Genetic characterization of native and introduced populations of the neotropical cichlid genus Cichla in Brazil

A molecular phylogenetic analysis based on mitochondrial 16S ribosomal DNA and Control Region sequences from native and introduced populations was undertaken, in order to characterize the introduction of Cichla (peacock bass or tucunaré) species in Brazil. Mitochondrial DNA haplotypes found in introduced fish from Minas Gerais state (southeastern Brazil) clustered only with those from native species of the Tocantins River (Cichla piquiti and C. kelberi), thereby suggesting a single or, at most, few translocation acts in this area, even though with fish from the same source-population. Our study contributes to an understanding of the introduction of Cichla in regions of Brazil outside the Amazon basin, and adds phylogenetic data to the recently describe Cichla species, endemic from the Tocantins-Araguaia basin.

Characterization of 23S rRNA domain V mutations in gastric biopsy patients from the eastern Amazon

Resistance of Helicobacter pylori to clarithromycin is characterised by simple point mutations in the 23S ribosomal RNA (rRNA) gene and is responsible for the majority of cases of failure to eradicate this bacterium. In this paper, we characterised the variability of the 23S rRNA gene in biopsies of patients with gastric pathologies in the eastern Amazon (Northern Region of Brazil) using PCR and sequencing. A total of 49 sequences of H. pylori strains were analysed and of those, 75.6% presented nucleotide substitutions: A2142G (3.3%), T2182C (12.9%), G2224A (6.45%), T2215C (61.3%), A2192G (3.3%), G2204C (6.4%) and T2221C (6.4%). Of the mutations identified, four are known mutations related to cases of resistance and 16.1% are not yet described, revealing a high prevalence of mutations in the H. pylori 23S rRNA gene among the strains circulating in the in the eastern Amazon. The high prevalence in individuals with gastric pathologies in the Northern Region of Brazil demonstrates the need for characterising the profile of these strains to provide correct therapy for patients, considering that mutations in this gene are normally associated with resistance to the primary medication used in controlling H. pylori infection.

A comparison of molecular markers to detect Lutzomyia longipalpis naturally infected with Leishmania (Leishmania) infantum

The aim of the present study was to detect natural infection by Leishmania (Leishmania) infantum in Lutzomyia longipalpis captured in Barcarena, state of Pará, Brazil, through the use of three primer sets. With this approach, it is unnecessary to previously dissect the sandfly specimens. DNA of 280 Lu. longipalpis female specimens were extracted from the whole insects. PCR primers for kinetoplast minicircle DNA (kDNA), the mini-exon gene and the small subunit ribosomal RNA (SSU-rRNA) gene of Leishmania were used, generating fragments of 400 bp, 780 bp and 603 bp, respectively. Infection by the parasite was found with the kDNA primer in 8.6% of the cases, with the mini-exon gene primer in 7.1% of the cases and with the SSU-rRNA gene primer in 5.3% of the cases. These data show the importance of polymerase chain reaction as a tool for investigating the molecular epidemiology of visceral leishmaniasis by estimating the risk of disease transmission in endemic areas, with the kDNA primer representing the most reliable marker for the parasite.

Novel 12S mtDNA findings in sloths (Pilosa, Folivora) and anteaters (Pilosa, Vermilingua) suggest a true case of long branch attraction

We sequenced 12S RNA mtDNA for the majority of the extant species of sloths and anteaters and compared our results with previous data obtained by our group using 16S RNA mtDNA in the same specimens and to GenBank sequences of the extinct giant sloth Mylodon. Our results suggest that pigmy-anteaters may be a case of the long-branch attraction phenomenon and also show the large genetic difference between the Amazonian and Atlantic forest three-toed sloths, contrasting with the small differences observed between the two non-Atlantic forest forms of sloths. These results have important implications for the taxonomy of sloths and anteaters and strongly suggest the placement of pigmy anteaters in their own family (Cyclopidae) and raising the taxonomic status of Bradypus torquatus to a genus.

Phylogenetic analysis of 16S mitochondrial DNA data in sloths and anteaters

We sequenced part of the 16S rRNA mitochondrial gene in 17 extant taxa of Pilosa (sloths and anteaters) and used these sequences along with GenBank sequences of both extant and extinct sloths to perform phylogenetic analysis based on parsimony, maximum-likelihood and Bayesian methods. By increasing the taxa density for anteaters and sloths we were able to clarify some points of the Pilosa phylogenetic tree. Our mitochondrial 16S results show Bradypodidae as a monophyletic and robustly supported clade in all the analysis. However, the Pleistocene fossil Mylodon darwinii does not group significantly to either Bradypodidae or Megalonychidae which indicates that trichotomy best represents the relationship between the families Mylodontidae, Bradypodidae and Megalonychidae. Divergence times also allowed us to discuss the taxonomic status of Cyclopes and the three species of three-toed sloths, Bradypus tridactylus, Bradypus variegatus and Bradypus torquatus. In the Bradypodidae the split between Bradypus torquatus and the proto-Bradypus tridactylus / B. variegatus was estimated as about 7.7 million years ago (MYA), while in the Myrmecophagidae the first offshoot was Cyclopes at about 31.8 MYA followed by the split between Myrmecophaga and Tamandua at 12.9 MYA. We estimate the split between sloths and anteaters to have occurred at about 37 MYA.

Filogenia molecular dos xenarthra (Mammalia): análise do grupo cingulata a partir de sequências nucleotídicas do gene mitocondrial rRNA 16S

Os Xenarthra são o grupo de mamíferos que inclui os tatus, os tamanduás e as preguiças. A América do Sul serviu de cenário para a história natural do grupo que, somente no fim do Cenozóico, dispersou-se para a América Central e, com uma perda de variedade, chegou à América do Norte e à algu-mas ilhas do Caribe. Trinta e uma espécies estão descritas dentro da linha-gem dos Xenarthra. Elas estão classificadas em 13 gêneros, quatro famílias (Bradypodidae, Megalonychidae, Myrmecophagidae e Dasypodidae) e duas ordens (Cingulata e Pilosa). A filogenia deste grupo tem sido alvo de diver-sas pesquisas que analisaram tanto dados morfológicos, quanto moleculares. Delsuc et al. (2003) analisaram seqüências de genes mitocondriais e nucleares e confirmaram a monofilia das três subfamílias (Dasypodinae, Euphacti-nae e Tolypeutinae) inclusas na família Dasypodidae. Delsuc et al. (2003) geraram a seguinte árvore: (((Bradypus, Choloepus)100, ((Myrmecophaga, Tamandua)100, Cyclopes)100), ((D. kappleri, D. novemcinctus)100, (Toly-pentes, (Priodontes, Cabassous)54)100, (Zaedyus, (Euphractus, Chaetophrac-tus)60)100)). Gaudin (2005) apresentou um trabalho que reviu e ampliou as análises morfológicas apresentadas até então, concluindo que os tatus atu-ais estão divididos em dois grupos, um mais basal (Dasypodinae) e outro mais derivado (Euphractinae), de acordo com o seguinte arranjo: (Bradypus, Tamandua), (Dasypus, (Priodontes, (Cabassous, (Tolypeutes, (Euphractus, Chaetophractus, (Zaedyus, Chlamyphorus)42)36)72)72)40)85). Neste traba-lho utilizou-se parte do gene mitocondrial rRNA 16S de 12 táxons atu-ais de Xenarthra para analisar a filogenia do grupo através do critério de máxima verossimilhança. Nossos resultados são apresentados analisando-se o gene 16S e analisando o banco de dados do 16S mais o de Delsuc et al. (2003). Nas duas situações, as filogenias apresentadas apóiam os resulta-dos de Delsuc et al. (2003): (Bradypus, (Choloepus, ((Cyclopes, (Myrme-cophaga, Tamandua)100)100, (Dasypus, (((Cabassous, Priodontes)68, Toly-peutes)100,((Chaetophractus, Euphractus)65, Zaedyus)100)100)100)100)100). Uma melhora nos valores de bootstrap nos ramos dentro das sub-famílias da família Dasypodidae é percebida em relação ao trabalho de Delsuc et al. (2003). Acreditamos que Elementos de Transposição do tipo (LINES) são os marcadores moleculares mais adequados para confirmar o arranjo obtido com as seqüências de genes mitocondriais e nucleares.

Karyotypic evolution of ribosomal sites in buffalo subspecies and their crossbreed

Domestic buffaloes are divided into two group based on cytogenetic characteristics and habitats: the “river buffaloes” with 2n = 50 and the “swamp buffaloes”, 2n = 48. Nevertheless, their hybrids are viable, fertile and identified by a 2n = 49. In order to have a better characterization of these different cytotypes of buffaloes, and considering that NOR-bearing chromosomes are involved in the rearrangements responsible for the karyotypic differences, we applied silver staining (Ag-NOR) and performed fluorescent in situ hybridization (FISH) experiments using 18S rDNA as probe. Metaphases were obtained through blood lymphocyte culture of 21 individuals, including river, swamp and hybrid cytotypes. Ag-NOR staining revealed active NORs on six chromosome pairs (3p, 4p, 6, 21, 23, 24) in the river buffaloes, whereas the swamp buffaloes presented only five NOR-bearing pairs (4p, 6, 20, 22, 23). The F1 crossbreed had 11 chromosomes with active NORs, indicating expression of both parental chromosomes. FISH analysis confirmed the numerical divergence identified with Ag-NOR. This result is explained by the loss of the NOR located on chromosome 4p in the river buffalo, which is involved in the tandem fusion with chromosome 9 in this subspecies. A comparison with the ancestral cattle karyotype suggests that the NOR found on the 3p of the river buffalo may have originated from a duplication of ribosomal genes, resulting in the formation of new NOR sites in this subspecies.