Wolbachia Surface Protein Research Articles

Effects of Wolbachia on mtDNA variation and evolution in natural populations of Tetranychus urticae Koch

We investigated the effects of Wolbachia infection on mtDNA variation in spider mites by sequencing a portion of the mitochondrial cytochrome oxidase I (COI) gene from 198 individuals of known infection status. Four Wolbachia strains were described in the current study, namely wUrtOri1, wUrtOri2, wUrtOri3 and wUrtCon1. As predicted, the haplotype and nucleotide diversity were lower in infected individuals than in uninfected individuals. However, these mtDNA haplotype data are not entirely concordant with the surface protein of wolbachia (wsp) sequence data and both infected and uninfected individuals were found of the same haplotype. Although values of Tajima's D and Fu & Li's F were consistently less than zero for most infected groups, McDonald-Kreitman tests suggested that the patterns of variation were different from those expected under neutrality in only the uninfected group. Thus, the neutrality tests do not show a clear effect of Wolbachia infection on patterns of mtDNA variation and substitution in spider mites.

Use of in situ Hybridization to Localize Wolbachia during embryogenesis in Brugia malayi

Wolbachia are intracellular bacteria that are required for development and reproduction in most filarial nematodes. New approaches are needed to improve understanding of the role of Wolbachia in filarial biology. Recent studies using fluorescent immunohistology revealed asymmetric segregation of Wolbachia in early embryos of Brugia malayi, and this helps to explain the restricted distribution of Wolbachia in adult worms. However, the distribution of Wolbachia in late stages of embryo development or in infective larvae (L3) is largely unknown. In this study we have explored the use of in situ hybridization (ISH) to localize Wolbachia and Wolbachia gene expression during embryogenesis and morphogenesis in B. malayi. ISH with a 16S rRNA probe revealed Wolbachia in the lateral cords, oocytes, and embryos of B. malayi female orms. This was consistent with prior studies that detected Wolbachia by immunohistology. ISH with probes for Wolbachia surface protein gene (wsp) and Wolbachia heme biosynthetic pathway gene (hemE) showed that these genes were strongly expressed in early embryos but not in later stage embryos (“comma”, “pretzel”, or stretched intrauterine microfilariae). A detailed study of the distribution of Wolbachia in B. malayi embryos using ISH with the 16S rRNA probe documented the asymmetric segregation of Wolbachia in early embryos (morulae) and showed that Wolbachia were only present in hypodermal cord precursor cells in later stage embryos (“comma” stage or later). Progressively restricted localization of Wolbachia during morphogenesis may explain why Wolbachia are absent from the genital primordium in L3. Wolbachia distribution and gene expression vary dramatically during filarial embryo development and across the worm’s life cycle. Additional research is needed to understand the tissue-specific population dynamics of Wolbachiaand to explore how signals from the nematode host might influence the growth, distribution, and gene expression in these bacteria.

Molecular detection and the phylogenetics of Wolbachia in Chinese spiders (Araneae)

Maternally inherited endosymbiotic bacteria Wolbachia pipientis have been shown to have wide-ranging effects on the reproduction of their hosts. This study presents the first survey and characterization of Wolbachia pipientis that have infected spiders collected from Wuhan, Hubei Province, China. First, we used universal primers of the wsp gene (Wolbachia Surface Protein, WSP) to examine the infection of Wolbachia in spiders. We found that, out of 31 spider species, 7 species were infected with Wolbachia. Then we used the specific primers of the A and B Wolbachia supergroups for the wsp gene to determine if there are super-infections in these infected spiders. Specimens of Nephila clavata were infected with strains of both A and B Wolbachia, while the others were infected with either strain A or B. Lastly, we aligned the sequences obtained with published ones to establish the phylogenetic relationships among Wolbachia found in spiders. The Wolbachia in Larinia argiopiformis Bosenberg & Strand 1906, Eriovixia cavaleriei (Schenkel 1963), Araneus ventricosus (L. Koch 1978), and Pholcus crypticolens Bosenberg & Strand 1906 belong to the A supergroup and the other three species, Nephila clavata (L. Koch 1878), Oxyopes sertatus L. Koch 1877 and Coleosoma octomaculatum Bosenberg & Strand 1906 belong to the B supergroup.

Accelerated microevolution in an outer membrane protein (OMP) of the intracellular bacteria Wolbachia

BackgroundOuter membrane proteins (OMPs) of Gram-negative bacteria are key players in the biology of bacterial-host interactions. However, while considerable attention has been given to OMPs of vertebrate pathogens, relatively little is known about the role of these proteins in bacteria that primarily infect invertebrates. One such OMP is found in the intracellular bacteria Wolbachia, which are widespread symbionts of arthropods and filarial nematodes. Recent experimental studies have shown that the Wolbachia surface protein (WSP) can trigger host immune responses and control cell death programming in humans, suggesting a key role of WSP for establishment and persistence of the symbiosis in arthropods.ResultsHere we performed an analysis of 515 unique alleles found in 831 Wolbachia isolates, to investigate WSP structure, microevolution and population genetics. WSP shows an eight-strand transmembrane β-barrel structure with four extracellular loops containing hypervariable regions (HVRs). A clustering approach based upon patterns of HVR haplotype diversity was used to group similar WSP sequences and to estimate the relative contribution of mutation and recombination during early stages of protein divergence. Results indicate that although point mutations generate most of the new protein haplotypes, recombination is a predominant force triggering diversity since the very first steps of protein evolution, causing at least 50% of the total amino acid variation observed in recently diverged proteins. Analysis of synonymous variants indicates that individual WSP protein types are subject to a very rapid turnover and that HVRs can accommodate a virtually unlimited repertoire of peptides. Overall distribution of WSP across hosts supports a non-random association of WSP with the host genus, although extensive horizontal transfer has occurred also in recent times.ConclusionsIn OMPs of vertebrate pathogens, large recombination impact, positive selection, reduced structural and compositional constraints, and extensive lateral gene transfer are considered hallmarks of evolution in response to the adaptive immune system. However, Wolbachia do not infect vertebrates. Here we predict that the rapid turnover of WSP loop motifs could aid in evading or inhibiting the invertebrate innate immune response. Overall, these features identify WSP as a strong candidate for future studies of host-Wolbachia interactions that affect establishment and persistence of this widespread endosymbiosis.

Serum antibody responses to Wolbachia surface protein in patients with human lymphatic filariasis

Wolbachia surface protein (WSP), which is the most abundantly expressed protein of Wolbachia from the human filarial parasite Brugia malayi, was chosen for the present study. B-cell epitope prediction of the WSP protein sequence indicates a high antigenicity, surface probability and hydrophilicity by DNA STAR software analysis. ProPred analysis suggests the presence of HLA class II binding regions in the WSP protein that contribute to T-cell responses and isotype reactivity. In order to validate these findings, the gene coding for endosymbiont WSP was PCR-amplified from the genomic DNA of the human filarial parasite Brugia malayi and cloned in T-7 expression vector pRSET-A. Western blot and ELISA at the total IgG level with recombiant WSP indicated a significantly elevated reactivity in CP compared to MF, EN and NEN individuals. Isotype ELISA also suggested an elevated reactivity in CP patients at the IgG1 level. In contrast, WSP-specific IgG4 levels were found to be elevated in MF patients compared to CP and EN. Besides this, WSP-specific IgE levels indicated an elevated reactivity in CP and MF patients compared to normals. Observations from ELISA supported the in silico predictions that indicate the presence of B- and T-cell epitopes. Hence, a combinatorial approach of in silico predictions and wet-lab studies provides interesting insights into the role of Wolbachia proteins in filarial pathogenesis.

Detection and identification of Wolbachia endosymbionts from laboratory stocks of stored-product insect pests and their parasitoids

Endosymbiotic bacteria belonging to the genus Wolbachia are maternally inherited and can cause various effects on the reproductive system of their invertebrate hosts. Owing to their critical effects on insect reproduction, Wolbachia are potential tools for future insect pest control strategies. Here, we explored Wolbachia infections among stock collections at the National Food Research Institute of Japan, where stored-product insect pests and their parasitoids are maintained. Diagnostic polymerase chain reactions (PCRs) and sequencing of a fragment of the Wolbachia surface protein ( wsp) gene revealed that, among 59 strains (38 species) belonging to the Coleoptera, Psocoptera, Lepidoptera and Hymenoptera, 18 strains (13 species) were infected with Wolbachia. Among them, 14 strains of Wolbachia were identified based on wsp gene sequences. Database searches showed that at least 6 Wolbachia strains were shared by multiple host species. Co-infections with two distinct Wolbachia strains were found in the drugstore beetle Stegobium paniceum and Angoumois grain moth Sitotroga cerealella. Detection and characterization of Wolbachia strains from these pests and their parasitoids provide us with basic information for future pest control strategies using these endosymbiotic bacteria.

HiddenWolbachiadiversity in field populations of the European cherry fruit fly,Rhagoletis cerasi(Diptera, Tephritidae)

The European cherry fruit fly Rhagoletis cerasi has been a field model for cytoplasmic incompatibility since the mid 1970s. Two Wolbachia strains were detected in this tephritid species and wCer2 was described as the CI inducing agent dividing European populations into two unidirectional incompatible groups, i.e. southern females produce viable offspring with northern males, whereas the reciprocal cross results in incompatibility. We detected three new Wolbachia strains by sequencing a multitude of plasmids derived from Wolbachia surface protein gene (wsp) polymerase chain reaction (PCR) products. Strain-specific primers were developed allowing individual diagnosis without need for cloning. Hybridization of specific PCR products with a wsp oligonucleotide enhanced the detection limit significantly and revealed the presence of low-titre infections in some strains, in different ontogenetic stages and in adults of different age. We then performed a survey of strain prevalence and infection frequency in eight European regions. wCer1 was fixed in all populations, whereas wCer2 was detected only in the South. wCer3 frequency was the lowest without a clear distribution pattern. The abundance of wCer4 was homogenous across Europe. Like wCer2, wCer5 showed significant differences in spatial distribution. Our new findings of previously undetected and recombinant Wolbachia strains in R. cerasi reveal a major caveat to the research community not to overlook hidden Wolbachia diversity in field populations. Low-titres and geographical variability in Wolbachia diversity are expected to influence the outcome of Wolbachia population dynamics and Wolbachia-based insect population control and may create invasion barriers for expanding and artificially introduced Wolbachia strains.

Superinfection of fiveWolbachiain the alnus ambrosia beetle,Xylosandrus germanus(Blandford) (Coleoptera: Curuculionidae)

Wolbachia bacteria are among the most common endosymbionts in insects. In Wolbachia research, the Wolbachia surface protein (wsp) gene has been used as a phylogenetic tool, but relationships inferred by single-locus analysis can be unreliable because of the extensive genome recombination among Wolbachia strains. Therefore, a multilocus sequence typing (MLST) method for Wolbachia, which relies upon a set of five conserved genes, is recommended. In this study, we examined whether the alnus ambrosia beetle, Xylosandrus germanus (Blandford), is infected with Wolbachia using wsp and MLST genes. Wolbachia was detected from all tested specimens of X. germanus (n=120) by wsp amplification. Five distinct sequences (i.e. five alleles) for wsp were found, and labeled as wXge1-5. MLST analysis and molecular phylogeny of concatenated sequences of MLST genes identified wXge3 and wXge5 as closely-related strains. The detection rate of wXge4 and wXge1 was 100% and 63.3%, respectively; wXge2, wXge3 and wXge5 were detected from less than 15% of specimens. We performed mitochondrial haplotype analyses that identified three genetic types of X. germanus, i.e. Clades A, B and C. Wsp alleles wXge1, wXge2 and wXge4 were detected in all clade A beetles; wXge2 allele was absent from Clades B and C. We concluded that (i) five wsp alleles were found from X. germanus, (ii) use of MLST genes, rather than the wsp gene, are more suited to construct Wolbachia phylogenies and (iii) wsp alleles wXge2 and wXge3/wXge5 would infect clade A and clade B/C of X. germanus, respectively.

A new case of Wolbachia dependence in the genus Asobara: evidence for parthenogenesis induction in Asobara japonica

Wolbachia is a maternally inherited bacterium that is widely distributed among arthropods, in which it manipulates the reproduction of its hosts. Although generally facultative for its hosts, Wolbachia has recently become obligatory in Asobara tabida (Hymenoptera: Braconidae) in which it is required for the completion of oogenesis. Here, we describe a new Wolbachia strain (wAjap) that is associated with the genus Asobara and infects Asobara japonica. wAjap was detected in all female-biased populations of A. japonica found in the main islands of Japan, but not in the arrhenotokous populations from the southern islands. Using phylogenetic analyses based on multi-locus sequence typing (MLST), we show that this strain is closely related to wAtab3 (the strain required for oogenesis in A. tabida), even though they differ on Wolbachia surface protein (WSP) and WO phage sequences. Using antibiotic treatments, we show that cured thelytokous females are not dependent on Wolbachia for oogenesis. However, they produced only sons, showing that wAjap induces thelytokous parthenogenesis. Analyses of mating behavior and offspring production of individuals from Wolbachia-infected populations showed that while males were still sexually functional, females no longer attract males, making Wolbachia an obligate partner for daughter production in thelytokous populations. The fact that Wolbachia has become independently obligatory in two species of the same genus tends to show that dependence evolution can be common and swift, although no clear benefit for the parasitoid can be attributed to this dependence. Although dependence should lead to co-divergence between Wolbachia and its hosts, the very few cases of co-speciation observed in host-Wolbachia associations question the stability of these obligatory associations.

Wolbachia in Dirofilaria repens, an Agent Causing Human Subcutaneous Dirofilariasis

Human subcutaneous dirofilariasis is an increasingly reported zoonosis caused by several filarial species, in particular by Dirofilaria (Nochtiella) repens. Like many filarial worms, D. repens harbors the bacterial endosymbiont Wolbachia that has been implicated in the inflammatory features of filarial infection. Immunohistochemical staining against the Wolbachia surface protein (WSP) was carried out on 14 skin nodules and showed numerous bacteria within the intact worms and occasional positive staining within the surrounding inflammatory infiltrate. Serum samples from 11 of these patients resulted positive for total immunoglobulin G titers against WSP as examined in enzyme-linked immunosorbent assay. This is the first description of Wolbachia distribution in D. repens and the first report of specific immune response to Wolbachia in patients with subcutaneous dirofilariasis.

Wolbachia in two populations of Melittobia digitata Dahms (Hymenoptera: Eulophidae)

We investigated two populations of Melittobia digitata Dahms, a gregarious parasitoid (primarily upon a wide range of solitary bees, wasps, and flies), in search of Wolbachia infection. The first population, from Xalapa, Mexico, was originally collected from and reared on Mexican fruit fly pupae, Anastrepha ludens Loew (Diptera: Tephritidae); the other, from Athens, Georgia, was collected from and reared on prepupae of mud dauber wasps, Trypoxylon politum Say (Hymenoptera: Crabronidae). PCR studies of the ITS2 region corroborated that both parasitoid populations were the same species; this potentially provides a useful molecular taxonomic profile since females of Melittobia species are superficially similar. Amplification of the Wolbachia surface protein gene (wsp) confirmed the presence of this endosymbiont in both populations. Sequencing revealed that the Wolbachia harbored in both populations exhibited a wsp belonging to a unique subgroup (denoted here as Dig) within the B-supergroup of known wsp genes. This new subgroup of wsp may either belong to a different strain of Wolbachia from those previously found to infect Melittobia or may be the result of a recombination event. In either case, known hosts of Wolbachia with a wsp of this subgroup are only distantly related taxonomically. Reasons are advanced as to why Melittobia - an easily reared and managed parasitoid - holds promise as an instructive model organism of Wolbachia infection amenable to the investigation of Wolbachia strains among its diverse hosts.

Galectin and aldolase‐like molecules are responsible for the specific IgE response in humans exposed to Dirofilaria immitis

Dirofilaria immitis is the agent of the heartworm disease in canids and felids, and of pulmonary dirofilariosis in man. Like other filariae, D. immitis harbours endosymbion Wolbachia bacteriae. In this work we analyse the response of specific IgE antibodies against both D. immitis antigens and the Wolbachia surface protein (WSP) in two groups of persons living in an area of canine endemia, one presenting high levels of total IgE (group 1) and other with normal levels (group 2). Infections with D. immitis were demonstrated by the presence of specific IgG in 228 individuals(48.8%) of the group 1 and only in one of the group 2. Specific IgE antibody response against D. immitis antigens was detected only in individuals of the group 1. IgE response against WSP was not detected in any group. The IgE response was directed mainly against two molecules of 33 and 42 kDa of the antigenic extract of D. immitis. These molecules were identified by mass spectrometry as a galectin and an aldolase, respectively. Their possible role in the survival mechanisms of the parasite and their contribution to development of allergic reactions in individuals resident in areas with heartworm disease are discussed.

Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis infection

Since the definitive identification in 1995 of the bacterial endosymbiont Wolbachia that resides in different tissues of the filarial worm Dirofilaria immitis, there has been increasing interest to understand whether and what role it plays in the pathogenesis of and immune response to heartworm infection. The present study evaluated the effects of treatments on lung pathology in 20 beagle dogs experimentally infected with D. immitis. Dogs in Group 1 were treated with doxycycline (10 mg/kg/day) orally from weeks 0–6, 10–12, 16–18, 22–26, and 28–34. Dogs in Group 2 served as infected, non-treated controls. Dogs in Group 3 were given doxycycline as described for Group 1 combined with weekly oral doses of ivermectin (6 mcg/kg) for 34 weeks and intramuscular (IM) melarsomine (2.5 mg/kg) at week 24, followed by two additional melarsomine injections 24 h apart 1 month later. Group 4 received only melarsomine as described for Group 3. Lung lesion criteria, scored by two independent blinded pathologists, included perivascular inflammation and endothelial proliferation. Doxycycline treatment alone had no effect on lesion scores, whereas the combination of doxycycline and ivermectin resulted in less severe perivascular inflammation. All lungs were evaluated for positive immunostaining for the Wolbachia surface protein (WSP). Control dogs showed numerous thrombi, intense perivascular and interstitial inflammation and, occasionally, positive staining for WSP. Interestingly, dogs receiving doxycycline/ivermectin/melarsomine showed significantly less severe arterial lesions and the virtual absence of thrombi.

Dirofilaria immitis and Wolbachia-derived antigens: Its effect on endothelial mammal cells

Antigens of both Dirofilaria immitis and Wolbachia symbiont bacteria are implicated in the inflammatory pathology of heartworm infection. The aim of the present study was to compare the stimulatory capacity of in vitro cultures of vascular endothelial cells by the adult somatic antigens of D. immitis (DiSA) and the recombinant form of the Wolbachia surface protein (rWSP), during the first 24 h of stimulation. Our results indicate a different stimulatory activity of the two antigens. Both the DiSA and rWSP stimulate the production of the enzymes responsible of the arachidonic acid metabolism, cyclooxygenase-2, 5-lipoxygenase (5-LO), and leukotriene B 4. Only DiSA stimulates the production of prostaglandin E 2. Related to the adhesion molecules, the DiSA stimulates the expression of intercellular adhesion molecule-1 (ICAM-1) and platelet endothelial cell adhesion molecule-1 (PECAM-1), whereas rWSP stimulates ICAM-1, PECAM-1, and vascular cell adhesion molecule-1 (VCAM-1). Expression of E-cadherin and vascular endothelial growth factor also were stimulated by rWSP. Neither of the two antigens altered the basic physiological mechanisms of endothelial cells, such as cell proliferation, cell cycle, or apoptosis. The biological and pathological significance of these finding are discussed.

Molecular identification of Wolbachia from the filarial nematode Mansonella perstans

Wolbachiae are bacterial endosymbionts of insects and many filarial nematodes whose products trigger inflammation in filarial infections. The dependence of the parasites on their endosymbionts has also led to the use of antibiotics directed against the Wolbachiae, therapy that has been demonstrated to have a profound salutary effect on filarial infections. The identification of Wolbachiae in Mansonella species has been conclusively shown for Mansonella ozzardi (Mo), but not for Mansonella perstans (Mp). Using primers known to amplify the 16S ribosomal DNA of other filarial Wolbachiae, an identical 1393bp band was found in all samples tested. Sequence analysis of these samples demonstrated a single consensus sequence for Mp Wolbachia 16S rDNA that was most similar to Wolbachia sequences from other filarial nematodes. When aligned with the only other Mansonella Wolbachia sequence (Mo) there were only 8 nucleotide differences in the 1369bp overlapping sequence. Phylogenetic dendrograms, examining the relationship of the Mp Wolbachia to other Wolbachia 16S rDNA, showed that the Wolbachia tracked almost identically to the 5S rRNA of their parasite host. Wolbachia surface protein (WSP) was also demonstrated in protein extracted from Mp-containing whole blood. In advance of a treatment trial of Mp, a method for the quantitation of Mp Wolbachia was developed and used to demonstrate not only a relationship between microfilarial numbers and Wolbachia copy numbers, but also to demonstrate the effect of antibiotic on ridding Mp of Wolbachia.

Wolbachia transmission dynamics in Formica wood ants

BackgroundThe role of Wolbachia endosymbionts in shaping the mitochondrial diversity of their arthropod host depends on the effects they have on host reproduction and on the mode of transmission of the bacteria. We have compared the sequence diversity of wsp (Wolbachia surface protein gene) and the host mtDNA in a group of Formica ant species that have diverged approximately 0.5 million years ago (MYA). The aim was to study the relationship of Wolbachia and its ant hosts in terms of vertical and horizontal transmission of the bacteria.ResultsAll studied ant species were doubly infected with two Wolbachia strains (wFex1 and wFex4) all over their geographical distribution area in Eurasia. The most common haplotypes of these strains were identical with strains previously described from a more distantly related Formica ant, with an estimated divergence time of 3.5 – 4 MYA. Some strain haplotypes were associated to the same or closely related mtDNA haplotypes as expected under vertical transmission. However, in several cases the wsp haplotypes coexisted with distant mtDNA haplotypes, a pattern which is more compatible with horizontal transmission of the bacteria.ConclusionTwo lines of evidence suggest that the sharing of Wolbachia strains by all F. rufa species is rather due to horizontal than vertical transmission. First, the fact that endosymbiont strains identical to those of F. rufa ants have been found in another species that diverged 3.5–4 MYA strongly suggests that horizontal transfer can and does occur between Formica ants. Second, the frequent sharing of identical Wolbachia strains by distant mitochondrial lineages within the F. rufa group further shows that horizontal transmission has occurred repeatedly. Nevertheless, our dataset also provides some evidence for longer-term persistence of infection, indicating that Wolbachia infection within this host clade has been shaped by both horizontal and vertical transmission of symbionts. The fact that all the ants were infected irrespective of the family structure of their societies gives no support to the proposed hypotheses that the spreading of Wolbachia in ants might be associated to the types of their societies.

In vitro infection of Wolbachia in insect cell lines

Bacteria of the genus Wolbachia are intracellular symbionts that are found in a broad range of insects and cause a variety of reproductive alterations, including male killing, parthenogenesis, feminization and cytoplasmic incompatibility. Although Wolbachia has not been cultured outside its host cells, it can be co-cultured with insect cell lines in vitro, and this technique would facilitate studies on Wolbachia. In this study, we inoculated five Wolbachia strains into two insect cell lines to examine the ability of the cell lines to harbor different infections. The Wolbachia strains used were wKue, derived from Ephestia kuehniella, wCauA and wCauB, from Cadra cautella, and wMel and wMelPop, from Drosophila melanogaster. The cell lines used were Sf9 of Spodoptera frugiperda (Lepidoptera) and AeAl2 of Aedes albopictus (Diptera). Immunofluorescence staining with an antibody against Wolbachia surface protein showed that wCauA was successfully maintained in both AeAl2 and Sf9 cells and that wCauB and wKue were maintained in AeAl2 cells. The infection levels, determined by real-time quantitative PCR targeting the Wolbachia 16S rRNA gene, varied among different infections.

Insight into the routes of Wolbachia invasion: high levels of horizontal transfer in the spider genus Agelenopsis revealed by Wolbachia strain and mitochondrial DNA diversity

The pandemic distribution of Wolbachia (alpha-proteobacteria) across arthropods is largely due to the ability of these maternally inherited endosymbionts to successfully shift hosts across species boundaries. Yet it remains unclear whether Wolbachia has preferential routes of transfer among species. Here, we examined populations of eight species of the North American funnel-web spider genus Agelenopsis to evaluate whether Wolbachia show evidence for host specificity and the relative contribution of horizontal vs. vertical transmission of strains within and among related host species. Wolbachia strains were characterized by multilocus sequence typing (MLST) and Wolbachia surface protein (WSP) sequences, and analysed in relation to host phylogeny, mitochondrial diversity and geographical range. Results indicate that at least three sets of divergent Wolbachia strains invaded the genus Agelenopsis. After each invasion, the Wolbachia strains preferentially shuffled across species of this host genus by horizontal transfer rather than cospeciation. Decoupling of Wolbachia and host mitochondrial haplotype (mitotypes) evolutionary histories within single species reveals an extensive contribution of horizontal transfer also in the rapid dispersal of Wolbachia among conspecific host populations. These findings provide some of the strongest evidence to support the association of related Wolbachia strains with related hosts by means of both vertical and horizontal strain transmission. Similar analyses across a broader range of invertebrate taxa are needed, using sensitive methods for strain typing such as MLST, to determine if this pattern of Wolbachia dispersal is peculiar to Agelenopsis (or spiders), or is in fact a general pattern in arthropods.

No Depletion of Wolbachia from Onchocerca volvulus after a Short Course of Rifampin and/or Azithromycin

Endosymbionic Wolbachia bacteria inside adult Onchocerca volvulus worms (causing river blindness) are necessary for female worm fertility. We evaluated whether rifampin and/or azithromycin used in a five-day course could kill Wolbachia. In an open-label trial in Guatemala, 73 patients with 134 palpable onchocercal nodules were randomized into four treatment groups: rifampin, azithromycin, a combination of the two drugs, and controls (multivitamins). After five days of antibiotic treatment, all participants received a single dose of ivermectin on day 6. Nine months after treatment, the nodules were removed and the worms were examined. Skin snips to determine microfilariae were obtained at baseline and nine months. There were no significant differences between any of the treatment groups in the condition of the worms in the nodules, the presence of Wolbachia surface protein, or the number of microfilariae in skin. Short courses with these antibiotics will not clear Wolbachia from O. volvulus.

Diversity and Phylogeny of <I>Wolbachia</I> Infecting <I>Bactrocera dorsalis</I> (Diptera: Tephritidae) Populations from China

Wolbachia are a common and widespread group of symbiotic bacteria found in the reproductive tissues of arthropods. Bactrocera dorsalis (Hendel) is an important pest causing considerable economic losses of fruits and vegetables in several southern provinces of China. In this study, polymerase chain reaction (PCR) with general Wolbachia surface protein (wsp) primers was used to test the presence of Wolbachia in 1,500 individuals of B. dorsalis from five geographical populations of China. We detected 19 individuals of B. dorsalis infected by Wolbachia, and the infection rates of different populations varied. Comparison of wsp gene sequences from 19 individuals and search of the GenBank identified four new sequences, probably representing four Wolbachia strains. Sequence comparison showed that the four Wolbachia strains from B. dorsalis in China belonged to three groups (Kue, Mel, and Cuc). Phylogenetic analysis of the wsp sequences suggests that geographical isolation of Wolbachia exists among the populations of B. dorsalis in China, and gene flow of Wolbachia might have occurred between B. dorsalis populations of China and Thailand. Phylogenetic analysis performed on the host mitochondrial cytochrome oxidase I (COI) gene and wsp gene suggests that host has coevolved with Wolbachia.