Internal Spatiotemporal Population Dynamics of Infection with Three Wolbachia Strains in the Adzuki Bean Beetle, Callosobruchus chinensis (Coleoptera: Bruchidae)

The adzuki bean beetle, Callosobruchus chinensis, is infected with three distinct lineages of endosymbiotic bacteria belonging to the genus Wolbachia, which were designated wBruCon, wBruOri, and wBruAus. In an attempt to understand the mechanisms underlying the infection with these three organisms, the spatiotemporal infection dynamics of the three Wolbachia strains was investigated in detail by using a quantitative PCR technique. During the development of C. chinensis, the wBruCon, wBruOri, and wBruAus infection levels consistently increased but the growth patterns were different. The levels of infection plateaued at the pupal stage at approximately 3 × 10⁸, 2 × 10⁸, and 5 × 10⁷ wsp copy equivalents per insect for wBruCon, wBruOri, and wBruAus, respectively. At the whole-insect level, the population densities of the three Wolbachia types did not show remarkable differences between adult males and females. At the tissue level, however, the total densities and relative levels of the three Wolbachia types varied significantly when different tissues and organs were compared and when the same tissues derived from males and females were compared. The histological data obtained by in situ hybridization and electron microscopy were concordant with the results of quantitative PCR analyses. Based on the histological data and the peculiar Wolbachia composition commonly found in nurse tissues and oocytes, we suggest that the Wolbachia strains are vertically transmitted to oocytes not directly, but by way of nurse tissue. On the basis of our results, we discuss interactions among the three coinfecting Wolbachia types, reproductive strategies of Wolbachia, and factors involved in the different cytoplasmic incompatibility phenotypes.     

Effects of wolbachia on genetic divergence between populations: mainland-island model

Cytoplasmic incompatibility (CI) induced by intracellular bacteriais a possible mechanism for speciation. Growing empirical evidencesuggests that bacteria of the group Wolbachia may indeed actas isolating factors in recent insect speciation. Wolbachiaare cytoplasmically transmitted and can cause uni- or bidirectionalCI. We present a mainland-island model to investigate how muchimpact Wolbachia can have on genetic divergence between populations.In the first scenario we assume that the island population hasdiverged at a selected locus and ask whether genetic divergencewill be maintained after introduction of migration from themainland. In the second we explore whether divergence will originateunder migration. For simplicity, the host organisms are modeledas haploid sexuals. Simulations show that if each populationis initially infected with a different strain of Wolbachia,then higher levels of divergence occur at the locally selectedlocus than in the absence of Wolbachia. A weaker effect is seenwhen there is only unidirectional CI caused by a single strainof Wolbachia on the island. CI increases divergence becauseit reduces effective migration between mainland and island.Migrants suffer from being confronted with the wrong CI systemand this also applies to their matrilineal descendants. Moreover,there is a strong linkage disequilibrium between host genotypeand infection state, which helps to maintain Wolbachia differencesbetween the populations in the face of migration A sex biasin migration can either increase or decrease the effect of Wolbachiaon divergence. Results support the view that Wolbachia has thepotential for increasing divergence between populations andthus could enhance probabilities of speciation.     

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.     

Wolbachia在我国广赤眼蜂种群内的感染

Wolbachia是广泛分布于节肢动物生殖组织内的一类细胞内共生菌,它属于原细菌的α亚类,能够通过调控寄主的生殖活动而促进其在寄主种群中的扩散。通过对wsp 基因的克隆及PCR-RFLP分析确定了Wolbachia在我国广赤眼蜂种群内的存在,并发现有2种Wolbachia 菌系的感染,命名为wEvaA和wEvaB。经过克隆分离得到了这2种Wolbachia的wsp基因序列,在GenBank的登录号为AY390279和 AY390280 ,并由基于wsp基因的聚类树中发现,这两种Wolbachia菌系均属于A组。     

Asymmetrical Interactions between Wolbachia and Spiroplasma Endosymbionts Coexisting in the Same Insect Host

We investigated the interactions between the endosymbionts Wolbachia pipientis strain wMel and Spiroplasma sp. strain NSRO coinfecting the host insect Drosophila melanogaster. By making use of antibiotic therapy, temperature stress, and hemolymph microinjection, we established the following strains in the same host genetic background: the SW strain, infected with both Spiroplasma and Wolbachia; the S strain, infected with Spiroplasma only; and the W strain, infected with Wolbachia only. The infection dynamics of the symbionts in these strains were monitored by quantitative PCR during host development. The infection densities of Spiroplasma exhibited no significant differences between the SW and S strains throughout the developmental course. In contrast, the infection densities of Wolbachia were significantly lower in the SW strain than in the W strain at the pupal and young adult stages. These results indicated that the interactions between the coinfecting symbionts were asymmetrical, i.e., Spiroplasma organisms negatively affected the population of Wolbachia organisms, while Wolbachia organisms did not influence the population of Spiroplasma organisms. In the host body, the symbionts exhibited their own tissue tropisms: among the tissues examined, Spiroplasma was the most abundant in the ovaries, while Wolbachia showed the highest density in Malpighian tubules. Strikingly, basically no Wolbachia organisms were detected in hemolymph, the principal location of Spiroplasma. These results suggest that different host tissues act as distinct microhabitats for the symbionts and that the lytic process in host metamorphosis might be involved in the asymmetrical interactions between the coinfecting symbionts.     

<Emphasis Type="Italic">Wolbachia</Emphasis>-induced cytoplasmic incompatibility in Japanese populations of <Emphasis Type="Italic">Tetranychus urticae</Emphasis> (Acari: Tetranychidae)

Intracellular bacteria of the genus Wolbachia (alpha Proteobacteria) induce cytoplasmic incompatibility (CI) in many arthropod species, including spider mites, but not all Wolbachia cause CI. In spider mites CI becomes apparent by a reduced egg hatchability and a lower daughter:son ratio: CI in haplodiploid organisms in general was expected to produce all-male offspring or a male-biased sex ratio without any death of eggs. In a previous study of Japanese populations of Tetranychus urticae, two out of three green-form populations tested were infected with non-CI Wolbachia strains, whereas none of six red-form populations harbored Wolbachia. As the survey of Wolbachia infection in T. urticae is still fragmentary in Japan, we checked Wolbachia infection in thirty green-form populations and 29 red-form populations collected from a wide range of Japanese islands. For Wolbachia-infected populations, we tested the effects of Wolbachia on the reproductive traits and determined the phylogenetic relationships of the different strains of Wolbachia. All but one green-form populations were infected with Wolbachia and all strains belonged to the subgroup Ori when the wsp gene was used to determine the phylogenetic relationships of different strains of Wolbachia. Six out of 29 red-form populations harbored Wolbachia and the infected strains belonged to the subgroups Ori and Bugs. Twenty-four of 29 infected green-form populations and five of six infected red-form populations induced CI among the hosts. Thus, CI-Wolbachia strains are widespread in Japan, and no geographical trend was observed in the CI-Wolbachia. Although three red-form populations harbored other intracellular bacteria Cardinium, they did not affect host reproduction.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Strains Typing in Different Geographic Population Spider, <Emphasis Type="Italic">Hylyphantes Graminicola</Emphasis> (Linyphiidae)

The Wolbachia endosymbiont of spiders has not been extensively examined. In order to investigate the distribution, evolutionary history, and reproductive phenotype of Wolbachia in spiders in China, we tested 11 geographic populations of Hylyphantes graminicola. Wolbachia infection has been detected in each population. 10 Wolbachia strains have been characterized by multilocus sequence typing (MLST). Phylogenetic analyses indicated that eight Wolbachia strains in H. graminicola belonged to supergroup B, and two belonged to supergroup A. No correlation existed between Wolbachia diversity and host’s geographic distance. The significant correlation was observed between pairwise distance of H. graminicola COI and genetic divergence of associated Wolbachia strains. We also found that Wolbachia infection frequencies in hosts varied over geographic space.     

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.     

High prevalence of Wolbachia infection does not explain unidirectional cytoplasmic incompatibility of Altica flea beetles

1. Wolbachia are intracellular bacteria found in a wide range of arthropods that can impact reproductive isolation of their hosts. Previous studies showed unidirectional cytoplasmic incompatibility (CI) and high infection rates by Wolbachia in the closely related leaf beetles Altica fragariae and Altica viridicyanea; however, whether this reproductive isolation was induced by Wolbachia remains unclear. 2. This study estimated the prevalence of Wolbachia in Altica beetles, assessed genetic diversity of Wolbachia strains infecting these beetles, and tested whether Wolbachia-induced CI explains reproductive isolation of A. fragariae and A. viridicyanea. 3. The results show that all of the 11 tested Altica species were infected by Wolbachia, and the infection rate was as high as 97.0% (n = 235). Multi-strain infections were common, being found in 10 of the 11 species tested and accounting for 23.0% of all screened specimens. In total, 35 Wolbachia strains were identified based on 208 wsp sequences obtained. Although a majority of A. fragariae and A. viridicyanea individuals from the Beijing population were infected with only one strain each, multi-strain infections did occur in both species. Antibiotic curing experiments did not change hatching success in either inter- or intraspecific crosses of A. fragariae and A. viridicyanea, indicating that these Wolbachia strains do not induce CI. These results were further corroborated by the lack of the Wolbachia cifB gene, which is responsible for causing CI. 4. These findings suggest that high prevalence of Wolbachia infection is unlikely to contribute to reproductive isolation and speciation in this system.     

In vitro cultivation of parthenogenesis-inducing Wolbachia in an Aedes albopictus cell line

Parthenogenesis‐inducing (PI) Wolbachia, infecting the parasitoid wasp Trichogramma kaykai Pinto and Stouthamer (Hymenoptera: Trichogrammatidae), were successfully maintained and cultivated in a mosquito, Aedes albopictus (Skuse) (Diptera: Culicidae), cell line, NIAS‐AeAl‐2. A parthenogenesis‐inducing (PI) Wolbachia strains with wasp ovaries were introduced into flasks with cultures of the cell line. The PI Wolbachia proliferated in NIAS‐AeAl‐2 as confirmed using a quantitative polymerase chain reaction method. Our final goal is to induce parthenogenesis in other hymenopteran species of commercial interest through the artificial transfer of PI Wolbachia. As a step towards this goal, we microinjected pupae of Trichogramma with PI Wolbachia culture in NIAS‐AeAl‐2 cells and PI Wolbachia was detected from recipient by PCR with Wolbachia specific primers (wsp gene).     

Wolbachia Strengthens Cardinium-Induced Cytoplasmic Incompatibility in the Spider Mite Tetranychus piercei McGregor

Wolbachia and Cardinium are maternally inherited intracellular bacteria that can manipulate the reproduction of their arthropod hosts, such as by inducing cytoplasmic incompatibility (CI). Although the reproductive alteration induced by Wolbachia or Cardinium have been well investigated, the effects of these two endosymbionts co-infecting the same host are poorly understood. We found that Tetranychus piercei McGregor is naturally infected with Wolbachia and Cardinium. We performed all possible crossing combinations using naturally infected and cured strains, and the results show that Wolbachia induced a weak level of CI, while Cardinium-infected and doubly infected males caused severe CI. Wolbachia and Cardinium could not rescue CI each other; however, Wolbachia boosted the expression of Cardinium-induced CI. Quantitative PCR results demonstrated that CI was associated with the infection density of Wolbachia and Cardinium.     

Contrasting effects of Wolbachia on cytoplasmic incompatibility and fecundity in the haplodiploid mite Tetranychus urticae

Recent studies on Wolbachia‐induced incompatibility in haplodiploid insects and mites have revealed a diversity of cytoplasmic incompatibility (CI) patterns among host species. Here, we report intraspecific diversity in CI expression among four strains of the arrhenotokous mite Tetranychus urticae and in T. turkestani. Variability of CI expression within T. urticae ranged from no CI to complete CI, and included either female embryonic mortality or male conversion types of CI. A fecundity cost attributed to the infection with the high‐CI Wolbachia strain was the highest ever recorded for Wolbachia (?80 to ?100% decrease). Sequence polymorphism at a 550‐bp‐portion of Wolbachia wsp gene revealed two clusters distant by 21%, one of which included three Wolbachia strains infecting mite populations sampled from the same host‐plant species, but showing distinct CI patterns. These data are discussed in the light of theoretical predictions on the evolutionary pathways followed in this symbiotic interaction.     

Culex torrentium mosquitoes from Germany are negative for Wolbachia

Wolbachia (Rickettsiales: Anaplasmataceae) infects a wide range of arthropods, including several mosquito species. The bacterium is known to induce a plethora of phenotypes in its host, examples being the reproductive phenotype cytoplasmic incompatibility or resistance against infection with arboviruses. The latter is especially relevant when assessing the vector competence of mosquito species for emerging arboviruses. Thus, knowledge of Wolbachia infection status is important for the assessment of vector competence. To facilitate Wolbachia screening in mosquito populations, a quantitative polymerase chain reaction (qPCR) assay was developed to enable high‐throughput analysis of mosquito samples. Using this assay, the Wolbachia infection status of the two most common Culex mosquito species in Germany, Culex pipiens biotype pipiens Linnaeus (Diptera: Culicidae) and Culex torrentium Martini (Diptera: Culicidae), was assessed. About 93% of all tested C. pipiens biotype pipiens individuals were positive for Wolbachia, whereas none of the C. torrentium samples was found to be infected. Furthermore, other applications of the qPCR assay were explored by assessing a potential link between the levels of Wolbachia and West Nile virus (WNV) infections in German C. pipiens biotype pipiens mosquitoes. No relationship was found between the two variables, indicating that a Wolbachia‐induced antiviral phenotype in this mosquito population is not exclusively attributable to the general level of bacterial infection.     

Wolbachia Divergence and the Evolution of Cytoplasmic Incompatibility in Culex pipiens

Many insect species harbor Wolbachia bacteria that induce cytoplasmic incompatibility (CI), i.e. embryonic lethality in crosses between infected males and uninfected females, or between males and females carrying incompatible Wolbachia strains. The molecular mechanism of CI remains unknown, but the available data are best interpreted under a modification–rescue model, where a mod function disables the reproductive success of infected males’ sperm, unless the eggs are infected and express a compatible resc function. Here we examine the evolution of CI in the mosquito Culex pipiens, harbouring a large number of closely related Wolbachia strains structured in five distinct phylogenetic groups. Specifically, we used a worldwide sample of mosquito lines to assess the hypothesis that genetic divergence should correlate with the divergence of CI properties on a low evolutionary scale. We observed a significant association of Wolbachia genetic divergence with CI patterns. Most Wolbachia strains from the same group were compatible whereas those from different groups were often incompatible. Consistently, we found a strong association between Wolbachia groups and their mod-resc properties. Finally, lines from the same geographical area were rarely incompatible, confirming the conjecture that the spatial distribution of Wolbachia compatibility types should be constrained by selection. This study indicates a clear correlation between Wolbachia genotypes and CI properties, paving the way toward the identification of the molecular basis of CI through comparative genomics.     

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.     

No evidence of Wolbachia among Great Lakes area populations of Daphnia pulex (Crustacea: Cladocera)

Wolbachia endobacteria are commonly associated with a varietyof arthropod species as hosts and induce known changes in theirhosts’ life-history traits. Despite exhibiting severalWolbachia-like life-history traits, and despite being a commonmodel organism, the zooplankton Daphnia pulex has not been formallytested for infection with Wolbachia. Among 203 isolates exhibitinga range of life-history phenotypes, we found no evidence ofWolbachia. This leaves the genes of D. pulex as the most likelycause of its own life-history traits.     

WOLBACHIA INFECTION IN DROSOPHILA SIMULANS: DOES THE FEMALE HOST BEAR A PHYSIOLOGICAL COST?

Fitness traits of three Drosophila simulans strains infected by endocellular bacteria belonging to the genus Wolbachia have been compared with those of replicate stocks previously cured from the infection by an antibiotic treatment. The traits measured were development time, egg-to-adult viability, egg hatch, productivity, fecundity, and the number of functional ovarioles. Individuals of the first strain were bi-infected by two Wolbachia variants, wHa and wNo. The second strain was infected by wHa, the third one by wNo. The Wolbachia studied here cause cytoplasmic incompatibility (CI), a high embryonic mortality (70% to > 90%) when an infected male is crossed with an uninfected female. Three generations after antibiotic treatment, we observed in all strains a significant drop in productivity in the cured stocks. This drop was not due to antibiotic toxicity and was associated with the loss of the Wolbachia. However the effect had disappeared in two of the three strains five generations after treatment, and could not be found in the third strain in a third measurement carried out 14 generations after treatment. The temporary nature of the productivity difference indicates that Wolbachia do not enhance productivity in infected strains. On the other hand, in all traits measured, our results show the absence of any negative effects of the Wolbachia on their host. This could be explained when considering Wolbachia evolution, as maternally transmitted parasites bear a strong selective pressure not to harm their female host. However, CI would allow the bacteria to be maintained even when harming the female. The apparent absence of deleterious effects caused by these Wolbachia might result from a trade-off, where a relatively low bacteria density would advantage the Wolbachia by suppressing any deleterious effects on the female host, at the cost of a weaker maternal transmission rate of the infection.     

Variable fitness and reproductive effects of Wolbachia infection in populations of the two-spotted spider mite Tetranychus urticae Koch in China

Maternally inherited Wolbachia bacteria are widely distributed among insects, and their presence usually causes modifications of the host. To understand the evolutionary history of diverse host-Wolbachia associations, we investigated the symbiosis between Wolbachia and the two-spotted spider mite Tetranychus urticae Koch in China. The cytoplasmic incompatibility (CI) level, fecundity, female ratio, host longevity and host development time were examined. Our results indicate that Wolbachia bacteria had variable effects on the reproduction and fitness of Chinese populations of T. urticae. Variability of CI expression within T. urticae ranged from no CI to a strong level of CI in spite of the low variability of the wsp gene. Relative to uninfected mites, infected females in one of the three populations showed enhanced fecundity associated with the infection of Wolbachia. This is the first report of a Wolbachia infection promoting the fecundity of infected females in T. urticae. Furthermore, we found both positive and negative effects of Wolbachia infection on longevity and the development time. The differences in ecological characters may be attributed to both Wolbachia and host genotype.     

Wolbachia Age-Sex-Specific Density in Aedes albopictus: A Host Evolutionary Response to Cytoplasmic Incompatibility?

Background

Wolbachia bacteria have invaded many arthropod species by inducing Cytoplasmic Incompatibility (CI). These symbionts represent fascinating objects of study for evolutionary biologists, but also powerful potential biocontrol agents. Here, we assess the density dynamics of Wolbachia infections in males and females of the mosquito Aedes albopitcus, an important vector of human pathogens, and interpret the results within an evolutionary framework.

Methodology/Principal Findings

Wolbachia densities were measured in natural populations and in age controlled mosquitoes using quantitative PCR. We show that the density dynamics of the wAlbA Wolbachia strain infecting Aedes albopictus drastically differ between males and females, with a very rapid decay of infection in males only.

Conclusions/Significance

Theory predicts that Wolbachia and its hosts should cooperate to improve the transmission of infection to offspring, because only infected eggs are protected from the effects of CI. However, incompatible matings effectively lower the fertility of infected males, so that selection acting on the host genome should tend to reduce the expression of CI in males, for example, by reducing infection density in males before sexual maturation. The rapid decay of one Wolbachia infection in Aedes albopictus males, but not in females, is consistent with this prediction. We suggest that the commonly observed reduction in CI intensity with male age reflects a similar evolutionary process. Our results also highlight the importance of monitoring infection density dynamics in both males and females to assess the efficiency of Wolbachia-based control strategies.     

Oxidative Stress Correlates with Wolbachia-Mediated Antiviral Protection in Wolbachia-Drosophila Associations

Wolbachia mediates antiviral protection in insect hosts and is being developed as a potential biocontrol agent to reduce the spread of insect-vectored viruses. Definition of the molecular mechanism that generates protection is important for understanding the tripartite interaction between host insect, Wolbachia, and virus. Elevated oxidative stress was previously reported for a mosquito line experimentally infected with Wolbachia, suggesting that oxidative stress is important for Wolbachia-mediated antiviral protection. However, Wolbachia experimentally introduced into mosquitoes impacts a range of host fitness traits, some of which are unrelated to antiviral protection. To explore whether elevated oxidative stress is associated with antiviral protection in Wolbachia-infected insects, we analyzed oxidative stress of five Wolbachia-infected Drosophila lines. In flies infected with protective Wolbachia strains, hydrogen peroxide concentrations were 1.25- to 2-fold higher than those in paired fly lines cured of Wolbachia infection. In contrast, there was no difference in the hydrogen peroxide concentrations in flies infected with nonprotective Wolbachia strains compared to flies cured of Wolbachia infection. Using a Drosophila mutant that produces increased levels of hydrogen peroxide, we investigated whether flies with high levels of endogenous reactive oxygen species had altered responses to virus infection and found that flies with high levels of endogenous hydrogen peroxide were less susceptible to virus-induced mortality. Taken together, these results suggest that elevated oxidative stress correlates with Wolbachia-mediated antiviral protection in natural Drosophila hosts.