Integration of physical, chemical and biological tactics against insect pests and virus diseases in horticultural crops

Actualmente, la gesti��n de sistemas de Manejo Integrado de Plagas (MIP) en cultivos hort��colas tiene por objetivo priorizar los m��todos de control no qu��micos en detrimento del consumo de plaguicidas, seg��n recoge la directiva europea 2009/128/CE ���Uso Sostenible de Plaguicidas��� (OJEC, 2009). El uso de agentes de biocontrol como alternativa a la aplicaci��n de insecticidas es un elemento clave de los sistemas MIP por sus innegables ventajas ambientales que se utiliza ampliamente en nuestro pa��s (Jacas y Urbaneja, 2008). En la regi��n de Almer��a, donde se concentra el 65% de cultivo en invernadero de nuestro pa��s (47.367 ha), MIP es la principal estrategia en pimiento (MAGRAMA, 2014), y comienza a serlo en otros cultivos como tomate o pepino. El cultivo de pepino, con 8.902 ha (MAGRAMA, 2013), tiene un protocolo semejante al pimiento (Robledo et al., 2009), donde la ��nica especie de pulg��n importante es Aphis gossypii Glover. Sin embargo, pese al continuo incremento de la superficie de cultivo agr��cola bajo sistemas MIP, los da��os originados por virosis siguen siendo notables. Algunos de los insectos presentes en los cultivos de hort��colas son importantes vectores de virus, como los pulgones, las moscas blancas o los trips, cuyo control resulta problem��tico debido a su elevada capacidad para transmitir virus vegetales incluso a una baja densidad de plaga (Holt et al., 2008; Jacas y Urbaneja, 2008). Las relaciones que se establecen entre los distintos agentes de un ecosistema son complejas y muy espec��ficas. Se ha comprobado que, pese a que los enemigos naturales reducen de manera beneficiosa los niveles de plaga, su incorporaci��n en los sistemas planta-insecto-virus puede desencadenar complicadas interacciones con efectos no deseables (Dicke y van Loon, 2000; Jeger et al., 2011). As��, los agentes de biocontrol tambi��n pueden inducir a que los insectos vectores modifiquen su comportamiento como respuesta al ataque y, con ello, el grado de dispersi��n y los patrones de distribuci��n de las virosis que transmiten (Bailey et al., 1995; Weber et al., 1996; Hodge y Powell, 2008a; Hodge et al., 2011). Adem��s, en ocasiones el control biol��gico por s�� solo no es suficiente para controlar determinadas plagas (Medina et al., 2008). Entre los m��todos que se pueden aplicar bajo sistemas MIP est��n las barreras f��sicas que limitan la entrada de plagas al interior de los invernaderos o interfieren con su movimiento, como pueden ser las mallas anti-insecto (��lvarez et al., 2014), las mallas fotoselectivas (Raviv y Antignus, 2004; Weintraub y Berlinger, 2004; D��az y Fereres, 2007) y las mallas impregnadas en insecticida (Licciardi et al., 2008; Martin et al., 2014). Las mallas fotoselectivas reducen o bloquean casi por completo la transmisi��n de radiaci��n UV, lo que interfiere con la visi��n de los insectos y dificulta o impide la localizaci��n del cultivo y su establecimiento en el mismo (Raviv y Antignus, 2004; Weintraub, 2009). Se ha comprobado c��mo su uso puede controlar los pulgones y las virosis en cultivo de lechuga (D��az et al., 2006; Legarrea et al., 2012a), as�� como la mosca blanca, los trips y los ��caros, y los virus que estos transmiten en otros cultivos (Costa y Robb, 1999; Antignus et al., 2001; Kumar y Poehling, 2006; Doukas y Payne, 2007a; Legarrea et al., 2010). Sin embargo, no se conoce perfectamente el modo de acci��n de estas barreras, puesto que existe un efecto directo sobre la plaga y otro indirecto mediado por la planta, cuya fisiolog��a cambia al desarrollarse en ambientes con falta de radiaci��n UV, y que podr��a afectar al ciclo biol��gico de los insectos fit��fagos (V��nninen et al., 2010; Johansen et al., 2011). Del mismo modo, es necesario estudiar la compatibilidad de esta estrategia con los enemigos naturales de las plagas. Hasta la fecha, los estudios han evidenciado que los agentes de biocontrol pueden realizar su actividad bajo ambientes pobres en radiaci��n UV (Chyzik et al., 2003; Chiel et al., 2006; Doukas y Payne, 2007b; Legarrea et al., 2012c). Otro m��todo basado en barreras f��sicas son las mallas impregnadas con insecticidas, que se han usado tradicionalmente en la prevenci��n de enfermedades humanas transmitidas por mosquitos (Martin et al., 2006). Su aplicaci��n se ha ensayado en agricultura en ciertos cultivos al aire libre (Martin et al., 2010; D��az et al., 2004), pero su utilidad en cultivos protegidos para prevenir la entrada de insectos vectores en invernadero todav��a no ha sido investigada. Los aditivos se incorporan al tejido durante el proceso de extrusi��n de la fibra y se liberan lentamente actuando por contacto en el momento en que el insecto aterriza sobre la malla, con lo cual el riesgo medioambiental y para la salud humana es muy limitado. Los plaguicidas que se emplean habitualmente suelen ser piretroides (deltametrina o bifentr��n), aunque tambi��n se ha ensayado dicofol (Martin et al., 2010) y alfa-cipermetrina (Martin et al., 2014). Un factor que resulta de vital importancia en este tipo de mallas es el tama��o del poro para facilitar una buena ventilaci��n del cultivo, al tiempo que se evita la entrada de insectos de peque��o tama��o como las moscas blancas (Bethke y Paine, 1991; Mu��oz et al., 1999). Asimismo, se plantea la necesidad de estudiar la compatibilidad de estas mallas con los enemigos naturales. Es por ello que en esta Tesis Doctoral se plantea la necesidad de evaluar nuevas mallas impregnadas que impidan el paso de insectos de peque��o tama��o al interior de los invernaderos, pero que a su vez mantengan un buen intercambio y circulaci��n de aire a trav��s del poro de la malla. As��, en la presente Tesis Doctoral, se han planteado los siguientes objetivos generales a desarrollar: 1. Estudiar el impacto de la presencia de parasitoides sobre el grado de dispersi��n y los patrones de distribuci��n de pulgones y las virosis que ��stos transmiten. 2. Conocer el efecto directo de ambientes pobres en radiaci��n UV sobre el comportamiento de vuelo de plagas clave de hort��colas y sus enemigos naturales. 3. Evaluar el efecto directo de la radiaci��n UV-A sobre el crecimiento poblacional de pulgones y mosca blanca, y sobre la fisiolog��a de sus plantas hospederas, as�� como el efecto indirecto de la radiaci��n UV-A en ambas plagas mediado por el crecimiento de dichas planta hospederas. 4. Caracterizaci��n de diversas mallas impregnadas en deltametrina y bifentr��n con diferentes propiedades y selecci��n de las ��ptimas para el control de pulgones, mosca blanca y sus virosis asociadas en condiciones de campo. Estudio de su compatibilidad con parasitoides. ABSTRACT Insect vectors of plant viruses are the main agents causing major economic losses in vegetable crops grown under protected environments. This Thesis focuses on the implementation of new alternatives to chemical control of insect vectors under Integrated Pest Management programs. In Spain, biological control is the main pest control strategy used in a large part of greenhouses where horticultural crops are grown. The first study aimed to increase our knowledge on how the presence of natural enemies such as Aphidius colemani Viereck may alter the dispersal of the aphid vector Aphis gossypii Glover (Chapter 4). In addition, it was investigated if the presence of this parasitoid affected the spread of aphid-transmitted viruses Cucumber mosaic virus (CMV, Cucumovirus) and Cucurbit aphid-borne yellows virus (CABYV, Polerovirus) infecting cucumber (Cucumis sativus L). SADIE methodology was used to study the distribution patterns of both the virus and its vector, and their degree of association. Results suggested that parasitoids promoted aphid dispersal in the short term, which enhanced CMV spread, though consequences of parasitism suggested potential benefits for disease control in the long term. Furthermore, A. colemani significantly limited the spread and incidence of the persistent virus CABYV in the long term. The flight activity of pests Myzus persicae (Sulzer), Bemisia tabaci (Gennadius) and Tuta absoluta (Meyrick), and natural enemies A. colemani and Sphaerophoria rueppellii (Weidemann) under UV-deficient environments was studied under field conditions (Chapter 5). One-chamber tunnels were covered with cladding materials with different UV transmittance properties. Inside each tunnel, insects were released from tubes placed in a platform suspended from the ceiling. Specific targets were located at different distances from the platform. The ability of aphids and whiteflies to reach their targets was diminished under UV-absorbing barriers, suggesting a reduction of vector activity under this type of nets. Fewer aphids reached distant traps under UV-absorbing nets, and significantly more aphids could fly to the end of the tunnels covered with non-UV blocking materials. Unlike aphids, differences in B. tabaci captures were mainly found in the closest targets. The oviposition of lepidopteran T. absoluta was also negatively affected by a UV-absorbing cover. The photoselective barriers were compatible with parasitism and oviposition of biocontrol agents. Apart from the direct response of insects to UV radiation, plant-mediated effects influencing insect performance were investigated (Chapter 6). The impact of UV-A radiation on the performance of aphid M. persicae and whitefly B. tabaci, and growth and leaf physiology of host plants pepper and eggplant was studied under glasshouse conditions. Plants were grown inside cages covered by transparent and UV-A-opaque plastic films. Plant growth and insect fitness were monitored. Leaves were harvested for chemical analysis. Pepper plants responded directly to UV-A by producing shorter stems whilst UV-A did not affect the leaf area of either species. UV-A-treated peppers had higher content of secondary metabolites, soluble carbohydrates, free amino acids and proteins. Such changes in tissue chemistry indirectly promoted aphid performance. For eggplants, chlorophyll and carotenoid levels decreased with supplemental UVA but phenolics were not affected. Exposure to supplemental UV-A had a detrimental effect on whitefly development, fecundity and fertility presumably not mediated by plant cues, as compounds implied in pest nutrition were unaltered. Lastly, the efficacy of a wide range of Long Lasting Insecticide Treated Nets (LLITNs) was studied under laboratory and field conditions. This strategy aimed to prevent aphids and whiteflies to enter the greenhouse by determining the optimum mesh size (Chapter 7). This new approach is based on slow release deltamethrin- and bifenthrin-treated nets with large hole sizes that allow improved ventilation of greenhouses. All LLITNs produced high mortality of M. persicae and A. gossypii although their efficacy decreased over time with sun exposure. It was necessary a net with hole size of 0.29 mm2 to exclude B. tabaci under laboratory conditions. The feasibility of two selected nets was studied in the field under a high insect infestation pressure in the presence of CMV- and CABYV-infected cucumber plants. Besides, the compatibility of parasitoid A. colemani with bifenthrin-treated nets was studied in parallel field experiments. Both nets effectively blocked the invasion of aphids and reduced the incidence of both viruses, however they failed to exclude whiteflies. We found that our LLITNs were compatible with parasitoid A. colemani. As shown, the role of natural enemies has to be taken into account regarding the dispersal of insect vectors and subsequent spread of plant viruses. The additional benefits of novel physicochemical barriers, such as photoselective and insecticide-impregnated nets, need to be considered in Integrated Pest Management programs of vegetable crops grown under protected environments.

Efecto de la infecci��n por virus persistentes y no persistentes sobre el comportamiento alimenticio, la preferencia, tasa de aterrizaje y asentamiento y la eficacia biol��gica de Aphis gossypii Glover

En el complejo de plagas que atacan a los principales cultivos hort��colas protegidos, destacan principalmente los Hem��pteros, y dentro de estos los pulgones, dada su importancia como vectores de virus que provocan considerables da��os y p��rdidas econ��micas. Debido a que la dispersi��n de la mayor��a de los virus de plantas puede ser eficaz con densidades bajas de vectores y su control es muy complicado al no existir m��todos curativos para su control, es necesario generar nuevos conocimientos sobre las interacciones virus-vector con el fin de desarrollar nuevas y eficaces estrategias de control. Por ello, el objetivo general de esta Tesis ha sido conocer el efecto de la infecci��n viral (directo-mediado por la presencia del virus en el vector- e indirecto-mediado por las alteraciones f��sico-qu��micas que se originan en la planta como consecuencia de la infecci��n viral-) sobre el comportamiento y eficacia biol��gica del vector Aphis gossypii Glover y sus posibles repercusiones en la epidemiolog��a de virosis de transmisi��n no persistente (Cucumber mosaic virus, CMV, Cucumovirus) y persistente (Cucurbit aphid-borne yellows virus, CABYV, Polerovirus). El primer objetivo de esta Tesis Doctoral, se centr�� en el estudio del efecto indirecto del virus de transmisi��n no persistente CMV sobre el comportamiento alimenticio y la preferencia del pulg��n A. gossypii en el cultivo de pepino. Los ensayos de despegue y aterrizaje mostraron que los pulgones que fueron liberados en las plantas de pepino infectadas con CMV tuvieron una mayor propensi��n en migrar hacia las plantas no infectadas (60, 120 y 180 minutos despu��s de la liberaci��n) que aquellos que fueron sometidos al tratamiento contrario (planta no infectada hacia planta infectada con CMV). El estudio de preferencia y asentamiento mostr�� que el vector A. gossypii prefiere asentarse en plantas infectadas con CMV en una etapa temprana de evaluaci��n (30 minutos despu��s de la liberaci��n). Sin embargo, este comportamiento se revirti�� en una etapa posterior (4 y 48 horas despu��s de la liberaci��n), donde los pulgones se asentaron m��s en las plantas no infectadas. A trav��s de la t��cnica de Gr��ficos de Penetraci��n El��ctrica (EPG) se observ�� un efecto indirecto del virus CMV, revelado por un cambio brusco en el comportamiento de prueba del pulg��n a lo largo del tiempo, cuando ��stos fueron expuestos a las plantas infectadas con CMV. Los primeros 15 minutos de registro EPG mostraron que los pulgones hicieron un n��mero mayor de punciones intracelulares (potencial drops - pds) y pruebas en las plantas infectadas con CMV que en las plantas no infectadas. Por otra parte, la duraci��n de la primera prueba fue m��s corta y la duraci��n total de las pds por insecto fue mucho m��s larga en las plantas infectadas con CMV. Se observaron diferencias significativas en el tiempo transcurrido desde el final de la ��ltima pd hasta el final de la prueba, siendo ese tiempo m��s corto para los pulgones que estaban aliment��ndose en plantas infectadas con CMV. En la segunda hora de registro los pulgones rechazaron las plantas infectadas con CMV como fuente de alimento, permaneciendo menos tiempo en las fases de prueba en floema (fase de salivaci��n ��� E1 y fase de ingesti��n del floema ��� E2). El comportamiento alimenticio observado sobre las plantas infectadas con CMV favorece la adquisici��n y posterior transmisi��n de los virus de transmisi��n no persistente, los cuales son adquiridos e inoculados durante la realizaci��n de pruebas intracelulares en las primeras pruebas de corta duraci��n. En el segundo objetivo de la Tesis se evalu�� el efecto directo e indirecto del virus de transmisi��n persistente CABYV en el comportamiento alimenticio y preferencia del pulg��n A. gossypii en cultivo de pepino, especie susceptible al virus, y algod��n, especie inmune al virus. No se observ�� un efecto directo del virus relevante en el comportamiento alimenticio del vector, ya que los resultados obtenidos a nivel floem��tico en plantas de pepino no se observaron en plantas de algod��n, inmune al virus CABYV. Esto sugiere que los resultados obtenidos en pepino, pueden deberse a un ���posible efecto indirecto��� originado por la infecci��n de las plantas susceptibles al virus durante la realizaci��n del ensayo, lo que indirectamente puede modificar el comportamiento del pulg��n durante la fase de evaluaci��n. Sin embargo, el virus CABYV modific�� indirectamente el comportamiento alimenticio de su vector a trav��s de cambios en la planta infectada. Los pulgones tardaron menos tiempo en llegar al floema, realizaron un mayor n��mero de pruebas floem��ticas y permanecieron durante m��s tiempo en actividades floem��ticas en plantas infectadas con CABYV. El comportamiento observado sobre las plantas infectadas con CABYV favorece la adquisici��n de virus persistentes, los cuales son adquiridos durante la alimentaci��n sostenida en floema. El estudio de preferencia y asentamiento de A. gossypii mostr�� que los pulgones virul��feros prefieren asentarse en plantas no infectadas a corto y largo plazo de evaluaci��n (2, 4 y 48 horas despu��s de la liberaci��n). Los ensayos de despegue y aterrizaje mostraron que los pulgones virul��feros que fueron liberados en las plantas de pepino infectadas con CABYV tuvieron una mayor propensi��n en migrar hacia las plantas no infectadas (3, 6, 24 y 48 horas despu��s de la liberaci��n) que aquellos que fueron sometidos al tratamiento contrario (planta no infectada hacia planta infectada con CABYV). Sin embargo, los pulgones no virul��feros no mostraron preferencia por plantas de pepino no infectadas o infectadas con CABYV en ninguno de los ensayos (preferencia o despegue) o periodos evaluados (corto y largo plazo). Los resultados indican que el virus CABYV es capaz de modificar indirectamente el comportamiento alimenticio de su vector a trav��s de cambios en la planta infectada, favoreciendo su adquisici��n por su principal vector, A. gossypii. Una vez que los pulgones tienen capacidad de transmitir el virus (virul��feros) se produce un cambio en su comportamiento prefiriendo asentarse sobre plantas no infectadas optimiz��ndose as�� la dispersi��n viral. El tercer objetivo de la Tesis, fue evaluar los efectos directos e indirectos del virus CABYV as�� como los efectos indirectos del virus CMV en la eficacia biol��gica del vector A. gossypii. Los resultados obtenidos en los ensayos realizados con el virus persistente CABYV indican que el virus parece no modificar directamente ni indirectamente la eficacia biol��gica del vector en plantas de pepino o algod��n, no observ��ndose diferencias estad��sticas en ninguno de los par��metros poblacionales evaluados (tiempo de desarrollo, tasa intr��nseca de crecimiento, tiempo generacional medio, tasa media de crecimiento relativo y ninfas totales). En cuanto a los ensayos realizados con el virus no persistente, CMV, los resultados muestran un efecto indirecto del virus sobre la biolog��a del vector. As�� result�� que tanto la tasa intr��nseca de crecimiento natural (rm) como la tasa media de crecimiento relativo (RGR) fueron m��s altas para pulgones crecidos sobre plantas infectadas con CMV que sobre plantas no infectadas, favoreciendo la reproducci��n y crecimiento poblacional del vector sobre plantas infectadas con CMV. Los resultados obtenidos en la presente Tesis, ofrecen un ejemplo de como los virus de plantas pueden manipular directa e indirectamente a su vector, maximizando as�� su dispersi��n entre las plantas. Esos nuevos conocimientos generados tienen implicaciones importantes en la transmisi��n, dispersi��n y en la epidemiolog��a de los virus y deben ser considerados para dise��ar o ajustar los modelos de simulaci��n existentes y patrones de dispersi��n que describen las epidemias de estos virus. ABSTRACT The main objective of this Thesis has been to understand the effect of the viral infection (direct-mediated by the presence of the virus in the vector and indirect mediated by the chemical and physical changes originated in the plant as a consequence of the viral infection) on the behaviour and biological efficacy of the vector Aphis gossypii Glover and its consequences in the epidemiology of two viral diseases, one with non-persistent transmission (Cucumber mosaic virus, CMV, Cucumovirus) and another with persistent transmission (Cucurbit aphid-borne yellows virus, CABYV, Polerovirus). The first objective of this Thesis was the study of the indirect effect of the nonpersistent virus CMV on the feeding behaviour and preference of the aphid A. gossypii in cucumber plants. The results of the alighting and settling behaviour studies showed that aphids exhibited no preference to migrate from CMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), but showed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min after release. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected over mockinoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage and aphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph (EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of short superficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (second hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much less time spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour including an early increase in the number of short superficial probes and intracellular punctures followed by a phloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in a NP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting, settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spread of the virus. The second objective of this work was to evaluate the effects that the persistently aphid transmitted Cucurbit aphid-borne yellows virus (CABYV) can induce directly and indirectly on the alighting, settling and probing behaviour activities of the cotton aphid A. gossypii. Only minor direct changes on aphid feeding behaviour was observed due to CABYV when viruliferous aphids fed on mock-inoculated plants. However, the feeding behaviour of non-viruliferous aphids was very different on CABYV-infected than on mockinoculated plants. Non-viruliferous aphids spent longer time feeding from the phloem when plants were infected by CABYV than on mock-inoculated plants, suggesting that CABYV indirectly manipulates aphid feeding behaviour through its shared host plant in order to favour viral acquisition. The vector alighting and settling preference was compared between nonviruliferous and viruliferous aphids. Viruliferous aphids showed a clear preference for mockinoculated over CABYV-infected plants at short and long time, while such behaviour was not observed for non-viruliferous aphids. Overall, our results indicate that CABYV induces changes in its host plant that modifies aphid feeding behaviour in a way that virus acquisition from infected plants is enhanced. Once the aphids become viruliferous they prefer to settle on healthy plants, leading to optimize the transmission and spread of the virus. The third objective was to evaluate the direct and indirect effects of CABYV and indirect effects of the CMV on the A. gossypii fitness. Obtained results for the persistent virus CABYV showed that the virus did not modify the vector fitness in cucumber or cotton plants. None of the evaluated variables was statistically significant (development time (d), intrinsic growth rate (rm), mean relative growth rate (RGR) and total number of nymphs). On the other hand, data obtained for the non-persistent virus (CMV) showed an indirect effect of the virus on the vector fitness. Thus, the rm and RGR were higher for aphids grown on CMV-infected plants compared to aphids grown on mock-inoculated plants. Overall, the obtained results are clear examples of how plant viruses could manipulate directly and indirectly vector behaviour to optimize its own dispersion. These results are important for a better understanding of transmission, dispersion and epidemiology of plant viruses transmitted by vectors. This information could be also considered to design or adjust simulation models and dispersion patterns that describe plant virus epidemics.

Optimization of Dengue immunoassay by label-free interferometric optical detection method

In this communication we report a direct immunoassay for detecting dengue virus by means of a label-free interferometric optical detection method. We also demonstrate how we can optimize this sensing response by adding a blocking step able to significantly enhance the optical sensing response. The blocking reagent used for this optimization is a dry milk diluted in phosphate buffered saline. The recognition curve of dengue virus over the proposed surface sensor demonstrates the capacity of this method to be applied in Point of Care technology.

Spatio-temporal dynamics of viruses are differentially affected by parasitoids depending on the mode of transmission by their insect vectors.

Relationships between agents in multitrophic systems are complex and very specific. Insect-transmitted plant viruses are completely dependent on the behaviour and distribution patterns of their vectors. The presence of natural enemies may directly affect aphid behaviour and spread of plant viruses, as the escape response of aphids might cause a potential risk for virus dispersal. The spatio-temporal dynamics of Cucumber mosaic virus (CMV) and Cucurbit aphid-borne yellows virus (CABYV), transmitted by Aphis gossypii in a non-persistent and persistent manner, respectively, were evaluated at short and long term in the presence and absence of the aphid parasitoid, Aphidius colemani. SADIE methodology was used to study the distribution patterns of both the virus and its vector, and their degree of association. Results suggested that parasitoids promoted aphid dispersion at short term, which enhanced CMV spread, though consequences of parasitism suggest potential benefits for disease control at long term. Furthermore, A. colemani significantly limited the spread and incidence of the persistent virus CABYV at long term. The impact of aphid parasitoids on the dispersal of plant viruses with different transmission modes is discussed.

A non-persistently transmitted-virus induces a pull?push strategy inits aphid vector to optimize transmission and spread

Plant viruses are known to modify the behaviour of their insect vectors, both directly and indirectly,generally adapting to each type of virus?vector relationship in a way that enhances transmissionefficiency. Here, we report results of three different studies showing how a virus transmitted in a non-persistent (NP) manner (Cucumber mosaic virus; CMV, Cucumovirus) can induce changes in its host plant,cucumber (Cucumis sativus cv. Marumba) that modifies the behaviour of its aphid vector (Aphis gossypiiGlover; Hemiptera: Aphididae) in a way that enhances virus transmission and spread non-viruliferousaphids changed their alighting, settling and probing behaviour activities over time when exposed toCMV-infected and mock-inoculated cucumber plants. Aphids exhibited no preference to migrate fromCMV-infected to mock-inoculated plants at short time intervals (1, 10 and 30 min after release), butshowed a clear shift in preference to migrate from CMV-infected to mock-inoculated plants 60 min afterrelease. Our free-choice preference assays showed that A. gossypii alates preferred CMV-infected overmock-inoculated plants at an early stage (30 min), but this behaviour was reverted at a later stage andaphids preferred to settle and reproduce on mock-inoculated plants. The electrical penetration graph(EPG) technique revealed a sharp change in aphid probing behaviour over time when exposed to CMV-infected plants. At the beginning (first 15 min) aphid vectors dramatically increased the number of shortsuperficial probes and intracellular punctures when exposed to CMV-infected plants. At a later stage (sec-ond hour of recording) aphids diminished their feeding on CMV-infected plants as indicated by much lesstime spent in phloem salivation and ingestion (E1 and E2). This particular probing behaviour includingan early increase in the number of short superficial probes and intracellular punctures followed by aphloem feeding deterrence is known to enhance the transmission efficiency of viruses transmitted in aNP manner. We conclude that CMV induces specific changes in a plant host that modify the alighting,settling and probing behaviour of its main vector A. gossypii, leading to optimum transmission and spreadof the virus. Our findings should be considered when modelling the spread of viruses transmitted in a NPmanner.

Coevoluci��n virus-planta: impacto de las infecciones virales en poblaciones silvestres de Chiltep��n (capsicum annuum var. Aviculare (dierbach) d���arcy & eshbaugh) en M��xico.

El impacto negativo que tienen los virus en las plantas hace que estos puedan ejercer un papel ecol��gico como moduladores de la din��mica espacio-temporal de las poblaciones de sus hu��spedes. Entender cu��les son los mecanismos gen��ticos y los factores ambientales que determinan tanto la epidemiolog��a como la estructura gen��tica de las poblaciones de virus puede resultar de gran ayuda para la comprensi��n del papel ecol��gico de las infecciones virales. Sin embargo, existen pocos trabajos experimentales que hayan abordado esta cuesti��n. En esta tesis, se analiza el efecto de la heterogeneidad del paisaje sobre la incidencia de los virus y la estructura gen��tica de sus poblaciones. Asimismo, se explora como dichos factores ambientales influyen en la importancia relativa que los principales mecanismos de generaci��n de variabilidad gen��tica (mutaci��n, recombinaci��n y migraci��n) tienen en la evoluci��n de los virus. Para ello se ha usado como sistema los begomovirus que infectan poblaciones de chiltep��n (Capsicum annuum var. aviculare (Dierbach) D��Arcy & Eshbaugh) en M��xico. Se analiz�� la incidencia de diferentes virus en poblaciones de chiltep��n distribuidas a lo largo de seis provincias biogeogr��ficas, representando el ��rea de distribuci��n de la especie en M��xico, y localizadas en h��bitats con diferente grado de intervenci��n humana: poblaciones sin intervenci��n humana (silvestres); poblaciones toleradas (lindes y pastizales), y poblaciones manejadas por el hombre (monocultivos y huertos familiares). Entre los virus analizados, los begomovirus mostraron la mayor incidencia, detect��ndose en todas las poblaciones y a��os de muestreo. Las ��nicas dos especies de begomovirus que se encontraron infectando al chiltep��n fueron: el virus del mosaico dorado del chile (Pepper golden mosaic virus, PepGMV) y el virus huasteco del amarilleo de venas del chile (Pepper huasteco yellow vein virus, PHYVV). Por ello, todos los an��lisis realizados en esta tesis se centran en estas dos especies de virus. La incidencia de PepGMV y PHYVV, tanto en infecciones simples como mixtas, aumento cuanto mayor fue el nivel de intervenci��n humana en las poblaciones de chiltep��n, lo que a su vez se asoci�� con una menor biodiversidad y una mayor densidad de plantas. Adem��s, la incidencia de infecciones mixtas, altamente relacionada con la presencia de s��ntomas, fue tambi��n mayor en las poblaciones cultivadas. La incidencia de estos dos virus tambi��n vari�� en funci��n de la poblaci��n de chiltep��n y de la provincia biogeogr��fica. Por tanto, estos resultados apoyan una de las hip��tesis XVI cl��sicas de la Patolog��a Vegetal seg��n la cual la simplificaci��n de los ecosistemas naturales debida a la intervenci��n humana conduce a un mayor riesgo de enfermedad de las plantas, e ilustran sobre la importancia de la heterogeneidad del paisaje a diferentes escalas en la determinaci��n de patrones epidemiol��gicos. La heterogeneidad del paisaje no solo afect�� a la epidemiolog��a de PepGMV y PHYVV, sino tambi��n a la estructura gen��tica de sus poblaciones. En ambos virus, el nivel de diferenciaci��n gen��tica mayor fue la poblaci��n, probablemente asociado a la capacidad de migraci��n de su vector Bemisia tabaci; y en segundo lugar la provincia biogeogr��fica, lo que podr��a estar relacionado con el papel del ser humano como agente dispersor de PepGMV y PHYVV. La estima de las tasas de sustituci��n nucleot��dica de las poblaciones de PepGMV y PHYVV mostr�� una r��pida din��mica evolutiva. Los ��rboles filogen��ticos de ambos virus presentaron una topolog��a en estrella, lo que sugiere una expansi��n reciente en las poblaciones de chiltep��n. La reconstrucci��n de los patrones de migraci��n de ambos virus indic�� que ��sta expansi��n parece haberse producido desde la zona central de M��xico siguiendo un patr��n radial, y en los ��ltimos 30 a��os. Es importante tener en cuenta que el patr��n espacial de la diversidad gen��tica de las poblaciones de PepGMV y PHYVV es similar al descrito previamente para el chiltep��n lo que podr��a dar lugar a la congruencia de las genealog��as del hu��sped y la de los virus. Dicha congruencia se encontr�� cuando se tuvieron en cuenta ��nicamente las poblaciones de h��bitats silvestres y tolerados, lo que probablemente se debe a una codivergencia en el espacio pero no en el tiempo, dado que la evoluci��n de virus y hu��sped han ocurrido a escalas temporales muy diferentes. Finalmente, el an��lisis de la frecuencia de recombinaci��n en PepGMV y PHYVV indic�� que esta juega un papel importante en la evoluci��n de ambos virus, dependiendo su importancia del nivel de intervenci��n humana de la poblaci��n de chiltep��n. Este factor afect�� tambi��n a la intensidad de la selecci��n a la que se ven sometidos los genomas de PepGMV y PHYVV. Los resultados de esta tesis ponen de manifiesto la importancia que la reducci��n de la biodiversidad asociada al nivel de intervenci��n humana de las poblaciones de plantas y la heterogeneidad del paisaje tiene en la emergencia de nuevas enfermedades virales. Por tanto, es necesario considerar estos factores ambientales a la hora de comprender la epidemiologia y la evoluci��n de los virus de plantas.XVII SUMMARY Plant viruses play a key role as modulators of the spatio-temporal dynamics of their host populations, due to their negative impact in plant fitness. Knowledge on the genetic and environmental factors that determine the epidemiology and the genetic structure of virus populations may help to understand the ecological role of viral infections. However, few experimental works have addressed this issue. This thesis analyses the effect of landscape heterogeneity in the prevalence of viruses and the genetic structure of their populations. Also, how these environmental factors influence the relative importance of the main mechanisms for generating genetic variability (mutation, recombination and migration) during virus evolution is explored. To do so, the begomoviruses infecting chiltepin (Capsicum annuum var. aviculare (Dierbach) D'Arcy & Eshbaugh) populations in Mexico were used. Incidence of different viruses in chiltepin populations of six biogeographical provinces representing the species distribution in Mexico was determined. Populations belonged to different habitats according to the level of human management: populations with no human intervention (Wild); populations naturally dispersed and tolerated in managed habitats (let-standing), and human managed populations (cultivated). Among the analyzed viruses, the begomoviruses showed the highest prevalence, being detected in all populations and sampling years. Only two begomovirus species infected chiltepin: Pepper golden mosaic virus, PepGMV and Pepper huasteco yellow vein virus, PHYVV. Therefore, all the analyses presented in this thesis are focused in these two viruses. The prevalence of PepGMV and PHYVV, in single and mixed infections, increased with higher levels of human management of the host population, which was associated with decreased biodiversity and increased plant density. Furthermore, cultivated populations showed higher prevalence of mixed infections and symptomatic plants. The prevalence of the two viruses also varied depending on the chiltepin population and on the biogeographical province. Therefore, these results support a classical hypothesis of Plant Pathology stating that simplification of natural ecosystems due to human management leads to an increased disease risk, and illustrate on the importance of landscape heterogeneity in determining epidemiological patterns. Landscape heterogeneity not only affected the epidemiology of PepGMV and PHYVV, but also the genetic structure of their populations. Both viruses had the highest level of genetic differentiation at the population scale, probably associated with the XVIII migration patterns of its vector Bemisia tabaci, and a second level at the biogeographical province scale, which could be related to the role of humans as dispersal agents of PepGMV and PHYVV. The estimates of nucleotide substitution rates of the virus populations indicated rapid evolutionary dynamics. Accordingly, phylogenetic trees of both viruses showed a star topology, suggesting a recent diversification in the chiltepin populations. Reconstruction of PepGMV and PHYVV migration patterns indicated that they expanded from central Mexico following a radial pattern during the last 30 years. Importantly, the spatial genetic structures of the virus populations were similar to that described previously for the chiltepin, which may result in the congruence of the host and virus genealogies. Such congruence was found only in wild and let-standing populations. This is probably due to a co-divergence in space but not in time, given the different evolutionary time scales of the host and virus populations. Finally, the frequency of recombination detected in the PepGMV and PHYVV populations indicated that this mechanism plays an important role in the evolution of both viruses at the intra-specific scale. The level of human management had a minor effect on the frequency of recombination, but influenced the strength of negative selective pressures in the viral genomes. The results of this thesis highlight the importance of decreased biodiversity in plant populations associated with the level of human management and of landscape heterogeneity on the emergence of new viral diseases. Therefore it is necessary to consider these environmental factors in order to fully understand the epidemiology and evolution of plant viruses.

Supervised classification of minimally invasive surgery psychomotor competence based on instruments motion analysis

Background Objective assessment of psychomotor skills has become an important challenge in the training of minimally invasive surgical (MIS) techniques. Currently, no gold standard defining surgical competence exists for classifying residents according to their surgical skills. Supervised classification has been proposed as a means for objectively establishing competence thresholds in psychomotor skills evaluation. This report presents a study comparing three classification methods for establishing their validity in a set of tasks for basic skills��� assessment. Methods Linear discriminant analysis (LDA), support vector machines (SVM), and adaptive neuro-fuzzy inference systems (ANFIS) were used. A total of 42 participants, divided into an experienced group (4 expert surgeons and 14 residents with >10 laparoscopic surgeries performed) and a nonexperienced group (16 students and 8 residents with <10 laparoscopic surgeries performed), performed three box trainer tasks validated for assessment of MIS psychomotor skills. Instrument movements were captured using the TrEndo tracking system, and nine motion analysis parameters (MAPs) were analyzed. The performance of the classifiers was measured by leave-one-out cross-validation using the scores obtained by the participants. Results The mean accuracy performances of the classifiers were 71 % (LDA), 78.2 % (SVM), and 71.7 % (ANFIS). No statistically significant differences in the performance were identified between the classifiers. Conclusions The three proposed classifiers showed good performance in the discrimination of skills, especially when information from all MAPs and tasks combined were considered. A correlation between the surgeons��� previous experience and their execution of the tasks could be ascertained from results. However, misclassifications across all the classifiers could imply the existence of other factors influencing psychomotor competence.