Phylogeny of snake trypanosomes inferred by SSU rDNA sequences, their possible transmission by phlebotomines, and taxonomic appraisal by molecular, cross-infection and morphological analysis

Blood examination by microhaematocrit and haemoculture of 459 snakes belonging to 37 species revealed 24% trypanosome prevalence in species of Viperidae (Crotalus durissus and Bothrops jararaca) and Colubridae (Pseudoboa nigra). Trypanosome cultures from C. durissus and P. nigra were behaviourally and morphologically indistinguishable. In addition, the growth and morphological features of a trypanosome from the sand fly Viannaniyia tuberculata were similar to those of snake isolates. Cross-infection experiments revealed a lack of host restriction, as snakes of 3 species were infected with the trypanosome from C. durissus. Phylogeny based on ribosomal sequences revealed that snake trypanosomes clustered together with the sand fly trypanosome, forming a new phylogenetic lineage within Trypanosoma closest to a clade of lizard trypanosomes transmitted by sand flies dagger. The clade of trypanosomes from snakes and lizards suggests an association between the evolutionary histories of these trypanosomes and their squamate hosts. Moreover, data strongly indicated that these trypanosomes are transmitted by sand flies. The flaws of the current taxonomy of snake trypanosomes are discussed, and the need for molecular parameters to be adopted is emphasized. To our knowledge, this is the first molecular phylogenetic study of snake trypanosomes.

Discriminação de ambientes aquáticos poluídos e não poluídos através da amplificação e clivagem do rDNA 16S

O crescimento desordenado da população e a expansão mal planejada das cidades, tem levado a precariedade das condições de saneamento e abastecimento hídrico, tornando a água um importante meio de desenvolvimento e transporte de microrganismos, muitos destes patogênicos. Desta maneira, torna-se necessário além da busca de soluções, o monitoramento eficiente e constante dos níveis e focos de contaminação. O presente trabalho teve como objetivo principal diferenciar ambientes aquáticos poluídos de não poluídos por contaminação de origem fecal, através de padrões moleculares. Para tanto foram escolhidos os arroios Feijó e Carvão, localizados na região metropolitana de Porto Alegre, Rio Grande do Sul, de onde foram coletadas amostras de água no verão e no inverno de 2001, submetidas a determinação do número mais provável (NMP) de coliformes totais e fecais, através da fermentação em tubos múltiplos. No Arroio Feijó foram observados índices de coliformes fecais superiores ao arroio Carvão, portanto o primeiro foi considerado modelo de ambiente aquático poluído e o segundo, não poluído. Seguiu-se a extração do DNA total das amostras brutas, amplificação do rDNA 16S pela PCR e clivagem destes com as enzimas de restrição Rsa I, Taq I e Alu I. Os padrões de clivagens observados com Rsa I sugerem a discriminação destes ambientes, uma variação sazonal foi observada entre as amostras de verão e inverno. O método mostrou-se sensível para uma análise comparativa entre estruturas de comunidades bacterianas em ambientes aquáticos.

Caracterização de fungos micorrízicos arbusculares autóctones de parreirais da Serra Gaúcha e otimização de métodos de multiplicação em espécies aromáticas para aplicação na fruticultura

Este estudo teve por objetivos realizar a caracterização morfológica e molecular dos fungos micorrízicos arbusculares (FMA) autóctones de parreirais da Serra Gaúcha; a otimização do método de produção de inóculos de FMA em plantas aromáticas; além de verificar a eficiência destes inóculos em porta-enxertos de plantas frutíferas. Coletouse solo rizosférico e raízes secundárias de videira em vinte parreirais distribuídos em cinco cidades da Serra Gaúcha (Bento Gonçalves, Caxias, Garibaldi, Nova Pádua e Farroupilha), amostrando-se quatro parreirais por município. A identificação morfológica dos esporos presentes nas amostras foi realizada através de microscopia óptica. A caracterizarão molecular foi realizada por PCR-TTG e seqüenciamento da região rDNA 18S dos esporos previamente identificados pela microscopia. Para a PCR foi utilizado DNA oriundo dos esporos isolados e também de macerado de raízes. Pelo método morfológico, identificaram-se 33 espécies distribuídas em 8 gêneros distintos de FMA. Obtiveram-se quatro perfis moleculares por PCR -TTGE do rDNA 18S de raízes e cinco perfis moleculares por PCR -TTGE do rDNA 18S de esporos das espécies. Através do alinhamento de seqüências obtidas da região de rDNA 18S com as seqüências depositadas no banco de dados NCBI foi possível identificar 7 espécies de FMA. Foram testadas três espécies de plantas aromáticas, hortelã pimenta (Mentha piperita L.), orégano (Origanum vulgare L.) e melissa (Melissa officinalis L.) como multiplicadoras de três espécies de FMA (Glomus clarum Nicol. & Schenck, Glomus etunicatum Becker & Gerd. e Acaulospora sp.) em dois volumes de recipiente (bandeja de isopor com alvéolo de 40 ml e bandeja de isopor com alvéolo de 100 ml). Verificouse a eficiência das plantas aromáticas para produzirem os inóculos destas três espécies de FMA, na colonização do sistema radicular e no desenvolvimento vegetativo de portaenxertos de videira (cv. SO4), citros (cv. Citrange Troyer) e, como dados complementares, em pessegueiro (cv. Okinawa). As plantas aromáticas estudadas multiplicaram com sucesso as espécies de FMA, sendo o inóculo gerado pelas mesmas eficiente em colonizar os porta-enxertos Citrange Troyer, SO4 e Okinawa, propiciando, inclusive, melhor desenvolvimento vegetativo aos dois últimos.

Parâmetros citogenéticos de espécies comerciais de carangidae (perciformes), com vistas a sua empregabilidade na conservação biológica e piscicultura marinha

Worldwide, families Carangidae and Rachycentridae represent one of the groups most important commercial fish, used for food, and great potential for marine aquaculture. However, the genetic bases that can underpin the future cultivation of these species, cytogenetic between these aspects are very weak. The chromosomal patterns have provided basic data for the exploration of biotechnological processes aimed at handling chromosomal genetic improvement, such as induction of polyploidy, androgenesis and ginogenesis, as well as obtaining monosex stocks and interspecific hybridizations. This paper presents a comprehensive cytogenetic survey in 10 species, seven of the family Carangidae and the monotypic family Rachycentridae. Classical cytogenetic analysis and in situ mapping of multigene sequences were employed, and additionally for the genus Selene and morphotypes of Caranx lugubris, comparisons were made using geometric morphometrics. In general, conservative species exhibit a marked chromosome number (2n=48). Although present in large part, different karyotypic form, retain many characteristics typical of chromosomal Order Perciformes, the high number of elements monobrachyal, Ag-NORs/18S rDNA sites and heterochromatin simply reduced, preferably centromeric. The main mechanisms involved in karyotypic diversification are the pericentric inversions, with secondary action of centric fusions. In addition to physical mapping and chromosome detail for the species are presented and discussed patterns of intra-and interspecific diversity, cytotaxonomic markers. This data set provides a better understanding of these patterns caryoevolutyonary groups and conditions for the development of protocols based on Biotechnology for chromosomal manipulation Atlantic these species

Mapeamento cromossômico de genes ribossomais em espécies estuarinas da família Gerreidae Identificação de uniformidade cariotípica e sua empregabilidade com fins biotecnológicos - REPROGEN

Perciformes are dominant in the marine environment, characterized as the largest and most diverse fish group. Some families, as Gerreidae, popularly known as silver jennies, carapebas, or mojarras have a high economic potential to marine fish farming, natural explotation and game fishing. Genetic information of these species are of fundamental importance for their management and production. Despite exist over 13,000 marine fish species described, only 2% were cytogenetically analyzed and less than 1% have some reproductive characteristics known. Induced breeding, cytogenetic characterization and cryopreservation of gametes, represent important areas in applied fish studies. In this project cytogenetic analyzes were performed to acess genetic aspects of Gerreidae species, distributed in coastal and estuarine regions of Northeast Brazil. Different methods for identifying chromosomal regions were employed using conventional techniques (Ag-NORs, C-banding), staining with base-specific fluorochromes (DAPI-CMA3), and physical mapping of ribosomal genes 18S and 5S rDNA, through hybridization in situ with fluorescent probes (FISH). The six species analyzed showed remarkable chromosome conservatism. The 18S and 5S ribosomal genes when analyzed in phylogenetic perspective demonstrate varied evolutionary dynamics, suggesting ocurrence of stasis process in some groups and greater dynamism in others. Double FISH with 18S and 5S probes showed both how efficient cytotaxonomic markers in the homogeneous karyotypes of this group of species. The karyotypic pattern identified in addition to the evolutionary aspects of karyotype, are suggestive of existence of low potential of post-zygotic barrier, prompting further research to prospect for artificial interspecific hybridization of these species of commercial importance

Low variation in ribosomal DNA and internal transcribed spacers of the symbiotic fungi of leaf-cutting ants (Attini: Formicidae)

Leaf-cutting ants of the genera Atta and Acromyrmex (tribe Attini) are symbiotic with basidiomycete fungi of the genus Leucoagaricus (tribe Leucocoprineae), which they cultivate on vegetable matter inside their nests. We determined the variation of the 28S, 18S, and 5.8S ribosomal DNA (rDNA) gene loci and the rapidly evolving internal transcribed spacers 1 and 2 (ITS1 and ITS2) of 15 sympatric and allopatric fungi associated with colonies of 11 species of leafcutter ants living up to 2,600 km apart in Brazil. We found that the fungal rDNA and ITS sequences from different species of ants were identical (or nearly identical) to each other, whereas 10 GenBank Leucoagaricus species showed higher ITS variation. Our findings suggest that Atta and Acromyrmex leafcutters living in geographic sites that are very distant from each other cultivate a single fungal species made up of closely related lineages of Leucoagaricus gongylophorus. We discuss the strikingly high similarity in the ITS1 and ITS2 regions of the Atta and Acromyrmex symbiotic L. gongylophorus studied by us, in contrast to the lower similarity displayed by their non-symbiotic counterparts. We suggest that the similarity of our L. gongylophorus isolates is an indication of the recent association of the fungus with these ants, and propose that both the intense lateral transmission of fungal material within leafcutter nests and the selection of more adapted fungal strains are involved in the homogenization of the symbiotic fungal stock.

Padrões cromossômicos e mapeamento de genes ribossomais 18S e 5S em peixes pelágicos Atlânticos

Cytogenetic studies in fish have been contributed significantly to a better understanding of the marine biodiversity, presenting information related to characterization, evolution and conservation of species e fisheries stocks. Among the marine species which cytogenetic data are less well known pelagic forms are detached, that despite the economic importance and conservation efforts have been suffering great pressure from the artisanal and industrial fisheries. The present work characterized cytogenetically six species of large pelagic fish in the Atlantic, belonging to the Order Perciformes, among them, four species of Scombridae, Thunnus albacares, T. obesus, Scomberomorus brasiliensis and Acanthocybium solandri and two Coryphaenidae, Coryphaena equiselis and C. hippurus using Classical cytogenetic methods as conventional staining, C-banding and Ag-NORs and molecular through staining fluorochromes AT and GC-specific and mapping of ribosomal multigene families, 18S and 5S. The identification of phylogenetic patterns and cytotaxonomic markers between the species and the presence of sex chromosomes in at least one species of Coryphaenidae, are particularly useful in the formulating of phylogenetic hypotheses, as well as comparisons between groups and populations

Diversidade de Phytophthora parasitica isolados de Citrus usando seqüências de nucleotídeos da região ITS-5.8S rDNA

Realizou-se estudo para caracterização e verificação da diversidade genética de Phytophthora parasitica, agente causador da gomose dos citros. Quatorze isolados de Phytophthora parasitica, provenientes do Estado de São Paulo, foram seqüenciados a partir das regiões internas transcritas (ITS1 e ITS2) do gene 5.8S. Obtiveram-se seqüências de 812 pb a 860 pb que foram comparadas com seqüências de outras espécies de Phytophthora spp depositadas no NCBI. Foram feitos estudos filogenéticos, utilizando-se o método neighbor-joining com 1000 bootstrap e construído o dendrograma mais representativo. Obtiveram-se os resultados de 98,88% a 100% de similaridade genética entre os 14 isolados paulistas, e 99,5% a 98,8% entre estes e a seqüência de P. nicotianae (gi| 8927482) obtida do GenBank NCBI.

Strain differentiation of Trichophyton rubrum by randomly amplified polymorphic DNA and analysis of rDNA nontranscribed spacer

Trichophyton rubrum is the most common pathogen causing dermatophytosis. Molecular strain-typing methods have recently been developed to tackle epidemiological questions and the problem of relapse following treatment. A total of 67 strains of T rubrum were screened for genetic variation by randomly amplified polymorphic DNA (RAPD) analysis, with two primers, 5'-d[GGTGCGGGAA]-3' and 5'-d[CCCGTCAGCA]-3', as well as by subrepeat element analysis of the nontranscribed spacer of rDNA, using the repetitive subelements TRS-1 and TRS-2. A total of 12 individual patterns were recognized with the first primer and 11 with the second. Phylogenetic analysis of the RAPID products showed a high degree of similarity (> 90 %) among the epidemiologically related clinical isolates, while the other strains possessed 60% similarity. Specific amplification of TRS-1 produced three strain-characteristic banding patterns (PCR types); simple patterns representing one copy of TRS-1 and two copies of TRS-2 accounted for around 85 % of all isolates. It is concluded that molecular analysis has important implications for epidemiological studies, and RAPID analysis is especially suitable for molecular typing in T. rubrum.

Classic and molecular study of Giardia duodenalis in children from a daycare center in the region of Presidente Prudente, São Paulo, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Phenotypic and genotypic characterization of Prototheca zopfii in a dog with enteric signs

This is a case report of enteric protothecosis caused by Prototheca zopfii in an eight-year-old male mixed breed dog with a history of chronic bloody diarrhea, loss of appetite and weight loss. Algae were isolated from rectal scrapings in defibrinated sheep blood agar and dextrose Sabouraud agar. Cytological evaluation showed the presence of globular and cylindrical organisms with a defined capsule and variable number of endospores, characteristic of the genus Prototheca, in the rectum of the animal. Scanning electron microscopy of P. zopfii strains at different development stages confirmed the diagnosis of algal infection. Molecular identification using a conserved 18S rDNA gene sequence determined that the strain belonged to genotype 2. This report describes success on treatment of canine protothecosis, diagnosed based on clinical, cytological, microbiological, scanning electron microscopy and genotypical findings. (c) 2009 Elsevier Ltd. All rights reserved.

5S rDNA characterization in twelve Sciaenidae fish species (Teleostei, Perciformes): depicting gene diversity and molecular markers

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Two 5S rDNA arrays in Neotropical fish species: is it a general rule for fishes ?

In this paper we describe Southern blot hybridization results probed with 5S rRNA genes for several Neotropical fish species representing different taxonomic groups. All the studied species showed a general trend with the 5S rDNA tandem repeats organized in two distinct size-classes. At the same time, data on 5S rDNA organization in fish genome were summarized. Previous information on the organization and evolution of 5S rRNA gene arrays in the genome of this vertebrate group are in agreement with the Southern results here presented. Sequences obtained for several fish species have revealed the occurrence of two distinct 5S rDNA classes characterized by distinct non-transcribed spacer sequences, which are clustered in different chromosomes in some species. Moreover, the 5S rDNA loci are generally distributed in an interstitial position in the chromosomes and they are usually not syntenic to the 45S rDNA. The presence of two classes of 5S rDNA in several non-related fish species suggests that this could be a common condition for the 5S rRNA gene organization in the fish genome.