Wolbachia infection in crustaceans: novel hosts and potential routes for horizontal transmission

Thirty‐five percent of isopods are estimated to be infected by Wolbachia, an intracellular maternally inherited α‐Proteobacterium. Previous studies have indicated that horizontal transfer of Wolbachia strains may occur, although the mechanisms are unclear. The wsp gene was sequenced from 17 Wolbachia strains harboured by crustacean host species and three from their associated predators and parasites. Two major clades of Wolbachia were found in crustacean, with relatives also found in insects, the other restricted to crustaceans. Highly divergent Wolbachia strains were found in a woodlouse‐eating spider and its prey, suggesting no intertaxon bacterial exchange via the predator–prey route. The phylogenetic proximity of Wolbachia from parasitoid flies or phoretic mites to those from isopods suggests that horizontal symbiont transmission may have occurred between those taxa. Two distant Wolbachia strains were detected in two intertidal amphipods; these strains were closely related to different coastal isopod symbionts, suggesting Wolbachia transmission may occur between distantly related crustacean hosts living under the same ecological conditions.     

The mushroom habitat as an ecological arena for global exchange of Wolbachia

Wolbachia infect a variety of arthropod and nematode hosts, but in arthropods, host phylogenetic relationships are usually poor predictors of strain similarity. This suggests that new infections are often established by horizontal transmission. To gain insight into the factors affecting the probability of horizontal transmission among host species, we ask how host phylogeny, geographical distribution and ecology affect patterns of Wolbachia strain similarity. We used multilocus sequence typing (MLST) to characterize Wolbachia strain similarity among dipteran hosts associated with fleshy mushrooms. Wolbachia Supergroup A was more common than Supergroup B in Diptera, and also more common in mycophagous than non‐mycophagous Diptera. Within Supergroup A, host family within Diptera had no effect on strain similarity, and there was no tendency for Wolbachia strains from sympatric host species to be more similar to one another than to strains from hosts in different biogeographical realms. Supergroup A strains differed between mycophagous and non‐mycophagous Diptera more than expected by chance, suggesting that ecological associations can facilitate horizontal transmission of Wolbachia within mycophagous fly communities. For Supergroup B, there were no significant associations between strain similarity and host phylogeny, biogeography, or ecology. We identified only two cases in which closely related hosts carried closely related Wolbachia strains, evidence that Wolbachia‐host co‐speciation or early introgression can occur but may not be a major contributor to overall strain diversity. Our results suggest that horizontal transmission of Wolbachia can be influenced by host ecology, thus leading to partial restriction of Wolbachia strains or strain groups to particular guilds of insects.     

<Emphasis Type="Italic">Wolbachia pipientis</Emphasis> in Australian Spiders

Wolbachia pipientis is an endosymbiotic bacterium common to arthropods and filarial nematodes. This study presents the first survey and characterization of Wolbachia pipientis that infect spiders. All spiders were collected from Queensland, Australia during 2002–2003 and screened for Wolbachia infection using PCR approaches. The Wolbachia strains present in the spiders are diverse, paraphyletic, and for the most part closely related to strains that infect insects. We have also identified several spider Wolbachia strains that form a lineage outside the currently recognized six main Wolbachia supergroups (A–F). Incongruence between spider and Wolbachia phylogenies indicates a history of horizontal transmission of the bacterium in these host taxa. Like other arthropods, spiders are capable of harboring multiple Wolbachia strains.     

Bacterial symbiont sharing in Megalomyrmex social parasites and their fungus‐growing ant hosts

Bacterial symbionts are important fitness determinants of insects. Some hosts have independently acquired taxonomically related microbes to meet similar challenges, but whether distantly related hosts that live in tight symbiosis can maintain similar microbial communities has not been investigated. Varying degrees of nest sharing between Megalomyrmex social parasites (Solenopsidini) and their fungus‐growing ant hosts (Attini) from the genera Cyphomyrmex, Trachymyrmex and Sericomyrmex allowed us to address this question, as both ant lineages rely on the same fungal diet, interact in varying intensities and are distantly related. We used tag‐encoded FLX 454 pyrosequencing and diagnostic PCR to map bacterial symbiont diversity across the Megalomyrmex phylogenetic tree, which also contains free‐living generalist predators. We show that social parasites and hosts share a subset of bacterial symbionts, primarily consisting of Entomoplasmatales, Bartonellaceae, Acinetobacter, Wolbachia and Pseudonocardia and that Entomoplasmatales and Bartonellaceae can co‐infect specifically associated combinations of hosts and social parasites with identical 16S rRNA genotypes. We reconstructed in more detail the population‐level infection dynamics for Entomoplasmatales and Bartonellaceae in Megalomyrmex symmetochus guest ants and their Sericomyrmex amabilis hosts. We further assessed the stability of the bacterial communities through a diet manipulation experiment and evaluated possible transmission modes in shared nests such as consumption of the same fungus garden food, eating of host brood by social parasites, trophallaxis and grooming interactions between the ants, or parallel acquisition from the same nest environment. Our results imply that cohabiting ant social parasites and hosts may obtain functional benefits from bacterial symbiont transfer even when they are not closely related.     

Vertical and horizontal transmission drive bacterial invasion

A huge variety of Arthropod species is infected with endosymbiotic Wolbachia bacteria that manipulate their host’s reproduction to invade populations. In addition to vertical transmission from mother to offspring through the egg cytoplasm, it has been demonstrated through phylogenetic analyses and natural transfer experiments that horizontal transmission of Wolbachia (i.e. contagion) can occur between Arthropod hosts. More recently, factors influencing horizontal transfer have also been explored. While it is clear that horizontal transmission between species plays a major role in the evolutionary history of Wolbachia infections among insects, its role in the spread of a new infection through a host population, notably through within‐species transfers, remained unknown. In this issue of Molecular Ecology, Kraaijeveld et al. (2011) present the first evidence that horizontal transmission played a key role in the early spread of parthenogenesis‐inducing Wolbachia through the parasitoid wasp Leptopilina clavipes. To support their finding, the authors studied genetic variation in three types of markers, including host nuclear DNA, mitochondrial DNA and Wolbachia DNA. Specifically, they examined potential associations between their diversity patterns. No diversity was detected in Wolbachia genes, indicating that a single Wolbachia strain must have infected and spread through L. clavipes. In addition, a correlation between substantial variation in mitochondrial and nuclear genotypes suggested that horizontal transmission played an important role in the current clonal genetic variation in this wasp. Such horizontal transmission could be facilitated by a specific host ecology (e.g. parasitoid wasps sharing the same host resource) and potentially impact co‐evolution between host and symbiont.     

Epidemiology in evolutionary time: the case of Wolbachia horizontal transmission between arthropod host species

Wolbachia are bacterial endosymbionts that manipulate the reproduction of their arthropod hosts. Although theory suggests that infections are frequently lost within host species due to the evolution of resistance, Wolbachia infect a huge number of species worldwide. This apparent paradox suggests that horizontal transmission between host species has been a key factor in shaping the global Wolbachia pandemic. Because Wolbachia infections are thus acquired and lost like any other infection, we use a standard epidemiological model to analyse Wolbachia horizontal transmission dynamics over evolutionary time. Conceptually modifying the model, we apply it not to transmission between individuals but between species. Because, on evolutionary timescales, infections spread frequently between closely related species and occasionally over large phylogenetic distances, we represent the set of host species as a small‐world network that satisfies both requirements. Our model reproduces the effect of basic epidemiological parameters, which demonstrates the validity of our approach. We find that the ratio between transmission rate and recovery rate is crucial for determining the proportion of infected species (incidence) and that, in a given host network, the incidence may still be increasing over evolutionary time. Our results also point to the importance of occasional transmission over long phylogenetic distances for the observed high incidence levels of Wolbachia. In conclusion, we are able to explain why Wolbachia are so abundant among arthropods, although selection for resistance within hosts often leads to infection loss. Furthermore, our unorthodox approach of using epidemiology in evolutionary time can be applied to all symbionts that use horizontal transmission to infect new hosts.     

First record of the bacterial endosymbiont Wolbachia for phytophagous hoverflies from genus Merodon (Diptera: Syrphidae)

Wolbachia is a widespread bacterial endosymbiont among arthropod species. It influences the reproduction of the host species and also mitochondrial DNA diversity. Until now there were only a few studies that detected Wolbachia infections in hoverflies (Diptera: Syrphidae), and this is the first broader study with the aim of examining the incidence of Wolbachia in the hoverfly genus Merodon. The obtained results indicate an infection rate of 96% and the presence of both Wolbachia supergroup A and B, which are characteristic for most of the infected arthropod species. Additionally, the presence of multiple Wolbachia strains in the Merodon aureus group species was detected and the mitochondrial DNA COI‐based relationships of the group are discussed in the light of infection. Finally, we discuss plant‐mediated horizontal transmission of Wolbachia strains among the studied hoverfly species.     

Tracing the history and ecological context of Wolbachia double infection in a specialist host (Urophora cardui)—parasitoid (Eurytoma serratulae) system

The endosymbiotic bacterium Wolbachia is the most widespread bacteria in insects, yet the ecology of novel acquisitions in natural host populations is poorly understood. Using temporal data separated by 12 years, I tested the hypothesis that immigration of a parasitoid wasp led to transmission of its Wolbachia strain to its dipteran host, resulting in double‐strain infection, and I used geographic and community surveys to explore the history of transmission in fly and parasitoid. Double infection in the fly host was present before immigration of the parasitoid. Equal prevalence of double infection in males and females, constant prevalence before and after immigration in two regions, and increase in one region of immigration indicate little if no competition between strains. Double infection was present throughout the fly's distribution range, but proportions varied highly (0–0.71, mean = 0.26). Two fly‐specific MLST strains, observed in Eastern and Western Europe, respectively, differed at hcpA only. Flies with either fly‐strain could be double infected with the parasitoid's strain. The geographic distribution of double infection implies that it is older than the fly host's extent distribution range and that different proportions of double infection are caused by demographic fluctuations in the fly. The geographic data in combination with community surveys of infections and strains further suggest that the parasitoid strain was the fly's ancestral strain that was transmitted to the parasitoid, that is, the reverse transmission route as first hypothesized. Based on these findings together with a comparison of oviposition strategies of other hosts harboring related Wolbachia strains, I hypothesize that trans‐infection during an insect host's puparial metamorphosis might be important in promoting horizontal transmission among diverse holometabolic taxa.     

Various infection status and molecular evidence for horizontal transmission and recombination of Wolbachia and Cardinium among rice planthoppers and related species

Wolbachia and Cardinium are widely distributed and are considered important for their ability to disturb reproduction and affect other fitness‐related traits of their hosts. By using multilocus sequence typing (MLST), RFLP (restriction fragment length polymorphism) and 16S ribosomal DNA gene sequencing methods, we extensively surveyed Wolbachia and Cardinium infection status of four predominant rice planthoppers and one kind of leafhopper in different rice fields. The results demonstrated that Sogatella furcifera (Horváth) and Laodelphax striatellus (Fallén) were infected with the same Wolbachia strain (wStri), while Nilaparvata lugens (Stål) and its closely related species Nilaparvata muiri China were infected with two phylogeneticlly distant strains, wLug and wMui, respectively. Three new Wolbachia strains (provisionally named wMfas1, wMfas2 and wMfas3) were detected in the leafhopper Macrosteles fascifrons (Stål). Only S. furcifera was co‐infected with Cardinium, which indicated that the distribution of Cardinium in these rice planthoppers was narrower than that of Wolbachia. Unambiguous intragenic recombination events among these Wolbachia strains and incongruent phylogenetic relationships show that the connections between different Wolbachia strains and hosts were more complex than we expected. These results suggest that horizontal transmission and host associated specialization are two factors affecting Wolbachia and Cardinium infections among planthoppers and their related species.     

The hitchhiker's guide to Europe: the infection dynamics of an ongoing Wolbachia invasion and mitochondrial selective sweep in Rhagoletis cerasi

Wolbachia is a maternally inherited and ubiquitous endosymbiont of insects. It can hijack host reproduction by manipulations such as cytoplasmic incompatibility (CI) to enhance vertical transmission. Horizontal transmission of Wolbachia can also result in the colonization of new mitochondrial lineages. In this study, we present a 15‐year‐long survey of Wolbachia in the cherry fruit fly Rhagoletis cerasi across Europe and the spatiotemporal distribution of two prevalent strains, wCer1 and wCer2, and associated mitochondrial haplotypes in Germany. Across most of Europe, populations consisted of either 100% singly (wCer1) infected individuals with haplotype HT1, or 100% doubly (wCer1&2) infected individuals with haplotype HT2, differentiated only by a single nucleotide polymorphism. In central Germany, singly infected populations were surrounded by transitional populations, consisting of both singly and doubly infected individuals, sandwiched between populations fixed for wCer1&2. Populations with fixed infection status showed perfect association of infection and mitochondria, suggesting a recent CI‐driven selective sweep of wCer2 linked with HT2. Spatial analysis revealed a range expansion for wCer2 and a large transition zone in which wCer2 splashes appeared to coalesce into doubly infected populations. Unexpectedly, the transition zone contained a large proportion (22%) of wCer1&2 individuals with HT1, suggesting frequent intraspecific horizontal transmission. However, this horizontal transmission did not break the strict association between infection types and haplotypes in populations outside the transition zone, suggesting that this horizontally acquired Wolbachia infection may be transient. Our study provides new insights into the rarely studied Wolbachia invasion dynamics in field populations.     

Closely Related Wolbachia Strains within the Pumpkin Arthropod Community and the Potential for Horizontal Transmission via the Plant

Phylogenetic studies have implicated frequent horizontal transmission of Wolbachia among arthropod host lineages. However, the ecological routes for such lateral transfer are poorly known. We surveyed the species of two arthropod communities, one on pumpkin and the other on loofah plants, for Wolbachia, constructed wsp gene phylogenies of those Wolbachia strains found to infect community members, and established ecological links among infected members. Four taxonomically diverse insects in the pumpkin arthropod community contained very closely related Wolbachia wsp sequences (<1.5% divergence by Kimura-2-parameter distances). These insects, namely, the whitefly Bemisia tabaci, the planthopper Nisia nervosa, the flea beetle Phyllotreta sp., and the fleahopper Halticus minutus, were all collected from pumpkin leaves. They were ecologically linked through feeding on the same leaf substrate. Unlike other infected leaf insects, the whitefly population appeared to have a permanent breeding relationship with pumpkin plants, and high and stable, but not fixed, monthly Wolbachia infection rates. Our findings suggest potential roles for the plant in Wolbachia transmission and for whiteflies in being an infection source for other pumpkin leaf-feeding insects.     

Tracing horizontal Wolbachia movements among bees (Anthophila): a combined approach using multilocus sequence typing data and host phylogeny

The endosymbiotic bacterium Wolbachia enhances its spread via vertical transmission by generating reproductive effects in its hosts, most notably cytoplasmic incompatibility (CI). Additionally, frequent interspecific horizontal transfer is evident from a lack of phylogenetic congruence between Wolbachia and its hosts. The mechanisms of this lateral transfer are largely unclear. To identify potential pathways of Wolbachia movements, we performed multilocus sequence typing of Wolbachia strains from bees (Anthophila). Using a host phylogeny and ecological data, we tested various models of horizontal endosymbiont transmission. In general, Wolbachia strains seem to be randomly distributed among bee hosts. Kleptoparasite‐host associations among bees as well as other ecological links could not be supported as sole basis for the spread of Wolbachia. However, cophylogenetic analyses and divergence time estimations suggest that Wolbachia may persist within a host lineage over considerable timescales and that strictly vertical transmission and subsequent random loss of infections across lineages may have had a greater impact on Wolbachia strain distribution than previously estimated. Although general conclusions about Wolbachia movements among arthropod hosts cannot be made, we present a framework by which precise assumptions about shared evolutionary histories of Wolbachia and a host taxon can be modelled and tested.     

Cover Caption

The endosymbiont Wolbachia is widely distributed among insects. It can manipulate the reproduction of its hosts and affect hosts' other fitness‐related traits as well. Revealing infection status and variation of Wolbachia within the natural populations of rice planthoppers will help understand the diversification, horizontal transmission and host‐associated specialization of the endosymbiont. The research may have implications for the biological control of planthoppers (see pages 329‐344). Here, the result of specific fluorescence in situ hybridization (FISH) detection of Wolbachia in nymph abdomen of the brown planthopper, Nilaparvata lugens (Stål) is showed. The Wolbachia‐specific probes are red and cell nuclei are shown in blue. (Photo provided by Xiao‐Yue Hong of Nanjing Agricultural University, China).     

Age,tissue, genotype and virus infection regulate Wolbachia levels in Drosophila

The bacterial symbiont Wolbachia can protect insects against viral pathogens, and the varying levels of antiviral protection are correlated with the endosymbiont load within the insects. To understand why Wolbachia strains differ in their antiviral effects, we investigated the factors controlling Wolbachia density in five closely related strains in their natural Drosophila hosts. We found that Wolbachia density varied greatly across different tissues and between flies of different ages, and these effects depended on the host–symbiont association. Some endosymbionts maintained largely stable densities as flies aged while others increased, and these effects in turn depended on the tissue being examined. Measuring Wolbachia rRNA levels in response to viral infection, we found that viral infection itself also altered Wolbachia levels, with Flock House virus causing substantial reductions in symbiont loads late in the infection. This effect, however, was virus‐specific as Drosophila C virus had little impact on Wolbachia in all of the five host systems. Because viruses have strong tissue tropisms and antiviral protection is thought to be cell‐autonomous, these effects are likely to affect the virus‐blocking phenomenon. However, we were unable to find any evidence of a correlation between Wolbachia and viral titres within the same tissues. We conclude that Wolbachia levels within flies are regulated in a complex host–symbiont–virus‐dependent manner and this trinity is likely to influence the antiviral effects of Wolbachia.     

Clonal genetic variation in a Wolbachia‐infected asexual wasp: horizontal transmission or historical sex?

Wolbachia are endocellular bacteria known for manipulating the reproductive systems of many of their invertebrate hosts. Wolbachia are transmitted vertically from mother to offspring. In addition, new infections result from horizontal transmission between different host species. However, to what extent horizontal transmission plays a role in the spread of a new infection through the host population is unknown. Here, we investigate whether horizontal transmission of Wolbachia can explain clonal genetic variation in natural populations of Leptopilina clavipes, a parasitoid wasp infected with a parthenogenesis‐inducing Wolbachia. We assessed variance of markers on the nuclear, mitochondrial and Wolbachia genomes. The nuclear and mitochondrial markers displayed significant and congruent variation among thelytokous wasp lineages, showing that multiple lineages have become infected with Wolbachia. The alternative hypothesis in which a single female became infected, the daughters of which mated with males (thus introducing nuclear genetic variance) cannot account for the presence of concordant variance in mtDNA. All Wolbachia markers, including the hypervariable wsp gene, were invariant, suggesting that only a single strain of Wolbachia is involved. These results show that Wolbachia has transferred horizontally to infect multiple female lineages during the early spread through L. clavipes. Remarkably, multiple thelytokous lineages have persisted side by side in the field for tens of thousands of generations.     

Co‐evolutionary patterns and diversification of ant–fungus associations in the asexual fungus‐farming ant Mycocepurus smithii in Panama

Partner fidelity through vertical symbiont transmission is thought to be the primary mechanism stabilizing cooperation in the mutualism between fungus‐farming (attine) ants and their cultivated fungal symbionts. An alternate or additional mechanism could be adaptive partner or symbiont choice mediating horizontal cultivar transmission or de novo domestication of free‐living fungi. Using microsatellite genotyping for the attine ant Mycocepurus smithii and ITS rDNA sequencing for fungal cultivars, we provide the first detailed population genetic analysis of local ant–fungus associations to test for the relative importance of vertical vs. horizontal transmission in a single attine species. M. smithii is the only known asexual attine ant, and it is furthermore exceptional because it cultivates a far greater cultivar diversity than any other attine ant. Cultivar switching could permit the ants to re‐acquire cultivars after garden loss, to purge inferior cultivars that are locally mal‐adapted or that accumulated deleterious mutations under long‐term asexuality. Compared to other attine ants, symbiont choice and local adaptation of ant–fungus combinations may play a more important role than partner‐fidelity feedback in the co‐evolutionary process of M. smithii and its fungal symbionts.     

No evidence for Wolbachia‐induced parthenogenesis in the social Hymenoptera

In some parasitoid wasps, infection by the micro‐organism Wolbachia leads to asexual reproduction. Within the Hymenoptera, the limits of distribution of parthenogenesis inducing Wolbachia have not yet been established. To address this issue, we screened all known thelytokous social hymenopteran species using a PCR assay. None was infected, and therefore we conclude that worker thelytoky evolves independently of Wolbachia in ants and bees. This supports the previously proposed hypothesis that a sex determining system based on heterozygosity may form a proximate limitation to Wolbachia‐induced parthenogenesis.     

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.     

Microsatellite primers for fungus‐growing ants

We isolated five polymorphic microsatellite loci from a library of two thousand recombinant clones of two fungus‐growing ant species, Cyphomyrmex longiscapus and Trachymyrmex cf. zeteki. Amplification and heterozygosity were tested in five species of higher attine ants using both the newly developed primers and earlier published primers that were developed for fungus‐growing ants. A total of 20 variable microsatellite loci, developed for six different species of fungus‐growing ants, are now available for studying the population genetics and colony kin‐structure of these ants.     

Effects of Wolbachia on fitness of the Mediterranean fruit fly (Diptera: Tephritidae)

Wolbachia pipientis is a widespread endosymbiont of insects and other arthropods exerting a wide range of biological effects on their hosts. A growing number of recent studies document the influence of Wolbachia on reproduction and lifespan of insect host species. However, little is known regarding effects of Wolbachia on the demographic traits of different host populations. Moreover, whether different Wolbachia strains exert different effects on fitness components of their hosts remains largely unknown. We studied the effects of (a) the Wolbachia strain wCer2 on fitness components of two laboratory lines of the Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae) and (b) two different Wolbachia strains (wCer2 and wCer4) on one of the Mediterranean fruit fly lines. Wolbachia infection (wCer2) shortens the egg‐to‐adult developmental duration of both C. capitata lines, although it prolongs embryonic development. In one of the two lines, egg‐to‐adult mortality increased. Wolbachia infection shortens adult lifespan (to a different extent in males and females) and reduces female fecundity. The different Wolbachia strains differentially affect both immature mortality and developmental duration, and adult longevity and female fecundity. Our findings demonstrate both differential response of two C. capitata lines to Wolbachia infection and differential effects of two Wolbachia strains on the same Mediterranean fruit fly line. Practical and theoretical implications of our findings are discussed.