A fundamental goal in ecology and evolutionary biology has been to understand how microevolutionary forces affect the origin and maintenance of mutualisms over ecological and evolutionary time scales. Mutualistic partners vary in the reciprocal benefits they provide, yet the role of partner variation on microevolutionary forces that impact the maintenance of mutualisms is unclear. Using the mutualism between legumes and nitrogen fixing symbionts, my dissertation investigated the ecological and evolutionary consequences of variation in partner quality. In the first experiment, I demonstrate how insect herbivory can change the costs and benefits of associating with exploiters, and that some degree of exploitation from non-beneficial rhizobia can reduce insect herbivory, thus removing the fitness advantage of associating purely with beneficial rhizobia. In the second study, I examine how rhizobia genotype modifies competition between hosts grown in kin and non-kin groups. I show that lower fitness in plant kin groups can simply be a by-product of genetic variance in plant size and non-linear relationships between plant size and fitness. I further show that the symbiotic community can change difference in fitness between kin and non-kin groups independent of these by-product effects. In my last chapter, I provide the first empirical evidence that an important mechanism for mutualism stability-- the ability for hosts to preferentially associate with beneficial rhizobia-- is genetically variable and can evolve in response to exploitation. I also show that host preference for beneficial rhizobia can be maintained in legume populations, even in the absence of exploitation. My dissertation provides insight into the potential evolutionary dynamics of stabilizing mechanisms by suggesting that the agents of selection that affect the level of host exploitation can come from biotic factors other than the exploiters themselves. My dissertation has also shown that inclusion of other ecological interactions, such as herbivory, can provide valuable perspective on fitness effects of symbionts on their hosts, and can even change our fundamental assumptions about the effects of exploitation on host fitness, which has formed the backbone of mutualism theory.

Transposons are selfish, mobile genetic elements, moving within the genome. The transposase genemakes this possible, as it codes for the enzyme that catalyzes the movement. In the case of bacteria,they can also move horizontally between individual bacteria, and sometimes even between species.By default, they are a burden for the host organism, coding for a protein that the host does not need.They also pose the risk of disabling the host’s crucial genes by inserting themselves into it.Transposons are under some pressure to benefit the host, to help propagate themselves moreeffectively. And some transposons have indeed evolved to benefit the host. Lactobacillus kunkeei is a bacterial species known to reside in honeybee guts. It is known for itsrole in honey preservation and wine spoilage. The genome of L. kunkeei is reduced because it is asymbiont, however it contains an unusually high amount of transposons in its genome. In this study, the transposase genes (transposon enzymes) found in L. kunkeei are studied andcategorized. The L. kunkeei have been extracted from honeybees (Apis mellifera). The honeybeesthemselves have been collected from the islands Åland and Gotland. This study focuses on the transposase genes that come in pairs, one after another in the genome.Transposase genes were identified using annotation software and orthology-based methods. Theannotation software provides numbering for the genes, which allows finding paired genes. Thepaired genes were categorized based on alignments and phylogenetic software. Pseudogenizedtransposons were identified based on length and/or clustering into triplets. A total of 766 paired transposase genes were found. The transposase genes were found to take up1.9% of the genome, on average. A low level of diversity has been found when performingalignments and generating phylogenetic trees. The positions of the transposase genes are generallyconserved within phylogenetic groups. Pseudogenization has been detected for some transposasegenes – 4.5 per genome, on average. All of the studied transposons belong to the IS3 family, whichis a family of Class I transposons.

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.

Les symbioses eucaryotes/micro-organismes constituent une importante source d’innovation évolutive et de diversité écologique. Ces associations sont très répandues chez les insectes, en particulier chez les insectes parasitoïdes (insectes parasites d’autres insectes) qui hébergent en particulier une grande diversité de virus transmis verticalement. Leurs effets directs sur les parasitoïdes ainsi que les effets indirects sur la structure des communautés sont à l’heure actuelle mal compris. Nous avons abordé ces questions au travers l’étude d’un virus héritable (LbFV) ayant la particularité de manipuler le comportement de superparasitisme de son hôte, l’hyménoptère parasitoïde de drosophiles Leptopilina boulardi. La mise au point d’un outil moléculaire diagnostic de l’infection nous a permis de montrer que ce virus, spécifique à L. boulardi, peut atteindre de fortes prévalences dans les populations d’hôtes. Nous avons également mis en évidence un effet de la présence du virus sur les interactions compétitives interspécifiques au sein de la communauté des parasitoïdes de drosophiles. L’approche intégrée de ce travail constitue une étape importante dans la connaissance du rôle des virus héritables sur l’écologie et l’évolution de leurs hôtes / Eukaryots/microorganisms symbiosis is an important source of evolutionary novelty and ecological diversification. These associations are widespread in insects, particularly in parasitoids (insects that parasitize other insects) where a broad diversity of vertically transmitted viruses are found. However, their direct and indirect effects on host community are poorly understood. In this thesis, we used a system involving a Drosophila parasitoid, Leptopilina boulardi and a heritable virus LbFV that manipulates the behaviour of the parasitoid by increasing its tendency to lay eggs in a host that is already parasitized (superparasitism). Using a viral molecular marker developed in this work, we showed very high prevalences of the virus in L. boulardi populations. Additionally, we found a strong effect of the virus on interspecific competition in the Drosophila parasitoid community. The integrative approach of this work is an important step in understanding the role of heritable viruses in parasitoid ecology and evolution

Les patrons de distribution chez les micro-organismes reposeraient sur leurs capacités à disperser dans le temps et dans l'espace, en lien avec des facteurs abiotiques comme les propriétés du sol, le climat, et des interactions biotiques, notamment avec l'hôte dans le cas des symbiontes, mais aussi sur les traits d'histoire de vie propres aux micro-organismes, telle que la capacité à sporuler. Frankia sp. est une actinobactérie sporulante et fixatrice d'azote à la biogéographie complexe, car vivant à la fois de façon saprophytique dans le sol, en symbiose racinaire (nodosité) avec les plantes actinorhiziennes dont les aulnes (Alnus, Betulaceae). Deux types de souches de Frankia génétiquement différentes ont été décrites dont la distinction phénotypique majeure réside dans la capacité à maintenir (Sp+) ou non (Sp-) leur sporulation in planta. Cette sporulation endophytique est à notre connaissance unique dans un contexte symbiotique et son implication dans la biogéographie de Frankia, reste peu connue. Ces travaux de thèse intègrent à la fois des approches descriptives et expérimentales, sur le terrain et au laboratoire, afin d'accroître la compréhension du rôle écologique de la sporulation in planta de Frankia. Dans un premier temps, nous avons étendu la description de la phylobiogéographie des souches de Frankia Sp+ afin de tester la validité du patron de distribution centré sur les milieux froids des zones de haute altitude et de haute latitude de l'hémisphère nord. Un intérêt tout particulier a été porté sur les aires géographiques où une plus forte diversité de Frankia était attendue, dans la zone d'origine de l'aulne et ses refuges glaciaires. Dans un second temps, nous avons étudié l'influence du partenaire végétal dans la distribution observée des Frankia Sp+ et l'implication du trait Sp+ dans la capacité d'association à l'hôte. Des croisements expérimentaux ont été réalisés au laboratoire afin de découpler les effets de l'espèce-hôte et du climat, et tester les implications du trait Sp+ en termes d'infectivité, compétitivité et spectre d'hôte. Enfin, nous avons étudié les conséquences écosystémiques de l'expansion subalpine du complexe symbiotique Alnus/Frankia, au niveau de la diversité microbienne et du fonctionnement du cycle de l'azote, en fonction du phénotype de sporulation des souches associées. Des analyses pédologiques, en association avec des mesures de nitrification, dénitrification et fixation d'azote, ainsi que des analyses de diversité microbienne (globale et fonctionnelle), ont été réalisées dans différentes aulnaies Sp+, Sp- ou mixte, à différents stades de colonisation de l'aulne. Les résultats obtenus démontrent une prédominance des souches Sp+ associées aux espèces d'aulne des milieux froids sur les 3 continents de la zone Holarctique, avec une diversité nouvelle dans l'aire d'origine et les zones refuges de l'aulne. Les croisements effectués révèlent une infectivité et compétitivité plus forte des Sp+ par rapport aux Sp-. De plus, contrairement aux Sp- à spectre d'hôte très large, les Sp+ présentent un spectre limité entraînant des incompatibilités d'association suggérant une dépendance forte à une espèce-hôte donnée. Les modifications des communautés microbiennes du sol en réponse à l'expansion du complexe symbiotique Alnus/Frankia ont été démontrées, en lien avec la stimulation du cycle de l'azote dans les milieux sub-/alpins. Les premiers résultats sur l'efficience comparée de la fixation d'azote in natura des souches Sp- par rapport aux Sp+ suggèrent que 100% de l'azote de l'aulne est obtenu par le biais de la fixation. Aucun patron n'est mis en évidence entre souches Sp+ et Sp-, suggérant un effet plus complexe de la saisonnalité, de l'âge de l'arbre et de celui de la nodosité. Les résultats obtenus nous permettent de mieux appréhender les facteurs guidant la biogéographie de Frankia et de discuter de l'évolution de ces patrons de distribution en réponse au réchauffement climatique / Microbial biogeography would be based on the ability of microorganisms to disperse across time and space, as a function of abiotic factors such as soil properties, climate, and of biotic interactions, in particular with the host in the case of symbionts, but also on life history traits such as the ability to sporulate. Frankia sp. is a spore-forming and nitrogen-fixing actinobacterium that has a complex biogeography given its abilities for both saprophytic life and root symbiotic interaction with actinorhizal plants such as alders (Alnus, Betulaceae). Two distinct groups of Frankia lineages have been described according to a major phenotypic divergence, based on the presence (Sp+) or the absence (Sp-) of spores in planta.. To the best of our knowledge, this endophytic sporulation is an original trait in a symbiotic context and very little is known about its incidence in Frankia biogeography. This work integrates descriptive and experimental approaches on both field and laboratory areas, in order to improve the understanding of the ecological role of Frankia in planta sporulation. First, we have extended the description of the phylobiogeography of Sp+ Frankia strains to validate the previously proposed distribution pattern focused on cold environements at high altitude or high latitude. A phylogeny has been computed using a large number of nodular strains coming from the 3 continents of the Northern Hemisphere and 10 different Alnus species. Special attention was paid to geographic areas where a higher diversity was expected, in Asia, and in its glacial refuges. Second, we studied the influence of the host-plant on the distribution of Fankia Sp+ and the incidence of Sp+ in the symbiotic interaction. Experimental crosses have been performed to disentangle host and climate effects and to test the incidence of the Sp+ trait in terms of infectivity, competitiveness and host-range. Finally, we studied the ecological consequences of the Alnus/Frankia symbiotic complex, on the microbial diversity and on the nitrogen cycle functionning, with respect to the sporulation of Frankia and to the Alnus expansion on sub-/alpine grasslands. Soils analyses were performed in association with measures of nitrification and denitrification, as well as global and functional microbial diversity analyses, in Sp+, Sp- or mixed alder stands and at different colonization stages. In each part of this work, alder ectomycorhizae were analyzed to compare the distribution pattern between the two symbionts and to highlight potential interactions with the Sp+ trait of Frankia. Our results show the dominance of Sp+ strains in nodules of alder species from cold environments over the 3 continents of the Holarctic zone, with original diversity patterns in alder area of origin and in glacial refuges. Even if these strains are genetically homogenous, host-specific clusters were observed in the phylogeny. Crosses revealed that Sp+ strains were more infective and competitive than Sp- strains. Moreover, unlike Sp- strains that harbor a wide host-range, Sp+ strains have a narrower specificity leading to association’s incompatibilities and suggesting strong host dependence. For the first time, modifications of microbial communities were revealed in response to the Alnus-Frankia symbiotic complex colonization and were linked to a stimulation of the nitrogen cycle in the sub-/alpine grasslands. The first comparative results of nitrogen fixation between Sp+ and Sp- strains in natura suggest a maximal efficiency of fixation, representing almost 100% of the alder nitrogen. However, unlike previous reports in literature, no pattern was observed between Sp+ and Sp- strains, suggesting a complex effect of seasonality, alder age as well as that of nodules. Altogether, the previous results contribute to a better understanding of the Frankia biogeography drivers and allow us to discuss the expected evolution of distribution pattern in response to the global warming

Os produtos naturais desempenham fundamental importância na descoberta de novos fármacos. Muitos destes fármacos originaram-se de produtos naturais microbianos o que demonstra uma extrema eficiência na habilidade dos micro-organismos de sintetizar substâncias químicas com elevado potencial biológico. Sabe-se que os insetos sociais estão sujeitos a condições climáticas e populacionais que aumentam sua susceptibilidade a parasitas. Assim, estes insetos desenvolveram um mecanismo de defesa evolutivo que consiste na associação simbiótica a bactérias capazes de biossintetizar produtos antimicrobianos contra os patógenos. Dispondo dessas informações, o presente trabalho contribuiu com o estudo dos produtos naturais biossintetizados pela bactéria Paenibacillus polymyxa ALLI-03-01, provável simbionte da espécie de abelha sem ferrão Melipona scutellaris. Os micro-organismos foram isolados de colônias mantidas no Departamento de Biologia da FFCLRP-USP. Todas as linhagens foram testadas contra dois fungos entomopatogênicos - Beauveria bassiana e Metarhizium anisopliae; os que apresentaram atividade antifúngica tiveram seus extratos em acetato de etila e metanol preparados e foram avaliados em ensaios antiparasitários contra Trypanosoma cruzi e Leishmania donovani. A bactéria ALLI-03-01, isolada do alimento larval, foi escolhida para os estudos químicos, pois apresentou satisfatória atividade antifúngica e proeminente atividade antiparasitária. Este micro-organismo foi identificado pelo sequenciamento do gene 16S rRNA como sendo a bactéria Paenibacillus polymyxa, conhecida pela alta capacidade de produção de metabólitos secundários. Para conhecer seu metabolismo, esta bactéria foi cultivada em meio ISP-2 ágar e líquido e do extrato em acetato de etila do cultivo fermentativo de P. polymyxa ALLI-03-01 foi isolado o ácido L-3-fenil lático. As atividades antifúngicas dos padrões comerciais dos ácidos D-(+)- e L-(-)- 3-fenil lático foram testadas frente ao fungo entomopatogênico B. bassiana e apenas o enantiômero L apresentou atividade antifúngica. Do extrato em metanol do cultivo em meio sólido do mesmo micro-organismo foram identificados em mistura, principalmente por MALDI-TOF MS/MS, nove diferentes ciclolipodepsipeptídeos, da classe das Fusaricidinas. As fusaricidinas são conhecidas por apresentarem alta atividade anti-microbiana, que foi comprovada quando duas frações compostas por diferentes fusaricidinas foram testadas frente ao fungo entomopatogênico Metarhizium anisopliae e contra a bactéria patogênica Paenibacillus larvae. As frações apresentaram resultados satisfatórios de inibição contra ambos os patógenos, dessa maneira as fusaricidinas e a produção seletiva do ácido L-3-fenil lático por esta bactéria podem ser vias de proteção da colônia, evitando que patógenos destruam-na e permitindo o desenvolvimento sadio de larvas. Sendo assim, este trabalho relata pela primeira vez a possível relação simbiótica existente entre o micro-organismo Paenibacillus polymyxa ALLI-03-01 e a colônia de abelhas Melipona scutellaris, bem como a produção por este micro-organismo de compostos com alta atividade antimicrobiana que podem exercer o controle de parasitas dentro da colônia, estabelecendo assim, uma possível relação ecológica entre o micro-organismo destacado e as abelhas Melipona scutellaris. / Natural products play an important role in drug discovery. Several drugs have been discovered or developed from microbial natural products hits and leads showing a great efficiency of microorganisms to synthesize compounds with high biological potential. It is known that social insects are subjected to climate and population conditions that increase their susceptibility to parasites. Therefore, these insects have developed an evolutionary defense mechanism consisting in symbiotic association with bacteria capable of biosynthesize antimicrobial products against pathogens. In this context, this study contributed to the knowledge of natural products biosynthesized by the bacterium Paenibacillus polymyxa ALLI-03-01, a probable symbiont of the stingless bee Melipona scutellaris. Microorganisms were isolated from colonies maintained at the Department of Biology of FFCLRP-USP. All strains were tested against two entomopathogenic fungi - Beauveria bassiana and Metarhizium anisopliae; strains that showed antifungal activity had their ethyl acetate and methanol extracts prepared and evaluated against the parasites Trypanosoma cruzi and Leishmania donovani. The bacterium ALLI-03-01, isolated from larval food, was selected for chemical studies, due to its satisfactory antifungal activity and outstanding antiparasitic activity. This microorganism was identified by gene 16S rRNA sequencing as Paenibacillus polymyxa, a bacterium recognized for its ability of biosynthesize secondary metabolities. This bacterium was cultivated in ISP-2 agar and liquid medium and from the ethyl acetate extract of P. polymyxa ALLI-03-01 cultivated in liquid medium L-3-phenyllactic acid was isolated. The antifungal activity of comercial standards of D-(+)- and L-(-)-phenyllactic acid were tested against entomopathogenic fungus B. bassiana and only the L enantiomer presented antifungal activity. Nine cyclo lipodepsipeptides known as fusaricidins were identified in mixture using MALDI-TOF-MS/MS from the methanol extract of ALLI-03-01. The fusaricidins are recognized for their high antimicrobial activity, which was confirmed when two fractions composed by different fusaricidins were tested against the entomopathogenic fungus Metarhizium anisopliae and the pathogenic bacterium Paenibacillus larvae. The fractions showed satisfactory inhibition against both pathogens. Therefore, fusaricidins and selective production of L-3-phenyllactic acid by the bacterium may be colony protection resources, preventing the action of pathogens and allowing healthy larvae development. Therefore, this study reports for the first time a possible symbiotic relationship between Paenibacillus polymyxa ALLI-03-01 and the colony of M. scutellaris bees, as well as production of compounds with high antimicrobial activity by this microorganism that might act as parasite control agents within the colony, establishing a possible ecological relationship between the bacterium and M. scutellaris bees.

Blood-sucking louse Polyplax serrata harbor two phylogenetically distinct symbiotic bacteria from the orders Legionellales (Gammaproteobacteria) and Neisseriales (Betaproteobacteria). Based on rigorous phylogenetic analyses, the first bacteria was confirmed as member of genus Legionella, and the name Candidatus Legionella polyplacis, sp. n. was proposed. Vertical transgeneration transmission was proved by visualisation of the symbiont in host tissues. Improved genome of latter symbiont is considerably larger and possesses more metabolic functions. In the genome, I found horizontally transferred operon, which encode synthesis of enzymes involved in urea cleavage.

Les associations symbiotiques entre microorganismes et eucaryotes sont omniprésentes dans le monde vivant. Ces microorganismes peuvent jouer un rôle crucial dans l’évolution et l’écologie de leurs porteurs en modifiant leur phénotype. Ces symbiotes étant le plus souvent héritables, les phénotypes étendus résultant de ces associations symbiotiques peuvent se transmettre aux générations suivantes. Certains microorganismes vont permettre l’accès à une ressource alimentaire, d’autres conférer une protection contre un ennemi. Une telle protection symbiotique est rencontrée chez le puceron du pois (Acyrthosiphon pisum) en interaction avec la bactérie Hamiltonella defensa. Cette symbiose confère au puceron une résistance face à l’attaque de son principal ennemi : le parasitoïde Aphidius ervi. Les populations de ce ravageur des Légumineuses sont structurées en biotypes (populations spécialisées sur des plantes hôtes). La distribution du symbiote protecteur au sein des populations de pucerons est singulière. De nombreux individus vivant sur la luzerne, la bugrane ou les genêts abritent ce symbiote alors qu’il est peu fréquent dans les populations d’autres biotypes d’A. pisum comme le pois ou le trèfle. Nous avons cherché à comprendre pourquoi H. defensa n’était pas retrouvé chez tous les biotypes du puceron du pois. Afin de prédire la dynamique de la symbiose protectrice et le potentiel de résistance dans les populations aphidiennes naturelles, nous nous sommes intéressés à plusieurs processus écologiques et évolutifs. L’incidence de la pression des parasitoïdes sur la composition des populations symbiotiques a été mesurée chez trois biotypes (luzerne, pois et trèfle) à travers une approche terrain. La distribution du symbiote H. defensa dans les populations est directement dépendante de la variabilité du phénotype associé exprimé dans les différentes populations, j’ai identifié les phénotypes associés au symbiote pour des pucerons issus de différents biotypes ainsi que l’influence du contexte local sur ces phénotypes. L’absence d’H. defensa chez certains individus peut s’expliquer par la redondance d’une fonction protectrice en place chez ces biotypes comme un alternative symbiotique autre que H. defensa ou encore une immunité forte. Enfin, nous avons testé si le cumule des protections symbiotiques conférées par deux bactéries du cortège du puceron du pois pouvaient se cumuler créant ainsi des super-organismes. Mon travail met en évidence l’implication de nombreux facteurs dans la prédiction des fréquences symbiotiques d’une bactérie facultative dans les populations d’hôte. / Symbiotic associations between microorganisms and eukaryotes are ubiquitous in the living world. These microorganisms can play a crucial role in the evolution and ecology of their hosts by altering their phenotypes. Since these symbionts are usually heritable, extended phenotypes resulting from these symbiotic associations may be transmitted to subsequent generations. Some microorganisms will allow access to a food source; others will provide protection against natural enemies. Such symbiotic protection is found in the pea aphid (Acyrthosiphon pisum) in its interaction with the bacteria Hamiltonella defensa. This symbiosis provides the aphid with a resistance against the attack of its main parasitoid enemy: Aphidius ervi. The populations of the pea aphid, a legume pest insect, are structured in different biotypes (specialized populations on host plants). The distribution of this protective symbiont within pea aphid populations is singular: many individuals living on Medicago sativa (alfalfa), Ononis spinosa or Genista sagittalis and G. tinctoria host plant with H. defensa while it is rarely found in other populations of A. pisum biotypes such as Pisum sativum (pea) or Trifolium sp. (clover). We sought to understand why H. defensa was not found in every pea aphid biotype. In order to predict the dynamics of the protective symbiosis and the resistance potential in natural aphid populations, we focused on several ecological and evolutionary processes. We measured the consequence of parasitoid stress in the composition of symbiotic populations in three different biotypes (alfalfa, clover and pea) using a field approach. The distribution of H. defensa symbiont in populations dependent directly on the variability of the associated phenotype expressed in different populations. We identified the phenotypes associated with this symbiont in aphids from different biotypes, and the influence of the local context on these phenotypes. The lack of H. defensa in some individuals can be explained by the redundancy of a protective function already in place in these biotypes, such as an alternative symbiotic species or a strong immunity. Finally, we tested whether the symbiotic protections provided by two different bacteria in the pea aphid could be cumulated, thus creating super-organisms. My work highlights the many factors involved in predicting the frequencies of facultative symbiotic bacteria in host populations.

Os produtos naturais desempenham fundamental importância na descoberta de novos fármacos. Muitos destes fármacos originaram-se de produtos naturais microbianos o que demonstra uma extrema eficiência na habilidade dos micro-organismos de sintetizar substâncias químicas com elevado potencial biológico. Sabe-se que os insetos sociais estão sujeitos a condições climáticas e populacionais que aumentam sua susceptibilidade a parasitas. Assim, estes insetos desenvolveram um mecanismo de defesa evolutivo que consiste na associação simbiótica a bactérias capazes de biossintetizar produtos antimicrobianos contra os patógenos. Dispondo dessas informações, o presente trabalho contribuiu com o estudo dos produtos naturais biossintetizados pela bactéria Paenibacillus polymyxa ALLI-03-01, provável simbionte da espécie de abelha sem ferrão Melipona scutellaris. Os micro-organismos foram isolados de colônias mantidas no Departamento de Biologia da FFCLRP-USP. Todas as linhagens foram testadas contra dois fungos entomopatogênicos - Beauveria bassiana e Metarhizium anisopliae; os que apresentaram atividade antifúngica tiveram seus extratos em acetato de etila e metanol preparados e foram avaliados em ensaios antiparasitários contra Trypanosoma cruzi e Leishmania donovani. A bactéria ALLI-03-01, isolada do alimento larval, foi escolhida para os estudos químicos, pois apresentou satisfatória atividade antifúngica e proeminente atividade antiparasitária. Este micro-organismo foi identificado pelo sequenciamento do gene 16S rRNA como sendo a bactéria Paenibacillus polymyxa, conhecida pela alta capacidade de produção de metabólitos secundários. Para conhecer seu metabolismo, esta bactéria foi cultivada em meio ISP-2 ágar e líquido e do extrato em acetato de etila do cultivo fermentativo de P. polymyxa ALLI-03-01 foi isolado o ácido L-3-fenil lático. As atividades antifúngicas dos padrões comerciais dos ácidos D-(+)- e L-(-)- 3-fenil lático foram testadas frente ao fungo entomopatogênico B. bassiana e apenas o enantiômero L apresentou atividade antifúngica. Do extrato em metanol do cultivo em meio sólido do mesmo micro-organismo foram identificados em mistura, principalmente por MALDI-TOF MS/MS, nove diferentes ciclolipodepsipeptídeos, da classe das Fusaricidinas. As fusaricidinas são conhecidas por apresentarem alta atividade anti-microbiana, que foi comprovada quando duas frações compostas por diferentes fusaricidinas foram testadas frente ao fungo entomopatogênico Metarhizium anisopliae e contra a bactéria patogênica Paenibacillus larvae. As frações apresentaram resultados satisfatórios de inibição contra ambos os patógenos, dessa maneira as fusaricidinas e a produção seletiva do ácido L-3-fenil lático por esta bactéria podem ser vias de proteção da colônia, evitando que patógenos destruam-na e permitindo o desenvolvimento sadio de larvas. Sendo assim, este trabalho relata pela primeira vez a possível relação simbiótica existente entre o micro-organismo Paenibacillus polymyxa ALLI-03-01 e a colônia de abelhas Melipona scutellaris, bem como a produção por este micro-organismo de compostos com alta atividade antimicrobiana que podem exercer o controle de parasitas dentro da colônia, estabelecendo assim, uma possível relação ecológica entre o micro-organismo destacado e as abelhas Melipona scutellaris. / Natural products play an important role in drug discovery. Several drugs have been discovered or developed from microbial natural products hits and leads showing a great efficiency of microorganisms to synthesize compounds with high biological potential. It is known that social insects are subjected to climate and population conditions that increase their susceptibility to parasites. Therefore, these insects have developed an evolutionary defense mechanism consisting in symbiotic association with bacteria capable of biosynthesize antimicrobial products against pathogens. In this context, this study contributed to the knowledge of natural products biosynthesized by the bacterium Paenibacillus polymyxa ALLI-03-01, a probable symbiont of the stingless bee Melipona scutellaris. Microorganisms were isolated from colonies maintained at the Department of Biology of FFCLRP-USP. All strains were tested against two entomopathogenic fungi - Beauveria bassiana and Metarhizium anisopliae; strains that showed antifungal activity had their ethyl acetate and methanol extracts prepared and evaluated against the parasites Trypanosoma cruzi and Leishmania donovani. The bacterium ALLI-03-01, isolated from larval food, was selected for chemical studies, due to its satisfactory antifungal activity and outstanding antiparasitic activity. This microorganism was identified by gene 16S rRNA sequencing as Paenibacillus polymyxa, a bacterium recognized for its ability of biosynthesize secondary metabolities. This bacterium was cultivated in ISP-2 agar and liquid medium and from the ethyl acetate extract of P. polymyxa ALLI-03-01 cultivated in liquid medium L-3-phenyllactic acid was isolated. The antifungal activity of comercial standards of D-(+)- and L-(-)-phenyllactic acid were tested against entomopathogenic fungus B. bassiana and only the L enantiomer presented antifungal activity. Nine cyclo lipodepsipeptides known as fusaricidins were identified in mixture using MALDI-TOF-MS/MS from the methanol extract of ALLI-03-01. The fusaricidins are recognized for their high antimicrobial activity, which was confirmed when two fractions composed by different fusaricidins were tested against the entomopathogenic fungus Metarhizium anisopliae and the pathogenic bacterium Paenibacillus larvae. The fractions showed satisfactory inhibition against both pathogens. Therefore, fusaricidins and selective production of L-3-phenyllactic acid by the bacterium may be colony protection resources, preventing the action of pathogens and allowing healthy larvae development. Therefore, this study reports for the first time a possible symbiotic relationship between Paenibacillus polymyxa ALLI-03-01 and the colony of M. scutellaris bees, as well as production of compounds with high antimicrobial activity by this microorganism that might act as parasite control agents within the colony, establishing a possible ecological relationship between the bacterium and M. scutellaris bees.

Lors de cette thèse, je me suis intéressée aux modèles et aux algorithmes pour étudier l'histoire évolutive commune des hôtes et des symbiotes. Le premier objectif était d'analyser la robustesse des méthodes de réconciliation des arbres phylogénétiques, qui sont très utilisées dans ce type d'étude. Celles-ci associent (ou lient) un arbre, d'habitude celui des symbiotes, à l'autre, en utilisant un modèle dit basé sur des évènements. Les évènements les plus utilisés sont la cospéciation, la duplication, le saut et la perte. Les phylogénies des hôtes et des symbiotes sont généralement considérés comme donnés, et sans aucune erreur. L'objectif était de comprendre les forces et les faiblesses du modèle parcimonieux utilisé et comprendre comment les résultats finaux peuvent être influencés en présence de petites perturbations ou d'erreurs dans les données en entrée. Ici deux cas sont considérés, le premier est le choix erroné d'une association entre les feuilles des hôtes et des symbiotes dans le cas où plusieurs existent, le deuxième est lié au mauvais choix de l'enracinement de l'arbre des symbiotes. Nos résultats montrent que le choix des associations entre feuilles et le choix de l'enracinement peuvent avoir un fort impact sur la variabilité de la réconciliation obtenue. Nous avons également remarqué que l'evènement appelé “saut” joue un rôle important dans l'étude de la robustesse, surtout pour le problème de l'enracinement. Le deuxième objectif de cette thèse était d'introduire certains evènements peu ou pas formellement considérés dans la littérature. L'un d'entre eux est la “propagation”, qui correspond à l'invasion de différents hôtes par un même symbiote. Dans ce cas, lorsque les propagations ne sont pas considérés, les réconciliations optimales sont obtenues en tenant compte seulement des coûts des évènements classiques (cospeciation, duplication, saut, perte). La nécessité de développer des méthodes statistiques pour assigner les coûts les plus appropriés est toujours d'actualité. Deux types de propagations sont introduites : verticaux et horizontaux. Le premier type correspond à ce qu'on pourrait appeler aussi un gel, à savoir que l'évolution du symbiote s'arrête et “gèle” alors que le symbiote continue d'être associé à un hôte et aux nouvelles espèces qui descendent de cet hôte. Le second comprend à la fois une invasion, du symbiote qui reste associé à l'hôte initial, mais qui en même temps s'associe (“envahit”) un autre hôte incomparable avec le premier, et un gel par rapport à l'évolution des deux l'hôtes, celui auquel il était associé au début et celui qu'il a envahi. Nos résultats montrent que l'introduction de ces evènements rend le modèle plus réaliste, mais aussi que désormais il est possible d'utiliser directement des jeux de données avec un symbiote qui est associé plusieurs hôtes au même temps, ce qui n'était pas faisable auparavant / In this Ph.D. work, we proposed models and algorithms to study the common evolutionary history of hosts and symbionts. The first goal was to analyse the robustness of the methods of phylogenetic tree reconciliations, which are a common way of performing such study. This involves mapping one tree, most often the symbiont’s, to the other using a so-called event-based model. The events considered in general are cospeciation, duplication, host switch, and loss. The host and the symbiont phylogenies are usually considered as given and without any errors. The objective here was to understand the strengths and weaknesses of the parsimonious model used in such mappings of one tree to another, and how the final results may be influenced when small errors are present, or are introduced in the input datasets. This may correspond either to a wrong choice of present-day symbiont-host associations in the case where multiple ones exist, or to small errors related to a wrong rooting of the symbiont tree. Our results show that the choice of leaf associations and of root placement may have a strong impact on the variability of the reconciliation output. We also noticed that the host switch event has an important role in particular for the rooting problem. The second goal of this Ph.D. was to introduce some events that are little or not formally considered in the literature. One of them is the spread, which corresponds to the invasion of different hosts by a same symbiont. In this case, as when spreads are not considered, the optimal reconciliations obtained will depend on the choice made for the costs of the events. The need to develop statistical methods to assign the most appropriate ones therefore remains of actuality. Two types of spread are introduced: vertical and horizontal. The first case corresponds to what could be called also a freeze in the sense that the evolution of the symbiont “freezes” while the symbiont continues to be associated with a host and with the new species that descend from this host. The second includes both an invasion, of the symbiont which remains with the initial host but at the same time gets associated with (“invades”) another one incomparable with the first, and a freeze, actually a double freeze as the evolution of the symbiont “freezes” in relation to the evolution of the host to which it was initially associated and in relation to the evolution of the second one it “invaded”. Our results show that the introduction of these events makes the model more realistic, but also that it is now possible to directly use datasets with a symbiont that is associated with more than one host at the same time, which was not feasible before