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La enfermedad de Chagas afecta a más de un millón y medio de personas en Argentina y es transmitida principalmente por la vinchuca Triatoma infestans (Klug, 1834). El control químico del vector con insecticidas piretroides constituye la herramienta más utilizada para reducir la incidencia de la enfermedad. En los últimos años se han detectado fallas de control a campo en zonas del Gran Chaco Argentino, corroborándose en el laboratorio la ocurrencia de resistencia. El objetivo de esta tesis fue avanzar en el conocimiento de la evolución de la resistencia a insecticidas en poblaciones de campo de T. infestans mediante el uso combinado de herramientas de la toxicología, la genética y la ecología. Se analizó la distribución de la susceptibilidad en 69 poblaciones recolectadas en relevamientos entomológicos realizados por el Programa Nacional de Chagas y criadas en el laboratorio. Como resultado del análisis toxicológico, el 83 % de las poblaciones resultaron susceptibles a deltametrina, todas ellas pertenecientes a las provincias de Mendoza, San Juan, Tucumán, Santiago del Estero, Formosa, Catamarca y Chaco. Sólo se hallaron poblaciones resistentes en el Departamento chaqueño de Gral. Güemes. En este lugar, se caracterizó un patrón toxicológico complejo, compuesto por un 23 % de poblaciones susceptibles, un 41 % de poblaciones con baja resistencia (sin fallas de control a campo) y un 36 % de poblaciones con alta resistencia, estas últimas con los grados de resistencia (GR) más elevados detectados hasta el momento. La totalidad de las poblaciones resistentes a deltametrina resultó susceptible a fenitrotión, remarcando la importancia de este insecticida como la única alternativa de control a campo. Los ensayos bioquímicos en las poblaciones del foco resistente evidenciaron el aumento en la actividad de las enzimas degradativas oxidasas P450 y esterasas como mecanismo contributivo de la resistencia observada. Sin embargo, frecuencias altas de la mutación puntual L925I en el sitio de acción de los piretroides (el canal de sodio dependiente de voltaje) estarían determinando los GR elevados. Finalmente, se logró explicar el 70 % de la variabilidad toxicológica del foco a partir de predictores de temperatura, precipitación y extensión del paraje. Las variables de rociado, usadas como indicadores de la presión de selección por parte del insecticida, no contribuyeron a explicar el patrón toxicológico. Se propone que el ambiente podría estar modulando la presión de selección del insecticida, promoviendo la variabilidad toxicológica en una zona de gran complejidad en cuanto al control de T. infestans. Este conocimiento busca contribuir a generar estrategias integrales de control vectorial que permitan reducir la incidencia de la enfermedad. In Argentina, Chagas disease affects more than one and a half million people and is mainly transmitted by the kissing bug Triatoma infestans (Klug, 1834). Chemical control of the vector with pyrethroid insecticides has been the most frequently used tool to reduce the disease incidence. Recently, failures of field control have been detected in areas of the Argentinian Gran Chaco, and then the emergence of resistance has being corroborated in the laboratory. The main goal of this thesis was to advance in the knowledge of the evolution of the resistance to insecticides in field populations of T. infestans by the integration of several fields like toxicology, genetics and ecology. The distribution of susceptibility was analyzed in 69 populations collected in entomological surveys carried out by the National Chagas Program. From the toxicological analysis, 83% were susceptible to deltamethrin and were from the provinces of Mendoza, San Juan, Tucumán, Santiago del Estero, Formosa, Catamarca and Chaco. The only resistant populations were found in Gral. Güemes Department (Chaco province). In this area, a complex toxicological pattern was found that consisted of 23% of susceptible populations, 41% of populations with low resistance (without field control failures) and 36% of populations with high resistance. It is interesting to note that these latter possessed the highest degrees of resistance (RR) detected of the moment. All the populations resistant to deltamethrin were susceptible to fenitrothion, highlighting the importance of this insecticide as the only field control alternative. The biochemical tests in the populations of the resistant focus showed the increase in the activity of the degradative enzymes P450 oxidases and esterases as contributive mechanism of the resistance characterized. The genetics study revealed high frequencies of the L925I mutation at the site of action of the pyrethroids (the voltage-gated sodium channel) might be the responsible of the high RRs. Finally, the ecological analysis showed that 70% of the toxicological variability of the focus could be explained from temperature, precipitation and village extension predictors. Spray variables, used as indicators of the selection pressure by the insecticide, did not contribute to explain the toxicological pattern. It is proposed that the environment could be modulating the selection pressure of the 6 insecticide, promoting toxicological variability in an area of great complexity regarding the control of T. infestans. This thesis contribute to the generation of comprehensive vector control strategies that reduce the incidence of the disease. Fil: Fronza, Georgina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Científicas y Técnicas para la Defensa. Centro de Investigación de Plagas e Insecticidas; Argentina

Se evaluó la resistencia de Haematobia irritans (Diptera: Muscidae) del ganado bovino por exposición a dosis letal (DL50 y DL99) a los insecticidas Cipermetrina (Piretroide) y Coumafos (Organofosforado) en ranchos de Tierra Caliente del estado de Guerrero, México. Se utilizaron 30 ranchos, colectando de cada uno, 240 moscas de la superficie corporal de los bovinos. Sub-muestras de 20 moscas en triplicado fueron desafiadas a las formulas por la técnica de papel filtro impregnado a dosis concentradas de 2,5; 6,0 y 0 μg/cm2 de Cipermetrina y de 0,01; 0,06 y 0 μg/cm2 de Coumafos durante dos horas. La resistencia de H. irritans a Cipermetrina y Coumafos fue del 100% en los ranchos evaluados, debido a que la mortalidad de moscas promedio fue baja, del 28% a exposición de Cipermetrina y del 10% a exposición de Coumafos. La mortalidad total de moscas fue mayor (P0,235) en la resistencia de H. irritans hacia las fórmulas. Se concluye que la resistencia generada por H. irritans hacia los insecticidas Cipermetrina y Coumafos en bovinos, de los ranchos de la región Tierra Caliente de Guerrero limita su eficacia para ser utilizados en su control. Resistance was assessed Haematobia irritans (Diptera: Muscidae) in cattle exposed to lethal dose (LD50 and LD99) to insecticides Cypermethrin (Pyrethroid) and Coumaphos (Organophosphate) on ranches of Tierra Caliente of Guerrero, Mexico. Thirty ranches were used, in each ranch were collected 240 flies from the body surface of cattle. Sub-samples of 20 flies were challenged in triplicate to insecticides by the technique of filter paper impregnated with concentrated doses of 2.5, 6.0 and 0.0 μg/cm2 of Cypermethrin and 0.01, 0.06 and 0.0 μg/cm2 of Coumaphos for two hours. The resistance of H. irritans to Cypermethrin and Coumaphos was 100% on ranches evaluated, and because to the average fly lower mortality was 28% Cypermethrin exposure and 10% to Coumaphos exposure. Total mortality of flies was higher (P0.235) in the resistance of H. irritans to insecticides. It is concluded that the resistance generated by H. irritans to Coumaphos and Cypermethrin in cattle ranches in the region of Tierra Caliente of Guerrero limits its efficacy for use in their control. 283-286 Olivaares@hotmail.com Bimestral

To investigate the kdr (knockdown resistance) resistance-associated gene mutation and determine its frequency in pyrethroid-resistant horn fly (Haematobia irritans) populations, a total of 1,804 horn flies of 37 different populations from all Brazilian regions (North, Northeast, Central-West, Southeast, and South) were molecular screened through polymerase chain reaction (PCR). The kdr gene was not detected in 87.08% of the flies. However, the gene was amplified in 12.92% of the flies, of which 11.70% were resistant heterozygous and 1.22% were resistant homozygous. Deviation from Hardy-Weinberg equilibrium (HWE) was found only in 1 ranch with an excess of heterozygous. When populations were grouped by region, three metapopulations showed significant deviations of HWE (Central-West population, South population and Southeast population). This indicates that populations are isolated one from another and kdr occurrence seems to be an independent effect probably reflecting the insecticide strategy used by each ranch. Although resistance to pyrethroids is disseminated throughout Brazil, only 48% of resistant populations had kdr flies, and the frequency of kdr individuals in each of these resistant populations was quite low. But this study shows that, with the apparent exception of the Northeast region, the kdr mechanism associated with pyrethroid resistance occurs all over Brazil. Com o objetivo de verificar a ocorrência e determinar a frequência da mutação kdr (knock down resistance) em populações de Haematobia irritans (mosca-dos-chifres) resistentes aos piretróides, foram analisados 1.804 indivíduos de 37 populações de todas as Regiões do Brasil. Com exceção da Região Nordeste, o kdr (knock down resistance gene) foi encontrado em populações de todas as regiões. A mutação não foi detectada em 87,08% dos indivíduos. Entretanto, o gene foi amplificado de 12,92% das moscas, das quais 11,70% se mostraram heterozigotas resistentes e 1,22% homozigotas resistentes. Em todas as populações verificou-se equilíbrio de acordo com a Lei de Hardy e Weinberg, exceto uma com excesso de heterozigotos. Entretanto, quando agrupamos diferentes populações numa metapopulação de acordo com a região geográfica, é possível observar um desvio nas populações Centro-Oeste, Sul e Sudeste, indicando isolamento populacional e que a ocorrência do kdr é provavelmente um efeito independente, talvez refletindo a estratégia de uso do inseticida de cada produtor. Apesar da resistência aos piretróides estar disseminada por todo o país, apenas 48% das populações resistentes apresentaram o kdr, e a frequência de indivíduos kdr nas populações resistentes se mostrou bastante baixa. À exceção da Região Nordeste, o mecanismo de resistência ligado ao kdr ocorre em todo o país.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T18:44:57Z No. of bitstreams: 1 S1984-29612009000300002.pdf: 484970 bytes, checksum: 899106ad13818fa65ec47969593ca1bb (MD5) Made available in DSpace on 2013-08-22T18:44:57Z (GMT). No. of bitstreams: 1 S1984-29612009000300002.pdf: 484970 bytes, checksum: 899106ad13818fa65ec47969593ca1bb (MD5) Previous issue date: 2009-09-01 Made available in DSpace on 2013-09-30T19:33:32Z (GMT). No. of bitstreams: 2 S1984-29612009000300002.pdf: 484970 bytes, checksum: 899106ad13818fa65ec47969593ca1bb (MD5) S1984-29612009000300002.pdf.txt: 27505 bytes, checksum: c2afaddb9a2cff55ca4e02a1728eb050 (MD5) Previous issue date: 2009-09-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:52:58Z No. of bitstreams: 2 S1984-29612009000300002.pdf: 484970 bytes, checksum: 899106ad13818fa65ec47969593ca1bb (MD5) S1984-29612009000300002.pdf.txt: 27505 bytes, checksum: c2afaddb9a2cff55ca4e02a1728eb050 (MD5) Made available in DSpace on 2014-05-20T13:52:58Z (GMT). No. of bitstreams: 2 S1984-29612009000300002.pdf: 484970 bytes, checksum: 899106ad13818fa65ec47969593ca1bb (MD5) S1984-29612009000300002.pdf.txt: 27505 bytes, checksum: c2afaddb9a2cff55ca4e02a1728eb050 (MD5) Previous issue date: 2009-09-01 Com o objetivo de verificar a ocorrência e determinar a frequência da mutação kdr (knock down resistance) em populações de Haematobia irritans (mosca-dos-chifres) resistentes aos piretróides, foram analisados 1.804 indivíduos de 37 populações de todas as Regiões do Brasil. Com exceção da Região Nordeste, o kdr (knock down resistance gene) foi encontrado em populações de todas as regiões. A mutação não foi detectada em 87,08% dos indivíduos. Entretanto, o gene foi amplificado de 12,92% das moscas, das quais 11,70% se mostraram heterozigotas resistentes e 1,22% homozigotas resistentes. em todas as populações verificou-se equilíbrio de acordo com a Lei de Hardy e Weinberg, exceto uma com excesso de heterozigotos. Entretanto, quando agrupamos diferentes populações numa metapopulação de acordo com a região geográfica, é possível observar um desvio nas populações Centro-Oeste, Sul e Sudeste, indicando isolamento populacional e que a ocorrência do kdr é provavelmente um efeito independente, talvez refletindo a estratégia de uso do inseticida de cada produtor. Apesar da resistência aos piretróides estar disseminada por todo o país, apenas 48% das populações resistentes apresentaram o kdr, e a frequência de indivíduos kdr nas populações resistentes se mostrou bastante baixa. À exceção da Região Nordeste, o mecanismo de resistência ligado ao kdr ocorre em todo o país. To investigate the kdr (knockdown resistance) resistance-associated gene mutation and determine its frequency in pyrethroid-resistant horn fly (Haematobia irritans) populations, a total of 1,804 horn flies of 37 different populations from all Brazilian regions (North, Northeast, Central-West, Southeast, and South) were molecular screened through polymerase chain reaction (PCR). The kdr gene was not detected in 87.08% of the flies. However, the gene was amplified in 12.92% of the flies, of which 11.70% were resistant heterozygous and 1.22% were resistant homozygous. Deviation from Hardy-Weinberg equilibrium (HWE) was found only in 1 ranch with an excess of heterozygous. When populations were grouped by region, three metapopulations showed significant deviations of HWE (Central-West population, South population and Southeast population). This indicates that populations are isolated one from another and kdr occurrence seems to be an independent effect probably reflecting the insecticide strategy used by each ranch. Although resistance to pyrethroids is disseminated throughout Brazil, only 48% of resistant populations had kdr flies, and the frequency of kdr individuals in each of these resistant populations was quite low. But this study shows that, with the apparent exception of the Northeast region, the kdr mechanism associated with pyrethroid resistance occurs all over Brazil. Universidade de São Paulo Instituto de Ciências Biomédicas Departamento de Parasitologia Universidade Estadual Paulista Instituto de Biociências Departamento de Parasitologia Empresa Brasileira de Pesquisa Agropecuária Centro de Pesquisa Agropecuária do Pantanal Agricultural Research Service United States Department of Agriculture Knipling - Bushland US Livestock Insects Research Laboratory Universidade Estadual Paulista Instituto de Biociências Departamento de Parasitologia

A study to evaluate the resistance of B. microplus to the active principles cypermethrin, deltamethrin and chlorpyriphos was performed together with the survey of the tick control practices used in 12 farms located in Vale do Paraíba Region, São Paulo, Brazil. Results obtained by the larval packet test (LPT) technique, adopted by FAO, showed established resistance to pyrethroids (cypermethrin 16.7% sensitive, 8.3% resistant level I and 75% resistant level II; deltamethrin 25% /sensitive, 33.3% resistant level II and 41.7% resistant level III) and emerging resistance to organophosphate compounds (chlorpiriphos 58.3% sensitive, 33.3% resistant level I and 8.4% resistant level II). The inquiry applied to the farmers showed that the combination of organophosphates and pyrethroids compounds products is the most commonly tick-killing compounds used in the region, followed by amitraz and macrocyclic lactones. RESUMO Foi realizada pesquisa da resistência do Boophilus microplus aos princípios ativos cipermetrina, deltametrina e clorpirifós e, concomitantemente, feito o levantamento sobre as práticas adotadas no controle do carrapato em 12 propriedades localizadas na região do Vale do Paraíba, São Paulo, Brasil. Os resultados obtidos por meio da técnica LPT (Larval Packet Test), segundo a classificação adotada, mostraram que, para a cipermetrina, 16.7% das fazendas apresentaram populações classificadas como sensíveis; 8.3% com resistência nível I e 75% com resistência nível II. Para a deltametrina, 25% sensíveis; 33.3% com resistência nível II e 41.7% com resistência nível III. Para o organofosforado clorpirifós 58.3% mostraram-se sensíveis; 33.3% com resistência nível I e 8.4% com resistência nível II. Os questionários aplicados aos produtores revelaram que os produtos à base da associação de organofosforados com piretróides são os mais usados na região, seguidos do amitraz e lactonas macrocíclicas.

Los objetivos de este trabajo de tesis están relacionados a la evaluación de la resistencia a insecticidas piretroides en los estados embrionarios t postembrionarios de poblaciones de Pediculus humanus capitis de Buenos Aires resistentes a permetrina. Durante el desarrollo de la tesis fueron planteados objetivos a posteriori necesarios para el desarrollo de los objetivos originales. Los objetivos fueron: Observar la posible expresión de resistencia a insecticidas en embriones en poblaciones de P. humanus capitis con resistencia detectada en estadios postembrionarios. Estudiar los mecanismos de resistencia a insecticidas expresados en embriones y estados postembrionarios. Estudiar los mecanismos de resistencia a insecticidas expresados en embriones y estados postembrionarios de P. humanus capitis. Los objetivos a posteriores fueron: Caracterizar morfológicamente el desarrollo embrionario de P. humanus capitis y P. humanus humanus a fin de trabajar con homogéneo en cuanto al tiempo de desarrollo. Determinar las condiciones ambientales óptimas (T°C y HR%) para el desarrollo de P. humanus capitis en laboratorio. Puesta a punto de los métodos de medición de actividad enzimática en los embriones. De acuerdo a estos objetivos se obtuvieron los siguientes resultados y conclusiones: Se caraterizó el desarrollo embrionario según caracteres morfológicos vistos a través de la transparencia del corion. Los caracteres morfológicos fueron: apéndices y manchas oculares. La ombinación de los distintos estados de desarrollo de estos caracteres permitieron determinar rtes etapas en la embriogénesis:etapa temprana, etapa media, y etapa tardía (manchas oculares negras). Se determinaron las condiciones ambientales optimas (temperatura y humedad relativa) en laboratorio para la embriogénesis de P. humanus capitis: 27-31°C de temperatura y 45-75% humedad relativa. Se observó que en poblaciones con resistencia a piretroides y DDT expresada en los estados postembrionarios los embriones también expresan resistencia a los mismos insecticidas. En estados postembrionarios, no se observó resistencia a carbaril ni a spinosad. En embriones no se observ+o resistencia a spinosad pero se determinó una incipiente resistencia a carbaril. Se midió actividad esterasa en embriones por la stres metodologías utilizadas (Gomori, Ellman y Fluorométrico), por el contrario no fue posible determinar actividad del complejo P450 por el método fluorométrico utilizado. Según los resultados de inhibición enzimática in vivo y la determinación de las actividades enzimáticas una mayor actividad esterasa no parece estar involucrada en la resistencia observada en adultos y en embriones. Según el análisis global de los resultados, la resistencia en las poblaciones de P. humanus capitis utilizadas en este trabajo se debería: 1) modificación de sitio de acción de piretroides y DDT (tipo-kdr) (estados embrionarios y postembrionarios), 2) aumento metabolismo oxidativo (estados postembrionarios). Se plantea al insecticida spinosad como posible activo en formulaciones pediculicidas. The aim of this thesis are related to the evolution of the pyrethroids insecticide resistance in embryonic and postembryonic stages of Pediculus humanus capitis populations from Buenos Aires resistant to permethrin. During the development of the thesis a posteriori objetives were settled necessary for the development of the original aims. The original aims were: To observe the possible expression of insecticide resistance in embryos in P. humanus capitis population with detected resistance in postembryonic stages. To study the insecticide resistance mechanism expressed in P. humanus capitis embryos and postembryonic stages. The a posteriori aims were: To characterize morphologically the embryonic development of P. humanus capitis and P. humanus in order to work with homogeneous material as regards development time. To determinate the optimal environmental conditions (T°C and HR%) for the P. humanus capitis in laboratory. To determinate the measure methods of enzimatic activity in embryos. According to these aims the following results and conclusions were drawn: The embryonic development according to morophological characters seen through the corion transparence were characterized. The morphological characters were: appendages and oculars spots. The combinations of different development stages of these characters allowed to determinate three periods of time in the embryogenesis: early period, middle period and late period. Optimal environmental conditions were determinated (T°C and HR%) in the laboratory for P. humanus capitis: 27-31°C temperature and 45-75 relative humidity. It was observed that in populations with resistance to pyrethoids and DDT expressed in postembryonic stages, the embryos also express resistance to the same insecticides. In postembryonic stages, carbaryl and spinosad resistance was not observed. Resistance to spinosad was not observed in embyos but an incipient carabryl resistance was determined. Esterase activity embryos was measured by the three used methods (Gomori, Ellman and Fluorometric) on the contrary it was not possible to determine P450 complex activity by the fluorometric method. According to in vivo enzimatic inhibition results and the enzimatic activities determination, an increased in esterase activity is not involved in the resistance observed in adults and embryos. According to the global analysis of results, the resistance in P. humanus capitis population used in this research would be due to: 1)pyrethroids and DDT site of action modification (kdr-like) (embryonic and postembryonic stages), 2) oxidative metabolism increase (postembryonic stage). Spinosad insecticide as a possible active in pediculicide formulations is questioned. Fil: Mougabure Cueto, Gastón Adolfo. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.

Como continuación de los trabajos sobre resistencia de la mosca blanca de los invernaderos a insecticidas en la Zona Andina de Colombia y Ecuador, se calcularon en laboratorio las líneas base, los valores CL,, y CL,, y las dosis diagnóstico para imidacloprid, monocrotofos, carbofuran, carbosulfan, bifentrin y cialotrina en adultos de una variedad susceptible de 7Pialeurodes vaporariorum (Westwood) mantenida en cría permanente en el CIAT. El imidacloprid fue evaluado en dosis comerciales mediante la metodología de trifolios de fruol puestos sobre agar noble en cajas petri. Los demás insecticidas se evaluaron en viales impregnados de insecticida en grado técnico. Las dosis diagnóstico para adultos fueron 300 pgívial (monocrotofos), 5 pgívial (carbofuran), 100 pg/vial (carbosulfan), 5 pgívial (bifentrin), 500 pgívial (cialotrina) y 40 ppm (imidacloprid). Se calcularon también las líneas base, los valores CL,, , CL,, y las dosis diagnóstico sobre ninfas de primer ínstar utilizando la metodología de inmersión del follaje y dosis comerciales de diafentiuron, buprofezin e imidacloprid. Las dosis diagnóstico para ninfas fueron 300 pprn (diafentiuron), 16 ppm (buprofezin) y 300 pprn (imidacloprid). Las dosis diagnóstico, así calculadas, se utilizaron para medir periódicamente la resistencia a insecticidas en adultos y ninfas de variedades de T. vaporariorum del Valle del Cauca colectadas en zonas con uso crítico de insecticidas. Se encontró resistencia alta a organofosforados, resistencia intermedia a piretroides, y susceptibilidad a carbamatos e imidacloprid en adultos. No se detectaron niveles de resistencia de ninfas a reguladores de crecimiento e imidacloprid. As a continuation to the work on insecticide resistance in whitefly populations in Colombia and Ecuador, line base data, LC,, and LC,, values, and diagnostic doses for imidacloprid, monocrotophos, carbofuran, carbosulfan, bifenthrin and lambdacyhalothrin were calculated. Adults of a susceptible variety of 'kialeurodes vaporariorum maintained at CIAT since 1991 were used. Disks of the treated foliage were placed on agar inside petri dishes. Al1 other insecticides were evaluated using glass vials coated with technical grade insecticides. Diagnostic doses for adults were 300 pgívial (monocrotophos), 5 pgívial (carbofuran), 100 pg/vial (carbosulfan), 5 pgívial (bifenthrin), 500 pgívial (lamdacyhalotrin), and 40 pprn (imidacloprid). Base line data, LC,, and LC,,values, and diagnostic doses were also established for first instar nymphs using the leaf-dip methodology with commercial dosages of diafenthiuron, buprofezin and imidacloprid. Diagnostic doses for nyrnphs were 300 pprn (diafenthiuron), 16 pprn (buprofezin) and 300 pprn (imidacloprid). The above mentioned diagnostic doses were used to monitor resistance to insecticides in adults and nymphs of T. vaporariorum variety in areas of the Cauca Valley, where excessive use of insecticides occurs. Adult populations showed high levels of resistance to organophosphates, intermediate resistance to pyrethroids, and susceptibility to carbamates and imidacloprid. No resistance to insect growth regulators and imidacloprid was detected among the nymph populations sampled.