One in four known species of fruit flies inhabit the Hawaiian Islands. From a small number of colonizing flies, a wide range of species evolved, some of which managed to reverse-colonize other continental environments. In order to explore the developmental pathways, which separate the Hawaiian Drosophila proper and the Scaptomyza group that contains reverse-colonized species, the transcriptomes of two better-known species in each group, Scaptomyza anomala and Drosophila grimshawi, were analyzed to find changes in gene expression between the two groups. This study describes a novel transcriptome for S. anomala studies as well as unusual changes in gene expression in D. grimshawi relative to other species, revealing priorities of both species in early development.

Sorghum crop has become attractive to breeders due to its drought tolerance, and many uses including a human food source, animal feed, industrial fibre and bioenergy crop. Sorghum, like any other plant, is a host to a variety of microbes that can have neutral, negative or positive effects on the plant. While the majority of microorganisms are beneficial, pathogens colonize plant tissues and overwhelm its defence mechanisms. This colonization is a direct threat to the sorghum productivity. The development of microbiome-based approaches for sustainable crop productivity and yield is hindered by a lack of understanding of the main biotic factors affecting the crop microbiome. Metabarcoding has proven to be a valuable tool which has been widely used for characterizing the microbial diversity and composition of different environments and has been utilized in many research endeavours. This study analyses the relationship between the microbiota and their response to natural pathogen infection in sorghum disease groups (R, MR, S and HS) and identifies the most dominant pathogen in the highly susceptible disease group. The study also, assesses the spore viability through the use of the automated cell counter and confirms Fusarium graminearum (dominant pathogen linked to the HS disease group) through sequencing of the marker genes, to subsequently characterize pathways likely to be involved in pathogen infection resistance. To achieve the objectives, a combination of 16S rRNA (V3/V4 regions) and ITS (ITS1/ITS4) of the internal transcribed spacer regions were amplified and sequenced using NGS technologies to study the microbiota in response to natural infection. Additionally, comparative transcriptional analysis of sorghum RILs in response to Fusarium graminearum infection was conducted through RNA-Seq. Upon natural infection, the foliar symptoms assessment of the RILs was conducted and four disease groups; resistant (R), moderately resistant (MR), susceptible (S) and highly susceptible (HS) were designated. The results of the present metabarcoding study indicate that resistant sorghum leaves (R group) supported a large diversity of fungal and bacterial microbes. The genera Methylorubrum, Enterobacter and Sphingomonas with reported plant growth promoting traits were more abundant and highly enriched in the R and MR group, with members of the latter genus significantly enriched in the R group. The resistant fungal group had a majority of OTUs showing similarity to well-known plant growth-promoting fungal genus including Papiliotrema (Tremellaceae family), which are known biocontrol agents. The yeast Hannaella was also highly linked with the resistant plants. Some Hannaella species are known to produce indole acetic acid (IAA) for promoting plant growth. Metabarcoding was also used to assess the major potential disease-causing taxa associated with the highly diseased group. It identified fungal pathogenic species, that have not previously been identified as pathogens of sorghum such as Ascochyta paspali and Ustilago kamerunensis (which are known pathogenic fungi of grass species) and were associated with the susceptible disease groups (S and HS). These analyses revealed the potential sorghum fungal pathogen Epicoccum sorghinum, and was highly linked with the S disease group. It further expanded the identification of a reportedly economically importance species causing sorghum related diseases Fusarium graminearum (anamorph Gibberella zeae). This species has also been identified in this study to be highly associated with the RILs showing major disease symptoms. Fusarium graminearum a significant pathogen in winter cereals and maize has been associated with stalk rot of sorghum and sorghum grain mould. The presence of Fusarium graminearum in sorghum can be a toxicological risk, since this species has the potential to produce mycotoxins. It was further shown that natural pathogen infection results in distinct foliar microbial communities in sorghum RILs. The co-occurrence taxa represented by Tremellomycetes and Dothiomycetes fungal classes and Bacillaceae and Sphingomonadaceae bacterial family had more central roles in the network. The modules which are located centrally on the network have been expected to play important ‘topological roles’ in interconnecting pairs of other fungal and bacterial taxa in the symbiont–symbiont co-occurrence network. These taxa having a central role, are considered to be keystone microbes, and have been suggested to be drivers of microbiome structure and functioning. The results of bacterial and fungal community composition, community co-occurrences further suggested the importance of keystone taxa which may disproportionately shape the structure of foliar microbiomes. The foliar disease symptom assessments revealed that sorghum RIL 131 was highly diseased and RIL 103 did not show any visible disease symptoms and were subsequently used for transcriptomic analysis. Gene expression patterns were studied between the identified RIL that did not show visible symptoms (resistant RIL no 103) and the RIL that showed major disease symptoms (susceptible RIL no 131). Fusarium graminearum the dominant potential pathogen found in this study to be associated with the highly susceptible plants was used to inoculate RILs at seedling stage in a greenhouse and samples were collected in triplicates at 24 hours post infection (hpi), 48 hpi, 7 days post infection (dpi) and 14 dpi. Prior to that, ITS and UBC genes confirmed the identity of Fusarium graminearum, and the automated haemocytometer confirmed the cell/spore viability. Using RNA-Seq analysis it was shown that the resistant RIL had defence related pathways from early response (24- 48 hpi) to late response (7-14 dpi). And the more the infection progressed, the more the defence related genes were up-regulated in terms of fragments per kilobase of exon model per million reads mapped (FPKM) and False Discovery Rate (FDR ≤ 0.05) values. Transcriptome time series expression profiling was used to characterize the plant response to Fusarium graminearum with the Dirichlet Process Gaussian Process mixture model software (DPGP) in susceptible and resistant RILs. The susceptible RIL (number 131) transcriptional response upon Fusarium graminearum infection presented differences of the closely related clustered expression profiles across all timepoints in both RILs. Group 2 exclusively clustered the genes encoding the sesquiterpene metabolism pathway, which is one of the major physiological change occurring in response to fungal infection and has been previously reported to produce the mycotoxins associated with Fusarium head blight (FHB) of cereals. This pathway presented an increase from the initial infection phase to the late infection phase in group 4, the genes encoding starch sucrose, metabolism and cyanoamino acid pathways presented a pattern that had a sharp decline from 48 hpi -14 dpi (at a later stage of infection). This could suggest that, as the time progresses in the susceptible RIL the pathways which are important in plant defence declines at a late infection stage. Group 3 presented a pattern increase of the 5-lipoxygenase (LOX 5) gene expressed from 48 hpi-14 dpi timepoints. The loss and silencing of LOX5 function have in the past described to be linked with enhanced disease resistance. In this study the LOX5 was expressed and this could suggest that LOX5 might have a function as a susceptibility factor in disease caused by Fusarium graminearum in sorghum RILs. CBL-interacting protein kinase 6 (CIPK6) gene was also associated with this group. This gene has been associated with negative regulation of immune response to Pseudomonas syringae in Arabidopsis as plants overexpressing CIPK6 were more susceptible to Pseudomonas syringae. Transcriptional response of a resistant RIL (number 103) to infection with Fusarium graminearum presented an increase in genes encoding metabolic and biosynthesis of metabolites pathways in group 1 and group 4 at early infection phase and a sharp decline in the late infection phase. An increase in the genes encoding pathways in earlier infection state could suggest the establishment of a beneficial energy balance for defence. Additionally, genes encoding phenylpropanoid (PAL), galactose and glycolysis pathway were amongst the genes increased at early stages of infection in group 1. Sugar can play a significant role in resistance to fungal pathogens through phenylpropanoid metabolism stimulation, and previous studies showed that the phenylpropanoid pathway could play a role in resistance of wheat to Fusarium graminearum and deoxynivalenol. Overall, this study represents a first step in understanding the molecular mechanisms involved in resistance to Fusarium graminearum. This analysis has also identified the reported beneficial microbes and defence related genes and pathways. Together, the current findings suggest that different ‘resident’ consortia found in naturally infected and uninfected sorghum plants may be viable biocontrol and plant-growth promoting targets. Cultivation studies may shed light on the nature of the putative symbiotic relationships between bacteria and fungi. These results have consequences for crop breeding, and the analysis of microbial diversity and community composition can be useful biomarkers for assessing disease status in plants. The transcriptome and metabarcoding data generated will help guide further research to develop novel strategies for management of disease in sorghum RILs through the integrative approach considering both beneficial microbes and defence related genes. This provides the baseline information and will positively impact in the development of Fusarium graminearum resistant genotypes in future through the integration/incorporation of beneficial microorganisms (bacteria and fungi) and resistant genes in breeding strategies. / Life and Consumer Sciences / D. Phil. (Life Sciences)

No description available.

Lake Baikal is the oldest, by volume, the largest, and the deepest freshwater lake on Earth. It is characterized by an outstanding diversity of endemic faunas with more than 350 amphipod species and subspecies (Amphipoda, Crustacea, Arthropoda). They are the dominant benthic organisms in the lake, contributing substantially to the overall biomass. Eulimnogammarus verrucosus, E. cyaneus, and E. vittatus, in particular, serve as emerging models in ecotoxicological studies. It was, then, necessary to investigate whether these endemic littoral amphipods species form genetically separate populations across Baikal, to scrutinize if the results obtained --~for example, about stress responses~-- with samples from one single location (Bolshie Koty, where the biological station is located), could be extrapolated to the complete lake or not. The genetic diversity within those three endemic littoral amphipod species was determined based on fragments of Cytochrome C Oxidase I (COI) and 18S rDNA (only for E. verrucosus). Gammarus lacustris, a Holarctic species living in water bodies near Baikal, was examined for comparison. The intra-specific genetic diversities within E. verrucosus and E. vittatus (13% and 10%, respectively) were similar to the inter-species differences, indicating the occurrence of cryptic, morphologically highly similar species. This was confirmed with 18S rDNA for E. verrucosus. The haplotypes of E. cyaneus and G. lacustris specimens were, with intra-specific genetic distances of 3% and 2%, respectively, more homogeneous, indicating no --or only recent disruption of-- gene flow of E. cyaneus across Baikal, and recent colonization of water bodies around Baikal by G. lacustris. The data provide the first clear evidence for the formation of cryptic (sub)species within endemic littoral amphipod species of Lake Baikal and mark the inflows/outflow of large rivers as dispersal barriers. Lake Baikal has provided a stable environment for millions of years, in stark contrast to small, transient water bodies in its immediate vicinity. A highly diverse endemic amphipod fauna is found in one but not the other habitat. To gain more insights and explain the immiscibility barrier between Lake Baikal and non-Baikal environments faunas, the differences in the stress response pathways were studied. To this end, exposure experiments to increasing temperature and a heavy metal (cadmium) as proteotoxic stressors were conducted in Russia. High-quality de novo transcriptome assemblies were obtained, covering multiple conditions, for three amphipod species: E. verrucosus and E. cyaneus -Baikal endemics-, and G. lacustris -Holarctic- as a potential invader. After comparing the transcriptomic stress responses, it was found that both Baikal species possess intact stress response systems and respond to elevated temperature with relatively similar changes in their expression profiles. G. lacustris reacts less strongly to the same stressors, possibly because its transcriptome is already perturbed by acclimation conditions (matching the Lake Baikal littoral). Comprehensive genomic resources are of utmost importance for ecotoxicological and ecophysiological studies in an evolutionary context, especially considering the exceptional value of Baikal as a UNESCO World Heritage Site. In that context, the results presented here, on the genome of Eulimnogammarus verrucosus, have been the first massive step to establish genomic sequence resources for a Baikalian amphipod (other than mitochondrial genomes and gene expression data in the form of de novo transcriptomes assemblies). Based on the data from a survey of its genome (a single lane of paired-end Illumina HiSeq 2000 reads, 3X) as well as a full dataset (two complete flow cells, 46X) the genome size was estimated as nearly 10 Gb based on the k-mer spectra and the coverage of highly conserved miRNA, hox genes, and other Sanger-sequenced genes. At least two-thirds of the genome are non-unique DNA, and no less than half of the genomic DNA is composed of just five families of repetitive elements, including low complexity sequences. Some of the repeats families found in high abundance in E. verrucosus seem to be species-specific, or Baikalian-specific. Attempts to use off-the-shelf assembly tools on the available low coverage data, both before and after the removal of highly repetitive components, as well as on the full dataset, resulted in extremely fragmented assemblies. Nevertheless, the analysis of coverage in Hox genes and their homeobox showed no clear evidence for paralogs, indicating that a genome duplication did not contribute to the large genome size. Several mate-pair libraries with bigger insert sizes than the 2kb used here and long reads sequencing technology combined with semi-automated methods for genome assembly seem to be necessary to obtain a reliable assembly for this species.

During the last years, important progress has been made in modeling early brain development using 3-dimensional in vitro systems, so-called cerebral organoids. These can be grown from pluripotent stem cells of different species such as our closest living relatives, the chimpanzees and from patients carrying disease mutations that affect brain development. This offers the possibility to study uniquely human features of brain development as well as to identify gene networks altered in neurological diseases. Profiling the transcriptional landscape of cells provides insights into how gene expression programs have changed during evolution and are affected by disease. Previously, studies of this kind were realized using bulk RNA-sequencing, essentially measuring ensemble signals of genes across potentially heterogeneous populations and thus obscured subtle changes with respect to transient cell states or cellular subtypes. However, remarkable advances during the last years have enabled researchers to profile the transcriptomes of single cells in high throughput. This thesis demonstrates how single-cell transcriptomics can be used to dissect human-specific features of the developing and adult brain as well as cellular subpopulations dysregulated in a malformation of the cortex.

In the Caribbean, Acropora cervicornis (staghorn coral) exemplifies the worldwide anthropogenic decline of reef-building corals. From the mid-Pleistocene through the mid-1900s, A. cervicornis was a dominant framework builder, providing complex habitat for reef organisms. Since the 1980s, populations of A. cervicornis have declined by as much as 98%. Despite the overall decline, scattered remnants persist, and some appear to be thriving. As in recent studies on other acroporids, if we can identify variation in traits related to resilience in the remaining A. cervicornis, and understand the genetic basis of such variation, we could better forecast the species’ future response to climate change, and inform ongoing restoration efforts. Here, I compare phenotypic and transcriptomic indicators of resilience in A. cervicornis from two nearby but environmentally-disparate habitats on Turneffe Atoll, Belize: Calabash Caye forereef and Blackbird Caye backreef. Blackbird exhibits significantly higher flow, light, average temperature, and temperature variation. Over four years, I conducted a longitudinal study of 122 tagged coral colonies. Corals from Blackbird and Calabash, which I confirmed to be genetically distinct based upon single nucleotide polymorphisms, exhibited pronounced differences in traits related to resilience including the proportion of healthy tissue, chlorophyll, growth, and wound-healing. By most measures, Blackbird corals displayed superior indicators of resilience. Through a two-year reciprocal transplant study involving 120 corals, I identified substantial environmental plasticity in these traits, e.g., Blackbird corals transplanted to Calabash exhibited higher chlorophyll levels and more rapid wound healing than when grown in Blackbird, exceeding the native Calabash corals. RNA sequencing and assembly of site-specific transcriptomes revealed greater diversity of transcripts and genes from photosynthetic symbionts at Blackbird but greater diversity of bacterial associates at Calabash. Single nucleotide polymorphism (SNP) analyses using RNAseq data determined that corals from the two sites were separate putative populations. Principal components analysis of gene expression in natives and transplants revealed a clear distinction based on site of origin, but also a clear effect of environment. Thousands of differentially expressed genes distinguished the sites, including many genes implicated in heat stress, oxidative stress and UV-light stress. This genetic and phenotypic diversity of remnant staghorn populations on Turneffe represents a potential basis for future re-expansion of this important framework builder through natural or assisted shifts toward resilient populations. / 2023-02-03T00:00:00Z

Les organes centraux du tractus urogénital sont le testicule et l’ovaire, qui assurent la production de gamètes et d’hormones, et donc la fertilité de l’individu. Ces deux organes, parfaitement distincts et complémentaires, ont pour origine une gonade bipotentielle qui s’engagera vers une trajectoire de différenciation masculine ou féminine au cours de la vie fœtale. Les deux gonades vont par la suite subir plusieurs phases de différenciation et de développement de leurs populations cellulaires, afin d’acquérir leurs fonctions propres qui leur permettront d’assumer leur rôle à l’âge adulte. Depuis plus d’une quinzaine d’années, le concept de syndrome de dysgénésie testiculaire fait état d’un lien entre l’exposition du fœtus à des composés environnementaux et des anomalies du tractus urogénital. Bien que sujette à de vifs débats au sein de la communauté scientifique, cette hypothèse a attiré l’attention de la recherche sur les conséquences de l’exposition des mères aux xénobiotiques sur l’enfant à naître. La différenciation et le développement des gonades fœtales sont gouvernés par des programmes d’expression spécifiques de chaque sexe, dont de nombreuses zones d’ombres subsistent, notamment concernant la fraction non-codante exprimée par le génome humain. La première partie de cette thèse de doctorat présente, pour la première fois, le paysage transcriptionnel contrôlant ces processus complexes entre la 6ième et 17ième semaine de développement chez l’Homme. Grâce à l’avènement des technologies de transcriptomique, il est désormais possible d’identifier et d’observer l’expression des gènes de manière sensible et sans a priori. Le RNA-seq m’a donc permis de décrire de manière exhaustive la dynamique d’expression des gènes, pendant les stades précoces de la différenciation sexuelle, jusqu’aux phénomènes plus tardifs conduisant aux linéages des différentes populations cellulaires spécifiques du testicule et de l’ovaire. Dans une deuxième partie, mon travail de recherche s’est attaché à étudier l’impact de deux perturbateurs endocriniens suspectés, l’ibuprofène et le chlordécone, sur le programme d’expression du testicule fœtal humain. L’utilisation du RNA-seq m’a permis de définir et de comparer la signature toxicogénomique de chaque molécule afin de contribuer à la compréhension de leur mécanisme d’action et d’identifier les populations cellulaires affectées. Enfin, face à l’essor des technologies ultra-haut-débit dans les sciences de la vie, y compris dans les domaines de la reproduction, j’ai activement participé au déploiement d’une nouvelle version du Reprogenomic Viewer dans la dernière partie de ma thèse (http://rgv.genouest.org). Cet outil nternet a pour vocation de centraliser et de rendre accessibles les données de séquençage accumulées au sein de la communauté de la reproduction via des outils de visualisation intuitifs. / Fetal life is a crucial period for sexual reproduction when bipotential gonads differentiate into either a testis or an ovary. Gaining insights into the complex molecular events underlying this process is central to a better understanding of disorders of sexual development. The present work intends to improve the knowledge on molecular pathways at play during gonad development in humans using RNA-sequencing. This project particularly seeks to identify early transcriptional events that may play critical role in the regulatory network driving human sexual differentiation. To address this issue, we defined the transcriptional landscape of fetal human gonads by sequencing total RNA extracted from testes and ovaries between 6 and 17 gestational weeks. The resulting paired-end reads were mapped on the human genome and then assembled into transcripts using the Tuxedo suite. We next defined a high-confidence set of transcripts showing differential expression across samples. Clusters of co-expressed genes were subjected to functional analysis. The analysis of this massive RNA-seq dataset has led to a high-confidence set of 35,194 assembled transcripts; among which 32,391 known and novel isoforms coding genes (mRNAs), 1,209 to long non-coding (lnc) RNAs and 318 to novel unannotated transcripts/genes (NUTs). The dynamic of transcriptional landscape occurring during human fetal gonads development has been described and new genes and interesting candidates, including new genes, have been highlighted as potential key genes governing this biological process. The second interest of this work was the study of the impact of two endocrine disruptors, ibuprofene and chlordecone, on human fetal testis using RNA-seq. The transcriptional alteration induced by these compound in the gonad allowed a deeper understanding of their mechanisms of action of endocrine disruption. The last part of this work was the development of a new version of the ReproGenomics Viewer (http://rgv.genouest.org), a web tool dedicated to the integration and accumulation of sequencing data from studies performed in the field of reproduction.

Naphthenic acids (NAs) are oil-derived mixtures of carboxylic acids and are aquatic contaminants of emerging concern. The objective of the research presented in this thesis was to investigate the toxicity of NAs in tadpoles of the frog Silurana (Xenopus) tropicalis. Using electrospray ionization high-resolution mass spectrometry (ESI-HRMS), I determined that the proportions of O2 (presumably carboxylic acid moiety) species were 98.8, 98.9 and 58.6% respectively, for two commercial extracts (S1 and S2), and acid extractable organics (AEOs) from oil sands process-affected water (OSPW). The rank order potency based on the lethal concentration fifty (LC50) and effect concentration fifty (EC50) with and without normalization for the quantity of O2 species was S1 > S2 > AEO. The main effects observed were reduced body size, edema, and cranial, cardiac, gut and ocular abnormalities. Oligonucleotide microarray technology was used to determine the transcriptomic responses in developing S. tropicalis embryos following exposure to S1 and S2 at a sub-lethal concentration of 2 mg/L. Some of the significantly enriched pathways (p < 0.05) included metabolism and cell membrane depolarization, and some were related to observed abnormalities including edema, gastrointestinal system, and cartilage differentiation. I established and validated a derivatization method for NAs using pentafluorobenzyl bromide (PFBBr) prior to gas chromatography-electron impact mass spectrometry (GC-EIMS) to increase chromatographic resolution, and sensitivity, compared to boron trifluoride-methanol (BF3/MeOH) and N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA). Solid-phase microextraction of volatiles originating from S1, S2, Merichem NAs and an AEO mixture led to the identification of 54, 56, 40 and 4 compounds, respectively. The compounds identified in the mixtures included aliphatic and cyclic hydrocarbons, carboxylic acids, alkyl-benzenes, phenols, naphthalene and alkyl-naphthalene, and decalin compounds. To determine the chemical nature of the toxic compounds in NA mixtures, the S2 and AEOs preparations were fractionated using open column chromatography. A non-polar and a polar fraction were obtained from S2. Overall, the toxicity of the polar fraction was not significantly different from whole S2 (p > 0.05). Six fractions of AEOs were obtained, however because of limited material, only the toxicities of F3 and F4 were assessed. The toxicity of F3 was significantly lower than AEOs (p < 0.05) and F4 was not toxic for S. tropicalis (p > 0.05). These results suggest that during fractionation, toxic compounds were lost or that the toxicity of AEOs results from the combined effects of the compounds present in the whole extract. The toxicological dose descriptors, morphometric, transcriptomic and chemical analysis herein presented may contribute to the development of environmental guidelines for NAs and AEOs.

No description available.

No description available.