Interações da proteína Vip3Aa20, Diatraea saccharalis (Fabricius) e seus parasitóides, Cotesia flavipes (Cameron) e Trichogramma galloi Zucchi

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Susceptibilidade das fases do ciclo de vida de Diatraea saccharalis (Lepidoptera: Crambidae) à ação de fungos entomopatogênicos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Desenvolvimento de Plutella xylostella (Linnaeus, 1758) (Lepidoptera: Plutellidae) em Brassicaceae ao longo de gerações

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

Divergência genética de cultivares de cana-de-açúcar quanto à resistência a Diatraea saccharalis

The objective was to evaluate the genetic diversity of cultivars in sugar cane for resistance to D. saccharalis. The experiment was carried out in the laboratory in completely randomized design with 11 treatments (one control and 10 treatments) in ten replications. The replications were made from artificial diets (food and refood) made with dry steam crushed from sugar cane cultivars stems, except for one of them considered standard diet. The cultivars used were: RB867515, RB855453, RB855536, CTC 15, CTC 9, SP80-1842, SP79-1011, SP89-1115, SP81-3250 and SP87-365. In the evaluation biological characteristics of the insect considered were: larval development (days), larval viability (%), pupal development (days), pupal weight (g), pupal viability (%), period of hatched larvae to adults emergence (days), total viability (%) and adults longevity without food (days). The generalized Mahalanobis distance (D-2) for the cluster analysis by the method of average linkage between groups (UPGMA) and Tocher's method optimization was determined. Four and five groups were formed, respectively, by the method of average linkage between groups (UPGMA) and Tocher's method optimization. We concluded that the cultivar CTC 15 standed out as highly susceptible to D. saccharalis, while the cultivar SP87-365 behaved as moderately resistant by antibiosis to D. saccharalis.

Desenvolvimento de Zaprionus indianus Gupta, 1970 (Diptera: Drosophilidae) em diferentes dietas

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

Metodologias de pesquisa e avaliação da resistência de genótipos de amendoim a Spodoptera albula (walker, 1857) (Lepidoptera: Noctuidae)

Pós-graduação em Agronomia (Entomologia Agrícola) - FCAV

Biology and reproductive capacity of Spodoptera eridania (Cramer) (Lepidoptera, Noctuidae) in different soybean cultivars

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biologia comparada de populações da lagarta–do–cartucho em folhas de milho e mandioca

The fall armyworm is a pest that feeds on various botanies species. The objective of this experiment was to study the biology of this pest in corn and cassava leaves. Caterpillars were collected in area under cultivation with cassava and maintained on artificial diet for two generations. Under controlled conditions in a climatic chamber (B.O.D) in the laboratory (25 degrees, 60 +/- 10% RH and photophase 14 hours) were evaluated daily 50 caterpillars in corn treatments and 50 in cassava, where duration and viability of the larval phase and pupal, weight of pupas were observed after 24 hours, deformation percentage of pupas and adults, longevity, fecundity and total life cycle. The viability of larvae fed on leaves of maize and cassava was 74% and 60%, respectively. The larval period of the insects was shorter in maize 16.89 days (seven instars) and cassava 20.08 days (six instars). The pupal phase lasted 11.42 days in cassava treatment and 10.87 in the maize. The pupal weight of females and males was higher in corn 204.91 mg and 198.97 mg, respectively. The biological cycle varied depending on the ingested food. Adult longevity lasted 9.88 days for insects fed on cassava leaves. Therefore, cassava affected the development of S. frugiperda.

The use of artificial neural networks in analysing the nutritional ecology of Chrysomya megacephala (F.) (Diptera: Calliphoridae), compared with a statistical model

Artificial neural networks (ANNs) have been widely applied to the resolution of complex biological problems. An important feature of neural models is that their implementation is not precluded by the theoretical distribution shape of the data used. Frequently, the performance of ANNs over linear or non-linear regression-based statistical methods is deemed to be significantly superior if suitable sample sizes are provided, especially in multidimensional and non-linear processes. The current work was aimed at utilising three well-known neural network methods in order to evaluate whether these models would be able to provide more accurate outcomes in relation to a conventional regression method in pupal weight predictions of Chrysomya megacephala, a species of blowfly (Diptera: Calliphoridae), using larval density (i.e. the initial number of larvae), amount of available food and pupal size as input data. It was possible to notice that the neural networks yielded more accurate performances in comparison with the statistical model (multiple regression). Assessing the three types of networks utilised (Multi-layer Perceptron, Radial Basis Function and Generalised Regression Neural Network), no considerable differences between these models were detected. The superiority of these neural models over a classical statistical method represents an important fact, because more accurate models may clarify several intricate aspects concerning the nutritional ecology of blowflies.

Bioatividade de derivados de Annonaceae sobre Helicoverpa armigera (Hübner, 1808) (Lepidoptera: Noctuidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Resistance to malathion and lambda-cyhalothrin in Ceratitis capitata (Wiedemann) (Diptera: Tephritidae)

La mosca mediterránea de la fruta, Ceratitis capitata (Wiedemann, 1824) (Diptera: Tephritidae), es una de las plagas de mayor incidencia económica en cítricos y otros frutales a nivel mundial. En España las medidas de control de esta plaga en cítricos, desde mediados de los 90 hasta 2009, se basaron principalmente en el monitoreo de las poblaciones y en la aplicación de tratamientos aéreos y terrestres con malatión cebo. Sin embargo, desde la retirada en la Unión Europea en 2009 de los productos fitosanitarios que contienen malatión, los insecticidas más utilizados para el control de esta plaga han sido lambda-cihalotrina y spinosad. En 2004-2005 se detectaron poblaciones españolas de C. capitata resistentes a malatión. Esta resistencia se ha asociado a una mutación (G328A) en la acetilcolinesterasa (AChE), a una duplicación del gen de la AChE (Ccace2) (una de las copias lleva la mutación G328A) y a resistencia metabólica mediada por esterasas (posiblemente aliesterasas). Sin embargo, cuando se secuenció la aliesterasa CcE7 en individuos de una línea resistente a malatión, no se encontró ninguna de las mutaciones (G137D y/o W251L/S/G) asociadas a resistencia en otras especies, si bien se encontraron otras mutaciones al compararlos con individuos de una línea susceptible. Asimismo, mediante la selección en laboratorio de una línea resistente a malatión (W-4Km) con lambda-cihalotrina, se ha podido obtener una línea resistente a lambda-cihalotrina (W-1K). Finalmente, se ha demostrado la capacidad de esta especie para desarrollar resistencia a spinosad mediante selección en laboratorio. Los múltiples mecanismos de resistencia identificados evidencian el potencial de esta especie para desarrollar resistencia a insecticidas con diferentes modos de acción. Los objetivos de esta tesis doctoral son: 1) evaluar la susceptibilidad de poblaciones españolas de campo de C. capitata a lambda-cihalotrina y dilucidar los mecanismos de resistencia en la línea W-1Kλ; 2) comparar la herencia, el coste biológico y la estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A); y 3) investigar el papel de las mutaciones identificadas en la aliesterasa CcαE7 en la resistencia a malatión. Estos estudios son de utilidad para el desarrollo de estrategias de manejo de la resistencia que puedan prevenir o retrasar la aparición de resistencia y aumentar la sostenibilidad de los insecticidas disponibles para el control de esta plaga. Nuestros resultados indican que las poblaciones españolas de C. capitata analizadas han desarrollado resistencia a lambda-cihalotrina. Los valores de CL50 estimados para las poblaciones recogidas en la Comunidad Valenciana, Cataluña y Andalucía oscilaron entre 129 ppm y 287 ppm, igualando o sobrepasando la concentración recomendada para los tratamientos de campo (125 ppm). Estos resultados contrastan con los obtenidos con tres poblaciones de campo recogidas en Túnez, cuya susceptibilidad fue similar a la de la línea control (C). La línea resistente a lambda-cihalotrina W-1K se continuó seleccionando en el laboratorio alcanzándose unos niveles de resistencia de 205 veces con respecto a la línea C, siendo su CL50 (4224 ppm) más de 30 veces superior a la concentración recomendada para los tratamientos de campo. Esta línea resistente mostró altos niveles de resistencia cruzada a deltametrina (150 veces) y a etofenprox (240 veces), lo que sugiere que el desarrollo de resistencia a lambda-cihalotrina podría comprometer la eficacia de otros piretroides para el control de esta plaga. Hemos demostrado que la resistencia de la línea W-1K a lambda-cihalotrina fue casi completamente suprimida por el sinergista PBO, lo que indica que las enzimas P450 desempeñan un papel muy importante en la resistencia a este insecticida. Sin embargo, tanto las moscas de la línea susceptible C como las de la línea resistente W-1K perdieron inmediatamente la capacidad de caminar (efecto “knock-down”) al ser tratadas tópicamente con lambda-cihalotrina, lo que sugiere que la resistencia no está mediada por alteraciones en la molécula diana (resistencia tipo “kdr”). La resistencia metabólica mediada por P450 fue analizada comparando la expresión de 53 genes CYP (codifican enzimas P450) de las familias CYP4, CYP6, CYP9 y CYP12 en adultos de la línea resistente W-1K y de la línea susceptible C. Nuestros resultados muestran que el gen CYP6A51 (número de acceso GenBank XM_004534804) fue sobreexpresado (13-18 veces) en la línea W-1K. Por otra parte, la expresión del gen CYP6A51 fue inducida tanto en adultos de la línea W-1K como de la línea C al ser tratados con lambda-cihalotrina. Sin embargo, no se obtuvieron diferencias significativas entre la línea susceptible C y la línea resistente W-1K al comparar la cantidad de P450 y la actividad NADPH-citocromo c reductasa presente en fracciones microsomales obtenidas a partir de abdómenes. Asimismo, no hemos podido correlacionar el metabolismo de deltametrina, estimado in vitro mediante la incubación de este insecticida con fracciones microsomales, con el nivel de resistencia a este piretroide observado en los bioensayos con la línea W-1K. Por otro lado, no se encontró ninguna alteración en la región promotora 5'UTR del gen CYP6A51 (-500 pb desde el inicio de la traducción) que pudiera explicar su sobreexpresión en la línea W-1K. Los datos obtenidos sugieren que la resistencia a lambda-cihalotrina en la línea W-1K está mediada por P450 y que la sobreexpresión de CYP6A51 puede desempeñar un papel importante, aunque se necesitan más evidencias para establecer una asociación directa de la resistencia con este gen. Hemos estudiado la herencia, el coste biológico y la estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A). La línea susceptible C, donde no se encuentra la mutación G328A (genotipo S/S), se cruzó con dos isolíneas establecidas para representar genotipos únicos correspondientes a los dos mecanismos de resistencia asociados a la molécula diana: 1) la isolínea 267Y (genotipo R/R) establecida a partir de una pareja que portaba la mutación G328A en homocigosis; 2) la isolínea 306TY (genotipo RS/RS) establecida a partir de una pareja que portaba en homocigosis la duplicación del gen Ccace2. No se realizaron cruces recíprocos, ya que mediante experimentos de hibridación in situ en cromosomas politénicos se pudo comprobar que el locus de la AChE y la duplicación (probablemente en tándem) se localizan en el cromosoma autosómico 2L. La susceptibilidad al malatión de los parentales resistentes (R/R o RS/RS) y susceptibles (S/S), los cruces F1 (S/R, S/RS y R/RS) y los retrocruzamientos indican que la resistencia a malatión es semi-dominante en ambos casos. Sin embargo, nuestros resultados no fueron concluyentes con respecto a la naturaleza monogénica de la resistencia a malatión en estas isolíneas. Por lo tanto, no podemos descartar que otros genes que contribuyan a la resistencia, además de la mutación G328A (isolínea 267Y) y de la duplicación del gen Ccace2 (isolínea 306TY), puedan haber sido seleccionados durante el proceso de selección de 267Y y 306TY. Varios parámetros biológicos fueron evaluados para determinar si estos dos mecanismos de resistencia a malatión suponen un coste biológico para los genotipos resistentes. Individuos con genotipo R/R mostraron un retraso en el tiempo de desarrollo de huevo a pupa, un peso de pupa reducido y una menor longevidad de los adultos, en comparación con los individuos con genotipo S/S. Sin embargo, el peso de pupa de los individuos con genotipo RS/RS fue similar al de los individuos S/S, y su desarrollo de huevo a pupa intermedio entre S/S y R/R. Estas diferencias en el coste biológico pueden estar relacionadas con la reducción de la eficiencia catalítica de la AChE mutada en los individuos R/R, y al efecto compensatorio que la copia no mutada del gen tiene en los individuos RS/RS que portan la duplicación. La estabilidad de la resistencia a malatión mediada por la mutación G328A y la duplicación se analizó mediante el seguimiento de los caracteres de resistencia en la progenie de retrocruzamientos S/R x R/R y S/RS x RS/RS a lo largo de varias generaciones en ausencia de presión de selección con insecticidas. Nuestros resultados muestran que la frecuencia del alelo que porta la mutación G328A disminuyó desde 67,5% en la primera generación del retrocruzamiento S/R x R/R (75% esperado, asumiendo segregación mendeliana y que sólo hay dos alelos: uno mutado y otro no mutado) a 12% después de 10 generaciones. Por el contrario, la frecuencia de la duplicación sólo disminuyó desde 75% en en la primera generación del retrocruzamiento S/RS x RS/RS (75% esperado, asumiendo segregación Mendeliana y que la duplicación segrega como un único alelo) a 50% en el mismo período, lo que indica que la duplicación es más estable que la mutación. Asimismo, se analizó la presencia de la mutación y de la duplicación en poblaciones de campo recogidas en seis localidades en 2004-2007, cuando todavía se usaba el malatión, y se comparó con poblaciones recogidas en los mismos campos en 2010, un año después de la prohibición del malatión en la Unión Europea. La frecuencia media del genotipo susceptible (S/S) aumentó del 55,9% en el período 2004-2007 a 70,8% en 2010, mientras que la frecuencia de los genotipos portadores de la mutación en homocigosis o heterocigosis (R/R y S/R) disminuyó del 30,4 al 9,2%, los que llevan la duplicación en homocigosis o heterocigosis (RS/RS y S/RS) aumentaron levemente desde 12,8 hasta 13,3%, y los que llevan a la vez la mutación y la duplicación (R/RS) también aumentaron del 1 al 6,7%. Estos resultados son consistentes con que la duplicación del gen Ccace2 (con una copia con la mutación G328A y la otra copia no mutada) es más ventajosa que la mutación G328A por si sola, ya que la duplicación mantiene los niveles de resistencia a la vez que limita el coste biológico. Para investigar la asociación entre la resistencia a malatión y las mutaciones encontradas previamente en CcE7, hemos generado isolíneas con mutaciones específicas seleccionadas por su ubicación próxima a la entrada al centro activo de la enzima. La isolínea Sm2 (procedente de una hembra heterocigota para la mutación V96L y un macho homocigoto para el alelo no mutado) mantuvo altos niveles de resistencia a malatión, incluso después de 30 generaciones sin presión de selección. Por el contrario, la isolínea 267Y (compuesta por individuos homocigotos para la mutación L267Y) y la línea 306TY (compuesta por individuos homocigotos para la doble mutación R306T-N307Y) mostraron una reducción significativa en los niveles de resistencia. También hemos encontrado que la resistencia a malatión de la línea Sm2 fue parcialmente revertida por DEF y TPP, y que Sm2 mostró una reducción significativa en la actividad MTB, como se ha descrito en otras especies que muestran resistencia específica a malatión mediada por aliesterases. Además, fue posible asociar la presencia de la mutación V96L en individuos de la línea Sm2 con supervivencia a una concentración discriminante de malatión (5,000 ppm) y con una baja actividad MTB. Estos resultados sugieren una posible relación entre la mutación V96L en la aliesterasa CcE7 y la resistencia a malatión, aunque todavía no se puede concluir que la resistencia es causada por esta mutación, siendo necesarios más estudios para comprobar su contribución a la resistencia. En conclusión, se ha encontrado por primera vez resistencia a lambda-cihalotrina en poblaciones de campo de C. capitata, y nuestros resultados indican que las P450 son el principal mecanismo de resistencia en la línea W-1K. Esta situación se suma al caso previamente descrito de resistencia en campo a malatión asociada a la mutación G328A, a la duplicación del gen Ccace2 (una de las copias lleva la mutación G328A) y a resistencia metabólica mediada por esterasas. Nuestros resultados también indican que la alteración de la molécula diana AChE parece ser responsable de un cierto nivel de resistencia a malatión en C. capitata, que puede ser estimada como aproximadamente 25-40 veces para la mutación G328A y 40-60 veces para la duplicación; mientras que la resistencia mediada por esterasas y que ha sido asociada en este estudio con la mutación V96L en CcE7 puede conferir un efecto multiplicativo (por un factor de 5 a 10) aumentando la resistencia a malatión a 200-400 veces. Por otra parte, hemos demostrado que los insectos resistentes que llevan la duplicación tienen un coste biológico menor y muestran una estabilidad mayor que aquellos con la mutación G328A en ausencia de presión de selección con insecticidas. Esto representa un escenario en el que los genotipos con la duplicación permanecerán en el campo en frecuencias bajas a moderadas, pero podrían ser seleccionados rápidamente si se utilizan malatión u otros insecticidas que muestren resistencia cruzada. Estos resultados tienen importantes implicaciones para los programas de manejo de la resistencia, ya que el repertorio de insecticidas eficaces para el control de C. capitata es cada vez más limitado. Además, la coexistencia de múltiples mecanismos de resistencia en poblaciones de campo ofrece el potencial para desarrollar resistencia frente a otros insecticidas disponibles para el control de esta plaga. Estrategias para de manejo de la resistencia basadas en la alternancia de insecticidas con diferentes modos de acción, y su combinación con otros métodos de control, deben ser implementadas para evitar el desarrollo de resistencia en campo. ABSTRACT The Mediterranean fruit fly (Medfly), Ceratitis capitata (Wiedemann, 1824) (Diptera: Tephritidae), is one of the most economically damaging pests of citrus and other fruit crops worldwide. Control measures in citrus crops in Spain from the mid 90's to 2009 were mainly based on field monitoring of population levels and aerial and ground treatments with malathion bait sprays. However, since the withdrawal of phytosanitary products containing malathion in the European Union in 2009, lambda-cyhalothrin and spinosad have become the most widely used insecticides for the control of this pest. Resistance to malathion was found in Spanish field populations of C. capitata in 2004-2005. This resistance has been associated with a mutation G328A in the acetylcholinesterase (AChE), a duplication of the AChE gene (Ccace2) (one of the copies bearing the mutation G328A), and metabolic resistance mediated by esterases (probably aliesterases). However, when the gene of the aliesterase CcE7 was sequenced in individuals from a malathion resistant strain of C. capitata, none of the known G137D and/or W251L/S/G mutations associated to resistance in other species were found, though other mutations were detected when compared with individuals from a susceptible strain. Noteworthy, a lambda-cyhalothrin resistant strain (W-1K) was obtained by selecting a field-derived malathion resistant strain (W-4Km) with lambda-cyhalothrin. Moreover, it has also been demonstrated the capacity of this species to develop resistance to spinosad by laboratory selection. The multiple resistance mechanisms identified highlight the potential of this species to develop resistance to insecticides with different modes of action. The objectives of this PhD Thesis are: 1) to assess the susceptibility of Spanish field populations of C. capitata to lambda-cyhalothrin and to elucidate the resistance mechanisms in the W-1Kλ strain; 2) to compare the inheritance, fitness cost and stability of the malathion resistance mediated by the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A); and 3) to investigate the role of the aliesterase CcαE7 mutations in malathion resistance. All these studies will be of use for devising proactive resistance management strategies that could prevent or delay resistance development and would increase the sustainability of the insecticides available for Medfly control. Our results indicate that Spanish field populations of C. capitata have developed resistance to lambda-cyhalothrin. The LC50 values estimated for populations collected at Comunidad Valenciana, Cataluña and Andalucía ranged from 129 ppm to 287 ppm, equaling or overpassing the recommended concentration for field treatments (125 ppm). These results contrast with those obtained with three different Tunisian field populations, whose susceptibility was similar to that of the control (C) strain. The lambda-cyhalothrin resistant W-1K strain has been further selected to achieve a 205-fold resistance compared to the C strain, being its LC50 (4,224 ppm) more than 30 times higher than the recommended concentration for field applications. This resistant strain showed high levels of cross-resistance to deltamethrin (150-fold) and etofenprox (240-fold), suggesting that the development of resistance to lambda-cyhalothrin may compromise the effectiveness of other pyrethroids for the control of this species. We have shown that the resistance of the W-1K strain to lambda-cyhalothrin was almost completely suppressed by the synergist PBO, indicating that P450 enzymes play a very important role in resistance to this insecticide. However, both susceptible C and resistant W-1K flies were knocked down after topical treatment with lambda-cyhalothrin, suggesting that kdr resistance mediated by alterations of the target site is not playing a major role. Metabolic resistance mediated by P450 was further analyzed by comparing the expression of 53 genes of the families CYP4, CYP6, CYP9 and CYP12 in adults flies from the resistant W-1K and the susceptible C strains. We found that the gene CYP6A51 (GenBank accession number XM_004534804) was overexpressed (13-18-fold) in the W-1K strain. Moreover, the expression of the CYP6A51 gene was induced when adults of the W-1K and C strains were treated with lambda-cyhalothrin. However, no significant differences were obtained between susceptible C and resistant W-1K strains for the quantity of P450 and for the activity of NADPH- cytochrome c reductase measured in microsomal fractions obtained from abdomens. Moreover, we failed to correlate the metabolism of deltamethrin, analyzed in vitro by incubating this insecticide with microsomal fractions, with the resistance level against this pyrethroid observed in bioassays with W-1K. The sequencing of the 5´UTR region of the CYP6A51 gene failed in finding an alteration in the promoter region (-500 bp from translation start site) that could explain overexpression in the W-1K strain. All data obtained suggest that resistance to lambda-cyhalothrin in the W- 1K strain is mediated by P450 and that overexpression of CYP6A51 may play a major role, although further evidences are needed to establish a direct association of resistance with this gene. We have studied the inheritance, fitness cost and stability of the malathion resistance mediated by the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A). The malathion-susceptible C strain where the G328A mutation is not found (S/S genotype) was crossed with two isolines established to represent unique genotypes corresponding to the two target-site resistance mechanisms: 1) the 267Y isoline (genotype R/R) was established from a couple bearing the mutation G328A in homozygosis; and 2) the 306TY isoline (genotype RS/RS) was established from a couple being homozygous for the duplication of the Ccace2 gene. Reciprocal crosses have not been performed, since in situ hybridization on polythene chromosomes showed that the AChE locus and the duplication (most probably in tandem) are placed at the autosomal chromosome 2L. Mortality responses to malathion of resistant isolines (R/R or RS/RS) and susceptible (S/S) genotypes, F1 crosses (S/R, S/RS, and R/RS), and the back-crosses indicated that resistance to malathion is inherited as a semi-dominant trait in both cases. However, our results were not conclusive about the monogenic nature of the resistance to malathion in these isolines. Thus, we can not discard that other genes contributing to resistance, in addition to the mutation G328A (isoline 267Y) and the duplication of the Ccace2 gene (isoline 306TY), may have been selected during the selection process of 267Y and 306TY. Several biological parameters were evaluated to determine if these two malathion resistance mechanisms impose a fitness cost for resistant genotypes. Individuals with genotype R/R have a reduced fitness in terms of developmental time from egg to pupa, pupal weight and adult longevity, when compared to susceptible individuals (genotype S/S). Interestingly, the fitness cost was substantially diminished in individuals with genotype RS/RS. These differences in fitness may be related to the reduction of the catalytic efficiency of mutated AChE in individuals R/R, and the compensatory effect that the non-mutated copy of the gene has on individuals RS/RS bearing the duplication. The stability of malathion reistance associated with the mutation G328A or the duplication was analyzed by following these resistant traits in the progeny of the back-crosses S/RS x RS/RS and S/R x R/R over consecutive generations in the absence of insecticide selection pressure. Our results show that the frequency of the allele bearing the mutation G328A decreased from 67.5% at the first generation of the back-cross S/R x R/R (75% expected, assuming Mendelian segregation and that there are only two alleles: one mutated and the other non-mutated) to 12% after 10 generations. By contrast, the frequency of the duplication only declined from 75% at the first generation of the back-cross S/RS x RS/RS (75% expected, assuming Mendelian segregation and that the duplication segregates as an unique allele) to 50% in the same period, indicating that the duplication is more stable than the mutation. The presence of the mutation and the duplication was analyzed in field populations collected in six localities in 2004-2007, when malathion was still used, and compared to populations collected in the same fields in 2010, one year after the prohibition of malathion in the European Union. The average frequency of the susceptible genotype (S/S) increased from 55.9% in the period 2004-2007 to 70.8% in 2010, whereas the frequency of those genotypes carrying the mutation in homozygosis or heterozygosis (R/R and S/R) declined from 30.4 to 9.2%, those carrying the duplication in homozygosis or heterozygosis (RS/RS and S/RS) increased slightly from 12.8 to 13.3%, and those carrying both the mutation and the duplication (R/RS) also increased from 1 to 6.7%. These results are consistent with the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A and the other copy non-mutated) being more advantageous than the G328A mutation alone by maintaining resistance while restoring part of the fitness. In order to investigate the association of malathion resistance with mutations previously found in the aliesterase CcE7, we have generated isolines bearing specific mutations selected by their putative location near the upper part of the active site gorge of the enzyme. The isoline Sm2 (originating from a female heterozygous for the mutation V96L and a male homozygous for the non-mutated allele) kept high levels of resistance to malathion, even after 30 generations without selection pressure. On the contrary, the isoline 267Y (composed by individuals homozygous for the mutation L267Y) and the strain 306TY (composed by homozygous for the double mutation R306T-N307Y) showed a significant reduction in the levels of resistance. We have found also that resistance to malathion in the Sm2 isoline was partially reverted by DEF and TPP, and that Sm2 showed a significant reduction in MTB activity, as reported for other species showing malathion-specific resistance mediated by aliesterases. Besides, it was possible to associate the presence of the mutation V96L in individuals from the Sm2 isoline with both survival to a discriminating concentration of malathion (5,000 ppm) and low MTB activity. Our results point out to a possible connection betwen the mutation V96L in the aliesterase CcE7 and resistance to malathion, though we can not yet conclude that the resistance is caused by the mutation, being needed further work to understand its contribution to resistance. In conclusion, resistance to lambda-cyhalothrin has been found for the first time in field populations of C. capitata, and metabolic resistance mediated by P450 appears to be the main resistance mechanism in the resistant strain W-1K. These findings add to the previously reported case of field resistance to malathion, associated to the G328A mutation and the duplication of the Ccace2 gene (with one of the copies bearing the mutation G328A) and to metabolic resistance mediated by esterases. Our results also indicate that altered target site AChE appears to be responsible for a certain level of resistance to malathion in C. capitata, that can be estimated as about 25-40-fold for the mutation G328A and 40-60-fold for the duplication; whereas metabolic resistance mediated by esterases and associated in this study with the mutation V96L in CcE7 may confer a multiplicative effect (by a factor of 5 to10) increasing malathion resistance to 200-400-fold. Moreover, we have shown that resistant insects carrying the duplication have better fitness and exhibit a higher stability than those with the mutation G328A in the absence of insecticide pressure. This represents a scenario where genotypes with the duplication will remain in the field at low to moderate frequencies, but could be rapidly selected if malathion or other insecticides showing cross-resistance are used. These findings have important implications for resistance management programs, as the repertoire of effective insecticides for C. capitata control is becoming very limited. Besides, multiple resistance mechanisms coexisting in field populations provide the potential to develop resistance to other available insecticides for the control of this pest. Appropriate resistance management strategies based on the alternation of insecticides with different modes of action, and their combination with other control methods, must then be implemented to avoid the evolution of resistance in the field.

Burrknots as food source for larval development of Grapholita molesta (Lepidoptera: Tortricidae) on apple trees.

Grapholita molesta (Busck) is one of the main pests of apple trees, and lives on their shoots and fruits. In southern Brazil, the insect is also found on old branches and structures similar to aerial roots, so-called burrknots. This study evaluated the development and population growth potential of G. molesta fed on burrknots, compared with apple fruit cultivar. Fuji and a corn-based artiÞcial diet. The study was carried out in the laboratory under controlled temperature (25 1C), relative humidity (7010%), and photophase (16 h). The biological parameters of the immature and adult stages were determined, and a fertility life table was constructed. Insects fed on burrknots showed a longer duration and a lower survival for the egg-to-adult period (29.3 d and 22.5%) compared with those that fed on apples (25.1 d and 30.0%) and artiÞcial diet (23.9 d and 54.8%). Insects reared on aerial roots had a lower pupal weight (10.0 mg) compared with those reared on either artiÞcial diet (13.7 mg) or apple cultivar. Fuji (12.4 mg). The fecundity and longevity of males and females did not signiÞcantly differ for the three foods. Based on the fertility life table, insects reared on burrknots had the lowest net reproductive rate (Ro), intrinsic rate of population growth (rm) and finite rate of increase, compared with insects reared on artiÞcial diet and apple fruit. Burrknots support the development of the complete cycle of G. molesta, which allows populations of this pest to increase in orchards.

Self-referencing and consumer evaluations of larger-sized female models : a weight locus of control perspective

In two experiments, we show that the beliefs women have about the controllability of their weight (i.e., weight locus of control) influences their responses to advertisements featuring a larger-sized female model or a slim female model. Further, we examine self-referencing as a mechanism for these effects. Specifically, people who believe they can control their weight (“internals”), respond most favorably to slim models in advertising, and this favorable response is mediated by self-referencing. In contrast, people who feel powerless about their weight (“externals”), self-reference larger-sized models, but only prefer larger-sized models when the advertisement is for a non-fattening product. For fattening products, they exhibit a similar preference for larger-sized models and slim models. Together, these experiments shed light on the effect of model body size and the role of weight locus of control in influencing consumer attitudes.

Longitudinal patterns of weight in women diagnosed with breast cancer [Conference Abstract]

Introduction: Weight gain is a common concern following breast cancer and has been associated with negative health outcomes. As such, prevention of weight gain is of clinical interest. This work describes weight change between 6- and 18-months following a breast cancer diagnosis and explores the personal, treatment and behavioural characteristics associated with gains in weight. Methods: Body mass index was objectively assessed, at three-monthly intervals, on a population-based sample of women newly diagnosed with unilateral breast cancer (n=185). Changes in BMI between 6- and 18-months post-diagnosis were calculated, with gains of one or more being considered clinically detrimental to future health. Results: Approximately 60% of participants were overweight or obese at 6-months post-diagnosis. While BMI remained relatively stable across the testing period (range=27.3-27.8), 24% of participants experienced clinically relevant gains in BMI (median gains=1.9). Following adjustment for potential confounders, younger age (<45 years; Odds ratio, OR=9.8), being morbidly obese at baseline (OR=4.6) and receiving hormone therapy (OR=4.8) were characteristics associated with an increased odds (p<0.05) of gaining BMI. Other characteristics associated with gains in BMI were more extensive surgery and having a history of smoking, although these relationships were not supported statistically. In contrast, caring for younger children was associated with reduced risk of gaining BMI (OR=0.3, p=0.20). Conclusions: Clinically relevant weight gain between 6- and 18-months post-breast cancer diagnosis is an issue for one in four women, with certain subgroups being particularly susceptible. However, the majority of women diagnosed with breast cancer are overweight or obese and gains in body weight are common. Thus, interventions that address the importance of achieving and sustaining a healthy body weight, delivered to all women with breast cancer, may have greater public health impact than interventions targeting any specific breast cancer subgroup.