940 resultados para Insect resistance management
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Pós-graduação em Biociências - FCLAS
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Pós-graduação em Agronomia (Produção Vegetal) - FCAV
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Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV
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Pós-graduação em Agronomia (Produção Vegetal) - FCAV
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Pós-graduação em Agronomia (Proteção de Plantas) - FCA
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The present study aimed to determine the susceptibility of 17 Rhipicephalus (Boophilus) microplus populations, originating in the Southeast and Southern regions of Brazil, to different ivermectin concentrations (200, 500 and 630 mu g/kg), administered through subcutaneous or topical (pour-on) routes. R. (B.) microplus populations from the states of Minas Gerais (seven populations), Sao Paulo (seven populations) and Parana (three populations) were chosen for the tests. The selected cattle were allocated to treatment groups on day 0, and block formation was based on the arithmetic mean of female ticks (4.5-8.0 mm long) counted on three consecutive days (-3, -2 and -1). To evaluate the therapeutic and residual efficacies of these formulations, tick counts (females ranging from 4.5 to 8.0 mm long) were performed on days 3,7 and 14 post-treatment, and continued on a weekly basis thereafter until the end of each experiment. The results obtained throughout this study, utilizing field efficacy studies, allowed us to conclude that the resistance of R. (B.) microplus against 200 and 500 mu g/kg ivermectin is widely disseminated because all tick populations that had contact with these specific concentrations were diagnosed as resistant. However, it is possible to infer that R. (B.) microplus resistance against 630 mu g/kg ivermectin was also widespread, diagnosed at six of ten analyzed properties. Resistance of these ectoparasites to 630 mu g/kg ivermectin is most likely emerging in three other populations of R. (B.) microplus. Strategies of resistance management need to be quickly determined to keep the selection pressure at a minimum level in Brazil. (C) 2014 Elsevier B.V. All rights reserved.
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Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV
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Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV
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Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV
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Diatraea saccharalis, is a major sugarcane pest, causing damage to the stalks of sugarcane plants. In this study, a trypsin inhibitor (ApTI) was purified from Adenanthera pavonina seeds and was tested for its insect growth regulatory effect. ApTI showed a dose-dependent effect on average larval weight and survival. 0.1% ApTI produced approximately 67% and 50% decreases in weight and survival larval, respectively. The results from dietary utilization experiments with D. saccharalis larvae showed a reduction in the efficiency of conversion of ingested food and digested food, and an increase in approximate digestibility and metabolic cost. The level of trypsin was significantly decreased (ca. 55%) in the midgut of larvae reared on a diet containing 0.05% ApTI and the trypsin activity in ApTI-fed larvae demonstrated sensitivity to ApTI. The action of ApTI on the development of D. saccharalis larvae shows that this protein may have great toxic potential. (C) 2011 Elsevier Ltd. All rights reserved.
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A cDNA coding for a digestive cathepsin L, denominated Sl-CathL, was isolated from a cDNA library of Sphenophorus levis larvae, representing the most abundant EST (10.49%) responsible for proteolysis in the midgut. The open reading frame of 972 bp encodes a preproenzyme similar to midgut cathepsin L-like enzymes in other coleopterans. Recombinant Sl-CathL was expressed in Pichia pastoris, with molecular mass of about 42 kDa. The recombinant protein was catalytically activated at low pH and the mature enzyme of 39 kDa displayed thermal instability and maximal activity at 37 degrees C and pH 6.0. Immunocytochemical analysis revealed Sl-CathL production in the midgut epithelium and secretion from vesicles containing the enzyme into the gut lumen, confirming an important role for this enzyme in the digestion of the insect larvae. The expression profile identified by RT-PCR through the biological cycle indicates that Sl-CathL is mainly produced in larval stages, with peak expression in 30-day-old larvae. At this stage, the enzyme is 1250-fold more expressed than in the pupal fase, in which the lowest expression level is detected. This enzyme is also produced in the adult stage, albeit in lesser abundance, assuming the presence of a different array of enzymes in the digestive system of adults. Tissue-specific analysis revealed that Sl-CathL mRNA synthesis occurs fundamentally in the larval midgut, thereby confirming its function as a digestive enzyme, as detected in immunolocalization assays. The catalytic efficiency of the purified recombinant enzyme was calculated using different substrates (Z-Leu-Arg-AMC, Z-Arg-Arg-AMC and Z-Phe-Arg-AMC) and rSl-CathL exhibited hydrolysis preference for Z-Leu-Arg-AMC (k(cat)/K-m = 37.53 mM S-1), which is similar to other insect cathepsin L-like enzymes. rSl-CathL activity inhibition assays were performed using four recombinant sugarcane cystatins. rSl-CathL was strongly inhibited by recombinant cystatin CaneCPI-4 (K-i = 0.196 nM), indicating that this protease is a potential target for pest control. (C) 2011 Elsevier Ltd. All rights reserved.
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La mosca mediterránea de la fruta Ceratitis capitata (Wiedemann, 1824) está considerada una de las plagas clave para la fruticultura. El malatión es un insecticida organofosforado que fue empleado mayoritariamente en España para el control de C. capitata hasta 2009, año en el que dejó de utilizarse por no estar incluido en el anexo I de la Directiva Europea 91/414/ECC. El incremento del uso del malatión, debido a las graves pérdidas económicas causadas por C. capitata, provocó la aparición de poblaciones de campo resistentes. El estudio de una población resistente a malatión, recogida en Castelló en 2004, permitió la identificación de dos mecanismos de resistencia: una mutación puntual (G328A) en la acetilcolinesterasa (AChE) y un mecanismo de resistencia metabólica, probablemente mediado por carboxilesterasas. Teniendo en cuenta estos antecedentes, nos propusimos estudiar los mecanismos implicados en la resistencia a malatión en C. capitata. Además, durante el desarrollo de esta Tesis, el malatión fue sustituido por otros insecticidas como el espinosad y la lambda-cialotrina para el control de la plaga. En este nuevo contexto, es extremadamente importante analizar la susceptibilidad de poblaciones de campo frente a espinosad y estudiar la posible existencia de resistencia cruzada a estos insecticidas, así como sentar las bases para el estudio de futuros mecanismos de resistencia. En primer lugar, analizamos mediante bioensayos con dosis discriminante la susceptibilidad a malatión y espinosad en doce poblaciones de C. capitata de Andalucía, Aragón, Cataluña, Comunidad Valenciana e Islas Baleares; y nuestros resultados sugirieron la presencia de individuos resistentes a malatión en la mayoría de las poblaciones analizadas. En el caso del espinosad, observamos que la susceptibilidad a este insecticida de origen biológico fue elevada en la mayoría de las poblaciones, sin embargo, la población recogida en Xàbia (Alicante) mostró un nivel de susceptibilidad unas dos veces menor al resto de poblaciones. Mediante la selección en laboratorio, obtuvimos dos líneas resistentes a malatión, W-4Km y W-10Km, con unos niveles de resistencia con respeto a la línea susceptible C de 178 y 400 veces, respectivamente. Además, se seleccionó por primera vez en C. capitata una línea altamente resistente a espinosad (Xàbia-W-100s), que actualmente es unas 500 veces más resistente que la línea de laboratorio C. Con el objetivo de escoger la estrategia más adecuada para el manejo de la plaga, estudiamos la susceptibilidad a diferentes tipos de insecticidas en la línea resistente a malatión W- 4Km. En esta línea detectamos resistencia cruzada moderada a los organofosforados fentión, diazinón, fosmet, triclorfón y metil-clorpirifos (de 7 a 16 veces) y frente al carbamato carbaril, al piretroide lambda-cialotrina y al quimioesterilizante lufenurón (de 4 a 6 veces). Por otra parte, la resistencia cruzada frente a espinosad fue baja (1,5 veces). Es importante destacar que los niveles de resistencia estimados frente a todos los insecticidas fueron de uno o dos órdenes de magnitud inferiores al observado en la línea W-4Km frente a malatión (178 veces), hecho que podría deberse, al menos, a dos posibles hipótesis: que la mutación AChE G328A confiera mayor insensibilidad al malaoxón (forma activa del malatión) que a otros insecticidas que tienen como diana la AChE y/o, en segundo lugar, que el mecanismo de resistencia mediado por carboxilesterasas hidrolice el malatión de manera más eficiente que los otros insecticidas analizados. En el estudio de nuevos mecanismos de resistencia en C. capitata, por un lado, analizamos la diversidad de enzimas citocromo P450, asociadas con resistencia metabólica en otras especies, y por otro lado, desarrollamos un sistema para la detección de nuevas mutaciones puntuales que pudiesen aparecer en los genes que codifican la AChE (Ccace2) y la aliesterasa (Ccae7). Mediante el empleo de cebadores degenerados obtuvimos 37 genes CYP, que codifican enzimas P450, pertenecientes a cinco familias. Posteriormente, en un estudio de inducción con fenobarbital, observamos que la expresión de cuatro de los seis genes analizados era susceptible de ser inducida. Por otro lado, se puso a punto un sistema que permite amplificar y secuenciar, a partir de DNA genómico, los exones de los genes Ccace2 y Ccae7 en los que se han encontrado mutaciones relacionadas con resistencia a insecticidas en otras especies. Los resultados obtenidos facilitarán el estudio de nuevos mecanismos de resistencia mediados por estas enzimas en C. capitata. Se diseñó un método PCR-RFLP para identificar los individuos portadores de la mutación AChE G328A (alelo de resistencia Ccace2R) sin la necesidad de realizar bioensayos y que, además, permite detectar resistencia cuando ésta se encuentra a baja frecuencia. Según el análisis realizado, el alelo Ccace2R se observó en 25 de las 27 localidades españolas muestreadas en el territorio español, incluyendo las Islas Baleares y Canarias. Sin embargo, este alelo no se detectó en poblaciones procedentes de once países y de cinco continentes. El análisis de la presencia del alelo Ccace2R en las líneas resistentes a malatión durante el proceso de selección en el laboratorio mostró una rápida disminución de los homocigotos, tanto para el alelo susceptible como para el alelo de resistencia, en favor de los individuos heterocigotos. Así, después de 52 generaciones de selección, se observó que la totalidad de los individuos analizados de la línea W-10Km presentaban un genotipo heterocigoto para la mutación AChE G328A. Este desequilibrio contradice la segregación mendeliana esperada para un gen con dos alelos pero podría ser explicado por la existencia de una duplicación del gen Ccace2. La demostración de la presencia de esta duplicación se realizó mediante: i) el cruzamiento de individuos heterocigotos de la línea W-10Km con homocigotos susceptibles de la línea C, que dio lugar a una descendencia en la que el 100% de los individuos eran heterocigotos; ii) la evaluación del número de copias del gen Ccace2 por PCR cuantitativa en tiempo real (qPCR), que resultó dos veces mayor en individuos de la línea W-10Km en comparación con los de la línea C; iii) el análisis del nivel de expresión de Ccace2, que fue el doble en la línea W-10Km con respecto a la línea C, y iv) el estudio de la actividad AChE, que resultó mayor en los individuos de la línea W-10Km. Según los resultados obtenidos, una duplicación del gen Ccace2 provoca la coexistencia en un mismo cromosoma del alelo silvestre y del alelo mutado y, además, las dos copias del gen Ccace2, al estar ligadas, producen una heterocigosis permanente (Ccace2RS). De esta manera se explica que el hecho de que 100% de los individuos de la línea W-10Km mostrasen un perfil de restricción correspondiente a un individuo heterocigoto ya que, en realidad, eran homocigotos estructurales para la duplicación (genotipo CCace2RS/RS). Se ha detectado un coste biológico asociado a la duplicación que consiste en un incremento en la mortalidad acumulada de los adultos a partir del séptimo día después de la emergencia. La descripción de la duplicación Ccace2RS supone la identificación de un nuevo mecanismo de resistencia a malatión en C. capitata. Finalmente, mediante el diseño de un método de doble PCR-RFLP se determinó la presencia de la duplicación Ccace2RS en la mayoría de las poblaciones españolas. La proporción de individuos portadores de la duplicación osciló entre el 5% y el 35%, observándose los mayores valores de frecuencia en las poblaciones de C. capitata recogidas en la cuenca mediterránea. Podemos por lo tanto concluir que la resistencia a malatión asociada a la mutación AChE G328A y a la duplicación Ccace2RS está ampliamente establecida en las poblaciones españolas de C. capitata. Nuestros resultados desaconsejan la utilización del malatión (si fuera de nuevo autorizado) o de otros organofosforados para el control de esta plaga. Además, una de las líneas resistentes a malatión mostró resistencia cruzada frente a insecticidas con diferentes modos de acción y que se utilizan actualmente para el control de C. capitata, tales como lambda-cialotrina y lufenurón. La alta susceptibilidad a espinosad observada en las poblaciones españolas, así como la reducida resistencia cruzada estimada para este insecticida, sugieren que su utilización es adecuada para el control de la plaga. Sin embargo, la utilización de un sólo insecticida puede entrañar riesgos por favorecer la selección de resistencia, de hecho, mediante selección en laboratorio se obtuvo una población altamente resistente a espinosad. Por tanto, es recomendable implementar programas de control integrado y de manejo de la resistencia en C. capitata utilizando distintos sistemas de control e insecticidas con diferentes mecanismos de acción que permitan su sostenibilidad en el tiempo. Los sistemas de detección de alelos de resistencia desarrollados en este trabajo permitirán la detección precoz de resistencia en campo, facilitando la decisión sobre el sistema de control más adecuado. Además, los conocimientos generados podrán contribuir al desarrollo de nuevos sistemas de detección para otros mecanismos de resistencia. Abstract. The Mediterranean fruit fly, Ceratitis capitata (Wiedemann, 1824), is considered one of the most harmful pests in fruit crops. Until 2009, when malathion use was banned due to its not inclusion in the Annex I of Directive 91/414/EEC, the application of this organophosphate (OP) insecticide in Spain increased gradually due to the large economic losses caused by C. capitata. The increase in the frequency of treatments resulted in the development of resistant field populations. The study of a malathion-resistant population, collected in 2004 in Castelló (Comunidad Valenciana), allowed the identification of two resistance mechanisms: a single point mutation (G328A) in the target acetylcholinesterase (AChE), as well as a metabolic resistance mechanism, most likely carboxylesterase-mediated. Taking all the preceding into account, we studied the malathion resistance mechanisms in C. capitata. During the development of this PhD Thesis malathion use was banned by the European Union, being replaced by other insecticides, such as spinosad and lambda-cyhalotrin. Within this new working frame, the need to analyse the possible existence of cross-resistance to these insecticides and the susceptibility to spinosad in field populations was raised. This would define the baseline for future studies on resistance mechanisms. Firstly, through discriminant dose bioassays, we analysed malathion and spinosad susceptibility in twelve C. capitata populations from Andalucia, Aragon, Cataluña, C. Valenciana and the Baleares Islands. Our results suggest the presence of malathion-resistant individuals in most of the populations analysed. Regarding spinosad, we noticed a high susceptibility to this biologically derived insecticide in most of the populations, but in the one collected in Xabia (Alicante), which had a susceptibility level two times lower than the rest of populations. Through laboratory selection, we obtained two malathion-resistant strains, W-4Km and W-10Km, with resistance levels 178- and 400-fold, respectively, compared to the control susceptible C strain. Besides, a strain highly-resistant to spinosad (Xabia-W-100s), 500-times more resistant than control C strain, was selected. In order to decide the most appropriate management strategy for the pest, we studied the susceptibility to different insecticides in the malathion-resistant W-4Km strain. We detected a moderated cross-resistance to the OPs fenthion, diazinon, phosmet, trichlorphon and methylchlorpyrifos (7- to 16-fold), and to the carbamate carbaryl, the pyretroid lambda-cyhalotrin and the chemosterilizer lufenuron (4- to 6-fold). On the other hand, cross-resistance to spinosad was low (1.5-fold). It is important to note that resistance levels to all insecticides were one or two orders of magnitude less than that observed against malathion in W-4Km strain (178-fold), a fact that might be due to, at least, two possible causes: mutation AChE G328A may provide a higher insensitivity to malaoxon (the active form of malathion) than to other insecticides having AChE as target, and/or, secondly, the carboxylesterase-mediated resistance mechanism hydrolyzes malathion more efficiently than all other analysed insecticides. To investigate new resistance mechanisms in C. capitata we analysed the diversity of the cytochrome P450 enzymes, which have been associated to metabolic resistance in insects, and we developed a new method to detect single point mutations in acetylcholinesterase (Ccace2) and aliesterase (Ccae7) genes that could appear. Using degenerate primers we obtained 37 CYP genes, coding P450 enzymes, included in five families. Afterwards, in a phenobarbital-induction study, we observed that the expression of 4 out of the 6 analysed genes could be induced. On the other hand, a system was set up to amplify and to sequence from genomic DNA the exons of genes Ccace2 and Ccae7 where mutations related to insecticide resistance have been found in other species. The results obtained could facilitate the study of new resistance mechanisms in C. capitata mediated by these enzymes. A PCR-RFLP method was designed to detect the presence of the mutation AChE G328A (resistance allele Ccace2R), with no need to perform bioassays and allowing detecting resistance at low frequency. According to the analysis, the resistance allele was found in 25 out of 27 sampled locations in Spain, including the Balearic and the Canary Islands. However, this allele was not detected in other populations collected in 11 countries from 5 continents. The follow-up of the presence of the allele Ccace2R in the malathion-resistant strains during the selection process in the laboratory showed a quick decrease in homozygous individuals, for both the susceptible and the resistant alleles, favouring heterozygous. Thus, after 52 generations of selection, all the individuals analysed from W-10Km strain showed a heterozygous genotype for mutation AChE G328A, contradicting mendelian segregation as expected for a gene with two alleles. Afterwards, we were able to demonstrate that this was caused by the presence of a duplication of the gene coding acetylcholinesterase by: i) crossing heterozygous individuals from W-10Km strain with susceptible homozygous from C strain, originating a F1 population in which 100% of individuals were heterozygous; ii) evaluating the number of copies of gen Ccace2 by quantitative PCR in real time (qPCR), that happened to be twice higher in individuals from W-10Km VII strain when compared with C strain; iii) analysing the level of expression of Ccace2, twice in W- 10Km strain when compared to C strain; iv) studying the acetylcholinesterase activity, that was higher in individuals from W-10Km strain. According to these results, duplication of gen Ccace2 originates the coexistence of the susceptible and the resistant allele in the same chromosome. The two linked copies of the gene Ccace2 provoke the existence of permanent heterozygosis (Ccace2RS). This explains why the 100% of individuals from W-10Km strain showed an heterozygous restriction pattern since, in fact, they were structural homozygotes for the duplication (genotype Ccace2RS/RS). A biological cost has been detected associated to this duplication, consisting in a rise in accumulated adult mortality from the seventh day after emergence. The Ccace2RS duplication described in this study represents a new resistance mechanism to malathion in C. capitata. Finally, by the design of a double PCR-RFLP method, the presence of Ccace2RS duplication was confirmed in most of the Spanish populations. We observed that the proportion of individuals carrying the duplication oscillated between 5 and 35%, the frequency being higher in those C. capitata populations collected in the area of the Mediterranean basin. Therefore, we can conclude that malathion resistance associated to mutation AChE G328A and to Ccace2RS duplication are widely distributed in Spanish populations of C. capitata. Our results advice against the use of malathion (if it came to be newly authorized for use) or other OPs for the control of this pest. Besides, one of the malathion-resistant strains showed cross-resistance against insecticides with diverse action modes that are currently used for pest control, such as lambdacyhalotrin and lufenuron. High susceptibility to spinosad in the Spanish populations, as well as the reduced cross-resistance estimated for this insecticide suggests its adequacy for Medfly control. However, the use of a single insecticide is a risky strategy since it favours the selection of resistance. In fact, a population highly resistant to spinosad was obtained through laboratory selection. Therefore, it is advisable to implement integrated pest management (IPM) and resistance management programs for C. capitata control. Using insecticides with different modes of action and diverse control systems would contribute to the sustainability of the pest control. The resistance allele detection systems developed through this work will allow the early detection of resistance in the field, making possible the selection of the most appropriate method for pest control. Besides, the generated knowledge may also contribute to the development of new detection systems for other resistance mechanisms.
Induction and transmission of Bacillus thuringiensis tolerance in the flour moth Ephestia kuehniella
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The use of Bacillus thuringiensis (Bt) endotoxins to control insect vectors of human diseases and agricultural pests is threatened by the possible evolution of resistance in major pest species. In addition to high levels of resistance produced by receptor insensitivity (5, 16, 17), several cases of tolerance to low to medium levels of toxin have been reported in laboratory colonies of lepidopteran species (3, 18). Because the molecular basis of some of these cases of tolerance to the toxin are not known, we explored alternative mechanisms. Here, we present evidence that tolerance to a Bt formulation in a laboratory colony of the flour moth Ephestia kuehniella can be induced by preexposure to a low concentration of the Bt formulation and that the tolerance correlates with an elevated immune response. The data also indicate that both immune induction and Bt tolerance can be transmitted to offspring by a maternal effect and that their magnitudes are determined by more than one gene.
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Both large and small scale migrations of Helicoverpa armigera Hübner in Australia were investigated using AMOVA analysis and genetic assignment tests. Five microsatellite loci were screened across 3142 individuals from 16 localities in eight major cotton and grain growing regions within Australia, over a 38-month period (November 1999 to January 2003). From November 1999 to March 2001 relatively low levels of migration were characterized between growing regions. Substantially higher than average gene-flow rates and limited differentiation between cropping regions characterized the period from April 2001 to March 2002. A reduced migration rate in the year from April 2002 to March 2003 resulted in significant genetic structuring between cropping regions. This differentiation was established within two or three generations. Genetic drift alone is unlikely to drive genetic differentiation over such a small number of generations, unless it is accompanied by extreme bottlenecks and/or selection. Helicoverpa armigera in Australia demonstrated isolation by distance, so immigration into cropping regions is more likely to come from nearby regions than from afar. This effect was most pronounced in years with limited migration. However, there is evidence of long distance dispersal events in periods of high migration (April 2001-March 2002). The implications of highly variable migration patterns for resistance management are considered.
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Analysis of gene flow and migration of Helicoverpa armigera (Hubner) in a major cropping region of Australia identified substantial genetic structuring, migration events, and significant population genotype changes over the 38-mo sample period from November 1999 to January 2003. Five highly variable microsatellite markers were used to analyze 916 individuals from 77 collections across 10 localities in the Darling Downs. The molecular data indicate that in some years (e.g., April 2002-March 2003), low levels of H. armigera migration and high differentiation between populations occurred, whereas in other years (e.g., April 2001-March 2002), there were higher levels of adult moth movement resulting in little local structuring of populations. Analysis of populations in other Australian cropping regions provided insight into the quantity and direction of immigration of H. armigera adults into the Darling Downs growing region of Australia. These data provide evidence adult moth movement differs from season to season, highlighting the importance of studies in groups such as the Lepidoptera extending over consecutive years, because short-term sampling may be misleading when population dynamics and migration change so significantly. This research demonstrates the importance of maintaining a coordinated insecticide resistance management strategy, because in some years H. armigera populations may be independent within a region and thus significantly influenced by local management practices; however, periods with high migration will occur and resistance may rapidly spread.