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The aim of this study was to develop a sensitive and specific method of detecting Toxoplasma gondii in the immunocompromised host which would reduce the need for other tests and would ensure the prompt initiation of the appropriate treatment, the effects of which could be monitored. Such a system would also be of benefit in theinvestigation of parasite/host interaction. Initial work investigated an antigen ELISA and the PCR using two different gene targets CB 1 and P30) to find the most sensitive system. The ELISA was insensitive but both PCR systems were capable of detecting parasite in blood, lymph and tissue samples from experimentally infected sheep. The B 1 PCR detected parasite earlier and over a significantly longer period than the P30 PCR, this greater sensitivity being due to the higher copy number of the B 1 gene. The PCR was applied to samples from patients with AIDS with the aim of finding an ideal sample for the diagnosis of infection. Parasite was detected in blood up to a month prior to clinical signs of infection, and therefore blood samples are ideal for monitoringpatients at risk of recrudescence of a chronic infection. This result indicates that recrudescence is not due to local reactivation, but is due to a more widespread parasitaemia. However, as parasitaemia was shown to be transient in cases of recrudescence, sampling time may be critical. Parasite was also detected in urine, biopsytissue and post mortem material, but was not detected in CSF.Dexamethasone was used to create a mouse model of recrudescence in the immunocompromised patient to further investigate interaction between the parasite and host. The PCR detected parasite in blood, brain and heart of chronically infected animals, however the detection rate was significantly higher in groups receiveing immunosuppressive therapy. Dexamethasone treatment mimicked the effects seen in the AIDS population where 30-35% of chronically infected individuals showed clinical signsof toxoplasmosis. However the PCR may also be detecting latent cysts in tissue samples, and blood samples were occasionally positive without clinical evidence of infection. This could be due to small amounts of parasite circulating intermittently, or to breakdownproducts from parasite degradation. There was therefore a need to differentiate between active and chronic infection, and this was carried out by developing a quantitative PCR based on competitive amplification. A novel Sma I restriction site was created within the P30 gene, and known amounts were co-amplified with samples. The amplified products were then digested with Sma I to differentiate between mutated and T. gondii DNA and the point at which product yield was equalled indicated the amount of original DNA present in the sample. The system was shown to work using human PM samples, and could be adapted to indicate a cut-off point where parasite DNA levels reveal active infection. In conclusion the B 1 PCR is the method of choice in detecting T. gondii in AIDS patients. Any patient in which active parasite is detected should be treated and closely monitored using the qPCR for any evidence of reactivation.

Host shifts -- where a pathogen jumps from one host species to another -- have been described as one of the main factors leading to emerging infectious diseases (EID). The harm that a pathogen causes to a host (virulence) varies following a host shift. Differences in susceptibilities among host species means that pathogens may be more likely to switch between certain groups of hosts. Factors that determine the variation in host susceptibility are still unknown, but one possible predictor is the host evolutionary history. Here, we examine how phylogenetically related hosts vary in susceptibility when dealing with infections of two viruses differing in pathogenicity. We infected 39 species of Drosophilidae with Drosophila A virus (DAV), a virus initially described as avirulent, and we measured host mortality (virulence) and virus replication (viral load). Then, we compared our results to previously collected data from the virulent Drosophila C virus (DCV) and we analysed the data of both viruses together. We found large variation in DAV virulence and viral load, with benign infections in some cases and high mortality in others. There was phylogenetic correlation in viral load, with species presenting similar viral load clustering together in the phylogeny. However, we did not find correlation for virulence, indicating that DAV virulence was not predictable based on viral load. Also, we did not find correlation between DAV and DCV results, indicating that variation in host susceptibility is not predictable by other pathogens infections. It is possible that hosts and parasites ecology or genetic traits may be also influencing susceptibility variation. These results suggest that although some traits are predicted by phylogeny, to determine the factors driving host susceptibility variation to different pathogens following host shifts is a very complex task / Trocas de hospedeiro -- quando um patógeno passa de uma espécie de hospedeiro para outra -- são descritas como um dos principais fatores causadores de doenças infecciosas emergentes (DIE). O dano que um patógeno causa a seu hospedeiro (virulência) varia quando trocas de hospedeiro ocorrem. Diferenças em susceptibilidade entre espécies de hospedeiros indicam que patógenos são mais propensos a realizar trocas entre determinados grupos de hospedeiros. Os fatores que determinam a variação na susceptibilidade do hospedeiro ainda são desconhecidos, porém um possível preditor é a história evolutiva do hospedeiro. Nesse estudo, nós examinamos como hospedeiros filogeneticamente relacionados variam em susceptibilidade ao lidar com duas infecções de vírus que variam em patogenicidade. Infectamos 39 espécies de Drosophilidae com o vírus A de Drosophila (DAV), inicialmente descrito como não virulento, e medimos mortalidade do hospedeiro (virulência) e replicação viral (carga). Em seguida, nós comparamos nossos resultados com informações do vírus C de Drosophila (DCV), um virulento, e analisamos os dados dos dois vírus conjuntamente. Encontramos uma grande variação nos dados de virulência e carga viral para DAV, com infecções benignas em alguns casos e alta mortalidade em outros. Encontramos correlação para carga viral, com espécies com cargas virais semelhantes aparecendo filogeneticamente próximas. Contudo, não há correlação filogenética para virulência, indicando que a virulência de DAV não pode ser predita com base na carga viral. Além disso, nós não encontramos correlação entre os resultados de DAV e DCV, indicando que a variação na susceptibilidade não pode ser predita por infecções de outros patógenos. É possível que fatores ecológicos e genéticos do hospedeiro e do parasita estejam influenciando a variação em susceptibilidade. Esses resultados sugerem que, apesar de alguns traços possam ser preditos pela filogenia, determinar os fatores causadores da variação na susceptibilidade a diferentes patógenos após trocas de hospedeiros é um trabalho extremamente complexo

Plant feeding insects comprise about 25% of all animal species on earth and play an important role in all ecosystems. Although we understand that their association with plants is a key-factor driving the diversification in this group, we still have large gaps in our knowledge of the underlying processes of this relationship. Female choice of host plant is an important event in the insect life-cycle, as it is a major determinant of the larval food plant. In this Thesis I studied different aspects of insect host plant choice and used butterflies from the family Nymphalidae as my study system. I found that butterflies have a well developed olfactory system and that they use odors when searching for food or host plants. However, the information obtained from the odor of host plants does not seem to be sufficient for the studied species to make a distinction between plants of different qualities. Interestingly, even when in full contact with the leaf they do not make optimal decisions. I show for example that a sub-optimal female choice may be mitigated by larval ability to cope with unfavorable situations. Moreover, species that utilize a broader set of host plants may not be very well adapted to all the hosts they use, but at the same time they may survive in areas where there is only a subset of the plants available. Lastly, differences in the evolution of life-history traits between species can account for differences in how each species realizes its lifestyle. Thus, by incorporating findings on mechanisms of host plant choice with the ecological and evolutionary context of a species, our ability to explain the dynamics of host plant choice and insect-plant interactions can be improved. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p>

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais / A agregação de parasitas em determinados indivíduos da população de hospedeiros está associada à exposição e à suscetibilidade diferenciais, que variam de acordo com o sexo, o tamanho corporal, a condição reprodutiva, o comportamento de uso do espaço (que influencia a probabilidade de capturas e recapturas em diferentes locais) e os fatores ambientais. Desse modo, o objetivo desse estudo foi avaliar a influência de tais fatores sobre a infecção de Eimeria spp. (endoparasita intestinal) em Gracilinanus agilis, um marsupial didelfídeo semélparo neotropical. O local de coletas foi um fragmento de cerrado sentido restrito na Estação Ecológica do Panga, MG, entre 2011 a 2015. Foi utilizado um método de capturamarcação- recaptura para os hospedeiros e avaliada a prevalência e a abundância do parasita, por meio de análises fecais laboratoriais. A abundância e a prevalência de Eimeria spp. foram associadas, principalmente, à condição reprodutiva dos indivíduos, com ambos os sexos apresentando maior prevalência e alta abundância parasitária durante o final do período reprodutivo, que coincide com a época chuvosa. Machos apresentaram mais parasitas do que as fêmeas durante a estação seca, época de início do período reprodutivo, no qual o acasalamento ocorre. Não foi comprovada a hipótese de que a maior abundância de parasitas nos machos seria decorrente de seu maior deslocamento espacial. No entanto, o número de recapturas de G. agilis correlacionou-se significativamente com a abundância de Eimeria spp. Esse padrão pode ser explicado pelo maior contato do marsupial com suas próprias fezes, as quais transmitem os parasitas, como também pelo aumento do estresse ocasionado pelo aprisionamento na armadilha, que afeta negativamente a eficiência do sistema imunológico. Assim, a presença e a abundância de Eimeria spp. ao longo do período reprodutivo em ambos os sexos, parece estar mais associada com a susceptibilidade do hospedeiro do que com sua exposição, visto que não houve relação entre mobilidade e infecção. Porém, as medidas de deslocamento nesse estudo devem ser avaliadas com cautela, dado o baixo número de recapturas utilizado para estimativa do uso do espaço em G. agilis. / The aggregation of parasites in certain individuals of the host population is associated with differential exposure and susceptibility, which vary according to sex, body size, reproductive condition, space use behavior (which influences the probability of catches and recaptures in different locations) and environmental factors. Thus, the objective of this study was to evaluate the influence of such factors on the infection of Eimeria spp. (intestinal endoparasite) in Gracilinanus agilis, a semelparous didelphid neotropical marsupial. The collection site was a restricted-sense cerrado fragment at the Panga Ecological Station, MG, between 2011 to 2015. A capture-mark-recapture method was used for the hosts identification and the prevalence and abundance of the parasite were evaluated by means of laboratory fecal analyzes. The abundance and prevalence of Eimeria spp. were associated mainly with the reproductive status of the individuals, with both sexes presenting higher prevalence and high parasitic abundance during the end of the reproductive period, which coincides with the rainy season. Males had more parasites than females during the dry season, when the breeding season begins, in which mating occurs. The hypotheses wasn’t confirmed that the highest abundance on males would be due to their greater spatial displacement. However, the number of recaptures of G. agilis was significantly correlated with the abundance of Eimeria spp. This pattern can be explained by the greater contact of the marsupial with its own feces, which transmit the parasites, as well as by the increased stress caused by the entrapment, that negatively influences the immunological system efficiency. Thus, the presence and abundance of Eimeria spp. during the reproductive period in both sexes seems to be due to behavioral differences that define distinctions in susceptibility to infection rather than host exposure, once we failed to find any correlation between host mobility and Eimeria infection. However, our estimates of G. agilis use of space shoud be viewed with caution, due to the low number of recaptures which could lead to underestimates, and consequently disrupts any existing relationship between these variables. / Dissertação (Mestrado)