Microorganisms are a goidmine for evolutionary genetics as their genomes can evolve at an extraordinary rate which results in some of the most extravagant adaptations in terms of genome structure and function as well as survival in the most unusual environments. One trend observed in several evolutionary scenarios is genome degeneration. It is most prominent in endosymbionts and obligate intracellular parasites and is a consequence of many constraints encountered in the intracellular environment. The process involves loss of many protein-coding genes, resulting in greater dependence on the host, and loss of non-coding DNA such as intergenic regions, which has a direct impact on regulation of genome function. I have chosen two evolutionarily distinct systems to analyze the stages and functional consequences of genome degeneration, namely the impact of genome compression on transcription in an obligate parasite Antonospora locustae (genus Microsporidia), and gene content in the mitochondrion of a diatom endosymbiont found in the dinoflagellate Durinskia baltica. I have successfully mapped transcriptional start and termination sites from 14 loci in Antonospora locustae, and cloned fragments of two genes that are part of the electron transport chain from the mitochondrion of the diatom endosymbiont in Durinskia baltica. My analysis reveals that transcription in A. locustae is always initiated immediately upstream of the open reading frame at a single point for every locus, whereas transcriptional termination can occur at several points for a single gene and, in some instances overlaps with a downstream reading frame. The identification ofNADH5 and ATPase9 from the mitochondrion of the endosymbiont in D. baltica is further evidence for the preservation of function in this enigmatic organelle.

Microorganisms are a goidmine for evolutionary genetics as their genomes can evolve at an extraordinary rate which results in some of the most extravagant adaptations in terms of genome structure and function as well as survival in the most unusual environments. One trend observed in several evolutionary scenarios is genome degeneration. It is most prominent in endosymbionts and obligate intracellular parasites and is a consequence of many constraints encountered in the intracellular environment. The process involves loss of many protein-coding genes, resulting in greater dependence on the host, and loss of non-coding DNA such as intergenic regions, which has a direct impact on regulation of genome function. I have chosen two evolutionarily distinct systems to analyze the stages and functional consequences of genome degeneration, namely the impact of genome compression on transcription in an obligate parasite Antonospora locustae (genus Microsporidia), and gene content in the mitochondrion of a diatom endosymbiont found in the dinoflagellate Durinskia baltica. I have successfully mapped transcriptional start and termination sites from 14 loci in Antonospora locustae, and cloned fragments of two genes that are part of the electron transport chain from the mitochondrion of the diatom endosymbiont in Durinskia baltica. My analysis reveals that transcription in A. locustae is always initiated immediately upstream of the open reading frame at a single point for every locus, whereas transcriptional termination can occur at several points for a single gene and, in some instances overlaps with a downstream reading frame. The identification ofNADH5 and ATPase9 from the mitochondrion of the endosymbiont in D. baltica is further evidence for the preservation of function in this enigmatic organelle.

Microorganisms are a goidmine for evolutionary genetics as their genomes can evolve at an extraordinary rate which results in some of the most extravagant adaptations in terms of genome structure and function as well as survival in the most unusual environments. One trend observed in several evolutionary scenarios is genome degeneration. It is most prominent in endosymbionts and obligate intracellular parasites and is a consequence of many constraints encountered in the intracellular environment. The process involves loss of many protein-coding genes, resulting in greater dependence on the host, and loss of non-coding DNA such as intergenic regions, which has a direct impact on regulation of genome function. I have chosen two evolutionarily distinct systems to analyze the stages and functional consequences of genome degeneration, namely the impact of genome compression on transcription in an obligate parasite Antonospora locustae (genus Microsporidia), and gene content in the mitochondrion of a diatom endosymbiont found in the dinoflagellate Durinskia baltica. I have successfully mapped transcriptional start and termination sites from 14 loci in Antonospora locustae, and cloned fragments of two genes that are part of the electron transport chain from the mitochondrion of the diatom endosymbiont in Durinskia baltica. My analysis reveals that transcription in A. locustae is always initiated immediately upstream of the open reading frame at a single point for every locus, whereas transcriptional termination can occur at several points for a single gene and, in some instances overlaps with a downstream reading frame. The identification ofNADH5 and ATPase9 from the mitochondrion of the endosymbiont in D. baltica is further evidence for the preservation of function in this enigmatic organelle. / Science, Faculty of / Botany, Department of / Graduate

Current bioinformatic methods such as molecular phylogenetics and phylogenomics provide us with good insight to symbiont evolution. Though modern science evolves rapidly, accelerates speed of acquiring novel discoveries and improves their quality, there is still endless row of questions waiting to be answered. This thesis focuses on origins of symbiosis between insects and Enterobacteria, and the mechanisms promoting association of bacteria with arthropods. The main emphasis is put on the secondary symbionts of the genus Sodalis (Enterobacteriaceae) and the pathogenic Anaplasma phagocytophilum (Anaplasmataceae) that seems to be undergoing first steps to become hereditary mutualist.

Abstract The evolution of sociality is often associated with close relatedness and genetic similarity of interacting individuals. However, colonies of advanced social insects (e.g. ants, bees and wasps) characterized by large colony size and division of tasks, are also shaped by acquisition of genetic diversity by polyandry, polygyny, recombination and even by hybridization. The balance between forces selecting for high relatedness on one hand and for improved colony performance though increased genetic diversity on the other hand forms an intriguing area of research. My study has produced the first genetic linkage maps for ants (Acromyrmex echinatior and Pogonomyrmex rugosus) and social wasps (Vespula vulgaris). Together with the findings of earlier honeybee research, it is shown that advanced eusocial insects have higher recombination rates than any other insect (or animal) studied so far. The estimates obtained here were 14 cM/Mb for P. rugosus, 9.7 cM/Mb for V. vulgaris and 6.2 cM/Mb for A. echinatior. Pogonomyrmex harvester ants have a genetic caste determination system in which workers arise from mating between two hybridizing lineages whereas sexuals are produced by within-lineage mating. I evaluated the origin of the lineages and the caste determination system by using 751 variable nuclear genetic markers. Fertile hybrids would lead to introgression, particularly in genomic regions characterized by a high recombination rate and lack of strongly selected loci. The hybridizing lineages (lineage pairs J1/J2 and H1/H2) showed many fixed differences. Nineteen of them were in the constructed linkage map, scattered in different linkage groups. The results suggest that there has been no recent introgression. As the hybrids are viable (as workers), caste differentiation can be affected by many loci scattered throughout the ant genome or by a small number of very strongly selected loci. Genetic diversity in colonies of the ant Formica cinerea is affected by varying levels of polygyny. I tested the hypotheses that the prevalence of endosymbiotic bacteria can vary in polygynous colonies but be either very low or very high in monogynous colonies. However, I found no association between the level of polygyny and endosymbiont prevalence. In addition to Wolbachia, I found two other endosymbiotic bacteria Cardinium and Candidatus Serratia symbiotica which have not been earlier reported from ants. Genetic diversity in insect colonies is affected by polyandry and polygyny. My results indicate that high a recombination rate is also an important factor influencing diversity. Genotypically diverse progenies can enhance colony success, e.g. through effects on division of labour or defence against pathogens. Recombination differs from the other factors in its effects on genetic relatedness among colony members.

Plant microbiomes are increasingly appreciated as major drivers of plant health and ecosystem services, and are of ever-greater interest for their potential in human applications. However, plant-associated microorganisms often live in complex associations in nature. Here, I characterize one of these microbial associations: the symbiosis between foliar fungal endophytes and their bacterial endosymbionts (endohyphal bacteria, EHB). EHB influence fungal phenotypes and can shape the outcomes of plant-fungal interactions. EHB are thought to form facultative associations with many foliar fungi in the species-rich Ascomycota, but little is known about how these symbioses are initiated and maintained, or how EHB shape the ecology of their fungal hosts. In this study, I assessed factors mediating the relationships between two foliar fungi (Microdiplodia sp., Dothideomycetes; Pestalotiopsis sp. Sordariomycetes) and their EHB. I first established methods for introducing EHB into axenic mycelia of their fungal hosts, providing an important step forward for understanding the establishment of EHB associations and a critical tool for experimental tests of the effects of EHB on fungal phenotypes. Through experiments in vitro, I found that both the identity of the fungal host and the conditions under which fungi and bacteria are grown influence the establishment of EHB/fungal associations. Moreover, I showed EHB of foliar fungi can be transferred across fungal classes, thus creating experimental strains that could be used for the first time to examine the contribution of each symbiont to important fungal traits. Using these strains I evaluated how EHB influence the capacity of foliar fungi to degrade plant material as saprotrophs. I found that the presence and identity of EHB significantly influenced fungal growth on particular media, cellulase and ligninase activity, and mass loss from senescent tissue of their native host plant species in a partnership-specific manner. Because EHB can be acquired horizontally, they may help shape plant-fungal interactions, resultant ecosystem services, and the functional diversification of plant-associated fungi along the saprotroph-endophyte continuum. By manipulating EHB/fungal interactions in new ways, we can potentially influence fungal phenotypes for diverse human applications.

A interação entre microrganismos endossimbiontes e mosquitos tem sido amplamente estudada. Tais estudos visam encontrar alternativas para o controle biológico de mosquitos vetores. Recentemente foi descrito que Acanthamoeba polyphaga é capaz de infectar Aedes aegypti, mosquito vetor do vírus da dengue. Visando entender melhor sobre esta interação, estudaramse aspectos comportamentais e de desenvolvimento de mosquitos alimentados com A. polyphaga, em condições de laboratório. Além disso, procurou-se localizar os sítios de infecção destas amebas no interior do corpo dos mosquitos, através de cortes histológicos. Nas condições em que os testes foram feitos, as amebas contribuíram para o aumento de produção de ovos de Ae. aegypti. Da mesma forma, não produziram patologias nos insetos, uma vez que não houve morte dos mesmos, durante o desenvolvimento dos bioensaios. Algumas amebas que serviram de alimentação às larvas, escaparam à barreira da membrana peritrófica e à resposta de defesa do mosquito, invadindo outras regiões do corpo das larvas e dos adultos. As amebas são encontradas nas formas de cistos e trofozoítos, indicando que o micro-habitat intestinal e do corpo gorduroso não parecem ser muito lesivos para as mesmas. Os resultados obtidos até a data indicam a importância da continuidade dos estudos. Também é importante estudar outros processos associados à resposta imune dos mosquitos quando as amebas conseguem escapar as barreiras de defesa e invadir outros locais do corpo. / The interaction between endosymbiont microorganisms and mosquitoes have been widely studied. Such studies have the aim to find alternatives for biological control of mosquitoes. Recently it was reported that Acanthamoeba polyphaga is able of infecting Aedes aegypti the vector mosquito of dengue viruses. In order to better understand such interaction, we studied some biological aspects and development of mosquitoes that fed on A. polyphaga, in laboratory conditions. Furthermore, we tried to clarifying the sites of infection of these amoebae inside the mosquitoes body, using histological sections. In conditions where the tests were done, the amoebas contributed to the increased of egg production of Ae. aegypti. Likewise, no pathologies were observed in the insects, since no death was observed, during the development of bioassays. Some amoebas served as food to the larvae escaped the barrier of the peritrophic membrane, as well as the defense response of the mosquito, invading other body regions of larvae and adults. Amoebas are found in the forms of cysts and trophozoites, indicating that the intestinal micro-habitat and body fat did not appear to be especially harmful to them. The results to date indicate the importance of continuing such studies. It is also important to study other processes associated with the immune response of mosquitoes when amoebae escape the defense barriers and invade other body sites.

A interação entre microrganismos endossimbiontes e mosquitos tem sido amplamente estudada. Tais estudos visam encontrar alternativas para o controle biológico de mosquitos vetores. Recentemente foi descrito que Acanthamoeba polyphaga é capaz de infectar Aedes aegypti, mosquito vetor do vírus da dengue. Visando entender melhor sobre esta interação, estudaramse aspectos comportamentais e de desenvolvimento de mosquitos alimentados com A. polyphaga, em condições de laboratório. Além disso, procurou-se localizar os sítios de infecção destas amebas no interior do corpo dos mosquitos, através de cortes histológicos. Nas condições em que os testes foram feitos, as amebas contribuíram para o aumento de produção de ovos de Ae. aegypti. Da mesma forma, não produziram patologias nos insetos, uma vez que não houve morte dos mesmos, durante o desenvolvimento dos bioensaios. Algumas amebas que serviram de alimentação às larvas, escaparam à barreira da membrana peritrófica e à resposta de defesa do mosquito, invadindo outras regiões do corpo das larvas e dos adultos. As amebas são encontradas nas formas de cistos e trofozoítos, indicando que o micro-habitat intestinal e do corpo gorduroso não parecem ser muito lesivos para as mesmas. Os resultados obtidos até a data indicam a importância da continuidade dos estudos. Também é importante estudar outros processos associados à resposta imune dos mosquitos quando as amebas conseguem escapar as barreiras de defesa e invadir outros locais do corpo. / The interaction between endosymbiont microorganisms and mosquitoes have been widely studied. Such studies have the aim to find alternatives for biological control of mosquitoes. Recently it was reported that Acanthamoeba polyphaga is able of infecting Aedes aegypti the vector mosquito of dengue viruses. In order to better understand such interaction, we studied some biological aspects and development of mosquitoes that fed on A. polyphaga, in laboratory conditions. Furthermore, we tried to clarifying the sites of infection of these amoebae inside the mosquitoes body, using histological sections. In conditions where the tests were done, the amoebas contributed to the increased of egg production of Ae. aegypti. Likewise, no pathologies were observed in the insects, since no death was observed, during the development of bioassays. Some amoebas served as food to the larvae escaped the barrier of the peritrophic membrane, as well as the defense response of the mosquito, invading other body regions of larvae and adults. Amoebas are found in the forms of cysts and trophozoites, indicating that the intestinal micro-habitat and body fat did not appear to be especially harmful to them. The results to date indicate the importance of continuing such studies. It is also important to study other processes associated with the immune response of mosquitoes when amoebae escape the defense barriers and invade other body sites.

A interação entre microrganismos endossimbiontes e mosquitos tem sido amplamente estudada. Tais estudos visam encontrar alternativas para o controle biológico de mosquitos vetores. Recentemente foi descrito que Acanthamoeba polyphaga é capaz de infectar Aedes aegypti, mosquito vetor do vírus da dengue. Visando entender melhor sobre esta interação, estudaramse aspectos comportamentais e de desenvolvimento de mosquitos alimentados com A. polyphaga, em condições de laboratório. Além disso, procurou-se localizar os sítios de infecção destas amebas no interior do corpo dos mosquitos, através de cortes histológicos. Nas condições em que os testes foram feitos, as amebas contribuíram para o aumento de produção de ovos de Ae. aegypti. Da mesma forma, não produziram patologias nos insetos, uma vez que não houve morte dos mesmos, durante o desenvolvimento dos bioensaios. Algumas amebas que serviram de alimentação às larvas, escaparam à barreira da membrana peritrófica e à resposta de defesa do mosquito, invadindo outras regiões do corpo das larvas e dos adultos. As amebas são encontradas nas formas de cistos e trofozoítos, indicando que o micro-habitat intestinal e do corpo gorduroso não parecem ser muito lesivos para as mesmas. Os resultados obtidos até a data indicam a importância da continuidade dos estudos. Também é importante estudar outros processos associados à resposta imune dos mosquitos quando as amebas conseguem escapar as barreiras de defesa e invadir outros locais do corpo. / The interaction between endosymbiont microorganisms and mosquitoes have been widely studied. Such studies have the aim to find alternatives for biological control of mosquitoes. Recently it was reported that Acanthamoeba polyphaga is able of infecting Aedes aegypti the vector mosquito of dengue viruses. In order to better understand such interaction, we studied some biological aspects and development of mosquitoes that fed on A. polyphaga, in laboratory conditions. Furthermore, we tried to clarifying the sites of infection of these amoebae inside the mosquitoes body, using histological sections. In conditions where the tests were done, the amoebas contributed to the increased of egg production of Ae. aegypti. Likewise, no pathologies were observed in the insects, since no death was observed, during the development of bioassays. Some amoebas served as food to the larvae escaped the barrier of the peritrophic membrane, as well as the defense response of the mosquito, invading other body regions of larvae and adults. Amoebas are found in the forms of cysts and trophozoites, indicating that the intestinal micro-habitat and body fat did not appear to be especially harmful to them. The results to date indicate the importance of continuing such studies. It is also important to study other processes associated with the immune response of mosquitoes when amoebae escape the defense barriers and invade other body sites.

Les bactéries symbiotiques facultatives, transmises verticalement de mère à descendants, sont connues pour avoir des effets importants sur la reproduction de leurs hôtes (incompatibilité cytoplasmique et biais de sex-ratio), sur l’adaptation à des situations de stress (spécialisation, résistance aux pathogènes et aux fortes températures) ainsi que sur l’évolution de l’ADN mitochondrial par l’induction de balayages sélectifs. Bemisia tabaci est un complexe d’espèces divisé en nombreux biotypes, principalement différenciés sur la base de marqueurs moléculaires (essentiellement mitochondrial). Cet insecte héberge 7 symbiotes, 1 symbiote nutritionnel obligatoire et 6 symbiotes facultatifs. Nos résultats ont montré une extrême diversité ainsi qu’une prévalence exceptionnelle des bactéries symbiotiques chez B. tabaci. Leur dynamique rapide a entraîné de nombreux balayages sélectifs sur l’ADN mitochondrial influençant nettement son évolution. La très forte fréquence des infections multiples chez cet insecte peut certainement être expliquée par la co-localisation de l’ensemble des bactéries au sein des mêmes cellules, avec le symbiote nutritionnelle. Enfin, la coexistence de différents cytotypes en sympatrie offre l’opportunité d’étudier les interactions qui se mettent en place entre différentes communautés symbiotiques et comment ces systèmes vont évoluer / Facultative endosymbiotic bacteria that are vertically transmitted from mother to offsprings, have important effects on their host reproduction (cytoplasmic incompatibilities and sex-ratio biais), on host adaptation to situation of stress (specialization, resistance against pathogens or high temperature) and also on the evolution of mtDNA, by inducing selective sweeps. Bemisia tabaci is a species complex divided into numerous, mainly differentiated based on molecular markers (essentially mtDNA markers). This insect is infected by 7 bacterial symbionts, one nutritionnal symbiont that is obligatory and 6 facultative symbionts. Our results show an extreme diversity of symbionts in this insect and a very high prevalence in B. tabaci populations. Moreover their rapid dynamic has strongly influenced mtDNA evolution by inducing recurrent selective sweeps. The colocalization of the whole symbiotic community in the same cells, with the nutritional symbiont, might certainly explain the very high frequency of multiple infections in B. tabaci. Finally, coexistence of very different cytotypes in sympatry allows to study the interactions that take place between different symbiotic communities and how these systems will evolve