Tesis sobre el tema "Wolbachia endosymbionts"

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.

Most animals contain chronic microbial infections that inflict no harm on their hosts. Recently, the gut microflora of humans and other animals have been characterized. However, little is known about the forces that shape the diversity of these bacterial communities. In this work, comparative genomics was used to investigate the evolutionary dynamics of host-adapted bacterial communities, using Wolbachia infecting arthropods and Lactobacteria infecting bees as the main model systems. Wolbachia are maternally inherited bacteria that cause reproductive disorders in arthropods, such as feminization, male killing and parthenogenesis. These bacteria are difficult to study because they cannot be cultivated outside their hosts. We have developed a novel protocol employing multiple displacement amplification to isolate and sequence their genomes. Taxonomically, Wolbachia is classified into different supergroups. We have sequenced the genomes of Wolbachia strain wHa and wNo that belong to supergroup A and B, respectively, and are present as a double-infection in the fruit-fly Drosophila simulans. Together with previously published genomes, a supergroup comparison of strains belonging to supergroups A and B indicated rampant homologous recombination between strains that belong to the same supergroup but were isolated from different hosts. In contrast, we observed little recombination between strains of different supergroups that infect the same host. Likewise, phylogenetically distinct members of Lactic acid bacteria co-exist in the gut of the honeybee, Apis mellifera, without transfer of genes between phylotypes. Nor did we find any evidence of co-diversification between symbionts and hosts, as inferred from a study of 13 genomes of Lactobacillus kunkeei isolated from diverse bee species and different geographic origins. Although Lactobacillus kunkeii is the most frequently isolated strain from the honey stomach, we hypothesize that the primary niche is the beebread where the bacteria are likely to contribute to the fermentation process. In the human gut, the microbial community has been shown to interact with the immune system, and likewise the microbial communities associated with insects are thought to affect the health of their host. Therefore, a better understanding of the role and evolution of endosymbiotic communities is important for developing strategies to control the health of their hosts.

Wolbachia are maternally transmitted endosymbionts that naturally infect approximately 40% of all insect species and impose a range of phenotypic effects upon their hosts, including feminization, parthenogenesis, cytoplasmic incompatibility (CI) and male-killing. The genetic basis and mechanisms of these phenotypes remain mostly undetermined. Investigating new strains of Wolbachia can provide important insight into the biology of the symbiosis. In Drosophila pandora, two distinct Wolbachia strains co-exist to manipulate host reproduction, which are wPanCI (causing CI) and wPanMK (causing male-killing) respectively. Furthermore, wPanMK seems to rescue CI induced by wPanCI. To understand the molecular basis of CI and male-killing, genome sequencing and comparison projects with wPanCI and wPanMK were undertaken. To investigate the mechanism of CI, both cifA and cifB were compared among wMel, wPanCI and wPanMK; the cifB protein in wPanMK is truncated and has one base pair deletion, which indicating that it may be non-functional in wPanMK. The similar cifA gene in wPanCI and wPanMK and wPanMK’s ability to recue CI provide evidence for a previously toxin (cifB)-antitoxin (cifA) model; however, wPanMK cannot induce CI due to the mutation of cifB provide evidence that CI mechanism need both cifA and cifB. For male-killing mechanism, a spaid gene has been described to affect the male-killing in another endosymbiotic bacterium, Spiroplasma poulsonii. By comparing a spaid-like protein in different Wolbachia strains, a common structure with a protein-protein interaction domain and a cell death domain has been described and a spaid-like gene, wPanMK_288, has been found in wPanMK, which may be the manipulator gene to induce the male-killing. The study explains both two models of CI mechanism and provides a common structure and suggests putative genes for the expression of MK in Wolbachia and Spiroplasma.
Thesis (Masters)
Master of Science (MSc)
School of Environment and Sc
Science, Environment, Engineering and Technology
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Wolbachia (Alphaproteobacteria) is an inherited endosymbiont of arthropods and filarial nematodes and was reported to be widespread across insect taxa. While Wolbachia’s effects on host biology are not understood from most of these hosts, known Wolbachia-induced phenotypes cover a spectrum from obligate beneficial mutualism to reproductive manipulations and pathogenicity. Interestingly, data on Wolbachia within the most species-rich order of arthropods, the Coleoptera (beetles), are scarce. Therefore, we screened 128 species from seven beetle families (Buprestidae, Hydraenidae, Dytiscidae, Hydrophilidae, Gyrinidae, Haliplidae, and Noteridae) for the presence of Wolbachia. Our data show that, contrary to previous estimations, Wolbachia frequencies in beetles (31% overall) are comparable to the ones in other insects. In addition, we used Wolbachia MLST data and host phylogeny to explore the evolutionary history of Wolbachia strains from Hydraenidae, an aquatic lineage of beetles. Our data suggest that Wolbachia from Hydraenidae might be largely host genus specific and that Wolbachia strain phylogeny is not independent to that of its hosts. As this contrasts with most terrestrial Wolbachia–arthropod systems, one potential conclusion is that aquatic lifestyle of hosts may result in Wolbachia distribution patterns distinct from those of terrestrial hosts. Our data thus provide both insights into Wolbachia distribution among beetles in general and a first glimpse of Wolbachia distribution patterns among aquatic host lineages.

Reciprocal and homologous crosses were carried out using pairs or groups of twenty males and females for three biotypes of the whitefly Bemisia tabaci complex. Crosses were undertaken for the A biotype-Arizona (AzA), the B biotype-Africa (AzB), and the monophagous, Jatropha (Jat) biotype-Puerto Rico. The maternal haplotype pedigree of parents and selected offspring (F₁, F₂) was determined using the mitochondria cytochome oxidase I sequence. All reciprocal crosses yielded viable female offspring, indicating reproductively compatibility, except for AzB♀ x AzA♂, or AzB♀ x Jat♂ crosses, which yielded females unidirectionally. As an unidirectional pattern was reminiscent of cytoplasmic-mediated incompatibility (CI), the possibility was investigated that the phenotype might be caused by CI-bacteria, instead of a germ line barrier. Using the 16S rRNA sequence three prospective CI-bacterial species were identified in whitefly colonies. A Cardinium spp. (Bacteroidetes) was present in the A biotype (isolates AzA, CulA, RivA, SalA), whereas the B biotype (isolates AzB, FlB1, FlB2) was infected with Rickettsia bellii (Proteobacteria), and a Wolbachia spp. (Proteobacteria) was associated with the Jat biotype. The unidirectional incompatible phenotypes were consistent with CI-bacterial infection of AzA (Cardinium) and Jat (Wolbachia), but no such association was apparent for B biotype-Rickettsia infections. The bidirectional compatibility for Jat x AzA suggested a CI-bacteria-mediated reciprocal rescue. However, that Wolbachia-infected Jat♀ and Cardinium-infected AzA♂ crosses yielded fewer females, compared to AzA-Cardinium♀ and Jat- Wolbachia♂ crosses, suggested that Cardinium could better counter Wolbachia-induced female mortality than Wolbachia. This suggested the possibility that these phylogenetically divergent bacteria might utilize similar CI-mechanisms. In this study, the suspect CI-bacteria were strongly associated with complete or partial obstruction of gene flow in certain crosses, and with sex bias in the AzA x Jat crosses. This is the first evidence that female offspring can be produced between phylogeographically divergent, and polyphagous and monophagous B. tabaci, for which gene flow barriers are widely reported, suggesting that hybridization is utilized as a means of diversification in B. tabaci. The inability to rid colonies of CI-bacteria has necessitated introgression experiments to investigate direct CI-causality over CI-association.

Eight Wolbachia variants were identified in the wasp community. Identical Wolbachia variants were identified in inquiline and parasitoid wasp species suggesting that horizontal transmission of Wolbachia occurs in this community

Orientador: Odair Correa Bueno
Banca: Ana Eugênia C. Campos Farinha
Banca: Denise Selivon Scheepmaker
Resumo: O gênero Solenopsis tem distribuição cosmopolita, mas espécies do grupo S. saevissima, são nativas da América do Sul e inclui as conhecidas formigas lava-pés. Elas foram introduzidas de forma acidental em diversas regiões biogeográficas do mundo. No Brasil possuem ampla distribuição, mas têm preferência por áreas de atividade humana. São formigas altamente agressivas e são responsáveis por acidentes que podem levar a choques anafiláticos e à morte. As formigas apresentam associações de diferentes tipos com outros organismos, inclusive com bactérias endossimbiontes como a Wolbachia, bactéria intracelular que também infecta as formigas do gênero Solenopsis. No presente trabalho, procurou-se caracterizar as populações de lava-pés (Solenopsis spp.) de ampla área do território brasileiro, analisando o parentesco dessas populações e inferindo sobre sua filogenia. Além disso, foi investigada a presença, frequência e distribuição do endossimbionte Wolbachia em populações de Solenopsis spp. no Brasil. A caracterização das lava-pés foi baseada na análise do gene citocromo oxidase I e em estudos filogenéticos. Observou-se desde completa coerência geográfica, até polifilia para as espécies S. invicta e S. saevissima, o que demonstra claramente a diversidade desse gênero de formigas no Brasil. Existe a possibilidade de ocorrer populações isoladas reprodutivamente, tendo como decorrência processos evolutivos de especiação. Além disso, clados com espécies divergentes agrupadas podem trazer evidências de espécies erroneamente identificadas morfologicamente, presente em bancos de dados. O levantamento da ocorrência de Wolbachia foi baseado no gene wsp do endossimbionte e análises filogenéticas foram realizadas para inferir a história evolutiva dessas bactérias nas populações de lava-pés do país. Foi encontrada uma grande... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: The genus Solenopsis has a cosmopolitan distribution, but species of S. saevissima group are native from South America and include the known fire ant. They were accidentally introduced in several countries of the world. In Brazil they have wide distribution with preference for areas of human activity. Ants are highly aggressive and responsible for accidents that can lead to anaphylactic shock and death. The ants have different associations with other organisms, including bacteria endosymbionts such as Wolbachia, intracellular bacteria that also infect the ants of the Solenopsis genus. In this study, we sought to characterize the populations of fire ants (Solenopsis spp.) in a wide area of Brazil, analyzing the relationship of these populations and inferring their phylogeny. Furthermore, we investigated the presence, frequency and distribution of the endosymbiont Wolbachia in those populations of Solenopsis spp. in Brazil. The characterization of fire ants was based on analysis of the cytochrome oxidase I gene and on phylogenetic studies. It was observed that there were complete geographical coherence and polyphyly for the species S. invicta and S.saevissima, which clearly demonstrate the diversity of this genus of ants in Brazil. There is the possibility to occur reproductively isolated populations, leading to evolutionary processes of speciation. Furthermore, clustered clades with divergent species can bring evidences of species wrong morphologically identified, presents in databases. The survey of the occurrence of Wolbachia was based on the wsp gene of the endosymbiont and the phylogenetic analyses were performed to infer the evolutionary history of these bacteria in the populations of fire ants. There was a great diversity of Wolbachia in the genus Solenopsis, with 51% of the analyzed colonies presenting infection and the highest incidence was found in populations from... (Complete abstract click electronic access below)
Mestre

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O gênero Solenopsis tem distribuição cosmopolita, mas espécies do grupo S. saevissima, são nativas da América do Sul e inclui as conhecidas formigas lava-pés. Elas foram introduzidas de forma acidental em diversas regiões biogeográficas do mundo. No Brasil possuem ampla distribuição, mas têm preferência por áreas de atividade humana. São formigas altamente agressivas e são responsáveis por acidentes que podem levar a choques anafiláticos e à morte. As formigas apresentam associações de diferentes tipos com outros organismos, inclusive com bactérias endossimbiontes como a Wolbachia, bactéria intracelular que também infecta as formigas do gênero Solenopsis. No presente trabalho, procurou-se caracterizar as populações de lava-pés (Solenopsis spp.) de ampla área do território brasileiro, analisando o parentesco dessas populações e inferindo sobre sua filogenia. Além disso, foi investigada a presença, frequência e distribuição do endossimbionte Wolbachia em populações de Solenopsis spp. no Brasil. A caracterização das lava-pés foi baseada na análise do gene citocromo oxidase I e em estudos filogenéticos. Observou-se desde completa coerência geográfica, até polifilia para as espécies S. invicta e S. saevissima, o que demonstra claramente a diversidade desse gênero de formigas no Brasil. Existe a possibilidade de ocorrer populações isoladas reprodutivamente, tendo como decorrência processos evolutivos de especiação. Além disso, clados com espécies divergentes agrupadas podem trazer evidências de espécies erroneamente identificadas morfologicamente, presente em bancos de dados. O levantamento da ocorrência de Wolbachia foi baseado no gene wsp do endossimbionte e análises filogenéticas foram realizadas para inferir a história evolutiva dessas bactérias nas populações de lava-pés do país. Foi encontrada uma grande...
The genus Solenopsis has a cosmopolitan distribution, but species of S. saevissima group are native from South America and include the known fire ant. They were accidentally introduced in several countries of the world. In Brazil they have wide distribution with preference for areas of human activity. Ants are highly aggressive and responsible for accidents that can lead to anaphylactic shock and death. The ants have different associations with other organisms, including bacteria endosymbionts such as Wolbachia, intracellular bacteria that also infect the ants of the Solenopsis genus. In this study, we sought to characterize the populations of fire ants (Solenopsis spp.) in a wide area of Brazil, analyzing the relationship of these populations and inferring their phylogeny. Furthermore, we investigated the presence, frequency and distribution of the endosymbiont Wolbachia in those populations of Solenopsis spp. in Brazil. The characterization of fire ants was based on analysis of the cytochrome oxidase I gene and on phylogenetic studies. It was observed that there were complete geographical coherence and polyphyly for the species S. invicta and S.saevissima, which clearly demonstrate the diversity of this genus of ants in Brazil. There is the possibility to occur reproductively isolated populations, leading to evolutionary processes of speciation. Furthermore, clustered clades with divergent species can bring evidences of species wrong morphologically identified, presents in databases. The survey of the occurrence of Wolbachia was based on the wsp gene of the endosymbiont and the phylogenetic analyses were performed to infer the evolutionary history of these bacteria in the populations of fire ants. There was a great diversity of Wolbachia in the genus Solenopsis, with 51% of the analyzed colonies presenting infection and the highest incidence was found in populations from... (Complete abstract click electronic access below)

Les bactéries intracellulaires Wolbachia ont développé une vaste gamme d’interactions symbiotiques, du parasitisme reproductif au mutualisme chez les arthropodes terrestres et les nématodes filaires, devenant ainsi les endosymbiotes les plus répandus sur terre. Bien qu’elles se développent lentement dans les cultures cellulaires d’insectes pour lesquelles les marqueurs sont limités et qu’elles ne sont génétiquement pas manipulables, il existe un intéret croissant de déchiffrer leur mode de vie intracellulaire pour 2 raisons. Premièrement, Wolbachia intervient dans le développement et la transmission des arbovirus et deuxièmement, les filarioses lymphatiques sont traitables grâce à la susceptibilité des Wolbachia qui infectent les nématodes filaires aux antibiotiques. Au début de ce projet, j’ai infecté 2 lignées cellulaires de Drosophila melanogaster qui sont transcriptomiquement divergentes par une même souche de Wolbachia pouvant naturellement infecter Drosophila melanogaster. J’ai utilisé ces 2 lignées cellulaires qui sont différentiellement permissive à l’infection pour explorer l’interaction de Wolbachia avec le réticulum endoplasmique. Les observations par microscopie à fluorescence en temps réel et par microscopie électronique prouvent que cet organite est une source de membranes pour Wolbachia et possiblement, une source de nutriments. Pourtant, les analyses d’expression génique et les approches d’immunofluorescence démontrent que Wolbachia n’induit ni un stress au niveau du réticulum endoplasmique ni une protéolyse via la voie de signalisation ERAD suggérant dès lors, que Wolbachia subvertissent d’autres mécanismes pour assurer leur besoin en acides aminés. Au cours de ce projet, j’ai commencé à mettre en place une technique pour transformer Wolbachia par biolistique. La validation de cette technique de transformation a ouvert la voie vers l’optimisation de la procédure de sélection des transformants pour enfin pouvoir génétiquement manipuler Wolbachia
The intracellular bacteria Wolbachia have developed a wide range of symbiotic interactions, from being opportunistic reproductive parasites to mutualists with terrestrial arthropods and filarial nematode species, making them the most common endosymbionts on earth. The discovery that they interfere with arboviruses development and transmission by mosquito vectors and that filarial diseases can be cured by targeting Wolbachia, have created a strong interest in deciphering the mechanisms underlying their intracellular lifestyle. However, being obligate intracellular endosymbionts, Wolbachia remain genetically intractable. They grow slowly in insect cell cultures, for which markers are limited. Despite these obstacles, and to limit cell line-specific phenotypes, I chose to infect 2 Drosophila melanogaster cell lines presenting different sets of expressed genes, with a unique Wolbachia strain, naturally hosted by Drosophila melanogaster. Using these 2 cell lines that are differently permissive to the infection, I explored the interaction of Wolbachia with the endoplasmic reticulum (ER). Through fluorescence time-lapse confocal and electron microscopy observations, I provide strong evidence that this organelle is the source of membrane for Wolbachia, and possibly a source of nutrients. However, gene expression analyses and immunofluorescence approaches demonstrate that Wolbachia do not induce ER stress nor an increased ERAD- induced proteolysis, suggesting; unlike previously reported, that Wolbachia salvage amino acids by other subversion mechanisms. Additionally, I pioneered biolistic bombardement of Wolbachia-infected cells and the validation of this transformation technique has paved the way towards optimization of transformant selection steps and ultimately to the genetic engineering of Wolbachia

Wuchereria bancrofli is a filarial parasite responsible for 90% of lymphatic filariasis worldwide. Currently a mass drug administration programme, The Global Programme for the Elimination of Lymphatic Filariasis (GPELF) is underway, through the administration of three drugs: diethylcarbamazine (DEC), albendazole and ivermectin, to eliminate lymphatic filariasis as a global he~lth problem. The major aims of the research described in this thesis, were to investigate the genetic diversity of W. bancrofli and their endosymbiotic bacteria Wolbachia and to investigate the presence of putative resistance benzimidazole alleles of W. bancrofli p-tubulin. Attempts were made to isolate W. bancrofli microsatellites using a novel magnetic bead isolation technique. This technique was not successful in identifying microsatellites of w.bancrofli due to overwhelming contamination with human microsatellite DNA. Wolbachia endosymbionts are vertically transmitted and show congruent phylogenies with their nematode hosts, therefore the diversity of W. bancrofli Wolbachia was explored as an indirect marker of w.bancrofli diversity. To investigate the diversity of Wolbachia, genes of the major surface protein (wsp) and cell-division protein (jisZ) were sequenced from W. bancrofli collected from different geographical endemic areas (Burkina Faso, Malawi, Sri Lanka) and were phylogeneticallyanalysed. Although sequence data generated in this study indicates that identical haplotypes of Wolbachia do exist in W. bancrofli samples from different geographical areas, a large amount of diversity was observed in both wsp and flsZ gene sequences in individual W. bancrofli and pooled samples from within these sites. Unweighted Pair Group Method with Arithmetic mean (upGMA) phylogenetic trees generated indicate that Wolbachia is divided into two distinct clades with elevated rates of sequence evolution, especially at non-synonymous sites. Due to the mass drug administration programme currently underway to eliminate lymphatic filariasis, combined with previous reports of benzimidazole resistance (as indicated by Schwab et al. (2006) by the presence of mutation TYR200 in Burkina Faso) experiments to screen for putative resistance benzimidazole alleles in W. bancrofli p-tubulin were investigated. Results demonstrate that the resistanceassociated mutation of benzimidazole at either codon 167 or 200 of the p-tubulin gene was not observed in W. bancrofli samples in patients with varying drug histories from Burkina Faso and Sri Lanka, in contrast to published reports from identical sample sites (village of Tangonko, Burkina Faso). Overall the absence of resistance observed in the study is encouraging for the future of mass drug administration, although further research is required to explain the dynamics of putative resistant alleles under pressure of MDA.

Onchocerciasis affects an estimated 37 million people in Sub-Saharan Africa and Latin America, causing debilitating skin and eye disease and accounting for a global loss of 1 million Disability-Adjusted Life-Years. The discovery of Wolbachia bacterial endosymbionts in filarial nematodes, including Onchocerca volvulus, has revolutionised the understanding of the parasite’s biology and immunopathogenic mechanisms of disease, and has offered a novel approach to its treatment and control with anti-Wolbachia antibiotics. Treatment with doxycycline is effective at sterilising and killing adult O. volvulus worms, proving superior to standard microfilaricidal treatment with ivermectin and of great potential as an alternative strategy for the control of onchocerciasis. Although the length of the required treatment has raised concerns about the use of doxycycline in Mass Drug Administration (MDA) strategies, a recent trial in Cameroon demonstrated the feasibility of a six week course of doxycycline MDA delivered with a community-directed approach. In the work presented here we found a significant reduction in microfilaridermia prevalence and loads four years after doxycycline MDA distribution, demonstrating its long-term effectiveness and supporting its implementation in existing control strategies. Wolbachia peptidoglycan-associated lipoprotein stimulates innate and adaptive immune responses, contributing to disease pathogenesis through the induction of pro-inflammatory cytokines and recruitment of neutrophils. These features together with the cytokine milieu induced by filarial nematodes could support the development of a pro-inflammatory Type-17 immune response. In this work, PBMC from patients with onchocerciasis were found to produce only minimal levels of IL-17 in response to filarial extracts. On the contrary, a rich IL-17+ cell infiltrate was found surrounding adult worms in Wolbachia positive onchocercomas using immunohistochemistry (IHC), which was depleted from onchocercomas following doxycycline treatment. Although a high percentage of Th17 cells were present in this infiltrate compared to other diseases, the majority of IL-17 producing cells in nodules were neutrophils, within an extracellular trap-like structure. This unexpected result was consistent with the reported IL-17 production by human neutrophils using IHC in the literature, but could not be confirmed at the protein or the transcription level in vitro in this work. Wolbachia is responsible for an abundant neutrophil infiltration in Onchocerca-infected tissues. However, the role of neutrophils in the immune response to filarial parasites and their interaction with Wolbachia is poorly known. The work presented here showed that Wolbachia lipoprotein activates human neutrophils in vitro, supporting previous studies demonstrating the link between Wolbachia and neutrophils in the host inflammatory responses to O. volvulus infection. These results also indentify Wolbachia lipoprotein as a key molecule driving human neutrophil recruitment and activation.

Parasitic reproductive endosymbionts are emerging as formidable threats to insect biodiversity. Wolbachia are prevalent maternally inherited intra-cellular bacteria found in >50% of arthropod species. These symbiotic bacteria interact with their hosts in diverse ways, most often they alter host reproduction causing four conditions that all selectively favor infected females: feminization, male killing, parthenogenesis, and cytoplasmic incompatibility (CI). Furthermore, depending on strain-type and host genetic background, Wolbachia are known to affect insect behavior, expand or shift host thermal tolerance ranges, and confer anti-viral protection to their hosts. Because Wolbachia both reside in and are transmitted with host cell cytoplasm, mitochondria and other cytoplasmically inherited genetic elements become linked with the bacteria. Thus, by enhancing their own transmission, Wolbachia-induced phenotypes can lead to mitochondrial selective sweeps, which may have profound impacts on vulnerable and small insect populations. Elucidating the extent to which endosymbionts influence biological and ecological functions is pivotal to making management decisions regarding imperiled insect species. My dissertation investigates biological and ecological impacts of host-endosymbiont interactions by examining Wolbachia infections in three different host systems. First, I used the federally threatened butterfly species Speyeria zerene hippolyta to determine whether the general reproductive success of local populations was affected by the introduction of CI-inducing Wolbachia-infected butterflies through implemented species recovery programs. Next, by characterizing the Wolbachia infections of parasitoids associated with the Eurema butterfly clade, I analyzed whether host-parasitoid interactions provide a path for interspecies horizontal transmission. Finally, I conducted a laboratory experiment using an isogenic Drosophila melanogaster line to determine whether Wolbachia influence host temperature preference. Together, my research examines how the individual level effects of host-endosymbiont interactions can expand into populations, have broader impacts on insect communities, and potentially impede the conservation and management of insects in nature. In chapter one, I screened S. z. hippolyta samples from three extant populations for Wolbachia infection. To examine the impacts of Wolbachia on small populations, I analyzed and compared infected and uninfected S. z. hippolyta reproductive data and showed that, in a population composed of infected and uninfected S. z. hippolyta, uninfected butterflies had reduced reproductive success (GLMM z = -8.067, P < 0.0001). I then developed a single-population demographic theoretical model using these same reproductive data to simulate and analyze different potential dynamics of small populations resulting from population supplementation with uninfected, CI-Wolbachia infected, or combined uninfected and infected butterflies. Analysis of model simulations revealed that supplementation with CI-inducing butterflies significantly suppressed host-population size (ANOVA F5,593 = 3349, PWolbachia-infected individuals (Tukey's post-hoc test P < 0.0001). In addition, supplementation by multiple releases using a combination of 50 infected and 300 uninfected butterflies has a less severe suppression effect, reducing the population by 75.8%, but the reduction occurs 42.6% faster than with the single release of 50 Wolbachia-infected butterflies (Tukey's post-doc test P < 0.0001). Parasitoid-host interactions have emerged as probable ecological relationships to facilitate horizontal transmission of Wolbachia. In chapter two, I addressed horizontal transmission using Eurema butterflies and their associated parasitoids. From four locations in Northern Queensland, Australia, I collected a total of 404 Eurema hecabe butterfly larvae. Twenty-three parasitoids emerged from the larvae of which 21 were Diptera and two were Hymenoptera. I amplified COI loci fragments from each parasitoid for BLAST query searches and found that 20 individual Diptera parasitoids matched to the genus Exorista and one to the genus Senometopia. One of the Hymenoptera parasitoids matched to the genus Microoplitis and the other to the genus Cotesia. To characterize Wolbachia infections, I used Wolbachia Multi Locus Sequencing Technique (MLST) and discovered that all 20 Exorista parasitoids were infected with an identical Wolbachia strain (ST-41), which is the same strain infecting their Eurema hecabe butterfly hosts. Although, further experiments are necessary to definitively determine that ST-41 Wolbachia are incorporated into germline cells of the parasitoids, this is the first study to provide ecological evidence for inter-ordinal Wolbachia transmission between Lepidoptera and Diptera. Furthermore, this discovery exposes the risk of population augmentation programs that move insects, potentially facilitating the spread of Wolbachia between species within a community through the accidental introduction of new Wolbachia-infected parasitoids. Finally, both Wolbachia and their insect hosts are temperature sensitive organisms. Wolbachia's replication behavior in their hosts is positively-temperature dependent, while environmental variation can have profound effects on insect's immune function, fitness, and fecundity. In chapter three, I conducted a laboratory experiment using a thermal gradient choice assay and an isogenic Drosophila melanogaster line with four different Wolbachia infection statuses -- uninfected, wMel, wMelCS, and wMelPop - to assess whether a relationship existed between Wolbachia infection and host temperature preference. Results from my laboratory experiment revealed that Wolbachia-infected flies preferred cooler temperatures compared to uninfected flies. Moreover, D. melanogaster temperature preferences varied depending on the Wolbachia strain variant with which they were infected; flies infected with the wMel strain had temperature preferences 2°C cooler compared to uninfected flies; flies infected with either wMelCS or wMelPop strains had preferred temperatures 8°C cooler compared to uninfected flies. Wolbachia-associated temperature preference variation within a species can lead to conspecifics occupying different microclimates, genetically adapting to different sets of specific environmental conditions, and may eventually result in ecological and reproductive isolation. While, reproduction isolation is recognized as one of the first stages in speciation, in small populations of endangered and threatened species, the inability to reproduce between conspecifics can drive species to extirpation or extinction. Collectively, the three chapters of my dissertation set precedent for future integration of host-endosymbiont research prior to implementing population supplementation or translocation programs for the conservation of imperiled insects.

Wolbachia é um gênero de bactérias endossimbiontes que são verticalmente transmitidas pelas fêmeas aos seus descendentes, embora transmissão entre táxons distintos também ocorra com certa freqüência. Entre os efeitos que essa bactéria causa nos sistemas de reprodutivos de seus hospedeiros destaca-se a incompatibilidade citoplasmática (IC). Wolbachia foi descrita em numerosas espécies de artrópodos, especialmente os insetos. Dentre os insetos, essa bactéria foi descrita em diversos gêneros das moscas-das-frutas da familia Tephritidae, tendo sido demonstrada a IC em Rhagoletis cerasi e Ceratitis capitata. Recentemente, essas bactérias foram identificadas nas moscas-das-frutas do gênero Anastrepha. No presente trabalho, procurou-se demonstrar se a Wolbachia estaria relacionada a IC entre Anastrepha sp.1 aff. fraterculus e Anastrepha obliqua. A amplificação e o seqüenciamento do gene wsp revelou que as populações das duas espécies, mantidas no laboratório, estavam infectadas por Wolbachia. A presença da bactéria foi, também, demonstrada citologicamente em embriões corados pelo DAPI, tendo-se, além disso, observado que elas se distribuem nos embriões em gradiente decrescente do pólo posterior para o anterior. Para testar a hipótese de que a bactéria é a causa da IC, foram estabelecidas populações das duas espécies onde a Wolbachia (e possivelmente outras bactérias não identificadas) foram eliminadas por tratamento térmico (30oC) aplicado às pupas. As taxas de eclosão de larvas nas progênies de cruzamentos entre indivíduos curados, foram menores que a eclosão nos cruzamentos entre indivíduos infectados, mas o tratamento não alterou a proporção sexual dos adultos. Cruzamentos intra-específicos entre indivíduos infectados e curados, produziu assimetria nas progênies dos cruzamentos recíprocos, ou seja, quando os fêmeas eram curadas e os machos infectados a taxa de eclosão foi significativamente menor que nos cruzamentos recíprocos demonstrando, assim, a existência de IC unidirecional nos cruzamentos dessas duas espécies de Anastrepha. Nesses cruzamentos, a proporção sexual das progênies adultas não divergiu significativamente do 1:1 esperado. Nas progênies híbridas de cruzamentos interespecíficos entre indivíduos infectados, observou-se um decréscimo significativo nas taxas de eclosão e alteração na proporção sexual entre os adultos, de acordo com a regra de Haldane. Quando as fêmeas eram de A. sp.1, as progênies eram constituídas apenas por fêmeas e quando eram de A. obliqua, machos também foram produzidos, mas em frequências menores que o esperado. Resultados similares foram obtidos quando indivíduos curados foram cruzados entre si. Cruzamentos interespecíficos de indivíduos infectados cruzados com indivíduos curados, produziram progênies com taxas de eclosão muito reduzidas e menores quando as fêmeas eram de A. sp.1 (infectadas ou curadas), do que nos cruzamentos onde as fêmeas eram de A. obliqua (infectadas ou não). Nestes últimos, quando a fêmea era infectada e os machos curados, a taxa de eclosão foi maior que no cruzamento recíproco, demonstrando a presença de IC entre as duas espécies. A proporção sexual também foi alterada nesses cruzamentos, segundo a regra de Haldane. Os resultados do presente trabalho demonstram, pela primeira vez, que a incompatibilidade citoplasmática mediada pela Wolbachia, ocorre nas duas espécies de Anastrepha e sugerem que deve existir uma interação entre a bactéria e os genomas das espécies hospedeiras.
The endosymbiont bacteria Wolbachia are vertically transmitted by the females to its progenies, although horizontal transmission between distinct taxa is also known. One of the most relevant effects caused by these bacteria in the reproductive behavior of its hosts is the cytoplasmic incompatibility (CI). Wolbachia was described in several groups of arthropods, in the insects including several genera of the tefritid fruit flies. Wolbachia-induced CI was reported in Rhagoletis cerasi and Ceratitis capitata. Recently, occurrence of these bacteria was described in several species of genus Anastrepha. Search for possible Wolbachia-induced CI in crosses of Anastrepha obliqua and A. sp.1 aff. fraterculus was the aim of the present study. Amplification and sequencing of the gene wsp of Wolbachia revealed that laboratory colonies of both species were infected with this bacteria. Cytological analysis of embryos stained with DAPI showed a multitude of bacteria distributed in decreasing gradients from the posterior to the anterior pole of the embryos. The hypothesis that there is CI between the two host species was tested using infected and cured individuals. The bacteria were removed by treating the pupae at the temperature of 30o C. Rate of egg hatch in progenies recovered from crosses of cured flies were lower than egg hatch in crosses of the infected strains, but no sex-ratio deviation from the expected 1:1 was observed. Intra-specific crosses between infected and cured individuals resulted in asymmetries in the progenies of reciprocal crosses. The egg hatch in crosses of cured obliqua females to infected sp.1 males was lower than in the reciprocal crosses, clearly indicating the presence of Wolbachia-induced CI in Anastrepha. In these crosses, sex-ratio among the adults does not deviate from the 1:1 ratio. In the hybrid progenies of inter-specific crosses, it was observed a decrease in the egg hatch rate, and deviation from 1:1 in the adult sex-ratio according to the Haldanes rule. From crosses of sp.1 females to obliqua males, only females were recovered while in crosses of obliqua females to sp.1 males, males were also produced but in lower frequencies than expected. Similar results were obtained when cured individuals were crossed. Very low egg hatch rates were observed in inter-specific crosses of infected to cured individuals, when sp.1 females (infected or cured) were used. In crosses of obliqua females (infected or cured), higher egg hatch rate was observed. Egg hatch was larger in crosses of infected female to cured males than in the reciprocal crosses, indicating a Wolbachia-induced IC between the two species of Anastrepha. The sex-ratios were also according to the Haldanes rule. This is the first report of Wolbachia-induced CI in Anastrepha. The existence of a possible interaction between the bacteria and the genomes of the host species is also suggested.

The aim of this thesis is to work towards establishing the molecular basis of the communication between the host nematode Brugia malayi and its host specific Wolbachia strain, wBma. The genome sequence of wBma was annotated and compared to the published annotated genome of the Wolbachia found in Drosophila melanogaster (wMel). Interesting genes discovered in the wBma genome include a family of seven ankyrin repeat containing (ANK) proteins. ANK proteins are known to be involved in many protein:protein interactions, more commonly in eukaryotes. A comparison of the wBma ANK proteins with the 23 ANK proteins present in the genome of wMel shows that two of the wMel ANK homologues in the wBma genome are non-functional pseudogenes. wBma has novel ANK proteins that may be involved in interaction with the host cellular machinery. RT-PCR of the wBma ANK genes shows that different ANK genes are expressed in different stages of the nematodes’ life cycles. Many interesting Wolbachia novel genes were identified in the genome of wBma, wMel and the Wolbachia strain from the filarial nematode that causes river blindness, Onchocerca volvulus (wOvo). Novel proteins predicted to be located to the bacterial surface or predicted to be secreted are good candidates for proteins that may be involved in communication between Wolbachia and its host. A real-time PCR technique was developed to calculate the number of wBma per B. malayi nucleus at different stages in the nematodes’ life cycle. gDNA samples prepared from different stages of the life cycle showed that the number of wBma per nematode nucleus was observed to increase ten-fold during development from microfilaria (MF) to larval fourth stage (L4). Post-L4, while adult male somatic nuclei had approximately six-fold that of L4, the adult female soma is estimated to have approximately 330-fold more. If the Wolbachia were multiplying only to ensure its passage to the next generation of MF, there would be little obvious adaptive advantage in multiplication within the soma. However, if the Wolbachia are supplying some essential metabolic need to the nematode, then their multiplication in the soma will reflect the importance of their role at different lifecycle stages. This data suggests that wBma are supplying a possible essential role, but that role is yet to be identified. Another way that Wolbachia and its host may be interacting is by gene transfer from the bacteria into the nuclear genome of the host organism. The transfer of genes from prokaryote to eukaryote is still considered an unusual event, but there is evidence that genes have been transferred from organelles of prokaryotic endosymbionts origin, such as chloroplasts and mitochondria to the cell’s nuclear genome. This thesis has identified two wOvo pseudogenes in an untranscribed region of an O. volvulus nuclear gene.

Les Wolbachia sont des α-Protéobactéries endocellulaires transmises maternellement et qui manipulent la reproduction des Arthropodes pour augmenter leur transmission. Chez le moustique Culex pipiens, Wolbachia induit l'incompatibilité cytoplasmique (IC) qui se traduit par une forte mortalité embryonnaire lors de croisements entre individus infectés par des souches incompatibles de Wolbachia. Ce moustique se caractérise par une forte diversité génétique de ses Wolbachia (nommées wPip) et par des patrons d'IC complexes. Nous avons examiné les mécanismes qui façonnent la dynamique de cette association symbiotique aux niveaux génomique, phénotypique et populationnel. Nous avons montré que les souches wPip ont une origine génétique commune récente et qu'elles s'organisent en groupes génétiques présentant une structuration géographique. Nous avons mis en évidence des évènements de recombinaison entre souches wPip qui pourraient jouer un rôle majeur dans la diversité génétique des Wolbachia et dans l'évolution rapide des patrons d'IC. En croisant des lignées de moustiques d'origines géographiques diverses et infectées par des souches de différents groupes génétiques, nous avons montré que les IC (i) évoluent très rapidement chez Cx. pipiens; (ii) sont contrôlées par plusieurs déterminants génétiques, et (iii) qu'il y a une relation entre les patrons d'IC et les groupes génétiques des Wolbachia. Dans les populations naturelles, il apparaît que les IC sont contre sélectionnées au sein d'une population mais qu'une zone de contact entre populations infectées par des souches incompatibles peut se maintenir de façon stable
Wolbachia are maternally inherited endocellular α-Proteobacteria that manipulate the reproduction of Arthropods to promote their own transmission. In the mosquito Culex pipiens, Wolbachia induce cytoplasmic incompatibility (CI) which results in high embryonic mortality in crosses between mosquitoes infected with incompatible Wolbachia strains. This mosquito is characterized by high genetic diversity of its Wolbachia (referred as wPip strains) and by complex CI patterns. We examined mechanisms that shape the dynamics of this symbiotic association at genomic, phenotypic and field population levels to understand how it evolves. We showed that wPip strains have a unique and recent evolutionary origin and that their diversity clusters into distinct genetic groups with a geographic structure. We revealed the existence of extensive recombinations among wPip strains, which could influence their adaptive dynamics by creating new wPip strains and thus allow the rapid emergence of new CI patterns. The analysis of crossing relationships between mosquito lines from different geographic origins and infected with wPip strains belonging to different genetic groups showed that CIs (i) evolve rapidly in Cx. pipiens; (ii) are controlled by several genetic factors, and (iii) there is a significant relationship between CI patterns and genetic divergence of wPip strains. In field populations, it appears that CIs are selected against within a population but a contact zone between populations infected by incompatible Wolbachia strains can be stably maintained

La symbiose est l'un des principaux moteurs de l'évolution. Le génotype du symbiote est capable d'altérer le phénotype de l'hôte, et vice-versa : c'est le « phénotype étendu ». Dans ce contexte, les endosymbioses à Wolbachia sont remarquables. Cette bactérie intracellulaire est un parasite de la reproduction capable d'induire la féminisation des mâles génétiques ou l'incompatibilité cytoplasmique chez ses hôtes crustacés isopodes terrestres. Actuellement, aucun mécanisme moléculaire régissant ces effets n'est connu. Dans le but d'identifier des gènes impliqués dans la féminisation, nous avons utilisé une approche intégrative qui combine à la fois des analyses génomiques, d'expression de gènes et phénotypiques. Nous avons tout d'abord analysé l'évolution moléculaire de la voie de la recombinaison homologue dans les génomes de Wolbachia, source importante de plasticité génomique pouvant être liée à la diversité des phénotypes. Ensuite, afin d'effectuer des études comparatives qui augmenteraient considérablement la compréhension des mécanismes de la féminisation, nous avons établi un système où la souche féminisante wVulC féminise deux hôtes isopodes (hôte naturel : Armadillidum vulgare : hôte hétérologue : Cylisticus convexus) présentant un timing différent de la différenciation sexuelle. En effet, l'effet féminisant étant supposé avoir lieu avant ou pendant la différenciation sexuelle, il est important de distinguer l'effet de Wolbachia dû à la différenciation sexuelle de celui dû au développement. Enfin, une approche par gènes candidats (du séquençage de génome bactérien à l'analyse comparative d'expression de gènes bactériens durant le développement de l'hôte) a permis de déterminer une liste réduite de 29 gènes (parmi les 1885 gènes de wVulC) dont la probabilité qu'ils soient impliqués dans la féminisation est élevée. Le rôle potentiel de ces gènes candidats comme effecteurs supposés de la féminisation induite par wVulC est ensuite discuté. Ce travail contribue grandement à l'identification de facteurs potentiels d'endosymbiotes qui ont un impact évolutif sur la détermination du sexe de leurs hôtes
Symbiotic interactions are a major driver of evolution. The symbiont genotype is able to alter the host phenotype, and the other way round: it is called "the extended phenotype". In this respect, Wolbachia endosymbiosis is remarkable. This intracellular bacterium is a well-known reproductive parasite able to induce feminization of genetic males or cytoplasmic incompatibility in its terrestrial isopod crustacean hosts. Currently, no molecular genetic basis of these reproductive manipulations has been described. In order to identify genes involved in feminization, we used an integrative approach that combines genomic, gene expression and phenotypic studies. We first analysed the molecular evolution of the homologous recombination pathway in Wolbachia genomes, an important source of genomic plasticity that can be linked with phenotypic diversity. Then, in order to perform comparative studies that will substantially improve the understanding of the molecular mechanisms of feminization, we established a system in which the feminizing strain wVulC feminizes two different isopod hosts (natural host: Armadillidium vulgare ; heterologous host Cylisticus convexus) that have a different sexual differentiation timing. Indeed, as feminization is thought to happen before or during sexual differentiation, it is important to distinguish the effect of Wolbachia due to sexual differentiation from that due to development. Finally, a gene candidate approach (from bacterial genome sequencing to comparative bacterial gene expression during host developement) allowed us to determine a reduced list of 29 genes (among the 1885 genes of wVulC) that have a high probability to be involved in feminization. The potential roles of these candidate genes as putative effectors of feminization induced by wVulC is then discussed. This work substantially contributes to the identification of putative endosymbiont factors that have an evolutionary impact on sex determination of their hosts

La présence de Wolbachia dans les ovogonies assure la transmission verticale de la bactérie à la descendance de l'hôte. Cependant, nous montrons que chez l'hôte Armadillidium vulgare, l'efficacité de l'infection des descendants tient à un enrichissement en Wolbachia au cours de la maturation des ovaires et des ovocytes dû à une sélection en faveur des ovocytes infectés et/ou à l'entrée secondaire de Wolbachia dans les ovocytes en cours de maturation via l'infection des tissus somatiques. Dans ces tissus, nous avons précisé la localisation de Wolbachia au niveau cellulaire et révélé des morphotypes typiques de chaque tissu. Nous avons également observé Wolbachia chez des hôtes très inattendus; des nématodes non filaires infectant les cloportes, posant la question d'une transmission horizontale, et les A. vulgare mâles, sans qu'ils soient féminisés. Etonnamment, nous avons observé l'infection des gonades mâles dans des lignées d'hôtes chez lesquelles les femelles sont infectées de manière cryptique mais sans que leurs ovocytes ne soient infectés. Le maintien de l'infection entre les générations d'hôtes pourrait alors être dû à une transmission paternelle, inédite pour Wolbachia, ou à une capacité de transmission horizontale très efficace de la bactérie. Par immersion de tissus directement dans des broyats d'organes infectés nous avons en effet démontré que Wolbachia infecte très rapidement des cellules de novo. Les mécanismes d'entrée de Wolbachia dans les cellules sont inconnus mais en monitorant des voies métaboliques clefs de l'hôte nos résultats montrent que l'infection entraine une réponse globale des tissus et implique notamment un détournement de la voie autophagique chez l'hôte
Wolbachia presence in oogonia ensures bacteria to be vertically transmitted to host offspring. However, in Armadillidium vulgare, we show that the proportion of infected oocytes increases in the course of both ovary and oocyte maturation to reach the transmission rate at the end of ovary maturation. This enrichment can be explained by a preferential selection of oocytes infected with Wolbachia and/or by a secondary acquisition of the bacteria by oocytes. We suspect an acquisition through infected somatic tissues. We localize Wolbachia at the cell level in these tissues and showed particular morphotypes for each tissue. We also observe Wolbachia in unexpected hosts; non filarial nematodes infecting woodlice (suggesting horizontal transmission), and in A. vulgare males (without a feminizing effect of the bacteria). We also observe lineages in which females are cryptically infected. Surprisingly, we observe infected male gonads in these lineages for which female oocytes are uninfected. The infection maintenance across host generations could be due to a paternal transmission of the bacteria (a transmission never described for Wolbachia), or due to an astonishing ability of horizontal transmission. Nevertheless, immersion of uninfected tissues in a solution of crushed infected tissues proves that Wolbachia can quickly infect new tissues. Cellular mechanisms that allow Wolbachia internalization into the cell are still unknown. Thus, we monitor key host metabolic pathways in ovaries and we denote that infection enhances a global response of the entire tissue. Additionally, Wolbachia infection especially implicates a high-jacking of the autophagic pathway

Cette étude vise à comparer l’histoire évolutive des parasitoïdes du genre Horismenus (Hymenoptera: Eulophidae) à celle de leurs hôtes bruches (Coleoptera: Bruchidae) et plante hôte (Phaseolus vulgaris L.) cultivée dans le contexte d’agriculture traditionnelle, au sein de son centre de domestication Mésoaméricain. Nous avons analysé la structure génétique de 23 populations de quatre espèces de parasitoïdes au Mexique, en utilisant un fragment du gène mitochondrial COI afin de les comparer aux structures précédemment publiées des hôtes bruches et du haricot commun. Nous avons prédit que les structures génétiques des populations d’hôtes (bruches et plante) et de parasitoïdes seraient similaires puisque également influencées par la migration entremise par l’humain (HMM) étant donnée que les parasitoïdes se développent telles que les bruches à l’intérieur des haricots. Compte tenu des stratégies de manipulation reproductive utilisées par l’alpha-protéobactérie endosymbionte Wolbachia spp. pour assurer sa transmission, la structure génétique des populations de parasitoïdes inférée à partir du génome mitochondrial devrait être altérée conséquemment à la transmission conjointe des mitochondries et des bactéries lors de la propagation de l’infection dans les populations de parasitoïdes. Les populations du parasitoïde H. missouriensis sont infectées par Wolbachia spp. Tel que prédit, ces populations ne sont pas différenciées (FST = 0,06), ce qui nous empêche d’inférer sur une histoire évolutive parallèle. Contrairement aux bruches, Acanthoscelides obtectus et A. ovelatus, la HMM n'est pas un processus contemporain qui influence la structure génétique des populations du parasitoïde H. depressus, étant donné la forte différenciation (FST = 0,34) qui existe entre ses populations. La structure génétique observée chez H. depressus est similaire à celle de sa plante hôte (i.e. dispersion aléatoire historique à partir d'un pool génique ancestral très diversifié) et est probablement le résultat d’un flux génique important en provenance des populations de parasitoïdes associées aux haricots spontanées à proximité des champs cultivés. L’étude de l’histoire évolutive intégrant plusieurs niveaux trophiques s’est avérée fructueuse dans la détection des différentes réponses évolutives entre les membres du module trophique face aux interactions humaines et parasitaires, et montre la pertinence d’analyser les systèmes écologiques dans leur ensemble.
This study aims to compare the evolutionary history of Horismenus parasitoids (Hymenoptera: Eulophidae) to that of their bruchid beetle hosts (Coleoptera: Bruchidae) and their domesticated host plant (Phaseolus vulgaris L.) in the context of traditional agriculture within their Mesoamerican center of domestication. We analyzed the genetic structure of 23 populations of four Horismenus species in Mexico using COI mitochondrial gene fragments and compared the structures to previously published data on bean plant and beetle hosts. We predicted that because parasitoids complete their development within their beetle hosts, within the bean, the genetic structure of both the host and the parasitoid would be similar and equally influenced by human-mediated migration (HMM). Furthermore, because of reproductive manipulation strategies often used by the alpha-proteobacteria endosymbionte Wolbachia spp. to ensure its transmission, the genetic structure of parasitoid populations inferred from mitochondrial genome would be bias consequently to the conjoint transmission of mitochondria and the bacteria according to propagation of the infection within parasitoids populations. The populations of H. missouriensis parasitoids are infected by Wolbachia spp. As predicted, these populations are not differentiated (FST = 0.06) which prevents us to infer on a parallel evolutionary history. Unlike their bruchids hosts, Acanthoscelides obtectus and A. ovelatus, the HMM is not a contemporary process influencing H. depressus population genetic structure according to the strong populations differentiation (FST = 0.34). The genetic structure observed within H. depressus populations is similar to that of its host plant (i.e. historical random dispersal from a highly diversified ancestral gene pool) and is probably the result of extensive gene flow from parasitoids associated with wild beans populations adjacent to crop fields. The study of evolutionary history integrating multitrophic levels has proved to be fruitful in detecting different evolutionary responses among members of the trophic module face to human and parasite interactions, but also points out the pertinence of analyzing ecological systems as a whole.

Many insect species are infected with endosymbiotic bacteria that live in the cytoplasm of their host cells. Most of these bacteria are maternally inherited and can manipulate reproduction of their insect hosts by inducing cytoplasmic incompatibility (CI), thelytokous parthenogenesis, male killing and feminisation, and some of these manipulations distort the sex ratio of their hosts. This PhD research studied Wolbachia infections in Eurema butterflies (Lepidoptera: Pieridae), the effects of Wolbachia on their sex determination system, and the effects on gene flow and genetic diversity across different Eurema species in Australia. The genus Eurema was chosen because Wolbachia had previously been identified in three species of this genus. In two species, E. hecabe and E. mandarina, Wolbachia induced CI and feminisation. CI is caused by the wCI strain and feminisation by the wFem strain. Both species are two out of only three known examples of Wolbachia caused feminisation in insects. Prior to this study details about the feminisation mechanisms were unknown except that it was expected that Wolbachia changes homogametic (ZZ) males into functional females that do not possess the female sex chromosome (W) found in uninfected heterogametic ZW females. It was also unknown whether Australian E. hecabe and other Australian Eurema species are infected with the feminising or any other Wolbachia strains (with the exception of previous preliminary and unpublished work on E. hecabe). Australia is home to six Eurema species of the two subgenera Terias and Eurema. In response to the Australian climate with seasonal and often unpredictable rainfall, these Australian Eurema species evolved different adaptation strategies such as reproductive diapause and migration behaviour.

碩士
高雄醫學大學
熱帶醫學碩士學位學程
106
Background. Wolbachia belongs to the order Rickettsiales and is considered a common intracellular bacteria within a variety of arthropods. As an endosymbiont, Wolbachia is not known to directly infect vertebrates and possesses an interesting ability to alter its host biology in diverse ways, such as suppression of vector population and interference of pathogen transmission. Previous studies have reported that Wolbachia plays a role in cytoplasmic incompatibility and is able to reduce dengue and Zika virus transmission in some mosquitoes. Hence, this symbiotic bacteria’s ability to manipulate its host may have potential applications in the vector control of tick-borne pathogens. Aim. To determine the presence, prevalence, and phylogenetic analysis of symbiotic Wolbachia in Rhipicephalus sanguineus ticks collected from Kaohsiung, Taiwan Methods. Ticks were collected from dogs throughout six districts of Kaohsiung, Taiwan. DNA samples were extracted from homogenized ticks using Qiagen DNeasy kit. Nested PCR assays were performed to detect wsp (Wolbachia surface protein) gene of Wolbachia supergroups A and B. Phylogenetic relationships were analyzed by Maximum likelihood method. Results. In general, Wolbachia DNA was detected in 46% (463/1004) of the Rhipicephalus sanguineus ticks collected from dogs. The prevalence of infection was detected in males, females, and nymphs with an infection rate of 44%, 40% and 56%, respectively. Phylogenetic analysis also verifies these ticks as being either singly or superinfected with groups A and B of Wolbachia. Conclusions. This study provides the first molecular evidence of Wolbachia infection in Rhipicephalus sanguineus ticks in Taiwan. Additionally, it is the first study to reveal the detection of Wolbachia in male Rhipicephalus sanguineus ticks and indicates the possible mechanism of vertical transmission within ticks. Based on the nested PCR assays, Rhipicephalus sanguineus ticks were found either singly or superinfected with groups A and B of Wolbachia.

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

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.

碩士
高雄醫學大學
醫學研究所碩士班
105
Background: The potential application of the endosymbiotic bacteria, Wolbachia, to the control of mosquito-borne infections has emerged as a novel biological control strategy against mosquitoes. Although Culex mosquitoes play the role as the primary vector for lymphatic filariasis and Japanese encephalitis in Indonesia, little is known about the composition and structure of their microbiota, specifically Wolbachia. Objectives: The objectives of this study are to determine the genetic identity of Culex mosquitoes collected from Sumatera Utara, Indonesia, and to identify the presence, frequency and phylogenetic analysis of its symbiotic Wolbachia. Methods: Field surveys were conducted to screen the infection status of Wolbachia in field-collected Culex mosquitoes from Sumatera Utara, Indonesia. We performed the PCR assays to identify the mosquito species based on the mitochondrial COI and ND5 genes, and to detect Wolbachia wsp (wolbachia surface protein) gene in Culex mosquitoes. The phylogenetic relationships were analyzed by neighbor-joining method. Results: Wolbachia DNA were detected in 33.8% (106/314) of wild-caught mosquitoes. The rate of infection in adult females were detected with 52.4% in urban area and 36.4% in rural area. While in males, the infections were detected with 9.4% in urban area and 20% in rural area. The Blast search and phylogenetic analysis of the COI and ND5 genes identify the genetic identity of Culex quinquefasciatus from Indonesia. The wsp gene sequence analysis revealed that the Wolbachia strain from Indonesian Cx. quinquefasciatus was identical to the Wolbachia strains of supergroup B previously reported in members of the Cx. pipiens complex and newly identified Wolbachia strains of supergroup A. Conclusions: This study provides the first molecular evidence of Wolbachia infection in Cx. quinquefasciatus populations of Indonesia. Based on wsp sequences, this study found Cx. quinquefasciatus were singly or superinfected with A and B Wolbachia strain. The phylogenetic analysis revealed a new discovery of Wolbachia group A in Cx. quinquefasciatus.

Tese de mestrado, Biologia Evolutiva e do Desenvolvimento, Universidade de Lisboa, Faculdade de Ciências, 2015
The complexity of life forms cannot be explained without the concept of symbiosis. Symbiotic relations exist abundantly in nature, particularly the ones between bacteria and eukaryotic hosts. One of the most widespread endosymbiotic bacteria described belongs to the genus Wolbachia, naturally infecting arthropods and nematodes. In arthropods, Wolbachia induces reproductive manipulations, to promote infected female’s fitness as they are vertically transmitted, maternally. Additionally, in Drosophila melanogaster, they have been shown to confer protection against viral infections. Being an obligatory vertically-transmitted endosymbiont, Wolbachia phylogeny should recapitulate that of its hosts. However, several studies show that host and symbiont phylogenies are not concordant, suggesting that horizontal transfers must have occurred throughout time. This has been confirmed, namely between parasitoid wasps and their hosts. Considering these observations, the present project aimed at uncovering if and at which rate horizontal transmission of Wolbachia could occur between Drosophila melanogaster and its natural parasitoid Leptopilina boulardi. Also, we tested if the viral protection phenotype induced in Drosophila would be passed on to the novel host, after this horizontal transfer event. Simultaneously we screened a wild-caught population of Leptopilina heterotoma for Wolbachia presence. We obtained infected individuals with which we established an isofemale line and derived Wolbachia-negative counterparts using antibiotic treatment. With this tool, we characterized the effects of Wolbachia presence upon viral systemic infection. Our results show that horizontal transmission of Wolbachia happens between Leptopilina boulardi and its host, but the infection is not stably maintained. We also see that these wasps do not appear to be susceptible to viruses that are pathogenic in Drosophila (specifically DCV and FHV) and appear to be mildly Detrimental to Wolbachia-infected Leptopilina heterotoma. Finally, we have verified that a natural endosymbiont infection appears to delay full development time in Leptopilina heterotoma, although no effect is detected for longevity. With this work we have established an experimental system for the controlled and systematic study of the complex interactions between Drosophila, parasitoid wasps, Wolbachia and viruses.
A diversidade de formas devida dificilmente é explicada sem ser tido em conta o conceito de simbiose. Relações simbióticas são abundantemente encontradas em ambientes naturais. Os primeiros eventos de endossimbiose conhecidos terão ocorrido há cerca de 1,5 milhares de milhão de anos atrás. Estes acontecimentos descrevem a incorporação de cianobactérias e proteobactérias ancestrais em organismos procariotas. Quando a simbiose se tornou mutualista obrigatória, estes organismos procariotas associados a endossimbiontes tornaram-se nos primeiros eucariotas do planeta, e os seus anteriormente designados endossimbiontes tornaram‐se organelos, nomeadamente cloroplastos e mitocôndrias. Atualmente existem incontáveis exemplos de organismos vivos que não existiriam se não se estabelecessem relações simbióticas entre diferentes entidades biológicas, constituindo a base para a enorme diversidade de formas de vida existentes. Alguns dos casos mais frequentes de interações entre diferentes organismos que moldam inquestionável e determinantemente a história evolutiva do planeta são os que ocorrem entre procariotas e hospedeiros eucariotas. Estes sistemas podem ser encontrados em todas As circunstâncias, quer em metazoários como plantas e animais, quer em organismos unicelulares. Por exemplo, o filo Porífera realiza incontáveis associações com microrganismos bacterianos que lhe permitem obter nutrientes mais eficazmente ou ainda os vários casos de plantas que formam associações com bactérias fixadoras de azoto. Numa tentativa de classificar os diferentes tipos de interações que podem ocorrer entre dois ou mais organismos, foram atribuídos nomes a categorias discretas que ocorrem no espectro de interações naturais possíveis. Relações simbióticas podem ser comensais, mutualistas ou parasíticas. Comensalismo descreve o tipo de interações em que um organismo beneficia da relação que desenvolve com outro, enquanto o segundo permanece indiferente. Relações mutualistas englobam situações em que a associação é mutualmente benéfica para ambos os envolvidos. Parasitismo implica que um dos envolvidos seja prejudicado, para benefício do outro. Estas duas últimas formas de interação estão intimamente relacionadas, na medida em que as medidas de adaptação que um endossimbionte tem de sofrer para poder invadir e colonizar um hospedeiro, qualquer que seja a interações que acabe por vingar, são semelhantes para uma relação mutualista ou parasita, especialmente se considerarmos as relações abundantes que se estabelecem entre bactérias e hospedeiros eucariotas. No entanto, e apesar das referidas classificações existirem e serem relevantes, é difícil atribuir qualidades discretas a endossimbiontes, uma vez que os efeitos que induzem no seu hospedeiro podem variar entre mutualistas ou patogénicos, consoante vários aspectos (como factores ambientais). Um exemplo privilegiado deste tipo de comportamento variável é o das bactérias do género Wolbachia. Estas são alfa-proteobactérias, membros do grupo Rickettsiales (que engloba todos as bactérias endossimbióticas obrigatórias conhecidas), que infectam artrópodes e algumas espécies de nematodes filariais. Este endossimbionte é um dos mais representados na natureza, infectando mais de 60% de todos os insectos conhecidos, para além de presente em ácaros, aranhas, escorpiões e isópodes. Sendo um dos organismos mais bem estudados atualmente no que diz respeito a relações de simbiose, existem vários estudos que descrevem algumas das adaptações que sofreu de modo a proliferar nas células dos hospedeiros. Nomeadamente, a bactéria utiliza mecanismos de transporte vesicular para viajar dentro das células do hospedeiro. No entanto, a característica deste género que é indubitavelmente mais estudada é a sua capacidade de manipular o sistema reprodutivo do hospedeiro em seu proveito. Existem diferentes formas segundo as quais Wolbachia consegue manipular a reprodução dos seus hospedeiros, nomeadamente: feminização, morte de machos, partenogénese e incompatibilidade citoplasmática (IC). Feminização descreve a transformação fenotípica em fêmeas de organismos geneticamente masculinos; morte de machos acontece quando machos infectados são inviabilizados, disponibilizando mais recursos para as irmãs que possam transmitir a infecção à geração seguinte; partenogénese descreve a produção de prole unicamente feminina contribuição parental masculina; por fim, a manipulação reprodutiva mais comum, a IC descreve o processo segundo o qual fêmeas infetadas geram menos prole viável quando fertilizadas por machos não infetados (ou se ambos hospedarem estirpes incompatíveis). Todos estes processos de manipulação reprodutiva têm como objectivo maximizar a dispersão e colonização de Wolbachia pelo maior número de indivíduos possível, o que por sua vez é conseguido através do favorecimento da descendência feminina. O principal motivo que explica estes processos prende‐se com a forma canónica de transmissão de Wolbachia entre hospedeiros, que é feita verticalmente por via materna. Uma outra influência que Wolbachia exerce sobre os seus hospedeiros prende‐se com a capacidade de proteger Drosophila melanogaster contra (algumas) infeções por vírus de RNA. Esta capacidade foi descrita recentemente (não só para Drosophila mas também para o mosquito Culex pipiens) e desde então múltiplos trabalhos têm sido desenvolvidos na tentativa de caracterizar e determinar os mecanismos subjacentes. É sabido que Wolbachia protege contra Drosophila C Virus (DCV) e Flock House Virus (FHV), entre outros vírus de RNA, mas que a proteção não se estende para vírus de DNA (que não se conhece infectarem naturalmente espécies de Drosophila) e que, inclusive, diferentes níveis de proteção estão associados a diferentes estirpes da bactéria. Outra peculiaridade deste género de endossimbiontes prende-se com a filogenia discordante que apresentam relativamente à dos seus hospedeiros. Tendo em conta que são verticalmente transmitidas, seria de esperar que a árvore filogenética das estirpes de Wolbachia espelhasse, com alguma exatidão, a árvore filogenética dos seus respetivos hospedeiros. Isto não se verifica sugerindo que, além da transmissão vertical, eventos de transmissão horizontal entre hospedeiros têm de ter ocorrido ao longo do tempo. Adicionalmente, estudos baseados nestas filogenias de Wolbachia permitem inferir que existem enormes semelhanças entre as estirpes albergadas por certos insectos filogeneticamente distantes, nomeadamente entre vespas parasitoides e os seus respectivos hospedeiros. Tendo isto em conta, foram conduzidos estudos que determinaram que a bactéria pode ser transmitida horizontalmente entre diferentes hospedeiros por canibalismo de animais infectados, por partilha próxima de nichos ecológicos e através de um vector como uma vespa parasitóide. Reunindo toda a informação apresentada acima, das características da Wolbachia, especificamente da sua capacidade de induzir proteção viral em Drosophila, da sua filogenia discordante (indicativa de eventos de transmissão horizontal) e da existência de casos reportados em que vespas parasitoides atuam como vector transportador de endossimbiontes, formulámos as questões que apresentamos de seguida. 1- Consegue a Wolbachia conferir proteção viral num hospedeiro parasitoide recem-adquirido, fruto de um evento de transmissão horizontal? 2- Está uma população natural de vespas parasitoides protegida contra infecções virais, pela sua estirpe nativa de Wolbachia? 2.1- Como responde esta população natural, em comparação com as vespas mantidas no laboratório? 2.2- Existem custos associados à manutenção de Wolbachia? Começámos por tentar responder à primeira questão estabelecendo linhas isogénicas da vespa Leptopilina boulardi não infectadas por Wolbachia e sujeitando-as a hospedeiros de Drosophila melanogaster contendo Wolbachia. Utilizando duas linhas de moscas infectadas com estirpes individuais diferentes, sujeitámos as vespas a estes hospedeiros infectados e recolhemos toda a descendência após este evento de parasitação. Com estas fêmeas estabelecemos linhas isogénicas, das quais recolhemos, na geração seguinte, indivíduos suficientes que nos permitisse extrair DNA e testar a presença de Wolbachia por PCR. Com estes dados pudemos inferir a taxa de transmissão horizontal entre Leptopilina boulardi e Drosophila melanogaster. Numa tentativa de avaliar se a proteção viral conferida em mosca era transmitida com o endossimbionte para o novo hospedeiro estabelecemos um protocolo de infecção viral sistémica em vespa. No entanto, verificámos que a espécie Leptopilina boulardi não parece ser susceptível aos vírus de RNA que são canonicamente testados em Drosophila. Atestámos também que, nesta espécie, uma transmissão horizontal de Wolbachia não origina uma infecção estável, que seja em última análise, verticalmente mantida. Para responder à questão 2, recolhemos indivíduos da natureza e testámos a presença de Wolbachia assim como determinámos a espécie em questão. Deparamo-nos com uma linha de Leptopilina heterotoma, infectada com Wolbachia. Para estabelecermos um controlo negativo, usámos um tratamento de antibiótico que tratou a infecção, proporcionando duas linhas semelhantes de vespa, uma com e outra sem Wolbachia. Posteriormente, infecção viral foi realizada para esta espécie, onde vimos um efeito pequeno da ação de DCV, apenas detectável na linha que continha a infecção bacteriana de origem mas não na linha tratada. Para determinar custos, realizámos ainda medições quanto ao tempo total de desenvolvimento e longevidade de ambas as linhas de Leptopilina heterotoma, onde detectámos um possível efeito da Wolbachia a atrasar ligeiramente o desenvolvimento de ovo até adulto. Sumariamente, os nossos resultados indicam que a transmissão horizontal de Wolbachia ocorre, embora o estabelecimento de uma infeção vertical estável seja seja, neste caso, indetectável. Concluímos também acerca do efeito que uma infecção viral pode ter (ou não) sobre uma espécie com a qual provavelmente partilha nichos ecológicos na natureza (considerando que tanto Leptopilina boulardi como Leptopilina heterótoma são parasitoides naturais de Drosophila melanogaster, o hospedeiro por excelência de DCV). Podemos ainda verificar que a presença deste endossimbionte pode induzir custos num hospedeiro nativo, como de resto é verificado noutras espécies.

Wolbachia is a maternally inherited, endosymbiotic bacterium that infects at least 40% of terrestrial arthropods. As a facultative symbiont in the majority of its hosts, Wolbachia commonly act as a reproductive parasite; however, there are a number of Wolbachia strains that do not cause reproductive manipulations in their hosts and have no apparent fitness enhancement, yet are stably maintained in populations at low to intermediate frequencies. How these strains of Wolbachia persist in nature has been a long-standing question and is still unresolved. One explanation for the persistence of such strains is that they provide a context-dependent fitness advantage to their hosts. In this thesis, I investigate one such strain of Wolbachia, wSuz, which infects the agricultural pest, Drosophila suzukii, also known as spotted wing Drosophila. To explore the possibility that wSuz may be involved in pathogen protection, I screened wild flies for Wolbachia and two naturally occurring RNA viruses, Teise Virus and a recently discovered virus related to Motts Mill Virus. I did not find an association between Wolbachia and virus infection. Additionally, I designed an experiment to test whether Wolbachia increases host fitness at high larval densities. Intriguingly, although there was no effect of density, the frequency of Wolbachia infection changed dramatically in just one generation, but in opposite directions in replicate experiments that were performed a month apart. These results support the hypothesis that Wolbachia frequencies can change quickly across generations and provide some type of condition-dependent benefit. The maintenance of Wolbachia remains a mystery, but my study provides some exciting clues about what conditions may be playing a role.
Graduate
2022-05-10

Conifer seed-infesting chalcids of the genus Megastigmus (Hymenoptera: Torymidae) are important forest pests. At least one species, M. spermotrophus Wachtl, has been shown to be able to manipulate the seed development of its host, Douglas-fir (Pseudotsuga menziesii) in remarkable ways, such as redirecting unfertilized ovules that would normally abort. The mechanism of host manipulation is currently unknown. Microbial associates and venoms are two potential mechanisms of host manipulation. Microbial associates are emerging as an important player in insect-plant interactions. There is also evidence that venoms may be important in gall-induction by phytophagous wasps. PCR and 16S rRNA pyrosequencing was used to characterize the microbial associates of Megastigmus and transcriptomic sequencing was used to identify putative venoms that were highly expressed in female M. spermotrophus. The common inherited bacterial symbionts Wolbachia and Rickettsia were found to be prevalent among several populations of Megastigmus spp. screened using a targeted PCR approach. A member of the Betaproteobacteria, Ralstonia, was identified as the dominant microbial associate of M. spermotrophus using 16S rRNA pyrosequencing. The transcriptome of M. spermotrophus was assembled de novo and three putative venoms were identified as highly expressed in females. One of these putative venoms, Aspartylglucosaminidase, (AGA) appears to have originated through gene duplication within the Hymenoptera and has been identified as a major venom component of two divergent parasitoid wasps. AGA was identified as a promising candidate for further investigation as a potential mechanism of early host manipulation by M. spermotrophus.
Graduate
0353
0410
0715
apaulson@shaw.ca