Certains symbiotes intracellulaires résident dans le cytoplasme des cellules et manipulent le système reproductif de leurs hôtes. Du fait de leur transmission maternelle, ces parasites sont sélectionnés pour optimiser la survie et la reproduction de leurs hôtes femelles. Chez les arthropodes, la bactérie Wolbachia infecte au moins 66% des espèces d’insectes mais peuvent aussi infecter des nématodes. Cette large distribution dans les populations hôtes confère à Wolbachia un potentiel important en tant que moteur d’évolution. En particulier, elle pourrait être utilisée comme vecteur transgène dans les espèces nuisibles. Mais la dynamique évolutive des infections à l’échelle des communautés est mal connue, en particulier la fréquence des transferts de parasites entre hôtes de différentes espèces et la stabilité évolutive des associations. Mon travail de thèse a porté sur la détection et dynamique des infections de Wolbachia à une échelle micro-évolutive, c’est-à-dire, dans des communautés d’arthropodes avec moins de 5 My. L’objectif de ce travail était à la fois la caractérisation des communautés géographiques d’arthropodes et celle des infections par Wolbachia de ces communautés. Nous avons également examiné l’existence de transferts horizontaux récents de ces symbiotes entre des taxa distantes ainsi que les routes écologiques potentielles pour ces transmissions / Sexual parasites are intracellular symbionts capable of manipulating the reproduction of their hosts. They are widespread in Arthropods where they display a wide range of reproductive manipulations; these can be potentially involved in the evolution of mating systems, speciation, gene acquisition and sex determination. In particular, Wolbachia is thought to infect more than 66% of insect species and is also found in nematodes. However, little is known about the dynamics of Wolbachia infections at the community level. Although at the intra-population level, invasion dynamics have been extensively studied, the same is not true at the community level where the turnover of infections remains largely uncharacterised. The question of how often are new infections acquired through horizontal transfers between distantly related hosts remains also open. Moreover, as Wolbachia is seen as a good candidate for a transgenic vector against pests, understanding its dynamic at the community level is crucial. We proposed to address them by performing an exhaustive characterisation of sexual parasites in simplified systems, using the opportunity offered by small arthropod communities in isolated islands

Originaire d'Asie du Sud et de l'Est, le moustique tigre Aedes albopictus est aujourd'hui implanté sur 5 des 6 continents et les moyens de lutte mis en place pour l'éliminer peinent à freiner son expansion. Ces dernières années, l'étude des communautés microbiennes associées aux insectes a permis de démontrer leur implication dans des fonctions clefs de la biologie de leurs hôtes (nutrition, immunité, résistance aux stress biotiques et abiotiques …). Ensemble, ils constituent un super-organisme appelé holobionte. Ainsi, une meilleure connaissance de l'écologie microbienne d'Ae. albopictus pourrait nous apporter de nouvelles perspectives dans la compréhension du fonctionnement du pathosystème vectoriel. C'est dans ce contexte que s'est inscrit mon projet de thèse qui a consisté à décrire le microbiote bactérien du moustique tigre en lien avec son écologie et la génétique de ses populations. Nos travaux se sont tout d'abord portés sur des exemples précis d'interactions avec des symbiotes d'intérêts puis nous avons élargi cette étude à l'ensemble des communautés bactériennes et leurs facteurs de variation, en bénéficiant du développement des nouvelles technologies de séquençage. Les résultats obtenus ouvrent la voie vers de nouvelles hypothèses sur le fonctionnement et la dynamique de l'holobionte moustique avec la prise en compte des interactions symbiotiques comme un élément majeur du pathosystème vectoriel / Originated from South East Asia, the Asian tiger mosquito Aedes albopictus is now established on 5 of the 6 continents. Control strategies to limit its introduction and expansion remain restricted. Those last years, studies on insect microbial communities highlighted the key role of symbionts in the biology of their hosts (nutrition, immunity, resistance to biotic and abiotic stresses…). Together, they constitute a super-organism called the holobiont. Therefore, a better knowledge of microbial ecology of Ae. Albopictus should increase the understanding of vectorial pathosystem. In this context, my thesis project consisted to improve the description of bacterial microbiota associated with the Asian tiger mosquito in relation with its ecology and population genetics. We first based our attention on specific models of symbiotic interactions and then we extended our study to the whole bacterial community and its variation factors using high throughput sequencing technologies. Our results open the way to new hypotheses about the function and dynamics of mosquito holobionte taking into account the symbiotic interactions as a major component of the vectorial pathosystem

A new strategy to fight mosquito-borne disease is based on infections of the maternally-transmitted, intracellular bacterium Wolbachia pipientis. Estimates predict that Wolbachia infects nearly half of all insect species, as well as other arthropods and some nematodes. Wolbachia manipulates the reproduction of its host to promote infection, most commonly causing a form of conditional sterility known as cytoplasmic incompatibility. Generally, Wolbachia infections are benign and do not inflict significant costs upon its host. However, studies demonstrate that some infections are associated with substantial costs to its host. These same infections can also induce pathogen interference and decrease vector competency of important disease vectors. Theory predicts that organisms that incur costs relative to conspecifics are less competitive and their competitive exclusion is expected. In the case of Wolbachia, the bacterium can influence reproduction such that phenotypes with lower fitness may still reach fixation in natural populations. In this dissertation, I describe theoretical and empirical experiments that aim to understand the invasion and stability of Wolbachia infections that impose costs on their host. Particular attention is paid to immature insect lifestages, which have been previously marginalized. These results are discussed in relation to ongoing vector control strategies that would use Wolbachia to manipulate vector populations. Specifically, I discuss the cost of novel Wolbachia infections in Aedespolynesiensis, which decreases larval survival and overall fitness relative to wild-type mosquitoes. Then, a theoretical framework was developed to determine the significance of reductions in larval viability in relation to the population replacement disease control strategy. Further theoretical studies determined that Wolbachia infections, once established, resist re-invasion by uninfected individuals despite relatively high costs associated with infection so long as the infection produces reproductive manipulations. Additional studies determined that larvae hatched from old eggs experience reduced survival in mosquito strains with novel Wolbachia infections when compared to the wild-type. To validate the theoretical studies, model predictions were tested empirically to determine the importance of the larval viability. Finally, a COPAS PLUS machine was evaluated and its role in understanding early larval development in mosquitoes is discussed. The importance of integrated research in disease control is highlighted.

The mosquito Aedes polynesiensis, a member of the Aedes scutellaris complex, is the main vector in the South Pacific region of the Wuchereria bancrofti parasite, the causative agent of lymphatic filariasis (LF), and is also a major nuisance biter. Decades of Mass Drug treatment (MDA) have not been successful in elimination LF. Two non-vector species in the Ae. scutellaris complex were introgressed with Ae. polynesiensis to attempt to obtain lines that would produce cytoplasmic incompatibility (CI) with wild populations and/or LF-refractoriness. Despite selection of progeny from Brugia-challenged, non-infective females at each backcross, no refractory line was acquired. However, three lines from crosses between aposymbiotic Ae. polynesiensis and Ae. riversi displayed CI and male mating competiveness suitable for the purpose of population suppression using the incompatible insect technique (IIT). A population study was conducted of potential release sites and the evaluation of monitoring tools for Ae. polynesiensis on Moorea and Tetiaroa, French Polynesia. There was no evidence of active migration between selected islets on the atoll of Tetiaroa, suggesting it is a suitable site for field releases of CI males. The BioGents Sentinel trap was shown to be an efficient and convenient trap suitable for Ae. polynesiensis monitoring. The effects of temperature and larval density on life-table parameters relevant to IIT were examined, including: larval survivorship, developmental time to pupation, male to female ratio, male pupae yield, male size and adult male survival. These findings were used to design and conduct a 14-week field experiment testing CI male strain against an isolated population, using optimized rearing conditions. Approximately 8000 males were released weekly on motu Onetahi, Tetiaroa atoll. Significant sterility was induced by Wolbachia in the targeted female population, supporting the development and scale-up of this approach toward Ae. polynesiensis nuisance and LF transmission reduction.

No description available.

Abstract The age of an adult female mosquito is a critical determinant of its ability to transmit a range of human pathogens such as the dengue viruses. Since most pathogens require a relatively long period of development in their mosquito vector before they can be transmitted to a new host, only older insects are of epidemiological importance. It has been proposed that life-shortening strains of the inherited bacterial symbiont Wolbachia may be potentially useful to shift mosquito population age structure towards younger individuals, thereby reducing pathogen transmission without eradicating the mosquito population. However, life-shortening Wolbachia strains do not occur in mosquitoes naturally. To facilitate the evaluation of this potential strategy, the major aim of this project was to transfer a life-shortening Wolbachia strain, wMelPop, from the fruit fly, Drosophila melanogaster, to the dengue vector, Aedes aegypti. After successful transinfection, I then characterized the wMelPop infection in this mosquito species, and the associated phenotypic effects of this Wolbachia strain on several aspects of Ae. aegypti life-history. The horizontal transfer of the obligate intracellular bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. Previous attempts to directly transfer wMelPop from D. melanogaster to Ae. aegypti have been unsuccessful, presumably due to mal-adaptation of wMelPop to this novel mosquito host. In an attempt to adapt wMelPop to the mosquito intracellular environment to facilitate transfer, we serially passaged this infection for 3 years in Aedes cell culture. After long-term serial passage in mosquito cell lines, wMelPop was then initially reintroduced into its native host, D. melanogaster, by embryonic microinjection to examine the phenotypic outcome of this process. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into Drosophila, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility (CI), when compared to the original wMelPop strain. These results were suggestive of adaptation of this Wolbachia strain to the mosquito intracellular environment. Subsequently, we successfully established wMelPop in Ae. aegypti using embryonic microinjection. Two independently transinfected Ae. aegypti lines, PGYP1 and PGYP2, were generated. Analogous to infection in Drosophila, the presence of wMelPop in both of these lines halved adult mosquito lifespan under a range of laboratory conditions. The new association is stable, and wMelPop is maternally inherited with high fidelity across Ae. aegypti life span. It is capable of inducing near perfect CI that does not diminish as male Ae. aegypti age, which should facilitate its invasion into natural field populations and persistence over time. Fecundity assays using young PGYP1 females revealed that wMelPop infection induced minimal costs for reproductive fitness for females during their first gonotrophic cycle. The phenotypic effects of wMelPop on several aspects of Ae. aegypti life-history were additionally characterized. Overall, minor costs of wMelPop infection for pre-imaginal survivorship, development and adult size were observed. However, wMelPop infection decreased the viability of quiescent Ae. aegypti eggs over time. Similarly, the reproductive fitness of wMelPop-infected Ae. aegypti females declined with age. These results revealed a general pattern associated with wMelPop pathogenesis in Ae. aegypti, where host fitness costs incurred by this infection increase during aging of both immature and adult life-history stages. We also discovered that wMelPop-infected Ae. aegypti have a reduced ability to utilise blood from non-human hosts for egg development. Blood feeding by wMelPop-infected females on mouse, guinea pig or chicken hosts resulted in a near complete abolishment of reproductive output associated with both a decline in the numbers of eggs oviposited as well as the hatching rate of successfully laid eggs. In contrast, the reproductive output of wMelPop-infected females fed human blood was normal, with intermediate effects observed with other vertebrate blood sources. Removal of Wolbachia from mosquitoes using antibiotic treatment restored egg development to standard levels on all blood sources. Further blood feeding assays over two reproductive cycles definitively illustrated a nutritional interaction between host blood source and egg development in wMelPop-infected Ae. aegypti. This dramatic phenotype may provide new insights into the nutritional basis of mosquito anthropophily. The results presented in this thesis highlight several important parameters required to theoretically model the infection dynamics of wMelPop, and its potential impacts on Ae. aegypti populations. Moreover, the successful establishment of wMelPop in Ae. aegypti forms the primary basis for further field-based evaluations, which will ultimately determine the viability of this Wolbachia-based strategy as an applied tool to reduce dengue transmission.

Much of our current mechanistic understanding of the innate immune response in animals has grown out of empirical work in insect models, especially the fruit fly Drosophila melanogaster. The mainstream understanding of the fly immune response to bacteria has been that it exists in two parts; a cellular and a humoral response. Drosophila also harbor substantial genetic variation for antibacterial defense and investment in immunity is thought to involve a costly trade-off with life history traits, including development, life-span and reproduction. My first study (chapter 2) aimed to understand the way in which insects invest in fighting bacterial infection. We selected for survival following systemic infection with the opportunistic pathogen, Pseudomonas aeruginosa in wild-caught D. melanogaster over 10 generations. We then examined genome wide changes in expression in the selected flies relative to unselected controls, both of which had been infected with the pathogen to specifically identify the genetic basis of the evolved immune response. In response to selection, population level survivorship to infection increased from 15% to 70%. The evolved capacity for defense was costly as evidenced by reduced longevity and larval viability and a rapid loss of the trait once selection pressure was removed. Counter to expectation, we observed more rapid developmental rates in the selected flies. Selection associated changes in expression of genes with dual involvement in developmental and immune pathways suggest pleiotropy as a possible mechanism for the positive correlation. We also found that both the Toll and Imd pathways work synergistically to limit infectivity and that cellular immunity plays a more critical role in overcoming P. aeruginosa infection than previously reported. Females usually produce a more robust immune response and are often less susceptible to infection. This female bias has been documented in humans, mice and some birds and reptiles. The most common explanation is that males increase their mating success at the cost of immune investment whilst females invest in immunity to maximize life-time egg production. In insects, however, there is growing evidence of male-biased immune performance. Using fly survival data from my first study, I found that males exhibited higher post-infection survival than females. In my second study (chapter 3), I related these differences in survival rate to changes in gene transcription. Firstly, we examined the expression of a set of immunity genes in both sexes in the presence and absence of infection. We found that male-biased survival may be partially attributable to a higher baseline expression of immune genes in males. Contrary to previous published work, we found that immune gene expression was readily induced in flies upon exposure to P. aeruginosa and that the two sexes responded in a similar manner. Lastly, we found that selection altered the expression of genes in males alone and only in the presence of infection. Together our findings suggest a superior immune response in male Drosophila. Wolbachia pipientis is an obligate intracellular bacterium capable of spreading itself through populations by manipulating the reproduction of its hosts. The Wolbachia strain wMelPop, which reduces longevity in D. melanogaster, has been introduced into the Dengue virus mosquito vector, Aedes aegypti, as a strategy to reduce disease transmission. The infecting Wolbachia halve the lifespan of the mosquito and induce numerous behavioral and physiological abnormalities including heightened locomotor activity and an age dependent reduction in blood feeding success. In my third study (chapter 4), we aimed to understand the mechanism underpinning these changes and hence chose to explore how Wolbachia may be interacting with the insect’s nervous and muscle tissue. Because wMelPop over-replicates in Drosophila, first we measured the bacterial density in A. aegypti. We found that there was a relationship between some of the feeding associated defects in the mosquito and the density of Wolbachia in the nervous and muscle tissue. Next, we carried out a series whole genome profiling experiments based on the head and muscle tissues to identify mosquito pathways affected by the microbe. Key findings that may relate to the phenotypes of interest include increased expression of genes relating to muscle contraction and synthesis of the neurotransmitter dopamine. Other novel findings that may not relate directly to the phenotypes of interest include evidence of a strong local tissue based immune response and widespread changes in expression of mosquito methylation and acetylation associated genes. We then used then amplification of inter-methylated sites (AIMS test) to obtain DNA fingerprints representative of the methylome of A. aegypti infected and uninfected with wMelPop. The presence of wMelPop caused hypermethylation in loci where they were not methylated in uninfected mosquitoes.

Wolbachia are widely spread, maternally transmitted insect endosymbiotic intracellular bacteria. They have been implicated in the control of several insect transmitted diseases, including dengue, yellow fever, Zika and malaria. Effective pathogen suppression in the insect host is shown to be proportional to the intracellular levels of bacteria. Therefore, understanding the molecular mechanisms underlying Wolbachia accumulation within organisms is extremely important for future epidemic control and research. Using Drosophila as a model insect, our lab has previously observed Wolbachia tropism to stem cell niches. Current work has identified polar cells as an additional site of Wolbachia tropism and demonstrated that Wg/Wnt signaling is important for Wolbachia intracellular accumulation in these somatic cells. In this thesis, we first observed that the Wg/Wnt pathway protein Armadillo also controls Wolbachia levels in the germline cells, indicating the possibility of having a conserved molecular mechanism controlling Wolbachia. Using RNAi and small molecule inhibitors of Shaggy, another component of the canonical Wg/Wnt pathway, we demonstrate that the canonical Wg/Wnt signaling is essential for Wolbachia intracellular accumulation. Our investigation provides fundamental insights into the mechanisms of Wolbachia intracellular accumulation. Furthermore, it offers novel strategies to modulate Wolbachia in non-model insect species, including various disease transmitting Anopheles, Culex, and Aedes. These findings potentially will increase the effectiveness of a Wolbachia-based vector transmitted disease suppression. / 2017-02-28

Obligately blood-sucking parasites harbour symbiotic bacteria providing them B-vitamins and cofactors missing from their blood diet. Within Hippoboscoidea group (parasites of birds and mammals), tsetse flies as medically important vectors have been studied extensively while bat flies and louse flies tend to be neglected. This thesis is composed of two complementary manuscripts focused on phylogeny and origin of bacterial symbionts in Hippoboscidae family (manuscript 1) and their genome evolution (manuscript 2). First, phylogenetic approach was employed to determine lineages of obligate and facultative symbionts present in this group. Second, genomic and phylogenomic analyses were carried out to better understand evolution of obligate endosymbionts from the Arsenophonus genus in this group. Results of the two studies indicate that relationships between Hippoboscoidea and their symbionts are extremely dynamic with frequent replacements of obligate symbionts. This hypothesis is supported by both phylogenetic and genomic evidence, in particular, Arsenophonus endosymbionts of Hippoboscidae represent several distinct lineages (of likely different ages) with noticeable differences in genome features and metabolic capabilities. The data presented in this thesis thus greatly extend our knowledge about evolution and genomics of symbiotic bacteria in Hippoboscidae and bloodsucking hosts in general.

Selfish genetic elements can distort the sex ratios of their hosts by increasing their own transmission to the next generation in a non-mendelian fashion. These elements can be either nuclear genes on a sex chromosome or cytoplasmically inherited microbes, and achieve an increased transmission by manipulating gametogenesis or host reproduction. Often these selfish elements benefit from a female biased population (for example heritable microbes are passed on maternally in the egg cytoplasm), while non-selfish, autosomal genes are selected to produce a balanced sex ratio. These differing reproductive strategies cause a genetic conflict that results in an “evolutionary arms race” that can promote the evolutionary change of sex determination systems. In this thesis, I investigate an extreme sex ratio distortion in a species of booklouse, Liposcelis sp. This species contains two distinct female types, one of which carries a maternally transmitted selfish genetic element that results in exclusively female offspring being produced. Recently, a candidate for the sex ratio distortion was identified as a horizontally transferred bacterial gene, that we have called Odile, and that is present in the genome of the (distorter) female carrying the distorting element. The gene originates from the endosymbiotic bacterium Wolbachia that is well known for its ability to distort the sex ratio of its hosts. I investigated this horizontal gene transfer event and attempt to characterize Odile. I provide evidence that this Wolbachia gene has been integrated into the genome of the distorter females and is not a bacterial contaminant. I found that the Odile gene has been duplicated and may have been horizontally transferred from Wolbachia independently to at least three other insect genomes. Additionally, I found that Odile is transcribed at low levels in a life-stage specific manner that is suggestive of a role in development. Additionally, I looked into male mate choice in this species as one aspect of the persistence of the distorting element. I found that male Liposcelis sp. do not discriminate between the two female types and do not spend more time mating with one female type over the other. These results contribute to ongoing research into the extreme sex ratio distortion found in this species and the candidate gene that may be the cause. Selfish genetic elements are an important driver of sex determination evolution, and Liposcelis sp. provides a unique and exciting system to investigate the implications of selfish elements in a genome further. / Graduate / 2019-12-17