Letteratura scientifica selezionata sul tema "Insect microflora"

Flaxseed (Linum usitatissimum L. 'McGregor') stored at 10, 20, 30, 40, and 50 °C and 35, 50, 60, and 70% relative humidity (RH) for up to 12 mo deteriorated in quality in < 1–3 mo at the two highest temperatures, although the seed was stored "dry" (≤ 10% moisture content, MC). Initial fatty acid values (FAV) of 41.1 mg KOH 100 g−1 dry seed (0.51% free fatty acids in oil) rarely increased more than 1.5-fold over 12 mo at 10 or 20 °C and up to 10% MC, or at 30 °C and 7 to 8% MC. Using FAV as a storage quality-loss criterion, flaxseed at 8–9% MC could be stored for 6 mo at 30 °C, 1–2 mo at 40 °C, or a few weeks at 50 °C with less than a 1.5-fold increase. A twofold increase in FAV was correlated to a discoloured or charred appearance of seeds and a rapid loss in seed germination. Seed germination did not decrease during 12 mo at 10 or 20 °C and 70% RH, or at 30 °C and 60% RH. The fungi Aspergillus glaucus group, A. candidus Link, and Penicillium spp. infected seed at some temperatures and relative humidities with low frequency by 6 mo, and A. flavus Link also occurred at 12 mo. Visible microflora were absent after 6 mo on seed at 40 and 50 °C. The beetles Oryzaephilus mercator (Fauvel), O. surinamensis L., Tribolium castaneum (Herbst), and T. confusum J. duVal survived and produced some larvae on both whole and ground flaxseed in 2 mo. McGregor was slightly more suitable for insect reproduction than NorMan or NorLin. Five other insect species could not survive. Extensive infestation of flaxseed by stored-product insects is unlikely. Key words: Flaxseed, storage, free fatty acids, germination, microflora, insects

RNA interference (RNAi) methods for insects are often limited by problems with double-stranded (ds) RNA delivery, which restricts reverse genetics studies and the development of RNAi-based biocides. We therefore delegated to insect symbiotic bacteria the task of: (i) constitutive dsRNA synthesis and (ii) trauma-free delivery. RNaseIII-deficient, dsRNA-expressing bacterial strains were created from the symbionts of two very diverse pest species: a long-lived blood-sucking bug, Rhodnius prolixus , and a short-lived globally invasive polyphagous agricultural pest, western flower thrips ( Frankliniella occidentalis ). When ingested, the manipulated bacteria colonized the insects, successfully competed with the wild-type microflora, and sustainably mediated systemic knockdown phenotypes that were horizontally transmissible. This represents a significant advance in the ability to deliver RNAi, potentially to a large range of non-model insects.

Analysis of microbial communities associated with plants insects and soil has been a significant challenge for plant protection researchers because of the lack of techniques with which to access these populations A new molecular community profiling technique 16S rRNA genebased PCR followed by denaturing gradient gel electrophoresis (DGGE) has been used to analyse microbial communities in a number of environments and has many potential uses in plant protection research The technique is currently being used for the analysis of insect gut microflora characterisation of phylloplane and rhizosphere microbial populations and in environmental assessment of the effects of biopesticides and new technologies on indigenous soil microbes

SUMMARYBased on the ability of bacterial associates of entomopathogenic nematodes to produce antibiotic compounds on artificial media, it has been commonly accepted thatXenorhabdussp. andPhotorhabdussp. inhibit a wide range of invading microorganisms in insects infected withSteinernemaspp. orHeterorhabditisspp. Therefore, the question of whether antibiotic compounds produced by the primary form of bacterial symbionts associated mutualistically withS. carpocapsaeandH. bacteriophoraexplain why insect carcasses do not putrefy but provide nutritional requirements for insect parasitic rhabditoid nematodes to complete their life-cycle was examined. Laboratory bioassays of anti-bacterial activity on nutrient agar and during parasitism in larvae ofGalleria mellonellahave confirmed earlier observations thatin vitrocolonies of the primary form ofX. nematophilusandP. luminescensproduced agar-diffusible antibiotic compounds of a broad spectrum of anti-bacterial activity; their role in parasitism seems doubtful, however. This hypothesis is supported by a low antibiotic potency of a limited spectrum of anti-bacterial activity throughout the life-cycle of the parasites, principally inGalleriainfected withS. carpocapsae. Since the lack of putrefaction cannot be explained simply by antibiotic inhibition of contaminating bacterial microflora, other competition mechanisms must be operating in parasitized insects. I postulated that a rapid and massive colonization of the insect body by nematophilic bacteria creates unfavourable conditions for the growth and multiplication of bacterial (proteolytic) contaminators making the insect carcass decay-resistant. In the case ofH. bacteriophora, low antibiotic activity at an early stage of parasitism could support the colonization byP. luminescensof the host.

The aim of the study is to identify fungi associated with fruit rot of pawpaw both in the field and in storage. Three experiments were conducted. In the first experiment, fungi growing on ripe pawpaw fruits randomly collected from plants on the UCC campus were identified in the laboratory. The position on the fruit the fungi appeared was recorded. The second experiment involved the storage of fruits in insect proof containers in the laboratory to indentify fungi growing onthe fruits and the position on the fruit the fungi appeared. In the third experiment, fruits were washed with 10% solution of Milton 2 before they were stored in insect proof containers. Fungi which grew on them and the position on the fruit the fungi appeared were identified. The survey revealed that Collectotricum gloesporiodes which cause anthracnose was the most frequently occurring fungi in the field in the study area. The most serious fungi diseases of pawpaw in storage were found to be anthracnose, stem end rot and black rot which were caused by Collectotricum gloesporiodes, Phomopsis caricae-papayae andPhoma caricae-papayae. It was demonstrated that surface sterilization with 10% Milton 2 solution increased the shelf life of the stored fruits for 2-3 days. Keywords: Carica papaya; Microflora; Milton 2; Surface sterilization; Frequency of occurrence.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 ìm2) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.

The perceived benefits of food grown under organic (low-input) management practices include lower levels of potentially harmful pesticides and an increased awareness of environmental protection. It is also believed that such crops are inherently more tolerant to biotic stresses, although there is little evidence to support this. The aims of this thesis are to investigate differential effects of conventional and organic management practices on soil microbial community structure and consequences for plant growth using Arabidopsis thaliana, as the model plant species. Soil used in the study was from ‘Palace Leas hay plots long term field trial’ where Plot 2 is fertilised using farmyard manure (FYM) and Plot 13 is fertilised using inorganic fertilisers. Changes in community structure of the two different fertiliser amendments were measured over time using T-RFLP. The laboratory addition of inorganic fertiliser had a significant effect on the bacterial community (p=0.047); however, neither initial field treatment nor time had any effect (p=0.263, p=0.842, respectively). The fungal community showed no significant effect from field treatment, laboratory amendment or time (p=0.342, p=0.131, p=0.219). A. thaliana plants grown in conventionally fertilised soil (Plot 13) grew slower and were smaller than those grown in FYM amended soil (Plot 2). After 28 days, rosette area was 552.1cm2 and 418.4cm2 for plants grown with FYM and mineral amendment, respectively (p=0.01). Dry weights of the rosette were 92.6mg and 72.8mg for FYM and conventionally grown plants respectively (p=0.044). Levels of boron (org=0.73ppm, conv=0.31ppm at 30 days), calcium (org=177.7ppm, conv=124.9ppm, at 30 days), potassium (org=331.0ppm, conv=161.5ppm at 30 days) and magnesium (org=29.5ppm, conv=18.3ppm at 30 days) were all significantly higher in FYM grown plants. The ratios of K:Ca and K:Mg in plants grown on FYM amended soil were higher than for those grown on mineral fertiliser amended soil, indicating that the latter plants were under increased stress. Analysis of the leaf proteome demonstrated that nitrilase 1, 14-3-3 like protein GF14, Heat Shock Cognate 70kDa Protein 1, and glutathione-S-transferases PM24 and ERD13 were up-regulated in conventionally grown plants, whilst lipoxygenase and Annexin D6 were downregulated. Again, this indicates that A.thaliana is under increased stress when grown in conventionally treated soil. Glucosinolates and volatiles, produced as part of the plant’s inducible defence system, were investigated by HPLC/MS. There were both qualitative and quantitative differences in glucosinolate profiles. Plants grown on FYM amended soils had higher levels of the alkyl glucoside gluconaprin, whilst those on the conventionally treated soil contained glucoraphanin and glucoerucin. Subsequent aphid infestation resulted in a small (7% and 8% for conventionally and FYM grown plants, respectively) increase in the indole glucosides. Those grown on the conventionally amended soil produced 26 different volatile chemicals compared to 18 from the FYM amended soil (Plot 2); average levels of volatiles released where higher from the former than the latter. The results indicate that there was little effect of amendment practice on direct defence mechanisms in A. thaliana, although indirect defence was altered by amendment practice. The interactions between fertiliser amendment, soil microbial community, and plant and aphid herbivores were investigated using Structural Equation Modelling. The only significant relationship was a positive correlation between organic FYM amendment and rate of plant growth (p=0.004), indicating that the use of FYM as an additional amendment increased plant growth. The effect of the rate of plant growth on the rate of aphid reproduction was almost significant and negative (p=0.067), indicating that the rate of plant growth negatively impacted the rate of aphid reproduction.

De nombreuses études ont jusqu'ici considéré les infections comme étant des interactions deux-à-deux, entre un hôte et un pathogène, minimisant ainsi la complexité du processus infectieux. En effet, les infections sont souvent très hétérogènes, menant à des interactions plus complexes. Au cours de ce travail, nous cherchons à répondre à deux questions: (i) La transmission d'un pathogène peut-elle être impactée lorsque de l'hétérogénéité phénotypique apparaît dans sa population au cours de l'infection ? (ii) Comment les pathogènes interagissent-ils avec la communauté bactérienne généralement associée à l'hôte avant l'infection ? Pour étudier ces questions, nous nous sommes intéressés à Xenorhabdus nematophila, une bactérie pathogène d'insectes transmise par un vecteur, le némaotde Steinernema carpocapsae. Au cours d'une infection par X. nematophila, différentes sous-populations ayant différentes caractéristiques phénotypiques sont produites. Nous avons cherché à déterminer les mécanismes moléculaires responsables de cette diversification phénotypique, ainsi que sa potentielle valeur adaptative pour X. nematophila. Nous avons montré que certaines de ces formes phénotypique sont des mutants qui semblent être sous forte sélection positive au cours de l'infection. À l'inverse, ces mutants ont un impact négatif sur la reproduction du vecteur nématode, ce qui réduit leur transmission. La dynamique d'hétérogénéité phénotypique chez X. nematophila semble donc déterminée par des pressions de sélections contraires à court terme et à long terme. La production de molécules anti-microbiennes chez X. nematophila devraient lui permettre de dominer la communauté bactérienne à l'intérieur de l'insecte et faciliter sa ré-association avec son vecteur. Nous avons donc décrit la composition de la communauté microbienne présente dans des insectes morts d'une infection par X. nematophila, et montré qu'en dépit de sa production d'antibiotiques, X. nematophila est loin de dominer la communauté microbienne après la mort de l'insecte. Elle cohabite avec des bactéries provenant à la fois du microbiote de l'hôte insecte, et de celui du vecteur nématode. Cela soulève de nombreuses questions sur le rôle d'autres microorganismes dans les interactions Xenorhabdus-Steinernema, et sur leur influence dans l'évolution de cette symbiose mututaliste
Numerous studies have considered infections as pairwise interactions between a single pathogen and its host, sometimes leading to an incomplete picture of infectious processes. In this work, we focused on more complex types of interactions that arise because infections are usually heterogeneous. More precisely, we have investigated two main issues: (I) how pathogen transmission is impacted by phenotypic heterogeneity which arises within the pathogen population during the infection, and (ii) how do pathogens interact with the bacterial community which is naturally associated to the host before infection? To assess these questions, we have been studying Xenorhabdus nematophila, an insect-killing bacterial pathogen which is transmitted by a nematode vector, Steinernema carpocapsae. One interesting feature of X. nematophila is that it produces different sub-populations during the course of an infection, each one having distinctive phenotypic features (e.g. one form produces antibiotics and is mobile, while the other does not produce antibiotics nor flagella). In this work, we first tried to identify the molecular mechanisms responsible for this diversification of phenotypes, and tested if phenotypic heterogeneity in X. nematophila has some adaptive value. We showed that some of these phenotypic forms were mutants, which seem to be under strong positive selection during infection. We also showed, however, that these mutants impair nematodes reproduction, which in turn reduces transmission. Therefore, the dynamics of phenotypic heterogeneity in X. nematophila seems to be determined by contradictory short-term and long-term selective pressures. A second interesting feature of X. nematophila is that it produces a lot of antimicrobial compounds which should allow it to dominate the bacterial community inside the insect it has killed. This can be key to ensure the re-association of X. nematophila with its nematode vector inside the insect cadaver. We investigated the bacterial composition of the microbial communities present in insects cadavers after infection by X. nematophila. We found that despite the numerous antibiotics it is able to secrete, X. nematophila is far from dominating microbial community after host death. It rather cohabits with microorganisms from the microbiota of both the insect host and the nematode vector. This raises numerous questions about the impact of these other microorganisms on Xenorhabdus-Steinernema interactions, and therefore on their potential influence on how this mutualistic association has evolved

Un grand nombre de paramètres environnementaux affectent les insectes, et l’identification des éléments impliqués dans l’adaptation aux stress est cruciale. La nutrition est une clé de cette adaptation : elle contrôle la santé des organismes, et par conséquent affecte leur tolérance au stress. Le microbiote intestinal module lui aussi les processus physiologiques des organismes, notamment en agissant sur le phénotype nutritionnel de l’hôte. Cependant, nous ignorons encore la capacité de ce microbiote à modifier la tolérance au stress de l’hôte. A travers la manipulation du microbiote et de la nutrition, nous avons testé les effets de l’interaction microbiote-nutrition chez Drosophila melanogaster en nous concentrant sur la tolérance au stress. Nous avons montré une nette dépendance des mouches à l’accès aux levures pour maintenir un développement et un métabolisme normal.A l’inverse, l’altération du développement générée par les substrats nutritionnellement pauvres ou par la surpopulation larvaire était bénéfique pour la tolérance au stress. Nous avons démontré que le microbiote peut partiellement compenser les effets d’une privation en levures, mais n’a pas d’effet quand elles sont en excès. Pour la thermotolérance, nous avons constaté que les effets du microbiote étaient très variables, et avons attribué cette variabilité à la présence ou l’absence des levures. Les assemblages de microorganismes environnementaux étaient modifiées par la composition du substrat, mais le microbiote intestinal était étonnamment stable. Ces résultats témoignent de l’importance des facteurs nutritionnels et du microbiote à moduler le développement et le phénotype des Drosophiles
A large number of environmental parameters constrain insect species, and identifying the elements involved in the adaptation to stressful conditions is crucial. Nutrition is a key factor of this adaptation: it largely controls health of organisms and therefore their stress tolerance. In addition, it is now established that microbiota (i.e. the whole microbial community associated with a host) is much involved in physiological processes of organisms. In particular, gut microbiota is able to modulate host’s nutritional phenotype, resulting in complex dietary-dependent effects. Whether or not gut microbiota can act on stress tolerance is not clear. Thus, using experimental manipulation of microbiota and nutrition, we investigated the outcome of the microbiota-nutrition relationship on Drosophila melanogaster, with a special focus on stress tolerance. We showed a clear dependency of flies on yeasts availability to maintain a normal development, metabolism, and size. Conversely, impaired development resulting from artificial poor diets or from larval crowding was often beneficial for stress tolerance. We demonstrated that microbiota could partially compensate for the absence of yeast resource, but no effects were found in rich food contexts. We observed variable importance of microbiota on thermal tolerance, and attributed this variation to the presence or absence of yeasts. Environmental microorganisms communities were modified by diet composition but gut microbiota was surprisingly stable. Together, these findings highlight the importance of nutritional factors and microbiota in developmental and phenotypical plasticity of fruit flies

Multicellular organisms are not single individuals but carry a complex natural microflora with them. This complex's diversity and function can be considered a distinct ecosystem. Traditional methods of isolation and identification miss >90% of the actual diversity. This study uses the gut microflora of the Madagascar hissing roach, Gromphadorhina portentosa, as a model to examine this ecosystem. Isolated cultured bacteria were used to establish methods for identifying members of the microflora based on ribosomal RNA sequences. Universal primers for Eubacterial, Archaeal, and Eukaryotic 16s/18s rRNA were then used for PCR amplification of total DNA isolated from gut contents. Sequences from isolates were compared using BLAST, ClustalW, and other programs to recognize the isolates' identities and place them using a phylogenetic tree analysis. Eubacterial, Archaeal, and Eukaryotic organisms were found present in the hissing roach gut which can serve as a model ecosystem since it houses Eubacterial, Archaeal, and Eukaryotic organisms.

Ces travaux de thèse reposent sur le double objectif de proposer des approches innovantes pour l’étude des relations entre un hôte et son microbiote, et de les appliquer à la description fine de l’holobionte du puceron du pois par des données métagénomiques. Les relations symbiotiques façonnent le fonctionnement et l’évolution de tous les organismes, mais restent décrites de manière imparfaite, notamment à cause de la difficulté à caractériser la diversité génomique des partenaires microbiens constitutifs des holobiontes. L’essor des technologies de séquençage métagénomique révolutionne l’étude de ces systèmes, mais pose également des problèmes méthodologiques pour analyser les jeux de données métagénomiques. La métagénomique est ici appliquée au puceron du pois, un modèle d’étude des relations symbiotiques qui abrite une communauté bactérienne d’une complexité modérée, idéale pour développer de nouvelles approches de caractérisation de la diversité microbienne. Cette thèse vise à mieux décrire la communauté de symbiotes qu’abrite cet holobionte, notamment en distinguant les différents niveaux de variabilité génomique en son sein. Nous présentons une démarche pour l’analyse métagénomique d’holobiontes, qui repose d’abord sur l’assignation taxonomiques des lectures par alignement à des génomes de référence ou préalablement assemblés, puis sur la recherche de variants génomiques. L’étude de génotypes complets de symbiotes permet de retracer l’histoire évolutive des relations hôte-microbiote avec une résolution élevée. Chez le puceron du pois, nous mettons en évidence des niveaux et structures de diversité génomique différents selon les symbiotes, que nous proposons d’expliquer par les modalités de transmission ou l’histoire évolutive propre à chacun des partenaires microbiens. Cette approche repose sur la disponibilité d’un génome de référence adapté, qui est souvent difficile à obtenir en métagénomique. Dans un second temps, nous présentons donc une méthode d’assemblage guidé par référence en contexte métagénomique. Cette méthode se déroule en deux temps : le recrutement et l’assemblage de lectures par alignement sur un génome de référence distant, puis l’assemblage de novo ciblé des régions manquantes, permis par des développements complémentaires apportés au logiciel MindTheGap. Comparativement à un assembleur métagénomique, cette méthode permet l’assemblage d’un seul génome en un temps réduit, et permet de détecter d’éventuels variants structuraux sur le génome ciblé. Appliqué au puceron du pois, MindTheGap a réalisé l’assemblage du symbiote obligatoire Buchnera en un seul contig, et a permis d’identifier différents variants structuraux du bactériophage APSE. Ces travaux ouvrent la voie à la fois à une caractérisation plus précise des relations hôte-microbiote chez le puceron du pois par des approches fonctionnelles et métaboliques, ainsi qu’à l’application des outils présentés à des systèmes plus complexes
The aim of this PhD thesis is to develop innovative approaches to characterize host-microbiota relationships, and to apply them to finely explore the pea aphid microbiota using metagenomic data. Symbiotic relationships play a major role in the life and evolution of all organisms, but are imperfectly described, essentially because of the difficult characterization of the genomic diversity of the microbial partners. The rise of high throughput metagenomic sequencing is a game changer for the study of those systems, but also raises methodological issues to analyze large metagenomic datasets. Metagenomic is here applied to the pea aphid holobiont, a model system for the study of symbiotic relationships, sheltering a moderately complex microbial community. This level of complexity seems to be ideal to develop new approaches for the strain-resolved characterization of host-microbiota relationships. This thesis aims at a better description of this symbiotic community by distinguishing several scales of metagenomic diversity. In a first part, we present a framework for the metagenomic analysis of holobionts, relying first on the taxonomic assignation of reads by alignment to reference or newly assembled genomes, and then on the detection of genomic variants. Whole genome variant profiles make possible to track the evolutionary history of host-microbiota associations with a high resolution. In the case of the pea aphid, we highlight different scales and structures for the metagenomic diversity of the different symbionts, accounting for different transmission modes or evolutionary histories specific to each microbial partner. This framework is based on the availability of a suitable reference genome, that may be hard to obtain in a metagenomic context. In a second part, we therefore present a novel method for reference guided genome assembly from metagenomic data. This method is based on two steps. First, the recruitment and assembly of reads by mapping metagenomic reads on a distant reference genome, and second, the de novo assembly of the missing regions, allowed by the development of an improved version of the software MindTheGap. Compared to a standard metagenomic assembler, this methods makes possible to assemble a single genome in a reasonable time, and allows to detect eventual structural variations within the targeted genome. When applied to the pea aphid holobiont, MindTheGap yielded single contig assemblie of the obligatory symbiont Buchnera aphidicola, and helped to identify different structural variants of the bacteriophage APSE. This works paves the way to a finer characterization of host-microbiota interactions, and to the application of the presented approaches to more complex systems

Les symbiotes d'insectes peuvent considérablement influencer leurs hôtes de diverses manières. Nous avons étudié ici la communauté de microbes de la mouche du chou (Delia radicum) et plus particulièrement le rôle de son microbiote intestinal et de Wolbachia, une bactérie intracellulaire. La transmission verticale et maternelle de Wolbachia était de 100% et nous n’avons trouvé aucune preuve de manipulation de la reproduction telles que l’incompatibilité cytoplasmique, la parthénogenèse thélytoque, la féminisation ou la dégénérescence des embryons mâles. Les effets de Wolbachia sur D. radicum étaient significatifs mais modérés, et se compensaient mutuellement (réduction du taux d’éclosion, meilleure survie larvo-nymphale, temps de développement plus long et augmentation de la mortalité des femelles en conditions de stress), ce qui suggère une infection quasi neutre chez cette espèce, même si nous avons observé une augmentation de la fréquence d’infection en conditions idéales. L'influence du microbiote intestinal a été étudiée en utilisant un antibiotique, la tétracycline, avec un protocole sur trois générations, ce qui a permis de discerner l’effet direct (toxique) de la tétracycline de ses effets indirects (perte de symbiotes) sur l’hôte. Le traitement antibiotique de D. radicum a eu de multiples effets, généralement négatifs, sur les traits d’histoire de vie des descendants, ces effets pouvant être détectés jusqu'à deux générations après le traitement. La perturbation du microbiote intestinal semble avoir un rôle plus important qu'un simple effet toxique de la tétracycline elle-même. De plus, l’étude suggère que le microbiote semble avoir un rôle bénéfique chez cette espèce, et qu’il est au moins partiellement hérité de la mère. Pour finir, nous avons étudié si Wolbachia pouvait modifier le dialogue plante-insecte entre D. radicum et l’une de ses plantes-hôtes, le colza (Brassica napus). La présence du symbiote a diminué les concentrations de glucosinolates dans les feuilles, ce qui suggère que Wolbachia pourrait améliorer la fitness de son hôte en diminuant les signaux chimiques de la plante pouvant être utilisés par les conspécifiques et/ou ennemis naturels de D. radicum. Cette étude a montré le potentiel d'une bactérie intracellulaire à influencer les relations plantes-insectes et a permis de discuter des interactions tri-trophiques entre les symbiotes, leurs insectes-hôtes et un troisième niveau trophique : la plante. Cette thèse démontre qu'il est maintenant nécessaire de prendre en compte les symbiotes dans de prochaines études, afin de mieux comprendre les relations possibles entre différents partenaires, ainsi que leurs implications écologiques ou évolutives
Microbial symbionts can deeply influence their animal hosts in various ways. Here, we studied the community of microbes of the cabbage root fly (Delia radicum) and more precisely the role of its gut microbiota and of Wolbachia, an intracellular bacterium. The vertical maternal transmission of Wolbachia was perfect, and we found no evidence of manipulation of reproduction such as cytoplasmic incompatibility, thelytokous parthenogenesis, feminization nor male killing. Wolbachia infection had significant but moderate and mutually compensating effects on D. radicum (reduced hatch rate, improved larvo-nymphal viability, longer development time and increased female mortality in stress conditions), suggesting that infection might be nearly neutral in this strain, although we observed an increase in infection frequency in ideal rearing conditions. The influence of the gut microbiota was studied using an antibiotic, tetracycline, with a protocol spanning three generations, which allowed to discriminate the possible direct (toxic) effect of tetracycline from its indirect effects (due to the loss of gut symbionts). Antibiotic treatment of adults led to multiple and mostly negative effects on life history traits of their offspring and grandchildren. Data suggested a larger role of gut microbiota perturbation than of a toxic effect, that the microbiota was partially inherited maternally, and that the “wild-type” gut microbiota was beneficial in this species. Finally, we investigated whether Wolbachia could modify the insect-plant dialogue between D. radicum larvae feeding on roots of oilseed rape (Brassica napus). The presence of the symbiont decreased glucosinolate concentrations in the leaves, suggesting that Wolbachia could increase the fitness of its host by decreasing plant cues used by D. radicum conspecifics and/or natural enemies. This study showed the potential of an intracellular bacteria to influence plant-insect relationships, and allowed to discuss the tri-trophic interactions between symbionts, their insect hosts and a third trophic level: the plant. This thesis demonstrates the necessity to consider intracellular and extracellular symbionts in further studies, in order to unravel all the possible relationships between different partners, as well as their ecological or evolutionary implications

Although Na+ homeostasis in vivo is essential for mammals, it is known that excessive salt (NaCl) intake has played a major role in the development of hypertension. In vivo, there is a hormonal system, the renin-angiotensin-aldosterone system (RAAS), that specializes in regulating Na+ retention, especially the amount of Na+ in plasma. Na+ homeostasis in vivo has been achieved mainly by the RAAS, through regulation of vascular tonus (blood pressure) and Na+ handling in the kidney (Na+ diuresis). Recent studies have revealed a third mechanism of Na+ homeostasis in vivo: regulation of interstitial Na+ levels in tissues, such as subcutaneous tissues, by tissue macrophage immunity. In the pathogenesis of salt-sensitive hypertension, Recent research have been revealed that three molecular axes (Ang II - Rho/NOX-eNOS system, Aldosterone-rac1 -ENaC system, and tissue Na+ − TonEBP in macrophage -VEGF-c) are significantly involved in maintaining Na+ homeostasis in salt induced hypertension. Furthermore, the mechanism by which salt causes hypertension via the immune system (intestinal, local mucosal, and tissue immunity) has also been reported. In this article, we would like to propose that three molecular dysfunctions are involved in the development of salt-sensitive hypertension through three immunological mechanisms in the maintenance of Na+ homeostasis. Next, I would like to explain the importance of gut-RAAS and abnormality of intestinal microflora (dysbiosis) in salt-sensitive hypertension. It has been known that the metabolites (e.g., short-chain fatty acid neural amino) produced by microflora are deeply involved in central (CNS) and sympathetic nervous system (SNS) activity. In addition, we would like to explain of the importance of brain-RAAS and cerebral inflammation in salt-sensitive hypertension. Moreover, recent research have revealed that the detection-mechanism in the brain for Na+ concentration([Na+]) in vivo and in the tongue for [Na+] in diet. These finding suggests that excessive salt intake may cause brain dysfunction, most delicate organ, before the onset of salt sensitive hypertension, and may also destroy brain structure after the onset of salt sensitive hypertension. Thus, we would like to insist that excessive salt intake might not only induce hypertension, but also be toxic especially for brain. Finally, we would like to explain that The DASH diet (Dietary Approaches to Stop Hypertension) is one of the universal diets for adult human, not only by reducing salt, but also by reducing metabolic stress and improving of dysbiosis.