Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-13T15:04:42Z No. of bitstreams: 2 DISSERTAÇÃO Marcos da Silveira Neto.pdf: 2631812 bytes, checksum: b75b5dd740260c8e5df8486b6a787c94 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-13T15:04:42Z (GMT). No. of bitstreams: 2 DISSERTAÇÃO Marcos da Silveira Neto.pdf: 2631812 bytes, checksum: b75b5dd740260c8e5df8486b6a787c94 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2014 / FACEPE / No Brasil as espécies D. flavipennella e D. saccharalis tem uma grande importância para o setor sucroalcooleiro por causar danos em cana-de-açúcar. Os estágios de ovo, larva, pupa e adulto são muito parecidos entre essas espécies e a identificação pode ser confusa. O Espaçador Interno Transcrito 2 (ITS2) do locus DNA ribossomal, que tem um papel importante no processamento do DNAr, vem sendo aplicado em estudos filogenéticos e populacionais por ter como característica divergência evolutiva. Este trabalho tem como objetivo compreender o grau de divergência evolutiva entre as espécies D. flavipennella e D. saccharalis com base no marcador ITS 2, analisar sua estrutura e características funcionais. O DNA foi extraído de amostras de ovo, lagarta e adulto. Foi realizada PCR e os amplicons foram purificados e sequenciados. As sequências foram analisadas com o programa MEGA 5.01. O tamanho do ITS 2 foi de 410 pb em D. flavipennella e 448 pb em D. saccharalis. O conteúdo de GC foi similar entre as duas espécies. As sequências apresentaram regiões conservadas intercaladas por regiões variáveis. Algumas regiões conservadas apresentavam motivos presentes também em espécies de diferentes grupos, provavelmente associados às estruturas participantes no processamento do RNA pós-transcricional. Três repetições microssatélites estavam presentes em D. saccharalis e ausentes em D. flavipennella, contribuindo para a diferença de tamanho entre as duas espécies. Apesar da divergência existente entre as sequências das espécies, existem regiões que são conservadas em níveis taxonômicos mais altos.

The molecular characterization of twenty six Theileria spp. isolates and one C. felis isolate were done on the small subunit ribosomal RNA (SSU rRNA) gene, the 5.8S gene, and the two internal transcribed spacer regions using gDNA. The SSU rRNA gene is increasingly accepted as a widely used marker for characterization, taxonomic classification, and phylogenetic analysis and this gene has been sequenced from a variety of different organisms, resulting in a large database for sequence comparisons (Chae et al. 1998; Chae et al., 1999 a,b,c; Stockham et al., 2000; Cossio-Bayugar et al., 2002; Gubbels et al., 2000). The genomic region consists of the internal transcribed spacer 1 (ITS 1), the 5.8S gene, and internal transcribed spacer 2 (ITS 2) (ITS 1-5.8S-ITS 2 gene region) and separates the SSU rRNA gene from the large subunit ribosomal RNA gene. The 5.8S rRNA gene is highly conserved in size and nucleotide sequence, is relatively constant in molecular weight, and has an average chain length of approximately 160 nucleotides and has proven useful in dividing subgenera of Gyrodactylus ((Nazar, 1984; Zietara et al., 2002). Pairwise comparisons were done between the clones of an individual isolate and among the clones of the different isolates. Phylogenetic trees were made from the resulting sequences. This study shows that different SSU rRNA genes may be associated with ITS 1-5.8S-ITS 2 gene regions of distinct sequence in the same isolate. This study also demonstrates that considerable ITS 1-5.8S-ITS 2 gene region sequence variation may exist within a species. This may be useful for subspeciation designation, or may simply reflect considerable variation within the population. This study shows that the ITS 1-5.8S-ITS 2 gene region may be a useful molecular marker for the taxonomy of Theileria spp.

Fascioliasis is a worldwide emerging snail-borne zoonotic trematodiasis with a great spreading capacity linked to animal and human movements, climate change, and anthropogenic modifications of freshwater environments. South America is the continent with more human endemic areas caused by Fasciola hepatica, mainly in high altitude areas of Andean regions. The Peruvian Cajamarca area presents the highest human prevalences reported, only lower than those in the Bolivian Altiplano. Sequencing of the complete rDNA ITS-2 allowed for the specific and haplotype classification of lymnaeid snails collected in seasonal field surveys along a transect including 2007–3473 m altitudes. The species Galba truncatula (one haplotype preferentially in higher altitudes) and Pseudosuccinea columella (one haplotype in an isolated population), and the non-transmitting species Lymnaea schirazensis (two haplotypes mainly in lower altitudes) were found. Climatic seasonality proved to influence G. truncatula populations in temporarily dried habitats, whereas L. schirazensis appeared to be more climatologically independent due to its extreme amphibious ecology. Along the southeastern transect from Cajamarca city, G. truncatula and L. schirazensis shared the same site in 7 localities (46.7% of the water collections studied). The detection of G. truncatula in 11 new foci (73.3%), predominantly in northern localities closer to the city, demonstrate that the Cajamarca transmission risk area is markedly wider than previously considered. Lymnaea schirazensis progressively increases its presence when moving away from the city. Results highlight the usefulness of lymnaeid surveys to assess borders of the endemic area and inner distribution of transmission foci. Similar lymnaeid surveys are still in need to be performed in the wide northern and western zones of the Cajamarca city. The coexistence of more than one lymnaeid transmitting species, together with a morphologically indistinguishable non-transmitting species and livestock movements inside the area, conform a complex scenario which poses difficulties for the needed One Health control intervention. / Ministerio de Economía y Competitividad / Revisión por pares

The aim of my study was to sum up the data about genetic variability of freshwater gastropods living in temporary ponds. My model species were moss bladder snail (Aplexa hypnorum) and the critically endangered ramshorn snail (Anisus vorticulus). Genetic variability was investigated by two mtDNA and two nuclear markers. The differences in genetic variation at the COI gene follow the pattern of different catchments. However, these findings do not agree with the patterns derived from genetic markers ITS1 and 16S. The reasons for the differences between the different markers are discussed in several ways involving few possible historical scenarios, but also possible errors of laboratory methods. In this study, there was only little success in amplification and the most likely reason is the inhibitory effect of snail mucus on DNA amplification. Another problem that occurred when this work was to obtain the sequence of the parasite (fluke) instead Anisus gene using gastropod specific primers. In addition to genetic and related methodological part of this work is the third part concerning the evaluation of environmental factors periodic pools in the presence and absence of the species studied. The fourth part sis focused on the phylogenetical analyses of the european and american Aplexa linneages. Powered by TCPDF...

This study devised a method to detect Ribeiroia ondatrae (class Trematoda) in water-bodies using environmental DNA (eDNA) collected from filtered water samples from selected ponds in the USA and Canada. Species-specific PCR primers were designed to target the Internal Transcribed Spacer-2 (ITS-2) region of the parasite’s genome. The qualitative PCR method was 70% (n=10) accurate in detecting R. ondatrae in ponds previously found to contain the parasite, while the qPCR method was 88.9% (n=9). To examine how the retinoic acid (RA) pathway gene expression may be perturbed during R. ondatrae infections, leading to limb development abnormalities in the wood frog (Lithobates sylvaticus). Multiple sequence alignments were used to design degenerate PCR primers to eight RA biosynthesis genes, but only two gene fragments were identified using this approach. Without effective primer sets it was not possible to measure changes in gene expression in infected frogs.

This study devised a method to detect Ribeiroia ondatrae (class Trematoda) in water-bodies using environmental DNA (eDNA) collected from filtered water samples from selected ponds in the USA and Canada. Species-specific PCR primers were designed to target the Internal Transcribed Spacer-2 (ITS-2) region of the parasite’s genome. The qualitative PCR method was 70% (n=10) accurate in detecting R. ondatrae in ponds previously found to contain the parasite, while the qPCR method was 88.9% (n=9). To examine how the retinoic acid (RA) pathway gene expression may be perturbed during R. ondatrae infections, leading to limb development abnormalities in the wood frog (Lithobates sylvaticus). Multiple sequence alignments were used to design degenerate PCR primers to eight RA biosynthesis genes, but only two gene fragments were identified using this approach. Without effective primer sets it was not possible to measure changes in gene expression in infected frogs.

The aim of this diploma thesis was to reveal the taxonomic position and phylogenetic relationships of the dominant planktonic algae in two acid metal-rich lakes (Hromnice Lake and Plešné Lake, Czech Republic) and to compare these isolates with other closely related strains with the focus on the tolerance to various toxic metals (Cr, Al, Cu, Mn, Zn, Hg). The phylogenetic analyses showed that both strains belong to species Coccomyxa simplex. It was the first evidence that specifically this species is capable to become the dominant phytoplankton alga in the extreme environment of acid lakes with increased supply of phosphorus. Based on 18S rDNA analysis, four independent phylogenetic lineages were revealed within the genus Coccomyxa with three of them containing isolates from acid freshwaters. Furthermore, new strains of the recently described species Coccomyxa polymorpha were found growing in various chemical solutions. The toxicity test revealed that Coccomyxa simplex strains isolated from Hromnice and Plešné lakes did not show any peculiar resistance to increased metal concentrations. A significantly strain-specific response was recorded in case of aluminum, however, it was not related to the concentration of this metal in the original habitat. The ability to thrive in extreme habitats is probably...