Comparación de tres métodos de extracción de ADN a partir de garrapatas duras (Acari: Ixodidae) / Comparison of three DNA extraction methods from hard ticks (Acari: Ixodidae)

RESUMEN Las garrapatas son el grupo de ectoparásitos de mayor relevancia en la transmisión de patógenos a los animales domésticos y a humanos. La detección de estos agentes, normalmente, se realiza por métodos basados en PCR y, por ello, se requiere de la obtención de ácidos nucleicos, para la amplificación selectiva de dianas moleculares. El objetivo de la presente investigación fue comparar el desempeño de tres métodos de extracción de ADN, sales, columnas y tiocianato de guanidina, a partir de garrapatas, para estudios de detección de patógenos y sistemática molecular de garrapatas. Se realizaron comparaciones múltiples del desempeño de extracción sobre 30 muestras de garrapatas y se valoró la calidad del extracto de ADN, mediante la amplificación de un fragmento del gen mitocondrial 16S de garrapatas, con la utilización de la técnica de PCR. Además, se evaluó la presencia de patógenos de los géneros Rickettsia, Ehrlichia, Anaplasma y Babesia. El método con mayor desempeño en la extracción de ADN fue el basado en tiocianato de guanidina (mdnR=160ng/uL), seguido por columnas (mdnR=4,7ng/uL) y luego sales (mdnR=1,6ng/uL). Se presentaron diferencias estadísticas en el rendimiento de la extracción; no obstante, no existieron diferencias respecto al éxito de amplificación, mediante PCR, de acuerdo con el método de extracción (p=0,1173). No se detectaron patógenos rickettsiales o piroplasmas en ninguno de los extractos de ADN de garrapatas evaluados. Considerando el costo/beneficio de los métodos comparados, la utilización del método de sales puede facilitar la masificación de trabajos sobre la detección de patógenos que son transmitidos por garrapatas, en laboratorios de discreto presupuesto.

ABSTRACT Ticks are the most important group of ectoparasites in the transmission of pathogens to domestic animals and humans. Detection of those pathogens is usually performed by PCR-based methods and therefore requires the extraction of nucleic acids for the selective amplification of molecular targets. The aim of the present investigation was to compare the performance of three DNA extraction methods (salts, columns and guanidine thiocyanate) from ticks for studies of pathogen detection and molecular systematics of ticks. DNA extraction performance was measured by multiple comparisons on 30 tick samples and assessment of quality of the DNA extract through PCR amplification of 16S mitochondrial ticks gene. The presence of Rickettsia, Ehrlichia, Anaplasma and Babesia were also evaluated. The guanidine thiocyanate was the method with highest performance (mdnR= 160ng/uL), then columns (mdnR= 4.7ng/uL) and finally salting out method (mdnR= 1.6ng/uL). Although there were statistical differences of performance among DNA extraction protocols, there were no differences regarding the success of PCR amplification according to the extraction method (p = 0.1173). No rickettsial pathogens or piroplasms were detected in any of the DNA extracts evaluated. Considering the cost/benefit ratio of the three methods, the use of the salting out method can facilitate the massification of studies on tick-borne pathogen in low-budget laboratories.

Advances in molecular identification and functional genes of Rehmannia glutinosa / 中草药

Rehmannia glutinosa is one of the most common bulk medicinal materials in China and it has a long history of medicinal use. In recent years, with the development of molecular biology, especially for the emergence of high-throughput sequencing technology, it not only provides new ways to identify R. glutinosa quickly, and reveal the genetic diversity and relationship of R. glutinosa, but also lays the vital foundation for explaining the mechanism on metabolism, root tuber growth, stress response and continuous cropping obstacles of R. glutinosa. The present paper reviews the recent study progress in molecular biology research of R. glutinosa from molecular systematics, molecular identification and functional genes, and puts forward three research prospects in order to provide a reference for further study on molecular biology of R. glutinosa.

Molecular phylogeny and biogeographic history of the Neotropical tribe Glandulocaudini (Characiformes: Characidae: Stevardiinae)

Although former studies on systematics and biogeography represent a progress on the knowledge of the tribe Glandulocaudini, none was grounded on molecular evidence. Thus, the first hypothesis of relationships for the tribe based on a multilocus analysis is presented, including all genera and most of the valid species. DNA sequences of Glandulocauda caerulea and Mimagoniates sylvicola were analyzed for the first time. A molecular clock analysis was used to estimate the origin of the Glandulocaudini and the approximate timing of cladogenetic events within the group. Glandulocaudini was recovered as monophyletic. No hypothesis recovered Glandulocauda as monophyletic, since G. melanopleura is sister to Lophiobrycon weitzmani while G. caerulea is closely related to Mimagoniates. The relationships within the latter genus were resolved. The molecular clock results indicate the origin of the Glandulocaudini during the Miocene with diversification in the group occurring from Neogene to Pleistocene. These results corroborated the hypothesis that its origin took place on the Brazilian crystalline shield with the subsequent occupation of the Atlantic Coastal drainages. Apparently, Pleistocene sea-level fluctuations might have shaped the distribution pattern of some species in Glandulocaudini.(AU)

Embora estudos prévios sobre sistemática e biogeografia representam um avanço no conhecimento da tribo Glandulocaudini, nenhum foi baseado em evidência molecular. Assim, a primeira hipótese de relações para a tribo com base em uma análise multilocus é apresentada, incluindo todos os gêneros e a maioria das espécies válidas. Sequências de DNA de Glandulocauda caerulea e Mimagoniates sylvicola foram analisadas pela primeira vez. Uma análise de relógio molecular foi utilizada para estimar a origem de Glandulocaudini e datas aproximadas de eventos cladogenéticos dentro do grupo. Glandulocaudini foi recuperada como monofilética. Nenhuma hipótese recuperou Glandulocauda como monofilético, uma vez que G. melanopleura é irmã de Lophiobrycon weitzmani e G. caerulea está proximamente relacionada a Mimagoniates. As relações dentro deste último gênero foram resolvidas. Os resultados do relógio molecular indicam que Glandulocaudini originou-se durante o Mioceno, com diversificação dentro do grupo ocorrendo desde o Neogeno até o Pleistoceno. Estes resultados corroboram a hipótese da sua origem no escudo cristalino brasileiro, com a subsequente ocupação das drenagens costeiras atlânticas. Aparentemente, as flutuações pleistocênicas do nível do mar podem ter moldado o padrão de distribuição de algumas espécies em Glandulocaudini.(AU)

Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state / Caracterização molecular de bactérias fixadoras de nitrogênio isoladas de plantas brasileiras no estado de São Paulo

Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.

Quatorze linhagens de bactérias fixadoras de nitrogênio foram isoladas de diferentes espécies de plantas, incluindo cassava, milho e cana-de-açúcar, usando condições seletivas desprovidas de nitrogênio. A capacidade de fixar nitrogênio foi verificada por ensaio de redução de acetileno. Todas as linhagens fixadoras de nitrogênio testadas apresentaram hibridização positiva com sonda de gene nifH derivada de Azospirillum brasilense. As linhagens foram caracterizadas por RAPD, ARDRA e sequenciamento do gene 16S rDNA. As análises de RAPD revelaram 8 genótipos, as 6 linhagens restantes foram agrupadas em 3 grupos de RAPD, sugerindo uma origem clonal. ARDRA e seqüências de 16S rDNA foram alocadas em 13 grupos conhecidos de bactérias fixadoras de nitrogênio, incluindo organismos dos gêneros Azospirillum, Herbaspirillum, Pseudomonas e Enterobacteriaceae. Duas linhagens foram classificadas como Stenotrophomonas ssp. Os resultados da identificação molecular baseados em sequencias de 16S rDNA corroboram com dados obtidos em testes morfológicos e bioquímicos.

Resurrection of the name Albula pacifica (Beebe, 1942) for the shafted bonefish (Albuliformes: Albulidae) from the eastern Pacific

The name Albula nemoptera (Fowler, 1911) is currently applied to the Shafted, or Threadfin, Bonefish (Albuliformes: Albulidae) inhabiting the tropical coastal waters of both the western Atlantic and eastern Pacific. In the present paper I provide a brief review of the taxonomy and nomenclature of A. nemoptera, and argue that the available morphological, biogeographical and molecular evidence supports resurrecting the name A. pacifica (Beebe, 1942) for the population of A. nemoptera from the eastern Pacific. Rev. Biol. Trop. 56 (2): 839-844. Epub 2008 June 30.

El nombre Albula nemoptera (Fowler, 1911) se aplica actualmente a las poblaciones del macabí de hebra (Albuliformes: Albulidae) de las aguas costeras tropicales del Atlántico Occidental y el Pacifico Oriental. En este artículo se presenta una revisión breve de la taxonomía y nomenclatura de A. nemoptera, y se sugiere que la evidencia morfológica, biogeográfica y molecular apoya el reestablecimiento del nombre A. pacifica (Beebe, 1942) para la población de A. nemoptera del Pacifico Oriental.

Phylogenetic analysis of the order Pleuronectiformes (Teleostei) based on sequences of 12S and 16S mitochondrial genes

The fish order Pleuronectiformes, composed of 14 families, has two suborders: Psettodoidei (with one family) and Pleuronectoidei (with thirteen families). The relationships among families of Pleuronectoidei and among the genera of their families have extensively been debated and a consensus has not yet been reached. In the present study, partial sequences of the 12S and 16S mitochondrial rRNA genes were obtained from 19 species belonging to the families Achiridae, Bothidae, Cynoglossidae, Paralichthyidae, Pleuronectidae, Scophthalmidae, and Soleidae. Additional sequences of 42 pleuronectiform species were obtained from GenBank. Phylogenetic analyses were conducted by the methods of maximum-parsimony, maximum-likelihood and Bayesian inference. Our results corroborate the monophyletic status of all families, excluding Paralichthyidae. In the family Achiridae, the genus Catathyridium (freshwater) was the sister group of Trinectes (saltwater), and Hypoclinemus (freshwater) was the sister group of Achirus (saltwater). Assuming that the putative ancestor of achirids lived in saltwater, it is suggested that the freshwater habitats in South America were colonized independently by different achirid lineages.

A phylogenetic analysis of Brycon and Henochilus (Characiformes, Characidae, Bryconinae) based on the mitochondrial gene 16S rRNA

The genus Brycon, the largest subunit of the Bryconinae, has 42 valid species distributed from southern Mexico to the La Plata River in Argentina. Henochilus is a monotypic genus, comprising a single species (H. wheatlandii) found in the upper Rio Doce basin. In the present study, partial sequences of the mitochondrial gene 16S were obtained for fifteen species of Brycon and for Henochilus wheatlandii. The results showed that the genus Brycon is paraphyletic, since Henochilus is the sister-group of B. ferox and B. insignis. The most basal species analyzed were the trans-Andean species B. henni, B. petrosus, and B. chagrensis.

Identification of Some Phellinus spp

Four strains of Phellinus spp. was identified based on internal transcribed spacer (ITS) region of rDNA sequence analysis and morphological characteristics. Basidiocarps of all strains were effused-reflexed and hymenial surface was poroid. Hyphal system was dimitic and basidiospore was globose to ellipsoid. The amplification of ITS regions produced a DNA fragment of 500 to 780 bp in all strains examined. The determined sequences were analyzed for the reconstruction of phylogenetic tree. From these results, Phellinus sp. KM-1, KM-2, and KM-4 was identified as P. hartigii, P. baumii, and P. linteus, respectively.

The Advances of Molecular Systematics on Morchella / 微生物学通报

This article provides a brief description of systematics on Morchella ,and reviews the advances of molecular systematics on Morchella over the world.