Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes

In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae). The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes). This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes) and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.

Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

A nuclear ribosomal DNA pseudogene in triatomines opens a new research field of fundamental and applied implications in Chagas disease

A pseudogene, designated as "ps(5.8S+ITS-2)", paralogous to the 5.8S gene and internal transcribed spacer (ITS)-2 of the nuclear ribosomal DNA (rDNA), has been recently found in many triatomine species distributed throughout North America, Central America and northern South America. Among characteristics used as criteria for pseudogene verification, secondary structures and free energy are highlighted, showing a lower fit between minimum free energy, partition function and centroid structures, although in given cases the fit only appeared to be slightly lower. The unique characteristics of "ps(5.8S+ITS-2)" as a processed or retrotransposed pseudogenic unit of the ghost type are reviewed, with emphasis on its potential functionality compared to the functionality of genes and spacers of the normal rDNA operon. Besides the technical problem of the risk for erroneous sequence results, the usefulness of "ps(5.8S+ITS-2)" for specimen classification, phylogenetic analyses and systematic/taxonomic studies should be highlighted, based on consistence and retention index values, which in pseudogenic sequence trees were higher than in functional sequence trees. Additionally, intraindividual, interpopulational and interspecific differences in pseudogene amount and the fact that it is a pseudogene in the nuclear rDNA suggests a potential relationships with fitness, behaviour and adaptability of triatomine vectors and consequently its potential utility in Chagas disease epidemiology and control.

First description of Candida nivariensis in Brazil: antifungal susceptibility profile and potential virulence attributes

This study evaluated the antifungal susceptibility profile and the production of potential virulence attributes in a clinical strain of Candida nivariensis for the first time in Brazil, as identified by sequencing the internal transcribed spacer (ITS)1-5.8S-ITS2 region and D1/D2 domains of the 28S of the rDNA. For comparative purposes, tests were also performed with reference strains. All strains presented low planktonic minimal inhibitory concentrations (PMICs) to amphotericin B (AMB), caspofungin (CAS), and voriconazole. However, our strain showed elevated planktonic MICs to posaconazole (POS) and itraconazole, in addition to fluconazole resistance. Adherence to inert surfaces was conducted onto glass and polystyrene. The biofilm formation and antifungal susceptibility on biofilm-growing cells were evaluated by crystal violet staining and a XTT reduction assay. All fungal strains were able to bind both tested surfaces and form biofilm, with a binding preference to polystyrene (p < 0.001). AMB promoted significant reductions (≈50%) in biofilm production by our C. nivariensis strain using both methodologies. This reduction was also observed for CAS and POS, but only in the XTT assay. All strains were excellent protease producers and moderate phytase producers, but lipases were not detected. This study reinforces the pathogenic potential of C. nivariensis and its possible resistance profile to the azolic drugs generally used for candidiasis management.

Identification and antifungal susceptibility of a large collection of yeast strains isolated in Tunisian hospitals.

Abstract In this study, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used as a rapid method to identify yeasts isolated from patients in Tunisian hospitals. When identification could not be exstablished with this procedure, sequencing of the internal transcribed spacer with 5.8S ribosomal DNA (rDNA) (ITS1-5.8S-ITS2) and D1/D2 domain of large-subunit (LSU rDNA) were employed as a molecular approach for species differentiation. Candida albicans was the dominant species (43.37% of all cases), followed by C. glabrata (16.55%), C. parapsilosis (13.23%), C. tropicalis (11.34%), C. dubliniensis (4.96%), and other species more rarely encountered in human diseases such as C. krusei, C. metapsilosis, C. lusitaniae, C. kefyr, C. palmioleophila, C. guilliermondii, C. intermedia, C. orthopsilosis, and C. utilis. In addition, other yeast species were obtained including Saccharomyces cerevisiae, Debaryomyces hansenii (anamorph known as C. famata), Hanseniaspora opuntiae, Kodamaea ohmeri, Pichia caribbica (anamorph known as C. fermentati), Trichosporon spp. and finally a novel yeast species, C. tunisiensis. The in vitro antifungal activities of fluconazole and voriconazole were determined by the agar disk diffusion test and Etest, while the susceptibility to additional antifungal agents was determined with the Sensititre YeastOne system. Our results showed low incidence of azole resistance in C. albicans (0.54%), C. tropicalis (2.08%) and C. glabrata (4.28%). In addition, caspofungin was active against most isolates of the collection with the exception of two K. ohmeri isolates. This is the first report to describe caspofungin resistant isolates of this yeast.

Combining molecular evolution and environmental genomics to unravel adaptive processes of MHC class IIB diversity in European minnows (Phoxinus phoxinus)

Host-pathogen interactions are a major evolutionary force promoting local adaptation. Genes of the major histocompatibility complex (MHC) represent unique candidates to investigate evolutionary processes driving local adaptation to parasite communities. The present study aimed at identifying the relative roles of neutral and adaptive processes driving the evolution of MHC class IIB (MHCIIB) genes in natural populations of European minnows (Phoxinus phoxinus). To this end, we isolated and genotyped exon 2 of two MHCIIB gene duplicates (DAB1 and DAB3) and 1665 amplified fragment length polymorphism (AFLP) markers in nine populations, and characterized local bacterial communities by 16S rDNA barcoding using 454 amplicon sequencing. Both MHCIIB loci exhibited signs of historical balancing selection. Whereas genetic differentiation exceeded that of neutral markers at both loci, the populations' genetic diversities were positively correlated with local pathogen diversities only at DAB3. Overall, our results suggest pathogen-mediated local adaptation in European minnows at both MHCIIB loci. While at DAB1 selection appears to favor different alleles among populations, this is only partially the case in DAB3, which appears to be locally adapted to pathogen communities in terms of genetic diversity. These results provide new insights into the importance of host-pathogen interactions in driving local adaptation in the European minnow, and highlight that the importance of adaptive processes driving MHCIIB gene evolution may differ among duplicates within species, presumably as a consequence of alternative selective regimes or different genomic context.

Metodologia para o Estudo de Fármacos Vegetais

O mecanismo de actuação, a toxicidade e os efeitos clínicos permanecem desconhecidos para a maioria dos fármacos vegetais, assentando o seu uso no conhecimento tradicional. É porém frequente encontrar plantas tóxicas que, quando ingeridas inapropriadamente e sem acompanhamento técnico, podem ser letais. A identificação micromorfológica é útil na inventariação da diversidade e no reconhecimento de caracteres diferenciadores de fármacos vegetais. O recurso a metodologias no âmbito da biologia molecular é uma mais-valia em relação ao complexo, moroso e dispendioso estudo fitoquímico. Usando quantidades de material muito pequenas, elas constituem também um avanço na certificação da qualidade, segurança e reprodutibilidade da composição de fármacos. Como exemplo da sua aplicação, apresentamos um estudo realizado com plantas endémicas de Cabo Verde, usadas na medicina tradicional, Tornabenea insularis e T. annua. Estas espécies apresentam problemas taxonómicos, o que também levanta questões na sua correcta utilização. Através da combinação dos microcaracteres foliares e dos frutos bem como da extracção de DNA e amplificação por PCR da região ITS e do gene 26S rDNA, os nossos resultados não permitem a separação dos dois taxa, não confirmando assim a actual classificação.

Caracterização Biofísica da Ilha de Santo Antão em Cabo Verde Através de Sistemas de Informação Geográfica e Deteção Remota

O mecanismo de actuação, a toxicidade e os efeitos clínicos permanecem desconhecidos para a maioria dos fármacos vegetais, assentando o seu uso no conhecimento tradicional. É porém frequente encontrar plantas tóxicas que, quando ingeridas inapropriadamente e sem acompanhamento técnico, podem ser letais. A identificação micromorfológica é útil na inventariação da diversidade e no reconhecimento de caracteres diferenciadores de fármacos vegetais. O recurso a metodologias no âmbito da biologia molecular é uma mais-valia em relação ao complexo, moroso e dispendioso estudo fitoquímico. Usando quantidades de material muito pequenas, elas constituem também um avanço na certificação da qualidade, segurança e reprodutibilidade da composição de fármacos. Como exemplo da sua aplicação, apresentamos um estudo realizado com plantas endémicas de Cabo Verde, usadas na medicina tradicional, Tornabenea insularis e T. annua. Estas espécies apresentam problemas taxonómicos, o que também levanta questões na sua correcta utilização. Através da combinação dos microcaracteres foliares e dos frutos bem como da extracção de DNA e amplificação por PCR da região ITS e do gene 26S rDNA, os nossos resultados não permitem a separação dos dois taxa, não confirmando assim a actual classificação.

PCR-RAPD and PCR-RFLP polymorphisms detected in Anopheles cruzii (Diptera, Culicidae)

The aim of the present study was to examine genetic variability in populations of An. cruzii by employing PCR-RAPD and PCR-RFLP markers. All analyses were carried out using individuals of the F1 generation of wild caught females obtained in Santa Catarina State (Florianópolis and São Francisco do Sul), Paraná State (Morretes, Paranaguá and Guaratuba) and São Paulo State (Cananéia). In the PCR-RAPD experiments, seven primers were used for comparisons within and among populations. The restriction profile of the ITS2 including a fragment of both 5.8S and 28S regions of the rDNA was obtained with the enzymes BstUI, HaeIII, TaqI, HhaI, Sau96I, HinfI, HincII and NruI. The PCR-RAPD method detected a large number of polymorphic bands. Genetic distance among populations of An. cruzii varied from 0,0214 to 0,0673, suggesting that all individuals used in the analyses belong to a single species. The number of migrants per generation (Nm) was 4.3, showing the existence of gene flow among populations. The restriction profile of the ITS2, 5.8S and 28S gene regions was similar in all An. cruzii samples, whereas the results obtained by using HhaI and NruI are indicative that the individuals analyzed have nucleotide sequences distinct from those of An. cruzii samples from Peruíbe and Juquiazinho deposited in GenBank.

Molecular analysis and mating behaviour of the Trichophyton mentagrophytes species complex.

Isolates of the Trichophyton mentagrophytes complex vary phenotypically. Whether the closely related zoophilic and anthropophilic anamorphs currently associated with Arthroderma vanbreuseghemii have to be considered as members of the same biological species remains an open question. In order to better delineate species in the T. mentagrophytes complex, we performed a mating analysis of freshly collected isolates from humans and animals with A. benhamiae and A. vanbreuseghemii reference strains, in comparison to internal transcribed spacer (ITS) and 28S rDNA sequencing. Mating experiments as well as ITS and 28S sequencing unambiguously allowed the distinction of A. benhamiae and A. vanbreuseghemii. We have also shown that all the isolates from tinea pedis and tinea unguium identified as T. interdigitale based on ITS sequences mated with A. vanbreuseghemii tester strains, but had lost their ability to give fertile cleistothecia. Therefore, T. interdigitale has to be considered as a humanized species derived from the sexual relative A. vanbreuseghemii.

Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA)

Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed

Avaliação da diversidade genética de fungos micorrízicos arbusculares em três estádios de estabilização de dunas

A diversidade genética de fungos micorrízicos arbusculares, recuperados de três estádios de estabilização de dunas, foi avaliada por técnicas moleculares e comparada com resultados obtidos anteriormente por técnicas baseadas na caracterização morfológica dos esporos. O uso da técnica de PCR-RFLP do rDNA, extraído de esporos, permitiu definir impressões características de espécies presentes nas dunas, evidenciar a presença de diferentes comunidades em cada estádio e identificar a anteduna como aquela com comunidades com maior polimorfismo. Esse estádio também apresentou maior diversidade, quando, no estudo das comunidades, foram utilizadas técnicas baseadas em aspectos morfológicos. A combinação de ambas as estratégias, molecular e baseada em aspectos morfológicos, forneceu importantes informações sobre a diversidade destes fungos, visando ao estudo do seu papel no ecossistema.

Caracterização do dna ribossômico do isolado de Scleroderma UFSMSc1 de Eucalyptus grandis W. Hill ex-maiden

O ácido desoxiribunocléico ribossomal (rDNA) é utilizado como uma ferramenta importante para caracterizar o polimorfismo entre os fungos. Existem muitas cópias de rDNA as quais são arranjadas por espaços não codificados. Essas cópias são altamente conservadas entre espécies de fungos. O objetivo deste trabalho foi estudar a região do Espaço Interno Transcrito (ITS) e analisar as diferenças no polimorfismo da seqüência dessa região no fungo Scleroderma UFSMSc1 com seqüências dos isolados de Scleroderma e Pisolithus do banco de dados GenBank. O DNA do isolado de Scleroderma UFSMSc1 foi extraído por meio da solução de extração à base de CTAB. A partir do DNA, foram feitas reações de PCR com os oligonucleotídeos iniciadores universais ITS1 e ITS4, cujo produto amplificado foi purificado e seqüenciado. A região do ITS do fungo mostrou uma banda simples de aproximadamente 650 pares de base. Na análise da seqüência dessa região em comparação com algumas depositadas no GenBank, observou-se a formação de agrupamento com espécies de Scleroderma. Os resultados mostraram que essa técnica favorece a identificação de espécies de Scleroderma, visto que tais fungos são difíceis de ser identificados apenas por seus caracteres morfológicos.

Biodiversity of rhizobia associated with cowpea cultivars in soils of the lower half of the São Francisco River Valley

The biodiversity of rhizobium in soils of the São Francisco Valley is unknown and can be studied using cowpea as trap plants. The objective of this study was to verify the diversity of diazotrophic bacteria that nodulate cowpea in soils of the lower half of the São Francisco River Valley by morphological and genotypic characterization. Seven soil samples (A1, A2, A3, A4, C1, C2 and MC) were collected to capture bacteria associated to five cowpea cultivars (IPA 206, BRS Pujante, BRS Marataoã, Canapu Roxo, and Sempre Verde), in a 5x7 factorial design with three replications. Thirty days after plant emergence, the nodules were collected and the bacteria isolated and analyzed in relation to their growth characteristics in YMA medium. The 581 isolates were grouped in 49 morphologic groups. Of this total, 62.3 % formed colonies in up to three days, 33.4 % grew from the 6th day on, and 4.3 % began to grow 4 to 5 days after incubation. Regarding the formation of acids and alkalis, 63 % acidified the medium, 12 % made it alkaline and 25 % maintained the medium at neutral pH. The highest diversity was observed in the A3 sample and in isolates associated with the cultivars Canapu Roxo and BRS Pujante. Thirty-eight representative isolates were chosen for the genotypic characterization, clustered in four groups based on the restriction analysis of 16s rDNA. This grouping was strongly correlated with the sampling site; 13 rhizobium isolates had an electrophoretic profile distinct from the standard rhizobium strains used in this study.