O Filo Microsporidia é composto por parasitas intracelulares obrigatórios que infectam de invertebrados a mamíferos. Alguns gêneros de microsporídeos entomopatogênicos são de extrema importância, visto que diversos estudos epizootiológicos indicam tais agentes como detentores de um papel fundamental na regulação da densidade populacional de seus hospedeiros. Além disso, esses patógenos são considerados como um dos principais obstáculos na manutenção da sanidade nas criações de insetos em laboratório. No Brasil, a partir de 1977, foram relatadas as primeiras ocorrências de nosemoses em criações de laboratório da broca-da-cana, Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae). Este problema agravou-se no decorrer dos anos em função de alterações nas técnicas de criação da broca visando aumentar a escala produtiva deste inseto e do parasitoide Cotesia flavipes (Cam.) (Hymenoptera: Braconidae). Os objetivos deste trabalho consistiram na: (i) caracterização morfológica e molecular do patógeno, abordando ciclo de vida, patogenicidade a outros lepidópteros de importância agrícola e prevalência em populações de laboratório e campo de D. saccharalis e C. flavipes; (ii) determinação da virulência de Nosema sp. a D. saccharalis em laboratório; (iii) caraterização da propagação do patógeno nas lagartas e sua disseminação pelas fezes; (iv) efeitos da infecção na biologia de D. saccharalis e de C. flavipes e no desempenho do parasitoide quanto à seleção de hospedeiros infectados e não infectados; (v) transmissão do patógeno por C. flavipes a sua progênie e seus hospedeiros e (vi) controle de Nosema sp. em D. saccharalis por produtos antimicrobianos. Foi confirmado que o microsporídeo isolado de D. saccharalis pertence ao gênero Nosema, sendo patogênico à todas as espécies-praga avaliadas. A prevalência do patógeno em C. flavipes foi maior que a prevalência nas criações de D. saccharalis. Os esporos de Nosema sp. foram liberados nas fezes de D. saccharalis, variando o tempo até a liberação e a quantidade de esporos de acordo com o inóculo inicial de infecção. Embora Nosema seja considerado por muitos cientistas como enzoótico e com baixo potencial como agente de controle microbiano para ser usado de forma inundativa, a DL50 para D. saccharalis de 1º ínstar foi estimada em 5,6 esporos, demonstrando como as lagartas recém-eclodidas são muito suscetíveis ao microsporídeo. Infecções por Nosema sp. afetaram negativamente a biologia de D. saccharalis, exceto a duração da fase de pupa, sendo transmitido horizontal e verticalmente. Não foram observadas diferenças significativas na mortalidade de D. saccharalis infectadas por Nosema sp. tratadas com os antimicrobianos utilizados neste estudo em comparação à mortalidade do tratamento controle. Os parâmetros biológicos e o comportamento de C. flavipes sofreram alterações causadas pelo patógeno Nosema sp., que foi transmitido pelo parasitoide para o hospedeiro e por pelo menos quatro geraçõe de C. flavipes. Estes resultados ressaltam a relevância dos estudos sobre o impacto de Nosema sp. em D. saccharalis e C. flavipes, justificando esforços futuros no manejo da doença, dada a importância dessas espécies no agroecossistema da cana-de-açúcar. / The phylum Microsporidia consists of obligatory intracellular parasites that infect from invertebrates to mammalians. Some genera of entomopathogenic microsporidia are extremely important, considering that several epizootiological studies indicate that these agents as having a key role in density population regulating of their hosts. In addition, these pathogens are considered a major obstacle in maintaining healthiness in laboratory insect rearing. In Brazil occurrences of nosemoses were first reported in laboratory rearing of sugarcane borer, Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae), in the 1977. This problem has worsened over the years due to changes in the rearing methods aiming to increase the production scale of this insect and the parasitoid Cotesia flavipes (Cam.) (Hymenoptera: Braconidae). The objectives of this project consisted of (i) morphological and molecular characterization of the pathogen, addressing life cycle, pathogenicity to other lepidopterans of agricultural importance and prevalence in D. saccharalis and C. flavipes in laboratory rearing and in sugarcane plantations in the State of São Paulo, (ii) virulence of Nosema sp. to D. saccharalis in laboratory, (iii) transmission of the pathogen in D. saccharalis larvae and their dissemination in feces, (iv) the effects of infection on D. saccharalis and C. flavipes biology and performance of the parasitoid on the selection of infected and uninfected hosts, (v) the transmission of the pathogen by C. flavipes to their progeny and their hosts and (vi) the control of Nosema sp. in D. saccharalis by antimicrobial products. It was confirmed that the microsporidium isolated from D. saccharalis belongs to the genus Nosema, being pathogenic to all pest species evaluated in the host specificity study. The prevalence of the pathogen in C. flavipes was higher than the one in the D. saccharalis rearing facilities. Nosema sp spores were disseminated in the feces of D. saccharalis, varying the time until their release and the quantity of spores according to the initial inoculum of infection. Although Nosema is considered by many scientists as enzootic and low potential as a microbial control agent to be used inundatively, the LD50 for 1st instar D. saccharalis was estimated as 5.6 spores, demonstrating how that the newly-hatched larvae are very susceptible to this pathogen. Nosema sp. infections negatively affected the biology of D. saccharalis, except in the duration of the pupal stage, being transmitted horizontally and vertically. There were no significant differences in mortality of D. saccharalis infected by Nosema sp. treated with the antibiotics used in this study compared with the mortality of the control treatment. The biological parameters and behavior of C. flavipes suffered adverse effects caused by the pathogen Nosema sp., which was transmitted to the host by the parasitoid and its progeny to four C. flavipes generation. These results underscore the relevance of studies on the impact of Nosema sp. in D. saccharalis and C. flavipes, justifying future efforts in managing the disease, given the importance of these species in the sugarcane ecosystem.

O Filo Microsporidia é composto por parasitas intracelulares obrigatórios que infectam de invertebrados a mamíferos. Alguns gêneros de microsporídeos entomopatogênicos são de extrema importância, visto que diversos estudos epizootiológicos indicam tais agentes como detentores de um papel fundamental na regulação da densidade populacional de seus hospedeiros. Além disso, esses patógenos são considerados como um dos principais obstáculos na manutenção da sanidade nas criações de insetos em laboratório. No Brasil, a partir de 1977, foram relatadas as primeiras ocorrências de nosemoses em criações de laboratório da broca-da-cana, Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae). Este problema agravou-se no decorrer dos anos em função de alterações nas técnicas de criação da broca visando aumentar a escala produtiva deste inseto e do parasitoide Cotesia flavipes (Cam.) (Hymenoptera: Braconidae). Os objetivos deste trabalho consistiram na: (i) caracterização morfológica e molecular do patógeno, abordando ciclo de vida, patogenicidade a outros lepidópteros de importância agrícola e prevalência em populações de laboratório e campo de D. saccharalis e C. flavipes; (ii) determinação da virulência de Nosema sp. a D. saccharalis em laboratório; (iii) caraterização da propagação do patógeno nas lagartas e sua disseminação pelas fezes; (iv) efeitos da infecção na biologia de D. saccharalis e de C. flavipes e no desempenho do parasitoide quanto à seleção de hospedeiros infectados e não infectados; (v) transmissão do patógeno por C. flavipes a sua progênie e seus hospedeiros e (vi) controle de Nosema sp. em D. saccharalis por produtos antimicrobianos. Foi confirmado que o microsporídeo isolado de D. saccharalis pertence ao gênero Nosema, sendo patogênico à todas as espécies-praga avaliadas. A prevalência do patógeno em C. flavipes foi maior que a prevalência nas criações de D. saccharalis. Os esporos de Nosema sp. foram liberados nas fezes de D. saccharalis, variando o tempo até a liberação e a quantidade de esporos de acordo com o inóculo inicial de infecção. Embora Nosema seja considerado por muitos cientistas como enzoótico e com baixo potencial como agente de controle microbiano para ser usado de forma inundativa, a DL50 para D. saccharalis de 1º ínstar foi estimada em 5,6 esporos, demonstrando como as lagartas recém-eclodidas são muito suscetíveis ao microsporídeo. Infecções por Nosema sp. afetaram negativamente a biologia de D. saccharalis, exceto a duração da fase de pupa, sendo transmitido horizontal e verticalmente. Não foram observadas diferenças significativas na mortalidade de D. saccharalis infectadas por Nosema sp. tratadas com os antimicrobianos utilizados neste estudo em comparação à mortalidade do tratamento controle. Os parâmetros biológicos e o comportamento de C. flavipes sofreram alterações causadas pelo patógeno Nosema sp., que foi transmitido pelo parasitoide para o hospedeiro e por pelo menos quatro geraçõe de C. flavipes. Estes resultados ressaltam a relevância dos estudos sobre o impacto de Nosema sp. em D. saccharalis e C. flavipes, justificando esforços futuros no manejo da doença, dada a importância dessas espécies no agroecossistema da cana-de-açúcar. / The phylum Microsporidia consists of obligatory intracellular parasites that infect from invertebrates to mammalians. Some genera of entomopathogenic microsporidia are extremely important, considering that several epizootiological studies indicate that these agents as having a key role in density population regulating of their hosts. In addition, these pathogens are considered a major obstacle in maintaining healthiness in laboratory insect rearing. In Brazil occurrences of nosemoses were first reported in laboratory rearing of sugarcane borer, Diatraea saccharalis (Fabr.) (Lepidoptera: Crambidae), in the 1977. This problem has worsened over the years due to changes in the rearing methods aiming to increase the production scale of this insect and the parasitoid Cotesia flavipes (Cam.) (Hymenoptera: Braconidae). The objectives of this project consisted of (i) morphological and molecular characterization of the pathogen, addressing life cycle, pathogenicity to other lepidopterans of agricultural importance and prevalence in D. saccharalis and C. flavipes in laboratory rearing and in sugarcane plantations in the State of São Paulo, (ii) virulence of Nosema sp. to D. saccharalis in laboratory, (iii) transmission of the pathogen in D. saccharalis larvae and their dissemination in feces, (iv) the effects of infection on D. saccharalis and C. flavipes biology and performance of the parasitoid on the selection of infected and uninfected hosts, (v) the transmission of the pathogen by C. flavipes to their progeny and their hosts and (vi) the control of Nosema sp. in D. saccharalis by antimicrobial products. It was confirmed that the microsporidium isolated from D. saccharalis belongs to the genus Nosema, being pathogenic to all pest species evaluated in the host specificity study. The prevalence of the pathogen in C. flavipes was higher than the one in the D. saccharalis rearing facilities. Nosema sp spores were disseminated in the feces of D. saccharalis, varying the time until their release and the quantity of spores according to the initial inoculum of infection. Although Nosema is considered by many scientists as enzootic and low potential as a microbial control agent to be used inundatively, the LD50 for 1st instar D. saccharalis was estimated as 5.6 spores, demonstrating how that the newly-hatched larvae are very susceptible to this pathogen. Nosema sp. infections negatively affected the biology of D. saccharalis, except in the duration of the pupal stage, being transmitted horizontally and vertically. There were no significant differences in mortality of D. saccharalis infected by Nosema sp. treated with the antibiotics used in this study compared with the mortality of the control treatment. The biological parameters and behavior of C. flavipes suffered adverse effects caused by the pathogen Nosema sp., which was transmitted to the host by the parasitoid and its progeny to four C. flavipes generation. These results underscore the relevance of studies on the impact of Nosema sp. in D. saccharalis and C. flavipes, justifying future efforts in managing the disease, given the importance of these species in the sugarcane ecosystem.

Microsporidia are obligate intracellular pathogens that cause severe disease in immunocompromised humans. While albendazole is the treatment of choice, no therapy exists that effectively treats all forms or causes of human microsporidiosis. Recent studies show that the microsporidian Encephalitozoon intestinalis binds glycosaminoglycans (GAGs) associated with the host cell surface, and that the divalent cations manganese (Mn2+) and magnesium (Mg2+) augment spore adherence to host cells by activating a constituent on the spore surface. These studies also illustrate a direct relationship between spore adherence and host cell infection; inhibition of spore adherence leads to reduced host cell infection while augmentation of spore adherence increases host cell infection. In light of recent studies, microsporidia spore adherence has become a promising target for the development of novel therapeutics to treat or even prevent human microsporidiosis. The goal of this study was to further characterize the molecular mechanisms governing spore adherence by identifying specific constituents on microsporidia spores that participate in spore adherence with host cells. A 40 kDa Encephalitozoon cuniculi host cell binding protein was discovered and identified as ECU01_0820, hereafter known as Encephalitozoon cuniculi microsporidia spore adherence protein (EcMsAP). The gene encoding EcMsAP has multiple heparin-binding motifs and an integrin-binding domain, which are characteristic of proteins that interact with constituents on the cell surface. Immuno-transmission electron microscopy reveals that native EcMsAP is located on the plasma membrane, endospore, exospore, and the anchoring disk of microsporidia spores. Recombinant EcMsAP and antibodies to recombinant EcMsAP both inhibit spore adherence and host cell infection. However, the deletion of heparin-binding motif #1 from the EcMsAP gene results in the loss of ability to inhibit spore adherence and infection. Host cell-binding assays reveal that recombinant EcMsAP binds Vero and CHO cell lines, but exhibits attenuated binding to glycan-deficient CHO cell lines. Finally, biomolecular interactions analysis provides direct evidence that EcMsAP is a glycan binding protein. This study not only identifies a potential microsporidial vaccine candidate, it further supports the assertion that microsporidia spore adherence is a critical step in the host cell infection process.

Objectives: The study aim was to analyse the kinetics of stem and transit cells in the crypts of jejunal mucosa infected with HIV and Microsporidia. Design: The size of villi, depth of crypts and proliferative activity of transit and stem cells in jejunal mucosa were measured using morphometric techniques. Methods: The surface area/volume ratio (S/V) of jejunal biopsies was estimated under light microscopy using a Weibel graticule. Crypt length was measured by counting enterocytes along the crypt side from the base to the villus junction, and the mean crypt length was calculated. The S/V and crypt lengths of the jejunal mucosa of 21 HIV and Microsporidia-infected test cases were compared with 14 control cases. The labelling index in relation to the crypt cell position of 10 of the test cases was analysed compared with 13 control cases. Results: Differences were found in the S/V and crypt length, and there was a negative correlation between S/V and crypt length in test and control cases combined. Cell labelling indices fell into low and high proliferation groups. There were significant differences in labelling indices between low proliferation test cases and controls, between high proliferation test cases and controls, and between high and low proliferation test cases. Conclusion: Villous atrophy induced by HIV and Microsporidia is attributed to crypt cell hyperplasia and the encroachment of crypt cells onto villi. These infections induce crypt hypertrophy by stimulating cell mitosis predominantly in transit cells but also in stem cells. Increased stem cell proliferation occurs only in high proliferation cases.

Nous étudions les interactions hôte/pathogène entre la drosophile et un parasite intracellulaire, la microsporidie Tubulinosema ratisbonensis.Les microsporidies dérivent du groupe des champignons possédant un génome réduit et pas de mitochondrie. Ces parasites dépendent fortement du métabolisme de leur hôte pour proliférer. Les interactions entre les microsporidies et leurs hôtes reste peu étudiées au niveau métabolique.In vivo, les spores ciblent beaucoup de tissues, en particulier le corps gras qui perd ces gouttelettes lipidiques. Les réserves métaboliques sont épuisées au cours de l’infection et les mouches présentent les caractéristiques de famine. La supplémentation en acide gras dans la nourriture des mouches infectées augmente la prolifération du parasite. Cet effet est bloqué lorsque l’on perturbe l’assimilation ainsi que le transport des lipides provenant de l’intestin. En conséquence, les mouches résistent mieux à l’infection lorsque la synthèse des lipides est bloquée. / We study the host/pathogen interactions between Drosophila and a natural intracellular parasite : the microsporidium Tubulinosema ratisbonensis. Microsporidia are highly derived group of fungi with a compact genome and no mitochondria. These obligate parasites thus rely intensively on host metabolism for growth and proliferation. The interactions between microsporidia and their hosts remain poorly understood at the metabolic level.In vivo, spores target many tissues especially the fat body that loses its lipid droplets. Metabolic reserves become depleted during the course of the infection and flies display the hallmarks of severe starvation. Fatty acids supplementation of the infected flies diet increases parasite proliferation and host susceptibility to the parasite. This effect is blocked when perturbing lipid assimilation and transport originating from the gut. Accordingly, flies resist better infection when lipid synthesis is blocked, as the parasite proliferation is hampered.

The aim of this thesis is to evaluate dogs introduced into the Arctic as a source of intestinal parasites infection both for wildlife and humans. To that aim coprological examination of faeces sampled in Svalbard and Greenland was performed. Microscopical and molecular detection of cryptosporidia, giardiae, microsporidia, roundworms and tapeworms in faeces was carried out. One sample from Svalbard was positive for Toxocara canis eggs and four samples from Svalbard were positive for unusual genotype of Enterocytozoon bieneusi.

Thelohania solenopsae are intracellular pathogens found in the red imported fire ant, Solenopsis invicta. These pathogens cause detrimental effects to their fire ant hosts. The present study revealed that the midgut and the meconium materials from pupating fourth instar larvae were possible vehicles for the horizontal transmission of the disease. The pathogen was further found to cause a reduction of humeral proteins. In SDS-PAGE stained with silver, several proteins were observed only in controls but not in infected fire ant queens. Different queens were found to have variable proteins reduced due to infection of this pathogen. Furthermore, vitellogenin titers were found to be significantly reduced in infected fire ant queens, although the infection rates of the fat body cells was found to be less than 20%. Finally, although the pathogens did not directly induce apoptosis in infected cells, there were more infected cells undergoing apoptosis than uninfected cells. There was no evidence to support the idea that infected fat body cells became more resistant to apoptosis inducers.

Thelohania solenopsae are intracellular pathogens found in the red imported fire ant, Solenopsis invicta. These pathogens cause detrimental effects to their fire ant hosts. The present study revealed that the midgut and the meconium materials from pupating fourth instar larvae were possible vehicles for the horizontal transmission of the disease. The pathogen was further found to cause a reduction of humeral proteins. In SDS-PAGE stained with silver, several proteins were observed only in controls but not in infected fire ant queens. Different queens were found to have variable proteins reduced due to infection of this pathogen. Furthermore, vitellogenin titers were found to be significantly reduced in infected fire ant queens, although the infection rates of the fat body cells was found to be less than 20%. Finally, although the pathogens did not directly induce apoptosis in infected cells, there were more infected cells undergoing apoptosis than uninfected cells. There was no evidence to support the idea that infected fat body cells became more resistant to apoptosis inducers.

Microsporidia are obligate intracellular pathogens of medical and ecological importance whose genomes have been studied extensively over the last decade. Their parasitic lifestyle has lead them to lose a great number of genes and, thus, biochemical pathways capacities, but these reductive processes have been often offset by the acquisition of several genes by means of horizontal gene transfer (HGT). First, in this thesis, we will describe the complete genomes of Encephalitozoon hellem and Encephalitozoon romaleae. Both species also were found to harbor a number of protein-coding genes absent in other microsporidia, which products assembled complete metabolic pathways. All these genes are functionally related to DNA and folate metabolism, and all appear to have been acquired from HGT events from different eukaryotic and prokaryotic donors. Interestingly in E. romaleae genes involved in de novo synthesis of folate are all pseudogenes, highlighting the transient nature of transferred genes. Secondly, we took a closer look at the ploidy and sexual status of Encephalitozoon cuniculi, a vertebrate pathogen, by mapping Illumina sequence reads against the genomes of four strains of this species. We identified the presence of low level of heterozygosity in all strains investigated; a feature that revealed the diploid nuclear state of the species. This reductive intra-individual genetic diversity could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce. Overall, the work presented in this thesis resulted in a much greater understanding of the genome evolution of a medically and economically important group of parasites.

Microsporidia are a unique phylum of ubiquitous fungal pathogens that are able to infect a wide variety of hosts, including economically and ecologically important organisms. Recently, global declines of the Western honeybee (Apis mellifera) have been associated with infections of the microsporidian pathogen Nosema ceranae. This species was originally described in the Asiatic honeybee (A. cerana), and its identification in global A. mellifera hives could result from a recent host transfer. Recent genome studies have found that global populations of this parasite from A. mellifera hives are polyploid and that humans may have fueled their global expansion. In this thesis, I investigate the genetic diversity of N. ceranae populations from within their native range (Thailand) and among different hosts (A. mellifera, A. cerana), putting them in context with other previously sequenced global populations. Using both PCR and genome-based methods, my findings reveal that Thai populations of N. ceranae exhibit interesting genetic differences from other global pathogen populations but also have some similarities. Thai N. ceranae populations share many single nucleotide polymorphisms (SNPs) with other global populations and appear to be clonal. However, in stark contrast with previous studies, these populations carry many SNPs not found in other global populations of this parasite, indicating that these populations have evolved in their current geographic location for some time. This genome analysis also indicates the potential presence of diploidy within Thai populations of N. ceranae and possible host-specific loss of heterozygosity. Overall, my findings begin to reveal interesting patterns of genetic diversity in N. ceranae populations that bring us one step closer to understanding the biology and genetics of this important honeybee pathogen.