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The human gut microbiome is home to many hundreds of different microbes which play a crucial role in human physiology. For most of them, little is known about how their genetic diversity translates into functional traits and how they interact with their host, which is to some extent due to the lack of isolate genomes. Cultivation-free metagenomic approaches yield extensive amounts of bacterial genetic data, and recently developed algorithms allow strain-level resolution and reconstruction of bacterial genomes from metagenomes, yet bacterial within-species diversity and transmission dynamics after fecal microbiota transplantation remain largely unexplored over cohorts and using these technological advances. To investigate bacterial within-species diversity I first undertook large-scale exploratory studies to characterize the population-level genomic makeup of the two key human gut microbes Eubacterium rectale and Akkermansia muciniphila , leveraging many hundreds of bacterial draft genomes reconstructed from short-read shotgun metagenomics datasets from all around the planet. For E. rectale , I extended previous observations about clustering of subspecies with geography, which suggested isolation by distance and the putative ancestral loss of four distinct motility operons, rendering a subspecies specifically found in Europe immotile. For A. muciniphila, I found that there are several closely related but undescribed Akkermansia spp. in the human gut that are all likely human-specific but are differentially associated with host body mass index, showcasing metabolic differences and distinct co-abundance patterns with putative cognate phages . For both species, I discovered distinct subspecies-level genetic variation in structural polysaccharide synthesis operons. Next, utilizing a complementary strain-resolved approach to track strains between individuals, I undertook a fecal microbiota transplantation (FMT) meta-analysis integrating 24 distinct clinical metagenomic datasets. I found that patients with an infectious disease or those who underwent antibiotic treatment displayed increased donor strain uptake and that some bacterial clades engraft more consistently than others. Furthermore, I developed a machine-learning framework that allows optimizing microbial parameters - such as bacterial richness - in the recipient after FMT based on donor microbiome features, representing first steps towards making a rational donor choice. Taken together, in my work I extended the strain-level understanding of human gut commensals and showcased that genomes from metagenomes can be suitable to conduct large-scale bacterial population genetics studies on other understudied human gut commensals. I further confirmed that strain-resolved metagenomics allows tracking of strains and thus inference of strain engraftment characteristics in an FMT meta-analysis, revealing important differences in engraftment over cohorts and species and paving the way towards better designed FMTs. I believe that my work is an important contribution to the field of microbiome research, showcasing the power of shotgun metagenomics, modern algorithms and large-scale data analysis to reveal previously unattainable insights about the human gut microbiome.

In human microbiome analysis, sequencing of bacterial 16S rRNA genes has revealed a role for the gut microbiota in maintaining health and contributing to various pathologies. Novel community analysis techniques must be evaluated in terms of bias, sensitivity, and reproducibility and compared to existing techniques to be effectively implemented. Next- generation sequencing technologies offer many advantages over traditional fingerprinting methods, but this extensive evaluation required for the most efficacious use of data has not been performed previously. Illumina libraries were generated from the V3 region of the 16S rRNA gene of samples taken from 12 unique sites within the gastrointestinal tract for each of 4 individuals. Fingerprint data were generated from these samples and prominent bands were sequenced. Sequenced bands were matched with OTUs within their respective libraries. The results demonstrate that denaturing gradient gel electrophoresis (DGGE) represents relatively abundant bacterial taxa (>0.1%) beta-diversity of all samples was compared using Principal Coordinates Analysis (PCoA) of UniFrac distances and Multi-Response Permutation Procedure (MRPP) was applied to measure sample cluster strength and significance; indicator species analysis of fingerprint bands and Illumina OTUs were also compared. The results demonstrate overall similarities between community profiling methods but also indicate that sequence data were not subject to the same limitations observed with the DGGE method (i.e., only abundant taxa bands are resolved, unable to distinguish disparate samples). In addition, the effect of stochastic fluctuations in ???????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????????? differ for DGGE and next-generation sequencing. I compared pooled and individual reactions for samples of high and low template concentration for both Illumina and DGGE using the combined V3-V4 region of the 16S rRNA gene, and demonstrated that template concentration has a greater impact on reproducibility than pooling. This research shows congruity between two disparate molecular methods, identifies sources of bias, and establishes new guidelines for minimizing bias in microbial community analyses.

La microbiologie judiciaire consiste à utiliser des microorganismes comme éléments de preuves dans une enquête judiciaire. Elle est appliquée dans plusieurs buts, dont l’identification de fluides biologiques et la compréhension des mécanismes de décomposition des cadavres entre autres. L’objectif de ce mémoire est de tester l’application de nouvelles méthodes de prédictions utilisant la microbiologie judiciaire et d’évaluer leur potentiel pour le domaine. Dans un premier temps, au chapitre 2, nous avons étudié une nouvelle méthode pour identifier les fluides vaginaux par le microbiome. Alors que plusieurs méthodes existent pour identifier des sources pures de fluides vaginaux, nous avons testé un modèle sur des mélanges de fluides pour voir s’il était possible de reconnaître la présence de sources vaginales dans ceux-ci. Nos résultats montrent que le modèle utilisé avec le seuil choisi a une spécificité de 100% en ce qui a trait à l’identification de fluides vaginaux dans des mélanges et une sensibilité de 70%. Ainsi, nos résultats démontrent qu’il serait possible d’utiliser éventuellement cette méthode en sciences judiciaires. Dans un deuxième temps, au chapitre 3, nous avons exploré le changement du microbiome épinécrotique sur une période de sept mois comprenant un hiver complet. Nous avons pu identifier les taxons spécifiques qui ont une différence d’abondance significative entre les saisons et ainsi développer un modèle de prédiction qui peut déterminer si un corps a passé l’hiver ou non. Notre modèle n’a jamais prédit qu’un corps n’avait pas passé l’hiver quand en réalité c’était le cas, mais a parfois surestimé les résultats. / Microbial forensics uses microorganisms as physical evidence in different investigations. It is used in multiple ways, such as the identification of body fluids or the understanding of the different mechanisms of body decomposition. The objectives of this research are to apply novel prediction methods using microbial forensics and assess their potential for forensic science. First, in chapter 2, we studied a novel method to identify vaginal fluids using the microbiome. Similar methods already exist to identify pure sources of body fluids, but we tested a model that could find vaginal fluids in mixture samples. Our results show that the model used with the chosen threshold has a specificity of 100% and a sensitivity of 70% for the identification of vaginal fluids in mixtures. Therefore, our results show that it would be possible to use this method in the future in forensic science. Second, in chapter 3, we explored the change of the epinecrotic microbiome over a 7-months period including a full winter. We could identify specific taxa that were differentially abundant before and after winter and developed a prediction model that would calculate if a body went through winter or not. The model never predicted that a body did not go through winter when it did, but it sometimes overestimated the results predicting it went through winter when it did not.

<p>All organisms live in complex habitats that shape the course of their evolution by altering the phenotype expressed by a given genotype (a phenomenon known as phenotypic plasticity) and simultaneously by determining the evolutionary fitness of that phenotype. In some cases, phenotypic evolution may alter the environment experienced by future generations. This dissertation describes how genetic and environmental variation act synergistically to affect the evolution of glucosinolate defensive chemistry and flowering time in Boechera stricta, a wild perennial herb. I focus particularly on plant-associated microbes as a part of the plant’s environment that may alter trait evolution and in turn be affected by the evolution of those traits. In the first chapter I measure glucosinolate production and reproductive fitness of over 1,500 plants grown in common gardens in four diverse natural habitats, to describe how patterns of plasticity and natural selection intersect and may influence glucosinolate evolution. I detected extensive genetic variation for glucosinolate plasticity and determined that plasticity may aid colonization of new habitats by moving phenotypes in the same direction as natural selection. In the second chapter I conduct a greenhouse experiment to test whether naturally-occurring soil microbial communities contributed to the differences in phenotype and selection that I observed in the field experiment. I found that soil microbes cause plasticity of flowering time but not glucosinolate production, and that they may contribute to natural selection on both traits; thus, non-pathogenic plant-associated microbes are an environmental feature that could shape plant evolution. In the third chapter, I combine a multi-year, multi-habitat field experiment with high-throughput amplicon sequencing to determine whether B. stricta-associated microbial communities are shaped by plant genetic variation. I found that plant genotype predicts the diversity and composition of leaf-dwelling bacterial communities, but not root-associated bacterial communities. Furthermore, patterns of host genetic control over associated bacteria were largely site-dependent, indicating an important role for genotype-by-environment interactions in microbiome assembly. Together, my results suggest that soil microbes influence the evolution of plant functional traits and, because they are sensitive to plant genetic variation, this trait evolution may alter the microbial neighborhood of future B. stricta generations. Complex patterns of plasticity, selection, and symbiosis in natural habitats may impact the evolution of glucosinolate profiles in Boechera stricta.</p> / Dissertation

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.

NTRODUCTION: Colonic bacteria produce short-chain fatty acids (SCFAs) by fermentation of dietary carbohydrates and fiber. The production of SCFAs is greatest in proximal colon where propulsion is likely to be highly dependent on chemical/nutrient stimuli. Unabsorbed SCFAs entering the distal colon are likely to modify peristalsis initiated by fecal pellet-induced distension. AIM: To determine the effect of individual SCFAs on propulsive contractions in guinea pig proximal colon and on pellet propulsion in distal colon. METHODS: Proximal colon was excised, cannulated and placed in Krebs buffer in an organ bath. After equilibration, the colon was distended with 1ml of Krebs buffer alone or containing sodium salts of acetate, butyrate, or propionate at 10-100mM. Motility was video recorded, spatiotemporal maps generated, and the number of full-length propulsive contractions during a 5 min period was determined. The distal colon was removed from guinea pig and placed in an organ bath containing Krebs buffer. Following equilibration, video-tracking software was used to measure the velocity of propulsion of a clay pellet placed in the orad end of a segment. Krebs buffer alone or containing individual SCFAs at 30mM was perfused caudad to the pellet at 0.1 ml/min. RESULTS: The basal rate of propulsive contractions in Krebs buffer was 2.9 ± 0.7 per 5 min. Butyrate and propionate had concentration-dependent and opposing effects on propulsive contractions; acetate had no effect. Butyrate significantly increased propulsive contractions (maximal increase of 207% (p ≤ 0.05) at 30 mM) whereas propionate abolished propulsive contractions (91-100% inhibition at 10-30mM). Control Krebs buffer containing 10- 100mM NaCl had no effect. The mean rate of pellet propulsion during perfusion of Krebs buffer alone was 1.5 ± 0.2 mm/sec. Addition of sodium butyrate (30 mM) to the perfusate increased the velocity of pellet propulsion by 40 ± 4% (p<0.05) whereas addition of 30mM sodium propionate decreased velocity of pellet propulsion by 75 ± 8%. Acetate had no significant effect in either proximal or distal colon. Mixtures of SCFAs showed a general decrease in overall motility. CONCLUSION: SCFAs have differential effects on propulsive contractions in the proximal and distal colon of the guinea pig, with butyrate being excitatory and propionate being inhibitory. These studies suggest that the movement of feces in the colon depends not only on distension but the chemical (i.e. dietary) composition of the feces.

Two vaginal phylotypes of the genus Megasphaera (phylotype 1 and phylotype 2) were recently associated with bacterial vaginosis (BV), an infection characterized by vaginal dysbiosis. Through an analysis of 16S rRNA profiles of 3,986 women enrolled in the Vaginal Human Microbiome Project, we confirmed that while both phylotypes were associated with BV, Megaspheara phylotype 1 had higher specificity for the condition. Megasphaera phylotype 2 was strongly associated with trichomoniasis. Previous studies have reported that BV-associated organisms are excluded in pregnancy. We observed that Megasphaera phylotype 1, which has been associated with adverse pregnancy outcomes, exhibited a trend of increased prevalence in the pregnant cohort. We sequenced the genomes of isolates of the two phylotypes and performed comparative analyses. We demonstrate that these two phylotypes have distinct genomic features and unique potential for metabolic processes that reveal niche specialization. These findings may provide insight into their differential associations with vaginal infections.

Recent advances in sequencing technologies have provided an unprecedented window in the unseen biological world. Accompanying this revolution is a growing appreciation for the ubiquity and diversity of beneficial interactions between animals and the microbes they carry. Given the symbiotic roles of microbes in host nutrition, immunity, behavior, development, and nearly every other facet of host biology, it is becoming increasingly clear that any understanding of hosts and their evolution would be incomplete without also considering the microbial dimension. Yet despite the growing body of evidence that many of these partnerships are rooted deep in evolutionary time, the majority of studies have tended to focus on how the composition of the present-day microbiome is shaped by present-day factors. In order to place the microbiome in the larger context of host biology, a more complete understanding of the evolutionary interplay between hosts and their microbial associates is needed. Here, I use Odontotaenius disjunctus, a large xylophagous beetle found throughout eastern North America, to explore how its present-day gut microbiome has both shaped and been shaped by evolutionary processes that have acted on the host. First, I show that recent evolution in O. disjunctus reflects the influence of Pleistocene glaciation on the host’s demographic history. Next, I show that the present-day gut microbiome of O. disjunctus reflects both the influence of this recent host evolution and the more ancient influence of natural selection that has acted on the host to maintain these beneficial partnerships over a much longer timescale. Finally, I show that the persistence of certain members of the gut microbiome over evolutionary time may be explained by their role in host lignocellulose digestion. My findings demonstrate that, much like host genomes, the microbiome has been, is being, and will continue to be shaped simultaneously by forces of selection and neutrality along the arc of evolutionary history shared by these intimate partners.

Insetos são os organismos multicelulares mais bem-sucedidos no ecossistema terrestre e sua riqueza se deve, em parte, à simbiose com microrganismos. Geradora de diversidade fenotípica, a comunidade microbiana associada a insetos permite a manutenção de fenótipos complexos capazes de colonizar novos nichos e de se adaptar a fatores de estresse. A relevância da relação de simbiose na ordem Lepidoptera, todavia, tem sido questionada em função da alta variabilidade da comunidade bacteriana associada aos seus representantes. Tendo como modelo de estudo lagartas do gênero Spodoptera (Lepidoptera: Noctuidae), esse trabalho teve por objetivo melhor compreender a composição e diversidade da microbiota associada ao intestino de lepidópteros, assim como verificar o efeito da pressão de seleção direcionada à resistência na sua estrutura e capacidade de utilização de inseticidas como fonte de carbono. Para isso, foi realizada a análise metagenômica da comunidade microbiana intestinal de quatro espécies do gênero Spodoptera assim como de cinco populações naturais de S. frugiperda e de linhagens suscetível e resistentes desse inseto praga. Foram avaliadas linhagens de S. frugiperda resistentes aos inseticidas spinosad, chlorpyrifos, lambda-cyhalothrin, flubendiamide, teflubenzuron e à toxina de Bacillus thuringiensis Cry1A.105+Cry2Ab2. A análise da composição, riqueza e capacidade da comunidade bacteriana utilizar inseticidas foi baseada na determinação de índices de diversidade alfa e beta, no isolamento de bactérias intestinais em meio seletivo e na análise do potencial de crescimento dos isolados em inúmeros inseticidas. Nossos resultados indicam a importância da microbiota intestinal para esse gênero de Lepidoptera. Bactérias do gênero Enterococcus foram predominantes em todas as populações de Spodoptera analisadas, independentemente da espécie, dieta ou pressões de seleção às quais as lagartas foram expostas. Populações naturais de S. frugiperda apresentaram um microbioma mais diverso e com maior número de bactérias capazes de metabolizar inseticidas. A diversidade do microbioma, assim como a presença e capacidade de bactérias utilizarem inseticidas como fonte de carbono para o seu crescimento foram influenciadas pelo nível de exposição a tais compostos, demonstrando que, assim como o hospedeiro, a microbiota intestinal também se encontra em pressão de seleção direcionada a resistência. / Insects are the most successful multicellular organisms in the terrestrial ecosystem and their richness is partially due to symbiosis with microorganisms. The microbial community associated with insects allows the maintenance of complex phenotypes capable of colonizing new niches and adapting to stressors. The relevance of symbiosis in Lepidoptera, however, has been questioned due to the high variability of the bacterial community associated with its representatives. Using Spodoptera larvae as a model, this work aimed to better understand the composition and diversity of the gut microbiota associated with Lepidoptera, as well as to verify the effects of the selection pressure with insecticides in the microbial gut structure and the ability of members of the community to metabolize insecticides as carbon source. Thus, we performed a metagenomic analysis of the gut microbial community of four species of the genus Spodoptera (Lepidoptera: Noctuidae), as well as for five natural populations of S. frugiperda and susceptible and resistant strains of this pest. Strains of S. frugiperda resistant to the insecticides spinosad, chlorpyrifos, lambda-cyhalothrin, flubendiamide, teflubenzuron and Bacillus thuringiensis toxin Cry1A.105 + Cry2Ab2 were evaluated. The analysis of the composition, richness and ability of the bacterial community to metabolize insecticides were carried out through alpha and beta diversity indexes, isolation of intestinal bacteria in selective medium and analysis of the growth potential of isolates in multiple insecticides. Our data indicate the importance of symbiosis for S. frugiperda. Enterococcus prevailed in all populations analyzed, regardless of the species of Spodoptera, food source or selection pressure that the larvae were exposed. Microbial diversity and ability to metabolize insecticides were higher in natural populations exposed to a range of stressors in the field. The diversity of the gut microbial community associated to Spodoptera frugiperda, as well as the ability of its members to metabolize insecticides, were influenced by the degree of exposure to insecticides, showing that, like the host, the gut microbiota is also under selection pressure to resistance.