Genus Paracoccidioides: Species Recognition and Biogeographic Aspects

Background: Paracoccidioidomycosis is a systemic mycosis caused by Paracoccidioides brasiliensis (species S1, PS2, PS3), and Paracoccidioides lutzii. This work aimed to differentiate species within the genus Paracoccidioides, without applying multilocus sequencing, as well as to obtain knowledge of the possible speciation processes. Methodology/Principal Findings: Single nucleotide polymorphism analysis on GP43, ARF and PRP8 intein genes successfully distinguished isolates into four different species. Morphological evaluation indicated that elongated conidia were observed exclusively in P. lutzii isolates, while all other species (S1, PS2 and PS3) were indistinguishable. To evaluate the biogeographic events that led to the current geographic distribution of Paracoccidioides species and their sister species, Nested Clade and Likelihood Analysis of Geographic Range Evolution (LAGRANGE) analyses were applied. The radiation of Paracoccidioides started in northwest South America, around 11–32 million years ago, as calculated on the basis of ARF substitution rate, in the BEAST program. Vicariance was responsible for the divergence among S1, PS2 and P. lutzii and a recent dispersal generated the PS3 species, restricted to Colombia. Taking into account the ancestral areas revealed by the LAGRANGE analysis and the major geographic distribution of L. loboi in the Amazon basin, a region strongly affected by the Andes uplift and marine incursions in the Cenozoic era, we also speculate about the effect of these geological events on the vicariance between Paracoccidioides and L. loboi. Conclusions/Significance: The use of at least 3 SNPs, but not morphological criteria, as markers allows us to distinguish among the four cryptic species of the genus Paracoccidioides. The work also presents a biogeographic study speculating on how these species might have diverged in South America, thus contributing to elucidating evolutionary aspects of the genus Paracoccidioides

Desenvolvimento de marcadores SNPs (Single Nucleotide Polymorphisms) para estudos populacionais do mosquito Aedes aegypti no Brasil

Aedes aegypti é o principal vetor dos vírus dengue e representa cada vez mais um problema de saúde pública. O uso de inseticidas de origem orgânica ou inorgânica é uma das metodologias mais adotadas como parte do controle de vetores, entretanto, processos de resistência têm sido detectados freqüentemente. O desenvolvimento de ferramentas moleculares tem facilitado o entendimento das relações entre o vetor, patógeno e o homem. Os marcadores genéticos mais utilizados em estudos de genética de populações geralmente representam um trabalho laboratorial intenso e, em alguns casos, são limitados pela quantidade de DNA genômico extraído por indivíduo. Recentemente, a tecnologia disponível, permitiu o desenvolvimento dos marcadores SNPs (Single Nucleotide Polymorphisms) que correspondem a diferenças de um único nucleotídeo na seqüência de DNA. Assim, o presente trabalho objetivou desenvolver marcadores SNPs localizados em genes nucleares, do mosquito Ae. aegypti, para aplicação na genotipagem de populações deste vetor no Brasil. Para tanto, foram investigada seqüencias em dezoito genes nucleares de dezesseis populações do Ae. aegypti, e nove destes genes foram selecionados para a presença de SNPs. Os oito marcadores mais polimórficos foram utilizados na caracterização de três populações do mosquito anteriormente analisadas utilizando-se as técnicas de RAPD (Random Amplified Polymorphic DNA) e seqüenciamento de genes mitocondriais. Os resultados obtidos com os SNPs mostraram um alto grau de polimorfismo intra e inter populacional, mais significativo quando comparado com os estudos realizados anteriormente, além da presença de duas diferentes linhagens genéticas entre as populações geográficas estudadas.Aedes aegypti is the primary vector of dengue fever virus and it represents a major problem of Public Health. The use of insecticides of organic or inorganic origin is one of the more accepted methodologies as part of this control, however, resistance processes have been detected constantly. The development of molecular tools has made possible the understanding of the relationships among vector, pathogen and man. The genetic markers more used in studies of genetics populations usually represent an intense laboratorial work and in some cases they are limited for the amount of genomic DNA extracted by individual. Recently, the available technology, allowed the development of the markers SNPs (Single Nucleotide Polymorphism) that correspond to differences of a single nucleotide in the sequence of DNA. Thus, this work aims to develop of SNPs markers in nuclear genes of the Ae. aegypti for application in genotyping of populations of this vector in Brazil. For so much, were investigated sequences in eighteen nuclear genes of sixteen populations of Ae. aegypti, and nine of this genes were selected to presence of the SNPs. Eight more polymorphic markers were used in the characterization of three populations of the mosquito previously analyzed using the techniques of RAPD (Random Amplified Polymorphic DNA) and sequencing of mitochondrial genes. Results obtained with the SNPs showed a high degree of polymorphic intra and interpopulations, more significantly when compared with the prior studies, besides the presence of two different genetic lineages distributed among the studied geographical populations.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Molecular and parasitological survey of Hepatozoon canis (Apicomplexa : Hepatozoidae) in dogs from rural area of São Paulo state, Brazil

Hepatozoon canis is a protozoan that infects dogs and is transmitted by the ingestion of the brown dog tick, Rhipicephalus sanguineus. Two distinct species of Hepatozoon genus can infect dogs, H. canis and H. americanum. Routine tests to detect the disease are based on direct examination of gametocytes on Giemsa-stained blood smears. The objectives of this study were the investigation of infection prevalence in rural area dogs, the comparison of diagnostics by blood smear examination and polymerase chain reaction (PCR), and the association of infection with tick infestation. Blood smears, collected by puncture of the cephalic vein and ear margin capillary bed from 150 dogs, were examined. This technique detected 17 positive animals (11.3%), with 14 (9.3%) in peripheral blood and seven (4.7%) in cephalic vein blood. PCR tests detected 80 (53.3%) positive animals. R. sanguineus and Amblyomma spp. were found in 36 of the dogs (24%), in equal proportions. The identified species for Amblyomma genus were A. cajennense and A. ovale. Data analysis showed that PCR was much more sensitive when compared to blood smear examination. Hepatozoon species was previously identified as closely related to H. canis

Babesia spp. infection in dogs from rural areas of São Paulo State, Brazil

The status of Babesia spp. infection in dogs from rural areas of São Paulo State, Brazil was Studied. For this, l 50 animals were examined by blood smears and by PCR; the presence of tick infestation was also investigated. By the blood smear examination, 3 animals (2%) were detected positive and by PCR for Babesia spp. 12 (8%) were positive, with bands Visualized in 450 bp. Rhipicephalus sanguineus or Amblyomma spp. were found on 36 (24%) of the 150 dogs. Amblyomma species found were A. cajennense (9/36-25%) and A. ovale (9/36-25%). It was not possible to correlate the presence of R. sanguineus and the infection with Babesia spp. The sequencing of four positive samples demonstrated close identity with B. canis vogeli already characterized in Brazil.A presença de infecção por Babesia spp. em cães de áreas rurais do Estado de São Paulo, Brasil foi investigada. Para tanto, 150 cães foram examinados por técnicas parasitológicas de esfregaços sanguíneos e moleculares (PCR), e também, foi verificada a presença de carrapatos nestes animais. Pela análise de esfregaços sanguíneos, 3 (2%) dos cães estavam infectados, enquanto pela PCR, 12 (8%) dos animais foram positivos com bandas aproximadas de 450 pares de base (pb). Foram observados 36 (24%) cães infestados com Rhipicephalus sanguineus ou com Amblyomma spp. As espécies de Amblyomma observadas foram A. cajennense (25%) e A. ovale (25%). Não foi possível correlacionar a presença de R. sanguineus com a infecção por Babesia spp. O seqüenciamento de quatro amostras positivas demonstrou alta identidade com B. canis vogeli, já caracterizada no Brasil

Seasonal population dynamics and the genetic structure of the mosquito vector Aedes aegypti in São Paulo, Brazil

Population genetic studies of insect vectors can generate knowledge to improve epidemiological studies focused on the decrease of pathogen transmission. In this study, we used nine SNPs across the Aedes aegypti genome to characterize seasonal population variations of this important dengue vector. Mosquito samples were obtained by ovitraps placed over Botucatu SP from 2005 to 2010. Our data show that, regardless of the large variation in mosquito abundance (deduced from the number of eggs obtained from ovitraps), the effective population size remained stable over the years. These results suggest that Ae. aegypti is able to maintain a sufficiently large active breeding population during the dry season to keep genetic frequencies stable. These results open new perspectives on mosquito survey and control methods.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Description of three new species of Hepatozoon (Apicomplexa, Hepatozoidae) from Rattlesnakes (Crotalus durissus terrificus) based on molecular, morphometric and morphologic characters

Hepatozoon spp. are commonly found infecting snakes. Since the latter are parasitized by diverse forms and data in the literature show divergence, we studied Hepatozoon spp. diversity on Crotalus durissus terrificus snakes using both molecular and morphological approaches. Naturally infected animals were employed. Blood was collected, blood smears were prepared and an aliquot was stored at -20. °C for DNA extraction. Five specimens of C. durissus terrificus were selected, each of them infected with one gamont type. Morphological and morphometric analyses of the found gamonts led to their grouping into three populations. For molecular characterization, seven oligonucleotide pairs that amplify distinct regions of rDNA gene were tested by adopting the PCR technique. Only the oligonucleotide pairs HepF300/Hep900 and HEMO1/HEMO2 were efficient in amplifying and distinguishing different isolates of Hepatozoon spp. from snakes. The better results were obtained when both oligonucleotide pairs were used in association. Based on the molecular and morphologic differences, three new species were proposed: Hepatozoon cuestensis sp. nov.; Hepatozoon cevapii sp. nov. and Hepatozoon massardii sp. nov. This is the first description of new Hepatozoon species from snakes, based on molecular characterization and morphological data, in South America. © 2013 Elsevier Inc

Characterization of Hepatozoon spp. in Leptodactylus chaquensis and Leptodactylus podicipinus from two regions of the Pantanal, state of Mato Grosso do Sul, Brazil

Hepatozoon sp. are parasites that commonly infect frogs and arthropod vectors. This species has variability in the morphological and morphometric characteristics. Due to these variations, the naming of the species is thus impaired and only by visualizing the sporogonic cycle in vector and by molecular studies this problem can be solved. Recently, the use of molecular genetics has helped the species denomination. In this work, we collected 145 frogs (68 Leptodactylus chaquensis and 77 Leptodactylus podicipinus) in different sampling sites, where were found 18 (26.47 %) L. chaquensis and 24 (31.17 %) L. podicipinus parasitized; besides of gamonts, schizogonic forms were also seen in animals organs. The positivity difference between the collection sites for both frog species was not significant (p=0.958). Comparing gamonts found in each species of anuran, we observed differences in morphology. The comparison in the molecular level for L. podicipinus was not possible due to small amount of blood obtained, just L. chaquensis had their parasites DNA sequenced. The amplified and sequenced samples, named HEP1 to HEP10, are presented in the phylogenetic tree as a different branch from other haemogregarines described on other hosts. Therefore, we have seen that, although the morphology and morphometry of the collected parasites at each site showed differences, the sequencing of these samples revealed identical species of Hepatozoon, and different compared to those from GenBank, thereby demonstrating that the species of Hepatozoon in L. chaquensis observed in this study probably represent a new species.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Morphological, morphometric, and molecular characterization of Hepatozoon spp. (Apicomplexa, Hepatozoidae) from naturally infected Caudisona durissa terrifica (Serpentes, Viperidae)

Hepatozoon spp. are the most frequent intracellular protozoa in snakes. Considering the variety of parasites infecting specimens of Caudisona durissa terrifica and the divergent data in literature where only two species, Hepatozoon romani and Hepatozoon capsulata, are described, the aim of this study was to morphologically, morphometrically, and molecularly characterize Hepatozoon spp. from some naturally infected specimens of C. durissa terrifica, and observe changes caused by these protozoa in parasitized erythrocytes. Four snakes were examined. Two of them had two morphological distinct gamonts, while the other two had only one type of gamont. The six distinct gamonts were provisionally named gamonts A, B, C, D, E, and F. Statistical analysis, however, confirmed the existence of only four parasite populations, those which were capable of inducing significant alterations in determined red blood cells variables. Attempts to infect Aedes aegypti and Culex quinquefasciatus mosquitoes were done for each snake specimen. Some mosquitoes became infected and oocysts were recovered and measured. The detection of Hepatozoon DNA was obtained with success but the molecular characterization was unable to differentiate species of the samples, with respect to the fragment studied.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES