La compréhension des processus microévolutifs ayant lieu dans la nature nécessite la quantification des principales forces sélectives s'exerçant sur les populations sauvages. Ces 10 dernières années, les études à long terme et l'écologie de la résurrection (qui fait revivre des stades en dormance) ont été les principales approches pour étudier l'évolution des traits d'histoire de vie sur plusieurs générations dans les populations sauvages. Mon travail consiste à comprendre comment des facteurs écologies simples (p. ex. la température) et des interactions interspécifiques ou intraspécifiques plus complexes (p. ex. les interactions antagonistes hôte-parasite ou mâle-femelle) façonnent le processus évolutif des populations sauvages. Dans cette optique, j'ai utilisé l'Artémia comme un organisme modèle, en combinant des études sur le terrain et en laboratoire. Premièrement, j'ai étudié l'évolution de la niche thermique avec une approche d'écologie de la résurrection, en utilisant une série temporelle d'œufs de dormance d'une population d' introduite à partir de marais salants de régions tempérées dans des marais salants tropicaux dans les années 80. Cette étude montre que la survie aux températures élevées (caractéristiques du nouvel environnement) a augmenté linéairement au cours du temps à partir de l'introduction, suggérant un taux d'adaptation constant sur plus de 100 générations. Deuxièmement, j'ai utilisé une approche similaire pour étudier l'adaptation entre males et femelles dans une autre population d'Artémia. Cette étude suggère que les conflits sexuels provoquent une dynamique de coévolution fluctuante dans la nature sur une échelle d'environ 100 générations. Troisièmement, j'ai étudié les impacts respectifs de différents parasites (une espèce de cestode et deux espèces de microsporidie) sur la compétition entre une espèce d'hôte autochtone asexuée et une espèce d'hôte invasive sexuée. Chacun des trois parasites étaient soit spécialiste d'une espèce ou de certains génotypes d'hôte. De plus, l'espèce de cestode dont l'effet castrateur chez l'hôte est bien connu infectait uniquement l'espèce autochtone, suggérant que ce parasite joue un rôle majeur dans la compétition entre les espèces d'hôte autochtones et invasives. Par ailleurs, les trois espèces de parasite semblaient manipuler le comportement d'agrégation de leur hôte, très probablement pour augmenter leur transmission à de nouveaux hôtes. Enfin, j'ai réalisé des études de génétiques des populations d'espèces asexuées diploïdes et polyploïdes d'Artemia et d'espèces sexuées asiatiques proches. Les espèces asexuées diploïdes produisent des mâles rares et il semblerait que ceux-ci permettent une faible fréquence de reproduction sexuée. De plus, l'hybridation d'espèces d'Artémia éloignée phylogénétiquement a donné naissance à au moins trois lignées polyploïdes indépendantes. / Getting a comprehensive understanding of microevolution in natural populations requires proper quantification of the important selective forces exerted on these populations. Over the last decade, long-term studies and resurrection-ecology (revival of resting stages) have been the main approaches to study life history trait evolution over many generations in the wild. My work aims at understanding how simple ecological factors (e.g. temperature) and complex interactions between and within species (host-parasite and male-females antagonistic interactions) shape evolutionary processes in natural populations. To this end, I used the brine shrimp Artemia as a model system and combined laboratory and field studies. First, I investigated thermal niche evolution with a resurrection ecology approach, using dormant-egg time series from an Artemia population introduced from temperate to tropical salterns in the mid-80's. This experiment shows that survival at the high temperatures typical of the new environment increased linearly through time after the introduction, suggesting a sustained rate of adaptation over more than 100 generations. Second, I used the same approach to study adaptation between sexes in another Artemia population. I found that sexual conflicts result in fluctuating male-female coevolutionary dynamics in natura, over a time scale of ~100 generations. Third, I studied the relative role of one cestode and two microsporidian parasites in mediating the competition between a native asexual host and an invasive bisexual host. I found that all three parasites were either host- or genotype-specific and that the castrating cestode parasite specifically infected the native species, suggesting that this parasite actually played a major role in the competition between native and invasive hosts. Interestingly, all three parasites manipulated the swarming behavior of their host, most likely to increase their transmission. Fourth, I performed population genetic studies of diploid and polyploid Artemia parthenogenetica and their Asian bisexual close relatives. Diploid asexual Artemia produce rare males and I found indication that these males allow some rare sex in this otherwise parthenogenetic species. In addition, hybridization between divergent Artemia species has led to the origin of at least three independent polyploid lineages.

Biological rhythms are thought to have evolved to enable organisms to organise their activities according to the Earth’s predictable cycles, but quantifying the fitness advantages of rhythms is challenging and data revealing their costs and benefits are scarce. More difficult still is explaining why parasites that exclusively live within the bodies of other organisms have biological rhythms. Rhythms exist in the development and traits of parasites, in host immune responses, and in disease susceptibility. This raises the possibility that timing matters for how hosts and parasites interact and, consequently, for the severity and transmission of diseases. Despite their obvious importance in other fields, circadian rhythms are a neglected aspect of ecology and evolutionary biology. The ambitions of this thesis are to integrate chronobiology, parasitology and evolutionary theory with mathematical models to obtain a greater understanding about how and suggest why malaria parasites have rhythms as well as the effect of infection on host rhythms. First, I identify how malaria parasites lose their developmental rhythms in culture, when they lack any potential time cues from the host. Next, I characterise parasite rhythms inside the mammalian host in terms of synchrony and timing and demonstrate there is genotype by environment interactions for characteristics of parasite rhythms. Then, I investigate the effect that parasite infection has on host rhythms and show there is variation between parasite genotypes in their effect on host locomotor activity and body temperature rhythms during infections. Finally, I explore which host rhythms may be driving parasite synchrony and timing and demonstrate the importance of peripheral host rhythms for the timing of malaria parasite developmental rhythms. The data presented here provides novel and important information on the role of rhythms during disease and opens up a new arena for studying host-parasite coevolution.

The trajectory of evolutionary adaptation can be influenced both by the interactions of organisms with their environments as well as by the biological characteristics of the organisms themselves. My dissertation research uses the New Zealand freshwater snail Potamopyrgus antipodarum to 1) gain important insight into how coevolutionary interactions between hosts and parasites influence patterns of gene expression and genetic differentiation of hosts and, 2) evaluate how reproductive mode, and ploidy level affect patterns of adaptive molecular evolution. Coevolutionary interactions between hosts and parasites are a primary source of strong natural selection that can lead to rapid evolutionary change. Here, I used evaluation of patterns of gene expression and genetic differentiation to take critical steps towards characterizing the genomic basis of coevolutionary interactions between P. antipodarum and Microphallus livelyi. I found that M. livelyi-infected P. antipodarum exhibit systematic downregulation of genes relative to uninfected P. antipodarum. The specific genes involved in response to parasites differ markedly across lakes, consistent with population-specific host-parasite interactions leading to population-specific evolutionary trajectories. I also identified a set of rapidly evolving loci that represent promising candidates for targets of parasite-mediated selection across lakes as well as within each lake population. These results constitute the first genomic evidence for population-specific responses to coevolving infection in the P. antipodarum-M. livelyi interaction and provide new insights into the genomic basis of coevolutionary interactions in nature. I also generated and characterized the first transcriptomic resources for Microphallus parasites collected from two species of Potamopyrgus snails (P. antipodarum and P. estuarinus). These data both revealed that these parasites appear to represent distinct genetic lineages, which is interesting in light of the tight coevolutionary interactions between P. antipodarum and M. livelyi, and lay the groundwork for future research. Polyploidy has the potential to facilitate adaptive evolution by providing redundant genome copies that are free to evolve new functions. By contrast, asexuality, with which polyploidy is often associated, is expected to restrict adaptive evolution by decreasing the efficacy of natural selection and access to new genetic variation. I evaluated whether and how ploidy level and reproductive mode influence patterns of adaptive molecular evolution in P. antipodarum to assess 1) the potential evolutionary genomic benefits of recent polyploidy, and 2) how patterns of adaptive molecular evolution in asexuals are influenced by polyploidy. I compared patterns of positive selection in 60 genes across 27 P. antipodarum lineages (10 diploid sexuals, 12 triploid asexuals, 5 tetraploid asexuals) and a diploid sexual outgroup, Potamopyrgus estuarinus. I found little evidence that ploidy level and/or reproductive mode influence patterns of positive selection in P. antipodarum. Even so, this study provides initial steps in evaluating whether ploidy level and reproductive mode influence patterns of adaptive molecular evolution. Taken together, my dissertation work contributes new insights to the field of host-parasite coevolutionary interactions and will inform future studies into how ploidy level and reproductive mode influence patterns of adaptive molecular evolution.

Social parasitism, the exploitation of a society by other social organisms, has evolved independently numerous times within social animals. In this thesis, I integrate behavioral, evolutionary and chemical analyses to elucidate the evolution of social parasitism in Megalomyrmex ants. I examine host-parasite interactions in two Megalomyrmex species, identify venom alkaloids, and reconstruct the phylogenetic relationships between species. In Chapter 1, I analyze nest architecture and behavioral interactions between the ant host Cyphomyrmex cornutus and its parasite Megalomyrmex mondabora. This is the first detailed account of the natural history of this host and its social parasite. In Chapter 2, I report a one-year-long fitness experiment that tests whether Trachymyrmex cf. zeteki colonies suffer reduced fitness from an association with the social parasite Megalomyrmex symmetochus. I show that M. symmetochus parasites negatively impact host fitness though several mechanisms, including a) manipulation of the host worker grooming behavior; b) castration of host queens produced by the host colony, which then become workers; and c) reduction of garden size, host worker number, and host reproductive output. In Chapter 3, I determine that five venom alkaloids of Megalomyrmex are taxonomically informative to help differentiate cryptic species within the M. mondabora complex; new species in this complex need to be described in a future taxonomic revision. In Chapter 4, I reconstruct phylogenetic relationships of the genus Megalomyrmex with DNA sequence information. I conclude that the genus is monophyletic and corroborate two of the four species groups proposed by Brandão (1990) in a previous morphological revision. I also find evidence in support of Darwin’s Predation Hypothesis on the origin of social parasitism, which postulates that socialparasitic behaviors evolve from predatory behaviors. Lastly, I discuss promising future research directions on the evolution of social parasitism in the ant genus Megalomyrmex, which could serve as a model for the study of social parasitism in other lineages of social insects. / text

Few empirical studies examine the influence that the maternal parasite environment can have on offspring fitness (maternal effects) in invertebrates. Several recent studies have found that mothers can adjust offspring phenotype to counter the negative effects of parasite infection. In this thesis I subjected the parental generation of the host species, Tenebrio molitor (Insecta: Coleoptera), to a high parasite, high density, or control treatment. Offspring were subsequently subjected to either the same stress, the alternate stress, or to the control, and fitness-related life history traits were measured in both generations. The results from this thesis do not support the hypothesis that T. molitor mothers influence offspring fitness in a positive way. Instead, maternal effects led to a reduction in offspring fitness under both types of stress. At least under some environmental conditions, females invest in their fitness at the expense of their offspring.

Few empirical studies examine the influence that the maternal parasite environment can have on offspring fitness (maternal effects) in invertebrates. Several recent studies have found that mothers can adjust offspring phenotype to counter the negative effects of parasite infection. In this thesis I subjected the parental generation of the host species, Tenebrio molitor (Insecta: Coleoptera), to a high parasite, high density, or control treatment. Offspring were subsequently subjected to either the same stress, the alternate stress, or to the control, and fitness-related life history traits were measured in both generations. The results from this thesis do not support the hypothesis that T. molitor mothers influence offspring fitness in a positive way. Instead, maternal effects led to a reduction in offspring fitness under both types of stress. At least under some environmental conditions, females invest in their fitness at the expense of their offspring.

Thesis (M.Sc.). / Written for the Dept. of Microbiology and Immunology. Title from title page of PDF (viewed 2008/05/28). Includes bibliographical references.

Made available in DSpace on 2015-07-22T20:49:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-03-25 / As membranas biológicas são formadas por uma mistura de várias classes de lipídeos, cujo empacotamento preferencial entre esfingolipídeos e esteróis formam junto com proteínas específicas, esta complexa organização conhecida como microdomínios de membrana. Com o intuito de verificar a presença de microdomínios de membrana em leveduras de fungos patogênicos como Paracoccidioides brasiliensis e Histoplasma capsulatum, as leveduras desses dois fungos foram lisadas com pérolas de vidro e incubadas com Brij 98 a 4°C. As frações de microdomínios de membrana foram separadas por ultracentrifugação em gradiente de sacarose e, seus componentes analisados por HPTLC, SDS-PAGE e “Western blotting”. As análises dos lipídeos de membrana mostram que aproximadamente 40% do ergosterol das duas espécies de leveduras analisadas estão presentes nos microdomínios de membrana. Enquanto as porcentagens de glicoesfingolipídeos presentes nessas frações correspondem a 42% e 25%, em leveduras de P. brasiliensis e H. capsulatum, respectivamente. Em conjunto com as análises lipídicas, verificou-se nas duas espécies, um enriquecimento protéico nas frações de microdomínios de membrana, entre estas proteínas podemos citar a Pma1p, uma proteína marcadora de microdomínios de fungos, e uma proteína de 30 kDa, capaz de se ligar a laminina. Para determinar a importância do ergosterol na manutenção da integridade dos microdomínios de membrana utilizou-se metil-beta-ciclodextrina (mβCD), que é capaz de complexar e retirar o ergosterol. Após o tratamento das leveduras com mβCD verificou-se a extração dos esteróis numa proporção de 80% e 70% para P. brasiliensis e H. capsulatum, respectivamente. No entanto, o perfil de distribuição dos glicoesfingolipídeos e fosfolipídeos, analisados por HPTLC, não apresentou mudanças significativas após o tratamento com a mβCD. As análises protéicas demonstraram o deslocamento de algumas proteínas para frações solúveis do gradiente de sacarose como Pma1p e uma proteína de 30 kDa. Por outro lado, a marcação com o anticorpo anti-α5-integrina mostra a presença de uma proteína de 50 kDa nos microdomínios de membrana, mesmo após o tratamento com a mβCD, sugerindo a existência de uma população de microdomínios de membrana que não depende do ergosterol para manutenção de sua integridade. A importância desses microdomínios de membrana foi testada na infectividade de macrófagos alveolares, onde uma redução de 53% na infectividade de macrófagos foi verificada após o tratamento das leveduras de H. capsulatum com mβCD. Os resultados apresentados nessa tese demonstram a existência de microdomínios de membrana em leveduras de P. brasiliensis e H. capsulatum, bem como sua importância para a interação levedura-macrófago. / Biological membranes are constituted by a mixture of several classes of lipids. In this enviroment, sphingolipids and sterols pack tightly to form together with specific proteins a complex membrane organization known as membrane microdomains. In order to detect the presence of membrane microdomains in yeast forms of pathogenic fungi, such as Paracoccidioides brasiliensis and Histoplasma capsulatum, yeast forms of both fungi were lysed by vortexing with glass beads and then incubated with Brij 98 at 4ºC. Fractions containing membrane microdomains were isolated by ultracentrifugation on sucrose gradient, and their components were analyzed by HPTLC, SDSPAGE and Western blotting. Analysis of membrane lipids showed that about 40% of ergosterol from both P. brasiliensis and H. capsulatum was present in the membrane microdomains, whereas the percentage of glycosphingolipids present in P. brasiliensis and H. capsulatum was 42% and 25%, respectively. Analysis by SDS-PAGE and Western blotting clearly showed a protein enrichment in the membrane microdomains fraction of both fungi. It is noteworthy the presence of Pma1p, a fungal microdomain marker, and also the presence of a 30 kDa (glyco)protein which binds to laminin. To investigate the requirement of ergosterol to mantain the integrity of membrane microdomains, it was used methylbeta- cyclodextrin (mβCD) an agent able to efficiently extract membrane sterols. After treatment of yeasts using mβCD, it was observed the removal of 80% and 70% of ergosterol in P. brasiliensis and H. capsulatum, respectively. After treatment with mβCD it was observed a shift of 25% of the glycosphingolipids from the insoluble to the soluble fraction, conversely the distribution profile of phospholipids remained unmodified after treatment with mβCD. The protein analysis showed the displacement of a few proteins to soluble fractions of the sucrose gradient, such as Pma1p and the (glyco)protein of 30 kDa. On the other hand, using an anti-α5-integrin antibody it was detected, even after the mβCD treatment, the presence of a 50 kDa protein in membrane microdomains, suggesting the existence of microdomains that do not depend on ergosterol for their integrity. These data strongly suggest the existence of two population of microdomains: i) dependent of ergosterol for integrity maintenance of microdomains and ii) microdomains non-dependent of ergosterol for the maintenance of their functional role. Furthermore, the biological importance of membrane microdomains was clearly demonstrated by a 53% reduction of infectivity of alveolar macrophage infectivity when yeast forms of H. capsulatum were treated with mβCD. / TEDE / BV UNIFESP: Teses e dissertações

Toxoplasma gondii é um parasito do filo Apicomplexa que infecta uma grande variedade de hospedeiros, incluindo os humanos. O parasito invade a célula hospedeira por penetração ativa, com a participação das proteínas de suas organelas secretoras durante esse processo. Até o momento, somente um número limitado de proteínas secretoras tem sido descoberto, além disso, as moléculas efetoras envolvidas na invasão e sobrevivência do parasito não estão completamente compreendidas. A osteopontina (OPN) é uma glicofosfoproteína adesiva secretada, multifuncional, que contém o domínio arginina-glicina-ácido aspártico (RGD) de ligação à integrina, que está envolvida em uma variedade de eventos fisiológicos e patológicos, incluindo sinalização e sobrevivência celular. Pela primeira vez, nós demonstramos pelas técnicas de imunofluorescência e imunocitoquímica ultraestrutural que há uma intensa marcação para uma proteína OPN-like nos grânulos densos de taquizoítos de T. gondii extracelulares. O western blotting e o RT-PRC confirmaram a expressão de OPN-like nos taquizoítos. Nossos resultados também mostram que após a invasão dos macrófagos, a proteína OPN-like está localizada na membrana do vacúolo parasitóforo. Esses dados sugerem que os grânulos densos secretam uma proteína OPN-like, e nós podemos especular que essa proteína participa durante o processo de interação do parasito com as células hospedeiras. . / Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional secreted adhesive glycophosphoprotein containing the arginine-glycine-aspartic acid (RGD) integrin-binding domain, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated by immunofluorescence and immunoelectron microscopy approaches that there is an intense labeling for an OPN-like protein in the dense granules of extracellular T. gondii tachyzoites. Western blotting and RTPCR confirmed this protein expression in tachyzoites. Our results also showed that after macrophage invasion the OPN-like protein is localized at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we can speculate that this protein participates during the parasite interaction process with host cells.

Toxoplasma gondii é um parasito do filo Apicomplexa que infecta uma grande variedade de hospedeiros, incluindo os humanos. O parasito invade a célula hospedeira por penetração ativa, com a participação das proteínas de suas organelas secretoras durante esse processo. Até o momento, somente um número limitado de proteínas secretoras tem sido descoberto, além disso, as moléculas efetoras envolvidas na invasão e sobrevivência do parasito não estão completamente compreendidas. A osteopontina (OPN) é uma glicofosfoproteína adesiva secretada, multifuncional, que contém o domínio arginina-glicina-ácido aspártico (RGD) de ligação à integrina, que está envolvida em uma variedade de eventos fisiológicos e patológicos, incluindo sinalização e sobrevivência celular. Pela primeira vez, nós demonstramos pelas técnicas de imunofluorescência e imunocitoquímica ultraestrutural que há uma intensa marcação para uma proteína OPN-like nos grânulos densos de taquizoítos de T. gondii extracelulares. O western blotting e o RT-PRC confirmaram a expressão de OPN-like nos taquizoítos. Nossos resultados também mostram que após a invasão dos macrófagos, a proteína OPN-like está localizada na membrana do vacúolo parasitóforo. Esses dados sugerem que os grânulos densos secretam uma proteína OPN-like, e nós podemos especular que essa proteína participa durante o processo de interação do parasito com as células hospedeiras. . / Toxoplasma gondii is an apicomplexan parasite infecting a broad host range, including humans. The parasite invades host cell by active penetration with the participation of its secretory organelles proteins during this process. Until now, only a limited number of secretory proteins have been discovered, and the effectors molecules involved in parasite invasion and survival are not well understood. Osteopontin (OPN) is a multifunctional secreted adhesive glycophosphoprotein containing the arginine-glycine-aspartic acid (RGD) integrin-binding domain, which is involved in various physiological and pathological events including cell signaling and survival. For the first time we demonstrated by immunofluorescence and immunoelectron microscopy approaches that there is an intense labeling for an OPN-like protein in the dense granules of extracellular T. gondii tachyzoites. Western blotting and RTPCR confirmed this protein expression in tachyzoites. Our results also showed that after macrophage invasion the OPN-like protein is localized at the parasitophorous vacuole membrane. These data suggest that dense granules secrete an OPN-like protein, and we can speculate that this protein participates during the parasite interaction process with host cells.