Dispersão de machos de Diatraea saccharalis (Fabricius) (Leipdoptera: Crambidae) em cana-de-açúcar

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

Seleção e caracterização de novos genes vip3A: genes inseticidas de segunda geração de Bacillus thuringiensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Seleção e caracterização de isolados de Bacillus thuringiensis para o controle de Thyrinteina arnobia (Stoll, 1782) (Lepdoptera: Geometridae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Interação de proteínas Cry1 e Vip3A de Bacillus thuringiensis para controle de lepidópteros-praga

O objetivo deste trabalho foi avaliar a suscetibilidade das lagartas Anticarsia gemmatalis (Lepidoptera: Erebidae) e Chrysodeixis includens (Lepidoptera: Noctuidae) às proteínas Cry1 e Vip3A, bem como determinar se há a interação entre essas proteínas no controle das duas espécies. Bioensaios com as proteínas isoladas e em combinações foram realizados, e as concentrações letais CL50 e CL90 foram estimadas para cada condição. As proteínas Cry1Aa, Cry1Ac e Vip3Af foram as mais efetivas no controle de A. gemmatalis, enquanto Cry1Ac, Vip3Aa e Vip3Af foram mais efetivas no de C. includens. As proteínas Cry1Ac e Cry1Ca causaram maior inibição do desenvolvimento das larvas sobreviventes à CL50, em ambas as espécies. Combinações entre Vip3A e Cry1 apresentam efeito sinérgico no controle das espécies e a combinação Vip3Aa+Cry1Ea destaca-se no controle de A. gemmatalis e C. includens. Essas proteínas combinadas são promissoras na construção de plantas piramidadas, para o controle simultâneo das pragas.

Marcação, voo tutorado e dispersão de Anticarsia gemmatalis (Hübner) (Lepidoptera: Erebidae) e Chrysodeixis includens (Walker) (Lepidoptera: Noctuidae)

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

Atividade larvicida dos óleos essenciais de Syzygium aromaticum e Citrus sinensis em populações de Aedes aegypti

Pós-graduação em Biociências - FCLAS

Interação de proteínas Vip3A e Cry1la10 de Bacillus thuringiensis com atividade inseticida a lepidópteros-praga

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Binding analysis of Bacillus thuringiensis Cry1 proteins in the sugarcane borer, Diatraea saccharalis (Lepidoptera: Crambidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Susceptibility of Rhipicephalus (Boophilus) microplus to ivermectin (200, 500 and 630 mu g/kg) in field studies in Brazil

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.

Estudos de dispersão de lagartas em milho e soja visando ao manejo de pragas no Brasil e EUA

Pós-graduação em Agronomia (Proteção de Plantas) - FCA

Interações moleculares das proteínas CRY1 e VIP3 no controle de lepidópteros em cana-de-açúcar

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic variability and demographic history of Heliothis virescens (Lepidoptera: Noctuidae) populations from Brazil inferred by mtDNA sequences

Intra-and inter-population genetic variability and the demographic history of Heliothis virescens (F.) populations were evaluated by using mtDNA markers (coxI, coxII and nad6) with samples from the major cotton-and soybean-producing regions in Brazil in the growing seasons 2007/08, 2008/09 and 2009/10. AMOVA indicated low and non-significant genetic structure, regardless of geographical scale, growing season or crop, with most of genetic variation occurring within populations. Clustering analyzes also indicated low genetic differentiation. The haplotype network obtained with combined datasets resulted in 35 haplotypes, with 28 exclusive occurrences, four of them sampled only from soybean fields. The minimum spanning network showed star-shaped structures typical of populations that underwent a recent demographic expansion. The recent expansion was supported by other demographic analyzes, such as the Bayesian skyline plot, the unimodal distribution of paired differences among mitochondrial sequences, and negative and significant values of neutrality tests for the Tajima's D and Fu's F-S parameters. In addition, high values of haplotype diversity ((H) over cap) and low values of nucleotide diversity (pi), combined with a high number of low frequency haplotypes and values of theta(pi)<theta(W), suggested a recent demographic expansion of H. virescens populations in Brazil. This demographic event could be responsible for the low genetic structure currently found; however, haplotypes present uniquely at the same geographic regions and from one specific host plant suggest an initial differentiation among H. virescens populations within Brazil.

Assessment of the high-dose concept and level of control provided by MON 87701 x MON 89788 soybean against Anticarsia gemmatalis and Pseudoplusia includens (Lepidoptera: Noctuidae) in Brazil

BACKGROUND: Genetically modified MON 87701 X MON 89788 soybean (Glycine max), which expresses the Cry1Ac and EPSP-synthase proteins, has been registered for commercial use in Brazil. To develop an Insect Resistance Management (IRM) program for this event, laboratory and field studies were conducted to assess the high-dose concept and level of control it provides against Anticarsia gemmatalis and Pseudoplusia includens. RESULTS: The purified Cry1Ac protein was more active against A. gemmatalis [LC50 (FL 95%) = 0.23 (0.150.34) mu g Cry1Ac mL-1 diet] than P. includens [LC50 (FL 95%) = 3.72 (2.654.86) mu g Cry1Ac mL-1 diet]. In bioassays with freeze-dried MON 87701X MON 89788 soybean tissue diluted 25 times in an artificial diet, there was 100% mortality of A. gemmatalis and up to 95.79% mortality for P. includens. In leaf-disc bioassays and under conditions of high artificial infestation in the greenhouse and natural infestation in the field, MON 87701X MON 89788 soybean showed a high level of efficacy against both target pests. CONCLUSIONS: The MON 87701X MON 89788 soybean provides a high level of control against A. gemmatalis and P. includes, but a high-dose event only to A. gemmatalis. Copyright (c) 2012 Society of Chemical Industry

Bases moleculares de la resistencia a insecticidas en la mosca mediterránea de la fruta Ceratitis Capitata (Wiedemann)

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.

Insect growth regulators as potential insecticides to control olive fruit fly (Bactrocera oleae Rossi): insect toxicity bioassays and molecular docking approach

Olive fruit fly, Bactrocera oleae (Rossi), is a key pest in olive orchards, causing serious economic damage. To date, the pest has already developed resistance to the insecticides commonly applied to control it. Thus, in searching for new products for an accurate resistance management programme, targeting the ecdysone receptor (EcR)might provide alternative compounds for use in such programmes. RESULTS: Residual contact and oral exposure in the laboratory of B. oleae adults to the dibenzoylhydrazine-based compounds methoxyfenozide, tebufenozide and RH-5849 showed different results. Methoxyfenozide and tebufenozide did not provoke anynegative effectsontheadults,but RH-5849 killed98-100%of the treated insects15 days after treatment. Theligand-binding domain (LBD) of the EcR of B. oleae (BoEcR-LBD) was sequenced, and a homology protein model was constructed. Owing to a restricted extent of the ligand-binding cavity of the BoEcR-LBD, docking experiments with the three tested insecticides showed a severe steric clash in the case of methoxyfenozide and tebufenozide, while this was not the case with RH-5849. CONCLUSION: IGR molecules similar to the RH-5849 molecule, and different from methoxyfenozide and tebufenozide, might have potential in controlling this pest.