Many insects and other arthropods have symbiotic microorganisms that may influence key facets of their biology. Cardinium hertigii is an intracellular bacterial symbiont, (phylum Bacteroidetes) of arthropods and nematodes. This versatile symbiont has been shown to cause three of four reproductive manipulations of their arthropod hosts known to be caused by symbionts: parthenogenesis induction (PI), where genetic males are converted into genetic females; feminization, where genetic males become functional females; and cytoplasmic incompatibility (CI), the symbiont-induced death of offspring from matings of infected males and uninfected females. Here, I explored the evolution of this symbiont and its reproductive manipulations, and found that closely related Cardinium strains have a tendency to associate with closely related hosts and the reproductive manipulations do not display a clear phylogenetic signal. To further understand the possible genes underlying these reproductive manipulations, I sequenced four Cardinium genomes and compared these with the two genomes analyzed in the literature. In these comparisons, I found that, although closely related Cardinium strains tend to reside in closely related hosts, there is no evidence for a suite of genes associated with host specificity, as few differences separate two strains residing in different host orders, suggesting that ecological opportunity for horizontal transmission may be more limiting to Cardinium than genomic capability. I additionally identify some genes that may be associated with the Cardinium’s ability to induce PI and CI in its wasp host. Overall, this dissertation has led to a better understanding of Cardinium and its effects on its hosts.

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¢H of the clones derived form the mature sponge and 15¢H 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.

Os insetos são o grupo de organismos multicelulares mais abundantes e diversos, habitando os mais variados ambientes. Muitas de suas adaptações a condições adversas podem estar relacionadas à sua associação a bactérias. O conhecimento da diversidade dessa associação pode elucidar a importância desses microrganismos nas respostas de insetos a fatores bióticos e abióticos. Bactérias também são o principal alvo de exploração para o desenvolvimento de produtos de interesse biotecnológico. Dada à sua diversidade e interações com o ambiente, insetos representam um novo nicho para a exploração de microrganismos com potencial biotecnológico. A longa história de associação dos insetos com bactérias e os dados recentes da participação da microbiota a eles associada na degradação de moléculas orgânicas, naturais e/ou sintéticas, indicam o potencial desses microrganismos de interferir na resposta do inseto a inseticidas. Adicionalmente, simbiontes bacterianos associados aos insetos também podem ser uma fonte promissora de compostos bioativos. A descoberta de novos compostos naturais vem decaindo, surgindo a necessidade de exploração de novos nichos de microrganismos e o uso de novas tecnologias para superar o número reduzido de novas moléculas identificadas. Assim, este trabalho buscou explorar simbiontes de insetos para o estudo da sua participação na metabolização de xenobióticos pelo hospedeiro e a identificação de novos compostos inseticidas, tendo como objetivos investigar i) os mecanismos envolvidos na degradação de inseticidas e sua contribuição na capacidade de sobrevivência do hospedeiro, e ii) a diversidade biológica na busca de novas moléculas inseticidas. Bactérias com potencial de degradação de xenobióticos isoladas da microbiota intestinal de insetos resistentes a inseticidas foram utilizadas para a colonização do trato intestinal de Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera: Noctuidae) suscetível à inseticidas e investigação da sua participação na sobrevivência do hospedeiro quando exposto a inseticidas, assim como a existência de custo adaptativo da associação a bactérias com capacidade de metabolização de inseticidas. O isolado IIL-Cl29 Leclercia adecarboxylata foi capaz de contribuir para a sobrevivência de lagartas expostas a chlorpyrifos ethyl, passando a exigir uma CL50 cerca de 2 vezes superior àquela de lagartas apossimbiontes. Estudo de biologia comparada entre a linhagem apossimbionte e aquela associadas ao isolado IIL-Cl29 demonstrou a existência de custo adaptativo para essa associação, quando na ausência da pressão de seleção do inseticida. A investigação dos mecanismos envolvidos na metabolização de inseticidas por bactérias simbiontes de S. frugiperda resistente ao organofosforado chlorpirifos ethyl, aos piretroides lambda-cyhalothrin e deltamethrin, a espinosina spinosad e a benzoilureia lufenuron revelou, por meio de análises químicas, que essas bactérias são capazes de metabolizar e bioacumular inseticidas interferindo ativamente na atividade de xenobióticos no hospedeiro. O isolado IIL-Luf14 Microbacterium arborescens foi selecionado para a realização de estudos mais aprofundados para a identificação de produtos de degradação e comprovação do mecanismo de bioacumulação de lufenuron. O potencial de simbiontes de insetos para o isolamento de bioinseticidas foi avaliado em estudos da diversidade de bactérias cultiváveis associadas a Acromyrmex coronatus (Fabricius, 1804) (Hymenoptera: Formicidae). Foram identificados 46 isolados pertencentes a Actinobacteria, Firmicutes e Proteobacteria associados a essa formiga e estudos da atividade inseticida in vivo e in vitro de extratos orgânicos de metabólitos mostraram grande variação nos resultados de atividade. Ensaios in vivo com lagartas de 1º instar de S. frugiperda mostraram-se mais adequados, e o isolado Asp77 Streptomyces drozdowiczii foi selecionado para a identificação de moléculas ativas com efeito inseticida. Dois compostos bioativos com atividade inseticida foram identificados e a atividade inseticida atribuída para duas classes químicas diferentes: um composto pirrolobenzodiazepino e um alcaloide. Nossos resultados comprovam a hipótese de que simbiontes participam da metabolização de xenobióticos em insetos, ao mesmo tempo que possuem potencial para exploração de novos compostos inseticidas. / The insects are the most diverse and abundant group of multicellular organisms, inhabiting a range of environments. Many of their adaptations to restricting conditions are related to their association with bacteria. The knowledge of the diversity of insect associations with bacteria can elucidate their role in insect response to biotic and abiotic factors. Bacteria are also the main target of exploitation for the development of biotechnological products. Insect´s diversity and interactions with the environment turn insects a new niche for the exploration of microorganisms with biotechnological potential. The long history of association of insects with bacteria and the participation of insect-associated bacteria in the degradation of natural and/or synthetic organic molecules indicate the potential of these microorganisms to interfere with insect response to insecticides. Additionally, bacterial symbionts associated with insects may also be a promising source of bioactive compounds. The discovery of new natural compounds has been declining, requiring the exploration of new niches of microorganisms and the use of new technologies to overcome the reduced number of molecules discovered. This work aimed to investigate the role of insect-associated bacteria in the metabolism of xenobiotics in the host and their potential to synthesize insecticidal compounds. Our objectives were to demonstrate i) the mechanisms involved in insecticide degradation by selected microbial symbionts, and to determine their contribution to host survival when exposed to insecticides, and ii) the biological and metabolic diversity of the microbiota associated with a leaf cutting ant. Bacteria that are able to degrade xenobiotics isolated from the gut microbiota of insecticide-resistant insects were used for the colonization of the gut of a susceptible strain of Spodoptera frugiperda (JE Smith, 1797) (Lepidoptera: Noctuidae). Comparisons of larval survival after insecticide exposure and the existence of adaptive costs of the association were done by using a aposymbiotic and infected susceptible lines. Suceptible larvae infected with IIL-Cl29 Leclercia adecarboxylata require twice the dose of chlorpyrifos ethyl to kill 50% of the larave (LC50) when compared to the aposymbiotic larvae. But the association with IIL-Cl29 was shown costly to S. frugiperda, particularly affecting female fecundity. However, no fitness costs were detected to infected larvae in the presence of the selection pressure (insecticide). GC-MS and LC-MS-MS analyses of the bacteria isolated from resistant larvae of S. frugiperda to organophosphate (chlorpirifos ethyl), pyrethroid (lambda-cyhalothrin and deltamethrin), spinosyn (spinosad) and benzoylurea (lufenuron) revealed they are able to hydrolyze, metabolyze and bioaccumulate insecticides, showing they can actively interfere with the efficacy of insecticides against the host. These processes were better characterized for the isolate IIL-Luf14 Microbacterium arborescens. The species and metabolic diversity of culturable bacteria associated with Acromyrmex coronatus (Fabricius, 1804) (Hymenoptera: Formicidae) were investigated to demonstrated their potential to produce bioinsecticides. A total of 46 isolates belonging to Actinobacteria, Firmicutes and Proteobacteria were identified. In vivo and in vitro assays to detect insecticide activity in organic extracts of fermentates indicated a great number of active extracts. In vivo assays with 1st instars of S. frugiperda were more adequate in detecting toxic molecules to insects, and the isolate Asp77 Streptomyces drozdowiczii was selected for the identification of active molecules with insecticidal effect. Two bioactive compounds with insecticidal activity were identified and the insecticidal activity attributed to two different chemical classes: pyrrolobenzodiazepine and alkaloid. Our results support the hypothesis that symbionts participate in the metabolization of xenobiotics in insects, at the same time that they have potential for exploration of new insecticides.

Sponge are primitive multi-cellular organisms. They are important sources of secondary metabolites. In the previous studies indicated that the sponges harbor stable symbiotic microbial consortia. The mechanisms for maintenance and transmission of microbial consortia to the next generations are still not fully understood. The sponge, Cinachyrella australinesis, was chosen to further investigate relationship of the symbiotic bacteria within to the host. Fluorescent in situ hybridization ¡]FISH¡^was employed with non-specific ¡]EUB338¡^and specific oligonucleotide probes for bacteria. The sponge was cryo-sectioned¡]1£gm¡^and hybridized with fluorescent probes. The distribution and ratios of the bacteria in the sponge agreed with those of previous studies indicating that the symbiotic bacteria of C. australiensis are stable and endosymbiotic in nature.

No description available.

Animal and plant genomes produce numerous small RNAs (smRNAs) that regulate gene expression post-transcriptionally affecting metabolism, development, and epigenetic inheritance. In order to characterize the repertoire of endogenous microRNAs and potential gene targets, we conducted smRNA and mRNA expression profiling over nine experimental treatments of cultures from the dinoflagellate Symbiodinium sp. A1, a photosynthetic symbiont of scleractinian corals. We identified a total of 75 novel smRNAs in Symbiodinum sp. A1 that share stringent key features with functional microRNAs from other model organisms. A subset of 38 smRNAs was predicted independently over all nine treatments and their putative gene targets were identified. We found 3,187 animal-like target sites in the 3’UTRs of 12,858 mRNAs and 53 plantlike target sites in 51,917 genes. Furthermore, we identified the core RNAi protein machinery in Symbiodinium. Integration of smRNA and mRNA expression profiling identified a variety of processes that could be under microRNA control, e.g. regulation of translation, DNA modification, and chromatin silencing. Given that Symbiodinium seems to have a paucity of transcription factors and differentially expressed genes, identification and characterization of its smRNA repertoire establishes the possibility of a range of gene regulatory mechanisms in dinoflagellates acting post-transcriptionally.

O branqueamento por estresse térmico é a principal causa do declínio de cnidários simbióticos nos oceanos tropicais. Contudo, este processo pode ser revertido. Assim, o objetivo deste trabalho é o estudo integrado dos processos fisiológico da perda e recuperação das zooxantelas relacionado ao fenômeno de branqueamento por aumento de temperatura em E. pallida, mediante o estudo da resposta do holobionte ao estresse térmico, com ênfase na troca de população simbiótica. Um dos mecanismos de recuperação da pigmentação investigados nesse estudo é a proliferação dos endosimbiontes remanescentes no cnidário, onde foi encontrando um padrão de repigmentação funcional em diferentes partes do pólipo. Neste contexto é proposto um mecanismo de ruptura da simbiose na qual os endosimbiontes participam ativamente de sua expulsão e dispersão no meio, abrindo uma nova discussão sobre o agente diretor da simbiose. Uma vez que o branqueamento tem sido relacionado com estresse oxidativo e a fotossíntese é um processo oxidante, avaliamos a atividade redox induzida pela fotossíntese da alga hospedada, assim como a atividade antioxidante e reparadora do hospedeiro. Descobrimos no hospedeiro um mecanismo preventivo para lidar com a atividade fotossintética da zooxantela abrigada. Por outro lado, a perda da alga a partir do branqueamento pode fornecer uma oportunidade para o hospedeiro ser repopulado por outros simbiontes com diferentes capacidades fisiológicas. Assim, encontramos que existe uma troca de alga ideal para o frio (clado F), e para o calor (clado E). Entretanto, alguns clados podem estar indisponíveis no ambiente por serem termosensíveis em vida livre. Concluímos assim que, dentro dos cenários das mudanças climáticas globais, o mais vantajoso para o animal e para a alga é manter uma endopopulação mista e trocar a população simbiôntica predominante segundo as condições ambientais. Aliás, encontramos que o holobionte estudado está melhor preparado para condições de aquecimento que de esfriamento. Por outro lado, considerando que o peróxido de hidrogênio é um derivado de filtros solares e que ao mesmo tempo é um precursor do branqueamento, investigamos se o branqueamento da anêmona Anemonia sulcata produzido pela exposição a peróxido de hidrogênio é um bom modelo como bioindicador ambiental através da avaliação da cor e de biomarcadores fisiológicos. Porém, descartamos A. sulcata como espécie bioindicadora por apresentar uma alta tolerância ao estresse oxidativo, provavelmente fornecido pela atividade fotossintética da alga e por ser uma espécie intermareal que suporta grandes intervalos de oxido-redução. Desta forma, os resultados obtidos no presente estudo apontam a relevância do Symbiodinium dentro da fisiologia do holobionte, tanto na recuperação do branqueamento quanto no estabelecimento e ruptura da simbiose. Consequentemente, futuros estudos podem aprimorar as técnicas de identificação dos clados hospedados para entender a eventual sucessão endopopulacional de acordo com as condições ambientais / Thermal bleaching is the main cause of simbiotic cnidarian decay in tropical oceans. However, some cnidarians have shown to recover the pigmentation from such events. The main aim of this study is the holistic approach of the physiological processes associated to zooxanthela loss and recovery after thermal stress in the holobiont E. pallida. Firstm we considered pigmentation recovey from the remaining zooxanthelae within the cnidarian host. We found a functional pattern of pigmentation recovery. In this context, we propose a mechanism of symbiosis break in which the endosymbionts participate actively on its release and dispersion to the environment, bringing a new discussion about the role of each symbiotic partner and the driver of bleaching and recovery. Since bleaching has been related to oxidative stress and photosynthesis is an oxidizing process, we evaluated the redox activity of the host as well as the antioxidant and restorative activity, induced by photosynthesis of the hosted algae. We found in the host a preventive mechanism to deal with the photosynthetic activity of the sheltered zooxanthella. On the other hand, loss of endosymbionts from bleaching may provide an opportunity for the host to be repopulated by other symbionts with different physiological capabilities. Thus, we found that there is an ideal clade exchange for the cold (clade F), and for the heat (clade E). However, some clades may not be available in the environment because they are thermosensitive in free-living form. We conclude that, within global climate change scenarios, the most beneficial for the animal and the alga is to maintain a mixed endopopulation and to exchange the predominant symbiotic population according to environmental conditions. In fact, we found that the studied holobiont is better prepared for high than low temperatures. On the other hand, considering that hydrogen peroxide is a derivative of sunscreens and at the same time is a precursor to bleaching, we investigated whether the bleaching of anemone Anemonia sulcata produced by exposure to hydrogen peroxide is a good model as an environmental bioindicator through color evaluation and physiological biomarkers. However, we discard A. sulcata as a bioindicator species because it presents a high tolerance to oxidative stress, probably provided by algae photosynthetic activity and because it is an intertidal species that supports large oxido-reduction intervals. Thus, the results obtained in the present study point to the relevance of Symbiodinium within holobiont physiology, both in the recovery of bleaching and in the establishment and rupture of symbiosis. Consequently, future studies may improve the identification techniques of the host clades to understand the eventual endopopulation succession according to the environmental conditions

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.

Nematodes are highly diverse animals capable of interacting with almost every other form of life on Earth from general trophic interactions to intimate and persistent symbiotic associations. Much of their recognition originates from their various parasitic lifestyles. From an agricultural standpoint, plant parasitic nematodes are widely known for the destruction they can cause to crop plants, such as the case of the root-knot nematode Meloidogyne incognita, or livestock animals, like the Trichinella spiralis, which infects pigs and other animals. From a human health perspective, nematodes can cause many debilitating diseases, for example Wuchereria bancrofti, which is a causative agent of lymphatic filariasis or elephantiasis. However, not all parasitic nematodes have bad implications for human health. For instance, the diverse interactions of insect parasitic nematodes can be used to our benefit. Many of these species have been considered as biological control alternatives to different insect pests that wreak havoc on human, animal, and plant health. There still remain many questions surrounding their evolution, ecology, and physiological capabilities. Many of these taxa are hard to cultivate in the lab due to their complex and intimate lifestyles. Entomopathogenic nematodes (EPNs) are of great interest in agriculture because they vector insect pathogenic bacteria, which are capable of causing death to an insect host within 48 hours post-infection. Much of the molecular underpinnings in this system still remain to be discovered, from understanding the basic ability of these two organisms to associate with one another to genetically engineering more robust and host specific pathogens for application in the field. The focus of the research presented herein is on Steinernematidae nematodes and their bacterial symbionts. Specifically, it focused on the relationship between Xenorhabdus bovienii and its Steinernema hosts. Bioassays were designed to investigate insect virulence of X. bovienii alone in two Lepidoptera insect species with known differential susceptibility to Steinernema-Xenorhabdus pairs. A comparative genomic analysis was performed to compare different Xenorhabdus bovienii strains with observed variation in insect virulence. Results from this analysis demonstrated that virulent strains possess a type VI secretion system (T6SS) locus that is completely absent in strains with attenuated virulence. Bacterial competition assays between T6SS+ and T6SS- strains suggest this locus is involved in bacterial competition. Additionally, symbiont preference assays were carried out to investigate whether Steinernema hosts are able to discern between virulent and attenuated X. bovienii strains. Results from these assays revealed that Steinernema nematodes are able to distinguish between cognate and non-cognate X. bovienii symbionts, giving preference to virulent strains over those with attenuated virulence. Altogether these results provide further evidence that supports the notion that symbiont-switching events have occurred over the Steinernema-Xenorhabdus co-evolutionary history. Specifically, the competitive virulence of certain X. bovienii strains may have conferred them the ability to be selected by different Steinernema hosts, therefore contributing to the success of the nematode-bacterium partnership in being pathogenic to diverse insect hosts.

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.