Introduction: This study investigates the role of Wolbachia bacteria in the pathogenesis of O. volvulus keratitis in a mouse model. Wolbachia bacteria are essential symbionts of most filarial nematodes of importance for mankind. Methods: Using a mouse model for river blindness in which soluble extracts of filarial nematodes are injected in the corneal stroma, changes in stromal thickness and haze of the cornea are observed by in vivo confocal microscopy, followed by immunohistochemical staining for neutrophils and PECAM-1, as well as ELISA of corneal chemokines. Reactions to filarial extracts containing Wolbachia are compared to those without the endosymbiont. Results: The approach of characterizing Wolbachia’s role in river blindness in this study is threefold. Firstly, Wolbachia-depleted extracts from doxycycline treated onchocerciasis patients led to a diminished inflammatory response in corneas of C57BL/6 mice compared to untreated, i.e. Wolbachia containing antigen. The decreased cell recruitment observed with doxycycline treated extracts involved neutrophils, but not eosinophils. This finding demonstrated that the presence of Wolbachia increases neutrophil recruitment. Secondly, extracts from Wolbachia-containing B. malayi revealed markedly more pathology than endosymbiont-free A. viteae antigen. This again pointed at the role of Wolbachia in development of disease. Thirdly, Toll-like Receptor 4 (TLR4) dependence was shown to exist for the inflammatory response to Wolbachia harboring O. volvulus antigen by looking at the corneal pathology in TLR4-mutant C3H/HeJ mice, compared to the wild-type C3H/HeN strain. Investigating further Wolbachia mediated mechanisms of neutrophil recruitment to the cornea, this study also showed that expression of the adhesion molecule PECAM-1 in limbal vessels, as well as upregulation of the CXC chemokines KC and MIP-2 were dependent on the presence of functional TLR4 and Wolbachia respectively. Conclusions: This study indicates that the innate immune system and Wolbachia endobacteria play an important role in the inflammatory response associated with the pathogenesis of onchocerca keratitis, suggesting a complete alteration in our understanding of the immunopathology of filariasis. / Einleitung: Diese Arbeit untersucht die Rolle des Bakteriums Wolbachia in der Pathogenese der Onchozerka volvulus Keratitis anhand eines Mausmodels. Wolbachia sind essentielle endosymbiontische Bakterien, die in den meisten Filariosen, die für die Menschheit von Bedeutung sind, existieren. Methoden: Mit Hilfe eines Mausmodels für die Flußblindheit, in dem lösliche Filarienextrakte in das korneale Stroma von Mäusen injiziert werden, lassen sich Veränderungen in der Stromadicke und –durchsichtigkeit mit in vivo konfokaler Mikroskopie beobachten, gefolgt von immunhistochemischer Färbung von Neutrophilen und PECAM-1, wie auch ELISA von kornealen Chemokinen. Dabei werden Entzündungsreaktionen nach Injektion von Filarienmaterial mit oder ohne Wolbachia verglichen. Resultate: Die Untersuchung von Wolbachia's Rolle in der Flußblindheit erfolgte in drei Schritten. Zunächst führte Antigenmaterial von Wolbachia-freien, mit Doxyzyklin behandelten Onchozerkosepatienten zu geringerer Entzündungsreaktion in der Kornea von C57BL/6 Mäusen verglichen mit Wolbachia-enthaltendem Material. Die verminderte Enzündungszellzahl bei Doxyzyklin-behandelten Extrakten umfasste Neutrophile, aber nicht Eosinophile Granulozyten. Die Anwesenheit von Wolbachia führt daher zu verstärkter Neutrophileneinwanderung. Zweitens erwiesen Wolbachia-enthaltende B. malayi Extrakte eine signifikant verstärkte korneale Pathologie verglichen mit Endosymbiont-freiem A. viteae Antigen. Dieses Ergebnis deutete erneut auf die Rolle von Wolbachia in der Krankheitsentstehung. Drittens wurde anhand von Toll-like Rezeptor 4 (TLR4) mutanten C3H/HeJ Mäusen gezeigt, dass die Entzündungsreaktion, die von Wolbachia-enthaltenden O. volvulus Extrakten hervorgerufen wird, von TLR4 abhängig ist. Weitere Untersuchungen Wolbachia-abhängiger Mechanismen der Neutrophileneinwanderung in die Kornea erwiesen, dass die Expression des Adhäsionsmoleküls PECAM-1 in limbischen Gefäßen, wie auch die Hochregulation der CXC Chemokine KC und MIP-2 von TLR4 und der Anwesenheit von Wolbachia abhängig sind. Konklusion: Diese Arbeit zeigt, dass das angeborene Immunsystem und Wolbachia eine wichtige Rolle in der Pathogenese der O. volvulus Keratitis spielen, was auf eine neue Verstehensweise der Filariosenimmunpathologie hinweist.

The endosymbiotic bacteria Wolbachia and the gut microbiome have independently been shown to affect several aspects of insect biology, including reproduction, development, lifespan, stem cell activity and resistance to human pathogens in insect vectors. This research shows that Wolbachia, which reside mainly in the fly germline, affect the microbial species present in the gut of a lab reared strain of Drosophila melanogaster. Fruit flies host two main genera of commensal bacteria – Acetobacter and Lactobacillus. Wolbachia-infected flies have significantly reduced titers of Acetobacter. Analysis of the microbiome of axenic flies fed with equal proportions of both bacteria shows that Wolbachia’s presence is a determinant in the microbiome composition throughout fly development. This effect of Wolbachia on the Drosophila microbiome is host genotype-dependent. To investigate the mechanism of microbiome modulation, the effect of Wolbachia on Imd and ROS pathways, the main regulators of immune response in the fly gut was measured. Wolbachia’s presence did not cause significant gene expression changes of the effector molecules in either pathway. It was also found that Wolbachia slightly reduce the relative length of the acidic region of the gut. However, this observation lacks the robustness necessary to provide a mechanism for the significantly reduced Acetobacter levels. Furthermore, microbiome modulation is not due to direct interaction between Wolbachia and the gut microbes,as confocal microscopy shows that Wolbachia is absent from the gut lumen. These results indicate that the mechanistic basis of the modulation of microbiome composition by Wolbachia is more complex than direct bacteria interaction or Wolbachia’s effect on fly immunity. The findings reported here highlight the importance of considering the gut microbiome composition and host genetic background during Wolbachia-induced phenotypic studies and microbial based-disease vector control strategies.

Arboviruses are blood-borne pathogens that threaten half of the world’s population. The recent outbreak of Zika virus (ZIKV) in Brazil has highlighted the importance of developing new strategies to limit virus spread. While vaccines are in development, one way to immediately suppress viral transmission is through biocontrol of mosquito vector. Novel biocontrol strategies utilize microbe – mosquito interactions to inhibit the transmission of pathogens. A powerful tool under investigation is the intracellular bacteria, Wolbachia pipientis, which are maintained in insect populations through maternal transmission. The Wolbachia strain wMel can be trans-infected into mosquitos limiting ZIKV transmission. However, thermal stress can hinder maternal transmission of the wMel strain of Wolbachia. For Wolbachia-based technologies of vector control, it is important to have additional strains with viral suppression capabilities available. We characterized alternative Wolbachia strains in A. albopictus mosquito cell lines and the underlying mechanisms of these interactions. We identified two novel Wolbachia strains with robust arbovirus repression. wAlbB, native to mosquitos blocked 90% of ZIKV growth. More strikingly, wStri, a nonnative symbiont, ablated ZIKV growth in A. albopictus cells below the limit of detection. After showing that ZIKV growth is rescued in wStri infected A. albopictus cells by the pharmacological removal of Wolbachia, we established these cells as an in vitro model for mechanistic studies. Using novel labeling and reporter techniques, we isolate a block in virus growth by Wolbachia at two stages of viral growth, entry and translation. We further show that cholesterol, which can partially rescue viral growth in Wolbachia wStri infected cells, aids in viral entry but does not promote viral growth post entry. Beyond our Wolbachia studies, we further investigated the limited arbovirus growth observed in many A. aegypti cell lines and identified two insect–specific viruses which interfere with arbovirus growth. To address the limited biocontrol tools in C. pipiens mosquitos, we characterized commensal microbiota that may be used as a direct competitor of viruses or as a tool to genetically enhance an antiviral response in the mosquito gut. Together this work expands our understanding of Wolbachia-mediated biocontrol strategies and offers novel resources to suppress arbovirus transmission. / 2020-07-18T00:00:00Z

No description available.

Thèse de doctorat : Génétique évolutive : Paris 12 : 2005. / Version électronique uniquement consultable au sein de l'Université Paris 12 (Intranet). Titre provenant de l'écran-titre. Bibliogr. : 213 réf.

Thesis (Ph. D.)--University of California, Riverside, 2009. / Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed March 16, 2010). Includes bibliographical references. Also issued in print.

Solitary bees are important pollinators of crops, with species in the family Megachilidae (mason bees) being used for orchard pollination. Commercial movement of these bees also moves their microbiota, including bacterial endosymbionts capable of reproductive manipulation. To test for presence of these bacteria, I screened commercially available species of US orchard pollinators and locally captured solitary bees from Kentucky. I also set up mason bee boxes in five apple orchards to examine recruitment of local pollinators. I conducted 454-pyrosequencing to determine bacterial diversity within four species followed by diagnostic PCR of 30 collected species (184 individuals) to determine infection frequency of selected endosymbionts. Consistent with literature, Wolbachia was abundant in these bees. I also found two other endosymbiotic bacteria, Sodalis (previously undetected in Hymenoptera), and Arsenophonus. Diagnostic screening demonstrated that Sodalis was present at moderate frequency in Osmia aglaia, whereas Arsenophonus was present at low frequency in Lasioglossum pilosum. Neither was found in other bees, but three bee species were infected with Sodalis-like endosymbionts. Although recruitment of bees to bee boxes was ineffective, I was able to independently collect native orchard pollinating Andrenidae species. My results demonstrate that other endosymbionts capable of reproductive manipulation, besides Wolbachia, are present in bees.

A large and extremely diverse number of insects harbour maternally transmitted bacterial symbionts. Some symbionts manipulate host reproduction in order to benefit their own fitness, and the most common of these reproductive manipulations is cytoplasmic incompatibility (CI). In CI, uninfected females produce few or no viable progeny when mated to infected males. The bacterial endosymbiont Cardinium causes CI in its host, Encarsia pergandiella (Hymenoptera: Aphelinidae). I used population cages with varying initial infection frequencies to test a model of CI invasion. Cardinium was found to spread rapidly in all populations, even in cases where the initial infection frequency was well below the predicted invasion threshold frequency. Male age can also be an important factor in CI dynamics. I tested the effect of male age on the level of incompatibility induced by Cardinium in E. pergandiella. Male age was found to have a negligible effect on CI strength.

Wolbachia are bacteria that infect and induce reproductive alterations in a large number of arthropods. The present study was undertaken to improve our understanding of the effects that Wolbachia have on the reproductive biology of the leaf beetle Chelymorpha alternans Boh, in Panama. Two strains of Wolbachia occur in C. alternans, both of which induce some degree of cytoplasmic incompatibility (CI), a reproductive problem occurring when individuals bearing different infection status are mated together resulting in lowered levels of egg fertility. I attempted to localize where Wolbachia occurs in highest density in male reproductive tissues and to see if variation in Wolbachia density was associated with levels of CI in crossing experiments involving males (1) differing in age, (2) carrying different strains and (3) belonging to different populations previously known to vary in their levels of CI. Wolbachia were found to occur only in the somatic cells surrounding the testis, rather than in the germ cells, and yet were capable of inducing strong CI. Male age, infection status and provenance all were factors affecting the level of CI induced.

Wolbachia is a maternally inherited α-proteobacteria that lives an obligate intracellular lifestyle in its eukaryotic hosts. Wolbachia pipientis was first identified in the 1920’s in the mosquito Culex pipiens; Wolbachia has since been detected in a broad range of hosts. Wolbachia induces a number of diverse reproductive manipulations in many of its hosts, implicating Wolbachia in control strategies for target species. However, changes in host bacterial interactions can evolve toward mutualism, and this requires investigation in target systems. / This first part of this work examines changes in the Drosophila simulans-Wolbachia Riverside system in California. This system, studied for a period of more than two decades, provides a rare example in nature of a symbiotic relationship shifting from parasitism to mutualism. In my study, no changes in overall estimates of infection frequency, maternal transmission or cytoplasmic incompatibility were found, however there appears to be heterogeneity in maternal transmission rates and the ability of males to induce cytoplasmic incompatibility (CI). The source of CI variation in one isofemale line was subsequently investigated through reciprocal backcrossing with an old Wolbachia-infected strain, Riv88, and the variation was attributed to the Wolbachia genome. Further investigations are required to determine the extent of CI variation in the wild. These findings significant implications for the many applied projects that intend to exploit Wolbachia for its ability to manipulate host reproductive systems. / The second part of this work investigates the extent to which evolution of a host-bacterial symbiosis can influence the effectiveness of control strategies using Wolbachia. A particular strain of Wolbachia, named popcorn (wMelPop), has been isolated for its ability to reduce host longevity. This bacterium has the capacity to alter the age structure of the target host populations. As many vector-borne diseases (eg: Dengue Fever, Malaria) require an incubation period within the vector prior to transmission to the final host, a reduction in the lifespan of vector populations has the potential to lower transmission levels. The long term stability of this bacterial strain has been investigated in its native host (Drosophila melanogaster), and a novel host (D. simulans). The longevity-shortening phenotype and several life-history traits that might influence the effectiveness of control strategies have been examined. Host genetic background and the indirect selection on the longevity phenotype (via early or late fecundity selection) were found to influence multiple life-history traits in these species. The source of this variation in D. melanogaster is attributed primarily to the host, but can also be influenced by Wolbachia. Strong host background effects were noted in D. simulans, which highlights the importance of testing multiple backgrounds for suitability of use in applied projects. / This work has allowed for greater understanding of the potential for evolution within host-symbiont systems, and can be used to provide a framework for undertaking projects that will use Wolbachia as biological control agents. Future projects should consider the potential for evolution in detail under laboratory conditions before control strategies are implemented in the wild.