The thesis brings new phylogenetic data on the rapidly growing bacterial genus Arsenophonus, and summarizes the current evolutionary picture of its symbiotic association with insects and other hosts. The study is prepared in form of a regular publication and will be submitted in microbiological journal.

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The prevailing use of chemical pesticides has generated several problems including insecticide resistance, outbreak of secondary pests, safety risks for humans and domestic animals, contamination of ground water and decrease in biodiversity among other environmental concerns (Webster, 1982). These problems and the nonsustainability of control programs based mainly on conventional insecticides have stimulated increased interest in the development and implementation of costeffective, environmentally safe alternatives to chemical pesticides for insect pest control. One of the most promising strategies to help minimize dependence on chemical pesticides has been the recent application of entomopathogenic nematodes (EPNs) as biocontrol agents. EPNs in the families Steinernematidae and Heterorhabdidae have been shown to have considerable potential as biological control agents. As a natural process, biological control has the potential to play an important role in the suppression of field crop pests in agriculture. EPNs as biocontrol agents have the following advantages: high virulence, safety to non target organisms, ability to search for hosts, high efficacy in favourable habitats, high reproductive potential, ease of mass production, ease of application (Ferron & Deguine, 1996). To isolate the EPNs in South African soil, 200 soil samples were randomly collected from 5 locations in the agricultural research council (ARC) Pretoria, Gauteng province in April 2006; and 5 locations in Brits, North West province in March, 2006. At the different collection sites, soil samples were obtained from soils associated with various types of vegetation. The nematodes were collected from sandy soil by the insect-baiting technique and maintained successfully in vivo for 12 months on Galleria mellonella (G. mellonella), 4 months on Tenebrio molitor (T.molitor); 2 months Pupae and in vitro (lipid agar) for 2 weeks in the laboratory. Out of a total of 200 soil samples that were baited, 2 were found to be positive for EPNs.EPNs. IV In addition to completing Koch’s postulates, the colour of cadavers infected by the putative EPNs were also used as a diagnostic characteristic for categorizing the nematode isolates. Characterization and identification of the EPN isolates were based on morphological characters, as well as on a molecular marker (18S rDNA). On the basis of the morphological and molecular data that was obtained both of the EPNs isolates were placed in the family Heterorhabdidae: Heterorhabditis bacteriophora (H. bacteriophora) and Heterorhabditis zealandica (H. zealandica). Also from the phylogenetic trees generated from the 18S rDNA sequence, the indigenous putative H. bacteriophora was shown to be closely related to H. bacteriophora (accession number EF690469) and indigenous putative H. zealandica to H. zealandica (accession number AY321481). The two EPNs were found associated with Gram negative rod-shaped bacteria. The bacterial symbionts of the two isolates were isolated and a region of the 16S rDNA gene was sequenced. National Center for Biotechnology Information (NCBI-BLAST) results of the 16S rDNA sequence obtained showed the endosybiotic bacteria to be Photorhabdus luminescens laumondii (P. laumondii) (H. bacteriophora) and Photorhabdus sp (H. zealandica). Results of the tree showed that isolates from H. bacteriophora appeared to be closely related to P. luminescens subsp laumondii strain TT01 Ay 278646. The isolates from H. zealandica appeared to be most closely related to Photorhabdus sp Accession number: Q 614 Ay 216500). Bioassays were used to determine the infectivity of the two EPNs. In this experiment different infective juvenile (IJs) concentrations (5, 10, 25, 50, 100,200 400 and 500) of the two EPNs were applied per G. mellonella; T. molitor larva and pupae. The bioassay was carried out in two parts. In the first part, mortality data was collected for H. bacteriophora and H. zealandica. The results showed that the degree of susceptibility of G. mellonella, T. molitor larvae and pupae to each nematode species was different. When 24 h post-exposure mortality data for larvae exposed to the IJs of H. bacteriophora and H. zealandica were analyzed, ANOVA showed no differences V in mortality between insects exposed to different H. bacteriophora IJ doses (Fig: 8.1 ABC). However, there were significant differences in mortality between insects exposed to different IJ doses of H. zealandica such as 5 and 500 IJs/insect (Fig: 8.2 ABC) Therefore, no differences were noted when mortality data was compared between IJ doses at both 72 h and 96 h following IJ application to the insects. The highest susceptibility was observed with G. mellonella followed by T. molitor pupae and then T. molitor larvae. According to Caroli et al., (1996), the total mortality of insect such as G. mellonella and other lepidopterans, was reached within 24-72 h of exposure to nematodes at concentrations such as those tested here. In this study similar results were observed with high concentration of nematodes (100, 200 and 500). In the second part of the dose response bioassay, the number of progeny IJs emerging from EPN-infected cadavers was determined for all two EPNs. The results indicate that IJ progeny production differed among the three insect hosts used, the IJ doses they were exposed to, as well as the EPN species (Figs 8.3 & 8.4). The highest number of emerged IJs of H. zealandica was produced by G. mellonella (mean ± SEM: 220500 ± 133933 IJs), followed by T. molitor larvae (mean ± SEM: 152133 ± 45466 IJs) and the lowest then T. molitor pupae (mean ± SEM: 103366 ± 56933 IJs).

>Magister Scientiae - MSc / Pharmaceutical research and development strategies rely on the constant discovery of novel natural products as potential drugs. Recent studies have shown that the microorganisms associated with sponges are the true producers of some previously isolated compounds. This study created a large collection of bacterial symbionts associated with the South African marine sponge, Hamacantha esperioides. The bioactivity assays performed, showed that 44 isolates produced compounds with antimicrobial or anti-inflammatory activity. The successful identification of novel species that produce potential natural products highlights the importance of cultivation-dependent methods. To further screen for natural products, a cultivation-independent approach was used. A sequenced-based method, based on the biosynthetic genes of polytheonamide, was developed to screen for proteusins in sponge metagenomic DNA and the genomes of bacterial symbionts. The degenerate primers could amplify the targeted genes from DNA known to contain homologues. Evaluation of the primers' specificity showed non-specific amplification of genes, some containing similar conserved domains as the target genes. This study demonstrated that the use and development of cultivation-dependent and -independent screens are important for the discovery of novel natural products from the symbiotic bacteria of South African sponges. / National Research Foundation (NRF)

Polymastigidae is one of the five families of oxymonads (Metamonada, Preaxostyla). It includes small flagellates inhabiting the digestive tract of insects. Recently obtained molecular phylogenetic data have questioned the monophyly of this group. The type genus of the family Polymastigidae is Polymastix. The phylogenetic position of this genus has not been studied yet, although this information could significantly help to clarify the relationships within this group. This work provides the first sequence data of this genus, which we have obtained using a single- cell genome amplification and subsequent whole genome sequencing on Illumina HiSeq X Ten (Macrogene) from three individuals of P. melolonthae inhabiting the posterior hindgut of crane fly larvae. Phylogenetic analysis based on the gene for 18S rRNA, EF-1α and HSP90 yielded several roughly equally probable hypotheses about the position of this genus within Oxymonadida. The first of them places the genus Polymastix in a close proximity to the genus Streblomastix, according to the second hypothesis, Polymastix is sister to Termitimonas travisae. A characteristic feature of this protist is ectosymbiotic bacteria attached to its surface. These were necessarily amplified and sequenced together with their hosts, which allowed us to obtain sequence...

During the evolution some groups of organisms have become coevolutionary associated with other groups, as is the case of host symbiont systems. To explore coevolutionary history of hosts and their associated symbionts, phylogenetic reconstruction of symbionts and phylogenetic reconstruction of hosts are usually compared. Coevolution is described by coevolutionary events (cospeciation, host switch, duplication, failure to diverge events and linage sorting events). The aim of this work was to test the suitability of MLST method for the complex of entomopathogenic nematodes from the genus Steinernema (with detailed analysis of Steinernema feltiae) and their symbiotic bacteria Xenorhabdus bovienii and subsequently use cophylogenetic comparative analysis to determine their level of cospeciation.

Since the introduction of advanced molecular methods the research on insect bacterial symbioses underwent a major focus shift towards large scale phylogenetics and comparative genomics. These new fields provided answers to several fundamental questions of symbiont evolution, functional capabilities of the host-associated bacteria, and the role of symbionts in the host?s biology. However, the vast diversity and complexity of symbiotic relationships still leaves gaps in our understanding to a rich mosaic of various symbiont types, effects and transitions from facultative association to obligate mutualism. The presented study focuses on distribution, diversity, phylogenetic patterns, evolutionary transitions and genome evolution of two less known but ecologically diverse bacterial genera, Arsenophonus and Sodalis. The thesis also takes advantage of the knowledge on a well established symbiotic model between aphids and Buchnera and reveals several evolutionary patterns in the host and symbiont.

Les Nématodes Entomopathogènes (NEP) de la famille des Steinernematidae et Heterorhabditidae sont des parasites pour les insectes. Ils hébergent dans leur intestin un symbiote bactérien (genres Xenorhabdus et Photorhabdus) essentiel au succès parasitaire. Les NEP sont présents dans les sols des cinq continents de la planète. Leur diversité génétique et leurs propriétés biologiques constituent une intéressante ressource biologique. Grâce à leur entomotoxicité, les NEP sont de bons outils de lutte biologique en agriculture et en culture ornementale un peu partout dans le monde. La diversité et la biogéographie des NEP dans les cinq continents de la Terre (à l'exception de l'Antarctique) ont été étudiées partout dans le monde mais le Liban est parmi les rares pays du moyen orient où aucune prospection de ces nématodes n'a été réalisée alors que des NEP en Turquie, Syrie, Jordanie, Palestine et Egypte ont déjà été trouvés et caractérisés. L'objet de la thèse est de procéder à une étude biogéographique dans le but de connaître la diversité des NEP au Liban. L'enjeu scientifique est donc de combler un « vide » dans la connaissance de la répartition et de la biodiversité mondiale des NEP. Pour cela, un échantillonnage à l'échelle des étages de végétation est mené au Liban. Des échantillons de sol sont ainsi prélevés, mis en contact avec des larves de Galleria mellonella pour isoler les nématodes entomopathogènes et leurs symbiotes. Les nématodes et leurs symbiotes sont par la suite identifiés morphologiquement et moléculairement. Par la suite, une approche à l'échelle de l'habitat fait l'objet de cette thèse également pour étudier les interactions biotiques et abiotiques influençant la présence des nématodes entomopathogènes dans le sol. Les enjeux technologiques, exposés au second volet de la thèse, sont liés aux propriétés biologiques des nématodes et de leurs symbiotes afin de valoriser leur entomotoxicité en lutte biologique. Dans ce cadre, la sensibilité des Cephalcia tannourinensis, ravageur des cédraies au Liban, par rapport aux nématodes entomopathogènes est exploitée in vitro ; différentes espèces de nématodes entomopathogènes sont testées pour suivre leur cycle à l'intérieur des Cephalcia. / Entomopathogenic nematodes (EPNs) are parasites of soil-dwelling insects that occur in natural and agricultural soils around the world. Thanks to their entomotoxicity, EPNs are good tools for biological control in agriculture almost everywhere in the world. They are ubiquitous, having been isolated from every inhabited continent (except Antartica) from a wide range of ecologically diverse soil habitats including cultivated fields, forests, grasslands, deserts, and even ocean beaches. Biogeographic assessments of EPNs in the Eastern Mediterranean basin have been conducted in several countries such as Turkey, Syria, Jordan, Israel, Palestine and Egypt. Lebanon is among the few countries of the Middle East for which no survey of EPNs has been done. The scientific stake is thus to fill a gap in our knowledge of EPNs distribution in the Mediterranean basin. Survey of EPNs was conducted in this framework to cover the different vegetation levels defined in Lebanon. Soil samples were removed placed in contact with Galleria mellonella to isolate entomopathogenic nematode and their symbiotic bacteria. EPNs and their bacteria were then identified morphologically and molecularly. On the other hand, despite the different national surveys conducted on EPNs distribution around the world, habitat preferences remain inadequately known for entomopathogenic nematodes. Therefore, a comprehensive understanding of their distribution and the various biotic and abiotic factors influencing their presence is also a second object of our work. Beside a technological approach related to the biological properties of the nematodes and their symbiotics: valorisation of the entomotoxicity in biological control will be part of the third shutter of the thesis. In this framework, the sensibility of cedar pests, Cephalcia tannourinensis against entomopathogenic nematodes is exploited in vitro; different EPNs species were tested to study their life cycle inside Cephalcia larvae.