Influence of new integrated pest management strategies on aphidophagous predators in horticultural crops

El 1 de enero de 2014 entró en vigor la Directiva Europea 2009/128/CE sobre uso sostenible de plaguicidas y el Real Decreto 1311/2012 por el cual se traspone dicha normativa comunitaria al ámbito nacional. Estos reglamentos establecen el marco legal por el que las explotaciones agrícolas deben cumplir los principios generales de la Gestión Integrada de Plagas (GIP). Los principios de la GIP dan preferencia a aquellos métodos de control que sean sostenibles y respetuosos con el medio ambiente, dando prioridad al control biológico, al físico y a otros de carácter no químico. Sin embargo, el uso de insecticidas selectivos con los enemigos naturales es necesario en ocasiones para el adecuado manejo de las plagas en cultivos hortícolas. Por ello, el objetivo general de esta Tesis ha sido aportar conocimientos para la mejora del control de plagas en cultivos hortícolas, mediante la integración de estrategias de lucha biológica, física y química. La primera de las líneas de investigación de esta Tesis se centró en el estudio del efecto de la presencia dos depredadores, larvas Chrysoperla carnea y adultos de Adalia bipunctata, en la dispersión del virus de transmisión no persistente Cucumber mosaic virus (CMV) y del virus de transmisión persistente Cucurbit aphid-borne yellows virus (CABYV), transmitidos por el pulgón Aphis gosypii en cultivo de pepino. La tasa de transmisión de CMV fue baja para los dos tiempos de evaluación ensayados (1 y 5 días), debido al limitado movimiento de su vector A. gossypii. Las plantas que resultaron infectadas se localizaron próximas a la fuente de inóculo central y la presencia de ambos enemigos naturales no incrementó significativamente el porcentaje de plantas ocupadas por pulgones ni la tasa de transmisión de CMV. Los patrones de distribución de A. gossypii y de CMV tan solo fueron coincidentes en las proximidades de la planta central infectada en la que se liberaron los insectos. En los ensayos con CABYV, la presencia de C. carnea y de A. bipunctata respectivamente provocó un incremento significativo de la dispersión de A. gossypii tras 14 días, pero no tras 7 días desde la liberación de los insectos. La reducción en el número inicial de pulgones en la planta central infectada con CABYV fue siempre mayor tras la liberación de C. carnea en comparación con A. bipunctata. Sin embargo, la tasa de transmisión de CABYV y su distribución espacial no se vieron significativamente modificadas por la presencia de ninguno de los depredadores, ni tras 7 días ni tras 14 días desde el inicio de los ensayos. Al igual que se estudió el efecto de la presencia de enemigos naturales en el comportamiento de las plagas y en la epidemiología de las virosis que transmiten, en una segunda línea de investigación se evaluó el posible efecto del consumo de pulgones portadores de virus por parte de los enemigos naturales. Este trabajo se llevó a cabo en el Laboratorio de Ecotoxicología del Departamento de Entomología de la Universidade Federal de Lavras (UFLA) (Brasil). En él se evaluó la influencia en los parámetros biológicos del enemigo natural Chrysoperla externa al alimentarse de Myzus persicae contaminados con el virus de transmisión persistente Potato leafroll virus (PLRV). El consumo de M. persicae contaminados con PLRV incrementó significativamente la duración de la fase larvaria, reduciendo también la supervivencia en comparación a otras dos dietas a base de M. persicae no contaminados con el virus y huevos del lepidóptero Ephestia kuehniella. La duración de la fase de pupa de C. externa no difirió significativamente entre las dietas a base de pulgones contaminados con PLRV y pulgones no contaminados, pero ambas fueron menores que con la dieta con huevos de E. kuehniella. Sin embargo, ni la supervivencia en la fase de pupa ni los parámetros reproductivos de los adultos emergidos mostraron diferencias significativas entre las dietas evaluadas. Por el contrario, la supervivencia de los adultos durante los 30 primeros días desde su emergencia sí se vio significativamente afectada por la dieta, siendo al término de este periodo del 54% para aquellos adultos de C. externa que durante su fase larvaria consumieron pulgones con PLRV. Dentro de la GIP, una de las estrategias de carácter físico que se emplean para el control de plagas y enfermedades en cultivos hortícolas protegidos es el uso de plásticos con propiedades fotoselectivas de absorción de la radiación ultravioleta (UV). Por ello, la tercera línea de investigación de la Tesis se centró en el estudio de los efectos directos e indirectos (mediados por la planta) de condiciones especiales de baja radiación UV sobre el crecimiento poblacional del pulgón A. gossypii y los parámetros biológicos del enemigo natural C. carnea, así como sobre las plantas de pepino en las que se liberaron los insectos. Los ensayos se realizaron en jaulones dentro de invernadero, utilizándose en el primero de ellos plantas de pepino sanas, mientras que en el segundo las plantas de pepino fueron previamente infectadas con CABYV para estudiar de qué manera afectaba la incidencia del virus en las mismas condiciones. Las condiciones de baja radiación UV (bajo plástico Térmico Antivirus®) ejercieron un efecto directo en las fases iniciales del cultivo de pepino, promoviendo su crecimiento, mientras que en fases más avanzadas del cultivo indujeron un aumento en el contenido en nitrógeno de las plantas. Las plantas de pepino que fueron sometidas a mayor intensidad de radiación UV (bajo plástico Térmico Blanco®) al inicio del cultivo mostraron un engrosamiento significativo de las paredes de las células epidérmicas del haz de las hojas, así como de la cutícula. El uso del plástico Térmico Antivirus®, utilizado como barrera fotoselectiva para crear condiciones de baja radiación UV, no alteró con respecto al plástico Térmico Blanco® (utilizado como control) el desarrollo poblacional del pulgón A. gossypii ni los parámetros biológicos evaluados en el depredador C. carnea. En el segundo experimento, realizado con plantas infectadas con CABYV, la incidencia de la virosis enmascaró las diferencias encontradas en experimento con plantas sanas, reduciendo aparentemente la influencia de las distintas condiciones de radiación UV. Por último, para el desarrollo de las estrategias de GIP es importante estudiar los posibles efectos secundarios que los plaguicidas pueden tener en los enemigos naturales de las plagas. Es por ello que en la Tesis se evaluaron la toxicidad y los efectos subletales (fecundidad y fertilidad) de flonicamida, flubendiamida, metaflumizona, spirotetramat, sulfoxaflor y deltametrina en los enemigos naturales C. carnea y A. bipunctata. Los efectos secundarios fueron evaluados por contacto residual tanto para larvas como para adultos de ambos enemigos naturales en condiciones de laboratorio. Flonicamida, flubendiamida, metaflumizona y spirotetramat fueron inocuos para larvas de último estadio y adultos de C. carnea y A. bipunctata. Por este motivo, estos insecticidas se presentan como buenos candidatos para ser incorporados dentro de programas de GIP en combinación con estos enemigos naturales para el control de plagas de cultivos hortícolas. Sulfoxaflor fue ligeramente tóxico para adultos de C. carnea y altamente tóxico para larvas de último estadio de A. bipunctata. Para A. bipunctata, sulfoxaflor y deltametrina fueron los compuestos más dañinos. Deltametrina fue también el compuesto más tóxico para larvas y adultos de C. carnea. Por tanto, el uso de deltametrina y sulfoxaflor en programas de GIP debería tomarse en consideración cuando se liberasen cualquiera de estos dos enemigos naturales debido al comportamiento tóxico que mostraron en condiciones de laboratorio. ABSTRACT On 1 January 2014 came into effect the Directive 2009/128/EC of the European Parliament about sustainable use of pesticides and the Royal Decree 1311/2012 that transposes the regulation to the Spanish level. These regulations establish the legal framework that agricultural holdings must adhere to in order to accomplish the general principles of Integrated Pest Management (IPM). The guidelines of IPM give priority to sustainable and eco-friendly pest control techniques, such as biological and physical measures. Nevertheless, the use of pesticides that are selective to natural enemies is sometimes a necessary strategy to implement accurate pest management programs in horticultural protected crops. Therefore, the general objective of this Thesis was to contribute to the improvement of pest management strategies in horticultural crops, by means of the integration of biological, physical and chemical techniques. The first research line of this Thesis was focused on the evaluation of the effects of two aphidophagous predators, Chrysoperla carnea larvae and Adalia bipunctata adults, on the spread of the non-persistently transmitted Cucumber mosaic virus (CMV, Cucumovirus) and the persistently transmitted Cucurbit aphid-borne yellows virus (CABYV, Polerovirus), by the aphid vector Aphis gossypii in a cucumber crop under greenhouse conditions. The CMV transmission rate was generally low, both after 1 and 5 days, due to the limited movement of its aphid vector A. gossypii. Infected plants were mainly located around the central virusinfected source plant, and the percentage of aphid occupation and CMV-infected plants did not differ significantly in absence and presence of natural enemies. The distribution patterns of A. gossypii and CMV were only coincident close to the central plant where insects were released. In the CABYV experiments, the presence of C. carnea larvae and A. bipunctata adults induced significant A. gossypii dispersal after 14 days but not after 7 days. The reduction in the initial aphid population established in the central plant was always higher for C. carnea than for A. bipunctata. Nevertheless, CABYV spread was not significantly modified by the presence of each predator either in the short term (7 days) or in the long term (14 days). Furthermore, the percentage of CABYV-infected plants did not significantly differ when each natural enemy was present in any evaluation period. It is important to evaluate the influence that natural enemies have on pest dynamics and on the spread of viral diseases, but it should be also taken into account the possible effect on the performance of natural enemies when they feed on preys that act as vectors of viruses. Thus, in a second research line developed in the Laboratory of Ecotoxicology, Department of Entomology, of the Universidade Federal de Lavras (UFLA) (Brazil), it was evaluated the performance of Chrysoperla externa under the condition of consuming Myzus persicae acting as vector of Potato leafroll virus (PLRV). The diet composed of PLRV-infected M. persicae significantly increased the length and reduced the survival rate, of the larval period in regard to the other two diets, composed of non-infected M. persicae and Ephestia kuehniella eggs. The lengths of the pupal stage were not significantly different between the aphid diets, but both were significantly shorter than that of E. kuehniella eggs. Neither pupal survival nor reproductive parameters revealed significant differences among the diets. Nevertheless, the adult survival curves during the first 30 days after emergence showed significant differences, reaching at the end of this interval a value of 54% for those C. externa adults fed on PLRVinfected aphids during their larval period. According to the IPM guidelines, one of the physical strategies for the control of pests and diseases in horticultural protected crops is the use of plastic films with photoselective properties that act as ultraviolet (UV) radiation blocking barriers. In this sense, the third research line of the Thesis dealt with the study of the direct and plant-mediated influence of low UV radiation conditions on the performance of the aphid A. gossypii and on the biological parameters of the natural enemy C. carnea, as well as on the cucumber plants where insects were released. The experiments were conducted inside cages under greenhouse conditions, using for the first one healthy cucumber plants, while for the second experiment the cucumber plants were previously infected with CABYV in order to assess the influence of the virus in the same conditions. The low UV radiation conditions (under Térmico Antivirus® plastic film) seemed to exert a direct effect in the early stages of cucumber plants, enhancing their growth, and in an increasing nitrogen content at further developmental stages. The higher UV radiation exposure (under Térmico Blanco® plastic film) in the early stages of the cucumber crop induced the thickening of the adaxial epidermal cell walls and the cuticle of leaves. The use of Térmico Antivirus® plastic film as a photoselective barrier to induce low UV radiation conditions did not modify, in regard to Térmico Blanco® plastic film (used as control), neither the population development of A. gossypii nor the studied biological parameters of the predator C. carnea. In the second experiment, done with CABYV-infected cucumber plants, the incidence of the virus seemed to mask the direct and plant-mediated influence of the different UV radiation conditions. In last term, for the development of IPM strategies it is important to study the potential side effects that pesticides might have on natural enemies. For this reason, in the Thesis were tested the toxicity and sublethal effects (fecundity and fertility) of flonicamid, flubendiamide, metaflumizone, spirotetramat, sulfoxaflor and deltamethrin on the natural enemies C. carnea and A. bipunctata. The side effects of the active ingredients of the insecticides were evaluated with residual contact tests for the larvae and adults of these predators under laboratory conditions. Flonicamid, flubendiamide, metaflumizone and spirotetramat were innocuous to last instar larvae and adults of C. carnea and A. bipunctata. Therefore, these pesticides are promising candidates for being incorporated into IPM programs in combination with these natural enemies for the control of particular greenhouse pests. In contrast, sulfoxaflor was slightly toxic to adults of C. carnea and was highly toxic to last instar larvae of A. bipunctata. For A. bipunctata, sulfoxaflor and deltamethrin were the most damaging compounds. Deltamethrin was also the most toxic compound to larvae and adults of C. carnea. In accordance with this fact, the use of sulfoxaflor and deltamethrin in IPM strategies should be taken into consideration when releasing either of these biological control agents, due to the toxic behavior observed under laboratory conditions.

Compatibilidad de Eretmocerus mundus Mercet (Hymenoptera: Aphelinidae) y Amblyseius swirskii Athias-Henriot (Acari: Phytoseiidae), importantes enemigos naturales de la mosca blanca Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) en cultivos hortícolas, con nuevas barreras físicas selectivas y modernos plaguicidas

Los programas de Gestión Integrada de Plagas (GIP) promueven el uso de estrategias de control que sean respetuosas con el medio ambiente, sin embargo el uso de insecticidas en los cultivos hortícolas sigue siendo necesario para el control de determinadas plagas, como es el caso de la mosca blanca Bemisia tabaci (Gennadius). Por ello, el objetivo de esta tesis es el estudio de la integración de las tres estrategias de control más empleadas hoy en día para el control de plagas: el control biológico, el físico y el químico. Una primera parte de este trabajo ha consistido en el estudio de los efectos letales y subletales de once insecticidas, aplicados a la dosis máxima de campo, sobre los enemigos naturales Eretmocerus mundus Mercet y Amblyseius swirskii Athias-Henriot, mediante ensayos de laboratorio y persistencia (laboratorio extendido). Para la evaluación de la toxicidad de los insecticidas sobre los estados de vida más protegidos de estos enemigos naturales, se trataron bajo la Torre de Potter las pupas de E. mundus y los huevos de A. swirskii. Además, se llevaron a cabo ensayos de contacto residual para determinar los efectos letales y subletales de estos insecticidas sobre el estado adulto de ambas especies de enemigos naturales. Para ello, los pesticidas se aplicaron sobre placas de cristal (laboratorio) o sobre plantas (laboratorio extendido: persistencia). Los resultados mostraron que los insecticidas flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen y spirotetramat eran compatibles con el estado de pupa de E. mundus (OILB 1: Inocuos). Sin embargo, abamectina, deltametrina y emamectina fueron categorizadas como ligeramente tóxicas (OILB 2) al causar efectos deletéreos. Los dos pesticidas más tóxicos fueron spinosad y sulfoxaflor, los cuales redujeron significativamente la emergencia de las pupas tratadas (OILB 4: Tóxicos). Flonicamida, flubendiamida, metoxifenocida y spiromesifen fueron compatibles con el estado adulto de E. mundus (OILB 1: Inocuos). Abamectina, deltametrina, emamectina, metaflumizona y spiromesifen pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Al contrario, spinosad y sulfoxaflor no resultaron ser compatibles (OILB D: Persistentes), aunque la realización de ensayos adicionales es necesaria para ver los efectos de los mismos en campo. Todos los insecticidas estudiados, excepto el spirotetramat (OILB 2: Ligeramente tóxico), fueron selectivos para el estado de huevo de A. swirskii (OILB 1: Inocuos). Flonicamida, flubendiamida, metaflumizona, metoxifenocida, spiromesifen, spirotetramat y sulfoxaflor, fueron compatibles con el estado adulto de A. swirskii (OILB 1: Inocuos). Abamectina, deltametrina, emamectina y spinosad pueden ser recomendados para su uso en programas de GIP, si se usan los plazos de seguridad apropiados, de acuerdo con la persistencia de cada uno de estos insecticidas, antes de la liberación del enemigo natural. Entre las nuevas estrategias de la GIP, los plásticos y mallas fotoselectivas han demostrado ser una herramienta importante para el control de plagas y enfermedades en cultivos hortícolas protegidos. Por ello, en una segunda parte de este trabajo, se estudiaron tanto los efectos directos, como la combinación de efectos directos y mediados por planta y plaga de ambientes pobres en luz UV, en presencia o ausencia del Virus del rizado amarillo del tomate (TYLCV), sobre E. mundus. En primer lugar, se realizó un ensayo al aire libre para la evaluación de la capacidad de vuelo de E. mundus en cajas tipo túnel (1 x 0,6 x 0,6 m) cubiertas con distintas barreras absorbentes de luz UV. Se detectó un efecto directo en la capacidad de orientación de E. mundus, debido a que este parasitoide utiliza estímulos visuales para localizar a sus huéspedes, únicamente en las barreras que bloqueaban más del 65% de la luz UV (malla G). En segundo lugar, bajo condiciones de invernadero, se evaluó la combinación de efectos directos y mediados por planta y plaga sobre E. mundus, usando plantas de tomate sanas o infectadas con el TYLCV y cajas (30 x 30 x 60 cm) cubiertas con los distintos plásticos fotoselectivos. En este caso, no se observó ningún efecto en la capacidad benéfica del parasitoide cuando este estaba en contacto con plantas de tomate infestadas con ninfas de B. tabaci, lo que demuestra que este insecto usa estímulos táctiles para encontrar a sus huéspedes a cortas distancias. Además, las diferentes condiciones de radiación UV estudiadas tuvieron cierto impacto en la morfología, fisiología y bioquímica de las plantas de tomate, infestadas o no con el virus de la cuchara, detectándose pequeñas alteraciones en alguno de los parámetros estudiados, como el peso fresco y seco, el contenido en H y el espesor de las cutículas y de las paredes celulares de la epidermis foliar. Por último, no se observaron efectos de la radiación UV mediados por planta, ni en B. tabaci ni en su parasitoide, E. mundus. En una tercera parte, se evaluaron los efectos de una malla tratada con bifentrin sobre ambos enemigos naturales, en ensayos de laboratorio, semicampo y campo. Las mallas tratadas fueron diseñadas originariamente para el control de mosquitos vectores de la malaria, y actualmente se está trabajando para su uso en agricultura, como una nueva estrategia de control de plagas. En ensayos de laboratorio, cuando adultos de E. mundus y A. swirskii se expusieron por contacto durante 72 horas con la malla tratada (cajas de 6 cm diámetro), se registró una alta mortalidad. Sin embargo, en el ensayo de preferencia, estos enemigos naturales no fueron capaces de detectar la presencia de bifentrin y, en aquellos individuos forzados a atravesar la malla tratada, no se observó mortalidad a corto plazo (72 horas). En estudios de semicampo, llevados a cabo bajo condiciones de invernadero en cajas de 25 x 25 x 60 cm de altura, la capacidad benéfica de E. mundus no se vio afectada. Finalmente, en ensayos de campo llevados a cabo en invernaderos comerciales (4000m2) en Almería, A. swirskii no se vio afectado por la presencia en el cultivo de la malla tratada con bifentrin y los niveles de infestación de B. tabaci y F. occidentalis detectados bajo dicha malla, fueron inferiores a los del control. Por último, se ha evaluado la composición de la microflora bacteriana de tres especies de parasitoides, E. mundus, Eretmocerus eremicus Rose & Zolnerowich y Encarsia formosa Gahan, y la influencia de la misma en su susceptibilidad a insecticidas. Se llevó a cabo una extracción total de ADN de los insectos y la región variable V4 del ARNr se amplificó usando cebadores universales bacterianos. Para identificar las secuencias de los géneros bacterianos presentes en los parasitoides, se realizó una Next Generation sequencing (Illumina sequencing). Una vez identificados los géneros bacterianos, el gen ADNr 16S de las Actinobacterias se amplificó del ADN extraído de los insectos, usando cebadores universales bacterianos y específicos de Actinobacterias, y los productos de la Nested PCR fueron clonados para identificar todas las especies del género Arthrobacter. Tres bacterias (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi y A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), próximas a las especies de Arthrobacter presentes en los parasitoides, se obtuvieron de la colección bacteriana del BCCMTM/LMG y se midió su actividad esterasa. Finalmente, se realizaron ensayos con antibióticos (tetraciclina) y de contacto residual con insecticidas (abamectina) para determinar la influencia de las especies de Arthrobacter en la susceptibilidad de E. mundus a insecticidas. Los resultados muestran que este género bacteriano puede afectar a la toxicidad de E. mundus a abamectina, mostrando la importancia de la comunidad microbiana en enemigos naturales, factor que debe ser considerado en los estudios de evaluación de los riesgos de los insecticidas. ABSTRACT Integrated Pest Management (IPM) programs promote the use of control strategies more respectful with the environment; however the use of insecticides in vegetable crops is still needed to control certain pests, such as the whitefly Bemisia tabaci (Gennadius). Therefore, the objective of this work is to study the integration of the three most commonly used pest control strategies nowadays: biological, physical and chemical control. Firstly, the lethal and sublethal effects of eleven insecticides, applied at their maximum field recommended concentration, on the parasitic wasp Eretmocerus mundus Mercet and the predator Amblyseius swirskii Athias-Henriot has been assessed in the laboratory and in persistence tests (extended laboratory). To test the effects of pesticides on the most protected life stage of these natural enemies, E. mundus pupae and A. swirskii eggs were sprayed under a Potter precision spray tower. Laboratory contact tests were therefore conducted to determine the lethal and sublethal effects of these pesticides on the adult stage of these natural enemies. In the residual contact tests the pesticides were applied on glass plates (laboratory) or plants (extended laboratory: persistence). The study showed that the insecticides flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen and spirotetramat were selective for E. mundus pupae (IOBC 1: Harmless). Nevertheless, abamectin, deltamethrin and emamectin were categorized as slightly harmful (IOBC 2) due to the deleterious effects caused. The two most harmful pesticides were spinosad and sulfoxaflor, which significantly reduced the adult emergence from treated pupae (IOBC 4: Harmful). Flonicamid, flubendiamide, methoxyfenozide and spiromesifen were compatible with E. mundus adults (IOBC 1: Harmless). Base on the duration of the harmful activity, abamectin, deltamethrin, emamectin, metaflumizone and spirotetramat could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. On the contrary, spinosad and sulfoxaflor were not compatible (IOBC D: persistent), although additional studies are required to determine their effects under field conditions. All the pesticides tested, except spirotetramat (IOBC 2: Slightly harmful), were selective for A. swirskii eggs (IOBC 1: Harmless). Flonicamid, flubendiamide, metaflumizone, methoxyfenozide, spiromesifen, spirotetramat and sulfoxaflor were compatible with A. swirskii adults (IOBC 1: Harmless). However, abamectin, deltamethrin, emamectin and spinosad could be recommended for use in IPM programs if appropriate safety deadlines are used before the natural enemy release. Among new IPM strategies, UV-absorbing photoselective plastic films and nets have been shown to be an important tool for the control of pests and diseases in horticultural protected crops. Because of that, we secondly studied the plant and pest insect-mediated and/or the direct effects on E. mundus under different UV radiation conditions, in presence or absence of the Tomato Yellow Leaf Curl Virus (TYLCV). In the first experiment, performed outdoors, the flight activity of E. mundus was studied in one-chamber tunnels (1 x 0.6 x 0.6 m) covered with different photoselective barriers. Because E. mundus uses visual cues for host location at a long distance, a direct effect on its host location ability was detected, but only in the UV-absorbing barriers blocking more than 65% of the UV light (G net). In a second experiment, the direct and plant and pest insect-mediated effects of different UV radiation conditions on E. mundus were studied, inside cages (30 x 30 x 60 cm) covered with the different UVplastic films and under greenhouse conditions, using healthy or TYLCV-virus infected tomato plants. In this case, not any effect on the beneficial capacity of this parasitoid was detected, proving that he uses tactile cues at a short distance of the host. Moreover, the different UV radiation conditions studied had a certain direct impact in the morphology, physiology and biochemistry of tomato plants infested or not with the TYLCV, and small alterations in some parameters such as fresh and dry weight, H percentage and cuticle and cell wall thickness of epidermal cells of the leaves, were detected. Finally, none plant-mediated UV effects neither in the whitefly B. tabaci nor in their parasitic wasp were found. Thirdly, the effects of a bifenthrin treated net were evaluated in different laboratory, semi-field and field experiments on the natural enemies studied. Treated nets were developed long time ago aiming at the control of the mosquitoes vectors of malaria, and nowadays, there is a great interest on assessing the possibility of their use in agriculture. In laboratory assays, a high mortality was recorded when E. mundus and A. swirskii adults were exposed by contact to the bifenthrin treated net for 72 hours in small cages (12 cm diameter). However, these natural enemies were not able to detect the presence of bifenthrin in a dual-choice test and no short-term mortality (72 hours) was recorded in those individuals that went through the treated net. In semi-field assays, performed under greenhouse conditions with cages of 25 x 25 x 60 cm high, the beneficial capacity of E. mundus was not affected. Finally, in field assays carried out in commercial multispan greenhouses (4000 m2) in Almería, A. swirskii was not affected by the presence of the bifenthrin treated net in the crop and the B. tabaci and F. occidentalis infestation levels were significantly lower than in the control. Finally, the composition of the microflora present in three species of parasitoids, E. mundus, Eretmocerus eremicus Rose & Zolnerowich and Encarsia formosa Gahan, and its influence in their susceptibility to insecticides, have been assessed. A total DNA extraction was performed on insects and universal bacterial primers were used to amplify the variable V4 region of the rRNA. A Next Generation sequencing (Illumina sequencing) was performed to identify the sequences of the bacterial genera present in the parasitic wasps. Once, the bacterial genera were identified, 16S rDNA gene of Actinobacteria were amplified from insects DNA extracts using the universal bacterial and actinobacterial primers, and the nested PCR products, were cloned to identify the Arthrobacter species. Three bacteria (A. aurescens Phillips, A. nicotinovarans Kodama, Yamamoto, Amano and Amichi and A. uratoxydans Stackebrandt, Fowler, Fiedler and Seiler), having the closest match with the Arthrobacter species present in the parasitic wasps, were obtained from the BCCMTM/LMG bacteria collection and its esterase activity was measured. Finally, antibiotic and residual contact tests were done to determine the influence of Arthrobacter species in the susceptibility of E. mundus to pesticides (abamectin). The results suggest that this bacterial genus can affect the toxicity of E. mundus to abamectin, which in turn supports the importance of the microbial community in natural enemies that it should be considered as a factor in risk assessment tests of pesticides.

Glucose intolerance caused by a defect in the entero-insular axis: A study in gastric inhibitory polypeptide receptor knockout mice

Mice with a targeted mutation of the gastric inhibitory polypeptide (GIP) receptor gene (GIPR) were generated to determine the role of GIP as a mediator of signals from the gut to pancreatic β cells. GIPR−/− mice have higher blood glucose levels with impaired initial insulin response after oral glucose load. Although blood glucose levels after meal ingestion are not increased by high-fat diet in GIPR+/+ mice because of compensatory higher insulin secretion, they are significantly increased in GIPR−/− mice because of the lack of such enhancement. Accordingly, early insulin secretion mediated by GIP determines glucose tolerance after oral glucose load in vivo, and because GIP plays an important role in the compensatory enhancement of insulin secretion produced by a high insulin demand, a defect in this entero-insular axis may contribute to the pathogenesis of diabetes.

Biogenesis of Tim Proteins of the Mitochondrial Carrier Import Pathway: Differential Targeting Mechanisms and Crossing Over with the Main Import Pathway

Two major routes of preprotein targeting into mitochondria are known. Preproteins carrying amino-terminal signals mainly use Tom20, the general import pore (GIP) complex and the Tim23–Tim17 complex. Preproteins with internal signals such as inner membrane carriers use Tom70, the GIP complex, and the special Tim pathway, involving small Tims of the intermembrane space and Tim22–Tim54 of the inner membrane. Little is known about the biogenesis and assembly of the Tim proteins of this carrier pathway. We report that import of the preprotein of Tim22 requires Tom20, although it uses the carrier Tim route. In contrast, the preprotein of Tim54 mainly uses Tom70, yet it follows the Tim23–Tim17 pathway. The positively charged amino-terminal region of Tim54 is required for membrane translocation but not for targeting to Tom70. In addition, we identify two novel homologues of the small Tim proteins and show that targeting of the small Tims follows a third new route where surface receptors are dispensable, yet Tom5 of the GIP complex is crucial. We conclude that the biogenesis of Tim proteins of the carrier pathway cannot be described by either one of the two major import routes, but involves new types of import pathways composed of various features of the hitherto known routes, including crossing over at the level of the GIP.

A novel modifier gene for plasma von Willebrand factor level maps to distal mouse chromosome 11

Type 1 von Willebrand disease (VWD), characterized by reduced levels of plasma von Willebrand factor (VWF), is the most common inherited bleeding disorder in humans. Penetrance of VWD is incomplete, and expression of the bleeding phenotype is highly variable. In addition, plasma VWF levels vary widely among normal individuals. To identify genes that influence VWF level, we analyzed a genetic cross between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold difference in plasma VWF level. DNA samples from F2 progeny demonstrating either extremely high or extremely low plasma VWF levels were pooled and genotyped for 41 markers spanning the autosomal genome. A novel locus accounting for 63% of the total variance in VWF level was mapped to distal mouse chromosome 11, which is distinct from the murine Vwf locus on chromosome 6. We designated this locus Mvwf for “modifier of VWF.” Additional genotyping of as many as 2407 meioses established a high resolution genetic map with gene order Cola1-Itg3a-Ngfr-Mvwf/Gip-Hoxb9-Hoxb1-Cbx·rs2-Cox5a-Gfap. The Mvwf candidate interval between Ngfr and Hoxb9 is ≈0.5 centimorgan (cM). These results demonstrate that a single dominant gene accounts for the low VWF phenotype of RIIIS/J mice in crosses with several other strains. The pattern of inheritance suggests a gain-of-function mutation in a unique component of VWF biosynthesis or processing. Characterization of the human homologue for Mvwf may have relevance for a subset of type 1 VWD cases and may define an important genetic factor modifying penetrance and expression of mutations at the VWF locus.

Land and resource management plan 1990 and beyond : Gifford Pinchot National Forest.

"RO-GIP-001-91"--P. [3] of cover.

Ebola virus glycoprotein GP is not cytotoxic when expressed constitutively at a moderate level

Transient expression of Ebola virus (EBOV) glycoprotein GP causes downregulation of surface proteins, cell rounding and detachment, a phenomenon believed to play a central role in the pathogenicity of the virus. In this study, evidence that moderate expression of GP does not result in such morphological changes was provided. It was shown that GP continuously produced in 293T cells from the Kunjin virus replicon was correctly processed and transported to the plasma membrane without affecting the surface expression of beta 1 and alpha 5 integrins and major histocompatibility complex I molecules. The level of GIP expression in Kunjin replicon GP-expressing cells was similar to that observed in cells infected with EBOV early in infection and lower than that produced in cells transfected with plasmid DNA, phCMV-GP(1) expressing GP from a strong promoter. Importantly, transient transfection of Kunjin replicon GIP-expressing cells with GIP-coding plasmid DNA resulted in overexpression of GP, which lead to the downregulation of surface molecules and massive rounding and detachment of transfected cells. Here, it was also demonstrated that cell rounding and downregulation of the surface markers are the late events in EBOV infection, whereas synthesis and massive release of virus particles occur at early steps and do not cause significant cytotoxic effects. These findings indicate that the synthesis of EBOV GP in virus-infected cells is controlled well by several mechanisms that do not allow GP overexpression and hence the early appearance of its cytotoxic properties.

Fatty acid derivatised analogues of glucose-dependent insulinotropic polypeptide with improved antihyperglycaemic and insulinotropic properties

C-terminal acylation of Lys(37) with myristic (MYR; tetradecanoic acid), palmitic (PAL; hexadecanoic acid) and stearic (octadecanoic acid) fatty acids with or without N-terminal acetylation was employed to develop long-acting analogues of the glucoregulatory hormone, glucose-dependent insulinotropic polypeptide (GIP). All GIP analogues exhibited resistance to dipeptidylpeptidase-IV (DPP-IV) and significantly improved in vitro cAMP production and insulin secretion. Administration of GIP analogues to ob/ob mice significantly lowered plasma glucose-GIP(Lys(37)MYR), N-AcGIP(Lys(37)MYR) and GIP(Lys(37)PAL) increased plasma insulin concentrations. GIP(Lys(37)MYR) and N-AcGIP(Lys(37)MYR) elicited protracted glucose-lowering effects when administered 24h prior to an intraperitoneal glucose load. Daily administration of GIP(Lys(37)MYR) and N-AcGIP(Lys(37)MYR) to ob/ob mice for 24 days decreased glucose and significantly improved plasma insulin, glucose tolerance and beta-cell glucose responsiveness. Insulin sensitivity, pancreatic insulin content and triglyceride levels were not changed. These data demonstrate that C-terminal acylation particularly with myristic acid provides a class of stable, longer-acting forms of GIP for further evaluation in diabetes therapy.

Control of the endocrine pancreas by gastrointestinal peptides

This thesis concerns the mechanism through which enteral delivery of glucose results in a larger insulin response than an equivalent parenteral glucose load. Preliminary studies in which mice received a glucose solution either intragastrically or intraperitoneally confirmed this phenomenon. An important regulatory system in this respect is the entero-insular axis, through which insulin secretion is influenced by neural and endocrine communication between the gastrointestinal tract and the pancreatic islets of Langerhans. Using an in vitro system involving static incubation of isolated (by collagenase digestion) islets of Langerhans, the effect of a variety of gastrointestinal peptides on the secretion of the four main islet hormones, namely insulin, glucagon, somatostatin and pancreatic polypeptide, was studied. The gastrointestinal peptides investigated in this study were the secretin family, comprising secretin, glucagon, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine (PHI) and growth hormone releasing factor (GRF). Gastrin releasing peptide (GRP) was also studied. The results showed that insulin release was stimulated by all peptides studied except PHI, glucagon release was stimulated by all peptides tested, except GRF which suppressed glucagon release, somatostatin release was stimulated by GIP and GRF but suppressed by VIP, PHI, glucagon and secretin, and PP release was stimulated by GIP and GRF, but suppressed by PHI. The insulinotropic effect of GRP was investigated further. A perifusion system was used to examine the time-course of insulin release from isolated islets after stimulation with GRP. GRP was shown to be insulinotropic only in the presence of physiologically elevated glucose concentrations and both first and second phases of insulin release were augmented. There was no effect at substimulatory or very high glucose concentrations. Studies using a cultured insulin-secreting islet cell line, the RINm5F cell line, were undertaken to elucidate the intracellular mechanism of action of GRP. This peptide did not enhance insulin release via an augmentation of glucose metabolism, or via the adenylate cyclase/cyclic AMP secondary messenger system. The pattern of changes of cytosolic free calcium in response to GRP, which involved both mobilization of intracellular stores and an influx of extracellular calcium, suggested the involvement of phosphatidylinositol bisphosphate breakdown as a mediator of the effect of GRP on insulin secretion.

Dipeptidyl peptidase IV (DPP IV) and related molecules in type 2 diabetes

Dipeptidyl peptidase IV (DPP IV) is a widely distributed physiological enzyme that can be found solubilized in blood, or membrane-anchored in tissues. DPP IV and related dipeptidase enzymes cleave a wide range of physiological peptides and have been associated with several disease processes including Crohn's disease, chronic liver disease, osteoporosis, multiple sclerosis, eating disorders, rheumatoid arthritis, cancer, and of direct relevance to this review, type 2 diabetes. Here, we place particular emphasis on two peptide substrates of DPP IV with insulin-releasing and antidiabetic actions namely, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). The rationale for inhibiting DPP IV activity in type 2 diabetes is that it decreases peptide cleavage and thereby enhances endogenous incretin hormone activity. A multitude of novel DPP IV inhibitor compounds have now been developed and tested. Here we examine the information available on DPP IV and related enzymes, review recent preclinical and clinical data for DPP IV inhibitors, and assess their clinical significance.

Recent advances in antidiabetic drug therapies targeting the enteroinsular axis

The enteroinsular axis (EIA) constitutes a physiological signalling system whereby intestinal endocrine cells secrete incretin hormones following feeding that potentiate insulin secretion and contribute to the regulation of blood glucose homeostasis. The two key hormones responsible are named glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Recent years have witnessed sustained development of antidiabetic therapies that exploit the EIA. Current clinical compounds divide neatly into two classes. One concerns analogues or mimetics of GLP-1, such as exenatide (Byetta) or liraglutide (NN2211). The other group comprises the gliptins (e.g. sitagliptin and vildagliptin) which boost endogenous incretin activity by inhibiting the enzyme dipeptidyl peptidase 4 (DPP 4) that degrades both GLP-1 and GIP. Ongoing research indicates that further incretin and gliptin compounds will become available for clinical use in the near future, offering comparable or improved efficacy. For incretin analogues there is the prospect of prolonged duration of action and alternative routes of administration. This review focuses on recent advances in pre-clinical research and their translation into clinical studies to provide future therapies for type 2 diabetes targeting the EIA. © 2009 Bentham Science Publishers Ltd.

Laparoscopic greater curvature plication in morbidly obese women with type 2 diabetes:effects on glucose homeostasis, postprandial triglyceridemia and selected gut hormones

Background: Laparoscopic greater curvature plication (LGCP) is an emerging bariatric procedure that reduces the gastric volume without implantable devices or gastrectomy. The aim of this study was to explore changes in glucose homeostasis, postprandial triglyceridemia, and meal-stimulated secretion of selected gut hormones [glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), ghrelin, and obestatin] in patients with type 2 diabetes mellitus (T2DM) at 1 and 6 months after the procedure. Methods: Thirteen morbidly obese T2DM women (mean age, 53.2 ± 8.76 years; body mass index, 40.1 ± 4.59 kg/m2) were prospectively investigated before the LGCP and at 1- and 6-month follow-up. At these time points, all study patients underwent a standardized liquid mixed-meal test, and blood was sampled for assessment of plasma levels of glucose, insulin, C-peptide, triglycerides, GIP, GLP-1, ghrelin, and obestatin. Results: All patients had significant weight loss both at 1 and 6 months after the LGCP (p≤0.002), with mean percent excess weight loss (%EWL) reaching 29.7 ;plusmn2.9 % at the 6-month follow-up. Fasting hyperglycemia and hyperinsulinemia improved significantly at 6 months after the LGCP (p<0.05), with parallel improvement in insulin sensitivity and HbA1c levels (p<0.0001). Meal-induced glucose plasma levels were significantly lower at 6 months after the LGCP (p<0.0001), and postprandial triglyceridemia was also ameliorated at the 6-month follow-up (p<0.001). Postprandial GIP plasma levels were significantly increased both at 1 and 6 months after the LGCP (p<0.0001), whereas the overall meal-induced GLP-1 response was not significantly changed after the procedure (p ;gt0.05). Postprandial ghrelin plasma levels decreased at 1 and 6 months after the LGCP (p<0.0001) with no significant changes in circulating obestatin levels. Conclusion: During the initial 6-month postoperative period, LGCP induces significant weight loss and improves the metabolic profile of morbidly obese T2DM patients, while it also decreases circulating postprandial ghrelin levels and increases the meal-induced GIP response. © 2013 Springer Science+Business Media New York.

Pontes para a (Des)inserção: inserção pelo trabalho no Concelho de Stª Mª da Feira

O presente relatório visa a obtenção do grau de Mestre em Serviço Social pelo Instituto Superior Miguel Torga. Motivada pela preocupação nascida na prática profissional quotidiana, no acompanhamento aos cidadãos beneficiários do Rendimento Social de Inserção (R.S.I.), procurámos compreender a relação entre a pobreza e o mercado de trabalho. Criado no ano de 1996 pela Lei nº 19-A/96 de 29 de Junho, como prestação de rendimento mínimo garantido, o R.S.I. tem introduzido formas cada vez mais apuradas de seleção das suas clientelas, quer através da redefinição do conceito de agregado e avaliação dos seus rendimentos, quer da contratualização da prestação, pelo reforço crescente das penalizações ao incumprimento relativo ao emprego e à formação. Objetivo geral é perceber como se efetiva o processo de colocação no mercado de trabalho e que oportunidades de (des)inserção social dai resultam para os beneficiários. Procurámos também analisar a configuração das propostas oferecidas, no âmbito do contrato de inserção, aos homens e às mulheres, aos “velhos” e “novos” pobres. Assim, foram efetuadas entrevistas exploratórias aos técnicos do Centro de Emprego e Formação Profissional Entre Douro e Vouga (CEFP-EDV); Gabinete de Inserção Profissional (GIP) e Núcleo Local de Inserção (NLI) de Stª Mª da Feira e aplicado um inquérito por questionário aos beneficiários do R.S.I. com Contrato de Inserção para colocação no mercado de trabalho. Os técnicos do CEFP-EDV e do GIP expressaram dificuldades no acompanhamento e gestão de carreira dos beneficiários, devido à sobrecarga e à natureza burocrática das tarefas exigidas nos seus organismos. Os beneficiários consideram igualmente que o CEFP-EDV não é eficiente nem eficaz, para a colocação no mercado de trabalho não cumprindo portanto a função que legalmente lhe está atribuída. Os homens são amplamente beneficiados na relação com o Centro de Emprego, comparativamente com as mulheres, porque recebem mais propostas de emprego e formação. A “velha pobreza” aparece instalada no desemprego e na prestação durante mais tempo que os “novos” pobres. Estes raramente são convocados pelo CEFP-EDV. São as redes informais que têm um papel mais ativo e preponderante no processo de inserção laboral. Na população inquirida a inserção pelo trabalho por si só não constituiu a solução para a saída da pobreza. / The present report aims to obtain a Master’s degree in Social Work from the Instituto Superior Miguel Torga. Motivated by concern aroused from the day to day professional practice, while monitoring citizens on social income benefits, “Rendimento Social de Inserção” (R.S.I.) (Social Insertion Income), we have sought to understand the relationship between poverty and the labour market. Introduced in the year 1996 by Law nº 19-A/96 of 29th June, as the provision of income support, the R.S.I. has introduced increasingly more refined forms of selection of its clientele, either by redefining the concept of aggregate and assessmento of their income, or the contractual provision, by increasingly strenthening the penalties for failure in relation to employment and training.The overall goal is to understand how the process of entering the work market is made and the opportunities of (un)inclusion arising from it for the beneficiaries. It was also sought to analyse the configuration of the proposals offered under the insertion contract to men and women, to the “old” and the “new” poor.Consequently, exploratory interviews were made to the technicians of Centro de Emprego e Formação Profissional Entre Douro e Vouga (CEFP-EDV) (Emloyment and Training Centre); Gabinete de Inserção Profissional (GIP) (Professional Insertion Office) and Núcleo Local de Inserção (Local Insertion Group) (NLI) of Stª Mª da Feira. The survey was carried out through a questionnaire to the beneficiaries of the R.S.I. with Insertion Contracts for placement in the labour market.The CEFP-EDV and GIP technicians expressed diffficulties in monitoring and career management of the benefeciaries due to overhead and bureaucratic nature of the tasks recquired in their institutions. The beneficiaries also considered that CEFP-EDV is neither efficient nor effective in placing people in the work market thus not fulfilling the function for which they are legally assigned. Men are largely benefitted in relation to the Job Centre, compared to women, because they get more job offers and training. The “old poverty” appears to be installed in unemployment and provision for longer than the “new” poor. These are rarely called up by CEFP-EDV. It is the informal networks that have a more active and leading role in the process of job placement. For the questioned population entering the job market does not itself constitute a solution to ending poverty.

Stable Incretin Mimetics Counter Rapid Deterioration of Bone Quality in Type 1 Diabetes Mellitus.

International audience

Gelatinas funcionais desenvolvidas com microsferas de alginato para aplicação nutracêutica

Os ingredientes bioativos são geralmente suscetíveis à degradação durante o armazenamento ou processamento alimentar, pois muitos deles são instáveis física, química ou enzimaticamente, o que leva à sua degração ou transformação com a consequente perda de bioatividade. Para ultrapassar estas limitações a microencapsulação emerge como uma resposta viável para proteger e estabilizar os bioativos, oferecendo também a possibilidade de uma libertação controlada e localizada [1]. Os materiais de encapsulação, processo de produção, morfologia da microesfera e, por último, as condições de aplicabilidade são os fatores mais importantes a ter em conta no desenho de um novo produto microencapsulado, juntamente com a questão da estabilidade e propriedades funcionais. Por outro lado, para obter um produto bem sucedido deve-se garantir um alto rendimento de encapsulação, a reprodutibilidade do processo e o perfil de libertação e, ainda, tentar evitar a agregação das microcápsulas.