Orientador: Odair Correa Bueno / Banca: Luiz Carlos Forti / Banca: Ana Paula Protti de Andrade Crusciol / Banca: Maria Santina de Castro Morini / Banca: Sulene Noriko Shima / Resumo: O presente trabalho teve como objetivo o estudo da evolução dos comportamentos de preparação do substrato, cuidado com a cria e cuidado com a rainha e alados em formigas cultivadoras de fungo. Estas formigas pertencem à tribo Attini, subfamília Myrmicinae, e ocorrem exclusivamente no continente americano. Esta tribo contém aproximadamente 230 espécies, porém pouco se conhece da biologia da maioria delas, graças ao fato de serem extremamente crípticas e de não apresentarem importância econômica, como as formigas cortadeiras, que são as mais estudadas. Foram utilizadas seis espécies, que representam bem os diversos níveis da filogenia da tribo: Acromyrmex disciger, Apterostigma pilosum, Mycetarotes parallelus, Myrmicocrypta sp., Trachymyrmex fuscus e Trachymyrmex sp. nov. Os comportamentos foram estudados em ninhos mantidos em laboratório, com o auxílio de micro-câmeras e um aparelho gravador de vídeo. Os comportamentos foram analisados, caracterizados e quantificados. Os resultados foram divididos em três capítulos de acordo com o tipo de comportamento. No capítulo sobre a evolução dos comportamentos de preparação do substrato foi observado que, basicamente, o processo evoluiu no sentido de aumentar a capacidade das operárias em decompor inicialmente o substrato. As espécies basais, A. pilosum, M. parallelus e Myrmicocrypta sp. apresentaram um processamento mais simples com um número menor de comportamentos e principalmente sem os comportamentos do tratamento químico, que é responsável pela fragmentação do substrato ao mesmo tempo em que ele é tratado com enzimas digestivas. As duas espécies do gênero Trachymyrmex apresentaram um processo mais complexo com grande participação do tratamento químico. Em A. disciger, que é uma cortadeira, há uma intensa especialização do sistema de castas para o aumento da eficiência... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work aims to study the evolution of the behaviors of substrate preparation, brood, queen and winged forms care in fungus growing ants. These ants are included in the tribe Attini, subfamily Myrmicinae, and occur exclusively in the american continent. This tribe contains approximately 230 species, but little is known about the biology of most of them, tanks to the fact that they show very cryptic habits and are not economically important, like the leaf cutting ants, which are the most studied. Six species were used in this work, which represent well all the levels of the phylogeny of the tribe: Acromyrmex disciger, Apterostigma pilosum, Mycetarotes parallelus, Myrmicocrypta sp., Trachymyrmex fuscus and Trachymyrmex sp. Nov. The behaviors were studied in laboratory nests, with a set of micro cameras and a video recording device. The behaviors were analyzed, characterized and quantified. The results were summarized and discussed in tree chapters, arranged by type of behavior. In the chapter about the evolution of the substrate preparation behaviors, basically it is assumed that this process evolved in order to develop the capacity of previously decompose the substrate by the workers. In the basal species, A. pilosum, M. parallelus and Myrmicocrypta sp. this process is very simple with a small number of behaviors and principally without the behaviors of chemical treatment, which are responsible for the fragmentation of the substrate as it is treated by with digestive enzymes. In the two species of the genus Trachymyrmex, the process became more complex and showed a greater participation of these behaviors. A. disciger, witch is a leaf cutting ant, showed an extensive specialization of physical castes in all the phases of the process which elevated it's efficiency. In the chapter about the evolution of the behaviors of brood care... (Complete abstract click electronic access below) / Doutor

Os pesticidas organofosforados são amplamente utilizados na agricultura, pois são muito eficazes no controle de pragas, promovendo um aumento na produtividade dos alimentos. Contudo, sua utilização indiscriminada provoca graves problemas ambientais e para a saúde humana, uma vez que são tóxicos também para as espécies que não são alvos e acumulam grandes quantidades de metabólitos tóxicos, como por exemplo, fenois. Os compostos fenólicos enquadram-se nos resíduos resultantes da degradação de compostos naturais e xenobióticos da atividade antrópica. Este trabalho teve por objetivo estudar as reações de conjugação de fase II em compostos fenólicos derivados da hidrólise de pesticidas organosfosforados (clorpirifós, metil paration e profenofós) e a biotransformação de outros fenois por enzimas provenientes de fungos de ambiente marinho. Primeiramente foi realizado um screening com os fungos de ambiente marinho Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, A. sydowii CBMAI 1241, Penicillium decaturense CBMAI 1234, P. raistrickii CBMAI 931, P. raistrickii CBMAI 1235 e Trichoderma sp. CBMAI 932 para avaliar a resistência destes microrganismos frente à toxicidade dos pesticidas organofosforados para posterior escolha da cepa mais resistente e melhor adaptada aos pesticidas testados nesse trabalho. O fungo selecionado para as reações em meio líquido de malte 2%, que melhor adaptou-se na presença dos pesticidas testados foi a cepa do fungo A. sydowii CBMAI 935. Foram realizadas curvas analíticas com o objetivo de estimar a extensão da biodegradação dos pesticidas clorpirifós, metil paration, profenofós e seus respectivos produtos de hidrólise, os fenois 3,5,6-tricloro-2-piridinol, 4-nitrofenol e 4-bromo-2-clorofenol, respectivamente. As reações de biotransformação em meio líquido de malte 2% foram avaliadas com 10, 20, 30 d de reação com concentração inicial dos pesticidas organofosforados de 50 mg.L-1. Todos os metabólitos encontrados nas reações de biotransformação dos pesticidas organofosforados com o fungo A. sydowii CBMAI 935 foram comparados com os seus padrões analíticos e sintéticos (metilação) com o objetivo de corroborar as reações de bioconjugação. Através deste estudo foi possível sugerir a presença de enzimas fosfotriesterases e enzimas metiltransferases provenientes do fungo A. sydowii CBMAI 935. Enzimas que promoveram a hidrólise e metilação dos pesticidas e compostos fenólicos testados nesse trabalho. Segundo a literatura, as reações de biotransforrmação e bioconjugação dos pesticidas orgafosforados, diminuem consideravelmente a toxicidade desses compostos recalcitrantes. / Organophosphate pesticides are widely used in agriculture, as they are very effective in pest control, promoting an increase in productivity of food. However, indiscriminate use causes serious problems environmental and for human health, since they are also toxic to non-target species and accumulate large amounts of toxic metabolites, such as phenols. Phenolic compounds are part of the waste resulting from the degradation of natural compounds and xenobiotics of human activity. This work aimed to study the phase II conjugation reactions in phenolic compounds derived from hydrolysis of pesticides organophosphates (chlorpyrifos, methyl parathion and profenofos) and the biotransformation of other phenols for enzymes from marine environment fungi. First was conducted a screening with the marine environment fungi. Aspergillus sydowii CBMAI 934, A. sydowii CBMAI 935, A. sydowii CBMAI 1241, Penicillium decaturense CBMAI 1234, P. raistrickii CBMAI 931, P. raistrickii CBMAI 1235 and Trichoderma sp. CBMAI 932 to evaluate the resistance of these microorganisms front the toxicity of organophosphate pesticides to later choose the most resistant strain and better adapted to pesticides tested in this work. The fungus selected to the reactions in liquid medium 2% malt, which best adapted in the presence of the pesticide tested was the fungal strain of A. sydowii CBMAI 935. Standard curves were performed in order to estimate the extent of biodegradation of pesticides chlorpyrifos, methyl parathion, profenofos and their hydrolysis products, phenols 3,5,6-trichloro-2-pyridinol, 4-nitrophenol and 4-bromo- 2-chlorophenol, respectively. The biotransformation reactions in liquid medium 2% malt were evaluated in 10, 20, 30 days reaction of with initial concentration of organophosphate pesticides of 50 mg.L-1. All metabolites found in the biotransformation reactions of organophosphate pesticides with the fungus A. sydowii CBMAI 935 were compared with their synthetic and analytical standards (methylation) in order to corroborate the bioconjugation reactions. Through this study was possible suggest the presence of enzymes phosphotriestesterases and methyltransferases from fungus A. sydowii CBMAI 935. Enzymes that promote hydrolysis and methylation of pesticides and phenolic compounds tested in this work. According to the literature, the reactions of biotransformation and biodegradation of organophosphate pesticides, greatly reduce the toxicity of recalcitrant compounds.

Some intracellular symbionts of insects confer host protection from a variety of bacterial, fungal and viral pathogens as well as from predators and parasitoids. Within the cryptic species complex of whiteflies known collectively as Bemisia tabaci is a cosmopolitan invasive agricultural pest, which is commonly infected with the symbiont Rickettsia sp. nr. bellii. Rickettsia swept rapidly through southwestern USA whitefly populations of the MEAM1 species and has been associated in a genotype-dependent manner with increased whitefly fitness and female biased sex ratios. Here we sought to determine whether Rickettsia in MEAM1 might have a defensive role against the general entomopathogenic fungus, Beauvaria bassiana. Nymphs from two lines of whitefly, each with Rickettsia positive (R+) and negative (R-) sublines were exposed to different doses of B. bassiana. The results provided evidence of protection by Rickettsia in one genetic line (MAC1) but not in the other (MAC2). In a third experiment, females of the four sublines were each outcrossed for two generations with males from an outbred whitefly culture, derived from the field within the year, and F2 nymphs from these four new sublines were exposed to the fungus. In this experiment, Rickettsia was protective in both MAC1-O and MAC2-O lines. Taken together, our results suggest the symbiont Rickettsia can confer protection against a generalist entomopathogenic fungus, B. bassiana, and that this protection is conditional on host genotype. To our knowledge, this is the first record of an insect symbiont conferring protection against a generalized and commercially available biological control agent. Bemisia tabaci MEAM1 is a global pest of warm temperate and tropical agriculture, and the prevalence of Rickettsia in many populations of this species could limit the predictability or efficacy of fungal pathogens as a potential management tool.

Endopolygalacturonases (PGs) are the first cell wall degrading enzymes that are produced when pathogenic fungi encounter the host cell wall (Albersheim and Anderson, 1971). The role that these enzymes play in pathogenicity has been investigated for numerous pathogenic fungi. Although the results are not conclusive, there is evidence for some fungi that these enzymes are significant for their pathogenecity. Furthermore, plants contain polygalacturonase inhibiting proteins (PGIPs) in their cell walls, which are able to inhibit PGs (De Lorenzo et al, 2001; 2002). Colletotrichum SHK2148 is a pathogenic fungus causing anthracnose of lupin plants in South Africa. The identity of the fungus has been described as Colletotrichum tortuosum (Koch, 1996). However, this was based on morphological evidence only. Thus, the classification of the South African lupin- associated Colletotrichum isolates was re-assessed by comparing Colletotrichum SHK2148 on a morphological and molecular level to the recently described Colletotrichum lupinispecies (Nirenberg et al, 2002) as well as previously described Colletotrichum acutatum lupin anthracnose isolates (Talhinas et al, 2002). Based on the culture morphology, ITS and <font face=”symbol”>b</font>â-tubulin sequence data, it was concluded that Colletotrichum SHK2148 groups with C. lupini, more specifically, C. lupini var. setosum. The fungus, renamed Colletotrichum lupini SHK2148, was evaluated for its PG activity in pectin media (pH 5) over a 12 day growth period by using an agarose diffusion assay. The specific PG activity reached its highest level after three days, whereafter it decreased. Previous studies performed at the ARC, revealed that the fungus produced PG activity and this crude activity was inhibited by a PGIP produced in apple. A study was launched to isolate and characterise the gene(s) responsible for PG production. PG gene sequences from Colletotrichum gloeosporioides f.sp. malvae and Colletotrichum lindemuthianum were compared and conserved regions were identified from which primers were designed to amplify a fragment of a PG gene from C. lupini SHK2148. Inverse PCR was used to resolve the 5’ and 3’ sequences of the PG gene whereafter a complete copy of the gene was isolated from the genome of the fungus and characterised. The isolated gene was approximately 1Kb, contained a single intron of 59 bp and was very similar to the PG gene from C. gloeosporioides f.sp. malvae (cmpgII) as well as one of the PG genes (clpg2) from C. lindemuthianum. Southern blot analyses revealed that the gene was present as a single copy in the genome of the fungus. The in vitro expression of the PG gene from C. lupini SHK2148, grown in pectin media (pH 5), was investigated via northern blot analyses as well as RT-PCR, which revealed that the gene was expressed in the same time period that the highest PG activity was observed. A full cDNA copy of the PG gene was isolated using mRNA harvested from mycelia that was grown for 4 days on pectin. The cDNA copy confirmed the predicted intron position of the previously isolated genomic PG gene. Due to the unavailability of a full cDNA copy of the C. lupini SHK2148 PG gene at the time when expression studies were initiated, a complete cDNA copy was constructed by swapping an internal cDNA PG fragment with its counterpart in the complete genomic PG gene copy. The resulting cDNA PG copy was used as a template from which PG constructs were prepared for expression in Pichia pastoris. Constructs containing the PG gene with its native signal peptide, the PG gene with the β-MF signal peptide factor as well as hybrid constructs where the N terminal part of the mature PG proteins of Fusarium moniliforme and C. lupini SHK 2148 were exchanged, were transformed into P. pastoris . No PG activity was observed with an agarose diffusion assay for any of the Pichia clones. SDS-PAGE analyses were used to evaluate total protein isolations from the P. pastoris clones. The supernatant and cells of the clones were subjected to western blot analyses using antibodies directed againstAspergillus niger PG as well as F. moniliforme PG. The only positive hybridisation signal was observed between the A. niger antibody and a protein in supernatant extracts of the P. pastoris clones. However, the size of the hybridising band was very large. This could be due to glycosylation of the C. lupini SHK 2148 PG in P. pastoris, although the size increase is unusually large. The results indicated that it is unlikely that the C. lupini SHK 2148 PG was expressed in P. pastoris transformed with any of these constructs. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / unrestricted

Asthma is a debilitating lung disease that affects nearly 300 million people worldwide. Environments with high humidity and subsequent mold exposure often trigger allergic asthma. Sex differences have been reported in the incidence, prevalence, and severity of asthma. B-lymphocytes are recruited in high numbers to the allergic lung in response to the inhalation of Aspergillus fumigatus mold spores (conidia). In this work, we used a mouse model of allergic fungal asthma to assess environmental humidity, sex, and B-lymphocytes in an inhalational model of allergic fungal asthma. Our results showed that animals sensitized in low humidity conditions had no airway hyperresponsiveness (AHR), inflammation, but an increase in IgG3 antibody production. Males weighed more than females, female mice had more fibrosis and produced more IgG3 Ab, but sex showed no impact on low humidity. C19+ B-lymphocytes differentially downregulated multiple genes related to allergic asthma returning the body to homeostasis.

In vitro studies indicate that the inflammatory response to zymosan, a fungal wall preparation, is dependent on Toll-like receptor (TLR) 2, and that this response is enhanced by the dectin-1 receptor. Complement may also play an important role in this inflammatory response. However, the relevance of these molecules within the in vivo pulmonary environment remains unknown. To examine pulmonary in vivo inflammatory responses of the lung to zymosan, zymosan was administered by intratracheal aerosolization to C57BL/6, TLR2- TLR4-, MyD88-, and complement-deficient mice. Outcomes included bronchoalveolar fluid cell counts. We next examined effects of dectin-1 inhibition on response to zymosan in alveolar macrophages in vitro and in lungs of C57BL/6, TLR2-, and complement-deficient mice. Finally, the effect of alveolar macrophage depletion on in vivo pulmonary responses was assessed. Marked zymosan-induced neutrophil responses were unaltered in TLR2-deficient mice despite a TLR2-dependent response seen with synthetic TLR2 agonists. TLR4, MyD88, and complement activation were not required for the inflammatory response to zymosan. Although dectin-1 receptor inhibition blocked the inflammatory response of alveolar macrophages to zymosan in vitro, in vivo pulmonary leukocyte recruitment was not altered even in the absence of TLR2 or complement. Depletion of alveolar macrophages did not affect the response to zymosan. Neither complement, macrophages, nor TLR2, TLR4, MyD88, and/or dectin-1 receptors were involved in the pulmonary in vivo inflammatory response to zymosan.

Hybridization refers to mating between species or between genetically differentiated populations of the same species. Although hybrid offspring may exhibit sterility and/or inviability, hybridization can generate novel genotypic and phenotypic diversities, leading to the origin of new traits and new species, the expansion into ecological niches outside of the parental range (e.g., host range), and altered virulence properties in pathogens. However, the relationship between parental genetic divergence and hybrid performance remains largely unknown. The human pathogenic Cryptococcus (HPC) is an ideal model to study the impacts of parental divergence on the genetic and phenotypic consequences of hybridization. HPC consists of a group of divergent lineages with various degrees of interfertility. These yeasts are the etiologic agents of cryptococcosis, a potentially lethal disease in humans and animals. In this thesis, I examined the effects of parental divergence on cryptococcal hybrids from multiple aspects. I conducted genetic crosses between different lineages to evaluate the mating success and the germination of sexual spores (i.e., basidiospores) under various environmental conditions. Then, I investigated the genotypic and phenotypic diversities among the hybrids under different environmental conditions. Furthermore, I examined the genome stability of diploid inter-lineage hybrids through laboratory experimental evolution and the effect of antifungal drug stress on the loss of heterozygosity (LOH) in these hybrids. We found that parental genetic divergence plays an important role in genotypic and phenotypic diversities among hybrid progeny in HPC. However, our results indicate that parental genetic di-vergence alone can’t explain most of the observed variations. Instead, genetic divergence along with specific parental strains, environmental factors, and their interactions all contributed to hybridization success and to hybrid genotypic and phenotypic variations. My findings will broaden the current understanding of the phenotypic and genotypic consequences of hybridization and explore the connection between genetic architecture and hybrid speciation in the human pathogenic Cryptococcus. / Thesis / Doctor of Science (PhD) / The role of hybridization in evolution can vary widely, giving rise to hybrid vigor and hybrid weakness. Hybridization plays an important role in plants and animals, especially crops, with advantages of increased yield and quality of products. However, the emergence of hybrid vigor in pathogens with increased virulence is an increasing threat to plant, animal, and human healths. My PhD thesis aimed at understanding the effects of parental divergence on hybridization and hybrids in the human pathogenic Cryptococcus. Here, I investigated basidiospore germination rate and hybrid progeny genotypes and phenotypes from diverse genetic crosses in this group of pathogens. My findings contribute towards understanding cryptococcal hybrids and establishing treatment plans against infections by these hybrids.

No description available.

No description available.

Recurrent Airway Obstruction (RAO)-affected horses experience bronchoconstriction and airway inflammation in response to inhalation of irritants including hay molds. Steaming hay reduces fungal content, but the effect on the antigenic potential has not been investigated. We tested the hypothesis that RAO-affected horses develop less severe clinical disease when fed steamed versus non-steamed hay and this reduction coincides with decreased hay fungal content. Six RAO-affected horses in clinical remission were divided in two groups and fed steamed or non-steamed hay for 10 days using a two-way cross-over design. Hay was steamed using a commercial hay-steamer. Clinical assessment was performed daily. Full assessment, including airway endoscopy, tracheal mucous scores and maximal change in pleural pressure, was performed on days 1, 5, and 10. Bronchial fluid sampling and cytology were performed on days 1 and 10. Hay core samples were collected pre- and post-steaming and cultured to determine fungal and bacterial concentrations. Statistical analysis was based on data distribution and quantity and performed using SAS®. P-value <0.05 was significant. Steaming significantly decreased the number of bacterial and fungal colony-forming-units in hay. Horses fed non-steamed hay experienced a significant increase in clinical score and a trend towards airway neutrophilia, while parameters were unchanged in horses fed steamed hay. Only horses fed non-steamed hay experienced a significant increase in tracheal mucous score. Horses fed steamed hay gained significantly more weight compared to horses fed non-steamed hay, even though the amount of hay consumed not greater on a dry matter basis. These results indicate that steaming reduces the RAO-affected horse's response to hay which coincides with a reduction in viable fungal content of hay. / Master of Science