Development of a bioassay method to test activity of cry insecticidal proteins against Diatraea spp. (Lepidoptera: Crambidae) sugarcane stem borers.

The genus Diatraea (Lepidoptera: Crambidae) includes stem borers representing the most critical sugarcane pests in the Americas. Colombia's most widely distributed and damaging Diatraea species include Diatraea saccharalis, D. indigenella, D. busckella, and D. tabernella. The reduced efficacy of biological tools commonly used in controlling several species highlights the importance of evaluating alternative management strategies, such as transgenic plants expressing insecticidal proteins from the bacterium Bacillus thuringiensis (Bt). The selection of optimal Bt insecticidal proteins for Diatraea control depends on bioassays with purified Bt proteins. Because there is no described artificial diet for borer species other than D. saccharalis and availability of most purified Bt toxins is restricted, this study aimed at developing a bioassay method using fresh corn tissue and providing proof of concept by testing susceptibility to the Cry1Ac insecticidal protein from Bt. Toxicity was evaluated with a single Cry1Ac dose applied directly to corn discs. Stem borer mortality after seven days was higher than 90% for all four tested Diatraea species, while control mortality was below 8%. In addition, we observed that Cry1Ac caused more than 90% weight inhibition in all survivors and delayed development. These results validate the use of this method to determine mortality and growth inhibition due to the consumption of the Cry1Ac protein in each of the Diatraea species. Furthermore, this method could be used to assess other entomopathogenic substances to control these insect pests.

Resistance to Diatraea (Lepidoptera: Crambidae) stem borers in sugarcane is independent of species.

Evaluation of host-plant resistance on sugarcane to the sugarcane stem borers of Diatraea spp. is normally conducted in Colombia under field conditions, where environmental variations make the study of the insect-plant relationships difficult. Additionally, several species (i.e., D. saccharalis, D. indigenella, D. tabernella, and D. busckella), which are predominant in Colombia, can overlap in their distribution, raising the question of whether different varieties have the same responses to different pest species. The present study conducted evaluations of host-plant resistance under screen house conditions using two contrasting varieties (CC 93-3895, resistant, and CC 93-3826, susceptible) that were infested with the above-mentioned borer species. Observations of pest injury were conducted on internodes, leaves, and spindles. Survival and size (body mass) of the individuals recovered were analyzed and a Damage Survival Ratio (DSR) was proposed. The resistant CC 93-3895 exhibited less stalk injury, less emergence holes on internodes, and lower DSR; additionally, recovery of pest individuals was lower in comparison with CC 93-3826, independent of the borer species. Insect-plant interactions are discussed, as no previous information was available for three of the species tested (i.e., D. tabernella, D. indigenella, and D. busckella). This screen house protocol is proposed to characterize host-plant resistance among several cultivars from the Colombian sugarcane germplasm bank, using CC 93-3826 and CC 93-3895 as contrasting controls and D. saccharalis as the species model.

Biology of the Most Widely Distributed Sugarcane Stem Borers, Diatraea spp. (Lepidoptera: Crambidae), in Colombia.

Most studies on insect biology and ecology of sugarcane borers have focused on Diatraea saccharalis (Fabricius), the most widely distributed species in the Americas. Little information is available on the biology of other borer species present in Colombia, such as D. indigenella Dyar & Heinrich, D. busckella Dyar & heinrich, and D. tabernella Dyar, that present greater expansion and damage in sugarcane-growing regions. The biology of all four species was accordingly studied under laboratory conditions. Diatraea saccharalis presented the shortest development time (39.4 days) and D. busckella the longest (58.2 days). Immature survival was higher for D. saccharalis (83%) and D. tabernella (77%), with the latter also presenting the highest pupal weight (256.6 mg). Observations on reproduction indicate that D. tabernella develops a larger number of egg masses per female (67.3) as compared with D. saccharalis (28.7). All three species spent more time in the pupal stage and resulted in greater pupal size than D. saccharalis; in particular, D. indigenella showed longer female longevity than D. saccharalis. High immature survival rate and greater reproductive success in D. tabernella could potentially generate a larger population in the field, whereas D. busckella takes longer to complete its development, thus increasing the chances of causing greater injury to sugarcane plants. Discussion on biology, ecology, and pest management of these little-known species is done using as model the better-known D. saccharalis.

Determinación de la Calidad Microbiológica del Río Toca-Boyacá, Sector Tuaneca abajo y el Centro / Determination of the microbiological quality of the Toca-Boyacá River, downstream Tuaneca and the center sectors / Determinação da qualidade Microbiológica do Rio Toca-Boyacá, Setores Tuaneca abaixo e Centro

Resumen Introducción: el agua apta para el consumo humano es aquella que no representa un riesgo para la salud del consumidor teniendo en cuenta sus características organolépticas, físicas, químicas y bacteriológicas, pero dicha calidad es afectada por vertimientos de actividades domésticos, industriales y económicas (Gamboa et al., 2015). Objetivo: este estudio se hizo con el fin de determinar la calidad microbiológica del río Toca, sector Tuaneca abajo y el Centro, Departamento de Boyacá, Colombia. Materiales y métodos: en la cuenca, se establecieron cuatro puntos de muestreo, M1, M2, M3 y M4, en cada punto se colectaron muestras de 50 mL de agua con 20 réplicas, en frascos de vidrio estériles, las cuales fueron refrigeradas a 4°C en neveras de icopor y procesadas en el menor tiempo posible en el laboratorio de Microbiología de la UPTC; en donde se realizó cuantificación de mesófilos aerobios, mohos y levaduras mediante recuento en placa; coliformes totales y fecales se evaluaron mediante la técnica de Número más Probable (NMP). Resultados: el punto de muestreo M1 presentó los mayores valores de coliformes totales con un valor de 1100 NMP/100mL, coliformes fecales: 43 NMP/100mL; así como de mohos y levaduras 61X103 UFC/mL; y el punto con mayor valor de mesófilos aerobios fue 13X104 UFC/ mL que corresponde al M2. Los puntos M3 y M4 presentaron ausencia de coliformes totales. Conclusiones: en contraste con el Decreto 1594 de 1984, sobre usos del agua y residuos líquidos, el río Toca presenta mala calidad de agua en los puntos de muestreo M1 y M2, los cuales presentan actividad ganadera y vertimientos de aguas residuales domésticas.

Abstract Introduction: water suitable for human consumption is the one that does not represent a risk to the health for the consumer, considering its organoleptic, physical, chemical and bacteriological characteristics, but this quality is affected by dumping of domestic, industrial and economic activities (Gamboa et al., 2015). Objective: this study was done in order to determine the microbiological quality of the Toca river, Tuaneca Abajo and the Center, department of Boyacá, Colombia. Materials and methods: in the basin, four sampling points were established, M1, M2, M3 and M4, at each point 50 ml samples of water were collected with 20 replicas, in sterile glass jars, refrigerated at 4 ° C in icopor coolers and processed in the shortest possible time in UPTC microbiology laboratory; where aerobic mesophiles, molds and yeasts were quantified by aerobic plate count; Total and fecal coliforms were evaluated using the Most Probable Number Technique (MPN). Results: the point sampling M1 had the highest of total coliforms values, equivalent to 1100 NMP/100mL, fecal coliforms: 43NMP/100mL. Likewise, of molds and yeasts 61X 103 CFU/mL; and the point with the highest value of mesophiles aerobic 13X104 CFU/mL corresponds to M2. Points M3 and M4 presented the absence of total coliforms. Conclusions: in contrast to Decree 1594 of 1984, on the use of water and liquid waste, the Toca River has poor water quality at sampling points M1 and M2, which present livestock activity and discharge of domestic wastewater.

Resumo Introdução: a água apta para o consumo humano é aquela que não representa um risco para a saúde do consumidor levando em conta suas características organolépticas, físicas, químicas e bacteriológicas, mas tal qualidade é afetada por vertimentos de atividades domésticas, industriais e econômicas (Spiro & Stigliani, 2003). Objetivo: determinar a qualidade microbiológica do rio Toca, setor Tuaneca Abajo e Centro, departamento de Boyacá, Colômbia. Materiais e métodos: na bacia, estabeleceram- se quatro pontos de amostragem, M1, M2, M3 e M4, em cada ponto coletaram-se amostras de 50 mL de água com 20 réplicas, em frascos de vidro estéreis, as quais foram refrigeradas a 4°C em caixas de isopor e processadas no menor tempo possível no laboratório de Microbiologia da UPTC; onde realizou-se a quantificação de mesófilos aeróbios, mofos e leveduras mediante contagem em placa; os coliformes totais e fecais avaliaram-se mediante a técnica de Número mais Provável (NMP). Resultados: os resultados obtidos indicam que o ponto de amostragem M1 apresentou os maiores valores de coliformes totais, equivalentes a 1100 NMP/100mL, coliformes fecais: 43 NMP/100mL Igualmente, de mofos e leveduras 61X 103UFC/ mL; e o ponto com maior valor de mesófilos aeróbios foi 13X104UFC/mL que corresponde ao ponto M2. Conclusão: ao contrário do Decreto 1.594 de 1984, sobre o uso de água e esgoto líquido, O rio Toca apresenta má qualidade de água nos pontos de amostragem M1 e M2. Os pontos M3 e M4 apresentaram ausência de coliformes totais.

Calcium-binding proteins that are type B″ regulatory subunits of phosphatase 2A in Giardia intestinalis.

Giardia intestinalis is a protozoan parasite that colonizes the upper part of the small intestine of its mammalian hosts. The trophozoite, which is the replicative stage, has a complex cytoskeleton that allows it to move and adhere to intestinal cells. It has been proposed that protein phosphatase 2A (PP2A) participates in the regulation of changes to the parasite cytoskeleton during its life cycle. However, how PP2A is involved in this regulation remains unclear since its substrates and regulators have not been characterized. In this work, we report the bioinformatic and experimental analysis of two potential regulatory B″ subunits of PP2A in Giardia, both of which are calcium-binding proteins. In this work, in silico and experimental evidence of the binding of both proteins to calcium is presented; the proteins are shown to interact with the catalytic PP2A subunit in the trophozoite stage, and they exhibit different subcellular localization patterns. Because PP2A is a heterotrimer, homology analysis of the different subunits of PP2A indicates that fewer holoenzyme combinations can be formed in this parasite than in other organisms. Our results suggest that the localization of PP2A may be associated with calcium-dependent signaling through its B″ type regulatory subunits.

In silico analysis of the EF-hand proteins in the genome of Giardia intestinalis assembly A.

Giardia intestinalis is a parasite that inhabits the small intestine of humans and other mammals, causing a disease that can manifest itself with acute diarrhea. This parasite is an early divergent eukaryote with a compact genome and a life cycle composed of two distinct cell types: the trophozoite, the replicative form, and the cyst, the infectious form. Signal transduction pathways implicated in differentiation processes of G. intestinalis are largely unknown. Calcium, considered an essential messenger in cell signaling, has been shown to regulate a myriad of key cell processes including metabolism, motility, and exocytosis, among other important functions, through calcium-binding proteins (CaBPs). The most important and largest family of CaBPs is the EF-hand protein family. To investigate the nature of calcium signaling pathways present in this protozoan, an in silico analysis of the genome to identify genes encoding EF-hand proteins was undertaken. Twenty-eight sequences containing EF-hand domains were found; most of which have only a pair of domains, and half of the sequences were divergent or unique to Giardia. In addition, the transcription pattern for eight genes encoding EF-hand proteins was assessed during encystation. It was found that all the genes were differentially transcribed suggesting a different function in this process. The in silico results suggest that in G. intestinalis, calcium is involved in the regulation of protein phosphorylation through kinases and phosphatases.

Experimental and bioinformatic characterization of CaBP2933 an EF-Hand protein of Giardia intestinalis.

Giardia intestinalis is a parasite that inhabits the small intestine of humans. This parasite is a divergent eukaryote with a compact genome. The calcium ion is an essential messenger in cell signaling. Calcium's role as a messenger is mediated through calcium-binding proteins (CaBPs) that decode the message. The most important family of CaBPs is the EF-Hand protein family. In this study we have explored the role of EF-Hand protein CaBP2933. We analyzed its location, confirmed its ability to bind calcium and identified some of its interacting proteins. Take together our results suggest that CaBP2933 is involved in vesicular trafficking during encystation, via an interaction with kinesin-3 motor protein.

Preliminary study of the enzyme ubiquitin carboxylterminal hydrolase 14 (UBP6) in Giardia intestinalis: structural bioinformatic analysis and transcriptional profile during encystation / Estudio preliminar de la enzima ubiquitina carboxiterminal hidrolasa 14 (UBP6) en Giardia intestinalis: análisis bioinformático de su estructura y perfil transcripcional durante la enquistación / Estudo preliminar da enzima ubiquitina carboxi-terminal hidrolasa 14 (UBP6) em Giardia intestinalis: análise bioinformática estrutural e perfil transcricional durante encistamento

This paper presents a combined approach with two aims. The first is to analyze the reported sequence of the enzyme ubiquitin carboxyl-terminal hydrolase 14 of Giardia intestinalis (UBP6) through computational methods to find components related with its hypothetical function. The second is to determine if the protein-coding gene is expressed in G. intestinalis and, if such is the case, also determine its transcription pattern along the life cycle of the parasite. It was established that the protein belongs to the family of Cys-dependent deubiquitinases and more specifically to ubiquitin specific proteases (USPs). Moreover, the catalytic center with the complete triad as well as typical features of the USP motif were also identified. Since the computational findings suggest that the enzyme could be functional, reverse transcription coupled to PCR was used as a first approach to establish if in fact the coding gene is expressed in the parasite. Interestingly, it was found not only that the gene is expressed, but also that there is a transcription variation along the life cycle of the parasite. These two findings are the starting point for further studies since they tentatively suggest that this enzyme could be involved in the protein turnover that occurs during parasite encystation. Although preliminary, this study is the first report concerning the study of a specific deubiquitinating enzyme in the parasite G. intestinalis.

En este trabajo se presenta una estrategia combinada que buscaba, primero, analizar por métodos computacionales la secuencia de la enzima ubiquitina carboxilo-terminal hidrolasa 14 de Giardia intestinalis (UBP6) reportada para buscar componentes relacionados con su función hipotética y segundo, determinar si el gen que codifica para la proteína se expresa en G. intestinalis y si lo hace, cómo es su patrón de transcripción a lo largo del ciclo de vida del parásito. Se encontró que la proteína pertenece a la familia de deubiquitinasas dependientes de cisteína y más específicamente a las proteasas específicas para ubiquitina (USPs por ubiquitin specific proteases). También se identificaron el centro catalítico con la triada completa así como características típicas del motivo USP. Teniendo en cuenta que los resultados computacionales sugieren que la enzima puede ser funcional, se usó la técnica de transcripción reversa acoplada a PCR como un primer acercamiento para establecer si el gen codificante se expresa en el parásito. De manera interesante, se determinó no solo que el gen se expresa sino que existe una variación de su transcripción a lo largo del ciclo de vida del parásito. Estos hallazgos son el punto de partida para posteriores estudios ya que sugieren de manera preliminar que esta enzima podría estar involucrada en el recambio de proteínas que ocurre en el parásito durante el proceso de enquistación. Aunque preliminar, este estudio es el primer reporte acerca de una enzima deubiquitinadora específica en el parásito G. intestinalis.

Este artigo apresenta uma abordagem combinada com dois objetivos. A primeira é analisar a sequência informou da enzima ubiquitina carboxil-terminal hidrolase 14 de Giardia intestinalis (UBP6) através de métodos computacionais para encontrar os componentes relacionados com a sua função hipotética. A segunda é para determinar se o gene de codificação da proteína é expressa em G. intestinalis e, se for o caso, também determinar o seu padrão de transcrição ao longo do ciclo de vida do parasita. Foi estabelecido que a proteína pertence à família de deubiquitinases Cys-dependentes e mais especificamente para proteases específicas de ubiquitina (USPs por ubiquitin specific proteases). Além disso, o centro catalítico com a tríade completo, bem como as características típicas do motivo USP também foram identificados. Uma vez que os resultados computacionais sugerem que a enzima poderia ser funcional, a transcrição reversa acoplada a PCR foi utilizado como uma primeira abordagem para determinar se, de facto, o gene codificante é expressa no parasita. Curiosamente, verificou-se não só que o gene é expresso, mas também que há uma variação de transcrição ao longo do ciclo de vida do parasita. Estes dois elementos são o ponto de partida para estudos posteriores, uma vez que tentativas sugerem que esta enzima pode estar envolvida no refill de proteínas que ocorre durante o parasita encistamento. Embora preliminares, este estudo é o primeiro relatório relativo ao estudo de uma enzima deubiquitinadora específica no parasita intestinalis.

Incidencia de Listeria spp. en carcasas de pollo congelado en un supermercado del nororiente de Bogotá / Incidence of Listeria spp., in frozen poultry carcasses at supermarket in northwest Bogotá

Listeria spp. es un género bacteriano que contamina alimentos de origen animal, incluido el pollo, yl puede permanecer viable durante las cadenas de producción y distribución. En el presente trabajo se analizaron 91 muestras de carcasas de pollo, obtenidas en una distribuidora en el nororiente de Bogotá; las muestras se tomaron en un período de 9 semanas y luego fueron procesadas. De las 91 carcasas, 40 (43.95%) resultaron positivas para Listeria spp. La presencia de este microorganismo puede estar asociada a deficiencias en los sistemas de tecnología de limpieza en las plantas de beneficio y/o contaminación con utensilios durante el desprese.

[Identification of the gene sequence of telomerase catalytic subunit in Plasmodium falciparum]. / Identificación de la secuencia del gen de la subunidad catalítica de la telomerasa en Plasmodium falciparum.

INTRODUCTION: The enzyme telomerase regulates telomere length by synthesis of telomeric repeats to compensate for telomeric loss in each DNA replication cycle. Therefore, telomerase is a potential target to block growth of cells with high replication rates. In Plasmodium falciparum, telomerase activity has been documented, but little information on its structure and role. METHODS: Herein, alignment of multiple sequences was undertaken comparing telomerase catalytic subunit sequences as found in existing databases. A consensus sequence was compared with the sequences in the P. falciparum genome project and as a result, a candidate sequence for a portion of the telomerase gene was recovered. Primer sets were designed for DNA and RNA amplifications. RESULTS: DNA fragments corresponding to telomerase conserved domains were amplified by using reverse transcription and PCR of cDNA. With a combination of bioinformatics and sequencing methods, the sequence of telomerase catalytic subunit gene (TERT) in P. falciparum was discovered, and its presence and transcription demonstrated.

Identificación de la secuencia del gen de la subunidad catalítica de la telomerasa en Plasmodium falciparum / Identification of the gene sequence of telomerase catalytic subunit in Plasmodium falciparum

Introducción. La enzima telomerasa participa en la regulación de la longitud de los telómeros al sintetizar nuevas repeticiones teloméricas que compensan las pérdidas en cada ronda de replicación del ADN. Por esta razón, el bloqueo de su actividad se plantea como un posible blanco de acción para detener el crecimiento de células con altas tasas de crecimiento. Tal es el caso de Plasmodium falciparum, parásito causante de la forma más grave de paludismo humano, en el cual se sabe que hay actividad de telomerasa pero no se tiene información sobre la enzima misma. Metodología. Para hacer un acercamiento al estudio de la telomerasa en P. falciparum, se realizó un alineamiento múltiple de las secuencias de la subunidad catalítica de la telomerasa disponibles en bases de datos y se obtuvo una secuencia consenso, la cual se comparó con las secuencias generadas en el proyecto de genoma de P. falciparum. Se encontró una secuencia que podría corresponder a parte del gen de la telomerasa de P. falciparum. Para comprobarlo, se diseñaron iniciadores que se utilizaron en ensayos de amplificación sobre el ADN y el ARN del parásito.Resultados. Se amplificaron fragmentos de ADN correspondientes a motivos conservados en las telomerasas y se detectó la presencia del ARNm mediante trascripción reversa y PCR sobre el ADNc generado. De esta manera, al combinar la utilización de herramientas de bioinformática y su posterior comprobación mediante técnicas de biología molecular

Detection of telomerase activity in Plasmodium falciparum using a nonradioactive method.

A simple, quick and sensitive method was used to detect telomerase activity in Plasmodium falciparum. The telomeric repeat amplification protocol (TRAP assay) was modified using electrophoresis and staining with SYBR-green I to detect telomerase activity in a range of 10 to 10(7) parasites. This might be a useful way to ascertain telomerase activity in different types of nontumor cells.

Detection of telomerase activity in Plasmodium falciparum using a nonradioactive method

A simple, quick and sensitive method was used to detect telomerase activity in Plasmodium falciparum. The telomeric repeat amplification protocol (TRAP assay) was modified using electrophoresis and staining with SYBR-green I to detect telomerase activity in a range of 10² to 10(7) parasites. This might be a useful way to ascertain telomerase activity in different types of nontumor cells

Genetic diversity of Plasmodium falciparum field samples from an isolated Colombian village.

Colombian field isolates of Plasmodium falciparum were analyzed for genetic diversity. Fifty-three samples were collected as thick smears from patients living in Panguí, an isolated area with low migration. While the samples were being collected, Panguí was experiencing an epidemic outbreak of malaria. The samples were typified using nested polymerase chain reaction (PCR) amplification of block 2 of the merozoite surface protein 1 (MSP1) gene and nested PCR with mutation-specific primers for position 108 of the dihydrofolate reductase enzyme gene. The results for the circulating population of parasites in Panguí show low diversity--four allelic forms--using MSP1 as a marker, a fact that contrasts with data reported for certain Asian and African zones. A high percentage of mixed infections was observed, as was high complexity of the infection. No differential distributions were found for any allelic type.