An initial survey for Wolbachia (Rickettsiales: Rickettsiaceae) infections in selected California mosquitoes (Diptera: Culicidae)

Knowledge of biogeographic variation in Wolbachia infection rates and inferred susceptibility to infection among different mosquito taxa has fundamental implications for the design and successful application of Wolbachia-based vector-borne disease control strategies. Using a Wolbachia-specific polymerase chain reaction assay, we tested 14 North American mosquito species in five genera (Aedes, Anopheles, Culiseta, Culex, and Ochlerotatus) for Wolbachia infection. Wolbachia infections were only detected in members of the Culex pipiens (L.) species complex.     

Wolbachia infections of phlebotomine sand flies (Diptera: Psychodidae)

Old and New World phlebotomine sand fly species were screened for infection with Wolbachia, intracellular bacterial endosymbionts found in many arthropods and filarial nematodes. Of 53 samples representing 15 species, nine samples offour species were found positive for Wolbachia by polymerase chain reaction amplification using primers for the Wolbachia surface protein (wsp) gene. Five of the wsp gene fragments from four species were cloned, sequenced, and used for phylogenetic analysis. These wsp sequences were placed in three different clades within the arthropod associated Wolbachia (groups A and B), suggesting that Wolbacia has infected sand flies on more than one occasion. Two distantly related sand fly species, Lutzomyia (Psanthyromyia) shannoi (Dyar) and Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho), infected with an identical Wolbachia strain suggest a very recent horizontal transmission.     

Characterization of Wolbachia infections and interspecific crosses of Aedes (Stegomyia) polynesiensis and Ae. (Stegomyia) riversi (Diptera: Culicidae)

Prior studies have identified a complicated pattern of interspecific hybridization between members of the Aedes (Stegomyia) scutellaris (Walker) mosquito group, which includes medically important vectors of bancroftian filariasis and dengue. Here, we report that two members of the group, Aedes polynesiensis Marks and Aedes riversi Bohart & Ingram, are both infected with intracellular Wolbachia bacteria. Sequencing of the Wolbachia wsp gene demonstrates that the infections differ from each other and from Wolbachia infections previously reported in mosquitoes. Aedes polynesiensis is the first mosquito identified with a wMel Wolbachia type. Intraspecific crosses of infected and aposymbiotic lines generated via antibiotic treatment show that the Wolbachia infections in both species cause high levels of cytoplasmic incompatibility. Interspecific crosses show that the two species are reproductively isolated. However, repeating the interspecific crosses with aposymbiotic mosquito strains demonstrates that the Wolbachia infections play a role in preventing hybrid offspring. We discuss Wolbachia infections in relation to better defining the evolutionary relationships and causes of speciation within the group, understanding the basis for the observed east-to-west gradient in filarial refractoriness, and developing novel genetic control measures.     

Molecular (sub) grouping of endosymbiont Wolbachia infection among mosquitoes of Taiwan

Wolbachia are maternally inherited bacteria that infect a wide range of arthropods as well as filarial worms. The infection usually results in reproductive distortions of the host, primarily cytoplasmic incompatibility, parthenogenesis, and feminization. This study showed that Wolbachia infection (15/29; 51.72%) was prevalent among field-caught mosquitoes in Taiwan. Three mosquito species were identified as having Wolbachia A infection, eight species as having Wolbachia B, and four other species were dually infected by both groups. Each Wolbachia isolate from different mosquitoes was further divided into a specific subgroup. However, there were still some isolates that did not belong to any known subgroup, suggesting that more subgroups remain to be identified. Investigation of tissue tropism in either Aedes albopictus (Skuse) or Armigeres subalbatus (Coquillett) revealed that Wolbachia were extensively distributed within the host, although the ovary was most susceptible to infection. This report provides preliminary features of molecular relationships among Wolbachia groups of mosquitoes from Taiwan.     

Distribution and diversity of Wolbachia infections in Southeast Asian mosquitoes (Diptera: Culicidae)

Wolbachia are a group of intracellular inherited bacteria that infect a wide range of arthropods. They are associated with a variety of reproductive alterations in their hosts, the best known being cytoplasmic incompatability. The Wolbachia pipientis assemblage has been divided into two major groups (A and B) and 12 subgroups. We report herein the first systematic survey of Wolbachia in mosquitoes, and the first survey classifying Wolbachia infections by subgroup. Wolbachia were detected in 28.1% of 89 wild-caught mosquito species, based on a polymerase chain reaction assay using ftsZ and wsp gene primers. Infections were found in all major disease vector genera except Anopheles. Nine of the 12 Wolbachia subgroups were represented. Group B Wolbachia strains showed more phylogenetic concordance with their host taxa than group A strains. Of the 25 positive mosquito species, five were superinfected with group A bacteria strains (AA), eight were superinfected with A and B strains (AB), and one was superinfected with group B strains (BB). The widespread distribution of Wolbachia among mosquito species further supports their potential importance in the genetic control of disease vectors.     

Wolbachia effects on Aedes albopictus (Diptera: Culicidae) immature survivorship and development

Wolbachia bacteria manipulate the reproduction of mosquito hosts via a form of sterility known as cytoplasmic incompatibility (CI), promoting the spread of infections into host populations. The rate at which an infection invades is affected by host fitness costs associated with the Wolbachia infection. Here, we examine for an effect of Wolbachia infection on the immature fitness of the Asian tiger mosquito Aedes albopictus (Skuse) (Diptera: Culicidae). In two experiments, we examine for a Wolbachia effect on immature survivorship and developmental rate, adult size, and an effect of larval nutrition on CI level. The highest survivorship can be observed in uninfected larvae, primarily because of reduced survivorship of Wolbachia-infected males. Although differences in the developmental rates are observed between the examined strains, the differences cannot be readily attributed to Wolbachia. An effect of Wolbachia on adult size is not observed. Poor male nutrition is associated with reduced fecundity and egg hatch of mates. The latter is hypothesized to explain the reduced egg hatch observed in CI crosses of malnourished males relative to well fed males. We discuss the results in relation to previously identified differences in adult fitness, naturally occurring invasions of Wolbachia, applied strategies of population replacement, and the need for additional modeling effort.     

Geographic distribution of Wolbachia infections in Cimex lectularius (Heteroptera: Cimicidae)

Endosymbiotic Wolbachia bacteria have been previously shown to infect laboratory colonies of the human bed bug, Cimex lectularius L. (Heteroptera: Cimicidae), but little information exists regarding the extent of infection in natural populations. We assayed C. lectularius populations from five North American regions (California, Connecticut, Florida, New York, and Toronto, Canada) and one African region (Macha, Zambia) for Wolbachia infection by the polymerase chain reaction (PCR). Wolbachia infections were prevalent in all populations assayed (83-100%). There were no significant differences in infection frequency between geographic regions, between sexes, or between life stages (adult versus nymph). The potential utility of Wolbachia for alternative bed bug control strategies is discussed.     

Wolbachia effects on host fitness and the influence of male aging on cytoplasmic incompatibility in Aedes polynesiensis (Diptera: Culicidae)

The endosymbiotic bacteria Wolbachia manipulate host reproduction by inducing a form of sterility known as cytoplasmic incompatibility (CI), promoting the invasion of infection into natural host populations. CI has received attention for use in applied strategies to control insect vectors of disease. Thus, to understand both naturally occurring Wolbachia invasions and evaluate potential applied strategies, it is important to understand Wolbachia interactions with its host, including impacts on fitness and the CI level. In this study, we examined for an effect of Wolbachia on survivorship, developmental time, sex ratio, longevity, fecundity, and egg hatch of Aedes polynesiensis Marks, which is the primary vector of Wuchereria bancrofti in the South Pacific. In this study, we have compared strains of A. polynesiensis that are naturally and artificially infected with Wolbachia and additional strains that are aposymbiotic (Wolbachia removed to generate an uninfected strain). Artificially infected strains were observed to have increased larval mortality and decreased adult longevity when compared with aposymbiotic strains. Naturally infected strains were observed to have decreased larval mortality, pupal mortality, increased adult longevity, and a larger adult size when compared with aposymbiotic strains. Artificially infected males that were 4 wk old were able to induce high rates of CI, similar to young males. We discuss the results in relation to the natural spread of Wolbachia and Wolbachia-based applied strategies to modify A. polynesiensis populations.     

A novel technique for removing Wolbachia infections from Aedes albopictus (Diptera: Culicidae).

Intracellular bacteria of the genus Wolbachia often behave as reproductive parasites by manipulating host reproduction to enhance the vertical transmission of infections. Wolbachia infections in Aedes albopictus (Skuse) cause a reproductive manipulation known as cytoplasmic incompatibility, which can reduce brood hatch. Because field populations of Ae. albopictus are naturally infected, studies of Wolbachia-induced effects on Ae. albopictus reproduction and fitness require that Wolbachia be artificially removed. Although simiple techniques for clearing Wolbachia infections from other host insects have been developed, removal of Wolbachia bacteria from Ae. abopictus is difficult. Here we describe an improved method for removing Wolbachia infections Ae. albopictus. This method differs from earlier techniques in that it relies upon the tetracycline treatment of adults instead of larvae. We demonstrate that tetracycline treatment of adult Ae. albopictus can predictably generate uninfected individuals, simplify the procedure required for Wolbachia removal, and reduce the level of inbreeding required to produce uninfected lines.     

Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness)

Endosymbiotic Wolbachia bacteria are abundant in the filarial nematodes that cause onchocerciasis (river blindness), including the larvae (microfilariae) that migrate into the cornea. Using a mouse model of ocular onchocerciasis, we recently demonstrated that it is these endosymbiotic bacteria rather than the nematodes per se that induce neutrophil infiltration to the corneal stroma and loss of corneal clarity (Saint Andre et al., Science 295:1892-1895, 2002). To better understand the role of Wolbachia organisms in the pathogenesis of this disease, we examined the fate of these bacteria in the cornea by immunoelectron microscopy. Microfilariae harboring Wolbachia organisms were injected into mouse corneas, and bacteria were detected with antibody to Wolbachia surface protein. Within 18 h of injection, neutrophils completely surrounded the nematodes and were in close proximity to Wolbachia organisms. Wolbachia surface protein labeling was also prominent in neutrophil phagosomes, indicating neutrophil ingestion of Wolbachia organisms. Furthermore, the presence of numerous electron-dense granules around the phagosomes indicated that neutrophils were activated. To determine if Wolbachia organisms directly activate neutrophils, peritoneal neutrophils were incubated with either parasite extracts containing Wolbachia organisms, parasite extracts depleted of Wolbachia organisms (by antibiotic treatment of worms), or Wolbachia organisms isolated from filarial nematodes. After 18 h of incubation, we found that isolated Wolbachia organisms stimulated production of tumor necrosis factor alpha and CXC chemokines macrophage inflammatory protein 2 and KC by neutrophils in a dose-dependent manner. Similarly, these cytokines were induced by filarial extracts containing Wolbachia organisms but not by Wolbachia-depleted extracts. Taken together, these findings indicate that neutrophil activation is an important mechanism by which Wolbachia organisms contribute to the pathogenesis of ocular onchocerciasis.     

Molecular phylogeny of Wolbachia endosymbionts in Southeast Asian mosquitoes (Diptera: Culicidae) based on wsp gene sequences

Wolbachia are maternally inherited intracellular bacteria that infect a wide range of arthropods and nematodes and are associated with various reproductive abnormalities in their hosts. Insect-associated Wolbachia form a monophyletic clade in the alpha-Proteobacteria and recently have been separated into two supergroups (A and B) and 19 groups. Our recent polymerase chain reaction (PCR) survey using wsp specific primers indicated that various strains of Wolbachia were present in mosquitoes collected from Southeast Asia. Here, we report the phylogenetic relationship of the Wolbachia strains found in these mosquitoes using wsp gene sequences. Our phylogenetic analysis revealed eight new Wolbachia strains, five in the A supergroup and three in the B supergroup. Most of the Wolbachia strains present in Southeast Asian mosquitoes belong to the established Mors, Con, and Pip groups.     

Onchocerciasis: the role of Wolbachia bacterial endosymbionts in parasite biology, disease pathogenesis, and treatment

The discovery of Wolbachia intracellular bacteria within filarial nematodes, including Onchocerca volvulus, the causative agent of onchocerciasis or "river blindness," has delivered a paradigm shift in our understanding of the parasite's biology, to where we now know that the bacterial endosymbionts are essential for normal development of larvae and embryos and may support the long-term survival of adult worms. The apparent mutualistic dependency has also offered a novel approach to the treatment of onchocerciasis through the use of antibiotics to eliminate Wolbachia, delivering for the first time a treatment which has significant macrofilaricidal efficacy. Studies with other filarial nematode species have also highlighted a role for Wolbachia in transmission and infection of the mammalian host through a fascinating manipulation of mast cell-mediated vasodilation to enhance infectivity of vector-borne larvae. Wolbachia has also been identified as the principal driver of innate and adaptive Th1 inflammatory immunity, which can either contribute to disease pathogenesis or, with the Wolbachia-mediated recruitment of mast cells, enhance infectivity. The Wolbachia activation of innate inflammation also drives inflammatory adverse events in response to chemotherapy with either diethylcarbamazine (DEC) or ivermectin. In this review we summarize the experimental and field trial data which have uncovered the importance of Wolbachia symbiosis in onchocerciasis.     

Infection of New- and Old-World Aedes albopictus (Diptera: Culicidae) by the intracellular parasite Wolbachia: implications for host mitochondrial DNA evolution

Wolbachia are cytoplasmically inherited, endosymbiotic bacteria known to infect a wide variety of arthropods. Polymerase chain reaction (PCR) amplification of the Wolbachia surface protein (wsp) gene was used to assay the infection of geographically disparate populations of Aedes albopictus (Skuse) by Wolbachia. Nine North American, four South American, one Hawaiian, and four Old World populations of A. albopictus were all doubly infected with both the wAlbA and wAlbB strains of Wolbachia. A 365-bp region of the wAlbA wsp gene was sequenced from seven geographically disparate host populations, and all sequences were identical. Similarly, a 474-bp region of the wAlbB wsp gene was sequenced from the same populations, and all sequences were identical. These results suggest a role for Wolbachia infection in causing the previously established pattern of low mitochondrial DNA variability, but average nuclear gene diversity, within and among populations of A. albopictus.     

Phylogenetic characterization of Wolbachia symbionts infecting Cimex lectularius L. and Oeciacus vicarius Horvath (Hemiptera: Cimicidae)

Wolbachia symbionts are obligate intracellular bacteria that cause host reproductive alterations in many arthropods and filarial nematodes. We identified Wolbachia symbionts in the cliff swallow bug (Oeciacus vicarious Horvath) and the human bed bug (Cimex lectularius L.) (Hemiptera: Cimicidae) by polymerase chain reaction (PCR) amplification and sequencing using Wolbachia-specific 16S rDNA and FtsZ primers. Phylogenetic analyses using Bayesian, maximum likelihood, and maximum parsimony algorithms indicated, with strong support, that (1) Wolbachia infections in these two cimicid hosts form a monophyletic group, and (2) the Wolbachia strains detected belong to the F clade, previously associated with termites, weevils, and filarial nematodes.     

No evidence of<Emphasis Type="Italic"> Wolbachia</Emphasis> endosymbiosis with<Emphasis Type="Italic"> Loa loa</Emphasis> and<Emphasis Type="Italic"> Mansonella perstans</Emphasis>

Endosymbiotic Wolbachia bacteria from different filarial species, including major pathogens of humans such as Wuchereria bancrofti, Brugia malayi and Onchocerca volvulus, seem to play an important role in the development, viability and fertility of these worms. Wolbachia trigger inflammatory host responses as well as adverse reactions against standard treatment regimens and are therefore under investigation as novel treatment targets. We investigated whether Wolbachia are also endosymbiotic in Loa loa and Mansonella perstans. In both male and female adult L. loa, we found no evidence of bacteria by light or transmission electron microscopy. Furthermore, Wolbachia-specific PCR was negative in both L. loa and M. perstans microfilariae. The absence of Wolbachia in both filarial species therefore discourages the use of antibiotics as an adjunct or alternative approach to current treatment concepts for both loiasis and mansonelliasis perstans.     

No evidence for bacteriophage WO orf7 correlation with Wolbachia-induced cytoplasmic incompatibility in the Culex pipiens complex (Culicidae: Diptera)

Gene flow between populations of Culex pipiens L. is relevant to observed differences in disease transmission, insecticide resistance, behavior, and physiology. Intracellular Wolbachia bacteria have been hypothesized to affect gene flow in insects. Specifically, Wolbachia cause a form of embryonic mortality known as cytoplasmic incompatibility (CI) in crosses between individuals with different Wolbachia types. Incompatibility in Culex is exceptional in that it represents the most complex CI pattern known, and yet Culex populations are not infected with divergent Wolbachia strains. This has led to the hypothesis that extrachromosomal factors such as phages or mobile genetic elements may be involved in determining CI phenotype. Recent molecular characterization of Culex laboratory strains has identified variation in the orf7 locus of the Wolbachia-associated bacteriophage WO. Here, crosses between eight Culex strains differing in their orf7 type were conducted to examine for the hypothesized involvement of bacteriophage WO in determining CI in Culex. Although crossing results show examples of compatibility, partial compatibility, and incompatibility, the results fail to show a correlation between the CI phenotypes and orf7 type. Specific examples include high egg hatch resulting in crosses between Culex strains that differ significantly in their orf7 type and low egg hatch resulting in crosses between Culex strains with similar orf7 types. Thus, the phage orf7 locus alone cannot predict CI type in the Culex strains examined in this study. However, rejection of the hypothesized role of WO phage in Culex CI will require the characterization of additional phage loci.     

Wolbachia- and Onchocerca volvulus-induced keratitis (river blindness) is dependent on myeloid differentiation factor 88

Endosymbiotic Wolbachia bacteria that infect the filarial nematode Onchocerca volvulus were previously found to have an essential role in the pathogenesis of river blindness. The current study demonstrates that corneal inflammation induced by Wolbachia or O. volvulus antigens containing Wolbachia is completely dependent on expression of myeloid differentiation factor 88.     

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.     

Immunohistochemical/immunogold detection and distribution of the endosymbiont Wolbachia of Dirofilaria immitis and Brugia pahangi using a polyclonal antiserum raised against WSP (Wolbachia surface protein)

Intracellular bacteria in filarial nematodes were described as early as the 1970s, yet it was only with the work on Dirofilaria immitis, the agent of canine and feline heartworm disease, that these microorganisms were identified as belonging to Wolbachia, a genus known for encompassing bacteria infecting insects and other arthropods. The implications for the presence of intracellular bacteria in filarial nematodes is now the subject of intense research, particularly regarding their role in the immunology and pathogenesis of disease in infected humans and animals and as a possible target for therapy. Here, the authors report results on the immunohistochemical and immunogold staining of Wolbachia in D. immitis and Brugia pahangi using polyclonal antibodies raised against the recombinant Wolbachia surface protein (WSP). The bacteria were present in the lateral hypodermal chords of both male and female worms and in the reproductive tract of adult females (oocytes, morulae, microfilariae). In D. immitis and B. pahangi from animals treated with tetracycline, positive staining was observed in the lateral chords of adult males and females, but was absent from the oocytes and morulae. These results indicate that Wolbachia endosymbionts can be identified immunohistochemically with anti-WSP polyclonal antibodies, that their distribution matches that already described for Wolbachia of other filarial worms, and that antibiotic treatment may impede the vertical transmission of these bacteria. Unequivocal detection of Wolbachia is essential for the study of this symbiont, in particular to monitor the effects of antibiotic treatment on worms. The use of a specific marker for bacteria in their nematode hosts represents an extremely useful tool in evaluating the pathogenic role and the effect of antibiotic treatment on these potential targets in the control of filarial disease.     

Wolbachia-induced cytoplasmic incompatibility in single- and superinfected Aedes albopictus (Diptera: Culicidae)

Maternally inherited bacteria of the genus Volbachia can cause cytoplasmic incompatibility resulting in the developmental arrest of early embryos. Previous studies have shown that both single- and superinfections of Wolbachia naturally occur in populations of Aedes albopictus (Skuse). Here, we report crossing experiments using three infection types occurring in Ae. albopictus: uninfected, single-infected, and superinfected individuals. Crosses were monitored over the lifetime of adults to detect possible effects of host age on cytoplasmic incompatibility levels and infection virulence. Both single- and superinfections induced high levels of cytoplasmic incompatibility throughout the lifetime of Ae. albopictus, demonstrating that both the single- and superinfections are well adapted for invasion of Ae. albopictus populations. Superinfected females were the longest lived and had the highest oviposition rates, whereas in males, uninfected individuals were the longest lived. These latter results demonstrate the need for additional experiments to better elucidate Wolbachia effects on host fitness in addition to cytoplasmic incompatibility.