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Biological and molecular characteristics of the pathogenic mollicute, Spiroplasma penaei, isolated from the hemolymph of infected Pacific white shrimp, Penaeus vannamei, were investigated. The doubling times of a S. penaei were 6.13 h (2% NaCl) and 3.43 h (no salt) under aerobic conditions, and 6.63 h (2% NaCl) and 3.22 h (no salt) under anaerobic conditions. Small diffuse white colonies with granular centers, surrounded by small satellite colonies that appeared embedded in the agar matrix, were detected on solid M1D medium (2% Noble agar) under aerobic conditions at 28°C. The genome size of the S. penaei was 1778 Kb, as determined by pulsed-field gel electrophoresis using undigested DNA. Reduction of virulence of S. penaei was not detected in serial passage 24 and 76 isolates but passage 131 isolate was attenuated as indicated by the number of surviving shrimp and histological findings of challenged P. vannamei. Toxicity was not detected in supernatant fractions of M1D medium cultures of S. penaei isolates. The most predominant host responses to the S. penaei reference isolate and to serial passage isolates were hemocytic nodules and hemocytic infiltration observed in hematoxylin and eosin-stained histological sections. Transmission electron microscopy of the lymphoid organ of experimentally infected P. vannamei depicted S. penaei without cell wall and free in the cytoplasm of lymphoid organ cells. The lesions observed in histological sections were verified by in situ hybridization using a digoxigenin (DIG)-labeled probe specific to the spiralin gene of Spiroplasma spp. Evolutionary relationship trees, based on five partial DNA sequences of 16S rDNA, 23S rDNA, 5S rDNA, gyrB, rpoB genes and two complete DNA sequences of 16S-23S and 23S-5S ISR, were constructed using the distance-based Neighboring-Joining method with Kimura-2-parameter substitution model. The NJ trees based on all DNA sequences investigated in this study positioned S. penaei in the Citri-Poulsonii clade and corroborates the observations by other investigators using the 16S gene. Pairwise genetic distance calculation between sequences of spiroplasmas showed S. penaei to be closely related to S. insolitum and distantly related to Spiroplasma sp. SHRIMP from China.

A família Rallidae é representada por aves cosmopolitas popularmente conhecidas como saracuras, sanãs, carquejas, galinhas-d`água e frangos-d`água. Compreende cerca de 135 espécies distribuídas em 33 gêneros, dos quais 13 são monotípicos. As relações filogenéticas baseadas em caracteres morfológicos e dados moleculares indicam diferentes afinidades entre os membros da família, principalmente na posição dos gêneros Rallus, Porphyrio, Gallinula e Fulica. Neste estudo, focado em espécies Neotropicais da família, uma nova análise filogenética baseada em caracteres osteológicos é proposta. Uma amostra de 100 esqueletos de 13 gêneros e 31 espécies foi analisada. No total 50 caracteres foram codificados, dos quais 17 são cranianos e 33 pós-cranianos para a construção de uma matriz e subseqüente análise filogenética de acordo com o princípio da parcimônia. Foram calculados árvores de consenso estrito e consenso de maioria. A primeira gerou 151 árvores igualmente parcimoniosas com 99 passos. A análise com método de pesagem sucessiva dos caracteres obteve melhores resoluções entre as espécies amostradas. A topologia do cladograma permite a validade de determinados gêneros como entidades monofiléticas, como Rallus, Porphyrio, Aramides, Gallinula e Fulica. O posicionamento de Porphyrio como um ramo basal dentro da subfamília Rallinae foi suportado e suas relações interespecíficas demonstram que as espécies do Novo mundo são mais proximamente relacionadas do que P. porphyrio, permitindo a inclusão taxonômica de Porphyrula. A relação próxima entre as espécies do gênero Gallinula e Fulica foi corroborada, no entanto, G. melanops é um ramo basal do clado que inclui as espécies de Fulica, indicando que uma mudança taxonômica é necessária. A relação entre as espécies de Rallus e Pardirallus é distante e não suporta a inclusão das espécies de Pardirallus em Rallus. As maiores discordâncias da filogenia proposta em comparação com estudos moleculares referem-se à posição interna dos membros de Porphyrio e suas relações com outros gêneros / The family Rallidae is represented by cosmopolitan birds commonly known as wood-rails, crakes, coots, gallinules and swamp hens. It comprises around 135 species, distributed in 33 genera, of which 13 are monotypics. The phylogenetic relationships based on morphological characters and molecular parameters indicate different affinities among family species, mainly the position of the genera Rallus, Porphyrio, Gallinula and Fulica. In this study, focused on the Neotropical species of the family, a new phylogenetic analysis based on osteological characters is proposed. A sample of 100 skeletons of 13 genera and 31 species was analyzed. A total of 50 characters was codified, of which 17 were cranial and 33 post-cranial to provide a matrix construction and a subsequent phylogenetic analysis according to the principle of parsimony. A strict consensus and a majority rule consensus tree were calculated. The former generated 151 equally parsimonious trees with 99 steps. The successive weighting approach analyses of characters obtained better resolutions around the sampled species. The cladogram topology allows the acceptance of some genera as valid monophyletic groups, such as Rallus, Porphyrio, Aramides, Gallinula and Fulica. The position of Porphyrio as a basal branch within the subfamily Rallinae was supported and the interspecific relationships show that New World species were more closely related than P. porphyrio, allowing the taxonomic inclusion of Porphyrula. The close relationship between the species of Gallinula and Fulica was corroborated, but G. melanops is a basal branch of a clade that includes Fulica species, indicating that a taxonomic change is needed. The relationship of Rallus and Pardirallus is distant and so does not support the inclusion of Pardirallus species in Rallus. The major discordances of the proposed phylogeny in comparison with molecular studies concern the internal position of the Porphyrio members and their relationships with other genera.

Foi realizado um estudo das relações filogenéticas de Stygarctidae e Digitopoda (Tardigrada, Archaeotardigrada, Macrocephala) seguindo-se os princípios e métodos henniguianos. Foram selecionados na literatura os caracteres morfológicos utilizados para os dois grupos: 61 caracteres (43 binários e 18 multiestado) para 17 táxons terminais, de Stygarctidae, ao nível de espécie, e 40 caracteres (31 binários e 9 multiestado) para 10 táxons superiores de Digitopoda. As análises manuais produziram cladogramas totalmente resolvidos com 101 passos evolutivos e índice de consistência 0,86 para Stygarctidae, e 79 passos evolutivos e índice de consistência 0,63 para Digitopoda. Também foram feitas análises numéricas, com o auxílio do programa Hennig 86, comparando-se os resultados destas com as análises manuais. O algoritmo utilizado foi o mhennig*. O consenso de duas árvores de Stygarctidae apresentou topologia idêntica à do cladograma supracitado, com 98 passos evolutivos, índice de consistência 0,77 e índice de retenção 0,82. No caso de Digitopoda, o consenso de duas árvores apresentou uma topologia radicalmente diferente do resultado manual, com 66 passos evolutivos, índice de consistência 0,68 e índice de retenção 0,63. Também foram analisados dois caracteres multiestado, cada um com duas condições apomórficas, para três táxons terminais (nível de espécie) de Orzeliscinae. Neste caso, o cladograma obtido apresentou quatro passos evolutivos e índice de consistência 1. Stygarctidae foi mantido como táxon monofilético, apresentando a seguinte topologia entre os seus gêneros: ((Parastygarctus + Stygarctus) + ((Mesostygarctus + Pseudostygarctus) + Megastygarctides)). Digitopoda foi subdividido em dois táxons monofiléticos, apresentando a seguinte topologia: (Neostygarctus + (Neostygarctus + Batillipes + (Batillipes + (Halechiniscinae + Orzeliscinae) + ((Halechiniscinae + Orzeliscinae) + Dipodarctus + (Dipodarctus + (Floractinae + Tanarctinae))))) + Renaudarctus + (Renaudarctus + (Euclavarctinae + Styraconyxinae))). Halechiniscidae não é monofilético e Halechiniscinae é um táxon mais restrito, que inclui apenas Halechiniscus e Chrysoarctus. Orzeliscinae é redefinido de forma a incluir Paradoxipus, grupo-irmão de Opydorscus + Orzeliscus. Styraconyxinae inclui também Archechiniscus. Archechiniscinae não é válido. Neostygarctidae, Renaudarctidae, Neoarctidae e Megastygarctidinae foram eliminados do sistema de Tardigrada / Relationships among the subgroups of Stygarctidae and Digitopoda (Tardigrada, Archaeotardigrada, Macrocephala) were invetigated according to hennigian principles and methods. Morphological characters were selected from the literature for these two groups: 61 characters (43 binary and 18 multistate) for 17 species of Stygarctidae and 40 characters (31 binary and 9 multistate) for 10 major groups of Digitopoda. Manual analysis produced fully resolved cladograms with 101 evolutionary steps and consistency index = 0,86 for Stygarctidae, and 79 evolutionary steps and consistency index = 0,63 for Digitopoda. Numerical analysis were done using the software Hennig 86, for comparison with manual analysis. The algoritm mhennig* was used. The consensus tree of two Stygarctidae trees showed identical topology with the manual cladogram, with 98 evolutionary steps, consistency index = 0,77 and retention index = 0,82. The consensus tree of two Digitopoda trees showed a complete different topology from the manual cladogram, with 66 evolutionary steps, consistency index = 0,68 and retention index = 0,63. There were also analysed two multistate characters, each one with two apomorphic conditions, for three Orzeliscinae species. In this case, another fully resolved cladogram with four evolutionary steps and consistency index = 1 was obtained. Stygarctidae was maintained as a monophyletic taxon, showing the following system for its genera: ((Parastygarctus + Stygarctus) + ((Mesostygarctus + Pseudostygarctus) + Megastygarctides)). Digitopoda, branched into two monophyleitc taxa, showed the following system: (Neostygarctus + (Neostygarctus + Batillipes + (Batillipes + (Halechiniscinae + Orzeliscinae) + ((Halechiniscinae + Orzeliscinae) + Dipodarctus + (Dipodarctus + (Floractinae + Tanarctinae))))) + Renaudarctus + (Renaudarctus + (Euclavarctinae + Styraconyxinae))). Halechiniscidae is not a monophyletic taxon and Halechiniscinae is a more inclusive taxon comprising only Halechiniscus e Chrysoarctus. Orzeliscinae includes Paradoxipus, the sistergroup of Opydorscus + Orzeliscus. Styraconyxinae is monophyletic with the inclusion of Archechiniscus. Archechiniscinae is not valid. Neostygarctidae, Renaudarctidae, Neoarctidae and Megastygarctidinae are not supported in the system of the Tardigrada

Reticuloendotheliosis virus (REV) is a retrovirus that causes a neoplastic disease in a wide range of avian hosts including chickens, turkeys, and ducks. In 1993, REV was detected in the endangered Attwater's prairie chicken (Tympanachus cupido attwateri), a subspecies of Tympanachus cupido. Subsequent infections of this prairie chicken have been identified at captive breeding facilities throughout Texas. The implications of these infections have severely hindered repopulation efforts by these facilities. This study focused on investigating REV infection of captive Attwater'€™s prairie chicken in order to better understand the disease affecting these endangered birds. The overall objective was to develop a means of eliminating this threat to the repopulation of the Attwater's prairie chicken. Several aspects of virus infection were investigated. Reagents capable of recognizing prairie chicken IgY and viral gag polypeptides were developed for use in assays for detection of antibody responses and titration of viral concentrations. Sequencing data of genomes collected from isolates of Texas prairie chickens and domestic chickens, as well as three REV prototype viruses, were compared to determine relationships among strains and identify the potential origin of the REV infecting Attwater'€™s prairie chicken. Additionally, a flow cytometry technique of segregating the lymphocyte population from peripheral blood mononuclear cells (PBMC) using a pan leukocyte monoclonal antibody was developed to more accurately measure changes within lymphocyte populations. This technique combined with intracellular labeling was used to deduce the target cells of REV infection. A nested polymerase chain reaction (PCR) test was developed for greater sensitivity in detecting infection in birds than the previous method of single amplification PCR. This greater sensitivity results in earlier identification of the virus in infected birds, which allows for earlier removal of infected birds to minimize transmission of the virus throughout the flock. The sensitivity of the nested PCR diagnostic test was determined in a dose response pathogenesis study, which was conducted on hybrid greater/Attwater's prairie chicken to observe the experimental development of disease in these birds. Finally, a vaccine was developed using plasmid DNA with REV encoded genes and tested on naturally infected prairie chickens to determine its efficacy in reducing viral load. Although no reduction in viral load was detected, the vaccine may be effective in providing prophylactic protection in future studies.

Phylogenetic analysis can produce easily tens of thousands of equally plausible evolutionary trees. Consensus trees and topological distance matrices are often used to summarize the evolutionary relationships among the trees of interest. However, current approaches are not designed to analyze very large tree collections. In this dissertation, we present two fast algorithms— HashCS and HashRF —for analyzing large collections of evolutionary trees based on a novel hash table data structure, which provides a convenient and fast approach to store and access the bipartition information collected from the tree collections. Our HashCS algorithm is a fast ( ) technique for constructing consensus trees, where is the number of taxa and is the number of trees. By reprocessing the bipartition information in our hash table, HashCS constructs strict and majority consensus trees. In addition to a consensus algorithm, we design a fast topological distance algorithm called HashRF to compute the × Robinson-Foulds distance matrix, which requires ( ^ 2) running time. A RF distance matrix provides plenty of data-mining opportunities to help researchers understand the evolutionary relationships contained in their collection of trees. We also introduce a series of extensions based on HashRF to provide researchers with more convenient set of tools for analyzing their trees. We provide extensive experimentation regarding the practical performance of our hash-based algorithms across a diverse collection of biological and artificial trees. Our results show that both algorithms easily outperform existing consensus and RF matrix implementations. For example, on our biological trees, HashCS and HashRF are 1.8 and 100 times faster than PAUP*, respectively. We show two real-world applications of our fast hashing algorithms: (i) comparing phylogenetic heuristic implementations, and (ii) clustering and visualizing trees. In our first application, we design novel methods to compare the PaupRat and Rec-I-DCM3, two popular phylogenetic heuristics that use the Maximum Parsimony criterion, and show that RF distances are more effective than parsimony scores at identifying heterogeneity within a collection of trees. In our second application, we empirically show how to determine the distinct clusters of trees within large tree collections. We use two different techniques to identify distinct tree groups. Both techniques show that partitioning the trees into distinct groups and summarizing each group separately is a better representation of the data. Additional benefits of our approach are better consensus trees as well as insightful information regarding the convergence behavior of phylogenetic heuristics. Our fast hash-based algorithms provide scientists with a very powerful tools for analyzing the relationships within their large phylogenetic tree collections in new and exciting ways. Our work has many opportunities for future work including detecting convergence and designing better heuristics. Furthermore, our hash tables have lots of potential future extensions. For example, we can also use our novel hashing structure to design algorithms for computing other distance metrics such as Nearest Neighbor Interchange (NNI), Subtree Pruning and Regrafting (SPR), and Tree Bisection and Reconnection (TBR) distances.

Purple nonsulfur bacteria are a group of extraordinary metabolic diverse bacteria. They can grow photoautotrophically, photoheterotrophically , chemoheterotrophically or chemoautotrophically. Under various conditions, they can enjoy exceptional flexibility within each of these modes of metabolism. Due to the special physical characteristics properties, they had attracted scientist¡¦s attention in resent years. These bacteria are widely distributed in nature such as lakes, water ponds, coastal lagoons or high concentration organic waste lagoons. Lotus Pond, located in northern Kaohsiung City, is a serious eutrophied artificial lake. Because of receiving sufficient light and having been polluted by significant amounts of soluble organic matter, the ecology of the lake is suitable for the growth of purple nonsulfur bacteria. In the study, the lake water and sediments by using a Winograsdsky column, we successfully isolated 16 strains bacteria from the Lotus Pond. We also amplified the 16S-rDNA fragments of these strains by PCR and sequenced these PCR products, then aligned these sequences with the data of GeneBank. We affirmed that the 16 isolated strains belong to purple nonsulfur bacteria. From phylogenetic analysis, these 16 strains belong to the following three groups of bacteria: Rhodopseudomonas palustris, Rubrivivax gelatinosus, and Rhodobacter sphaeroides. Characteristic studies of these strains, we found that all isolated strains are Gram negative bacteria and contain bacteriochlorophyll a. The strains that belong to R. palustris and R. sphaeroides group can use several different types of short chain organic acid as their carbon source and have denitrification ability. However, only the strains belong to R. palustris group are able to use the aromatic compound benzoate. From salt tolerant studies, we found the strains in R. sphaeroides group can grow well in 3% NaCl, and both R. palustris and R. gelatinosus group can only grow in 1% NaCl.

Tzukuan is an HBV-, HCV-, HDV- endemic township in southern Taiwan. Based on a mass screening on 2909 residents age of 45 years or more in 1997, the prevalence rates of HBsAg and anti-HCV were 12.8% and 41.6% respectively. Of HBsAg carriers, 15.3% were positive for anti-HDV. Tzukaun was divided into coastal area and inland area. The prevalence of anti-HCV of coastal area was two times higher than that of inland area (61.4% v.s. 29.1%) and genotype 1b and 2a are the main two subtypes. We wish to find the causes of discrepancy in these nearby areas by phylogenetic analysis. Stratified by the living areas, coastal or inland, 27 samples were picked up (ingroup). HCV sequence of NS5B region could be detected by RT-PCR then a nested PCR in eight males and ninteen females with mean age of 54.8 years old (range: 45-70). None of these 27 residents came from the same family. Another 10 HCV infected persons whose living townships also in southern Taiwan but other than Tzukuan were enrolled as local controls. From GenBank, 30 different HCV isolates were included. Phylogenic analysis unequivocally confirmed the simultaneous spread of two different HCV strains in this township clusters according to their subtypes were noted. A trend of the spreading from coastal to land area or an ultra-aggregation phynomenon which according to their living area, as we suspected, were not noted between Tzukuan¡¦s residents. In ingroup, the short genetic distance between the isolates of C hepatitis virus which came from different villages might be caused from the wide-spreading of HCV in this endemic area (the maximal and minimal genetic distance in 1b or 2a isolates are 0.0869 vs. 0.0098 and 0.0996 vs. 0.0334). Besides, according to the contacting history to foreigner by our aborigine tribes, from genebank, all isolates from different countries were included and three possible origins of HCV genotype 1b were noted in Tzukuan. All these findings might be caused from frequently HCV inflow in this endemic area and wide-spreading of HCV between different countries.

Reticuloendotheliosis virus (REV) is a retrovirus that causes a neoplastic disease in a wide range of avian hosts including chickens, turkeys, and ducks. In 1993, REV was detected in the endangered Attwater's prairie chicken (Tympanachus cupido attwateri), a subspecies of Tympanachus cupido. Subsequent infections of this prairie chicken have been identified at captive breeding facilities throughout Texas. The implications of these infections have severely hindered repopulation efforts by these facilities. This study focused on investigating REV infection of captive Attwater'€™s prairie chicken in order to better understand the disease affecting these endangered birds. The overall objective was to develop a means of eliminating this threat to the repopulation of the Attwater's prairie chicken. Several aspects of virus infection were investigated. Reagents capable of recognizing prairie chicken IgY and viral gag polypeptides were developed for use in assays for detection of antibody responses and titration of viral concentrations. Sequencing data of genomes collected from isolates of Texas prairie chickens and domestic chickens, as well as three REV prototype viruses, were compared to determine relationships among strains and identify the potential origin of the REV infecting Attwater'€™s prairie chicken. Additionally, a flow cytometry technique of segregating the lymphocyte population from peripheral blood mononuclear cells (PBMC) using a pan leukocyte monoclonal antibody was developed to more accurately measure changes within lymphocyte populations. This technique combined with intracellular labeling was used to deduce the target cells of REV infection. A nested polymerase chain reaction (PCR) test was developed for greater sensitivity in detecting infection in birds than the previous method of single amplification PCR. This greater sensitivity results in earlier identification of the virus in infected birds, which allows for earlier removal of infected birds to minimize transmission of the virus throughout the flock. The sensitivity of the nested PCR diagnostic test was determined in a dose response pathogenesis study, which was conducted on hybrid greater/Attwater's prairie chicken to observe the experimental development of disease in these birds. Finally, a vaccine was developed using plasmid DNA with REV encoded genes and tested on naturally infected prairie chickens to determine its efficacy in reducing viral load. Although no reduction in viral load was detected, the vaccine may be effective in providing prophylactic protection in future studies.

Novel photosynthetic protozoan phylum from caorals eas investigated using molecular biology tools to infer phylogenetic position. According to the data, isolates RM11-26 are also photosynthetic relatives of apicomplexan parasites representing an independent lineage from Chromera velia