Thesis (M.S.)--State University of New York at Binghamton, Biological Sciences Department, 2007. / Includes bibliographical references.

Les endosymbioses racinaires sont des associations bénéfiques établies entre les racines des plantes et des micro-organismes du sol. Ces symbioses ont un intérêt agronomique et écologique puisque les plantes fournissent à leurs partenaires microbiens une niche écologique et des sucres issus de la photosynthèse et en retour, les micro-organismes associés aux racines vont fournir à la plante des nutriments minéraux qui sont actuellement apportés dans l’agriculture conventionnelle sous forme d’engrais. Durant ma thèse, j’ai particulièrement étudié la symbiose endomycorhizienne à arbuscules (AMS). Elle implique des champignons du groupe des Gloméromycètes et plus de 80 % des plantes terrestres. Ainsi cette symbiose est la plus répandue sur terre connue à l’heure actuelle. Plusieurs étapes importantes pour l’établissement de l’AMS ont été définies. La première de ces étapes est la reconnaissance mutuelle entre le champignon endomycorhizien et la plante hôte. Le champignon est capable de percevoir les plantes par les exsudats racinaires qu’elles sécrètent dans la rhizosphère. Dans le mélange complexe de molécules que sont les exsudats racinaires, des phytohormones appelées strigolactones activent le métabolisme des champignons endomycorhizien, la ramification des leurs hyphes et la production de molécules fongiques appelée facteurs Myc. La perception des facteurs Myc par la plante active des processus permettant la colonisation des racines par le champignon. Ce dialogue moléculaire entre champignons endomycorhiziens et plantes hôtes reste toutefois méconnu. Des molécules de type Lipo-chitooligosaccharides (LCO) ou chito-oligosaccharides (CO) ont été identifiées dans les exsudats de spores ou d’hyphes de champignons et activent la voie de signalisation symbiotique chez les plantes mais leurs rôles respectifs dans l’établissement de l’AMS restent mal compris. Du côté de la plante, des récepteurs potentiels aux LCOs et aux COs sont codés par les gènes de la famille des Lysin Motif Receptor-Like Kinase (LysM-RLK) qui sont capables de lier les constituants structuraux des LCOs et des COs. Cependant aucune preuve n’avait été apportée, au commencement de ma thèse, permettant de conclure sur le rôle des LCOs, des COs, et des LysM-RLKs dans la mise en place de l’AMS. C’est ce que je me suis attachée à démontrer durant ma thèse. Pour cela, j’ai travaillé sur une dicotylédone (la tomate : Solanum lycopersicum) et sur une monocotylédone (Brachypodium distachyon, un modèle pour le blé). Pour identifier les récepteurs aux LCOs dans ces plantes et déterminer leur rôle dans l’AMS nous avons mis en place des techniques de génétique inverse. Nous avons ensuite déterminé l’affinité de ces récepteurs pour les LCOs. Ainsi, nous avons montré que la perception des LCOs dans la tomate est importante pour la mise en place de l’AMS. Par ailleurs, je me suis intéressée à la symbiose entre des bactéries du type rhizobium et des plantes principalement de la famille des légumineuses. La mise en place de cette symbiose nécessite la synthèse de LCOs par les rhizobia et leur perception par la plante via des récepteurs de la famille des LysM-RLKs. Ces similarités que la symbiose rhizobium-légumineuses partage avec l’AMS nous ont conduits à poser la question de savoir si les récepteurs de LCOs impliqués dans l’AMS (beaucoup plus ancienne que la symbiose rhizobium-légumineuse) ont été recrutés durant l’évolution pour jouer un rôle dans la symbiose rhizobium-légumineuse. J’ai pu montrer que les récepteurs de LCOs impliqués dans l’AMS chez les espèces non-légumineuses susmentionnées sont fonctionnels l’établissement de la symbiose rhizobium-légumineuse chez une légumineuse. / Root endosymbioses are beneficial associations established between plant roots and soil microorganisms. These symbioses have an agronomic and ecological interest as plants provide their microbial partners with an ecological niche and carbohydrates from photosynthesis. In return, the root-associated microorganisms provide the plant with minerals that are currently being delivered in conventional agriculture as fertilizers. During my thesis, I particularly studied the arbuscular mycorrhizal symbiosis (AMS). It involves fungi of the Glomeromycota group and more than 80 % of land plants. This is the currently known most widespread symbiosis on earth. Important steps for the AMS establishment have been defined. The first step is the mutual recognition between the endomycorrhizal fungus and the host plant. Fungi can perceive plants through the root exudates. In the complex mixture of molecules in the root exudates, phytohormones called strigolactones activate the endomycorrhizal fungal metabolism, the branching of their hyphae and the production of fungal molecules called Myc-Factors. Myc-Factors are perceived by the plant and activate a signaling pathway allowing root colonization by the fungus. However, parts of the molecular dialogue between endomycorrhizal fungi and host plants remain unknown. Lipo-chitooligosaccharide (LCO) or chito-oligosaccharides (CO) molecules have been found in exudates of fungal spores or hyphae and were shown to activate the plant symbiotic signaling pathway, however their respective roles in the AMS establishment are unclear. Putative plant receptors for LCOs and COs are encoded by genes from the Lysin Motif Receptor-Like Kinase family (LysM-RLK) which are able of binding the structural LCO and CO components. However, at the beginning of my PhD, we had no evidence allowing to conclude about the involvement of LCOs, COs, or LysM-RLKs in the AMS establishment. During my thesis, I aimed to understand the role the LCOs and their plant receptors in AMS. For this, I used on a dicotyledon (the tomato: Solanum lycopersicum) and on a monocotyledon (Brachypodium distachyon that is a model for wheat). In order to identify the LCO receptors in these two species, I used a reverse genetic approach. Then I determined these receptors affinity for various LCO structures. I showed that in tomato, LCO perception is important for AMS establishment. In addition, I have studied the symbiosis between rhizobium-type bacteria and plants of the legume family. Interestingly, the establishment of this symbiosis requires LCO synthesis by rhizobia and LCO perception by the plant via receptors of the LysM-RLK family. The fact that rhizobium-legume symbiosis shares similarities with the AMS led us to ask whether the LCO receptors involved in AMS (a much more ancient symbiosis than the rhizobium-legume symbiosis) have been recruited during evolution for a role in the rhizobium-legume symbiosis. I demonstrated that the LysM-RLKs involved in AMS in the above mentioned non-legume species are functional for the rhizobium-legumes establishment in a legume species.

Crayfish throughout North America, Europe, and Asia host assemblages of obligate ectosymbiotic annelid worms called branchiobdellidans. The work presented here is a detailed experimental and observational study of the ecological interactions between crayfish and their worms. In a comprehensive literature review, I show that branchiobdellidans have complex and context-dependent effects on their hosts, serving as both beneficial cleaners and tissue-consuming parasites. Using a field survey and laboratory experiments, I provide novel evidence for age-specific resistance as an adaptation to maximize life-long benefits of a mutualism. Specifically, I show that Cambarus crayfish display a consistent ontogenetic shift in resistance to the colonization of branchiobdellidans and this shift likely reflects underlying changes in the costs and benefits of symbiosis. I then show that this change in host resistance creates predictable patterns of symbiont diversity and composition throughout host ontogeny. Host resistance limits within-host symbiont communities to a few weakly interacting species, whereas relaxed resistance leads to more diverse symbiont communities that have strong interactions among symbiont taxa. Thus, host resistance has direct effects on within-host symbiont community structure by selectively filtering colonizing species, and indirect effects by moderating the strength of interactions among symbionts. Lastly, in a detailed study of the worm Cambarincola ingens, I depict a symbiont dispersal strategy that yields highly predictable transmission dynamics during pairwise host-host encounters and shows that variation in transmission dynamics can be explained by the fitness outcomes for dispersing symbionts. Field observations revealed that worm reproduction is contingent on host size and intraspecific competition for preferred microhabitats. Using a predictive model that assumes transmission of symbionts only when current conditions yield fitness below a minimum threshold, I was able to predict individual transmission events much more accurately than a comparable null model that assumed a fixed probability of transmission. My work provides empirical support for the emerging trend among researchers that advocates the adaptation of general ecological frameworks to understand symbiont population structure and diversity, but my work also emphasizes the value of detailed natural history studies to uncover system-specific ecological and co-evolutionary processes such as partner control mechanisms, symbiont microhabitat selections, and symbiont dispersal strategies. / Ph. D.

Bacterial endosymbionts of eukaryotes are generally studied in terms of their benefit or detriment to their hosts. The constraints that the host's life history imposes on its endosymbionts are rarely considered, although bacterial genome content and size are influenced by both the biotic and abiotic factors in the environment. The host organism is the primary habitat of the endosymbiont. Thus, desecribing the environment a host provides its endosymbiont is essential for understanding the evolution of endosymbiotic bacteria. I propose a system to classify the endosymbiotic environment by three characteristics: 1) host life cycle 2) host metabolism, and 3) endosymbiont location relative to host tissues. Insect-bacterial mutualisms have been classified in terms of endosymbiont environment. The majority of insect-bacterial mutualisms currently studied involve monophagous, hemimetabolous hosts that provide a relatively constant endosymbiotic enviroment. A relatively constant environment may explain the extremely reduced genomes of their endosymbionts. In contrast, polyphagous, holometabolous hosts provide the most variable endosymbiotic environment. In this work, I examined the interactions between the polyphagous, holometabolous insect, Bactrocera oleae (Rossi), and the enteric gammaproteobacterium, Candidatus Erwinia dacicola, throughout host development. Candidatus Erwinia dacicola was found in the digestive system of all life stages of wild olive flies. PCR and microscopy demonstrated that Ca. Erwinia dacicola resided intracellularly in the gastric caeca of the larval midgut, but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. I document the widespread distribution and high frequency of Ca. Er. dacicola in ten populations of wild olive flies sampled in four countries (3 Old World and 1 New World). The relative abundance of the bacterium was highest in adults and less prevalent in the egg and pupal stages. Among adult flies, the bacterium was most common in ovipositing females. These results suggest that Ca. Er. dacicola is a persistent, autochthonous endosymbiont of the olive fly. Finally, mating initiation was examined to study the influence of Ca. Er. dacicola on mating between a laboratory and a wild population of olive flies from Israel. Behavioral differences between the two populations, not presence of the endosymbiont, explained mating initiation.

Bacterial endosymbionts occur in diverse fungi, including members of many lineages of Ascomycota that inhabit living plants. These endosymbiotic bacteria (endohyphal bacteria, EHB) often can be removed from living fungi by antibiotic treatment, providing an opportunity to assess their effects on functional traits of their fungal hosts. We examined the effects of an endohyphal bacterium (Chitinophaga sp., Bacteroidetes) on substrate use by its host, a seed-associated strain of the fungus Fusarium keratoplasticum, by comparing growth between naturally infected and cured fungal strains across 95 carbon sources with a Biolog((R)) phenotypic microarray. Across the majority of substrates (62%), the strain harboring the bacterium significantly outperformed the cured strain as measured by respiration and hyphal density. These substrates included many that are important for plant-and seed fungus interactions, such as D-trehalose, myoinositol, and sucrose, highlighting the potential influence of EHB on the breadth and efficiency of substrate use by an important Fusariurn species. Cases in which the cured strain outperformed the strain harboring the bacterium were observed in only 5% of substrates. We propose that additive or synergistic substrate use by the fungus bacterium pair enhances fungal growth in this association. More generally, alteration of the breadth or efficiency of substrate use by dispensable EHB may change fungal niches in short timeframes, potentially shaping fungal ecology and the outcomes of fungal-host interactions.

Drosophila melanogaster and many other insects harbor intracellular bacterial symbionts that are transmitted vertically from infected host mothers to their offspring. Many of these bacteria alter host reproductive developmental processes in order to increase their transmission success. For example, Spiroplasma, a spirochete that naturally infects D. melanogaster, selectively kills males during mid-embryogenesis while sparing females. Previous studies suggested that Spiroplasma interacts genetically with the male-specific dosage compensation pathway, which causes ~2-fold up-regulation of most genes located on the male’s single X chromosome so that their expression matches the levels found in females who have two Xs. To further test this idea, I used confocal microscopy to visualize dosage compensation complex (DCC) localization and activity in infected as well as uninfected embryos. In the presence of Spiroplasma, the DCC became abnormally mis-localized across the nucleus. This pattern was accompanied by abnormal acetylation of histone H4K16, a mark induced by DCC activity and needed for proper X chromatin remodeling. My results imply that Spiroplasma directly targets the DCC by misdirecting it to uncompensated regions of the genome, an effect that leads to abnormal gene mis-regulation and consequent lethality (work from other members in our group). To further investigate this interaction, we transgenically expressed low levels of MSL-2 in both Spiroplasma infected and uninfected embryos in order to cause ectopic formation of the DCC in the female sex. I found that when infected, female embryos expressing the DCC showed significantly reduced viability in comparison to uninfected transgenic females. This result supports the notion that Spiroplasma uses the DCC in a dominant gain-of-function manner to kill embryos.

The determination of a symbiotic association as beneficial requires good assessment of the costs and benefits involved in the maintenance and transmission of these microbes across generations. In social insects, symbiotic associations are complex as they may involve a network of interactions between individual and colony that result in stable associations over evolutionary time. My goal was to investigate the roles of behavior and morphology as integrators that have enabled the benefits of harboring gut microbes to reach both adult and growing brood in a colony. To achieve this goal, I used turtle ants (Cephalotes), a group that has co-evolved with their gut microbes since the Eocene (Sanders et al. 2014) and that shows a variety of morphological and behavioral specializations likely connected to this symbiotic association. In my dissertation I present evidence that the specialized behavior and morphology of Cephalotes are indeed strongly associated with mechanisms that ensure stability of ant-gut microbe interactions over evolutionary time. In Appendix A, I show that a valve between the crop and midgut (proventriculus) of C. rohweri works as a filtration organ, capable of excluding possible pathogens from the mostly liquid diet consumed by turtle ants. In addition, the proventricular filter is also associated with the structuring of the gut microbiota, dividing it in at least two great groups: one upstream and another downstream of the proventriculus. Through behavioral observation and microscopy, we also suggest that the formation of the proventricular filter is only complete after young and sterile workers (callows) are inoculated with the core group of symbiotic bacteria. In Appendix B, I present results confirming that the compartmentalization of gut microbiota is also present in the congener C. varians. I compare these results with previously published data, defining the meta-communities of the gut microbiota, and demonstrate that the previously recognized core microbiota is composed of compartment-specific microbial communities and lineages. This compartmentalization of the gut microbiota is similar to the one found in highly specialized herbivores, both vertebrates and invertebrates. In addition, I also sampled the infrabuccal pocket, a characteristic oral cavity found in ants and that has largely been ignored in studies of gut symbiosis. Based on my results, I provide compelling evidence that hindgut microbes are inoculated into food particles trapped in the infrabuccal pocket, aiding in digestion of this substrate. Moreover, I suggest that trophallaxis olays a central role in inoculation of food and individuals, and might be responsible for the transmission of nutrients that are predicted to result from the gut bacteria metabolism. Finally, in Appendix C I characterize abdominal trophallaxis in C. rohweri to gain insight on its role in the context of symbiotic associations with gut microbes. I show that the hindgut contents, including bacteria, can be transmitted via abdominal trophallaxis. This interaction is found to occur between all combinations of major and minor workers, in addition to callows. The rate of solicitation of abdominal trophallaxis is higher when individuals are protein starved, indicating that hindgut content may also be nutritive. Using shotgun metagenomic data, we show that the microbiota present in the infrabuccal pocket (mostly hindgut bacteria) are indeed capable of re-utilizing nitrogen and synthesizing essential amino acids, in addition to breaking down plant material. We also report that oral trophallaxis is a possible route for transmission of crop-specific bacteria for callows, as this group has performed oral trophallaxis at a relatively higher rate than older workers. Put together, these results highlight the importance of nestmate interactions and gut morphology in the establishment and maintenance of symbiotic microbes in a social insect, introducing a new model for explaining the evolution and functioning of ant-gut microbe symbiosis.

Recent advances in sequencing technologies have provided an unprecedented window in the unseen biological world. Accompanying this revolution is a growing appreciation for the ubiquity and diversity of beneficial interactions between animals and the microbes they carry. Given the symbiotic roles of microbes in host nutrition, immunity, behavior, development, and nearly every other facet of host biology, it is becoming increasingly clear that any understanding of hosts and their evolution would be incomplete without also considering the microbial dimension. Yet despite the growing body of evidence that many of these partnerships are rooted deep in evolutionary time, the majority of studies have tended to focus on how the composition of the present-day microbiome is shaped by present-day factors. In order to place the microbiome in the larger context of host biology, a more complete understanding of the evolutionary interplay between hosts and their microbial associates is needed. Here, I use Odontotaenius disjunctus, a large xylophagous beetle found throughout eastern North America, to explore how its present-day gut microbiome has both shaped and been shaped by evolutionary processes that have acted on the host. First, I show that recent evolution in O. disjunctus reflects the influence of Pleistocene glaciation on the host’s demographic history. Next, I show that the present-day gut microbiome of O. disjunctus reflects both the influence of this recent host evolution and the more ancient influence of natural selection that has acted on the host to maintain these beneficial partnerships over a much longer timescale. Finally, I show that the persistence of certain members of the gut microbiome over evolutionary time may be explained by their role in host lignocellulose digestion. My findings demonstrate that, much like host genomes, the microbiome has been, is being, and will continue to be shaped simultaneously by forces of selection and neutrality along the arc of evolutionary history shared by these intimate partners.

Les plantes terrestres vivent en association avec des champignons du sol, formant ce que l’on appelle des symbioses mycorhiziennes. Elles échangent du carbone (photosynthétats) contre de l’eau et des minéraux. Ce mutualisme est toutefois troublé par certaines plantes, appelées mycohétérotrophes, capables de soutirer du carbone à leurs symbiontes fongiques. Le plus souvent non photosynthétiques, elles dépendent alors entièrement des champignons mycorhiziens. Certaines ont en revanche conservé la photosynthèse et obtiennent leur carbone par ces deux voies, on les appelle mixotrophes. Cette thèse est consacrée à l’étude des plantes mycohétérotrophes et mixotrophes chez les orchidées, avec des éléments de comparaison chez les éricacées. Les différents travaux qui la structurent précisent la phylogénie de certains groupes clés, s’intéressent aux évolutions génomiques, métaboliques et physiologiques accompagnant ces modes de nutrition originaux, et à leur sensibilité face aux conditions environnementales. / Terrestrial plants live in collaboration with soil fungi, forming associations called mycorrhizal symbioses. They exchange carbon (photosynthates) for water and nutrients. This mutualism is however disrupted by some plants, called mycoheterotrophs, which are able to obtain carbon from their fungal symbionts. Non-photosynthetic most of the time, then they entirely depend on mycorrhizal fungi. Some yet have retained photosynthesis and acquire carbon from these two ways, we called them mixotrophs. This PhD thesis is dedicated to the study of mycoheterotrophic and mixotrophic plants in orchids, with points of comparison in Ericaceae. This dissertation is structured around different kinds of work, which clarify the phylogeny of some key lineages, provide insights into the genomic, metabolic and physiologic evolution going along with these unusual nutrition types, and question how they respond to environmental parameters.

Os crustáceos estão entre os invertebrados marinhos mais diversos e tendem a se associar com outros organismos a fim de encontrar abrigo, oferta de alimento e um lugar seguro para reprodução. Aspectos reprodutivos como fecundidade, tamanho dos embriões e energia investida na reprodução são os principais parâmetros para a compreensão da história natural de uma espécie e/ou população. Assim, esse trabalho foi desenvolvido no sentido de investigar tais parâmetros e a influência do tipo de substrato ocupado. A espécie utilizada como modelo de estudo foi o caranguejo porcelanídeo Pachycheles monilifer coletado em dois substratos biológicos diferentes, no briozoário Schizoporella errata no píer do Itaguá e no poliqueto Phragmatopoma caudata na praia Grande, ambas as paias localizadas em Ubatuba, litoral norte de SP. Somente os embriões em estágio inicial de desenvolvimento foram utilizados para os cálculos de fecundidade e investimento reprodutivo (RO). Os embriões foram contados e medidos sob esteromicroscópio. Para obtenção do RO foram utilizados os pesos secos das fêmeas ovígeras e das massas de embriões, previamente secos em estufa. Além dos aspectos reprodutivos, análises de distância genética também foram realizadas pelo sequenciamento de genes mitocôndrias (16S e COI) de exemplares de P. monilifer. A estimativa da porcentagem de variação genética intra e interespecífica, a possibilidade de fluxo gênico entre indivíduos de diferentes praias do estado de São Paulo foram verificadas. A partir desses dados confirmou-se a validade da espécie, assim como o fluxo gênico contínuo entre indivíduos dessas duas praias. A baixa divergência genética permite concluir que as diferenças reprodutivas encontradas são, portanto um reflexo das condições proporcionadas pelo ambiente. Os animais simbiontes do briozoário, assim como as fêmeas ovígeras, são maiores que os associados ao poliqueta e também possuem maior fecundidade e investimento reprodutivo, porém não foram encontradas diferenças em relação ao tamanho dos embriões. No substrato do briozoário o estresse reprodutivo parece ser menor que no substrato do poliqueto, já que aquele substrato proporciona um ambiente fisicamente mais estável para os organismos simbiontes. No substrato do poliqueto, além do alto hidrodinamismo local, o espaço disponível na colônia parece ser mais restrito e pode limitar o tamanho de seus habitantes. Os animais simbiontes do briozoário parecem não serem influenciados por fatores externos à colônia, entretanto o mesmo não foi observado nos simbiontes do poliqueto. Tais diferenças reprodutivas refletem o tipo de substrato ocupado, evidenciando a plasticidade fenotípica da espécie para uma melhor adaptação local. / The crustaceans are the most diverse of the marine invertebrates and many of them tend to associate with other organisms where they can find shelter, food supply and a safe place to reproduce. Reproductive traits as fecundity, egg size and reproductive output (RO) are the most important aspects to understand the species life history. This study aimed to investigate the reproductive traits in relation to the substrate that the organisms live. The studied species used as model was the porcellanid crab Pachycheles monilifer that was collected in two different substrates, one of them is the bryozoan Schizoporella errata at the Itaguá beach and the other is the polychaete Phragmatopoma caudata at Grande beach, both are located in Ubatuba, northern coast of São Paulo. Only the initial stage of eggs was used to estimate the crab fecundity and RO. The eggs were counted and measured in a stereomicroscope. To obtain the RO the female and the egg mass dry weight, which were previously dried in an oven, were used. Besides the reproductive aspects, the genetic distance between the organisms was valued with partial sequences of the mitochondrial genes 16S and COI. The estimate of the intra and interspecific genetic variation and the possibility of gene flow between the individuals from the beaches in SP were analyzed too. Therefore, with all these analyses it was possible to confirm that the species is valid and that there is gene flow among the populations. The low genetic divergence allowed us to conclude that the different reproductive traits found among the populations were due to the different substrates that the organisms inhabit. The bryozoan symbionts are larger than the polychaete symbionts as well as the females fecundity and the RO, but there is no difference in egg size. In the bryozoan substrate it seems like the physical stress is smaller than in polychaete and it promotes a more stable habitat for the crabs reproduce. In the polychaete substrate, beyond the high hydrodynamic location, the available space in the colony appears to be narrower, which could limit the size of its inhabitants. The bryozoan symbionts do not seem to be influenced by the external factors of the colony, though the same was not observed in the polychaete colony. Such differences in reproduction reflect the kind of substrate inhabited, highlighting the phenotypic plasticity of the species to a local better adaptation.