Bactérias simbiontes foram fundamentais no processo evolutivo de eucariotos. Em insetos, essa associação permitiu a exploração de novos nichos ecológicos pela íntima associação estabelecida com bactérias que fornecem nutrientes essenciais a hospedeiros cuja dieta apresenta limitação em sua composição nutritiva, assim como proteção a condições de estresse e fatores bióticos e abióticos. Um grande número de associações facultativas, aquelas em que o simbionte não é essencial para a sobrevivência do hospedeiro, mas pode impactar na aptidão biológica, tem sido descobertas. Pentatomídeos estão comumente associados a bactérias extracelulares facultativas presentes nas invaginações da região final do intestino médio, sendo alvos de investigação para a determinação da diversidade de simbiontes e, principalmente, sua função na interação com o inseto hospedeiro. Porém, na ausência destes simbiontes, o desenvolvimento e a aptidão reprodutiva do hospedeiro podem ser prejudicados. Adicionalmente, bactérias livres associadas a insetos podem se associar a outros tecidos, como as estruturas reprodutivas, e causar efeitos nocivos ao hospedeiro. A intensidade dos efeitos induzidos por essa microbiota depende de vários fatores, como a sua diversidade, a idade e o tecido do hospedeiro. Nesta tese utilizamos como modelo de estudo o percevejo Euschistus heros (F.) (Hemiptera: Pentatomidae), uma importante praga de diversas culturas agrícolas, para investigar i) a diversidade de simbiontes cultiváveis associada às estruturas reprodutivas (ovários, espermateca e vesícula seminal) e digestivas (criptas da região final do intestino médio); ii) a diversidade de simbiontes associada a embriões e ventrículos do intestino médio de ninfas de último ínstar e adultos; o efeito de tratamentos para a eliminação de bactérias associadas iii) à vesícula seminal e iv) região V4 do intestino médio na aptidão reprodutiva de E. heros. A região V4 do intestino médio, ovário, espermateca e vesícula seminal apresentou Proteobacteria (Enterobacteriaceae) e Firmicutes (Enterococcaceae e Staphylococcaceae) como simbiontes cultiváveis, sendo a região V4 do intestino médio a mais diversa entre as estruturas avaliadas. Entre os filotipos isolados, foram encontrados filotipos comuns a todas as estruturas analisadas, mas também aqueles associados exclusivamente ao trato reprodutivo e aparelho digestivo. A análise de metagenômica indicou o embrião de E. heros como sendo o mais diverso, seguido pelas regiões V1 e V2 do intestino médio de ninfas e adultos. A região V4 foi a menos diversa, sendo habitada praticamente por uma Enterobacteriaceae. A espermateca e a vesícula seminal são naturalmente infectadas por simbiontes e a eliminação dessas bactérias após tratamento com antibióticos não resultou em alterações na capacidade reprodutiva de fêmeas. O tratamento de adultos com antibióticos e a esterilização superficial dos ovos não interferiu no desenvolvimento e reprodução de E. heros, ocorrendo, porém, redução da fertilidade e na mortalidade de ninfas quando os parentais foram alimentados com antibióticos. / Bacterial symbionts are fundamental in the evolutionary process of eukaryotes. In insects this association allowed the exploitation of new ecological niches, because the bacteria provide essential nutrients for those whose diet is nutritionally limited. Bacterial symbionts also provide protection against biotic and abiotic factors, allowing insects to exploit a range of food types. In recent years, a large number of facultative associations have been discovered. In such associations, symbionts are not essential for host survival, but may have a significant impact on insect host fitness. Pentatomids are commonly associated with facultative extracellular bacteria present in the invaginations of the posterior region of the midgut and have been targeted in studies for the determination of the diversity and function of these symbionts. However, associations with symbionts can impose adaptive costs to the host; therefore, in the absence of these symbionts, the development and reproduction of the host may be impaired. In addition, facultative symbionts can be potentially pathogenic bacteria, and can infect the host\'s reproductive system. The intensity of the effects induced by this microbiota depends of several factors, such as its diversity, age and host tissue infected. Transmission of the microbiota usually occurs vertically by specific mechanisms shortly after egg hatching, such as the contamination of the surface of the eggs by a gelatinous mass during deposition and by the deposition of bacterial capsules next to the eggs. Symbionts can be acquired from the environment at each generation, depending on the host species. In here, we used Euschistus heros (F.) (Hemiptera: Pentatomidae), an important agricultural pest of several crops, as a model to investigate: i) the diversity of culturable symbionts of E. heros (ovaries, spermatheca and seminal vesicle) and digestive tract (crypts of the posterior region of the midgut); ii) the diversity of non-culturable symbionts present along the midgut, in embryos, last instar and adults; the effects of symbionts associated with the iii) seminal vesicle and iv) the V4 region of the midgut on the reproductive fitness of E. heros. Our data show that the diversity of culturable symbionts associated with the V4 region of the midgut, ovary, spermatheca and seminal vesicle is represented by Proteobacteria (Enterobacteriaceae) and Firmicutes (Enterococcaceae and Staphylococcaceae). The V4 region of the midgut was the most diverse among the evaluated structures. In addition, unique culturable phylotypes were found in the reproductive and digestive tract. Metagenomic analysis indicated the embryo of E. heros carried the most diverse microbiota, followed by the V1 and V2 region of the gut. The V4 region was mosly inhabited by an Enterobacteriaceae. Symbionts associated with the spermatheca and the seminal vesicle naturally infect E. heros and do not affect the reproductive capacity of females. The sterilization of the egg surface did not interfere directly with the development and reproduction of E. heros, but the progenie of couples treated with antibiotics presented reduced fertility and nymphal mortality.

Medicago truncatula forme une association symbiotique avec Sinorhizobium meliloti qui conduit à la formation de nodosités fixatrices d’azote. Les cellules symbiotiques végétales accueillent des centaines de bactéries qui restent viables dans la nodosité et se différencient en bactéroïdes fixateurs d’azote. Dans le but de mieux comprendre les mécanismes moléculaires nécessaires à la mise en place de cette interaction, nous avons recherché de nouveaux gènes de plante requis pour une symbiose effective en utilisant des approches de génétique directe et inverse. Des méthodes de biologie cellulaire et moléculaire ont été utilisées pour caractériser le phénotype des mutants et mieux comprendre la fonction biologique de ces gènes.Le gène symbiotique DNF2 code une phosphatidylinositol phospholipase C putative. Les nodosités formées par le mutant dnf2 contiennent une zone de fixation qui est réduite et dans laquelle les rhizobia ne se différencient pas complètement en bactéroïdes. De plus ces nodosités sénescent rapidement et présentent des réactions similaires à des réponses de défense. Sous certaines conditions d’expérimentation, le phénotype sauvage peut être restauré chez ce mutant ce qui montre le caractère conditionnel du phénotype.Le gène symbiotique SYMCRK code un récepteur kinase riche en cystéine. Le phénotype du mutant symCRK est similaire à celui de dnf2, ce qui suggère que ces deux gènes sont impliqués dans des processus aboutissant à des réponses similaires, probablement la persistance des bactéries dans les cellules végétales ou l’inhibition des réactions de défense de la plante. Les phénotypes Fix- atypiques des mutants dnf2 et symCRK suggèrent que les gènes correspondants sont impliqués dans les processus de répression des défenses de la plante et de persistance des bactéroïdes. / Medicago truncatula and Sinorhizobium meliloti form a symbiotic association resulting in the formation of nitrogen-fixing nodules. In the nodules, symbiotic plant cells home and maintain hundreds of viable bacteria which are differentiated into bacteroids, the nitrogen-fixing form of rhizobia. In order to better understand the molecular mechanism sustaining this phenomenon, we used a combination of forward and reverse genetics approaches to identify genes required for nitrogen fixation. In addition we have used cell and molecular biology to characterize the phenotype of the corresponding mutants and to gain an insight into the genes functions.The symbiotic gene DNF2 encodes a putative phosphatidylinositol phospholipase C-like protein. Nodules formed by the mutant contain a zone of infected cells reduced to a few cell layers. In this zone, bacteria do not differentiate properly into bacteroids. Mutant nodules senesce rapidly and they exhibit defense-like reactions. The dnf2 symbiotic phenotype has been shown to be dependent on the experimental conditions.The symbiotic gene SYMCRK encodes a cystein-rich receptor kinase. The symCRK phenotype is similar to dnf2 suggesting that the two genes SYMCRK and DNF2 are participating in similar processes. This atypical phenotype amongst Fix- mutants unravels DNF2 and SYMCRK as new actors of bacteroid persistence inside symbiotic plant cells and repression of plant defense.

Les récents progrès des technologies de séquençage et des techniques de biologie moléculaire ont permis aux chercheurs de révéler l'ubiquité et la diversité des virus. Malgré ces progrès, la découverte des virus s'est surtout concentrée sur les virus de vertébrés. De plus, les virus connus sont très peu étudiés en terme d'écologie virale. Les arthropodes de par leur histoire évolutive ancienne représentent 80% de la diversité animale sur notre planète. L'omniprésence de l'endosymbiose chez les arthropodes et leur capacité d’héberger des symbiotes (parfois multiples) suggèrent que leurs interactions avec leurs virus pourraient également être de nature symbiotique, allant des interactions durables de type mutualiste à celles de type antagoniste. Dans ce contexte, mon travail de thèse s’est concentré sur un densovirus de moustiques, le Culex pipiens densovirus (CpDV), en étudiant leur prévalence et leur diversité dans les populations naturelles de moustiques Culex pipiens et leurs interactions avec les bactéries endosymbiotiques Wolbachia dans un système naturel hôte-bactérie-virus (Cx. pipiens -Wolbachia - CpDV), tant dans des populations naturelles que des colonies de laboratoire. Nous avons révélé une forte prévalence et une grande diversité de CpDV dans les populations mondiales de Cx. pipiens. De plus, le CpDV infecte de façon persistante les lignées de laboratoire et est transmis verticalement avec l'endosymbiote Wolbachia. Nous montrons une prévalence et une diversité élevées du CpDV en Turquie et en Tunisie, et nous nous sommes concentrés davantage sur ces deux pays pour étu-dier les interactions Wolbachia-CpDV dans les populations naturelles. En Tunisie, dans une zone de contact étroite où deux groupes de Wolbachia génétiquement distincts coexistent, ces différents groupes de Wolba-chia influencent la prévalence et la diversité du CpDV. Nous montrons également une corrélation positive entre la densité de Wolbachia et la densité de CpDV. D’une manière globale, nos résultats suggèrent que le système CpDV-Cx.pipiens -Wolbachia et plus généralement les densovirus sont de bons modèles pour étudier les interactions écologiques entre ces partenaires et leur évolution dans la nature et au laboratoire. / Virus discovery has long depended on the observation of outbreaks, and the research to understand their pathogenicity depended on their culture. The advances of sequencing technology and molecular biology tech-niques allowed researchers to discover the ubiquity and the diversity of viruses. Despite these advances, virus discovery remains mainly focused on vertebrate viruses. Furthermore, even the known diversity of these virus-es is poorly studied in term of virus ecology. Arthropods with their ancient evolutionary history represent 80% of animal diversity on our planet. The ubiquity of endosymbiosis in arthropods and their ability to support (sometimes multiple) symbionts suggest that their interactions with their viruses might be of symbi-otic nature, intimate long-term associations with outcomes ranging from mutualistic to antagonistic. In this context, this PhD dissertation focuses on a mosquito densovirus, Culex pipiens densovirus (CpDV), to in-vestigate their prevalence and diversity in natural populations of Culex pipiens mosquitoes and their inter-actions with endosymbiotic bacteria Wolbachia in a natural host-bacteria-virus system (Cx. pipiens -Wolbachia - CpDV) both in natural populations and laboratory colonies. We reveal a high prevalence and diversity of CpDV in worldwide Cx. pipiens populations. Moreover, CpDV persistently infects laboratory lines and are vertically transmitted along with their hosts’ endosymbiont Wolbachia. We reveal high CpDV prevalence and diversity in Turkey and Tunisia and further focus on these two countries to investigate Wolbachia-CpDV interactions in natural populations. In Tunisia, in a narrow contact zone where two ge-netically distinct Wolbachia groups coexist, different Wolbachia groups influences CpDV prevalence and diversity. Moreover, we show a positive correlation between Wolbachia density and CpDV density. Overall, our results suggests that CpDV-Cx.pipiens -Wolbachia system is a good model system to study the ecologi-cal interactions between these partners and their evolution together both in the nature and in laboratory conditions. Densoviruses with their diversity and long-term evolution with their hosts, and their easily ma-nipulated small genomes, are good models to study complex host-virus-bacteria interactions as well as virus ecology and evolution in a symbiotic context.

Les Wolbachia sont des alpha-protéobactéries intracellulaires transmises verticalement de la mère aux descendants via les ovocytes. Du fait de ce mode de transmission, des stratégies de manipulation de la reproduction favorisant leur propagation ont été sélectionnées chez ces bactéries. La plus communément utilisée par Wolbachia s’appelle l’incompatibilité cytoplasmique (IC). L’IC a lieu lorsque des mâles infectés copulent avec des femelles non infectées ou infectées par des Wolbachia incompatibles et se traduit par la mort des descendants avant l’éclosion. L’IC est généralement conceptualisée comme un modèle mod/resc ou toxine/antidote dans lequel les Wolbachia présentes chez les mâles introduiraient dans les spermatozoïdes une toxine (fonction mod) qui, après la fécondation, entrainerait la mort des embryons, sauf si les Wolbachia présentes dans l’œuf produisent un antidote (fonction resc). C’est chez l’espèce de moustique Culex pipiens que la plus grande diversité de phénotypes d’IC a été décrite. Cette diversité repose uniquement sur la diversité des souches de Wolbachia hébergées par C. pipiens. Dans cette thèse, nous avons mené la première étude des mécanismes cytologiques responsables de la mort des embryons dans les croisements incompatibles chez C. pipiens. Nous avons montré que des défauts de condensation et de ségrégation de la chromatine paternelle lors de la première division embryonnaire entraînent la mort des embryons dans tous les croisements incompatibles étudiés. Ces défauts cellulaires sont les seuls observés, ce qui indique que la diversité de relations d’IC décrite chez C. pipiens ne repose pas sur une diversité de défauts cellulaires. L’étude, chez plusieurs souches de wPips, de l’opéron cidA/cidB dont l’implication fonctionnelle dans l’IC a récemment été mise en évidence chez la drosophile, nous a permis de montrer que cet opéron est amplifié et polymorphe dans tous les génomes de wPips séquencés. L’exploration des variants de cet opéron dans les génomes de Wolbachia infectant des populations naturelles de C. pipiens, à l’aide de plus de 250 lignées isofemelles, a permis d’associer de manière robuste une variation de cidB avec un changement dans le phénotype mod de certains mâles. En outre, la présence d’un variant ubiquitaire de cidA, supporte le rôle de ce gène dans la fonction resc. Ainsi, chez C. pipiens, l’opéron cidA/cidB grâce à son amplification et sa diversification est impliqué dans la diversité des phénotypes d’IC et fonctionnerait comme un système toxine-mod /antidote-resc : cidB étant impliqué dans la fonction mod et cidA dans la fonction resc. / Wolbachia are intracellular alpha-proteobacteria vertically transmitted from mothers to their offspring through oocytes. As a consequence of this transmission mode, reproductive manipulation strategies that promote bacteria spread in host populations have been selected. The most common manipulation used by Wolbachia is called cytoplasmic incompatibility (CI). CI occurs when infected males mate with uninfected or incompatible Wolbachia-infected females and results in the death of offspring before hatching. CI is generally conceptualized as a mod/resc or toxin/antidote model in which paternal Wolbachia would introduce a toxin (mod function) in sperms which would, after fertilization, induce embryonic death unless an antidote produced by maternal Wolbachia in the egg counteracts its effect (resc function). A to date unique diversity of CI phenotypes has been described in the mosquito species Culex pipiens. This diversity is based solely on the diversity of Wolbachia strains hosted by C. pipiens. In this PhD, we conducted, in C. pipiens, the first study of the cytological mechanism behind embryonic mortality in CI crosses. We showed that paternal chromatin condensation and segregation defects during the first embryonic division were responsible for embryonic death in all CI crosses. These CI defects were the only ones observed indicating that the diversity of CI phenotypes in C. pipiens is not based on a diversity of cellular mechanisms. We then studied the cidA/cidB operon in several wPip strains as the functional involvement of this operon in CI was recently demonstrated in Drosophila. We showed that this operon is amplified and polymorphic in all genomes of sequenced wPip. Investigation of cidA/cidB variants in Wolbachia genomes infecting natural populations of C. pipiens, using more than 250 isofemale lines, enabled us to reveal a robust association between cidB variations and change in mod phenotype. In addition, the presence of an ubiquitous cidA variant supports the role of this gene in the resc function. In C. pipiens, the cidA/cidB operon, through its amplification and diversification, is involved in the CI phenotypes diversity and would operate as a toxin-mod / antidote-resc system: cidB being involved in the mod function and cidA in the resc function.

Les champignons ont développé diverses interactions avec les végétaux. Ces interactions peuvent être bénéfiques pour la plante dans le cas des champignons établissant des symbioses mutualistes ou néfastes si le champignon est pathogène. Elles reposent sur des mécanismes moléculaires mal élucidés. Des études réalisées sur le champignon mutualiste Hebeloma cylindrosporum associé au pin Pinus pinaster ont permis de créer une collection de mutants affectés dans leur capacité à interagir avec les plantes et à former l’organe mixte de la symbiose, l’ectomycorhize. L’objectif de ma thèse a été d’étudier un mutant affecté dans le gène codant une alpha-tubuline Hctubα2. Les tubulines sont des protéines présentes chez tous les Eucaryotes et permettent la formation des microtubules, des éléments clés du cytosquelette. Chez les champignons, on trouve une ou deux alpha tubuline(s). H. cylindrosporum en possède deux. J’ai étudié l’expression de ces deux tubulines lors l’établissement de l’interaction avec les racines de l’hôte. Les résultats indiquent que ces deux gènes sont différentiellement exprimés lors de l’interaction. J’ai étudié au niveau protéomique l’impact de la mutation en comparant les protéomes intracellulaires des deux souches. On retrouve deux alpha-tubulines chez certains champignons phytopathogènes comme Botrytis cinerea. L’hypothèse de l’implication de l’alpha-tubuline 2 dans l’établissement de la pathogénie a été émise. J’ai donc construit des mutants de Botrytis cinerea dans lesquels ce gène a été inactivé. J’ai également tenté de localiser à l’aide de fusions traductionnelles chacune des alpha-tubulines chez le champignon mycorhizien et chez le pathogène / In all terrestrial ecosystems, plants live in close interaction with numerous fungi. The interaction has a negative or positive effect on host plant depending upon the pathogenic or symbiotic status of the fungus. The establishment of these interactions is based on a tightly regulated molecular dialog between symbiotic partners. Previous studies on the ectomycorrhizal fungi, Hebeloma cylindrosporum associated with maritime pine (Pinus pinaster), created a collection of mutants affected in their mycorrhizal abilitiy. The aim of my thesis was to characterize one of these mutants affected in a gene, Hctubα2, encoding an alpha tubulin. Tubulins are eukaryotic cytoskeletal proteins involved in microtubules formation. Fungi have one or two alpha-tubulin. For example, H.cylindrosporum has two alpha-tubulin. The site of mutagenic DNA insertion in fungal genome was characterized. I studied the expression of both alpha-tubulins during the establishement of mycorrhizal interaction. Results showed that the two genes are differentially expressed during the interaction with host plant. At proteomic level, I studied the impact of the mutation comparing the two strains using 2D gel electrophoresis and sequencing differentially accumulated spots. Pathogenic fungi also bear two alpha-tubulins, as Botrytis cinerea. The hypothesis of the involvement of the alpha-tubulin 2 in pathogenesis was investigated. I created Botrytis cinerea mutants deleted for this gene. I also created translational fusions in order to visualize both alpha-tubulins in Hebeloma cylindrosporum and in Botrytis cinerea

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Nestes casos o depósito deve ser feito pessoalmente na Biblioteca Central, conforme orientações em http://biblioteca.ufam.edu.br/servicos/teses-e-dissertacoes Dúvidas? ddbc@ufam.edu.br on 2018-11-12T21:02:24Z (GMT) / Submitted by SABRINA MEIRELES (sabmeireles@gmail.com) on 2018-11-21T16:28:32Z No. of bitstreams: 3 Ata de defesa_SABRINA.pdf: 137071 bytes, checksum: b6dc6ae572159917e96c3353244d7fea (MD5) CartaEncaminhamentoAutodepósito-SABRINA-MEIRELES-Dissertação.pdf: 104760 bytes, checksum: 8b5e7685916e53325a10bf89174a2564 (MD5) Dissertação_Enviado TEDE.pdf: 2362346 bytes, checksum: 1eda835ac27bb468843b802a36d93c70 (MD5) / Approved for entry into archive by PPGBIOTEC Biotecnologia (ppg_biotec.ufam@yahoo.com.br) on 2018-11-21T17:55:02Z (GMT) No. of bitstreams: 3 Ata de defesa_SABRINA.pdf: 137071 bytes, checksum: b6dc6ae572159917e96c3353244d7fea (MD5) CartaEncaminhamentoAutodepósito-SABRINA-MEIRELES-Dissertação.pdf: 104760 bytes, checksum: 8b5e7685916e53325a10bf89174a2564 (MD5) Dissertação_Enviado TEDE.pdf: 2362346 bytes, checksum: 1eda835ac27bb468843b802a36d93c70 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-11-21T19:41:17Z (GMT) No. of bitstreams: 3 Ata de defesa_SABRINA.pdf: 137071 bytes, checksum: b6dc6ae572159917e96c3353244d7fea (MD5) CartaEncaminhamentoAutodepósito-SABRINA-MEIRELES-Dissertação.pdf: 104760 bytes, checksum: 8b5e7685916e53325a10bf89174a2564 (MD5) Dissertação_Enviado TEDE.pdf: 2362346 bytes, checksum: 1eda835ac27bb468843b802a36d93c70 (MD5) / Made available in DSpace on 2018-11-21T19:41:17Z (GMT). No. of bitstreams: 3 Ata de defesa_SABRINA.pdf: 137071 bytes, checksum: b6dc6ae572159917e96c3353244d7fea (MD5) CartaEncaminhamentoAutodepósito-SABRINA-MEIRELES-Dissertação.pdf: 104760 bytes, checksum: 8b5e7685916e53325a10bf89174a2564 (MD5) Dissertação_Enviado TEDE.pdf: 2362346 bytes, checksum: 1eda835ac27bb468843b802a36d93c70 (MD5) Previous issue date: 2018-07-25 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Native stingless bees (Apidae: Meliponini), as well as the other social insects of the order hymenoptera, present symbiotic interactions with the yeasts, indicated as the main responsible for the processing of pollen, fermented food preferred by the meliponids, and by the fermentation of other vegetal materials taken to these nests. In this project, it was proposed to identify yeasts associated with the nest of two Amazon native bees and establish a reference database for Amazonian strains. For this, the substrates (pollen, honey, larval food and wax) were collected from the nest of Melipona interrupta and Cephalotrigona femorata in three meliponaria, located in Iranduba, Parintins and Paraná de Parintins, (n = 12 samples). The methodological procedures adopted in this work are as follows: (1) isolation in YPD, (2) screening in CHROMágar for separation of the isolates in groups of colors, (3) DNA extraction, (4) screening for RFLP standards based on the amplification of the 18S, ITS1-5,8S-ITS2 and D1 / D2 regions of the 26S rRNA gene and digestion of the amplicon with the Dde I restriction enzyme. The amplicons of the different profiles were sequenced and the identification was achieved comparing the sequences against the nucleotide databases of the INSDC consortium using the BLASTN tool. A total of 749 nest bees associated yeasts were isolated, among these 15 lines were identified, namely: SM01 Candida sp., SM02 Hyphopichia sp., SM03 Rhodotorula sp., SM04 Wickerhamiella versalitis, SM05 Debaryomyces sp., SM06 Candida orthopsilosis, SM07 Candida apícola, SM08 Metschnikowia sp., SM09 Zygosaccharomyces siamensis, SM10 Hanseniaspora opuntiae, SM11 Pichia sp., SM12 Pichia kluyveri, SM13 Trichosporon asahii, SM14 Kodamaea ohmeri and SM15 Aureobasidium sp. This work contributed to (1) the identification of yeast associated to the natives stingless bees, (2) contributed to the databases through the deposit of complete nucleotide sequences, useful for yeast identification, (3) established thirteen new RFLP standards, (4) in addition to compose a database of yeasts, which from this bank, future works may prospect processes and biomolecules of biotechnological interest. / As abelhas nativas sem ferrão (Apidae: Meliponini) apresentam interações simbióticas com as leveduras, apontadas como as principais responsáveis pela fermentação do pólen, alimento preferido pelos meliponídeos, e pela fermentação de outros materiais de origem vegetal coletados pelas abelhas. Neste projeto, foi proposto identificar leveduras associadas ao ninho de duas abelhas nativas da Amazônia e estabelecer um banco de estirpes amazônicas de referência. Para isso, os substratos (pólen, mel, alimento larval e cera) foram coletados do ninho de Melipona interrupta e Cephalotrigona femorata em três meliponários, localizados em Iranduba, Parintins e Paraná de Parintins, (n=12 amostras). Os procedimentos metodológicos adotados neste trabalho seguem a seguinte ordem: (1) isolamento em YPD, (2) triagem em CHROMágar para separação dos isolados em grupos de cores, (3) extração de DNA, (4) triagem de padrões moleculares por PCR/RFLP baseado na amplificação das regiões 18S, ITS1-5,8S-ITS2 e D1/D2 do gene 26S rRNA e digestão do amplicon com a enzima Dde I (5) sequenciamento dos amplicons dos diferentes padrões obtidos na RFLP e a identificação mediante comparação das sequências obtidas contra os bancos de dados de sequências nucleotídicas do consórcio INSDC, com auxílio da ferramenta BLASTN. Foram isoladas 749 leveduras associadas ao ninho das abelhas Melipona interrupta e Cephalotrigona femorata, das quais, 15 linhagens foram identificas, a saber: SM01 Candida sp., SM02 Hyphopichia sp., SM03 Rhodotorula sp., SM04 Wickerhamiella versalitis, SM05 Debaryomyces sp., SM06 Candida orthopsilosis, SM07 Candida apícola, SM08 Metschnikowia sp., SM09 Zygosaccharomyces siamensis, SM10 Hanseniaspora opuntiae, SM11 Pichia sp., SM12 Pichia kluyveri, SM13 Trichosporon asahii, SM14 Kodamaea ohmeri e SM15 Aureobasidium sp. Este trabalho resultou em (1) dados inéditos a respeito das leveduras associadas às abelhas sem ferrão da Amazônia, (2) contribuiu com os bancos de dados por meio do depósito de sequências nucleotídicas completas de regiões gênicas usadas para identificação de leveduras, (3) estabeleceu 14 novos padrões de RFLP, (4) forneceu quinze linhagens a serem exploradas em estudos futuros, das quais cinco podem ser espécies ainda não descritas na literatura, que visem prospectar processos e biomoléculas de interesse biotecnológico.

O conhecimento das associações entre os microrganismos componentes da microbiota dos solos é de grande interesse no meio científico, principalmente relacionado aos microrganismos que se associam de forma benéfica com as plantas. Neste contexto, destaca-se a micorriza arbuscular, que é a associação entre os fungos micorrízicos arbusculares (FMAs) e uma amplitude de espécies vegetais. Além desta íntima interação entre estes organismos, torna-se necessário conhecer a importância dos demais componentes do sistema solo, como a microbiota formada por fungos e bactérias dos solos, para o estabelecimento desta interação. Os objetivos dos estudos componentes desta tese se concentraram na avaliação de FMAs (D. heterogama, R. clarus e Gi. rosea) inoculados em sistemas alterados quanto à composição das comunidades microbianas dos solos, promovidas pela metodologia de \'diluição para extinção\'. No primeiro estudo foram encontradas respostas diferenciais na capacidade de colonização micorrízica (%CM) de plantas de cana-de-açúcar pelos FMAs inoculados nos diferentes sistemas, além do efeito diferencial dos FMAs quanto às alterações nas comunidades de fungos e bactérias do solo. Num estudo mais detalhado, desenvolvido apenas com R. clarus em plantas de milho, foi verificado que a maiores diversidades microbianas do solo resultaram em maior colonização do hospedeiro, principalmente no período inicial de desenvolvimento das plantas. Neste experimento foi descrita a correlação direta da capacidade de colonização do FMA com a riqueza e a diversidade filogenética da microbiota dos solos. A descrição dos perfis metabólicos dos solos contendo diferentes comunidades microbianas revelou a capacidade diferencial destes solos em utilizar diferentes fontes de carbono, além de demonstrar um aumento no metabolismo (evidenciado pelo consumo total de fontes de carbono) devido à inoculação do FMA. Em conjunto, os dados obtidos nos dois estudos indicam que a micorrização das plantas depende da ação de outros microrganismos do sistema solo, que atuam como um terceiro fator nesta simbiose e que a microbiota do solo responde a inoculação de um organismo exógeno, primordialmente aumentando seu metabolismo. Deve-se, portanto, considerar que a degradação biológica dos solos, com perda de sua biodiversidade, pode ter papel determinante no funcionamento de interações específicas e benéficas às plantas. / Knowledge about the associations between microbial components of soil microbiota is of great interest in the scientific community, primarily related to microorganisms that are associated beneficially with plants. In this context, there is the arbuscular mycorrhiza, which is made of the association between mycorrhizal fungi (AMF) and a range of plant species. In this close interaction between these organisms, it is necessary to describe the importance of other components of the soil system, such as the microbiota formed by fungi and bacteria from the soil, to establish this interaction. The objectives of the studied that compose this thesis focused on the evaluation of AMF (D. heterogama, R. clarus and Gi. rosea) fitness, when inoculated in modified systems on the composition of the microbial communities in the soil, with alterations promoted by the \'dilution to extinction\' methodology. In the first study, differential responses were found in root colonization capacity (% CM) of sugarcane by AMF inoculated in different systems, and the differential effect of AMFs, changing the communities of fungi and bacteria of soil. In a more detailed study, designed only to R. clarus in corn plants, it was found that higher microbial diversity of soil resulted in higher colonization of the host, especially in the initial period of plant development. In this experiment it was described the direct correlation of AMF colonization capacity with richness and phylogenetic diversity of soil microbiota. The description of the metabolic profiles of soil containing various microbial communities revealed the differential ability of these soils utilize different carbon sources, in addition to demonstrating an increase in metabolism (as evidenced by the total consumption of carbon sources) due to inoculation of the AMF. Together, the data from the two studies indicate that colonization of plants by AMF depends on the action of other microorganisms soil system, which act as a third factor in this symbiosis, and that the soil microbes respond to inoculation of an exogenous organism, primarily increasing its metabolism. One should therefore consider that the biological degradation of the soil, with loss of biodiversity, may have crucial role in the functioning of specific and beneficial interactions to plants.

Submitted by Jorge Silva (jorgelmsilva@ufrrj.br) on 2018-05-18T17:44:09Z No. of bitstreams: 1 2014 - Dayana da Silva Correia.pdf: 2038600 bytes, checksum: 031920d278558b902310d93f752920d7 (MD5) / Made available in DSpace on 2018-05-18T17:44:09Z (GMT). No. of bitstreams: 1 2014 - Dayana da Silva Correia.pdf: 2038600 bytes, checksum: 031920d278558b902310d93f752920d7 (MD5) Previous issue date: 2014-03-26 / CAPES / CNPq / The symbiosis between soil invertebrates and micro"organisms is a major ally in promoting the decomposition of plant residues in the soil. The micro"organisms in turn, have an immense genetic diversity and play crucial roles in maintaining ecosystems. One of these functions is the production of extracellular enzymes that assist the mineralization of organic matter. The possibility of developing new biotechnological processes based on the exploration of microbial diversity is immense, due to the great variability that exists between biological systems and it can be optimized to improve the agricultural production systems in a sustainable manner. The objective of this work was to study the profile of the bacterial community and cellulolytic potential of bacteria isolated from three different species of invertebrates saprophages. The experiments were performed at the Experimental Station of Embrapa Agrobiology, Serop?dica, Rio de Janeiro State. Millipede, of the Trigoniulus corallinus species, were collected in piles of plant compounds from local experimental field sites, which were subsequently incubated for 60 days under six different diets. Bacterial diversity in the intestinal tract of invertebrates was analyzed by PCR"DGGE of 16S rDNA gene amplification by PCR electrophoresis in denaturing gradient gel (DGGE); two domains were used, Bacteria and Actinobacteria. Some bands of the DGGE gel were extracted and sequenced. To assess the potential for production of cellulases in response to the presence of carboxy methyl cellulose (CMC) of isolates, the technique Congo red stain was used and the values were expressed as means (Ie) enzymatic index. From the highest values of (Ie) twenty" three bacteria were selected for the analysis of 16S rDNA. After the phylogenetic identification, the cellulolytic potential was rated, through cellulolytic activity of endoglucanase (CMCase), and endo" and exoglucanases (FPase) tests. To determine the molecular weight and activity of the enzymes polyacrylamide gels (SDS"PAGE) and zymography were performed. The results obtained in the technique of DGGE, the profiles of DGGE bands, showed that the intestinal microbiota of the invertebrates has distinct bacterial groups. It is possible to infer that, despite the communities having similar abundance, such as in the Trigoniulus corallinus and Cubaris murine species, the groups that make up this abundance were different among the invertebrate?s species. From the clones of the incised bands, three phyla members, Proteobacteria, Firmicutes and Bacteroidetes, were sequenced. Through phylogenetic analysis, it was possible to identify the 23 species of bacteria. Presenting two distinct Actinomycetes and Firmicutes phylum, the largest genus identified was Streptomyces, followed by an isolated Bacillus, Paenibacillus, and Staphylococcus. The intestinal tract of the three species of saprophages invertebrates showed to be an adequate environment for prospection of bacteria with cellulolytic efficiency, with high potential for future biotechnological studies. / A simbiose entre invertebrados do solo e microrganismos ? um grande aliado no auxilio da decomposi??o de res?duos vegetais presentes no solo. Os microrganismos por sua vez, apresentam uma imensa diversidade gen?tica e desempenham fun??es cruciais na manuten??o dos ecossistemas, uma dessas fun??es ? a produ??o de enzimas extracelulares que auxiliam na mineraliza??o da mat?ria org?nica. A possibilidade de desenvolver novos processos biotecnol?gicos com base na prospec??o da diversidade microbiana ? imensa, em decorr?ncia da grande variabilidade que existe entre os sistemas biol?gicos e que podem ser aperfei?oados para melhorar os sistemas de produ??o agr?colas de forma sustent?vel. O objetivo deste trabalho foi estudar o perfil da comunidade bacteriana e potencial celulol?tico de bact?rias isoladas de tr?s diferentes esp?cies de invertebrados sapr?fagos. Os experimentos foram montados no Campo Experimental da Embrapa Agrobiologia, Serop?dica, Rio de Janeiro. Gong?los, da esp?cie Trigoniulus corallinus, foram coletados em pilhas de compostos vegetais presentes em torno do campo experiemental, que posteriormente foram incubados durante 60 dias, sob seis diferentes dietas. A diversidade bacteriana do trato intestinal dos invertebrados foi analisada atrav?s da t?cnica de PCR"DGGE por amplifica??o do gene 16S rDNA PCR por eletroforese em gel com gradiente desnaturante (DGGE); foram utilizados dois dom?nios Bacteria e Actinobact?ria. Algumas bandas do gel de DGGE foram extra?das e sequenciadas. Para avaliar o potencial quanto ? produ??o de celulases em resposta ? presen?a de carboxi"metil"celulose (CMC) das bact?rias isoladas, foi utilizada a t?cnica de colora??o vermelho Congo, e os valores foram expressos atrav?s de (Ie) ?ndice enzim?tico. A partir dos maiores valores de (Ie), foram selecionadas vinte e tr?s bact?rias para a an?lise de sequenciamento do gene 16S rDNA. Ap?s a identifica??o filogen?tica, foi avaliado o potencial celulol?tico, atrav?s de testes de atividade celulol?tica de endoglucanases (CMCase) e endo e exoglucanases (FPase). Para determinar a massa molecular e atividade das enzimas foram realizados g?is de poliacrilamida (SDS"PAGE) e zimograma. Os resultados obtidos na t?cnica de DGGE, os perfis de bandas de DGGE mostrou que a microbiota intestinal dos invertebrados, det?m grupos bacterianos distintos. Pode"se inferir neste ponto, que apesar das comunidades possu?rem abund?ncia similar, como as esp?cies de Trigoniulus corallinus e Cubaris murina, os grupos que comp?em esta abund?ncia foram diferentes entre as esp?cies de invertebrados. A partir dos clones de bandas incisadas, foram sequ?nciados membros de tr?s filos, Proteobacteria, Firmicutes e Bacteroidetes. Atrav?s da an?lise filogen?tica, foi poss?vel identificar as 23 esp?cies de bact?rias. Apresentando dois filos distintos Actinomicetos e Firmicutes, o maior g?nero identificado foi Streptomyces, seguido de um isolado para Bacillus, Paenibacillus e Staphylococcus. O trato intestinal das tr?s esp?cies de invertebrados sapr?fagos revelou ser um ambiente h?bil ? prospec??o de bact?rias com efici?ncia celulol?tica, com alto potencial para futuros estudos biotecnol?gico.

Low nutrition availability in the soil can be a major limitation of plant growth. To improve nutrient acquisition, the majority of land plants engage in symbiosis with arbuscular mycorrhizal (AM) fungi. The accommodation of fungal colonisation structures in the roots requires their radical reprogramming. This starts during pre-symbiotic communication, where signals are exchanged between the fungus and plant across the rhizosphere. The receptor D14-LIKE emerged as a vital component of this pre-symbiotic communication when it was found to be absolutely required for symbiosis in rice. However, the broader relevance of the receptor, both in terms of functional conservation across plant species and its relation to other pre-symbiotic plant signalling components, remained unclear. The aim of this thesis was to elucidate these two key points. To address the fragmented picture of fungal signals, plant receptors and signalling pathways, a large scale transcriptomic experiment in rice was conducted to tie D14L together with other distinct pre-symbiotic components. In the absence of D14L-mediated signalling, rice was found to be compromised in the perception of germinated spore exudates, as well as specific chitinaceous signals, meaning that normal transcriptional reprogramming could not be achieved in response to any of these treatments. In addition, the functional conservation of D14L signalling was explored using trans-species complementation experiments. It was found that the Arabidopsis homolog AtKAI2 could complement the developmental phenotype of the d14l rice mutant, but not symbiosis. Likewise, D14La from early diverging Marchantia polymorpha and Marachantia paleacea could rescue developmental phenotypes in d14l rice, but again failed to complement symbiosis. This demonstrated a functional separation between developmental and symbiotic signalling. The data generated during my PhD foster D14L as a central node for multiple inputs to pre-symbiotic reprogramming, and provides new insights into pre-symbiotic communication mechanisms which are required for the successful establishment of symbiosis.

Dormancy, a state of severely decreased or suspended metabolism, is a widespread survival strategy in nature. In the Foraminifera, one of the most studied groups of marine organisms, its presence had been suggested by circumstantial evidence, but rarely studied directly until recently. Despite the lack of research, stressor-induced dormancy can significantly alter the way in which foraminiferal ecology is understood, especially in marginal environments. In this dissertation, I reviewed the evidence for dormancy in the foraminiferal literature, concluding that evidence for dormancy is widespread across the Phylum. I then explored the role of dormancy in the survival of the diatom-bearing foraminifer Amphistegina gibbosa d’Orbigny when exposed to toxic chemicals, and when kept in dark conditions for extended periods of time. I developed methods for utilizing CellTracker Green™, a fluorescent probe, to explore metabolic activity in symbiont-bearing foraminifers, finding that it can be used in some situations, such as bioassay experiments or other cases of toxic chemical exposure, to distinguish dead from dormant individuals. The results of the associated experiments demonstrated that reduced metabolism occurred in individuals that survived toxic chemical exposure for over two months in darkness, as well as indicating that metabolic recovery can begin to occur within 30 minutes of removal from darkness. Fluorescence microscopy of symbiont autofluorescence also demonstrated that the diatom symbionts are also capable of surviving aphotic conditions, recovering when returned to lighted conditions. Further experiments showed that A. gibbosa and its associated symbionts are capable of surviving up to 20 months in darkness. Although survival decreased as the length of time in darkness increased, 80% of the specimens survived a 20-month treatment. In addition, all treatment lengths showed color recovery, indicating survival of the diatom symbionts, which give A. gibbosa its characteristic golden-brown color. However, patterns of color recovery indicated that extended periods in darkness increased the photosensitivity of the A. gibbosa holobiont, despite entering dormancy.