Genetic similarities and phylogenetic analysis of Muntjac (Muntiacus spp. ) by comparing the nucleotide sequence of 16S rRNA and cytochrome B genome / Semelhanças genéticas e análise filogenética de muntjac (Muntiacus spp. ) comparando a sequência de nucleótidos de rRNA 16S e genoma citocromo B

This study aimed to identify the phylogenetic similarities among the muntjac (Muntiacus spp.). The phylogenetic similarities among seven major muntjac species were studied by comparing the nucleotide sequence of 16s rRNA and cytochrome b genome. Nucleotide sequences, retrieved from NCBI databases were aligned by using DNASTAR software. A phylogenetic tree was created for the selected species of muntjac by using the maximum likelihood method on MEGA7 software. The results of nucleotide sequences (16s rRNA) showed phylogenetic similarities between, the M. truongsonensis and M. rooseveltorum had the highest (99.2%) while the lowest similarities (96.8%) found between M. crinifrons and M. putaoensi. While the results of nucleotide sequences (Cty b) showed the highest similarity (100%) between M. muntjak and M. truongsonensis and the lowest s (91.5%) among M. putaoensis and M. crinifrons. The phylogenetic tree of muntjac species (16s rRNA gene) shows the main two clusters, the one including M. putaoensis, M. truongsonensis, M. rooseveltorum, and M. muntjak, and the second one including M. crinifrons and M. vuquangensis. The M. reevesi exists separately in the phylogenetic tree. The phylogenetic tree of muntjac species using cytochrome b genes shows that the M. muntjak and M. truongsonensis are clustered in the same group.

Este estudo visou identificar as semelhanças filogenéticas entre os muntjac (Muntiacus spp.). As semelhanças filogenéticas entre sete grandes espécies muntjac foram estudadas comparando a sequência de nucleótidos de 16s rRNA e genoma citocromo b. As sequências de nucleótidos, obtidas a partir de bases de dados NCBI, foram alinhadas utilizando o software DNASTAR. Foi criada uma árvore filogenética para as espécies selecionadas de muntjac utilizando o método de probabilidade máxima no software MEGA7. Os resultados das sequências de nucleótidos (16s rRNA) mostraram semelhanças filogenéticas entre o M. truongsonensis e o M. rooseveltorum tiveram o maior número (99,2%) enquanto as semelhanças mais baixas (96,8%) encontradas entre M. crinifrons e M. putaoensi. Enquanto os resultados das sequências de nucleótidos (Cty-b) apresentaram a maior semelhança (100%) entre M. muntjak e M. truongsonensis e os mais baixos (91,5%) entre M. putaoensis e M. crinifrons. A árvore filogenética das espécies muntjac (gene rRNA 16s) mostra os dois principais aglomerados, o que inclui M. putaoensis, M. truongsonensis, M. rooseveltorum e M. muntjak, e o segundo incluindo M. crinifrons e M. vuquangensis. O M. reevesi existe separadamente na árvore filogenética. A árvore filogenética das espécies muntjac usando genes citocromo b mostra que os M. muntjak e M. truongsonensis estão agrupados no mesmo grupo.

Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene

BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

Evaluation of cytochrome b sequence to identify Leishmania species and variants: the case of Panama

BACKGROUND The genetic heterogeneity of Leishmania parasites is a major factor responsible for the wide variety of Leishmania-associated manifestations. Consequently, understanding the genetic make-up of Leishmania species using suitable molecular markers is an important component of realising local and regional scale disease risk. The cytochrome b (cytb) is frequently used to type New World Leishmania species. However, its potential to discriminate Leishmania species and variants requires further evaluation. OBJECTIVES To explore the capacity of cytb gene to identify New World Leishmania species and variants and to develop an approach able to type local Leishmania species and variants. METHODS We retrieved 360 partial and complete Leishmania cytb gene sequences publicly available in GenBank database to study all single nucleotide polymorphisms (SNPs) across the cytb gene that differentiate New World Leishmania species. This information was used to develop an approach based upon the polymorphisms found in a DNA segment of 948bp. We also compared the typing results found with this technique with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) profiling obtained using HSP70 gene as target. One hundred Panamanian isolates were used to both typed Leishmania species and assess local genetic variability. FINDINGS We found complete agreement between our cytb approach and the PCR-RFLP profiling method based on HSP70 for Leishmania species identification. Ninety-two isolates were identified as L. panamensis, although other Viannia species were found circulating at a lower frequency. Three L. panamensis haplotypes were identified in Panamanian provinces. We also provide an initial report of L. guyanensis haplotypes circulating in Panama. MAIN CONCLUSIONS Cytb gene sequence encompasses key main SNPs that aid to identify Leishmania species. The cytb approach developed with this information was able to identify and assess genetic variability of local Leishmania species found in this study.

Allergens of the urushiol family promote mitochondrial dysfunction by inhibiting the electron transport at the level of cytochromes b and chemically modify cytochrome c1

BACKGROUND: Urushiols are pro-electrophilic haptens that cause severe contact dermatitis mediated by CD8+ effector T-cells and downregulated by CD4+ T-cells. However, the molecular mechanism by which urushiols stimulate innate immunity in the initial stages of this allergic reaction is poorly understood. Here we explore the sub-cellular mechanisms by which urushiols initiate the allergic response. RESULTS: Electron microscopy observations of mouse ears exposed to litreol (3-n-pentadecyl-10-enyl-catechol]) showed keratinocytes containing swollen mitochondria with round electron-dense inclusion bodies in the matrix. Biochemical analyses of sub-mitochondrial fractions revealed an inhibitory effect of urushiols on electron flow through the mitochondrial respiratory chain, which requires both the aliphatic and catecholic moieties of these allergens. Moreover, urushiols extracted from poison ivy/oak (mixtures of 3-n-pentadecyl-8,11,13 enyl/3-n-heptadecyl-8,11 enyl catechol) exerted a higher inhibitory effect on mitochondrial respiration than did pentadecyl catechol or litreol, indicating that the higher number of unsaturations in the aliphatic chain, stronger the allergenicity of urushiols. Furthermore, the analysis of radioactive proteins isolated from mitochondria incubated with 3H-litreol, indicated that this urushiol was bound to cytochrome c1. According to the proximity of cytochromes c1 and b, functional evidence indicated the site of electron flow inhibition was within complex III, in between cytochromes bL (cyt b566) and bH (cyt b562). CONCLUSION: Our data provide functional and molecular evidence indicating that the interruption of the mitochondrial electron transport chain constitutes an important mechanism by which urushiols initiates the allergic response. Thus, mitochondria may constitute a source of cellular targets for generating neoantigens involved in the T-cell mediated allergy induced by urushiols.

Técnicas moleculares integradas a la vigilancia entomológica de vectores de la enfermedad de Chagas: Estudio del vector secundario Triatoma sordida en la Región Oriental del Paraguay / Molecular techniques integrated to the entomological surveillance of Chagas disease vectors: Study of the secondary vector Triatoma sordida in the Eastern Region of Paraguay

Las técnicas moleculares para la detección de infección natural y fuente de alimentación en vectores secundarios de la enfermedad de Chagas cuando son aplicadas a ejemplares capturados en áreas endémicas, históricamente ocupadas por Triatoma infestans, proporcionan a las investigaciones epidemiológicas respuestas más exactas con relación a la transmisibilidad de la enfermedad. El presente estudio tiene como objetivo emplear biomarcadores moleculares para evaluar el impacto de la infestación intra y peridomicilar de Triatoma sordida en viviendas bajo vigilancia entomológica de departamentos de la Región Oriental del Paraguay en el período 2007 al 2015. Un total de 559 ejemplares de T. sordida capturados en 253, 91 y 52 viviendas de los departamentos Paraguarí, San Pedro y Cordillera, respectivamente fueron analizados. La infestación detectada fue del 24% al 48% así como una elevada colonización intradomiciliar del 5% al 36% en los tres departamentos. La detección molecular de infección natural osciló entre el 14% y 44%; y en 111 ejemplares se determinó la fuente de alimentación. El marcador molecular citocromo b permitió demostrar por vez primera un elevado porcentaje de triatominos con sangre humana como fuente de alimentación, principalmente en Cordillera con un 82% (28/34 T. sordida capturados). Estos hallazgos dejan en evidencia el avance del T. sordida en la ocupación del nicho ecológico de T. cruzi y la capacidad de esta especie secundaria como vector en la transmisión de T. cruzi en comunidades de la Región Oriental

When molecular techniques for the detection of natural infection and blood meal source in secondary vectors of Chagas disease are applied to specimens captured in endemic areas, historically occupied by Triatoma infestans, provide more accurate answers to questions about transmissibility of the illness and further contribute to the epidemiological studies. The aim of this study was to evaluate the impact of intra and peridomiciliary infestation of Triatoma sordida in households from the departments of the Eastern Region of Paraguay, under entomological surveillance during the period 2007 to 2015, by using the molecular biomarkers technology. A total of 559 specimens of T. sordida captured in 253, 91 and 52 households from Paraguarí, San Pedro and Cordillera departments, respectively, were analyzed. The infestation detected was from 24% to 48% as well as a high intradomicialiary colonization from 5% to 36% in the three departments. The molecular detection of natural infections ranged from 14% to 44% and in 111 specimens the meal source was identified. The molecular marker cytochrome b allowed to demonstrate, for the first time, high frequency of triatomines with human blood as a food source, mainly in Cordillera as it was determined in 82% (28/34) of the T. sordida captured. These findings demonstrate a progress of T. sordida into the ecological niche of T. cruzi and the abillity of this secondary species as a vector of the transmission of T. cruzi in communities from the Eastern Region of Paraguay

Morphological and molecular characterization of Haemoproteus coatneyi and Haemoproteus erythrogravidus (Haemosporida: Haemoproteidae) in Passeriformes in Brazils Atlantic Forest / Caracterização morfológica e molecular de Haemoproteus coatneyi e Haemoproteus erythrogravidus (Haemosporida: Haemoproteidae) em Passeriformes da Mata Atlântica, Brasil

Abstract Haemoproteus spp. are protozoan parasites found in birds around the world. These parasites are identified through the morphology of gametocytes, phylogenetic analysis based on the mitochondrial cytb gene, and the parasite's geographic distribution. The absence of erythrocytic merogony, high intraspecific genetic variation and low parasitemia in wild birds makes it essential to use integrative approaches that assist in the identification of these parasites. Thus, microscopic and molecular analyses, combined with spatial distribution, were carried out to verify the presence of Haemoproteus spp. in wild birds in Brazil. Light microscopy revealed one Tangara sayaca bird was parasitized by Haemoproteus coatneyi and, two specimens of Zonotrichia capensis presented Haemoproteus erythrogravidus. The morphology of the gametocytes of these two parasitic species showed high similarity. The molecular analysis revealed the presence of one lineage of H. coatneyi and two lineages of H. erythrogravidus, one of which is considered a new lineage. These lineages were grouped phylogenetically in separate clades, with low genetic divergence, and the H. erythrogravidus lineage emerged as an internal group of the lineages of H. coatneyi. The geographic distribution demonstrated that the two species occur in the American continent. This is the first report of H. erythrogravidus in Brazil.

Resumo Haemoproteus spp. são protozoários parasitos encontrados em aves de todo o mundo. A identificação desses parasitos é realizada por meio da morfologia dos gametócitos, da análise filogenética, baseada no gene mitoncodrial cytb e na distribuição geográfica do parasito. A ausência de merogonia eritrocítica, a alta variação genética intraespecífica e a baixa parasitemia em aves silvestres, tornam essencial a utilização de abordagens integrativas que auxiliem na identificação desses parasitos. Assim, análises microscópicas e moleculares, aliadas à distribuição espacial, foram realizadas para verificar a presença de Haemoproteus spp. em aves silvestres no Brasil. A microscopia óptica demonstrou que uma ave Tangara sayaca estava parasitada por Haemoproteus coatneyi, e dois espécimes de Zonotrichia capensis apresentavam Haemoproteus erythrogravidus, cujas morfologias dos gametócitos apresentaram alta similaridade. A análise molecular recuperou uma linhagem de H. coatneyi e duas linhagens de H. erythrogravidus, sendo uma dessas considerada nova linhagem. Essas linhagens se agruparam filogeneticamente em clados separados, apresentando baixa divergência genética, sendo que as linhagens de H. erythrogravidus emergiram como grupo interno às linhagens de H. coatneyi. A distribuição geográfica demonstrou que as duas espécies estão ocorrendo no continente americano. Este é o primeiro relato de H. erythrogravidus no Brasil.

Estimación del tiempo límite de detección del gen citocromo b de humanos en hembras de Lutzomyia evansi / Estimation of time detection limit for human cytochrome b in females of Lutzomyia evansi

Resumen Introducción. Las técnicas de biología molecular han permitido ampliar el conocimiento sobre las fuentes de ingestión de sangre de los insectos vectores. Sin embargo, la utilidad de estas técnicas depende de la cantidad de sangre ingerida y del proceso de digestión en el insecto. Objetivo. Determinar el tiempo límite de detección del gen citocromo b (Cyt b) de humanos en hembras de Lutzomyia evansi alimentadas experimentalmente. Materiales y métodos. Se evaluaron ocho grupos de hembras de L. evansi alimentadas con sangre humana, las cuales fueron sacrificadas en intervalos de 24 horas desde el momento de la ingestión sanguínea. Se extrajo el ADN total de cada hembra y se amplificó un segmento de 358 pb del gen Cyt b. Los productos amplificados fueron sometidos a un análisis de polimorfismos en la longitud de los fragmentos de restricción (Restriction Fragment Length Polymorphism, RFLP), con el fin de descartar falsos positivos. Resultados. El segmento del gen Cyt b de humanos fue detectado en 86 % (49/57) de las hembras de L. evansi a partir de las 0 horas y hasta 168 horas después de la ingestión de sangre. En 7 % (4/57) de los individuos se amplificó el ADN del insecto y en el 7 % restante no se amplificó la banda de interés. No se encontraron diferencias estadísticas en cuanto a la amplificación del segmento del gen Cyt b de humanos ni al número de muestras amplificadas entre los grupos de hembras sacrificadas a distintas horas después de la ingestión. Conclusión. El segmento del gen Cyt b de humanos fue detectable en hembras de L. evansi hasta 168 horas después de la ingestión de sangre.

Abstract Introduction: Molecular biology techniques have allowed a better knowledge of sources of blood meals in vector insects. However, the usefulness of these techniques depends on both the quantity of ingested blood and the digestion process in the insect. Objective: To identify the time limit for detection of the human cytochrome b (Cyt b) gene in experimentally fed females of Lutzomyia evansi. Materials and methods: Eight groups of L. evansi females were fed on human blood and sacrificed at intervals of 24 hours post-ingestion. Total DNA was extracted from each female and a segment of 358 bp of Cyt b was amplified. In order to eliminate false positives, amplification products were subjected to a restriction fragment length polymorphism (RFLP) analysis. Results: The human Cyt b gene segment was detected in 86% (49/57) of the females of L. evansi, from 0 to 168 hours after blood ingestion. In 7% (4/57) of the individuals we amplified insect DNA, while in the remaining 7%, the band of interest was not amplified. We did not find any statistical differences between groups of females sacrificed at different times post-blood meal regarding the amplification of the human Cyt b gene segment or the number of samples amplified. Conclusion: The human Cyt b gene segment was detectable in L. evansi females up to 168 hours after blood ingestion.

Phylogenetic signal at the Cytb-SertRNA-IG1-ND1 mitochondrial region in Anopheles (Kerteszia) neivai Howard, Dyar & Knab, 1913 / Señal filogenética de la región Cytb- SertRNA -IG1-ND1 en Anopheles (Kerteszia) neivai Howard, Dyar & Knab, 1913

Abstract Introduction: Mitochondrial DNA has proven its utility for the study of insect evolution. Genes such as cytochrome b (Cytb) and the transfer gene for serine (SertRNA) can be used to compare closely related organisms. Objective: The phylogenetic utility of Cytb-SertRNA-IG1-ND1 was tested for polymorphisms, and secondary structure modeling in SertRNA was done to detect possible cryptic species in Anopheles neivai. Materials and methods: Specimens from Colombia, Guatemala, and the type locality in Panamá were collected and sequenced for specimen comparison based on DNA polymorphisms, and secondary structure modeling for the SertRNA gene. Results: Thirty-six sequences for A. neivai and A. pholidotus were obtained. Conclusions: Polymorphic variants were detected in A. neivai for Cytb-SertRNA-IG1- ND1. Despite this variation in A. neivai, cryptic species could not be detected.

Resumen Introducción. El ADN mitocondrial ha demostrado su utilidad para el estudio de la evolución en los insectos. Existen algunos genes mitocondriales como el citocromo b (Cytb) y el gen de transferencia para el aminoácido serina (SertRNA) que pueden usarse en el diagnóstico de especies estrechamente relacionadas. Objetivo. Explorar la utilidad filogenética de la región Cytb-SertRNA-IG1-ND1 para detectar posibles especies crípticas en Anopheles neivai. Materiales y métodos. Se recolectaron especímenes en Colombia, Guatemala y en la localidad tipo en Panamá, los cuales se secuenciaron y se compararon mediante el polimorfismo de ADN en toda la región y mediante la simulación de estructuras secundarias del gen SertRNA. Resultados. Se obtuvieron las secuencias de especímenes de A. neivai (34) y A. pholidotus (2). Conclusiones. Se detectaron algunos polimorfismos para la regiónCytb-SertRNA-IG1-ND1 en A. neivai, pero no así especies crípticas.

Assessing the mitochondrial DNA diversity of the Chagas disease vector Triatoma sordida (Hemiptera: Reduviidae)

Triatoma sordida is a species that transmits Trypanosoma cruzi to humans. In Brazil, T. sordida currently deserves special attention because of its wide distribution, tendency to invade domestic environments and vectorial competence. For the planning and execution of control protocols to be effective against Triatominae, they must consider its population structure. In this context, this study aimed to characterise the genetic variability of T. sordida populations collected in areas with persistent infestations from Minas Gerais, Brazil. Levels of genetic variation and population structure were determined in peridomestic T. sordida by sequencing a polymorphic region of the mitochondrial cytochrome b gene. Low nucleotide and haplotype diversity were observed for all 14 sampled areas; π values ranged from 0.002-0.006. Most obtained haplotypes occurred at low frequencies, and some were exclusive to only one of the studied populations. Interpopulation genetic diversity analysis revealed strong genetic structuring. Furthermore, the genetic variability of Brazilian populations is small compared to that of Argentinean and Bolivian specimens. The possible factors related to the reduced genetic variability and strong genetic structuring obtained for studied populations are discussed in this paper.

Diagnostic Cytochrome b gene profiles for the identification of paca (Cuniculus paca) bushmeat: implications for the monitoring of illegal hunting and wildlife trade / Perfis diagnósticos do gene Citocromo b para a identificação molecular de paca (Cuniculus paca): implicações para a detecção e monitoramento de caça e comércio ilegal

Abstract Paca (Cuniculus paca Linnaeus, 1766) is the second largest rodent found in Brazil. The quality of the meat and a long tradition of hunting have contributed to the decline of the natural populations of this species. Hunting of paca is strictly prohibited in Brazil, but in spite of this restriction, no forensic tools are available for the identification of the meat. We describe an efficient method, based on single nucleotide polymorphisms of the cytochrome b gene, that can be used to differentiate biological material derived from paca from those of domestic species commonly used as sources of meat. The identification of the presence of C. paca in the samples was 100% reliable.

Resumo Paca (Cuniculus paca Linnaeus, 1766) é o segundo maior roedor brasileiro. A qualidade da carne e a forte tradição da caça de subsistência são fatores que contribuem significativamente para o declínio das populações. Apesar da proibição a caça no Brasil, no momento ainda não há ferramentas disponíveis para identificar a carne e seus produtos como prova forense. Neste trabalho propomos um método eficaz de identificação, baseado em polimorfismos de único nucleotídeo no gene Citocromo b, objetivando diferenciar material biológico de paca das espécies domésticas comumente utilizadas como alimento no Brasil. A identificação das amostras de paca foram possíveis em 100% das amostras analisadas.

A sensitive, specific and reproducible real-time polymerase chain reaction method for detection of Plasmodium vivax and Plasmodium falciparum infection in field-collected anophelines

We describe a simple method for detection of Plasmodium vivax and Plasmodium falciparum infection in anophelines using a triplex TaqMan real-time polymerase chain reaction (PCR) assay (18S rRNA). We tested the assay on Anopheles darlingi and Anopheles stephensi colony mosquitoes fed with Plasmodium-infected blood meals and in duplicate on field collected An. darlingi. We compared the real-time PCR results of colony-infected and field collected An. darlingi, separately, to a conventional PCR method. We determined that a cytochrome b-PCR method was only 3.33% as sensitive and 93.38% as specific as our real-time PCR assay with field-collected samples. We demonstrate that this assay is sensitive, specific and reproducible.

PCR em tempo real para caracterização de fontes alimentares de flebotomíneos (Diptera: Psychodidae) / Real-time PCR for the characterization of feeding sources of sand flies (Diptera: Psychodidae)

Os flebotomíneos são insetos hematófagos de grande importância médica e veterinária atuando como vetores de parasitas como Leishmania. O estudo do padrão alimentar desses vetores pode ajudar a compreender a sua interação com potenciais reservatórios de Leishmania. Neste estudo, desenvolvemos ensaios de PCR em tempo real para identificação de sangue em flebotomíneos. Seis pares de primers foram desenhados com base no gene citocromo b de sequencias disponíveis no GenBank dos seguintes hospedeiros potenciais: cão, gato, cavalo, galinha, rato e humano. Primeiramente, os ensaios de PCR em tempo real utilizando SYBR Green foram conduzidos usando uma curva padrão com oito concentrações diferentes (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg e 1 fg por 2 µl) de amostras do DNA extraído do sangue com EDTA a partir de cada espécie de animal. Em seguida, o DNA foi extraído de 100 fêmeas de flebotomíneos ingurgitadas de campo pertencentes a três espécies (i.e., Lutzomyia longipalpis, L. migonei e L. lenti) foram testadas pelos protocolos aqui padronizados. Fêmeas de flebotomíneos foram experimentalmente alimentadas em um rato (Rattus rattus) e utilizadas para avaliar a detecção do ensaio. Os protocolos funcionaram de forma eficiente com limites de detecção de 10 pg a 100 fg. Fêmeas de flebotomíneos ingurgitadas coletadas no campo estavam alimentadas de humanos...

Estandarización de la técnica PCR-RFLP del gen mitocondrial cyt b como herramienta para la identificación de fuentes de alimentación de insectos hematófagos / Standardization of a PCR-RFLP technique based on RESUMEN mitochondrial cyt b gen as a tool to identify feeding sources of hematophagous insects

La identificación de la fuente de alimentación de insectos hematófagos puede proporcionar información sobre la capacidad vectorial, patrones de alimentación en condiciones naturales y proveer indirectamente datos sobre probables reservorios de enfermedades. Varias técnicas de identificación son empleadas, entre ellas las más utilizadas son las basadas en reacciones antígeno-anticuerpo. Actualmente, se han desarrollado ensayos moleculares, algunos de ellos permiten detectar e identificar solo sangre humana. Otros como la reacción en cadena de la polimerasa (PCR) del gen mitocondrial citocromo b (cyt b), que ha mostrado alto grado de sensibilidad y especificidad, permite detectar e identificar otras especies de vertebrados. El objetivo del trabajo fue estandarizar la técnica PCR-RFLP del gen mitocondrial citocromo b (cyt b) para determinar la fuente de alimentación sanguínea de insectos. Inicialmente se realizó un análisis bioinformático para la búsqueda y alineamiento de secuencias del gen cyt b de los potenciales huéspedes, con secuencias que están disponible en el GenBank. Se utilizaron 10 muestras de sangre de potenciales huéspedes vertebrados (humano, perro, gallina y roedor) y para la reacción de PCR se empleó un par de cebadores universales que amplifican una región del gen cyt b, seguido de cortes con dos enzimas de restricción (RFLP) (Hae III y Mwo I), generando patrones de electroforesis específicos para los diferentes vertebrados. Se logró la estandarización de la técnica de PCR del gen cyt b que fue capaz de detectar ADN de al menos 1 μL de sangre observándose el producto de amplificación de 358 pb. El análisis de los patrones de bandas obtenidos con el corte de las enzimas mostró los tamaños de fragmentos esperados para humano, gallina, perro y roedor. Estos resultados muestran la utilidad de la técnica PCR-RFLP del gen cyt b que, con un simple par de cebadores seguido del corte con dos enzimas de restricción...

Identification of feeding sources of hematophagous insects can provide informationabout the vectorial capacity,feeding patternsin natural conditionsand indirectlyprovidedataon possible disease reservoirs preferences.Several identificationtechniquesare used,most of them based on antigen-antibody reactions.Recently molecular assayshave been developedand, some ofthese assayscandetect andidentificateonlyhuman blood. Otherassays,likethepolymerase chain reaction (PCR)basedonmitochondrialcytochromebgene,have shown high sensitivity and specificityallowingdetectionand identificationofothervertebrate species. The aim of this studywas tostandardizea PCR-RFLP basedonmitochondrialcytochrome bgene(cyt b) inorder to determineblood mealfrom insects.Initially bioinformatic analysiswasperformed forsearching andalignmentofcyt bsequencesofpotential hosts availableattheGenBankdatabase.Blood samplesfrom potentialvertebrate hostswere usedand PCR was performed using specificprimersthat amplify aregionofcyt bgene. Theproducts were digestedwithrestriction enzymes (RFLP),generating specificelectrophoresis patterns forseveral vertebrates. The PCR technique forcyt bgene wasstandardized allowing the detection of at least 1μLof blood.The 359 bp band wascorrectly amplified and the profiles obtained after the enzyme digestion withHaeIIIandMwoI were the expected for human, chicken, dog and rodents. These resultsshowed the usefulness of the PCR-RFLP ofcyt bgenethat,with a single pair of primersfollowed digestionusing two restriction enzymes,allowed the differentiationof thevertebrate species of our interestthrough the patterns obtained without sequencinghavingalso the advantage of detecting small volumesof blood sample.

Leishmaniosis cutanea: manifestación clínica inusual / Cutaneous leishmaniosis: unusual clinical manifestation

Las manifestaciones clínicas de la leishmaniosis son variables y están relacionadas con la especie infectante, su relación con el medioambiente y con la respuesta inmune del hospedero. Se presenta un caso de leishmaniosis andina cutánea tardía con una manifestación extensa. El caso se confirmó a través de estudios microbiológicos e inmunológicos, la identificación se realizó mediante secuenciamiento del gen del citocromo b, determinándose la especie como Leishmania (Leishmania) amazonensis. La paciente recibió tratamiento con estibogluconato sódico y al término de la terapia, mostró mejoría clínica de las lesiones. Se recomienda considerar a la leishmaniosis en el diagnóstico diferencial cuando se atienda ulceras crónicas dermatológicas atípicas...

Clinical manifestations of leishmaniasis are diverse and related to the infecting species, its relationship with the environment and the host immune response. A case of late Andean cutaneous leishmaniasis with extensive manifestation is presented. The case was confirmed through microbiological and immunological studies; identification was performed by cytochrome b gene sequencing and the species was determined as Leishmania (Leishmania) amazonensis. The patient was treated with sodium stibogluconate and at the end of therapy the patient showed clinical improvement of the lesions. It is recommended to consider leishmaniasis in differential diagnosis when treating atypical dermatological chronic ulcers...

Mesoamerican tree squirrels evolution (Rodentia: Sciuridae): a molecular phylogenetic analysis / Evolución de las ardillas arborícolas mesoamericanas (Rodentia: Sciuridae): un análisis filogenético molecular

The tribe Sciurini comprehends the genera Sciurus, Syntheosiurus, Microsciurus, Tamiasciurus and Rheinthrosciurus. The phylogenetic relationships within Sciurus have been only partially done, and the relationship between Mesoamerican species remains unsolved. The phylogenetic relationships of the Mesoamerican tree squirrels were examined using molecular data. Sequence data publicly available (12S, 16S, CYTB mitochondrial genes and IRBP nuclear gene) and cytochrome B gene sequences of four previously not sampled Mesoamerican Sciurus species were analyzed under a Bayesian multispecies coalescence model. Phylogenetic analysis of the multilocus data set showed the neotropical tree squirrels as a monophyletic clade. The genus Sciurus was paraphyletic due to the inclusion of Microsciurus species (M. alfari and M. flaviventer). The South American species S. aestuans and S. stramineus showed a sister taxa relationship. Single locus analysis based on the most compact and complete data set (i.e. CYTB gene sequences), supported the monophyly of the South American species and recovered a Mesoamerican clade including S. aureogaster, S. granatensis and S. variegatoides. These results corroborated previous findings based on cladistic analysis of cranial and post-cranial characters. Our data support a close relationship between Mesoamerican Sciurus species and a sister relationship with South American species, and corroborates previous findings in relation to the polyphyly of Microsciurus and Syntheosciurus’ paraphyly. Rev. Biol. Trop. 62 (2): 649-657. Epub 2014 June 01.

La tribu Sciurini comprende los géneros Sciurus, Syntheosciurus, Microsciurus, Tamiasciurus y Rheinthrosciurus. Las relaciones filogenéticas de Sciurus han sido resueltas parcialmente mientras que las relaciones de las especies Mesoamericanas permanecen sin resolverse. Las relaciones filogenéticas de las ardillas arborícolas mesoamericanas fueron estudiadas empleando datos moleculares. Datos de secuencias disponibles de forma pública (genes mitocondriales CYTB, 12S, 16S y gen nuclear IRBP) en conjunto con secuencias nuevas para el gen del Citocromo B de 4 especies mesoamericanas del genero Sciurus, fueron analizadas empleando un modelo bayesiano de coalescencia multi-especie. Los análisis filogenéticos del conjunto de datos multilocus mostraron que las especies neotropicales forman un clado monofilético. El género Sciurus resulto ser parafilético debido a la inclusión de las especies de Microsciurus (M. alfari y M. flaviventer). Las especies suramericanas S. aestuans y S. stramineus presentaron una relación de especies hermanas. El análisis de un solo locus basado en el conjunto de datos más compacto y completo (secuencias del gen del citocromo B), apoyó la naturaleza monofilética de las especies suramericanas y recuperó un clado mesoamericano que incluye a S. aureogaster, S. granatensis y S. variegatoides. Estos resultados corroboran los descubrimientos previos que emplearon datos morfológicos craneales y pos-craneales. Nuestros datos apoyan la relación cercana entre las especies de Sciurus Mesoamericanas y la relación hermana de estas con las especies de Suramérica, así como también corroboran la relación polifilética de Microsciurus y parafilética de Syntheosciurus previamente reportadas.

Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene

An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

Genetic variation in the Cytb gene of human cerebral Taenia solium cysticerci recovered from clinically and radiologically heterogeneous patients with neurocysticercosis

Neurocysticercosis (NC) is a clinically and radiologically heterogeneous parasitic disease caused by the establishment of larval Taenia solium in the human central nervous system. Host and/or parasite variations may be related to this observed heterogeneity. Genetic differences between pig and human-derived T. solium cysticerci have been reported previously. In this study, 28 cysticerci were surgically removed from 12 human NC patients, the mitochondrial gene that encodes cytochrome b was amplified from the cysticerci and genetic variations that may be related to NC heterogeneity were characterised. Nine different haplotypes (Ht), which were clustered in four haplogroups (Hg), were identified. Hg 3 and 4 exhibited a tendency to associate with age and gender, respectively. However, no significant associations were found between NC heterogeneity and the different T. solium cysticerci Ht or Hg. Parasite variants obtained from patients with similar NC clinical or radiological features were genetically closer than those found in groups of patients with a different NC profile when using the Mantel test. Overall, this study establishes the presence of genetic differences in the Cytb gene of T. solium isolated from human cysticerci and suggests that parasite variation could contribute to NC heterogeneity. .

Rhodnius barretti, a new species of Triatominae (Hemiptera: Reduviidae) from western Amazonia

Rhodnius barretti , a new triatomine species, is described based on adult specimens collected in rainforest environments within the Napo ecoregion of western Amazonia (Colombia and Ecuador). R. barretti resembles Rhodnius robustus s.l. , but mitochondrial cytochrome b gene sequences reveal that it is a strongly divergent member of the “robustus lineage”, i.e., basal to the clade encompassing Rhodnius nasutus , Rhodnius neglectus , Rhodnius prolixus and five members of the R. robustus species complex. Morphometric analyses also reveal consistent divergence from R. robustus s.l. , including head and, as previously shown, wing shape and the length ratios of some anatomical structures. R. barretti occurs, often at high densities, in Attalea butyracea and Oenocarpus bataua palms. It is strikingly aggressive and adults may invade houses flying from peridomestic palms. R. barretti must therefore be regarded as a potential Trypanosoma cruzi vector in the Napo ecoregion, where Chagas disease is endemic.

Natural Leishmania sp. reservoirs and phlebotomine sandfly food source identification in Ibitipoca State Park, Minas Gerais, Brazil

Leishmania spp are distributed throughout the world and different species are associated with varying degrees of disease severity. However, leishmaniasis is thought to be confined to areas of the world where its insect vectors, sandflies, are present. Phlebotomine sandflies obtain blood meals from a variety of wild and domestic animals and sometimes from humans. These vectors transmit Leishmania spp, the aetiological agent of leishmaniasis. Identification of sandfly blood meals has generally been performed using serological methods, although a few studies have used molecular procedures in artificially fed insects. In this study, cytochrome b gene (cytB) polymerase chain reaction (PCR) was performed in DNA samples isolated from 38 engorged Psychodopygus lloydi and the expected 359 bp fragment was identified from all of the samples. The amplified product was digested using restriction enzymes and analysed for restriction fragment length polymorphisms (RFLPs). We identified food sources for 23 females; 34.8% yielded a primate-specific banding profile and 26.1% and 39.1% showed banding patterns specific to birds or mixed restriction profiles (rodent/marsupial, human/bird, rodent/marsupial/human), respectively. The food sources of 15 flies could not be identified. Two female P. lloydi were determined to be infected by Leishmania using internal transcribed spacer 1 and heat shock protein 70 kDa PCR-RFLP. The two female sandflies, both of which fed on rodents/marsupials, were further characterised as infected with Leishmania (Viannia) braziliensis. These results constitute an important step towards applying methodologies based on cytB amplification as a tool for identifying the food sources of female sandflies.