Systems epidemiology to devise new interventions for multi-host tuberculosis

Animal tuberculosis (TB) is an infectious disease of livestock and wildlife mainly caused by Mycobacterium bovis and, in a minor extent, Mycobacterium caprae. In Portugal, animal TB is maintained in a multi-host system involving livestock and wild ungulates. The understanding of the processes driving transmission at this interface is key to inform control. In this work, M. caprae isolates (n=55) from Portugal were characterized by spoligotyping and MIRU-VNTR, supporting the clonal structure, co-infection and in vivo microevolution of this ecotype. M. bovis (n=948) from cattle, red deer and wild boar from TB hotspot regions were genotyped. Bayesian inference identified five ancestral populations and associated the most probable ancient M. bovis subpopulation with cattle and Beja, providing clues on the epidemics origin. A multinomial spatiotemporal probability model identified two significant TB clusters: one that persisted in 2004-2010 with Barrancos (Beja) at the centre, highlighting a significant higher risk associated to cattle; a second cluster, predominant in 2012-2016, holding the county Rosmaninhal (Castelo Branco) at the centre, for which wild boar contributed the most in relative risk. Whole-genome sequences (WGS) of 44 representative M. bovis distinguished five genetic clades and supported sustained transmission and multiple introductions in this multi-host system. Exploratory evolutionary analysis gave further support to pathogen transition between different hosts. Comparative genomics applied to M. bovis (n=70) representing the global clonal complex diversity predicted an open pan-genome and showed diversification of discrete subpopulations through core and accessory genomes. Consistent non-synonymous SNPs illustrated clade-specific virulence landscapes correlating with disease severity. Positive selection and weaker effects of recombination compared with mutation were evidenced as predominant evolutionary forces. Altogether, our results provide novel evidence on the population structure and evolution of M. caprae and M. bovis, delivering insights that could be used to inform adaptive TB control choices in different hosts and regions

Gamete differentiation : a genetic, biochemical and evolutionary study using Ectocarpus siliculosus

Despite its prevalence among eukaryotes, there are still many questions awaiting answers and plenty of discoveries to be made with regard to the mechanisms and maintenance of sexual reproduction. Brown algae (Phaeophyceae) represent a very interesting group to study the evolution of sexual reproduction, owing to the rich variation of life cycles, fertilization modes and sex determination systems displayed by this lineage. Particularly unique are various degrees of sexual dimorphism between gametes of closely related species, ranging from isogamy through anisogamy to oogamy. Therefore, the opportunity to compare these systems on the molecular level in Phaeophyceae promises advancement in our understanding of the evolution of sexual reproduction not only in algae, but in all living organisms. In this thesis we investigated the molecular basis of sexual reproduction and its link to reproductive isolation using a brown algal model organism Ectocarpus siliculosus. We constructed whole transcriptome libraries of male and female gametes to explore their functional differentiation at the gene expression level. We sequenced sex biased genes in a representative number of strains from distant geographical locations and varying stages of reproductive isolation, to search for signatures of positive selection. This thesis has offered the first insight into protist gametes transcriptomes, revealing complex, functional organization and differentiation according to the adopted sexual roles, as well as evolutionary variation of sex biased genes. Our study supports the relevance of algal systems in the research of sexual reproduction and provides a starting point for more exciting discoveries in that field

The role of visual adaptation in cichlid fish speciation

D. Shane Wright (1) , Ole Seehausen (2), Ton G.G. Groothuis (1), Martine E. Maan (1) (1) University of Groningen; GELIFES; EGDB(2) Department of Fish Ecology & Evolution, EAWAG Centre for Ecology, Evolution and Biogeochemistry, Kastanienbaum AND Institute of Ecology and Evolution, Aquatic Ecology, University of Bern.In less than 15,000 years, Lake Victoria cichlid fishes have radiated into as many as 500 different species. Ecological and sexual sel ection are thought to contribute to this ongoing speciation process, but genetic differentiation remains low. However, recent work in visual pigment genes, opsins, has shown more diversity. Unlike neighboring Lakes Malawi and Tanganyika, Lake Victoria is highly turbid, resulting in a long wavelength shift in the light spectrum with increasing depth, providing an environmental gradient for exploring divergent coevolution in sensory systems and colour signals via sensory drive. Pundamilia pundamila and Pundamilia nyererei are two sympatric species found at rocky islands across southern portions of Lake Victoria, differing in male colouration and the depth they reside. Previous work has shown species differentiation in colour discrimination, corresponding to divergent female preferences for conspecific male colouration. A mechanistic link between colour vision and preference would provide a rapid route to reproductive isolation between divergently adapting populations. This link is tested by experimental manip ulation of colour vision - raising both species and their hybrids under light conditions mimicking shallow and deep habitats. We quantify the expression of retinal opsins and test behaviours important for speciation: mate choice, habitat preference, and fo raging performance

Detecting Selection on Noncoding Nucleotide Variation: Methods and Applications

There has been a long tradition in molecular evolution to study selective pressures operating at the amino-acid level. But protein-coding variation is not the only level on which molecular adaptations occur, and it is not clear what roles non-coding variation has played in evolutionary history, since they have not yet been systematically explored. In this dissertation I systematically explore several aspects of selective pressures of noncoding nucleotide variation: The first project (Chapter 2) describes research on the determinants of eukaryotic translation dynamics, which include selection on non-coding aspects of DNA variation. Deep sequencing of ribosome-protected mRNA fragments and polysome gradients in various eukaryotic organisms have revealed an intriguing pattern: shorter mRNAs tend to have a greater overall density of ribosomes than longer mRNAs. There is debate about the cause of this trend. To resolve this open question, I systematically analysed 5’ mRNA structure and codon usage patterns in short versus long genes across 100 sequenced eukaryotic genomes. My results showed that compared with longer ones, short genes initiate faster, and also elongate faster. Thus the higher ribosome density in short eukaryote genes cannot be explained by translation elongation. Rather it is the translation initiation rate that sets the pace for eukaryotic protein translation. This work was followed by modelling studies of translation dynamics in a yeast cell. Chapter 3 concerns detecting selective pressures on the viral RNA structures. Most previous research on RNA viruses has focused on identifying amino-acid residues under positive or purifying selection, whereas selection on RNA structures has received less attention. I developed algorithms to scan along the viral genome and identify regions that exhibit signals of purifying or diversifying selection on RNA structure, by comparing the structural distances between actual viral RNA sequences against an appropriate null distribution. Unlike other algorithms that identify structural constraints, my approach accounts for the phylogenetic relationships among viral sequences, as well the observed variation in amino-acid sequences. Applied to Influenza viruses, I found that a significant portion of influenza viral genomes have experienced purifying selection for RNA structure, in both the positive- and negative-sense RNA forms, over the past few decades; and I found the first evidence of positive selection on RNA structure in specific regions of these viral genomes. Overall, the projects presented in these chapters represent a systematic look at several novel aspects of selection on noncoding nucleotide variation. These projects should open up new directions in studying the molecular signatures of natural selection, including studies on interactions between different layers at which selection may operate simultaneously (e.g. RNA structure and protein sequence)

Simulations and Modelling for Biological Invasions

Biological invasions are characterized by the movement of organisms from their native geographic region to new, distinct regions in which they may have significant impacts. Biological invasions pose one of the most serious threats to global biodiversity, and hence significant resources are invested in predicting, preventing, and managing them. Biological systems and processes are typically large, complex, and inherently difficult to study naturally because of their immense scale and complexity. Hence, computational modelling and simulation approaches can be taken to study them. In this dissertation, I applied computer simulations to address two important problems in invasion biology. First, in invasion biology, the impact of genetic diversity of introduced populations on their establishment success is unknown. We took an individual-based modelling approach to explore this, leveraging an ecosystem simulation called EcoSim to simulate biological invasions. We conducted reciprocal transplants of prey individuals across two simulated environments, over a gradient of genetic diversity. Our simulation results demonstrated that a harsh environment with low and spatially-varying resource abundance mediated a relationship between genetic diversity and short-term establishment success of introduced populations rather than the degree of difference between native and introduced ranges. We also found that reducing Allee effects by maintaining compactness, a measure of spatial density, was key to the establishment success of prey individuals in EcoSim, which were sexually reproducing. Further, we found evidence of a more complex relationship between genetic diversity and long-term establishment success, assuming multiple introductions were occurring. Low-diversity populations seemed to benefit more strongly from multiple introductions than high-diversity populations. Our results also corroborated the evolutionary imbalance hypothesis: the environment that yielded greater diversity produced better invaders and itself was less invasible. Finally, our study corroborated a mechanical explanation for the evolutionary imbalance hypothesis – the populations evolved in a more intense competitive environment produced better invaders. Secondly, an important advancement in invasion biology is the use of genetic barcoding or metabarcoding, in conjunction with next-generation sequencing, as a potential means of early detection of aquatic introduced species. Barcoding and metabarcoding invariably requires some amount of computational DNA sequence processing. Unfortunately, optimal processing parameters are not known in advance and the consequences of suboptimal parameter selection are poorly understood. We aimed to determine the optimal parameterization of a common sequence processing pipeline for both early detection of aquatic nonindigenous species and conducting species richness assessments. We then aimed to determine the performance of optimized pipelines in a simulated inoculation of sequences into community samples. We found that early detection requires relatively lenient processing parameters. Further, optimality depended on the research goal – what was optimal for early detection was suboptimal for estimating species richness and vice-versa. Finally, with optimal parameter selection, fewer than 11 target sequences were required in order to detect 90% of nonindigenous species

Genome-wide estimation of recombination, mutation and positive selection enlightens diversification drivers of Mycobacterium bovis

Genome sequencing has reinvigorated the infectious disease research feld, shedding light on disease epidemiology, pathogenesis, host–pathogen interactions and also evolutionary processes exerted upon pathogens. Mycobacterium tuberculosis complex (MTBC), enclosing M. bovis as one of its animal-adapted members causing tuberculosis (TB) in terrestrial mammals, is a paradigmatic model of bacterial evolution. As other MTBC members, M. bovis is postulated as a strictly clonal, slowly evolving pathogen, with apparently no signs of recombination or horizontal gene transfer. In this work, we applied comparative genomics to a whole genome sequence (WGS) dataset composed by 70 M. bovis from diferent lineages (European and African) to gain insights into the evolutionary forces that shape genetic diversifcation in M. bovis. Three distinct approaches were used to estimate signs of recombination. Globally, a small number of recombinant events was identifed and confrmed by two independent methods with solid support. Still, recombination reveals a weaker efect on M. bovis diversity compared with mutation (overall r/m= 0.037). The diferential r/m average values obtained across the clonal complexes of M. bovis in our dataset are consistent with the general notion that the extent of recombination may vary widely among lineages assigned to the same taxonomical species. Based on this work, recombination in M. bovis cannot be excluded and should thus be a topic of further efort in future comparative genomics studies for which WGS of large datasets from diferent epidemiological scenarios across the world is crucial. A smaller M. bovis dataset (n= 42) from a multi-host TB endemic scenario was then subjected to additional analyses, with the identifcation of more than 1,800 sites wherein at least one strain showed a single nucleotide polymorphism (SNP). The majority (87.1%) was located in coding regions, with the global ratio of non-synonymous upon synonymous alterations (dN/dS) exceeding 1.5, suggesting that positive selection is an important evolutionary force exerted upon M. bovis. A higher percentage of SNPs was detected in genes enriched into “lipid metabolism”, “cell wall and cell processes” and “intermediary metabolism and respiration” functional categories, revealing their underlying importance in M. bovis biology and evolution. A closer look on genes prone to horizontal gene transfer in the MTBC ancestor and included in the 3R (DNA repair, replication and recombination) system revealed a global average negative value for Taijima’s D neutrality test, suggesting that past selective sweeps and population expansion after a recent bottleneck remain as major evolutionary drivers of the obligatory pathogen M. bovis in its struggle with the host.info:eu-repo/semantics/publishedVersio