Study of Expression of Indigenous Bt cry2AX1 Gene in T3 Progeny of Cotton and its Efficacy Against Helicoverpa armigera (Hubner)

Abstract Development of transgenic Bt crops with stable and high level of Bt protein expression over generations under different environmental conditions is critical for successful deployment at field level. In the present study, progenies of transgenic cotton Coker310 event, CH12 expressing novel cry2AX1 gene were evaluated in T3 generation for stable integration, expression and resistance against cotton bollworm, Helicoverpa armigera. The cry2AX1 gene showed stable inheritance and integration in the T3 progeny plants as revealed by PCR and Southern blot hybridization. The expression of Cry2AX1 protein on 90 days after sowing (DAS) was in the range of 1.055 to 1.5 µg/g of fresh leaf tissue except one plant which showed 0.806 µg/g of fresh leaf tissue and after 30 days (i.e., on 120 DAS) three plants recorded in between 0.69 to 0.82 µg/g and other plants are in range of 0.918 to 1.058 µg/g of fresh leaf tissue. Detached leaf bit bioassay in T3 progeny on 110 DAS recorded mortality of 73.33 to 93.33 per cent against H. armigera and severe growth retardation in surviving larvae. These results indicate that the expression of chimeric cry2AX1 is stable and exhibits insecticidal activity against H. armigera in T3 progeny of CH12 event of transgenic cotton.

Selection of Bacillus thuringiensis strains toxic to cotton boll weevil (Anthonomus grandis, Coleoptera: Curculionidae) larvae / Selección de cepas de Bacillus thuringiensis tóxicas para larvas del picudo del algodonero (Anthonomus grandis, Coleoptera: Curculionidae)

Preliminary bioassays with whole cultures (WC) of 124 Bacillus thuringiensis strains were performed with neonate larvae of Anthonomus grandis, a major cotton pest in Argentina and other regions of the Americas. Three exotic and four native strains were selected for causing more than 50% mortality. All of them were β-exotoxin producers. The native strains shared similar morphology of parasporal crystals, similar protein pattern and identical insecticidal gene profiles. These features resembled Lepidoptera-toxic strains. Furthermore, these strains showed a Rep-PCR pattern identical to lepidoptericidal strain HD-1, suggesting that these strains may belong to serovar kurstaki. However, some differences were observed in the plasmid profiles and in the production of β-exotoxin. To determine the culture fractions where the insecticidal metabolites were present, bioassays including resuspended spore-crystal pellets, filtered supernatants (FS) were compared with those of WC. Both fractions tested showed some level of insecticidal activity. The results may suggest that the main toxic factors can be found in FS and could be directly correlated with the presence of β-exotoxin. Based on the bioassays with FS and autoclaved FS, the participation of thermolabile virulence factors such as Cry1I in toxicity is neither discarded. In the selected strainsβ-exotoxin would be the major associated virulence factor; therefore, their use in biological control of A. grandis should be restricted. Nevertheless, these strains could be the source of genes (e.g., crylla) to produce transgenic cotton plants resistant to this pest.

Se realizaron ensayos preliminares con cultivos completos de 124 cepas de Bacillus thuringiensis utilizando larvas neonatas de Anthonomus grandis, una plaga principal del algodón en Argentina y otras regiones de América. Se seleccionaron 3 cepas exóticas y 4 nativas por producir mortalidad superior al 50%, todas ellas productoras de β-exotoxina. Las cepas nativas presentan la misma morfología de cristales, un perfil de proteínas similar y los mismos genes insecticidas. Estas características hacen que se parezcan a cepas tóxicas para lepidópteros. Además, mostraron un perfil de Rep-PCR idéntico al de la cepa lepidoptericida HD-1, lo que indica que podrían pertenecer al serovar kurstaki. Sin embargo, se observaron diferencias en el perfil plasmídico y en la producción de β-exotoxina. Para determinar en qué fracción del cultivo se encontraban los metabolitos responsables de la toxicidad, se compararon los resultados de bioensayos en los que se utilizó biomasa, sobrenadante filtrado (SF) o cultivos completos. Ambas fracciones mostraron cierto grado de toxicidad. Los resultados indican que los principales factores tóxicos se encuentran en el sobrenadante y estarían directamente relacionados con la presencia de β-exotoxina. De acuerdo con los bioensayos de SF y SF autoclavado, no se descarta también la participación en la toxicidad de factores de virulencia termolábiles, como CrylIa. En las cepas seleccionadas, el principal factor de virulencia es la β-exotoxina, por lo que su uso debería restringirse para el control biológico de A. grandis. No obstante, estas podrían ser fuente de genes (p. ej., crylIa) para la producción de plantas de algodón transgénicas resistentes a dicha plaga.

Diversity and distribution of lepidopteran-specific toxin genes in Bacillus thuringiensis strains from Argentina / Diversidad y distribución de genes de toxinas insecticidas Lepidoptera-específicos en cepas de Bacillus thuringiensis de Argentina

A total of 268 Bacillus thuringiensis strains obtained from different sources of Argentina were analyzed to determine the diversity and distribution of the cryl, cry2, cry8, cry9 and vip3A genes encoding for lepidopteran-specific insecticidal proteins. Twin strains were excluded. Ten different profiles were detected among the 80 selected B. thuringiensis strains. Two of these profiles (cry1Aa, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (35/80), and cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa (25/80)) pooled 75% of the strains. The existence of this low diversity is rare, since in most of the studied collections a great diversity of insecticidal toxin gene profiles has been described. In addition, the most frequently detected profile was also most frequently derived from soil (70%), stored product dust (59%) and spider webs (50%). In contrast, the cry1Aa, cry1Ab, cry1Ac, cry1Ia, cry2Aa, cry2Ab and vip3Aa profiles were mainly detected in strains isolated from leaves (40%) and dead insect larvae (50%). Six of the identified insecticidal toxin gene profiles were discovered in strains isolated from stored product dust and leaves indicating higher diversity of profiles in these kinds of sources than in others. Some strains with high insecticidal activity against Epinotia aporema (Lepidoptera) larvae were identified, which is important to explore future microbial strategies for the control of this crop pest in the region.

Se analizaron 268 cepas de Bacillus thuringiensis obtenidas de diferentes fuentes de Argentina con el objeto de determinar la diversidad y distribución de genes cryl, cry2, cry8, cry9 y vip3A, que codifican proteínas insecticidas lepidóptero-específicas. Se excluyeron las cepas gemelas. Se detectaron solo diez perfiles diferentes entre los 80 B. thuringiensis seleccionados. Dos de estos perfiles, el cry1Aa, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (35/80) y el cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa (25/80), comprendieron el 75% de las cepas seleccionadas. La existencia de esta baja diversidad es una rareza, ya que en la mayor parte de las colecciones estudiadas se ha descrito una gran diversidad de perfiles de genes de toxinas insecticidas. El perfil detectado con mayor frecuencia se obtuvo principalmente de cepas procedentes de suelo (el 70% de los de esa fuente lo tenían), también fue mayoritario entre los procedentes de polvo de producto almacenado (59%) y en los que procedían de telas de arana (50%). En cambio, el perfil cry1Aa, cry1Ab, cry1Ac, crylIa, cry2Aa, cry2Ab y vip3Aa se detectó principalmente en las cepas aisladas de hojas (40%) y de larvas de insectos muertos (50%). Seis de los perfiles identificados fueron encontrados en cepas aisladas de polvo de producto almacenado y de hojas, lo que indica una mayor diversidad de perfiles en estas fuentes que en otras. Se identificaron algunas cepas con alta actividad insecticida contra larvas de Epinotia aporema (Lepidoptera), hallazgo importante para explorar en el futuro estrategias microbianas para el control de esta plaga en la región.

Perfil de suscetibilidade da população de Aedes aegypti (Diptera: Culicidae) da ilha de Santiago, Cabo Verde, a inseticidas / Susceptibility profile of Aedes aegypti from Santiago Island, Cape Verde, to insecticides

Em Cabo Verde, arquipélago situado na Costa Ocidental Africana, os primeiros casos de dengue ocorreram em 2009, com a notificação de mais de 21.000 casos, a maioria desses registrados na Ilha de Santiago. O mosquito Aedes aegypti foi identificado como vetor, e ações para seu controle, usando os inseticidas temephos (larvicida) e a deltametrina (adulticida), têm sido implementadas. Objetiva-se com esse trabalho avaliar o atual status de suscetibilidade a inseticidas e caracterizar os mecanismos de resistência nessa população. Amostras de A. aegypti da ilha de Santiago foram coletadas através de armadilhas de oviposição, para o estabelecimento de uma população a ser analisada. Foram realizados bioensaios do tipo dose diagnóstica, usando garrafas impregnadas com doses únicas dos adulticidas malathion (organofosforado), deltametrina (piretróide) e cipermetrina (piretróide), e bioensaios do tipo dose resposta, usando múltiplas concentrações dos inseticidas temephos (organofosforado), Bacillus thuringiensis sorovariedade israelensis (bactéria entomopatogênica) e diflubenzuron (inibidor de síntese de quitina). Para a investigação dos mecanismos de resistências, foram realizados testes bioquímicos com substratos específicos para quantificar a atividade das enzimas glutationa S-transferases, esterases (alfa, beta e PNPA) e oxidases de função mista, ligadas a detoxificação de xenobióticos, e a taxa de inibição da acetilcolinesterase ligada a insensibilidade do sítio alvo...

Cape Verde, an archipelago located on the West African Coast, recorded the first cases of dengue in 2009 in an epidemic with more than 21,000 reportedcases. The worst affected area was Santiago Island...

Extended genetic analysis of Brazilian isolates of Bacillus cereus and Bacillus thuringiensis

Multiple locus sequence typing (MLST) was undertaken to extend the genetic characterization of 29 isolates of Bacillus cereus and Bacillus thuringiensis previously characterized in terms of presence/absence of sequences encoding virulence factors and via variable number tandem repeat (VNTR). Additional analysis involved polymerase chain reaction for the presence of sequences (be, cytK, inA, pag, lef, cya and cap), encoding putative virulence factors, not investigated in the earlier study. MLST analysis ascribed novel and unique sequence types to each of the isolates. A phylogenetic tree was constructed from a single sequence of 2,838 bp of concatenated loci sequences. The strains were not monophyletic by analysis of any specific housekeeping gene or virulence characteristic. No clear association in relation to source of isolation or to genotypic profile based on the presence or absence of putative virulence genes could be identified. Comparison of VNTR profiling with MLST data suggested a correlation between these two methods of genetic analysis. In common with the majority of previous studies, MLST was unable to provide clarification of the basis for pathogenicity among members of the B. cereus complex. Nevertheless, our application of MLST served to reinforce the notion that B. cereus and B. thuringiensis should be considered as the same species.

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.

Characterization of Bacillus thuringiensis soil isolates from Cuba, with insecticidal activity against mosquitoes

Chemical insecticides may be toxic and cause environmental degradation. Consequently, biological control for insects represents an alternative with low ecological impact. In this work, three soil isolates (A21, A51 and C17) from different regions of the Cuban archipelago were identified, characterized and evaluated against Aedes aegypti and Culex quinquefasciatus. The new isolates were compared with reference IPS82 strain and two strains isolated from biolarvicides Bactivec and Bactoculicida, respectively. The differentiation was done by morphological, biochemical, bioassays activity and molecular methods (SDS-PAGE, plasmid profile and random amplified polymorphic analysis). All isolates were identified as Bacillus thuringiensis. The A21, A51 and C17 isolates showed higher larvicide activity than Bactivec’s isolated reference strain, against both A. aegypti and C. quinquefasciatus. A21 isolate had a protein profile similar to IPS82 and Bactivec strain. A51 and C17 isolates produced a characteristic proteins pattern. A21 and A51 isolates had plasmid patterns similar to IPS82 standard strain, while C17 isolate had different both plasmid profile and protein bands. All the studied isolates showed a diverse RAPD patterns and were different from the strains previously used in biological control in Cuba. Rev. Biol. Trop. 59 (3): 1007-1016. Epub 2011 September 01.

El uso prolongado de insecticidas ha conducido al desarrollo de resistencia en diferentes especies de mosquitos y al incremento de la degradación del ambiente. El control biológico de insectos ha devenido como una alternativa útil y de bajo impacto ambiental. En nuestro estudio fueron identificados, caracterizados tres aislamientos de suelos procedentes de diferentes regiones del archipiélago cubano y comparados con cepas de referencia: aisladas de los biolarvicidas Bactivec y Bactoculicida, además de IPS82. La diferenciación de los mismos se llevó a cabo mediante métodos morfológicos, bioquímicos y moleculares (SDSPAGE, perfil plasmídico, RAPD). Los aislamientos fueron identificados como Bacillus thuringiensis; A21, A51 y C17 mostraron una mayor actividad contra larvas de Aedes aegypti and Culex quinquefasciatus que la cepa aislada del biolarvicida Bactivec, utilizada como referencia en este estudio. Dos de los aislamientos poseían perfiles proteicos y plasmídicos similares al de la cepa control IPS82, pero el restante difería de ellos. Los tres mostraron patrones de RAPD diferentes lo que nos permitió su diferenciación. Estos patrones de RAPD también diferían del observado para las cepas utilizadas comúnmente en el control biológico en nuestro país.

Bacillus thuringiensis: avances y perspectivas en el control biológico de Aedes aegypti / Bacillus thuringiensis: Advances and perspectives in the biological control of Aedes aegypti

Bacillus thuringiensis (Bt) se presenta como una alternativa atractiva para el control de Aedes aegypti por sus claras ventajas sobre insecticidas químicos, a saber: alta especificidad, inocuidad sobre el medio ambiente y lento desarrollo de resistencia. Las nuevas tendencias en formulaciones que utilizan agentes encapsulantes como almidón, o adsorción a partículas de arcilla, ayudan a proteger los productos Bt de factores que afectan su actividad, tales como la radiación UV, la temperatura y la degradación microbiana, mejorando la persistencia del producto, al tiempo que pueden actuar como fago-estimulantes. No obstante, es necesario evaluar estas propuestas en el contexto del control de Aedes aegypti, sobretodo en relación a la manipulación humana de los criaderos y el estado nutricional de la larva. Bt también ofrece la posibilidad de obtener productos variados que permitan la alternancia de aplicaciones y, posiblemente, productos que, de ser necesario, se adecuen a las necesidades específicas de cada región

Bacillus thuringiensis is an attractive alternative for the control of Aedes aegypti for its clear advantages over chemical insecticides, high specificity, safety on the environment and slow development of resistance. New trends in formulations using encapsulating agents such as starch, or adsorption to clay particles that help protect the products of Bt factors affecting their activity, such as UV radiation, temperature and microbial degradation, enhance the persistence of the product, which can act as phage-type stimulants. However, it is necessary to evaluate these proposals in the context of the control of Ae. aegypti, especially in relation to the human manipulation of breeding sites and nutritional status of the larva. Bt also offers the possibility of obtaining products that enable a variety of alternate applications, and possibly products, if necessary, to suit the specific needs of each region

Conducta de oviposición de Aedes aegypti (L.) en presencia de Macrocyclops albidus (J.) y Bacillus thuringiensis var. israelensis en condiciones de laboratorio / Conduct of the oviposition of Aedes aegypti (L.) in the presence of Macrocyclops albidus ( J.), and Bacillus thuringiensis var. israelensis under lab conditions

Se evaluó la conducta de oviposición de hembras grávidas de Aedes aegypti en presencia de los agentes biológicos: Macrocyclops albidus (Copepoda: Cyclopoidea) o Bacillus thuringiensis var. israelensis (Bti) en condiciones de laboratorio. Se colocaron recipientes con 225 mL de agua declorada para la puesta de huevos con 3 variantes: agua con copépodos, agua con Bti y agua. El promedio de huevos depositados en los recipientes con copépodos y Bti fueron de 1 227,9 y 1 200,8 respectivamente, superiores al de 887,4 huevos del recipiente que contenía solo agua, aunque las diferencias observadas no fueron estadísticamente significativas (p> 0,05). Los Indices de la Actividad de Oviposición (IAO) calculados mostraron valores mayores que 0 para copépodos y para Bti, indicando que las hembras no evitaron ovipositar en estos recipientes. Los resultados sugieren que en la naturaleza los recipientes que contienen estos controladores biológicos pudieran constituir sitios para la oviposición de hembras grávidas de Aedes aegypti, lo que tendría implicaciones positivas en su eficacia para controlar este vector.

The behaviour of the oviposition of pregnant females of Aedes aegypti in the presence of biological agents: Macrocyclops albidus (Copepoda: Cyclopoidea) or Bacillus thuringiensis var. israelensis (Bti) was evaluated under lab conditions. Recipients containing 225 mL of dechlorinated water were placed for oviposition with three variants: water with copepods, water with Bti, and water. The average of eggs layed in the recipients with copepods and Bti were 1 227.9 and 1 200.8, respectively, a figure higher than the 887.4 eggs of the recipient containing only water, although the differences observed were not statistically significant (p >0.05). The Oviposotion Activity Index (OAI) estimated showed values over 0 for copepods and for Bti, which indicated that females did not avoid to deposit their eggs in these recipients. The results suggested that in nature, these recipients with biological controllers may be oviposition sites for pregnant females of Aedes aegypti, which may have positive implications on its effectiveness to control this vector.

Interação das toxinas Cry do Bacillus thuringiensis svar. israelensis com o mesêntero de larvas do vetor Aedes aegypti (Diptera: Culicidae) / Interaction of Cry toxins from Bacillus thuringiensis svar. israelensis with the mesenteron of the larvae of the vector Aedes aegypti (Diptera: Culicidae)

O Bacillus thuringiensis svar. israelensis (Bti) é um importante entomopatógeno utilizado na produção de larvicidas para o controle do Aedes aegypti, vetor da dengue. A toxicidade do Bti está baseada no cristal, produzido durante a esporulação, que contém quatro protoxinas Cry11Aa (70 kDa), Cry4Aa (125 kDa), Cry4Ba (130 kDa) e Cyt1A (28 kDa). Sua ação ocorre através da ingestão dos cristais que são solubilizados no mesêntero, onde as protoxinas são liberadas e clivadas por serina-proteases em toxinas ativas que agem em sinergia no epitélio intestinal e provocam a morte das larvas. Apesar da alta seletividade do Bti, ainda não foi completamente elucidado como as toxinas Cry interagem com os receptores específicos presentes no epitélio das larvas. O objetivo principal do trabalho foi caracterizar, através de ensaios in vitro de natureza quantitativa, a capacidade de ligação de cada toxina Cry (4Aa, 4Ba e 11Aa) às preparações de microvilli intestinal (BBMF) de larvas de Ae. aegypti. Para tal, cada componente Cry foi produzido a partir de cepas recombinantes, Bt cepa 4Q2-81, para produção de biomassas. A atividade inseticida das biomassas para larvas do 3º/4º estádios foi determinada através de bioensaios e, outra parte da biomassa foi utilizada para a obtenção dos cristais. Os cristais contendo cada protoxina foram processados in vitro e uma amostra de cada uma delas foi marcada com iodo (I125). Para realizar os estudos de ligação foram feitas preparações BBMF, a partir de larvas do 3º/4º estádios. Os estudos da capacidade de ligação da toxina foram realizados através de ensaios de competição, de saturação e de cinética, através de incubações entre a toxina- I125 e preparações de BBMF, na ausência ou na presença de um competidor. (...) Os resultados obtidos mostraram que as toxinas Cry competem pelos mesmos sítios e partilham receptores presentes na BBMF. Em todos os casos estudados, a afinidade do complexo toxinareceptor não foi elevada, e não foi detectada sinergia entre as toxinas Cry para a ligação à BBMF. A ligação entre as toxinas-I125 e a BBMF é irreversível, e observou-se uma forte tendência à oligomerização nos três casos. Os resultados obtidos nesse trabalho sugerem que a toxicidade das toxinas Cry para larvas de Aedes está relacionada à etapa irreversível de ligação com os receptores, e não é caracterizada por um padrão elevado de afinidade do complexo toxina-receptor ...

Potentiation of insecticidal activity of Bacillus thuringiensis subsp. kurstaki HD-1 by proteinase inhibitors in the American bollworm, Helicoverpa armigera (Hübner).

The effect of crude proteinase inhibitor extracts from seeds of different crop plants (black gram, chickpea, chickling vetch, finger millet, French bean, green gram, horse gram, lentil, pea and soybean) on the insecticidal activity of B. thuringiensis var. kurstaki HD-1 was investigated against neonate larvae of H. armigera by diet incorporation method. The larval mortality due to crude proteinase inhibitors alone (5% seed weight equivalent) ranged from 4.1 to 19.1%; the maximum mortality with finger millet and the minimum with pea var. DDR-23. A mixture of B. thuringiensis var. kurstaki HD-1 (10 ppm) and proteinase inhibitor (5% seed weight equivalent) was synergistic in larval mortality with respect to proteinase inhibitors of pea var. DMR-16, chickling vetch var. RLK-1098 and B101-212, lentil var. ILL-8095 and L-4076, soybean var. PK-1042, PK-416 and Pusa-22, chickpea var. Pusa-413, French bean (Chitra) and black gram; and antagonistic with respect to those of finger millet, horse gram and kidney bean. The larval growth reduction with crude proteinase inhibitors alone ranged from 17.9 to 53.1%; the maximum growth reduction with soybean var. PK-1042 and minimum with lentil var. L-4076. A mixture of B. thuringiensis var. kurstaki and proteinase inhibitor was synergistic in growth reduction with respect to proteinase inhibitors of lentil var. ILL-8095, and L-4626 and antagonistic with respect to that of finger millet. The midgut proteinase inhibition with crude seed extracts (3.3% seed weight equivalent) ranged from 9.3 to 60.9% and was negatively correlated with larval mortality. These results showed that interactive effect of B. thuringiensis var. kurstaki HD-1 and proteinase inhibitors in the larvae of H. armigera depended upon the quality and quantity of proteinase inhibitors, which vary widely in different plants.

Effect of sublethal concentration of Bacillus thuringiensis var. kurstaki on food and developmental needs of the american bollworm, Helicoverpa armigera (Hübner).

Effect of sublethal concentration of B. thuringiensis on the first, third, fourth and fifth instar larvae of the American bollworm, H. armigera was investigated to study their response to food consumption, digestion, utilization, and their development till adult formation. The young larvae surviving B. thuringiensis treatment in their first instar and third instar delayed larval period by two to three days, but did not consume more food as compared to control. However, they showed higher digestibility of food as compared to control, which was compensated by their reduced ability to utilize the digested food for body substance. Contrary to the effect on first and third instar larvae, the fifth instar larvae surviving B. thuringiensis treatment in its fourth instar consumed less food, showed less absorption efficiency in digesting food, but compensated by increase in the utilization of ingested and digested food into body substance. Insects surviving B. thuringiensis HD-1 sublethal toxicity adapted to normal larval growth when fed on untreated food, depending upon insect growth prior to treatment. The moths emerging from B.thuringiensis treated larvae had sex ratio favouring females, and adult pairs laid less fertile eggs than those from the untreated ones. The response of B. thuringiensis treated larvae to their food and developmental needs is discussed.

Application of bacillus thuringiensis in poultry houses as a biological control agent against the house fly, musca domestica sorbens

The efficiency of Bacillus thuringiensis [Bt] as a microbial housefly control agent was evaluated in one of the natural maggot breeding medium; namely, poultry feces. Two commercial formulations [a wettable powder and a liquid concentrate] were assayed against Musca domestica sorbens in synthetic fly breeding media and in chicken feces. The latter favored maggot breeding when compared with the synthetic one. Both formulations resulted in a significant reduction in maggot numbers, pupal development and adult emergence. The concentration significantly affected the adult emergence. The liquid concentrate induced higher larvicidal activity than dry powder formulation. Contaminated feces produced from orally fed chickens were significantly toxic to the breeding maggots reaching maximum larvicidal activity till the 4th day post-feeding feces and continued in less activity till the 6th day

Effectiveness of "Bacillus thuringiensis" strains against "Spodoptera frugiperda" (Lepidoptera: Noctuidae)

The fall armyworm ("Spodoptera frugiperda") is one of the most important pests of maize, causing up to 20(per cent) production losses when defoliation occurs near to flowering, or even complete destruction of plants. Among the alternatives to control this pest, the use of "Bacillus thuringiensis" ("Bt") has gained attention due to its efficiency and low impact on natural enemies. Strains "Bt dendrolimus" HD 37, "Bt aizawai" HD 68, "Bt kurstaki" HD 73, "Bt darmstadiensis" HD 146, and "Bt thuringiensis" 4412 were tested against second instar larvae in "in vivo" assays. Suspensions of "Bt aizawai" HD 68 and "Bt thuringiensis" 4412, containing 3 x 10 8 cells/ml, induced mortality of 100(per cent) and 80.4(per cent), respectively. To test virulence, cell concentrations of 8x10(5) to 3x10(8) cells/ml of strains "Bt aizawai" HD 68 and "Bt thuringiensis" 4412 were applied on the second instar larvae: LC(50) were 6.7x10(6) and 8.6x10(6) cells/ml, respectively.

Use of endophytic diazotrophic bacteria as a vector to express the cry3A gene from "Bacillus thuringiensis"

The goal of this study was to evaluate the potential of endophytic diazotrophic bacteria as a vector to express a 'cry' gene from "Bacillus thuringiensis", envisaging the control of pests that attack sugarcane plants. The endophytic nitrogen-fixing bacteria "Gluconacetobacter diazothophicus" strain BR11281 and "Herbaspirillum seropedicae" strain BR11335 were used as models. The 'cry3A' gene was transferred by conjugation using a suicide plasmid and recombinant strains were selected by their ability to fix nitrogen in semi-solid N-free medium. The presence of the 'cry' gene was detected by Southern-blot using an internal fragment of 1.0 kb as a probe. The production of (delta)-endotoxin by recombinant "H. seropedicae" strain was detected by dot blot while for "G. diazotrophicus" the Western-blot technique was used. In both cases, a specific antibody raised against the "B. thuringiensis" toxin was applied. The (delta)-endotoxin production showed by the "G. diazotrophicus" recombinant strain was dependent on the nitrogen fixing conditions since the 'cry3A' gene was fused to a 'nif' promoter. In the case of "H. seropedicae" the (delta)-endotoxin expression was not affected by the promoter ('rhi') used. These results suggest that endophytic diazotrophic bacteria can be used as vectors to express entomopathogenic genes envisaging control of sugarcane pests

Resistência do Aedes fluviatilis (Lutz, 1904) ao Bacillus thruringiensis israelensis (Goldberg e Margalit, 1977) e a infecção pelo Plasmodium gallinaceum (Brumpt, 1935)

A família Culicidae é de fundamental importância médica porque transmite vários patógenos ao homem incluindo aqueles que causam a malária. O Aedes fluviatilis infectado com Plasmodium gallinaceum tem sido usado como modelo para estudar a interação entre os parasitas da malária e os mosquitos vetores. Foi analisado o uso do Bacillus thuringiensis var. isralensis (B.t.i.), da resistência aos inseticidas convencionais e dos problemas no meio ambiente causado pelo seu uso prolongado. Contudo, poucos estudos têm considerado o potencial de resistência dos insetos aos patógenos naturais. Para verificar essa possibilidade e entender como o B.t.i. pode afetar a taxa de infecção da malária no mosquito vetor em áreas tratadas, foi testada a habilidade vetorial da população de Ae. fluviatilis selecionada para resistência em condições laboratoriais. Em nossos bioensaios, a DL50 apresentou um aumento de 7,5 vezes de resistência na 2° geração (F2). Os intestinos médios de adultos tratados com B.t.i. e processados para SDS-PAGE não demonstraram a presença de uma banda 206 kDa presente nos mosquitos adultos não tratados com o patógeno.

A análise estatística de Ae. fluviatilis tratados e não tratados com B.t.i. infectados com P. gallinaceum e insetos não tratados infectados não mostraram diferenças significativas nas taxas de infecção, avaliadas pelo número de oocistos presentes nos intestinos médios. A análise dos estômagos dos mosquitos revelou que os perfis moleculares, obtidos pela análise de LS-PCR dos indivíduos não tratados diferem dos daqueles insetos expostos ao B.t.i., já que a LS-PCR amplificou uma banda de aproximadamente 620 pb nos mosquitos controles. Esses perfis se mantiveram os mesmos tanto para um indivíduo quanto para os “pools”, na microvilosidade dos intestinos de adultos e larvas tratados com B.t.i. Portanto, o Ae. fluviatilis exposto ao B.t.i. exibiu certas alterações bioquímicas, fisiológicas, e moleculares e de resistência, porém os nossos estudos não foram capazes de demonstrar alteração na capacidade vetorial.

Resistência do Aedes fluviatilis (Lutz, 1904) ao Bacillus thruringiensis israelensis (Goldberg e Margalit, 1977) e a infecção pelo Plasmodium gallinaceum (Brumpt, 1935) / Resistance of Aedes fluviatilis (Lutz, 1904) to Bacillus thruringiensis israelensis (Goldberg and Margalit, 1977) and infection by Plasmodium gallinaceum (Brumpt, 1935)

A família Culicidae é de fundamental importância médica porque transmite vários patógenos ao homem incluindo aqueles que causam a malária. O Aedes fluviatilis infectado com Plasmodium gallinaceum tem sido usado como modelo para estudar a interação entre os parasitas da malária e os mosquitos vetores. Foi analisado o uso do Bacillus thuringiensis var. isralensis (B.t.i.), da resistência aos inseticidas convencionais e dos problemas no meio ambiente causado pelo seu uso prolongado. Contudo, poucos estudos têm considerado o potencial de resistência dos insetos aos patógenos naturais. Para verificar essa possibilidade e entender como o B.t.i. pode afetar a taxa de infecção da malária no mosquito vetor em áreas tratadas, foi testada a habilidade vetorial da população de Ae. fluviatilis selecionada para resistência em condições laboratoriais. Em nossos bioensaios, a DL50 apresentou um aumento de 7,5 vezes de resistência na 2° geração (F2). Os intestinos médios de adultos tratados com B.t.i. e processados para SDS-PAGE não demonstraram a presença de uma banda 206 kDa presente nos mosquitos adultos não tratados com o patógeno. A análise estatística de Ae. fluviatilis tratados e não tratados com B.t.i. infectados com P. gallinaceum e insetos não tratados infectados não mostraram diferenças significativas nas taxas de infecção, avaliadas pelo número de oocistos presentes nos intestinos médios. A análise dos estômagos dos mosquitos revelou que os perfis moleculares, obtidos pela análise de LS-PCR dos indivíduos não tratados diferem dos daqueles insetos expostos ao B.t.i., já que a LS-PCR amplificou uma banda de aproximadamente 620 pb nos mosquitos controles. Esses perfis se mantiveram os mesmos tanto para um indivíduo quanto para os “pools”, na microvilosidade dos intestinos de adultos e larvas tratados com B.t.i. Portanto, o Ae. fluviatilis exposto ao B.t.i. exibiu certas alterações bioquímicas, fisiológicas, e moleculares e de resistência, porém os nossos estudos não foram capazes de demonstrar alteração na capacidade vetorial.

Influence of pH on the toxicity and survival of total cells and spores of Bacillus thuringiensis

Entre los grupos de microorganismos que se estudian por su potencial entomopatógeno, B. thuringiensis (Bt) es sin lugar a dudas la bacteria de mayor importancia en el control de insectos, plagas y vectores. En este trabajo se estudió la patogenicidad y sobrevivencia de células totales y de esporas de tres cepas de B. thuringiensis autóctonas de suelos argentinos (A61, A27 y A68) y una cepa comercial HD-1, a distintas concentraciones de ión hidrógeno (pH=4,4; 5,4;7,4; 8,4; 9,4 y 10,4), bajo condiciones de laboratorio. No se observó disminución de la toxicidad de las cepas desarrolladas a distintos valores de pH en bioensayos contra Cydia pomonella. El mayor efecto antimicrobiano sobre el número de esporas y células totales se detectó a pH 4,4. El pH 5,4 fue el que permitió un mayor índice de sobrevivencia en los serotipos investigados. Tres cepas de Bt (A61,A27,HD-1) mostraron alta toxicidad contra C. pomonella. El serotipo sotto, sin embargo, no produjo ese efecto

Assignment of the crystal toxin genes of the mosquitocidal bacterium, bacillus thuringiensis israelensis to a specific plasmid

Bacillus thuringiensis 4Q1-WT and the prepared mutants, 4Q1-72 and 4Q1-81, were bioassayed against Aedes caspius larvae. The strain-4Q1-WT, which contains all plasmid arrays and the strain 4Q1-72 which contains the 108 kb plasmid gave 100% mortality, while stain 4Q1-81, which contains no plasmids, gave 0% mortality. Crystals from all tested strains were isolated, solubilized and characterized using PAGE to detect any homology or difference in crystal production, properties, and the relatedness to the plasmid profile

Susceptibilidade de populaçöes de Simulium (Chirostilbia) pertinax Kollar, 1832 (Culicomorpha, Simulidae) ao temephos e a um formulado à base de Bacillus thuingiensis var. israelensis / Susceptibility of populations of Simulium (Chirostilia) pertinax Kollar, 1832 (Culicomorpha, Simuliidae) to temephos and to Bacillus thuringiensis var. israelensis formulation

O uso de pequenas calhas de madeira no leito de riachos, manualmente colonizadas com borrachudos, é proposto para avaliaçöes da eficiência de larvicidas 3 ou 4 horas após as aplicaçöes. A susceptibilidade larval também foi avaliada pelo critério de TL50. Em três ensaios no litoral do Estado de Säo Paulo e Rio de Janeiro, populaçöes de Simulium (C.) pertinax Kollar, 1832 mostram-se resistentes ao temephos, mesmo em altas concentraçöes. Vectobac 12 AS, à base de Bacillus thuringiensis var. israelensis mostrou-se mais potente contra larvas dos últimos estádios e eficiente para controle em concentraçöes a partir de 7.200 UIP/l (10 min.) O TL50 para 3.744 UIP/l (10 min.) foi de 70,9 minutos