Dissertationen zum Thema „Transmission du symbiote“

De nombreuses bactéries entrent en symbiose avec les métazoaires colonisant les habitats à base chimiosynthétique dans l'océan profond. Les hôtes auxquels elles apportent le carbone organique deviennent ainsi dépendants des bactéries sulfo-oxydantes et/ou méthanotrophes. Pour assurer la continuité de ces associations et la colonisation de nouveaux sites, des stratégies diverses de dispersion ont été développées par les animaux. Deux types de transmission des bactéries symbiotiques existent : la transmission verticale où elles sont transmises via les gamètes, et la transmission horizontale où elles sont acquises à partir de l¿environnement, depuis des populations de bactéries libres ou relarguées par d'autres hôtes. Cette thèse vise à répondre à des questions concernant les cycles de vie des symbiontes. La diversité et la distribution des formes libres de symbiontes et des bactéries apparentées dans plusieurs habitats réduits ont été étudiées par l¿analyse des séquences de l'ARNr 16S des bactéries colonisant des substrats organiques déployés dans l'Atlantique et la Méditerranée. La transmission maternelle des symbiontes chez le bivalve vésicomyidé Isorropodon bigoti du golfe de Guinée a été confirmée par l'observation et l'identification des symbiontes dans les ovocytes et dans les stades post-larvaires. Enfin, l'effet des stress chimique, thermique et de la remontée sur la dynamique des populations de symbiontes sulfo-oxydants et méthanotrophes chez la moule Bathymodiolus azoricus a été analysé par les techniques de FISH 3D et de pyroséquençage. Cette thèse a permis de mieux comprendre des cycles de vie des bactéries symbiotiques dans et en dehors de leur hôtes animaux
Metazoans colonizing deep-sea reducing habitats often employ chemosymbiotic bacterial associations. Hosts become dependent upon their sulfur-oxidizing and/or methanotrophic symbionts, which provide organic carbon compounds. Various larval dispersal strategies have evolved in the hosts, ensuring the colonization of new sites. The continuity of the symbiotic association is maintained by symbiont transmission. Symbionts may pass directly to the host’s progeny via gametes (vertical transmission) or may be acquired from the environment as free-living forms or as those released from other hosts (horizontal transmission). This work answers several questions about the lifecycles of symbionts regarding the diversity of symbiont-related bacteria in environmental bacterial communities; the localization and dynamics of symbionts in host tissues depending on their transmission mode; or after abiotic stresses applied to the holobiont.The diversity and the distribution of free-living symbionts and their close relatives from several chemosynthesis-based habitats has been analysed by 454 pyrosequencing of the 16S rRNA of bacteria colonizing plant-derived substrates in the Northern Atlantic and Mediterranean. The trans-ovarial transmission has been detailed in the clam Isorropodon bigoti by the identification of symbionts within oocytes and in the forming gills of their post-larvae. Finally, the influence of several abiotic stresses in Bathymodiolus azoricus mussels on the dynamics of their symbionts has been investigated by FISH and pyrosequencing. This PhD presents new data regarding various aspects of the life cycle of chemosynthetic symbionts inside and outside their metazoan hosts

Les symbioses mutualistes sont ubiquistes et jouent un rôle majeur dans le fonctionnement des écosystèmes. Chez les insectes, il est souvent décrit que la spécificité et la stabilité évolutive de ces symbioses mutualistes est due à une transmission verticale stricte des symbiotes des parents à la descendance. Pour tester le rôle de la transmission verticale dans le maintien évolutif des symbioses mutualiste, son exclusivité et son efficacité doivent être étudiées à une échelle évolutive (d’une lignée hôte à une autre) ainsi qu’à une échelle écologique (d’une génération à la suivante). Ce projet de thèse a pour but d’étudier la transmission verticale comme mécanisme de stabilisation évolutive dans les associations nutritionnelles entre les termites souterrains du genre Reticulitermes et leurs symbiotes mutualistes intestinaux. Pour cela, des outils de biologie moléculaire ont été développés afin d’étudier la diversité et la composition de la communauté microbienne intestinale totale des termites (approche de méta-code barre), mais également en se focalisant sur les protistes du genre Trichonympha qui jouent un rôle essentiel dans la dégradation de la lignocellulose (approches de barcoding ADN et de PCR quantitative). Le premier axe de cette thèse vise à étudier les patterns de transmission des symbiotes à l’échelle évolutive du genre Reticulitermes en testant l’hypothèse que la phylogénie de l’hôte est un facteur majeur expliquant la composition du microbiote intestinal des termites. L’étude menée sur les Trichonympha de termites a permis de montrer que la diversification de ces associations n’est pas uniquement expliquée par des événements de co-spéciation, mais que des événements de changement d’hôte et de perte de symbiotes ont eu lieu. L’étude menée sur l’ensemble du microbiote intestinal des termites permettra de déterminer ces patterns de diversification sur l’ensemble des taxa microbiens. Le second axe de cette thèse est d’évaluer le niveau de fidélité entre partenaires à une échelle intergénérationnelle en testant l’hypothèse que la transmission verticale des symbiotes est stricte d’une génération à une autre. L’efficacité de la transmission verticale a été mesurée en deux étapes : (i) en comparant les symbiotes présents dans les ouvriers de la colonie avec ceux portés par les reproducteurs primaires (i.e. les alates) lorsqu’ils quittent leur colonie natale pour en fonder une nouvelle, et (ii) en comparant les symbiotes présents dans les ouvriers des deux colonies parentales avec ceux portés par la nouvelle colonie fondée. Les résultats préliminaires de l’étude sur les deux Trichonympha présents chez R. grassei semblent indiquer qu’ils sont co-transmis dans les alates. L’étude sur l’ensemble du microbiote intestinal permettra de déterminer si tous les taxa microbiens sont co-transmis ou si une transmission aléatoire des symbiotes a lieu dans les alates. Une troisième étude sur l’ensemble du microbiote permettra de déterminer si tous les taxa microbiens sont transmis par les parents à la descendance. Ensemble, ces différentes études permettront de tester la transmission verticale comme mécanisme induisant une fidélité entre partenaires dans les systèmes termitesmicrobiote intestinal. A l’échelle des protistes du genre Trichonympha, ce mécanisme ne semble pas le seul impliqué dans la stabilité évolutive de ces associations
Many animals including humans live in symbiotic interaction with gut microorganisms contributing to essential functions (nutrition, immunity). The ‘vertical’ way of transmission of symbionts (i.e., from parents to offspring) must stabilise these symbioses, notably by strengthening partner fidelity. However, the efficiency of vertical transmission has rarely been studied, especially in the case where hosts harbour a complex microbial community (or ‘microbiota’) composed by many microbial taxa interacting between them and with the host.The objective of this work was to study the mode and efficiency of transmission of gut microorganisms (protists and bacteria) helping the wood-feeding termite Reticulitermes grassei to digest ingested wood (lignocellulose fibres). Our results revealed contrasted situations between microorganisms. While protists are efficiently vertically transmitted, the majority of bacterial taxa is not only vertically transmitted but seems to be acquired by the environment

Six especes de bivalves tropicaux de la famille des lucinidae (codakia orbicularis, c. Orbiculata, c. Pectinella, linga pensylvanica, lucina nassula et l. Pectinata) hebergeant des bacteries endosymbiotiques sulfo-oxydantes dans des cellules specialisees des branchies ont ete etudiees. L'etude ultrastructurale des filaments branchiaux a mis en evidence des particularites propres a chacune des especes pouvant soit intervenir dans les relations avec leurs symbiontes, soit constituer des adaptations au milieu. L'etude phylogenetique des endosymbiontes, effectuee par sequencage de l'adnr 16s, a notamment montre que les symbiontes de c. Orbicularis, c. Orbiculata, c. Pectinella et l. Pensylvanica appartiennent a la meme espece. A l'oppose, le symbionte de l. Pectinata semble representer la lignee la plus eloignee des bacteries symbiotiques des membres de la superfamille des lucinacea. L'hypothese de la transmission verticale des symbiontes chez les especes etudiees a pu etre rejetee. Cependant, le mode environnemental n'a ete demontre que pour c. Orbicularis, seule espece dont le developpement est maitrise. Le symbionte de c. Orbicularis est transmis aux nouvelles generations d'hote par l'intermediaire d'une forme libre associee au sediment, qui penetrerait par endocytose au niveau du pole apical des cellules indifferenciees de la zone laterale des filaments branchiaux de juveniles aposymbiotiques, qui se differencient alors en bacteriocytes. Le developpement larvaire de c. Orbicularis est de type planctotrophe facultatif; la metamorphose se deroule en deux etapes, avec un blocage a la fin du stade plantigrade qui peut etre letal en l'absence de substrat adequat. Le developpement de juveniles aposymbiotiques obtenu en presence de sable sterile montre que la symbiose n'est indispensable ni au developpement larvaire ni a la metamorphose de c. Orbicularis. Les cellules indifferenciees de la zone laterale des juveniles aposymbiotiques gardent leur capacite de differenciation de nombreux mois apres la metamorphose. De plus, l'infestation des branchies par les bacteries de l'environnement n'est pas limitee a une periode determinee du developpement ni a un stade determine de differenciation des filaments branchiaux.

Endosymbiosis is a pervasive phenomenon that has been a powerful force in insect evolution. In many well studied insect-bacterial associations, the bacteria can serve as reproductive manipulators, nutritional mutualists or defenders of their hosts. Fungi are also frequently associated with insects, and initial estimates suggest that these fungi are hyperdiverse. Saving a handful of examples, however, the functions of these fungi within insect hosts are largely unknown. This dissertation begins with a review that lays the conceptual groundwork for understanding bacterial and fungal endosymbiosis in insects. I make predictions about why one versus the other microbe might serve the insect, given any unique physiological, ecological or evolutionary conditions. I then aim to derive insights about microbial symbiosis by focusing on a particular system, that of brownbanded cockroaches, Supella longipalpa (Blattaria: Blattellidae) and their specialist wasp parasitoids, Comperia merceti (Hymenoptera: Encyrtidae). Here, I identify the symbiotic community of these two insects by using both culture-dependent and independent methods to characterize the vertically transmitted bacterial and fungal associates. Finally, I show that a heritable fungus in C. merceti, long presumed to be a mutualist, is parasitic under laboratory conditions: infected wasps incur fitness costs for housing the fungal symbiont relative to uninfected wasps. Additionally, although the fungus is not horizontally transmitted sexually, it is readily horizontally transmitted from the offspring of infected females to those of uninfected females that are using the same host.

Mutualistic symbioses have enabled the colonization of novel habitats and niches in a large array of eukaryotic and bacterial taxa. Reliable mechanisms of symbiont transmission between host generations are necessary to stabilize these associations over evolutionary time. Historically, symbionts have been categorized as either vertically transmitted from the parents to offspring or horizontally transmitted through the environment. The route between hosts influences how symbiont populations are connected between hosts and between geographic localities. Over evolutionary time vertical transmission leads to gene loss and genome erosion. Growing evidence from diverse associations suggests that modes utilizing both horizontal and vertical strategies exist, raising the question of how these “mixed modes” influence symbiont genome evolution. The overarching goal of my dissertation was to determine whether the mode of symbiont transmission in an obligate mutualism is consistent over evolutionary time and what impact transmission mode fidelity has on symbiont genome evolution. To test these questions, I used the chemosynthetic symbiosis between the marine bivalve Solemya velum and its gammaproteobacterial symbionts, which has been reported to transmit its symbionts vertically through the ovary, but bears none of the genomic hallmarks of strict vertical transmission. In this work, I used population genomics of S. velum subpopulations sampled from five localities along the New England coast, from Massachusetts to North Carolina, to test for evidence of horizontal transmission in the evolutionary history of this species. These analyses revealed that symbionts and mitochondria do not exhibit concordant genealogies, divergent symbiont lineages have come into contact and recombined, and symbiont genomes have experienced large-scale structural changes mediated by mobile elements and horizontal gene transfer. In total, these lines of evidence indicate that a substantial amount of horizontal transmission has occurred in the recent history of this association. The vertical transmission route through host tissues was characterized via in situ hybridization to symbiont 16S rRNA in adult host tissues and by qPCR of the rhlE gene in spawned eggs. These data showed that symbionts are present at low abundance in the ovary, associated with the supportive cells and mature oocytes, and each spawned egg contains approximately 50-100 copies of the symbiont genome. Lastly, I tested for evidence of mixed transmission modes in symbiont populations contained within host tissues (each host gill contains more than a billion symbiont cells), by deep-coverage whole genome sequencing. Using a novel variant-calling procedure, I detected low amounts of genetic variation among symbiont genomes within a host relative to between hosts. However, the variant sites that were present were correlated in position along the genome, present on the same chromosome, and segregating in the symbiont population at large, suggesting that these variants arose via recombination with a variant symbiont genotype introduced by horizontal transmission. In total, this work supports the existence of mixed transmission modes in symbiotic associations and indicates they have distinct consequences for symbiont evolution. Mixed modes may provide a best-of-both-worlds strategy to ensure that hosts acquire symbionts every generation while maintaining opportunities for recombination and acquisition of novel genetic elements. These results are relevant to understanding the impact of symbiont transmission mode on genome evolution in associations ranging from mutualisms to pathogenic infections.
Biology, Organismic and Evolutionary

Interactions among living organisms fall along the mutualism-parasitism continuum; where mutualistic interactions benefit one or both organisms and parasitic interactions harm them. Mutualisms are a particularly interesting form of interaction as their evolutionary stability is constantly at risk of destabilisation by cheaters which take greater advantage of their partners than other symbionts in the population. This has resulted in organisms involved in mutualistic interactions have evolved many mechanisms to prevent destabilisation by cheaters. Insects are involved in mutualistic interactions with a myriad of organisms, in particular micro-organisms. Some of the most well documented insect–micro–organism interactions are those of the fungus–farming insects; Attine ants, termites and ambrosia beetles, and the obligate mutualistic fungi they cultivate. These mutualisms have remained stable over millions of years. Another, less well studied, apparently stable, obligate insect-fungus mutualism is the interaction between Sirex woodwasps and Amylostereum fungi. In this review we examine the evolution of mutualisms from initial interaction, through to maintenance of a stable obligate interaction, and explore the mechanisms that act to stabilise them. We explore the evolutionary and ecological factors necessary for the maintenance of the Sirex–Amylostereum mutualism in the context of work that has been done on the evolution of other more extensively studied insect-fungus mutualisms.
Dissertation (MSc)--University of Pretoria, 2014.
Genetics
MSc
Unrestricted

Chez les Mammifères, le recrutement du microbiote débute in utero, ce qui restait à démontrer chez d'autres classes de Vertébrés. L’objectif général du projet était de tester si un tel recrutement se produit chez un Vertébré non Mammifère. Nous avons testé, chez le Poisson vivipare Brachyistius frenatus, l'hypothèse selon laquelle la poche utérine est colonisée par un microbiote transmissible aux alevins, conférant à leur propre microbiote une ontogenèse semblable à celle des Mammifères. Le projet visait l’atteinte des objectifs suivant: i) caractériser le mode de transmission du microbiote, ii) établir la composition, la diversité et les relations des communautés bactériennes du microbiote des femelles, des juvéniles et de leur environnement et iii) déterminer l’ontogenèse du microbiote chez B. frenatus. Ce projet a permis de caractériser le mode de transmission du microbiote, sa séquence de recrutement, ainsi que la contribution respective de différentes communautés sources en caractérisant la diversité bactérienne du microbiote des femelles, des juvéniles et de leur environnement avec une approche métagénomique de type code barre. La région V4 du gène de l'ARNr 16S a été ciblée comme marqueur taxonomique bactérien pour identifier les taxons des différents échantillons.Cette étude nous a permis d’identifier le premier cas d’une transmission verticale du microbiote in utero chez un vivipare non Mammifère et les résultats sous-entendent que B. frenatus est peut-être un tout nouveau modèle d’ontogenèse du microbiote. Cette étude a permis l’acquisition des connaissances sur la transmission du microbiote et, dans le contexte de convergence évolutive de la viviparité, elle ouvre à de nouvelles perspectives quant aux avantages évolutifs d'une telle transmission de symbiotes microbiens.
In Mammals microbial recruitment starts in utero, something that had not been shown in any other Vertebrate class. The main goal of this project was to test whether this type of recruitment happens in a non-mammalian Vertebrate. We tested in the viviparous fish Brachyistius frenatus the hypothesis under which the uterine pouch is colonized by a microbiome transmissible to the juveniles, conferring them an ontogeny similar to Mammals. This project also aimed to i) characterize the mode of transmission of the microbiota, ii) establish the composition, diversity and relationships between the microbial communities of pregnant females, juveniles and their environment and iii) determine the ontogeny of the microbiota in B. frenatus. We characterized the mode of transmission of the microbiome, explored its recruitment and the contribution of different source communities with a metagenomic approach (bar coding). We targeted the hyper variable region V4 of the small subunit (16S) rRNA gene to determine the presence of a vertical transmission of the microbiome In this study, we confirmed the presence of a vertically transmissible microbiome in the viviparous fish B. frenatus. We documented for the first time an in utero transmission of themicrobiota in a non-mammalian viviparous species. Our results also hint that B. frenatus might be a new model of microbiota ontogeny. This study contributes to the acquisition of knowledge on microbiome transmission and, in the context of evolutionary convergence of viviparity, allows the formulation of hypotheses concerning the evolutionary advantages of in utero microbiome transmission.

Dissertação de Mestrado Integrado em Medicina Veterinária
Esta dissertação teve como objetivo principal caracterizar a fauna de simbiontes de chimpanzés (Pan troglodytes), e colobos pretos e brancos (Colobus sp.) de diferentes localidades Africanas (Parque Nacional de Sapo, na Libéria e em Bulindi, no Uganda), comparar resultados entre eles e estudos prévios e teorizar sobre as possíveis implicações zoonóticas dos simbiontes encontrados. Recorrendo a técnicas de coprologia como sedimentação MIF modificada e a flutuação de Sheather, e um teste de imunofluorescência direta para deteção de oocistos de Crypstosporidium e cistos de Giardia, 47 amostras fecais preservadas de chimpanzé e 15 de colobos pretos e brancos foram examinadas. Os protozoários detetados incluíram Troglodytella abrassarti, Entamoeba spp., Blastocystis sp. e Iodamoeba buetschlii; e nematodes, Trichuris sp, Colobenterobius sp., strongylideos e várias larvas de nematodes. Todas as amostras foram negativas no teste de imunofluorescência directa. Em geral, o espectro de simbiontes encontrado no presente estudo está de acordo com o que é regularmente descrito em estudos que abordam chimpanzés e colobos pretos e brancos de vida selvagem. Contudo, pequenos microrganismos aquáticos denominados Rotifera ou rotíferos foram observados pela primeira vez em amostras fecais de chimpanzé e colobos pretos e brancos. Considerando o contato próximo entre os primatas estudados e seres humanos, é importante realizar futuras pesquisas recorrendo a técnicas moleculares nos simbiontes assinalados por este trabalho e avaliar o seu real potencial zoonótico.
ABSTRACT - Gastrointestinal symbionts of wild chimpanzees and sympatric colobus monkeys living in close proximity of humans in Liberia and Uganda - This dissertation aimed to characterize the symbiont fauna of chimpanzees (Pan troglodytes) and black and white colobus (Colobus sp.) from different African localities, (Sapo National Park in Liberia and Bulindi in Uganda), compare results between them and previous studies, and theorize about possible zoonotic implications of the found symbionts. Using coprologic techniques as modified MIF sedimentation and Sheather’s flotation, and a direct immunofluorescence teste for detection of Crypstosporidium oocysts and Giardia cysts, 47 chimpanzee and 15 black and white colobus preserved fecal samples were examined. Detected protozoans included Troglodytella abrassarti, Entamoeba spp., Blastocystis sp. and Iodamoeba buetschlii; and nematodes, Trichuris sp., Colobenterobius sp., strongylids and various nematodes larvae. All samples were negative for the direct immunofluorescence test. Overall, the spectrum of symbionts found in the present study is in accordance with what is regularly described in studies addressing the free ranging chimpanzees and black and white colobus. However, small aquatic microorganisms denominated Rotifera or rotifers were observed for the first time in chimpanzee and in black and white colobus faecal samples. Considering the close contact between the studied primates and humans, it is important to conduct further research using molecular techniques in the found symbionts to assess the real zoonotic potential.
N/A

Wolbachien sind weitverbreitete bakterielle Symbionten von Arthropoden. Sie werden überwiegend durch maternale Vererbung übertragen, können aber auch horizontal von Art zu Art übertragen werden. Wolbachien sind berüchtigt dafür, die Wirtsreproduktion zu manipulieren, können aber auch Mutualismen mit ihren Wirten evolvieren. In dieser Arbeit untersuche ich, welche Rolle horizontale Transmission und mutualistische Effekte bei der Wolbachien-Pandemie unter Arthropoden spielen. Zunächst schätze ich, dass Millionen Arthropodenarten mit Wolbachien infiziert sind. Um diese erstaunliche Verbreitung zu verstehen, entwickele ich ein Modell zur horizontalen zwischenartlichen Transmission von Wolbachien, das auf epidemiologischer und Netzwerk-Theorie aufbaut. Die Ergebnisse weisen auf die Bedeutung von horizontaler Transmission über große phylogenetische Distanzen hin. Da eine erfolgreiche Transmission wahrscheinlich durch symbionteninduzierte Wirtsvorteile begünstigt wird, betrachte ich dann umfassend und kritisch Wolbachien-Arthropoden-Mutualismen und finde diese in vielfältigen Kontexten, aber nur begrenzt Hinweise auf Wolbachien-induzierten Wirtsschutz. Mithilfe eines populationsgenetischen Modells untersuche ich dann den Einfluss von Wirtsvorteilen auf die Infektionsdynamik von Wolbachien. Erstmalig leite ich Invasionsbedingungen und Gleichgewichtsfrequenzen für Wolbachien-Doppelinfektionen her. Die Ergebnisse bestätigen, dass Wirtsvorteile die Invasion von Wolbachien in neue Wirte erheblich erleichtern. Schließlich untersuche ich die Wechselwirkungen zwischen einer Wolbachien-Infektion und dem Immunsystem des Wirtes, wobei ein Schwerpunkt auf reaktiven Sauerstoffspezies liegt. Ich schlage eine Hypothese vor, die unterschiedliche Immunantworten in neuen und ko-evolvierten Assoziationen erklärt. Insgesamt sprechen die Ergebnisse dieser Arbeit für einen wesentlichen Anteil von horizontaler Transmission und mutualistischen Effekten an der Wolbachien-Pandemie in Arthropoden.
Wolbachia are widespread bacterial symbionts of arthropods. They are transmitted predominantly via maternal inheritance, but are also able to move between different species (horizontal transmission). Wolbachia are notorious for selfishly interfering with host reproduction, but they can also evolve mutualistic associations with their hosts. In this thesis, we analyze the role of horizontal transmission and mutualistic effects in the Wolbachia pandemic among arthropods. First, we derive an estimate of the number of Wolbachia-infected arthropod species and find that millions of species are infected. In order to explain this striking distribution, we develop a model of Wolbachia horizontal transmission between species, building on epidemiological theory and network theory. Our findings point to the importance of transmission over large phylogenetic distances. Given that successful horizontal transmission is likely to be facilitated by symbiont-induced host benefits, we then perform a comprehensive review of Wolbachia-arthropod mutualisms and find that these occur in diverse contexts, although the evidence of Wolbachia-induced host protection in nature is limited so far. By means of a population genetic model, we then analyze the influence of host benefits on the infection dynamics of Wolbachia. For the first time, we derive invasion conditions and equilibrium frequencies for Wolbachia double infections. Our results corroborate that host benefits substantially facilitate invasion of Wolbachia into novel hosts. Finally, we examine the interactions between Wolbachia infection and the host immune system, with a focus on reactive oxygen species. We propose a hypothesis that explains differential immune responses in novel and coevolved associations. Taken together, the findings presented in this thesis argue for a significant involvement of horizontal transmission and mutualistic effects in the Wolbachia pandemic among arthropods.

We live in an era of global change, where emerging infectious diseases such as Ebola, Zika, bird flu, and white nose syndrome are affecting humans, wildlife, and domesticated species at an increasing rate. To understand and predict the dynamic spread of these infectious agents and other symbionts through host populations and communities, we need dynamic mathematical models that accurately portray host-symbiont transmission. But transmission is an inherently difficult process to measure or study, because it is actually a series of interacting processes influenced by abiotic and biotic factors at multiple scales, and thus empirical tests of the transmission function within epidemiological models are rare. Therefore, in this dissertation, I explore factors at the individual, population, and community-levels that influence host contact rates or symbiont transmission success in a common snail-symbiont system, providing a detailed description of the multi-faceted nature of symbiont transmission. From a review of the ecological literature, I found that most models assume that transmission is a linear function of host population density, whereas most empirical studies describe transmission as a nonlinear function of density. I then quantified the net nonlinear transmission-density relationship in a system where ectosymbiotic oligochaetes are directly transmitted among snail hosts, and I explored the ecological mechanisms underlying the nonlinear transmission-density relationship observed in the field via intraspecific transmission success and contact rate experiments in the laboratory. I found that the field results could be explained by heterogeneity in transmission success among snails with different characteristics and nonlinear contact-density relationships caused by non-instantaneous handling times. After I 'unpacked'population-level transmission dynamics into those individual-level mechanistic processes, I used this same approach to examine higher-level ecological organization by describing the mechanistic underpinnings of interspecific or community-level transmission in the same snail-symbiont system. I found that low interspecific transmission rates in the field were the product of opposing interactions between high population densities, high prevalences of infection, and very low interspecific transmission success caused by strong symbiont preferences for their current host species. Unpacking transmission in this way resulted in one of the most detailed empirical studies of transmission dynamics in a wildlife system, and yielded many surprising new insights in symbiont ecology that would not have been discovered with a purely phenomenological or holistic view of transmission. Though simple, linear, and holistic epidemiological models will always be important tools in disease ecology, 'unpacking'transmission rates and adding heterogeneity and nonlinearity to models, as I have done here, will become increasingly important as we work to maximize model prediction accuracy in this era of increased disease emergence.
Ph. D.

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.

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 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

This PhD thesis is the outcome of a wider project focused on studying the biology, genomics and infectivity of bacteria ascribed to the Midichloriaceae family. Midichloriaceae are a group of bacteria extremely diversified in terms of the ecological and taxonomic diversity of their hosts. Members of the family Midichloriaceae can be considered as a wide bacterial group, still to be investigated under several aspects. In particular, the association of members of the family Midichloriaceae with parasites (such as ticks, fleas and parasitic amoebae) suggests that these bacteria could be potentially infectious to vertebrates (including humans) and even responsible for medical and veterinarian diseases. The researchers conducted in this PhD project have been focused on three main topics: i) possible involvement of a midichloriacea in a disease (red mark syndrome; RMS) affecting rainbow trout; ii) in-depth analysis of different aspects concerning the hard tick Ixodes ricinus and its intramitochondrial symbiont Midichloria mitochondrii (i.e. antibiotic treatment of the arthropod, salivary glands vs. ovaric tissue comparative proteomics, transmission of the symbiont to the vertebrate host); iii) presence of M. mitochondrii-related bacteria in different tick species. A brief description of each topic of the work done is here reported: i) RMS is a chronic skin disease of unknown etiology affecting farmed rainbow trout Oncorhynchus mykiss in Europe. It consists of single or multiple skin lesions usually localized on the trunk of fish approaching market size. Many authors suppose that a bacterial infection could be the cause of the disease. Some studies have reported a possible correlation between the presence of a Rickettsia-like organism (subsequently ascribed to the family Midichloriaceae) and RMS skin lesions. Our work concerning the possible involvement of a midichloriacea in RMS in rainbow trout resulted in the detection of bacteria of this family, besides in skin lesions, in various internal organs and in the bloodstream. No bacteria were found in unaffected fish and in healthy skin samples from affected individuals. Bacteria were detected also in fish ponds water were RMS-affected fish are farmed. These results give support to the hypothesis of the involvement of these bacteria in developing RMS. It is possible that some free-living eukaryotes could host or be the vector of this midichloriacea. ii) M. mitochondrii is the most prevalent symbiont of the hard tick I. ricinus, present in 100% of eggs and adult females of wild I. ricinus populations. This bacterium is intracellular and is the only known symbiont able to invade the mitochondria of the host cells. However, the role that M. mitochondrii plays in the host metabolism has yet to be elucidated. In addition, multiple lines of evidence indicate the possibility of the transmission of this bacterium to the vertebrate host during the tick blood meal. In order to investigate the role of M. mitochondrii in the biology of the tick host, we performed an antibiotic treatment on I. ricinus individuals, with the aim of reducing/eliminating the symbiont, and to potentially observe the dynamic of bacterial infection in the tick host. We microinjected engorged adult females of I. ricinus with tetracycline, and we allowed the resulting larvae to feed on gerbils treated with the same antibiotic. The amount of M. mitochondrii was evaluated at different stages of the experiment using molecular techniques. In addition we evaluated the presence/absence of the symbiont DNA in the blood of gerbils used for the larval feeding. The performed treatments did not allow to eliminate the symbiont population from the host tick, however it allowed to reduce the multiplication that occurs after the larval blood meal. These results open the way for future experiments, using different antibiotic molecules, different administration methods and antibiotic administration on subsequent tick stages, to fulfill the goal of eliminating M. mitochondrii from the host I. ricinus, a major step in our understanding of the impact of this bacterium on ticks. In order to investigate, from a proteomic point of view, the tick I. ricinus and its symbiont, the protein profile of the ovary tissue (OT) and of salivary glands (SG) of adult females of this tick species were generated. To compare the OT and SG profiles, 2-DE profiling followed by LC-MS/MS protein identification were performed. We detected 21 spots showing significant differences in the relative abundance between the OT and SG, ten of which showed 4- to 18-fold increase/decrease in density. This work allowed to establish a method to characterize the proteome of I. ricinus, and to detect multiple proteins that exhibit a differential expression profile in OT and SG. Additionally, it was possible to use an immunoproteomic approach to detect a protein from the symbiont. Finally, the method developed will pave the way for future studies on the proteomics of I. ricinus, with the goals of better understanding the biology of this vector and of its symbiont M. mitochondrii. The transmission of M. mitochondrii after I. ricinus bite has been evidenced through direct and indirect evidences in human and other vertebrates by different authors. However, the time of seroconversion against the antigens of this symbiont is still unknown, as well as the kinetic of the antibody response against M. mitochondrii. In this work we performed an experimental infestation of rabbits with wild I. ricinus ticks (harboring M. mitochondrii) and I. ricinus from a lab colony (free of the symbiont). We were able to show the first seroconversion of a vertebrate model against M. mitochondrii. The seroconversion occurs approximately around the first and the second week after tick infestation, with duration of at least one month after infestation. It has been observed that M. mitochondrii represent an actual pack of antigens that can persist in the vertebrate host for a prolonged time. M. mitochondrii DNA was also observed circulating in blood of infested rabbits. The flagellar protein FliD of M. mitochondrii can be considered an interesting marker for I. ricinus bite, at least concerning adult females bite. However, the antibody response against this protein suggests that a possible screening for this marker should be considered within a limited time after tick infestation. Additional markers both from M. mitochondrii and I. ricinus should be considered in view of developing a possible marker for I. ricinus bite detection. iii) The multiple screening of 17 tick species allowed the detection and quantification of bacteria of the family Midichloriaceae in seven of them, including the first report of a representative of this family in a soft tick species (Argasidae), Ornithodoros maritimus. Based on sequence identity and phylogenetic analysis we propose that all these bacterial symbionts of ticks could be members of the genus Midichloria. The performed screening highlights different prevalence levels and variable bacterial loads in different tick species including one, Ixodes aulacodi, where the bacterium is present in all examined individuals, like in I. ricinus. This result prompts us to hypothesize different roles of Midichloria bacteria in different tick species.

(. . . ) Dans un premier temps, nous avons vérifié l'organisation fine des endomycorhizes VA qui a montré des caractères communs à toutes les mycorhizes de ce type. Puis nous avons effectué la synthèse in vitro de ces mycorhizes en utilisant comme plante-hôte des semis ou des vitroplants (. . . ) Par des contrôles cytologiques et par un suivi dans le temps, nous avons vérifié que les systèmes mycrohiziens sont conformes et se maintiennent dans les plantes infectées (. . . ) Nous nous sommes attachés à mettre au point une méthode simple et fiable de production d'inoculum endomycorhizien VA (. . . ) Nous avons reconstitué, en conditions contrôlées, des systèmes mycorhiziens doubles (endo- ectomycorhizes) et nous en avons étudié l'évolution (. . . )

博士
國立中興大學
昆蟲學系
85
Transovarian transmission of the yeast-like symbiote and vitellogenin transport in the brown planthopper, Nilaparvata lugens.Dor-Jih Cheng Transovarian transmission of the yeast- like symbiote (YLS) in the brown planthopper, Nilaparvata lugens, was studied using histological techniques. YLS was not found in testes and spermathecae in mated females. The ovaries of virgin females had only few eggs containing YLS. Therefore the sperm is not involved in YLS transmission. Lipid in fat body cells of females reduced with increase in adult age, and is then replaced with protein in situ. Electron micrographs showed a large number of endoplasmic reticula and granules in the fat body cells of reproductive females and the absence of nucleus and membranous boundary in mycetocytes. YLS became enclosed with membranous structures formed by fat body cells and was removed from the membranous structures by pinocytosis followed by extruding outward, and finally released into hemocoel. Then YLS moved into the posterior pole of the primary oocyte in the ovariole through epithelia plug by follicle cell endocytosis and formed a symbiote ball in mature eggs. The symbiote ball could move upward with the developing embryo and enclosed into the abdominal rudiment. Finally YLS invaded into nymphal mycetocytes by endocytosis. Electrophoretic profiles of hemolymph, body and ovarian homogenates from 5th instar nymphs, reproductive female and male adults showed a female-specific protein of 175kD, with an isoelectric point of 7.3. The Western blots using antibodies against this protein extracted exhibited immunological reaction with the 175kD protein from hemolymph and ovarian homogenates of females but did not react with proteins obtained from nymphs and male adults. Therefore, this female- specific protein in hemolymph proves to be vitellogenin (Vg), and that in ovarian homogenates to be vitellin(Vn). The antibodies against female and nymph YLS could react with 175kD and 75kD proteins from hemolymph of females, and 72kD protein from ovarian homogenates but did not react with nymph and male adult proteins. Determination with endo-β-N- acetylglucosaminidase H (Endo-H) showed that both Vg and Vn were glycosylated, the molecular weight being reduced from 175kD (Vg and Vn) to 150kD and a 50kD Vn protein reduced to 39kD. Analysis of protein concentration using enzyme-linked immunosorbent assay showed Vg and/or Vn concentration rose with increase in adult age. The maximum was found at 4 days after emergence, being ca. 23.7 μg/insect (i. e., 7.9μg /mg total protein), which is about 700 times higher than that of the newly emerged adults. The maximal Vg and/or Vn content is estimated to be about 6.83% of the total protein in females. Detection of tissues with immunofluorescence labelling using the antibody against Vg demonstrated that yolk granules, follicular patency, hemolymph, and epithelial plug of ovarioles were reactive. In addition, the YLSs in mycetocytes of nymphs, male and female adults, and those free in hemocoel and entering ovarioles all exhibited immunofluorescence. Ultrastructural studies were carried out using immunogold labelling. Electron micrographs showed that the YLS in every developmental stage contained most gold particles distributing over its cytoplasm and cell wall. Fat body cells contained lesser gold particles mainly in nucleolus, cytoplasmic vacuoles and granules, whereas yolk granules and microvilli between follicle cells and oocytes also had gold particles. These results indicate at least two physiological interactions between the YLS and Vg in this insect; that is, first, the YLS seems to be involved in Vg production; and second, Vg could protect the YLS from host defence response.

Ticks are one of the main vectors of disease to animals worldwide. Mainly due to anthropogenic impact on our planet, environmental changes are expected to impact tick and tick-borne diseases ecology and distribution. Rhipicephalus ticks are widely distributed throughout Southern Europe. Due to their unclarified taxonomic status, hidden diversity and unclear tick-host-pathogens interactions, assessments regarding the transmission potential of different species lineages and populations are not routinely performed. Following the premise that different tick species have different potential to transmit tick-borne diseases, the main aim of this study was to clarify if different Rhipicephalus sanguineus s.l. lineages or species distributed throughout Southern Europe show a differentiated potential for pathogens transmission to their hosts. First, tick collections from European and African countries were used to assess Rhipicephalus genus diversity using morphologic and genetic-based methods. Screening techniques were then applied to investigate their microbiota composition. By last, a spatio-temporal variation study of tick-borne symbionts with different transmission routes was performed in a four years database from the Netherlands to assess if the bacteria composition of ticks could affect pathogens and endosymbionts prevalence in the vector. Regarding the R. sanguineus s.l. diversity, our results evidence that the temperate lineage distributed in Southern Europe has at least two different morphotypes: “R. sanguineus” and “R. turanicus”. This result supports that a direct correlation between morphotype and transmission potential cannot be made based on the traditional paradigm, and additional methods should be used to confirm taxa identifications. Furthermore, our findings suggest that Coxiella-like endosymbiont diversity follow the Rhipicephalus species and lineages phylogeny, supporting not only a differentiated co-evolution as a different microbiota composition between lineages. By last, the results evidence significant associations between pathogens and endosymbionts, suggesting that microbiota composition can affect tick-borne pathogens prevalence within a tick, which ultimately could affect their transmission potential.

The evolutionary success of insects is believed to be at least partially facilitated by symbioses between insects and prokaryotes. Bacterial endosymbionts confer various fitness advantages to their hosts, for example by providing nutrients lacking from the insects’ diet thereby enabling the inhabitation of new ecological niches. The Florida carpenter ant Camponotus floridanus harbours endosymbiotic bacteria of the genus Blochmannia. These primary endosymbionts mainly reside in the cytoplasm of bacteriocytes, specialised cells interspersed into the midgut tissue, but they were also found in oocytes which allows their vertical transmission. The social lifestyle of C. floridanus may facilitate the rapid spread of infections amongst genetically closely related animals living in huge colonies. Therefore, the ants require an immune system to efficiently combat infections while maintaining a “chronic” infection with their endosymbionts. In order to investigate the immune repertoire of the ants, the Illumina sequencing method was used. The previously published genome sequence of C. floridanus was functionally re-annotated and 0.53% of C. floridanus proteins were assigned to the gene ontology (GO) term subcategory “immune system process”. Based on homology analyses, genes encoding 510 proteins with possible immune function were identified. These genes are involved in microbial recognition and immune signalling pathways but also in cellular defence mechanisms, such as phagocytosis and melanisation. The components of the major signalling pathways appear to be highly conserved and the analysis revealed an overall broad immune repertoire of the ants though the number of identified genes encoding pattern recognition receptors (PRRs) and antimicrobial peptides (AMPs) is comparatively low. Besides three genes coding for homologs of thioester-containing proteins (TEPs), which have been shown to act as opsonins promoting phagocytosis in other insects, six genes encoding the AMPs defesin-1 and defensin-2, hymenoptaecin, two tachystatin-like peptides and one crustin-like peptide are present in the ant genome. Although the low number of known AMPs in comparison to 13 AMPs in the honey bee Apis mellifera and 46 AMPs in the wasp Nasonia vitripennis may indicate a less potent immune system, measures summarised as external or social immunity may enhance the immune repertoire of C. floridanus, as it was discussed for other social insects. Also, the hymenoptaecin multipeptide precursor protein may be processed to yield seven possibly bioactive peptides. In this work, two hymenoptaecin derived peptides were heterologously expressed and purified. The preliminary antimicrobial activity assays indicate varying bacteriostatic effects of different hymenoptaecin derived peptides against Escherichia coli D31 and Staphylococcus aureus which suggests a functional amplification of the immune response further increasing the antimicrobial potency of the ants. Furthermore, 257 genes were differentially expressed upon immune challenge of C. floridanus and most of the immune genes showing differential expression are involved in recognition of microbes or encode immune effectors rather than signalling components. Additionally, genes coding for proteins involved in storage and metabolism were downregulated upon immune challenge suggesting a trade-off between two energy-intensive processes in order to enhance effectiveness of the immune response. The analysis of gene expression via qRT-PCR was used for validation of the transcriptome data and revealed stage-specific immune gene regulation. Though the same tendencies of regulation were observed in larvae and adults, expression of several immune-related genes was generally more strongly induced in larvae. Immune gene expression levels depending on the developmental stage of C. floridanus are in agreement with observations in other insects and might suggest that animals from different stages revert to individual combinations of external and internal immunity upon infection. The haemolymph proteome of immune-challenged ants further established the immune-relevance of several proteins involved in classical immune signalling pathways, e.g. PRRs, extracellularly active proteases of the Toll signalling pathway and effector molecules such as AMPs, lysozymes and TEPs. Additionally, non-canonical proteins with putative immune function were enriched in immune-challenged haemolymph, e.g. Vitellogenins, NPC2-like proteins and Hemocytin. As known from previous studies, septic wounding also leads to the upregulation of genes involved in stress responses. In the haemolymph, proteins implicated in protein stabilisation and in the protection against oxidative stress and insecticides were enriched upon immune challenge. In order to identify additional putative immune effectors, haemolymph peptide samples from immune-challenged larvae and adults were analysed. The analysis in this work focussed on the identification of putative peptides produced via the secretory pathway as previously described for neuropeptides of C. floridanus. 567 regulated peptides derived from 39 proteins were identified in the larval haemolymph, whereas 342 regulated peptides derived from 13 proteins were found in the adult haemolymph. Most of the peptides are derived from hymenoptaecin or from putative uncharacterised proteins. One haemolymph peptide of immune-challenged larvae comprises the complete amino acid sequence of a predicted peptide derived from a Vitellogenin. Though the identified peptide lacks similarities to any known immune-related peptide, it is a suitable candidate for further functional analysis. To establish a stable infection with the endosymbionts, the bacteria have to be transmitted to the next generation of the ants. The vertical transmission of B. floridanus is guaranteed by bacterial infestation of oocytes. This work presents the first comprehensive and detailed description of the localisation of the bacterial endosymbionts in C. floridanus ovaries during oogenesis. Whereas the most apical part of the germarium, which contains the germ-line stem cells, is not infected by the bacteria, small somatic cells in the outer layers of each ovariole were found to be infected in the lower germarium. Only with the beginning of cystocyte differentiation, endosymbionts are exclusively transported from follicle cells into the growing oocytes, while nurse cells were never infected with B. floridanus. This infestation of the oocytes by bacteria very likely involves exocytosis-endocytosis processes between follicle cells and the oocytes. A previous study suggested a down-modulation of the immune response in the midgut tissue which may promote endosymbiont tolerance. Therefore, the expression of several potentially relevant immune genes was analysed in the ovarial tissue by qRT-PCR. The relatively low expression of genes involved in Toll and IMD signalling, and the high expression of genes encoding negative immune regulators, such as PGRP-LB, PGRP-SC2, and tollip, strongly suggest that a down-modulation of the immune response may also facilitate endosymbiont tolerance in the ovaries and thereby contribute to their vertical transmission. Overall, the present thesis improves the knowledge about the immune repertoire of C. floridanus and provides new candidates for further functional analyses. Moreover, the involvement of the host immune system in maintaining a “chronic” infection with symbiotic bacteria was confirmed and extended to the ovaries
Der evolutionäre Erfolg von Insekten wird zumindest teilweise Symbiosen zwischen Insekten und Prokaryonten zugeschrieben. Dabei übertragen bakterielle Symbionten verschiedenste Fitnessvorteile an ihre Wirte. Beispielsweise ermöglicht die Bereitstellung von Nährstoffen, welche in der Nahrung des Insekts fehlen, die Erschließung neuer ökologischer Nischen. Die Florida Rossameise Camponotus floridanus trägt endosymbiontische Bakterien der Gattung Blochmannia. Diese primären Endosymbionten kommen hauptsächlich im Zytoplasma von spezialisierten Zellen des Mitteldarms, den sogenannten Bakteriozyten, vor. Blochmannien wurden aber auch in Oozyten und Eiern gefunden, was ihre vertikale Übertragung an Individuen der nächsten Generation ermöglicht. Als soziale Insekten leben C. floridanus in großen Kolonien von nah verwandten Individuen. Ihre Lebensweise begünstigt möglicherweise die schnelle Ausbreitung von Infektionen, weshalb erwartet werden müsste, dass die Ameisen ein effizientes Immunsystem besitzen. Gleichzeitig muss jedoch die „chronische“ Infektion mit den bakteriellen Symbionten aufrechterhalten werden. In der vorliegenden Arbeit wurde das Immunrepertoire der Ameisen mittels Illumina Sequenzierung charakterisiert. Zunächst wurde das vor kurzem publizierte Genom von C. floridanus funktionell re-annotiert. Dabei wurden 0.53% der annotierten Proteine der GO-Unterkategorie “Prozesse des Immunsystems” zugeordnet. Basierend auf Homologieanalysen wurden Gene identifiziert, die für 510 Immunproteine kodieren. Die Genprodukte spielen eine Rolle bei der Erkennung von Mikroben und in den Signalwegen des Immunsystems, sind jedoch auch an Prozessen der zellulären Immunantwort, wie beispielsweise Phagozytose und Melanisierung, beteiligt. Dabei sind Komponenten der Hauptsignalwege hoch konserviert. Obwohl die Anzahl der identifizierten Proteine, die Fremdorganismen erkennen (PRRs), und die Anzahl an antimikrobiellen Peptiden (AMPs) vergleichsweise gering ist, verfügt C. floridanus insgesamt über ein umfangreiches Immunrepertoire. Neben drei Genen, die für Thioester-enthaltende Proteine (TEPs) kodieren und wie in anderen Insekten möglicherweise eine Rolle als Opsonine bei der Phagozytose spielen, wurden sechs AMP-Gene identifiziert. Diese kodieren für Defesin-1 und Defensin-2, Hymenoptaecin, zwei Tachystatin-ähnliche und ein Crustin-ähnliches Peptid. Die geringe Anzahl an bekannten AMPs im Vergleich zur Honigbiene Apis mellifera (13 AMPs) und Wespe Nasonia vitripennis (46 AMPs) könnte ein möglicherweise geringeres Potential des Immunsystems anzeigen. Allerdings könnten zusätzliche Maßnahmen, die unter dem Begriff „Soziale Immunität“ zusammengefasst werden, das Immunrepertoire von C. floridanus ergänzen, wie es schon für andere Insekten diskutiert wurde. Zudem könnten durch proteolytische Prozessierung des Hymenoptaecin Multipeptid Präkursormoleküls sieben mögliche antimikrobielle Peptide freigesetzt werden. Für die vorliegende Arbeit wurden zwei verschiedene dieser Hymenoptaecin Peptide heterolog exprimiert und aufgereinigt. Die vorläufige funktionelle Charakterisierung der Peptide zeigt, dass diese Peptide möglicherweise bakteriostatische Wirkung mit einem unterschiedlichen Wirkspektrum gegen Escherichia coli D31 und Staphylococcus aureus entfalten. Dies erlaubt die Annahme, dass die Expression des Hymenoptaecins zu einer funktionellen Amplifikation der Immunantwort führt und das Immunrepertoire der Ameisen erweitert. Nach Injektion von bakteriellem Material in die Ameisen wurde die Expression von 257 Genen reguliert. Viele dieser Gene kodieren für Proteine zur Erkennung von Pathogenen oder kodieren für Effektoren des Immunsystems. Komponenten der Signalwege zeigten dagegen kaum Veränderungen in ihrer Expression auf. Außerdem zeigten Gene, die für Speicherproteine oder Proteine des Metabolismus kodieren, generell eine geringere Expression nach Stimulierung des Immunsystems auf. Dies lässt einen Ausgleich zwischen zwei energieintensiven Prozessen vermuten, um eine effektive Immunantwort zu ermöglichen. Darüber hinaus zeigt die Validierung der Expressionsdaten mittels qRT-PCR eine Abhängigkeit der Expression mehrerer Gene vom Entwicklungsstadium der Ameisen auf. Generell wurden die gleichen Tendenzen in der Regulation der Expression dieser Gene nach Immunstimulierung beobachtet. Allerdings wurde die Expression mehrerer immunrelevanter Gene in Larven weit stärker induziert als in Adulten. Wie es auch schon für andere Insekten gezeigt wurde, scheinen C. floridanus Larven und Arbeiterinnen auf individuelle Kombinationen externer und interner Immunfaktoren zurückzugreifen. Die vorher beschriebenen Transkriptomdaten wurden durch die Charakterisierung des Hämolymph-Proteoms von C. floridanus nach Immunstimulation ergänzt, wodurch die Immunrelevanz vieler Faktoren auch auf Proteinebene bestätigt werden konnte. Beispielsweise wurden zahlreiche PRRs und extrazellulär aktive Proteasen des Toll-Signalwegs, aber auch Immuneffektoren wie AMPs, Lysozyme und TEPs in der Hämolymphe identifiziert. Zusätzlich führte die Immunstimulation in Larven und Adulten zur Anreicherung nicht-kanonischer Proteine mit möglicher Immunfunktion, beispielsweise Vitellogenine, NPC2-ähnliche Proteine und Hemocytin. Aus einer früheren Arbeit ist bekannt, dass septische Verwundungen zusätzlich die transkriptionelle Aktivierung von Genen der Stressantwort hervorrufen können. So wurden auch in der Hämolymphe Proteine entdeckt, die eine Rolle bei der Stabilisierung von Proteinen, und dem Schutz gegen oxidativen Stress und Insektizide spielen. Zur Identifizierung weiterer möglicher Peptideffektoren wurden Hämolymphpeptid-Proben von immunstimulierten Larven und Adulten analysiert. Der Fokus der Analyse lag dabei auf der Identifizierung von Peptiden, die auf dem sekretorischen Weg gebildet werden, wie es zuvor für Neuropeptide von C. floridanus beschrieben worden war. 567 differentiell regulierte Peptide, die von 39 Proteinen abstammen, wurden in Larvenhämolymphe identifiziert, wohingegen in der Hämolymphe von Adulttieren 342 derartige Peptide, die 13 Proteinen zugeordnet werden können, gefunden wurden. Die meisten dieser Peptide können Hymenoptaecin oder bisher noch nicht charakterisierte Proteinen zugeordnet werden. Jedoch wurde ein Peptid in larvaler Hämolymphe identifiziert, dessen Aminosäuresequenz vollständig mit der Sequenz eines vorhergesagten, von Vitellogenin stammenden Peptids übereinstimmt. Weil dieses Peptid keine Ähnlichkeiten zu anderen bereits charakterisierten antimikrobiellen Peptiden aufweist, stellt es einen geeigneten Kandidaten für weitere funktionelle Analysen dar. Die bakterielle Infektion von Oozyten ermöglicht die transovariale Übertragung von B. floridanus und ermöglicht damit die Etablierung einer stabilen Infektion in der nächsten Wirtsgeneration. Die vorliegende Arbeit beinhaltet die erste umfassende und detaillierte Beschreibung der Lokalisation bakterieller Endosymbionten in Ovarien von C. floridanus. Im apikalen Germarium, in welchem sich die Keimbahn-Stammzellen befinden, liegt noch keine bakterielle Infektion des Gewebes vor. In späteren Segmenten des Germariums jedoch können Blochmannien das erste Mal in kleinen somatischen Zellen der äußeren Schicht jeder Ovariole detektiert werden. Mit beginnender Zystozytendifferenzierung werden die Endosymbionten von Follikelzellen ausschließlich in die heranwachsenden Oozyten transportiert, wobei sehr wahrscheinlich Exozytose-Endozytose-Prozesse involviert sind. Nährzellen zeigen zu keinem Zeitpunkt während der Oogenese eine bakterielle Infektion auf. Da in einer früheren Studie vorgeschlagen wurde, dass eine signifikant reduzierte Anregung der Immunantwort im Mitteldarmgewebe zur Toleranz der Endosymbionten beitragen könnte, wurde auch die Expression ausgewählter Immungene in den Ovarien durch qRT-PCR untersucht. Die relativ geringe Expression von Genen des Toll- und des IMD-Signalwegs und die zusätzlich vergleichsweise starke Genexpression negativer Regulatoren des Immunsystems, wie PGRP-LB, PGRP-SC2 und tollip, sind Indikatoren einer reduzierten Immunantwort in den Ovarien von C. floridanus. Wie schon für den Mitteldarm der Tiere vorgeschlagen, könnte dies möglicherweise sowohl zur Toleranz von Blochmannia als auch zur vertikalen Übertragung der Endosymbionten beitragen. Die vorliegende Doktorarbeit erweitert das Wissen über das Immunrepertoire von C. floridanus und es konnten vielversprechende Kandidaten für weitere funktionelle Analysen von möglichen Immunfaktoren identifiziert werden. Darüber hinaus konnten weitere Hinweise auf die Bedeutung von Immunfaktoren der Ameisen bei der Toleranz gegenüber den symbiontischen Bakterien gefunden und auf die Ovarien der Tiere ausgeweitet werden

Thesis (M.S.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008.
"Advisor, Todd A. Ciche"--Acknowledgements. Title from PDF t.p. (viewed on Aug. 5, 2009) Includes bibliographical references. Also issued in print.

Symbiosis between bacteria and single celled eukaryotes, such as ciliates and amoebae, is a widespread phenomenon. In the past years, the number of species characterizations of symbiotic bacteria, has constantly grown. Especially members of the order Rickettsiales (Alphaproteobacteria), which comprises obligate intracellular human and animal pathogens as well as other non-pathogenic intracellular species, got into the focus. However, biodiversity of both, protists and associated bacterial symbionts, is still largely underestimated. Thus, I hypothesised that different kind of bacteria occur naturally in the ciliate Paramecium, that paramecia can serve as potential reservoir for human and animal pathogens, and that the species distribution of Paramecium and its endosymbionts shows geographical patterns. In the present thesis, I provide data on the distribution and diversity of Paramecium species and their endosymbionts isolated from freshwater and brackish habitats from North and South America. The most frequent species were Paramecium biaurelia, Paramecium caudatum and Paramecium multimicronucleatum for the North American samples as well as P. multimicronucleatum and Paramecium calkinsi for the South American samples indicating geographical differences in the species distribution of Paramecium between the two continents. Monoclonal cultures were screened for the presence of endosymbionts by fluorescence in situ hybridization (FISH) using universal and specific probes. I detected bacterial endosymbionts belonging to different taxa of Rickettsiales sensu lato in Paramecium-cultures isolated from eight different habitats. Moreover, I characterized the four novel endosymbiont species “Candidatus Fokinia solitaria”, “Candidatus Fokinia cryptica”, “Candidatus Bealeia paramacronuclearis” and “Candidatus Jurandia parameciophila”, respectively, presenting detailed descriptions. Thus, I did not only detect various bacterial symbionts in different host species from two continents, but I also identified bacteria related to human and animal pathogens. Moreover, phylogenetic information provided by the four novel bacterial species helped me to revise the taxonomic organisation of the order Rickettsiales, which I split into the two orders Rickettsiales sensu stricto and Holosporales ord. nov. The importance and severity of a symbiosis for its host can be estimated, among others, by studying the symbionts transmission modes. Most host-symbiont systems in nature show a mixed mode of transmission, even if one transmission type occurs only rarely. Moreover, evolution of exclusively vertically or horizontally transmitted symbionts is scarce. However, circumstances for horizontal transmission of many symbiont species so far described as vertically transmitted, are completely unknown and still need to be revealed. Therefore, I assumed that both, vector organisms and environmental stress, can trigger horizontal transmission. I performed different sets of infection experiments using vector organisms and environmental stress factors as driving forces to trigger horizontal transmission. In experiments focused on vector-induced transmission, I was not able to confirm vector properties of Holospora towards Caedibacter, as described towards food bacteria. However, I rarely observed vector-independent horizontal transmission of Caedibacter taeniospiralis towards symbiont-free Paramecium species. The here tested genotypes of C. taeniospiralis might still take advantage of additional horizontal transmission, even if it occurs rarely. In addition, I tested the effect of salinity and temperature stress on the transmission of the recently described bacterial symbionts “Candidatus Megaira polyxenophila” and “Ca. Bealeia paramacronuclearis” as potentially interesting representatives. The involved symbiont and host genotypes differed in their response to the induced environmental stress. Results of horizontal transmission turned out to be ambiguous. While temperature stress did not show any effect on transmission, salinity stress could possibly induce horizontal transmission of “Ca. Megaira polyxenophila” at slightly increased salinity concentrations in regard to normal conditions indicating the significance of environmental stress for the selection of the preferred transmission mode.
Symbiosen zwischen Bakterien und einzelligen Eukaryoten wie Ciliaten und Amöben sind weitverbreit. In den vergangenen Jahren stieg die Anzahl der Artbeschreibungen symbiotisch lebender Bakterien stetig an. Besonders Vertreter der Ordnung Rickettsiales (Alphaproteobacteria), welche obligat intrazelluläre Menschen- und Tierpathogene sowie andere nicht-pathogene, intrazelluläre Arten beinhaltet, rückten in den Fokus. Die Biodiversität von Protisten und mit ihnen assoziierte, bakterielle Symbionten wird jedoch weitestgehend unterschätzt. Daher untersuchte ich, welche verschiedenen Bakterienarten im Ciliaten Paramecium vorkommen, ob Paramecien als potentielles Reservoir für menschen- und tierpathogene Bakterien dienen können und ob die Artverbreitung von Paramecium und seiner Symbionten geographische Muster aufweist. In der vorliegenden Arbeit, präsentiere ich Verbreitungs- und Diversitätsdaten von Paramecium-Arten sowie deren Endosymbionten, welche aus nord- und südamerikanischen Süß- und Brackwasserproben isoliert wurden. Die am häufigsten erfassten Arten in nordamerikanischen Proben waren Paramecium biaurelia, Paramecium caudatum und Paramecium multimicronucleatum, sowie P. multimicronucleatum und Paramecium calkinsi in südamerikanischen Proben. Die identifizierten Arten weisen auf geographische Unterschiede in der Artverteilung der beiden Kontinente hin. Monoklonale Massenkulturen wurden unter Verwendung universeller und spezifischer Sonden mittels Fluoreszenz in situ Hybridisierung auf das Vorhandensein von Endosymbionten überprüft. Dabei entdeckte ich bakterielle Endosymbionten verschiedener Rickettsiales sensu lato-Vertreter in Paramecienkulturen aus acht verschiedenen Habitaten. Darüber hinaus charakterisierte ich die vier neuen Endosymbiontenarten “Candidatus Fokinia solitaria”, “Candidatus Fokinia cryptica”, “Candidatus Bealeia paramacronuclearis” und “Candidatus Jurandia parameciophila” und lieferte detaillierte Artbeschreibungen. Somit erfasste ich nicht nur verschiedene Symbionten in unterschiedlichen Paramecium-Arten der beiden Kontinente, sondern identifizierte auch Bakterien, welche nahe mit relevanten Pathogenen verwandt sind. Die phylogenetischen Informationen, die mir diese vier neuen Bakterienarten lieferten, halfen mir die taxonomische Organisation der Ordnung Rickettsiales zu revidieren, welche ich in die zwei Ordnungen Rickettsiales sensu stricto und Holosporales ord. nov. aufspaltete. Die Bedeutung einer Symbiose für seinen Wirt kann unter anderem durch die Erforschung der Übertragungswege abgeschätzt werden. Die meisten natürlichen Wirt-Symbionten-Systeme zeigen gemischte Übertragungswege, auch wenn eine der Übertragungsarten nur wenig genutzt wird. Ferner tritt eine Evolution hin zu sich ausschließlich vertikal oder horizontal übertragenden Symbionten selten auf. Die Bedingungen, unter denen eine horizontale Übertragung der meisten sich als vertikal übertragend beschriebenen Symbionten stattfindet, sind unbekannt und müssen nach wie vor untersucht werden. Deshalb legte ich meiner Arbeit die Annahme zugrunde, dass sowohl Vektororganisen als auch Umweltstress eine horizontale Übertragung begünstigen können. Ich führte verschiedene Infektionsexperimente durch, in denen ich die Effekte von Vektororganismen und verschiedener abiotischer Stressfaktoren als potenzielle Auslöser für eine horizontale Übertragung untersuchte. In den Experimenten, die sich mit einer Vektor-induzierten Übertragung beschäftigten, konnte ich keine möglichen Vektoreigenschaften von Holospora gegenüber Caedibacter nachweisen, wie sie gegenüber Futterbakterien bekannt sind. In seltenen Fällen beobachtete ich jedoch eine Vektor-unabhängige, horizontale Übertragung von Caedibacter taeniospiralis auf Symbionten-freie Paramecium-Arten. Die hier getesteten C. taeniospiralis-Genotypen sind möglicherweise immer noch dazu in der Lage, die Vorteile einer zusätzlichen horizontalen Übertragung zu nutzen. Zusätzlich testete ich die Effekte von Salz- und Temperaturstress auf die Übertragung der kürzlich beschriebenen, bakteriellen Symbionten “Candidatus Megaira polyxenophila” und “Ca. Bealeia paramacronuclearis” als potentiell interessante Vertreter. Die getesteten Symbionten- und Wirtsgenotypen unterschieden sich hinsichtlich ihrer Reaktion auf den ausgelösten abiotischen Stress. Die erzielten Ergebnisse zur horizontalen Übertragung erscheinen jedoch nicht eindeutig. Während Temperaturstress keine Effekte auf die Übertragung hatte, führte leichter Salzstress zu einer möglichen horizontalen Übertragung von “Ca. Megaira polyxenophila”, was auf die Bedeutung abiotischer Stressfaktoren bei der Wahl des bevorzugten Übertragungsweges hinweist.
Le simbiosi fra batteri ed eucarioti unicellulari, come i ciliati e le amebe, è un fenomeno molto diffuso. Negli anni passati, il numero di descrizioni di specie di batteri simbionti è cresciuto costantemente. In particolare, sono venuti alla ribalta i membri dell’ordine Rickettsiales (Alphaproteobacteria), che comprende patogeni intracellulari obbligati umani e di animali, oltre ad altre specie di batteri intracellulari non patogeni. Comunque, la biodiversità dei protisti e dei batteri simbionti a loro associati è ancora largamente sottostimata. Perciò, è stato ipotizzato che ci siano diversi tipi di batteri che vivono in associatione con il ciliato Paramecium in natura, che I parameci possano fungere da possibili serbatoi naturali per patogeni umani e animali, e che la distribuzione delle specie di Paramecium e dei suoi simbionti presenti dei pattern geografici. Nel presente lavoro di tesi, sono presentati dati sulla distribuzione e diversità di specie del genere Paramecium e dei loro batteri endosimbionti, isolate da ambienti dulciacquicoli e salmastri dal nordamerica e sudamerica. Le specie più frequenti sono state Paramecium biaurelia, Paramecium caudatum e Paramecium multimicronucleatum per i campioni nordamericani, e P. multimicronucleatum e Paramecium calkinsi per i campioni sudamericani, il che indica differenze geografiche nella distribuzione della specie di Paramecium fra I gue continenti. Colture monoclonali sono state saminate per la presenza di endosimbionti tramite ibridazione in situ a fluorescenza (FISH), impiegando sonde universali e specifiche. Sono stati rilevati batteri endosimbionti appartenenti a diversi taxa di Rickettsiales sensu lato in colture di paramecio isolate da otto diversi habitat. Inoltre, sono state caratterizzate quattro nuove specie di endosimbionti, rispettivamente, “Candidatus Fokinia solitaria”, “Candidatus Fokinia cryptica”, “Candidatus Bealeia paramacronuclearis” e “Candidatus Jurandia parameciophila”), fornendo descrizioni dettagliate. Perciò non solo sono stati caratterizzati diversi batteri simbionti in differenti specie di ospiti da due continenti, ma sono anche stati identificati batteri affini a patogeni umani e animali. I dati filogenetici forniti dalle quattro nuove specie mi hanno permesso di revisionare l’organizzazione tassonomica dell’ordine Rickettsiales, che è stato diviso nei due ordini Rickettsiales sensu stricto e Holosporales ord. nov. L’importanza e la severità di una simbiosi per l’ospite può essere stimata studiando le modalità di trasmissione del simbionte. La maggior parte dei sistemi ospite-simbionte in natura presentano una modalità mista di trasmissione, anche se una tipo di trasmissione avviene solo occasionalmente. Inoltre, l’evoluzione di simbionti trasmessi eclusivamente per via verticale o orizzontale è scarsa. Comunque, le circostanze per la trasmissione orizzontale di molti simbionti finora descritti come trasmessi orizzontalmente sono completamente sconosciute, e necessitano di essere rivelate. Perciò, è stato ipotizzato che sia organismi vettori che stress ambientali possano indurre la trasmissione orizzontali. Sono stati eseguiti diversi set di esperimenti di infezione, impiegando organismi vettori e fattori di stress ambientali come agenti che potessero indurre tramissione orizzontale. In esperimenti incentrati sulla trasmissione indotta da vettori, non è stato possibile confermare capacità di vettore da parte di Holospora nei confronti di Caedibacter, come invece descritto nei confronti di batteri a vita libera. Comunque, in rari casi è stata osservata trasmissione orizzontale di Caedibacter taeniospiralis verso specie di Paramecium indipendente dal vettore. I genotipi testati di C. taeniospiralis potrebbero comunque trarre un vantaggio dalla possibilità alternativa di trasmissione orizzontale, anche se questa avviene solo di rado. Inoltre, è stato testato l’effetto dello stress da salinità e temperatura sulla trasmissione dei batteri simbionti recentemente descritti “Candidatus Megaira polyxenophila” e “Ca. Bealeia paramacronuclearis” come rappresentanti potenzialmente interessanti. I genotipi coinvolti di ospiti e simbionti hanno risposto diversamente a fattori indotti di stress ambientale. I risultati sono stati ambigui in termini di trasmissione orizzontale. Mentre lo stress da temperatura non ha mostrato un effetto sulla trasmissione, è stata rilevata una possibile induzione della trasmissione di “Ca. Megaira polyxenophila” in presenza di basso stress salino rispetto alle condizioni normali, indicando l’importanza degli stress ambientali per la selezione della modalità di trasmissione.

碩士
國立中山大學
海洋生物科技暨資源學系研究所
95
The sponge Cinachyrella australiensis (Demospongiae: Spirophorida) is widely distributed in Indian ocean, West Pacific ocean, and Australian waters. It also can be found in the intertidal pools of Wun-Li-Ton in southern Taiwan. The sponge can propagate asexually by budding. According to the previous studies, this sponge was suspected to be symbiotic with sulfur-oxidizing chemoautotrophic bacteria. How the generation do obtain this symbiont is still unknow. In this study, PCR was used to amplify the DNA extracted from buds and sponges to obtained the 16S rDNAs. A total of 20 clones from each bud and mature sponge samples were randomly selected and sequenced. The results indicated that the major symbiotic bacteria constitute 65％ of the clones derived form the mature sponge and 15％ from the buds. The dominant symbionts contain RubisCO gene and are related to the sulfur-oxidizing chemoautotrophic bacteria, associated with the tube worms of the deep sea hydrothermal vents. The location of the sulfur-oxidizing chemoautotrophic bacteria was observed by fluorescence in situ hybridization (FISH). It was found that the sulfur-oxidizing chemoautotrophic bacteria were intracellular symbiosis within the cells of cortex, archaeocytes of mesoglial, and bud. Similar results were also observed at the junction of a developing bud and mature sponge. Apparently, the symbionts are transmitted from sponge to bud vertically. Furthermore, in this study, we also found several other intracelluar symbionts besides the major symbiotic bacterium,some of them are autotrophic in nature.