El género Attalea (Arecaceae) de Bolivia: Afinidades con sistemas ecológicos regionales

La documentación de las especies neotropicales de la familia Arecaceae, basada en los recientes aportes a su taxonomía y su relación con los paisajes naturales, actualiza los patrones espaciales a los cuales se adaptan en su rango de distribución. En este caso se relevan 121 registros de especímenes de las 11 especies del género Attalea de Bolivia y su relación con 30 sistemas ecológicos que aproximan su ámbito de distribución a nivel regional. Para ello se sistematizó, se verificó y corrigieron las coordenadas geográficas vs. localidades de todos los especímenes coleccionados del género Attalea con el fin de cotejarlos con los sistemas ecológicos, utilizando las herramientas del ArgGis. Seguidamente elaboramos un dendrograma (especies vs. sistemas ecológicos) utilizando el método de distancia mínima en el programa R. El análisis de la relación de las especies con los sistemas ecológicos resalta una especie que no compone al sudoeste amazónico: A. eichleri y que procede de sistemas ecológicos del Cerrado. Entre las especies de Attalea amazónicas, A. blepharopus (endémica de Bolivia) se aísla de las demás y el resto subagrupa a especies según su presencia afín en bosques y sabanas, además del subandino y aluvial, como es para A. princeps, que se encuentra en 17 sistemas (57%). Ocho especies de Attalea son comunes con Perú y 10 con Brasil. Es importante relacionar la agrupación jerárquica de las especies de Attalea con los sistemas ecológicos en función a dinámicas paisajísticas para documentar sus patrones de espacio y también para su conservación.

The documentation of the Neotropical species of the Arecaceae family, based on the recent contributions to its taxonomy and its relationship with natural landscapes, updates the spatial patterns to which they adapt in their range of distribution. In this case 121 records of specimens of the 11 species of the genus Attalea of Bolivia and their relationship with 30 ecological systems that approximate their scope of distribution at regional level are released. To this end, the geographical coordinates were systematized, verified and corrected. Localities of all the specimens collected from the genus Attalea in order to compare them with ecological systems, using the ArgGis tools. We then elaborate a dendrogram (species vs. ecological systems) using the minimum distance method in the R program. The analysis of the relation of the species with the ecological systems highlights a species that does not compose to the southwest amazon: A. eichleri and that is native to ecological systems of the Cerrado. Among the SW Amazonian Attalea species, A. blepharopus (endemic to Bolivia) is isolated from the others and the rest subgroup species according to their presence in forests and savannas, in addition to the subandean and alluvial, as it is for A. princeps, which is found in 17 systems (57%). Eight species of Attalea are common with Peru and 10 with Brazil. It is important to relate the hierarchical grouping of the Attalea species with ecological systems in function of landscape dynamics to document their space patterns and also for their conservation.

Genes con islas CpG amplificados con la mezcla formamida, albúmina sérica bovina y dimetilsulfóxido / CG-rich genes amplified with the formamide, bovine seric albumin y dimethylsulfoxide mix / Genes com Ilhas CpG amplificados com a mistura formamida, albumina sérica bovina, e dimetilsulfóxido

Diversos aditivos químicos han sido utilizados para garantizar la polimerización de genes con islas CpG. El objetivo de este trabajo fue diseñar una mezcla potenciadora de PCR para amplificar genes con islas CpG. Con ese fin se analizaron fragmentos de los genes IRS2 y HNF1a con el programa EMBOSS CpG Report. Los iniciadores se diseñaron con el programa Primer 3 y se analizaron con el programa e-PCR. Se usaron tres aditivos químicos: Albúmina sérica bovina (0,1µg/µL), dimetilsulfóxido (5%) y formamida (5%) para 5 ensayos de PCR: dos usando un solo aditivo, dos combinando dos aditivos y uno combinando tres aditivos. Las amplificaciones con las mezclas se realizaron con las enzimas Taq Nativa, taq Recombinante y Taq Platinum. La calidad de los amplicones se probó por secuenciación. Fragmentos sin islas CpG (HNF-1a) amplificaron con las tres enzimas, sin el uso de los aditivos pero presentaron problemas de pureza en la secuenciación. Los fragmentos del gen IRS2 con islas CpG amplificaron sólo con la combinación de tres aditivos dimetilsulfóxido, albúmina sérica bovina y formamida, independientemente de la enzima usada, las secuencias fueron limpias. Se concluye que la mezcla de tres aditivos es una solución que permite obtener amplicones de alta calidad en genes con islas CpG, con cromatogramas limpios en la secuenciación.

Several chemical additives have been used to assure polymerization in CpG islands.The aim of the present work was to design a PCR enhancer mixture in order to amplify GC-rich genes. Fragments of IRS2 and HNF1a genes were analyzed using EMBOSS CpG Report Software. Primers were designed with the Primer3 Software and were tested with ePCR Software. Three additives were used: BSA (0.1µg/µL), DMSO (5%) and formamide (5%), in five PCR assays, two using one additive, two combining two additives and one with all additives. DNA sequences were amplified with the following enzymes: Native Taq, recombinant Taq and platinum Taq DNA polymerase. Amplicon quality was examined by sequencing. HNF1a gene was amplified without additives; however, the sequences were not amplified and showed purity problems in sequencing. The gene fragments IRS2 with CpG islands were amplified with additives DMSO, BSA and Formamida mixture, notwithstanding the enzyme used. These sequences were clean. DMSO-BSA-Formamide mixture can be a solution to obtain GC-rich DNA amplicons with such a high quality that it generates neat chromatograms during sequencing.

Diversos aditivos químicos têm sido utilizados para garantir a polimerização de genes com ilhas CpG. O objetivo do trabalho foi desenhar uma mistura que potencie PCR para amplificar genes com ilhas CpG. Para esta finalidade foram analisados fragmentos dos genes IRS2 e HNF1a com o programa EMBOSS CpG Report. Os iniciadores foram desenhados com o programa Primer 3 e se analisaram com o programa e-PCR. Foram utilizados três aditivos químicos: Albumina sérica bovina (0,1µg/µlL, Dimetilsulfóxido (5%) e formamida (5%) para 5 ensaios de PCR: dois usando um único aditivo, dois combinando dois aditivos e um combinando três aditivos. As amplificações com as misturas se realizaram com as enzimas, Taq Nativa, taq Recombinante e Taq Platinum. A qualidade das ampliações foi provada por sequenciação. Fragmentos sem ilhas CpG (HNF-1a) amplificaram com as três enzimas, sem o uso dos aditivos porém apresentaram problemas de pureza na sequenciação. Os fragmentos do gene IRS2 com ilhas CpG ampli­ficaram apenas com a combinação de três aditivos dimetilsulfóxido, albumina sérica bovina e formamida, independentemente da enzima usada, as sequências foram limpas. A conclusão que a mistura de três aditivos é uma solução que permite obter ampliações de alta qualidade em genes com ilhas CpG, com cromatogramas limpos na secuenciação.

Molecular comparison of topotypic specimens confirms Anopheles (Nyssorhynchus) dunhami Causey (Diptera: Culicidae) in the Colombian Amazon

The presence of Anopheles (Nyssorhynchus) dunhami Causey in Colombia (Department of Amazonas) is confirmed for the first time through direct comparison of mtDNA cytochrome c oxidase I (COI) barcodes and nuclear rDNA second internal transcribed spacer (ITS2) sequences with topotypic specimens of An. dunhami from Tefé, Brazil. An. dunhami was identified through retrospective correlation of DNA sequences following misidentification as Anopheles nuneztovari s.l. using available morphological keys for Colombian mosquitoes. That An. dunhami occurs in Colombia and also possibly throughout the Amazon Basin, is of importance to vector control programs, as this non-vector species is morphologically similar to known malaria vectors including An. nuneztovari, Anopheles oswaldoi and Anopheles trinkae. Species identification of An. dunhami and differentiation from these closely related species are highly robust using either DNA ITS2 sequences or COI DNA barcode. DNA methods are advocated for future differentiation of these often sympatric taxa in South America.

Incrimination of Anopheles (Nyssorhynchus) rangeli and An. (Nys.) oswaldoi as natural vectors of Plasmodium vivax in Southern Colombia

Malaria transmission in the Southern Colombian state of Putumayo continues despite the absence of traditional vector species, except for the presence of Anopheles darlingi near the southeastern border with the state of Amazonas. In order to facilitate malaria vector incrimination in Putumayo, 2445 morphologically identified Anopheles females were tested for natural infection of Plasmodium vivax by ELISA. Specimens tested included An. apicimacula (n = 2), An. benarrochi B (n = 1617), An. darlingi (n = 29), An. mattogrossensis (n = 7), An. neomaculipalpus (n = 7), An. oswaldoi (n = 362), An. peryassui (n = 1), An. punctimacula (n = 1), An. rangeli (n = 413), and An. triannulatus (n = 6). Despite being overwhelmingly the most anthropophilic species in the region and comprising 66.1 percent of the mosquitoes tested, An. benarrochi B was not shown to be a vector. Thirty-five An. rangeli and one An. oswaldoi were naturally infected with P. vivax VK210. Sequence data were generated for the nuclear second internal transcriber space region of 31 of these 36 vivax positive mosquitoes (86.1 percent) to confirm their morphological identification. An. oswaldoi is known to be a species complex in Latin America, but its internal taxonomy remains unresolved. Herein we show that the An. oswaldoi found in the state of Putumayo is genetically similar to specimens from the state of Amapá in Brazil and from the Ocama region in the state of Amazonas in Venezuela, and that this form harbors natural infections of P. vivax. That An. rangeli and this member of the An. oswaldoi complex are incriminated as malaria vectors in Putumayo, is a novel finding of significance for malaria control in Southern Colombia, and possibly in other areas of Latin America.

Molecular differentiation of Anopheles (Nyssorhynchus) benarrochi and An. (N.) oswaldoi from Southern Colombia

Anopheles (Nyssorhynchus) benarrochi, An. (N.) oswaldoi, and An. (N.) rangeli are the most common anthropophilic mosquitoes in the southern Colombian state of Putumayo. Adult females are most commonly collected in epidemiological studies, and this stage poses significant problems for correct identification, due to overlapping inter-specific morphological characters. Although An. rangeli is easy to identify, the morphological variant of An. benarrochi found in the region and An. oswaldoi are not always easy to separate. Herein we provide a rapid molecular method to distinguish these two species in Southern Colombia. Sequence data for the second internal transcribed spacer (ITS2) region of rDNA was generated for link-reared progeny of An. benarrochi and An. oswaldoi, that had been identified using all life stages. ITS2 sequences were 540 bp in length in An. benarrochi (n = 9) and 531 bp in An. oswaldoi (n = 7). Sequences showed no intra-specific variation and ungapped inter-specific sequence divergence was 6.4 percent. Species diagnostic banding patterns were recovered following digestion of the ITS2 amplicons with the enzyme Hae III as follows: An. benarrochi (365, 137, and 38 bp) and An. oswaldoi (493 and 38 bp). This polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay provides rapid, accurate, and inexpensive species diagnosis of adult females. This will benefit future epidemiological studies and, as PCR amplification can be achieved using a single mosquito leg, the remaining specimen can be either retained as a morphological voucher or further used in vector incrimination studies. That An. benarrochi comprises a complex of at least two species across Latin America is discussed.