phylogeny,ecology and population structure

We surveyed for the presence and identity of Wolbachia in 44 species of chalcid wasps associated with 18 species of Panamanian figs. We used existing detailed knowledge of the population structures of the host wasps, as well as the ecological and evolutionary relationships among them, to explore the relevance of each of these factors to Wolbachia prevalence and mode of transmission. Fifty-nine per cent of these wasp species have Wolbachia infections, the highest proportion reported for any group of insects. Further, neither the presence nor the frequency of Wolbachia within hosts was correlated with the population structure of pollinator hosts. Phylogenetic analyses of wsp sequence data from 70 individuals representing 22 wasp species show that neither the close phylogenetic relationship nor close ecological association among host species is consistently linked to close phylogenetic affinities of the Wolbachia associated with them. Moreover, no genetic variation was detected within any Wolbachia strain from a given host species. Thus, the spread of Wolbachia within host species exceeds the rate of horizontal transmission among species and both exceed the rate of mutation of the wsp gene in Wolbachia. The presence and, in some cases, high frequency of Wolbachia infections within highly inbred species indicate that the Wolbachia either directly increase host fitness or are frequently horizontally transferred within these wasp species. However, the paucity of cospeciation of Wolbachia and their wasp hosts indicates that Wolbachia do not persist within a given host lineage for long time-periods relative to speciation times.     

Presence and distribution of the endosymbiont Wolbachia among Solenopsis spp. (Hymenoptera: Formicidae) from Brazil and its evolutionary history

Wolbachia are intracellular bacteria that commonly infect arthropods. Its prevalence among ants of the genus Solenopsis is high. In the present study, the presence and distribution of these endosymbionts was examined among populations of Solenopsis spp. from Brazil. A phylogenetic analysis based on the wsp gene was conducted to infer the evolutionary history of Wolbachia infections within the populations surveyed. A high frequency of Wolbachia bacteria was observed among the genus Solenopsis, 51% of the colonies examined were infected. Incidence was higher in populations from southern Brazil. However, little genetic variability was found among different Wolbachia strains within supergroups A and B. Our findings also suggest that horizontal transmission events can occur through the social parasite S. daguerrei.     

Natural Wolbachia infections in the Drosophila yakuba species complex do not induce cytoplasmic incompatibility but fully rescue the wRi modification

In this study, we report data about the presence of Wolbachia in Drosophila yakuba, D. teissieri, and D. santomea. Wolbachia strains were characterized using their wsp gene sequence and cytoplasmic incompatibility assays. All three species were found infected with Wolbachia bacteria closely related to the wAu strain, found so far in D. simulans natural populations, and were unable to induce cytoplasmic incompatibility. We injected wRi, a CI-inducing strain naturally infecting D. simulans, into the three species and the established transinfected lines exhibited high levels of CI, suggesting that absence of CI expression is a property of the Wolbachia strain naturally present or that CI is specifically repressed by the host. We also tested the relationship between the natural infection and wRi and found that it fully rescues the wRi modification. This result was unexpected, considering the significant evolutionary divergence between the two Wolbachia strains.     

Making (good) use of Wolbachia: what the models say

Wolbachia, probably the most common animal endosymbiont, infects a wide range of arthropods as well as filarial nematodes. Generally vertically transmitted from mothers to offspring, it has evolved various strategies, ranging from brutal male-killing to mutualism, which facilitate invasion and persistence of the infections within host populations. Current interest in Wolbachia as a potential control agent against harmful nematodes and arthropods makes it important to be able to predict Wolbachia epidemiology and evolutionary trajectory. Here we highlight recent theoretical developments and suggest future modelling and empirical directions for basic and applied research in this domain.