Producción y formulación de un bioinsecticida de Bacillus thuringiensis subsp. israelensis H-14 para el control de mosquitos culicidos en el noreste de México

Tesis (Doctorado en Ciencias con Especialidad en Biotecnología) UANL

Mode of Action of a Purified Antitumor Protein from the Proteinaceous Crystal of Bacillus thuringiensis subsp. thuringiensis on Yoshida Ascites Sarcoma Cells

A purified antitumor protein from the proteinaceous crystal of Bacillus thuringiensis subsp. thuringiensis inhibits the growth of Yoshida ascites sarcoma both in vivo and in vitro. Exogenous respiration of the tumor cells was unaffected by the protein at a concentration as high as 500 µg/ml. The antitumor protein inhibits the uptake and incorporation of labeled precursors into macromolecules. However, the ratio of incorporation over uptake is not affected by the protein. Further, the protein brings about the leakage of 260-nm-absorbing material, proteins, and 32P-labeled cellular constituents from the Yoshida ascites sarcoma cells. The results show that the action of the antitumor protein appears to alter the cellular permeability of the tumor cells.

Sobrevivencia y actividad de Bacillus thuringiensis var. israelensis en ambientes naturales contra larvas de mosqui to [por] Maribel Leal Castillo

Tesis (Maestro en Ciencias con especialidad en microbiolo gia) U.A.N.L.

Diseño y evaluación de formulados de Bacillus thuringiensis var israelensis en laboratorio y campo en Culex pipiens quinquefasciatus (Say) y Aedes aegypti (Linnaeus)

Tesis (Maestría en Ciencias con Especialidad en Entomología Médica) U.A.N.L.

Desarrollo de un medio de cultivo a base de subproductos agroindustriales para la producción de delta-endotoxina de B. thuringiensis VAR. israelensis H-14 [por] Ma. Guadalupe Maldonado Blanco

Tesis (Maestría en Ciencias con Especialidad en Microbiología) U.A.N.L. Facultad de Ciencias Biológicas

Bacillus thuringiensis var. israelensis SPS1: caracterização da região promotora de genes cry e efeito em larvas de Aedes aegypti (L.) (Diptera: Culicidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Obtenção de bioinseticida a partir de Bacillus thuringiensis var. israelensis e aplicação de bioensaios em culicídeos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Comparative Studies of Bacillus thuringiensis var. israelensis Metabolism in Different Concentrations of Cassava Flour Processing Waste Based Media

Techniques of production of enthomopatogenic bacteria are developed aiming to increase the productivity and to reduce the costs of the fermentative process. Like this, it has been using agroindustrial wastes or by-products as nutrient sources in culture medium, having been used, in this study, the manipueira, a by-product of the processing of the cassava flour. Fermentations were performed in flasks of Erlenmeyer of 500 mL containing 250 mL of culture media, conditioned in shaker at 180 r.p.m. and 28°C, and the media were composed by manipueira, in concentrations that varied between 400 and 1000 mL/L. The time of the process varied between 48 and 120 hours. They appraised the following parameters: cellular growth, the production of spores, the reduction of organic matter (COD analysis) and the variation of reduction sugar. Although there was a proportional cellular growth to the manipueira concentration, the production of spores was similar in all the cases, at the end of the process, in spite of the smallest speed of production of the same ones in the highest concentrations. In relation to the variation of COD, it has, also, a percentile minor of reduction in the highest concentrations. In the analysis of variation of reduction sugars, the higher concentrations are the ones that they present larger slowness in the reduction of this.

Analysis of expression of the binary toxin genes from Bacillus sphaericus in Anabaena and the potential in mosquito control

Anabaena strains expressing the binary toxin genes of Bacillus sphaericus produce high larvicidal activity with living cells. Western blot analysis showed that the 51-kDa and 42-kDa toxin proteins were stable in Anabaena. When a DNA fragment upstream of the 51-kDa protein gene was deleted, the toxicity was reduced by over a hundred-fold, whereas deletions at the coding regions showed that the cooperation of the two proteins expressed in Anabaena is essential for the larvicidal activity. Outdoor tests showed that the genetically altered Anabaena could keep containers with natural water from being inhabited by Culex larvae for over 2 months.

Oxygen supply in Bacillus thuringiensis fermentations: bringing new insights on their impact on sporulation and delta-endotoxin production

The growth kinetics, sporulation, and toxicity of Bacillus thuringiensis var. israelensis were evaluated through the analysis of batch cultures with different dissolved oxygen (DO) profiles. Firstly, DO was maintained constant at 5%, 20%, or 50% throughout fermentation in order to identify the most suitable one to improve the main process parameters. Higher biomass concentration, cell productivity, and cell yield based on glucose were obtained with 50% DO. The higher aeration level also resulted in higher spore counts and markedly improved the toxic activity of the fermentation broth, which was 9-fold greater than that obtained with 5% DO (LC50 of 39 and 329 mg/L, respectively). Subsequently, using a two-stage oxygen supply strategy, DO was kept at 50% during the vegetative and transition phases until the maximum cell concentration was achieved. Then, DO was changed to 0%, 5%, 20%, or 100% throughout sporulation and cell lysis phases. The interruption of oxygen supply strongly reduced the spore production and thoroughly repressed the toxin synthesis. On the contrary, when DO was raised to 100% of saturation, toxic activity increased approximately four times (LC50 of 8.2 mg/L) in comparison with the mean values reached with lower DO levels, even though spore counts were lower than that from the 50% DO assay. When pure oxygen was used instead of normal air, it was possible to obtain 70% of the total biomass concentration achieved in the air assays; however, cultures did not sporulate and the toxin synthesis was consequently suppressed.

Methodology for fast evaluation of Bacillus thuringiensis crystal protein content

O desenvolvimento da produção e uso do Bacillus thuringiensis no Brasil em escala comercial enfrenta certas dificuldades, entre elas o estabelecimento de metodologias para a quantificação de produtos tóxicos a serem comercializados. Atualmente, a quantidade de toxinas é expressa como porcentagem do total de proteínas presentes em amostras em consideração. Tal metodologia, entretanto, não mede a quantidade real de uma determinada proteína presente em um produto qualquer, além do fato de diferentes linhagens bacterianas possuírem diferentes genes codificadores para endotoxinas e mesmo para b-toxina. Desde que os diferentes tipos de toxinas apresentam diferentes características antigências, este trabalho tem como objetivo a utilização de técnicas imunológicas para quantificar específicamente o conteúdo de proteína cristal presente em diferentes amostras. A proteína cristal produzida pela subespécie B. thuringiensis var. israelensis foi purificada por ultracentrifugação e utilizada para imunizar coelhas e produzir soros hiperimunes. Tais soros foram posteriormente usados para avaliar o nível de proteína cristal em bioinseticidas comerciais e em culturas de laboratório desta bactéria utilizando-se a técnica do imunodot. Os resultados foram obtidos por comparação de reações com concentrações conhecidas de proteína cristal permitindo assim avaliar com segurança os níveis desta proteína em várias preparações.

Avaliação de dois diferentes meios de cultura para o desenvolvimento de biopesticidas à base de Bacillus thuringiensis e sua aplicação em larvas de Aedes aegypti

The bacteria Bacillus thuringiensis var. israelensis (Bti) generates certain toxins with pesticide action, which can be used on the control of transmissible diseases by culicides, specially Aedes aegypti, the dengue vector. This biopesticide has been produced by submerged fermentation and, in Brazil, this production has been made by very little research centers and, more recently, by a unique small enterprise. For the implementation of a viable vectors control program through biopesticides, some studies about culture media are essential in order to join efficiency and low costs. In this way, agroindustrial wastes or by-products have been used as a nutrient source for the culture media production. In this study, corn steep liquor, a corn industrial processing by-product and tryptose, both with/without sugar addition, were compared as culture media. Cellular growth was evaluated by optical density at 620 nm, spore production by total viable cell count and LC50 by bioassays against 4th instar larvae. Among the four examined substrates, the medium composed by glucose plus corn steep liquor presented the best spore production and bioassay results.

Caracterização de isolados de Bacillus thuringiensis patogênicos à Spodoptera frugiperda (Lepidoptera: Noctuidae)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Quality assurance of aerial applications of larvicides for mosquito control : effects of granule and catch tray size on field monitoring programs

Aerial applications of granular insecticides are preferable because they can effectively penetrate vegetation, there is less drift, and no loss of product due to evaporation. We aimed to 1) assess the field efficacy ofVectoBac G to control Aedes vigilax (Skuse) in saltmarsh pools, 2) develop a stochastic-modeling procedure to monitor application quality, and 3) assess the distribution of VectoBac G after an aerial application. Because ground-based studies with Ae. vigilax immatures found that VectoBac G provided effective control below the recommended label rate of 7 kg/ha, we trialed a nominated aerial rate of 5 kg/ha as a case study. Our distribution pattern modeling method indicated that the variability in the number of VectoBac G particles captured in catch-trays was greater than expected for 5 kg/ha and that the widely accepted contour mapping approach to visualize the deposition pattern provided spurious results and therefore was not statistically appropriate. Based on the results of distribution pattern modeling, we calculated the catch tray size required to analyze the distribution of aerially applied granular formulations. The minimum catch tray size for products with large granules was 4 m2 for Altosid pellets and 2 m2 for VectoBac G. In contrast, the minimum catch-tray size for Altosid XRG, Aquabac G, and Altosand, with smaller granule sizes, was 1 m2. Little gain in precision would be made by increasing the catch-tray size further, when the increased workload and infrastructure is considered. Our improved methods for monitoring the distribution pattern of aerially applied granular insecticides can be adapted for use by both public health and agricultural contractors.

苏云金杆菌以色列变种防治水处理中摇蚊幼虫研究

研究了苏云金杆菌以色列变种(Bacillus thuringiensis var.israelensis de Barjac,简称B.t.i)用于摇蚊幼虫(即红虫)防治的菌种发酵条件.结果表明,发酵时间为36 h,温度为30℃,摇床转速为250 rpm,三角瓶培养基量为50 mL时,B.t.i对红虫的毒力最强.