Characterization of Cry toxins from autochthonous Bacillus thuringiensis isolates from Mexico / Caracterización de toxinas Cry de aislados de Bacillus thuringiensis autóctonos de México

Abstract: Background: Chemical pesticides, widely used in agriculture and vector-borne disease control, have shown toxic effects on the environment and the people in contact with them. Bacillus thuringiensis is a widely used bacterium for alternative and safer control of insect pests. Its toxins are specific for insects but innocuous for mammals and may be used as powerful adjuvants when applied with vaccines. The objective of this work was to characterize some autochthonous B. thuringiensis strains, which could be used for the control of a local pest (Diatraea considerata Heinrich) that affects sugar cane crops in Sinaloa, Mexico. Also, to evaluate these strains as a source of Cry toxins, which may be used in the future as adjuvants for some vaccines. Methods: Eight strains from field-collected dead insects were isolated. These were microbiologically identified as B. thuringiensis and confirmed by amplification and sequencing of 16S rDNA. Bioassays were performed to evaluate their pathogenicity against D. considerata, and Cry toxins were identified by proteomic analyses. Results: An increased mortality among larvae infected with strain Bt-D was observed, and its toxin was identified as Cry1Ac. Conclusions: The observed data showed that the selected strain was pathogenic to D. considerata and seemed to produce Cry1Ac protein, which has been reported as an adjuvant in different types of immunization.

Resumen: Introducción: Los pesticidas químicos, ampliamente usados en agricultura y en el control de vectores transmisores de enfermedades, han mostrado efectos tóxicos sobre el medio ambiente y las personas expuestas a ellos. Bacillus thuringiensis es una bacteria ampliamente utilizada como una alternativa segura y eficaz en el control biológico de plagas agrícolas. Sus toxinas son específicas de insectos, pero inocuas para mamíferos, e incluso poseen gran potencial para ser usadas como adyuvantes en vacunas. El objetivo de este trabajo fue caracterizar cepas autóctonas de B. thuringiensis con efectividad contra el gusano barrenador (Diatraea considerata Heinrich) de la caña de azúcar en cultivos del estado de Sinaloa, México, y como fuente de proteínas Cry, con potencial de utilizarse como adyuvantes en vacunas. Métodos: Se lograron aislar ocho cepas a partir de insectos muertos en campos agrícolas, las cuales fueron identificadas microbiológicamente como B. thuringiensis, lo que se confirmó por amplificación y secuenciación del 16S rDNA. La efectividad de los aislados para el control del gusano barrenador fue evaluada mediante bioensayos y las toxinas Cry fueron identificadas por análisis proteómico. Resultados: Se observó una mortalidad elevada en las larvas infectadas con las cepas de estudio. Particularmente, la cepa Bt-D, de la cual el análisis molecular mostró que posee una toxina tipo Cry1Ac. Conclusiones: Los resultados mostraron que la cepa Bt-D posee un elevado potencial patogénico hacia D. considerata y produce la proteína Cry1Ac, de la cual existen reportes de su aplicación como adyuvante en diferentes formas de inmunización.

Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Isolation of Bacillus thuringiensis strains that contain Dipteran-specific cry genes from Ilha Bela (São Paulo, Brazil) soil samples / Isolamento de linhagens de Bacillus thuringiensis contendo genes cry díptero-específicos a partir de amostras de solo oriundas de Ilha Bela-São Paulo, Brasil

The entomophatogenic bacterium Bacillus thuringiensis produces crystal proteins, named Cry proteins which are encoded by the cry genes. This bacterium is used on biological control of important economical pests, as well as in the control of disease´s vectors, such as Aedes aegypti, a mosquito that transmits the dengue viruses. Isolates of this bacterium can be characterized by the content of cry genes and this prediction helps target different insect orders. In this research, we isolated 76 colonies of B. thuringiensis from 30 soil samples that were taken from Ilha Bela (SP, Brazil), a place where simulids are already biologically controlled by B. thuringiensis, to find bacterial isolates that were capable of controlling A. aegypti. The 16S ribosomal subunit genes of the selected isolates were sequenced, and the isolates were molecularly characterized based on their Dipteran-specific cry gene contents. Eight of the 76 isolates (10.52%) contained the cry4Aa, cry4Ba or cry10Aa genes, these isolates were carried out against A. aegypti larvae on bioassay. The presence or absence of specific cry genes was associated with the observed average larval mortalities. From the 76 isolates, seven (9.2%) were potentially able to control A. aegypti larvae. Therefore these are promising isolates for the biological control of A. aegypti larvae.

Bacillus thuringiensis é entomopatogênica, por produzir proteínas cristais, denominadas proteínas Cry, as quais são codificadas pelos genes cry. Essa bactéria atua no controle biológico de insetos-praga de culturas economicamente importantes, bem como no controle de insetos vetores causadores de doenças, como o Aedes aegypti, mosquito transmissor do vírus da dengue. Os isolados dessa bactéria podem ser caracterizados pelo conteúdo de genes cry que possuem e, assim, predizer o alvo de controle dos mesmos às diferentes ordens de insetos. Com o objetivo de encontrar isolados eficientes no controle do vetor A. aegypti, o presente trabalho isolou 76 colônias de B. thuringiensis a partir de 30 amostras de solo oriundas de Ilhabela-SP, município que se caracteriza por realizar controle biológico de simulídeos com essa bactéria. Os 76 isolados foram sequenciados na região da subunidade ribossomal 16S e caracterizados molecularmente quanto ao conteúdo de genes cry díptero-específicos. No total, oito isolados (10,52% do total) apresentaram bandas para os genes cry4Aa, cry4Ba e cry10Aa, sendo os mesmos testados contra larvas de A. aegypti por meio de bioensaios. A presença e/ou ausência dos genes cry foi associada à mortalidade média de larvas. Dentre os isolados estudados, sete (9,2% do total) apresentaram elevado potencial de controle às larvas de A. aegypti, sendo assim considerados como promissores para o manejo do controle biológico de larvas de A. aegypti com a bactéria B. thuringiensis.

Two new Brazilian isolates of Bacillus thuringiensis toxic to Anticarsia gemmatalis (Lepidoptera: Noctuidae) / Dois novos isolados brasileiros de Bacillus thuringiensis tóxicos para Anticarsia gemmatalis (Lepidoptera: Noctuidae)

Bacillus thuringiensis is a bacterium used for biopesticides production and pest-resistant plants due to the synthesis of protein crystals by cry genes, which are effective in controlling several insect orders such as Lepidoptera. This work aimed at the evaluation and characterisation of two new B. thuringiensis isolates active against A. gemmatalis (Hübner 1818) larvae, which is the soybean major pest. The results showed that Bt117-4 isolate amplified fragments corresponding to cry2 and cry9 genes, and synthesised protein fragments equivalent to 130, 90 and 45 kDa. The Bt3146-4 isolate amplified DNA fragments corresponding to cry9 gene and synthesised protein fragments of 70, 58 and 38 kDa. Transmission electron microscopy revealed the presence of protein crystals in both isolates. CL50 with Cry purified proteins from Bt117-4 and Bt3146-4, corresponded to 0.195 and 0.191 µg larvae-1, respectively. The two B. thuringiensis isolates selected in this study were effective to control velvetbean caterpillar at laboratory conditions. Field tests should be carried on to develop new biopesticides formulation as well for cry genes resource for Anticarsia gemmatalis resistant transgenic plants.

Bacillus thuringiensis é uma bactéria utilizada na produção de biopesticidas e de plantas resistentes às pragas por causa da síntese de cristais proteicos pelos genes cry, os quais são eficazes no controle de diversas ordens de insetos, como os lepidópteros. O presente trabalho objetivou a avaliação e a caracterização de dois novos isolados de B. thuringiensis ativos contra lagartas de A. gemmatalis (Hübner 1818), que é a principal praga da cultura da soja. Os resultados obtidos revelaram que o isolado Bt117-4 amplificou fragmentos correspondentes aos genes cry2 e cry9, sendo que os fragmentos proteicos sintetizados foram equivalentes a 130, 90 e 45 kDa. O isolado Bt3146-4 amplificou fragmentos de DNA que correspondem ao gene cry9 e sintetizou fragmentos proteicos de 70, 58, e 38 kDa. Os dados de microscopia eletrônica de transmissão revelam a presença de cristais proteicos em ambos os isolados. A CL50, com proteínas Cry purificadas de Bt117-4 e Bt3146-4, correspondeu a 0,195 e 0,191 µg lagarta-1, respectivamente. Os dois isolados de B. thuringiensis selecionados neste trabalho mostraram-se eficientes no controle da lagarta-da-soja em laboratório, sendo recomendada sua avaliação a campo para posterior aplicação na formulação de biopesticidas ou como fonte de genes cry para a obtenção de plantas geneticamente modificadas resistentes à Anticarsia gemmatalis.

Presence and significance of Bacillus thuringiensis Cry proteins associated with the Andean weevil Premnotrypes vorax (Coleoptera: Curculionidae)

The Andean weevil Premnotrypes vorax represents an important cause of damage to Colombian potato crops. Due to the impact of this plague on the economy of the country, we searched for new alternatives for its biological control, based on the entomopathogenic bacteria Bacillus thuringiensis. A total of 300 B. thuringiensis strains obtained from potato plantations infested with P. vorax were analyzed through crystal morphology, SDS-PAGE, PCR and bioassays. We used site- directed mutagenesis to modify the Cry3Aa protein. Most of the B. thuringiensis isolates had a bipyramidal crystal morphology. SDS-PAGE analyses had seven strains groups with σ-endotoxins from 35 to 135 kDa. The genes cry 2 and cry 1 were significantly more frequent in the P. vorax habitat (PCR analyses). Three mutant toxins, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), and 3 (Q482A, S484A, R485A), were analyzed to assess their activity against P. vorax larvae. Toxicity was low, or absent, against P. vorax for isolates, wild type cry 3Aa and cry 3Aa mutants. The genetic characterization of the collection provides opportunities for the selection of strains to be tested in bioassays against other insect pests of agricultural importance, and for designing Cry proteins with improved insecticidal toxicity. Rev. Biol. Trop. 57 (4): 1235-1243. Epub 2009 December 01.

El gorgojo andino Premnotrypes vorax es una causa importante de daño en los cultivos colombianos de este tubérculo. Debido al impacto que esta plaga tiene sobre la economía del país, nos interesamos en buscar alternativas nuevas para el control biológico de P. vorax, basadas en la bacteria entomopatógena Bacillus thuringiensis. Se recolectaron un total de 300 cepas de B. thuringiensis a partir de plantaciones de papa infestadas con P. vorax, las cuales fueron analizadas por medio de la morfología del cristal, SDS-PAGE, PCR y ensayos biológicos. La mayoría de los aislamientos de B. thuringiensis presentaron cristales bipiramidales. Los análisis de SDS-PAGE indicaron la presencia de siete grupos de cepas con σ- endotoxinas que variaban entre 35 a 135 kDa. Las pruebas con PCR demostraron que los genes cry 2 y cry 1 fueron significativamente más frecuentes en el medioambiente de P. vorax. Además, se utilizó la mutagénesis sitio-dirigida para modificar la proteína Cry3Aa. Se analizaron tres toxinas mutantes, 1 (D354E), 2 (R345A, ∆Y350, ∆Y351), y 3 (Q482A, S484A, R485A), para determinar su actividad contra larvas de P. vorax. Los ensayos de toxicidad señalaron escasa, o nula, actividad hacia P. vorax tanto para las cepas, la toxina Cry3Aa de referencia y las proteínas Cry3Aa mutantes. La caracterización genética de la colección puede proveer oportunidades para la selección de cepas que pueden evaluarse por medio de bioensayos contra otros insectos-plaga de importancia agrícola, y para el diseño de proteínas Cry con actividad toxica mejorada.

Traslocación bacteriana en un modelo de autotrasplante de intestino delgado en perros / Bacterial translocation in a small intestine auto transplantation model in dogs

Sepsis is the commonest complication of small bowel transplantation. These infections are presumibly caused by bacterial translocation, due to splachnic ischemia. To study bacterial translocation in the inmediate postoperative period after small bowel transplantation in dogs and to relate it to splanchnic ischemia. Three groups of dogs were studied. In group A (n=6) spontaneous episodes of splanchnic schemia were monitored in the first 18 hrs of the postoperative period. In group B (n=5) a 60 min ischemia was induced by superior mesenteric artery occlusion, 2 hours after small bowel transplantation. In group C (n=5) a 60 min ischemia was induced by occlusion of mesenteric vein, 2 hrs after transplantation. Bacterial translocation was assessed through bacterial cultures from the mesenteric vein and splanchnic ischemia with intramucosal pH measurement (a pH<7.2 was considered indicative of ischemia). 28 of 83 cultures were positive, specially for Gram negative bacilli. The incidence of positive cultures was 14 percent for group A, 17 percent for group B and 79 percent for group C (p<0.01 cpmpared to groups A and b). The higher incidence of bacterial translocation occurred during the first 2 hours after transplantation, when the lower intramucosal pH recording were obtained. The percentage of positive cultures was 39 percent during periods of ischemia, compared to 24 percent during periods without ischemia (p=NS). Bacterial translocation occurs during the first 2 hours after intestinal transplantation in concomitance with the lower intramucosal pH readings