Ecotoxicology of Pesticides on Natural Enemies of Olive Groves. Potential of Ecdysone Agonists for Controlling Bactrocera Oleae (Rossi) (Diptera: Tephritidae)

UNIVERSIDAD POLITÉCNICA DE MADRID

Escuela Técnica Superior de Ingenieros Agrónomos

Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae)

TESIS DOCTORAL

Paloma Bengochea Budia

Ingeniera Agrónoma

2012 DEPARTAMENTO DE PRODUCCIÓN VEGETAL: BOTÁNICA Y PROTECCIÓN VEGETAL

Escuela Técnica Superior de Ingenieros Agrónomos

Ecotoxicology of pesticides on natural enemies of olive groves. Potential of ecdysone agonists for controlling Bactrocera oleae (Rossi) (Diptera: Tephritidae)

TESIS DOCTORAL

Paloma Bengochea Budia

Ingeniera Agrónoma

Directora: Mª del Pilar Medina Vélez

Dra. Ingeniera Agrónoma

Madrid, 2012 Tribunal nombrado por e Magfco. Y Excmo. Sr rector de la Universidad Politécnica de Madrid, el día de de 2012.

Presidente D.

Vocal D.

Secretario D.

Suplente D.

Realizada la lectura y defensa de la Tesis el día de de 2012 en Madrid, en la Escuela Técnica Superior de Ingenieros Agrónomos.

Calificación:

El Presidente Los Vocales

El Secretario

A mis padres, a mi hermano y a mis abuelas Gracias….

A todos aquellos que me habéis apoyado y/o ayudado durante antes y durante la elaboración de esta Tesis…espero no olvidarme de ninguno…

A la Universidad Politécnica de Madrid, porque sin la beca que me concedieron no hubiese realizado esta Tesis, y sobre todo…

A todo el personal de la Unidad de Protección de Cultivos, sin cuyo apoyo y ayuda no hubiese podido realizar este trabajo ni sobrevivir a todos los “problemillas” surgidos durante estos años. Por todas esas comidas terapéuticas en las que arreglamos el mundo, nos reímos y nos desahogamos. Por vuestra amistad. Gracias…

A mi tutora, Pilar Medina, por su apoyo incondicional y su ánimo en todos estos años. Por estar siempre dispuesta a ayudarme y aclarar todas mis dudas existenciales. Por animarme a escribir esta Tesis en inglés, que ha sido un reto. Por dejarme hacer millones de cursos y llevarme a tantos Congresos. Por escucharme cuando lo he necesitado.

A Flor, Ángeles, Elisa y Pedro, por transmitirnos a todos vuestro entusiasmo por la entomología, vuestras sugerencias y aportaciones.

A Luis, por tener siempre a punto a mis “pobres” Psyttalias para los ensayos.

A mis compañeros de laboratorio: los siguieron otros caminos: Sara, Guille, Edu, Raquel, Cherre (tu consejo para las larvas de la Ceratitis no tiene precio) y con los que sigo codo con codo compartiendo horas de laboratorio y se han convertido en buenos amigos: al incondicional equipo desayuno con los que empezar el día es otra cosa, y a los que se unen de vez en cuando: Mar, Yara, Nacho, Andrea, Jader, Agus y Rosa. A Fermín, que además es un gran apoyo y un grandísimo amigo; porque siempre está dispuesto a echarme una mano y a escucharme; por todos los momentos en que en el laboratorio nos hemos reído a más no poder (la fuga de las chrysopas, el pobre piticli…)

A Román Zurita, el “guardián de los campos”, por resolver todos los problemas tecnológicos que nos surgen y por habernos ayudado a transformar la enfermería del águila en el invernadero en que mis olivos se han refugiado durante estos años.

A Ángela Alonso y Ezequiel Cabrera, que me ayudaron a identificar los hongos que aparecía en la dieta de las pobres Bactroceras.

Al equipo INIA, especialmente a Manuel por resolver las dudas que me surgían sobre el olivar y a Ismael, por ayudarme con todas mis dudas estadísticas.

A Jose Luis Porcuna y Mamen Alaurín, por permitirme visitar en insectario de Silla y darme las primeras calabazas para comenzar mi cría de Aspidiotus.

A Manuel Ruiz-Torres y Bárbara Castellanos, que me mandaron aceitunas desde Jaén y Cáceres para poder hacer mis ensayos. A Andrew Jessup, de la sede de la FAO/IAEA de Seibersdorf, por dejarme visitar las instalaciones para aprender cómo criar la mosca y enviarnos material siempre que necesitábamos… y aun así la mosca se resistió…

A Carmen Calleja por resolver las dudas moleculares.

A Ian, Mª José, Olivier, Pieter y a todos aquellos que han contribuido a que esta Tesis tenga unas pocas menos de faltas de ortografía…

A Josep Jacas y Alberto Urbaneja por aceptarme dos meses en el IVIA. A todas las personas que conocí allí y que me acogieron como una más: Laura, Francesc, Sara, Alejandro, Óscar, Óscar, Pablo, Joel, Elena, María, Paco, Miquel, Consuelo… (mil perdones si me olvido de alguien). A Tati, que me introdujo en el mundo de los ácaros y además fue un gran apoyo el tiempo que estuve allí. A Pili, Poli, César y Helga que me adoptaron para ir a San Sebastián. Moltes gracies a tots!

A Guy Smagghe por acogerme por dos veces en la Universidad de Gante y permitirme introducirme en el “mundo molecular”. A todos aquellos que me ayudaron en el laboratorio el primer año, el segundo o los dos y que con paciencia me traducían las conversaciones en dutch: Marteen, Rick, Didier, Patrick, Luck, Peter, Dorin, Ivan, Hanneke, Jisheng, Ruben, Moises, Astrid, Sara… (y alguno más de los que espero me perdonen pero se me ha olvidado el nombre…). A Jochem, el “spanishsitter”, que además intentó ayudarme a distinguir los Chilocorus de las Chilocoras (aunque no tuviésemos mucho éxito), a Pieter, por los buenos momentos, y sobre todo a Olivier: sin tu ayuda (y vigilancia) quién sabe qué habría salido de las PCR, que al llegar a Gante me parecían máquinas dificilísimas de entender. Gracias por enseñarnos Flandes y compartir tantos ratos buenos con nosotros. Dankjewel!

A Marta y de nuevo a Fermín, el resto del “spanish team”, porque sin vosotros las estancias en Gante hubiesen sido muy diferentes y probablemente menos divertidas. Por haberme hecho poner cada mañana al mal tiempo buena cara (en el sentido más literal de la frase).

A todos los jolgorianos, con quienes paso tan buenos momentos sea donde sea

A Eva, Marta, Juli, Jesús, Eva, Isa e Irene, por una amistad que viene de lejos.

A mis frijos, Cris y Ángeles, porque son un apoyo incondicional pese a la distancia.

A Ana, Bea y Marta, que me animaron tanto a seguir con esta beca. A Yuse, Sergio, Bea, Eva, Sergio y demás agrónomos.

A Sara, Susi, Berta y Gema, mis compis agronómicas desde el primer día de carrera.

A Miguel, Cris, Alberto, Elena, Inés, Irene, Yoli, Mariano, Javi y demás ruteros con los que siempre parece que he quedado ayer aunque pueda haber pasado un año. A Ceci, Freya, Vero y Vera, las amigas al otro lado del charco.

A mis padres y mi hermano. Por haberme animado a realizar el doctorado cuando yo no tenía las cosas muy claras, pero sobre todo por su gran apoyo diario y su cariño. A mis abuelas, que son todo un ejemplo, y al resto de mi familia.

La paciencia es la madre de la ciencia… Index

Index

INDEX i

RESUMEN vii

SUMMARY ix

1. INTRODUCTION 1 1.1 The olive tree 1 1.1.2 The origin of the crop 2 1.1.3 Geographical distribution 3 1.1.4 Importance of the crop 3 1.1.4.1 Economic importance 4 1.1.4.2 Social importance 4 1.1.4.3 Environmental importance 5 1.1.5 Olive growing in Spain 5 1.1.6 Pests and diseases: characteristics of the most important pests and diseases of olive groves 6 1.1.6.1 The olive fruit fly (Bactrocera oleae) 10 1.1.6.2 The olive moth (Prays oleae) 14 1.1.6.3 The black scale (Saissetia oleae) 15 1.1.6.4 The olive leaf spot (Spilocaea oleagina) 16 1.2 Control of pests and diseases 17 1.2.1 Integrated Pest Management 18 1.2.2 Organic farming 21 1.2.3 Integrated Protection in olive groves 23 1.2.4 Organic olive farming 28 1.3 Side-effects of pesticides on non-target organisms 28 1.4 Natural enemies used in the experiments 30 1.4.1 Psyttalia concolor 30 1.4.2 Chilocorus nigritus 35

Index

2. OBJECTIVES 41

3. GENERAL MATERIALS AND METHODS 43

3.1 Environmental conditions of insect rearing and laboratory experiments 43 3.2 Insect rearing 44 3.2.1 Psyttalia concolor 44 3.2.1.1 Mass-rearing of Ceratitis capitata 45 3.2.1.1.1 Adults’ cage 45 3.2.1.1.2 Eggs handling 45 3.2.1.1.3 Larvae rearing 46 3.2.1.2 Mass-rearing of Psyttalia concolor 47 3.2.1.2.1 Parasitization 47 3.2.2 Chilocorus nigritus 48 3.2.2.1 Mass-rearing of scales 49 3.2.3 Bactrocera oleae 51 3.3 Common characteristics of the experiments 52 3.4 Parameters evaluated 54 3.4.1 Mortality 54 3.4.2 Life span 54 3.4.3 Effects on reproductive parameters 54 3.5 Statistical analysis 56

Index

4. LETHAL AND SUBLETHAL EFFECTS OF KAOLIN PARTICLE FILMS AND COPPER-BASED COMPOUNDS ON THE NATURAL ENEMIES PSYTTALIA CONCOLOR AND CHILOCORUS NIGRITUS 59

4.1 Introduction and objectives 59 4.2 Material and methods 60 4.2.1 Chemicals 60 4.2.1.1 Kaolin 62 4.2.1.2 Copper 64 4.2.2 Laboratory tests 65 4.2.2.1 Residual contact on glass surfaces 65 4.2.2.2 Oral toxicity 67 4.2.2.3 Treatment of parasitized pupae 68 4.2.2.4 Treatment of the parasitization surface 69 4.2.3 Extended-laboratory experiments 71 4.2.3.1 Treatment of olive tree leaves 71 4.2.3.2 Treatment of the parasitization surface and olive tree leaves 72 4.2.4 Semi-field experiment 73 4.2.5 Dual choice and no-choice experiments 75 4.2.5.1 Psyttalia concolor 75 4.2.5.2 Chilocorus nigritus 76 4.3 Results 79 4.3.1 Direct mortality 79 4.3.2 Life span 80 4.3.3 Emergence 84 4.3.4 Beneficial capacity 85 4.3.5 Dual choice and no choice experiments 88 4.3.5.1 Psyttalia concolor 88 4.3.5.2 Chilocorus nigritus 92

iii

Index

4.4 Discussion 95 4.4.1 Lethal and sublethal effects of kaolin, Bordeaux mixture and copper oxychloride 96 4.4.2 Effects of kaolin treated surfaces in dual choice and no- choice experiments 102 4.5 Appendix (tables of results) 104

5. ECDYSONE AGONISTS: EFFICACY AND ECOTOXICOLOGY ON BACTROCERA OLEAE AND PSYTTALIA CONCOLOR. INSECT TOXICITY BIOASSAYS AND MOLECULAR DOCKING APPROACHES 111

5.1 Introduction 111 5.1.1 The ecdysone receptor 112 5.2 Objectives and procedures 113 5.3 Materials and methods 114 5.3.1 Insect bioassays 114 5.3.2 EcR-LBD sequence 116 5.3.3 Confirmation of expression of EcR in the ovaries 122 5.3.4 Modeling of PcEcR-LBD and docking studies 123 5.4 Results 124 5.4.1 Efficacy and toxicological effects of methoxyfenozide, tebufenozide and RH-5849 124 5.4.2 BoEcR-LBD sequence 127 5.4.3 PcEcR-LBD sequence, phylogenetic tree and expression in the ovaries 130 5.4.4 Modeling of BoEcR-LBD and PCEcR-LBD and docking studies 135 5.5 Discussion 142 5.5.1 Effciacy and toxicology of insect gorwth regulators on Bactrocera oleae and Psyttalia concolor, respectively 143 5.5.2 Molecular docking studies 148 5.6 Appendix (tables of results) 151

6. CONCLUSIONS 155

Index

7. REFERENCES 157

APPENDIX 183 Index of figures 183 Index of tables 189 Acronyms 191

Index

Resumen /Summary

RESUMEN

Los tratamientos fitosanitarios en el olivar siguen siendo hoy en día uno de los métodos de control más empleados en la lucha contra las principales plagas y enfermedades que afectan a este cultivo: la mosca del olivo, Bactrocera oleae (Rossi), el prays, Prays oleae (Bernard), la cochinilla del olivo, Saissetia oleae (Olivier), y el repilo, provocado por el hongo Spilocaea oleagina Fries. Sin embargo, y como la nueva legislación en materia fitosanitaria se dirige hacia una gestión integrada de las plagas y enfermedades, continúa siendo importante evaluar y conocer los efectos que los pesticidas tienen sobre los enemigos naturales presentes en los diferentes agrosistemas.

Una parte de este trabajo ha consistido en el estudio de los efectos directos e indirectos del caolín y dos formulados a base de cobre (caldo bordelés y oxicloruro de cobre), mediante diferentes ensayos de laboratorio, laboratorio extendido y semicampo en los enemigos naturales Psyttalia concolor (Szèpligeti)., parasitoide de la mosca del olivo, y Chilocorus nigritus (F.), depredador de diaspídidos. Este depredador se ha utilizado en lugar de C. bipustulatus (L.), que es la especie que se encuentra en los olivares. El caolín actúa fundamentalmente como repelente de los insectos y/o disuade la oviposición. En el olivar se emplea para el control de la mosca y el prays. El cobre se emplea en el control de enfermedades fúngicas y bacterianas, como el repilo y otras enfermedades del olivar. En ninguno de los ensayos realizados se encontraron diferencias estadísticamente significativas con respecto a los controles, excepto cuando se evaluó la toxicidad oral de los productos en las hembras de P. concolor. En este caso, el caolín y el oxicloruro de cobre causaron una mortalidad mayor de las hembras a las 72 horas del tratamiento, y tanto el caolín como las dos formulaciones de cobre redujeron la supervivencia. Los parámetros reproductivos sólo se vieron vii

Resumen /Summary

afectados negativamente por la ingesta de caolín. Además de los ensayos anteriores, en el caso del caolín, por su particular modo de acción, se plantearon un ensayo de elección y otro de no elección. Tanto las hembras de P. concolor como los adultos de C. nigritus mostraron una clara preferencia por las superficies no tratadas con el producto cuando se les ofrecía la posibilidad de elegir entre una superficie tratada y otra sin tratar. Cuando esa posibilidad no existía, no se detectaron diferencias estadísticamente significativas entre los tratamientos y los controles.

Además se ha comprobado también la eficacia y la selectividad de tres insecticidas reguladores del crecimiento (metoxifenocida, tebufenocida y RH-5849) sobre B. oleae y P. concolor, respectivamente. Además de estudios para evaluar la toxicidad en laboratorio de los insecticidas, se extrajo RNA de los insectos y con el cDNA obtenido se secuenció y clonó el dominio de unión a ligando (LBD) del receptor de ecdisona de ambos insectos. Posteriormente, se obtuvo la configuración en tres dimensiones del LBD de ambas proteínas y se estudió el acoplamiento de las moléculas de los tres insecticidas en la cavidad que forman las 12 α-hélices que constituyen el LBD de cada una de las proteínas. Tanto los ensayos de toxicidad como las técnicas moleculares han demostrado que metoxifenocida y tebufenocida no tienen ningún efecto nocivo ni en B. oleae ni en P. concolor. RH-5849, sin embargo, resultó inocuo para el parasitoide pero redujo notablemente la supervivencia de los adultos de la mosca, especialmente cuando entraron en contacto con el residuo fresco. El estudio del acoplamiento de la molécula de este insecticida ha mostrado que podría más o menos encajar en la cavidad que forman las hélices del LBD de la proteína de B. oleae, por lo que la búsqueda de nuevos insecticidas para el control de la mosca del olivo podría realizarse tomando como modelo la molécula de RH-5849.

viii

Resumen /Summary

SUMMARY

Pesticide applications are still one of the most common control methods against the main olive grove pests and diseases: the olive fruit fly, Bactrocera oleae (Rossi), the olive moth, Prays oleae (Bernard), the black scale, Saissetia oleae (Olivier), and the olive leaf spot, caused by the fungus Spilocaea oleagina Fries. However, and because the new pesticide legislation is aimed at an integrated pest and disease management, it is still important to evaluate and to know the ecotoxicology of pesticides on the natural enemies of the different agrosystems.

A part of this work has been focusses on evaluating the direct and indirect effects of kaolin particle films and two copper-based products (Bordeaux mixture and copper oxychloride) through different laboratory, extended laboratory and semi-field experiments. Two natural enemies have been chosen: Psyttalia concolor (Szèpligeti), a parasitoid of the olive fruit fly, and Chilocorus nigritus (F.), predator of Diaspididae. This predator has been used instead of C. bipustulatus (L.), which is the species found in olive orchards. Kaolin mainly acts as a repellent of insects and/or as an oviposition deterrent. It is used in olive groves to control the olive fruit fly and the olive moth. Copper is applied against fungal and bacterial diseases. In olive groves it is used against the olive leaf spot and other diseases. No statistical differences were found in any of the experiments performed, compared to the controls, except when the oral toxicity of the products was evaluated on P. concolor females. In this case, kaolin and copper oxychloride caused a higher mortality 72 hours after the treatments, and both kaolin and the two copper formulations decreased females’ life span. Reproductive parameters were only negatively affected when kaolin was ingested. Apart from these experiments, due to the uncommon mode of action of kaolin, two extra experiments were carried out: a dual choice and a no-choice experiment. In this case, both P. ix

Resumen /Summary

concolor females and C. nigritus adults showed a clear preference for the untreated surfaces when they had the possibility of choosing between a treated surface and an untreated one. When there was no choice, no statistical differences were found between the treatments and the controls.

Furthermore, the efficacy and the selectivity of three insect growth regulators (methoxyfenozide, tebufenozide and RH-5849) on B. oleae and P. concolor, respectively, have also been evaluated. In addition to laboratory experiments to evaluate the toxicity of the insecticides, also molecular approaches were used. RNA of both insects was isolated. cDNA was subsequently synthesized and the complete sequences of the ligand biding domain (LBD) of the ecdysone receptor of each insect were then determined. Afterwards the three dimensional structures of both LBDs were constructed. Finally, the docking of the insecticide molecules in the cavity delineated by the 12 α-helix that composed the LBD was performed. Both toxicity assays and molecular docking approaches showed that either methoxyfenozide or tebufenozide had no negative effects nor on B. oleae nor on P. concolor. In contrast, RH-5849 had no deleterious effect to the parasitoid but decreased olive fruit fly adults’ life span, especially when they were in contact with the fresh residue of the insecticide applied on a glass surface. The docking study of RH-5849 molecule has shown a very light hindrance with the wall of the LBD pocket. This means that this molecule could more or less adjust in the cavity. Thus, searching of new insecticides for controlling the olive fruit fly could be based on the basic lead structure of RH-5849 molecule.

Introduction

Chapter 1

INTRODUCTION

1.1 The olive tree

The olive tree, Olea europaea L., is a treelike species of the Oleaceae family, within cultivated olive trees, belonging to the sativa subspecies, and wild olive trees, subspecies sylvestris, are included. Plants of this family are mainly trees and bushes Figure 1: An olive grove in Castile‐La Mancha and some of them produce essential oils in their flowers or fruits; only olive tree fruits are edible (Lombardo, 2003; Rapoport, 2008).

Its taxonomical classification is: class Angiosperms, subclass Ranunculidae, superorder Lamianae, order Lamiales, familiy Oleaceae, subfamily Oleoideae, genus Olea, species O. europaea L. (Devesa, 2005).

It is a long‐cycle crop, as it takes a long time to begin to produce olives, to reach its peak yield and to start its decline (Civantos, 2008). Its irregular production depends on climatic conditions, pests, diseases and the alternate bearing (named “vecería” in Spanish) (Orenga and Giner, 1998). According to Cirio (1997), olive growing is described as a considerable environmental‐variability depending crop, which is highly influenced by climatic, soil, biologic, agronomic, socioeconomic and cultural conditions.

Introduction

The genetic homogeneity of every cultivar is high because of the vegetative propagation techniques that have traditionally been used. On one hand, first olive farmers from every region used to select, among the wild olive trees, those which were the most productive, had the biggest fruits and the highest oil‐content, allowing the conservation of the characteristics of those original cultivars. On the other hand, the spreading of these first local cultivars allowed its hybridization with other from different regions, achieving the stability of the selected individuals through vegetative reproduction techniques (Barranco, 2008).

1.1.2 The origin of the crop

The olive crop was one of the first fruit trees cultivated by man. It has been claimed that its cultivation dated back to 4,000‐3,000 years BC in the area of Palestine. After that, it spread out to all the countries of the Mediterranean region. As a consequence of Columbus’s, Magellan’s and Elcano’s voyages, it started to be cultivated in the New World. Nowadays it is also grown in North America, South Africa, China, Japan and Australia (Lombardo, 2003; Civantos, 2008), although it is considered that around 98% of olive oil world heritage is located in the Mediterranean area (Civantos, 2008).

It is not clear when olive cultivation started in Spain, but the most accepted hypothesis pointed to Phoenicians and Greeks as its introducers. During the Roman period, Hispania olive oil trade spread out all over the western Roman Empire (Pajarón, 2007), which resulted in the expanse of the crop in the Betis Valley (nowadays known as Guadalquivir area), getting up to Sierra Morena. The railway‐ building during the 19th Century favoured olive cultivation in the interior areas of the country and filled out the Spanish olive crop map (AAO, 2011).

At the beginning of the second half of the last century, the olive growing system changed from a traditional into a modern one, due to the increase of labour salaries. This fact caused the replacement of the labor by machinery and the introduction of monoculture crops. However, concerning olive groves, these changes were not as big

2 Introduction as in other crops because of the longevity of the trees, mostly affecting cultural work, while the structure of the plantation and the variety of the trees were maintained (Pajarón, 2007).

1.1.3 Geographical distribution

The olive tree habitat is located between 30º and 45º, both in the north and in the south, in regions with a Mediterranean climate (characterized by hot and dry summers), and up to 1,000 metres above sea level. In the southern hemisphere it is also founded in more tropical latitudes whose climate is modified by altitude (Cirio, 1997; Rotundo and De Cristofaro, 2003; Civantos, 2008).

Optimal climatic conditions are those whose minimum temperatures are not lower than ‐5ºC, the average precipitation is no more than 500‐550 mm and the soil has a balanced composition, is rich in organic matter and its pH is neutral or slightly basic. Thanks to its huge adaptation capacity, it is able to grow also in very poor soils and dry locations (<250mm yearly) (Cirio, 1997).

1.1.4 Importance of the crop

The olive tree is the iconic tree of the Mediterranean area where, along with vines and cereals, it helps define the most striking features of the agricultural landscape (Duarte et al., 2008). Apart from its economic, social and cultural importance, its environmental value must also be taken into account, because of the high level of biodiversity and low rates of soil erosion found in this agrosystem (Pajarón, 2007).

Introduction

1.1. 4. 1 Economic importance

World olive growing is estimated at around 1,000 million of olive trees, occupying a surface of 10 million hectares. 98% is located in the Mediterranean basin, 1.2% in America, 0.4% in Asia and the other 0.4% in Oceania. Average world olive fruit production is estimated in 16 million tons, of which 90% goes to olive oil production and the other 10% to table olives (Civantos, 2008).

The main olive oil producing countries in the world are Spain (39%), Italy (22%), Greece (16%), Tunisia (6%), Turkey (5%), Syria (4%), Morocco (2%), Portugal (2%), Algeria (1%) and Jordanian (1%) (Civantos, 2008).

According to the data of the “International Olive Council” (IOC), the world olive oil production during the 2009/2010 campaign was 3,024,000 t, and the provisional figure for 2011, 2,948,000 t (IOOC, 2011).

1.1.4.2 Social importance

Traditional olive growing has a significant socio‐economical role, as it provides an important source of income and employment, particularly in marginal regions, strongly dependent upon agricultural activities (Duarte et al., 2008). As olive‐growing areas, many villages are trying to offer different activities for rural tourism, in order to earn money not only by selling their olives. In Spain, different initiatives are being encouraged, such as the opening of the “Museo de la Cultura del Olivo” (Olive Tree Culture Museum) in Baeza (Jaén) or the recognition of labels granted to some “guarantee of origin and quality” of some varieties. The promotion of the oleo‐tourism allows people to know more about the crop, the villages and their inhabitants, the properties of olive oil and its culinary uses (Anonymous, 2009a,b).

Some studies have been carried out about the olive oil benefits on human health. This product has been not only the basis of the Mediterranean diet for ages, but of

4 Introduction plenty more besides: it is used to make cosmetics, in religious rituals, in medicine and it also has an important role in mythology. Furthermore, during the last few decades it has been shown that diet is the most important environmental factor affecting the quality of life, and olive oil is necessary in order to reach a healthy old age and to prevent the most important causes of mortality all over the world (CIAS, 2008).

1.1.4.3 Environmental importance

Olive trees are essential in the Mediterranean ecosystem because their fruits are important foodstuffs for the fauna related to it. Moreover, because trees do not loose their leaves, and thanks to the shelter that foliage provides, a special microclimate is created within the olive canopy during the winter. This makes it warmer and more attractive than the outside (exposed to the wind and to low temperatures). Indigenous and wild flora, which includes around one thousand of herbaceous and woody species (Cirio, 1997), benefit from these special conditions as well (Saavedra, 1998).

1.1.5 Olive growing in Spain

Spain is the first olive oil and table olive producer and exporting country in the world (Civantos, 2008), having the longest olive grove surface (2,580,577 ha) and the largest number of olive trees (282,696,000) (AAO, 2011; MARM, 2011a). Because of its wider territorial spreading and its economical, social, environmental and health importance, the olive growing is one of the main sectors of the Spanish agricultural system. Olive trees can be found all over Spain, except in Galicia, Asturias and Cantabria (Civantos, 2008; AAO, 2011).

According to the data published by the Ministry of the Environmental and Rural and Marine Environs (Ministerio de Medio Ambiente y Medio Rural y Marino); from December 2011, Ministerio de Agricultura, Alimentación y Medio Ambiente), Spanish olive oil production in 2011 was estimated at 1,357,400 t and 485,300 t of table olives

(MARM, 2011b). 5

Introduction

During the last years, the Spanish olive grove system has undergone an updating of techniques which are increasing its productivity. In the 60’s and the 70’s, old trees or trees planted in marginal areas were pulled out and replaced with other crops which were more suitable or more profitable. Simultaneously, the Spanish administration established the “Plan of the restructuring of productivity and conversion of olive groves” (Plan de Reestructuración Productiva y de Reconversión del Olivar) in which proceedings to improve or increase the olive grove productivity figured. As a consequence, areas well adapted to this crop increased the surface dedicated to them and their production, while the less adapted ones cut down on it (Civantos, 2008). There are also other changes concerning this crop, such as the increase of irrigated olive groves, the use of higher denseness plantation (2,000 – 3,000 trees per hectare), the choosing of the trees whose trunks are better adapted for mechanical harvesting and the development of a nursery industry dedicated to obtain plants with just one trunk and an early fruiting. Furthermore, farmers are nowadays aware of the importance of using the suitable growing and oil‐making techniques that guarantee a better quality of the olive oil produced (Rallo, 1998).

1.1.6 Pests and diseases: characteristics of the most important pests and diseases of olive groves

From a phytosanitary approach, olive groves can be considered as simple agricultural systems. This is due to their environmental stability, the orientation of its production, the small number of really harmful parasitic, the tolerance to produced damages, and the abundance of natural enemies (Cirio, 1997). Because of these facts, the number of chemical treatments remains still low compared to other crops (Alvarado et al., 2008). However, the olive tree grows closely related to several biotic and abiotic factors which not only establish the specificity of the present organisms, but also determine their population changes. A single change in one of them affects the whole agrosystem balance (Iannotta, 2003; Alvarado et al., 2008). For example, although more than 225 potential damage organisms have been cited since olive tree cultivation started (Haniotakis, 2005), the most important pests affecting this crop are the olive fruit fly and the olive moth. However, from the 1960’s up to nowadays, as a

6 Introduction consequence of the abuse in the use of pesticides to fight against these two pests, another one, the black scale, has increased its population, causing different damage. Troubles brought on by other pests, like other scales, mites, etc., have also risen up due to this traditional pest management system (Alvarado et al., 2008).

Losses caused by pests, diseases and weeds are estimated to reach 30% of the crop, of which 15% are due to the action of insects. Amongst them, 10% are attributed to the main olive grove pests (Haniotakis, 2005).

According to this author, four pest categories have been established in the Mediterranean area: ‐ Key or major pests: they are the most damage‐causing ones. An annual monitoring of them is required. The olive fruit fly, Bactrocera oleae (Rossi) (Diptera, Tephritidae), is the only one considered in this category. ‐ Secondary important pests: their losses have an occasional or local importance. The olive moth Prays oleae (Bernard) (Lepidoptera, Yponomeutidae) and the black scale Saissetia oleae (Olivier) (Homoptera, Coccidae) are included in this category. ‐ Pests of a limited economic or localized importance: pests that change over time and cause locally and/or occasionally economic losses. ‐ Pests without much economic importance: they rarely cause damage or losses.

The main olive grove phytophagous and pathogens are included in Tables 1 and 2.

Introduction

1,2 Table 1: Main olive grove phytophagous and their eating habits

Key pests and secondary important pests Olive fruit fly (mosca del olivo): Bactrocera oleae (Rossi) Monophagous Olive moth (polilla, prays): Prays oleae (Bernard) Olygophagous Black scale (cochinilla de la tizne, caparreta): Saisseta oleae (Olivier) Polyphagous Pests of economically moderate importance Olive bark beetle (barrenillo, palomita): Phloeotribus scarabaeoides Olygophagous (Bernard) Olive bark borer (barrenillo negro): Hylesinus oleiperda (F.) Olygophagous Olive pyralid, jazmines moth, olive leaf moth (polilla del jazmín, glifodes): Olygophagous Palpita unionalis (Hübner) Olive pyralid moth (abichado): Euzophera pingüis (Haworth) Olygophagous Olive gall mite (sarna, erinosis o acariosis del olivo): Aceria oleae (Nalepa) Monophagous Secondary pests with local or temporary importance Apple mussel scale, oystershell scale (serpeta o coma del manzano): Polyphagous Lepidosaphes ulmi (L.) Olive parlatoria scale, (parlatoria, piojo violeta): Parlatoria oleae (Colvee) Polyphagous Olive psyllid (algodón, tramilla): Euphyllura olivina (Costa) Monophagous Olive weevil, oziorrinco (otiorrinco): Otiorhynchus cribricollis (Gyllenhal) Polyphagous White grubs, European cockchafer (gusanos blancos): Polyphagous Melolontha papposa Illiger, Ceramida cobosi (Bagena) Olive thrips (arañuelo): Liothrips oleae (Costa) Monophagous Olive midge (mosquito de la corteza): Resseliella oleisuga (Targioni‐ Olygophagous Tozzetti) Cicada (cigarra): Cicada barbara (Stal) Polyphagous Leopard moth (zeuzera o taladro amarillo): Zeuzera pyrina (L.) Polyphagous Birds, rodents, rabbits and hares Polyphagous (Oryctolagus cuniculus (L.) and Lepus europaeus Pallas) 1Phytophagous have been arranged in groups, according to their economic importance and their diet habits (monophagous, olygophagous or polyphagous). 2The Spanish name of the pests is indicated in brackets.

Sources: (Iannotta, 2003; Alvarado et al., 2008).

8 Introduction

Table 2: Olive grove pathogenic agents and abiotic diseases. Significance of the damage caused by them1,2 Bacteria Olive knot disease, tuberculosis of olive tree (tuberculosis del olivo): Moderate‐ Pseudomonas savastanoi pv. savastanoi High Foliar diseases‐Fungus Olive leaf spot (repilo): Spilocaea oleagina Fries (= Cyclonium oleaginum Cast.) High Anthracnose (antracnosis, aceituna jabonosa): Moderate Colletotrichum gloeosporioides Penz. (= Gloeosporium olivarum Alm.) Dalmatian disease (escudete de la aceituna): Low Camarosporium dalmaticum (= Sphaeropsis dalmatita Thüm.) Cercosporosis or leaf spot disease on olive (emplomado de la aceituna): Moderate Pseudocercospora cladosporioides (= Cercospora cladosporioides Sacc.) Coin canker (lepra): Phlyctema vagabunda Desm.(= Gloeosporium olivae Petri) Low Sooty mould, fumagina (fumagina, negrilla): Capnodium elaeophilum Prill. Low Other fruit rots (otras podredumbres del fruto): Low Fusarium, Alternaria, Cladiosporium… Not Other foliar fungal (otras micosis foliares): Stictis, Leveillula, Phylactina important Trunk decay (caries del tronco): Fonus, Phellinus, Polyporus, Stereum Low Chancre (chancro) Low Root fungus Verticillium wilt, soil borne fungus (verticilosis): Verticillum dahliae High Thick root rot fungus (podredumbre de raíces gruesas): Low Armillaria, Rosellinia, Omphalotus Thin root rot fungus (podredumbre de raicillas): Moderate‐ Phytophtora, Cylindrocarpon, Fusarium Low Virus Malformation, yellowish (malformaciones, amarilleo): unidentified virus Not important Latent infections, yellowish (infecciones latentes, amarilleo): Not important Nepovirus, Cucumovirus, Oleavirus Nematode Root lesions: nodes, galls (nódulos, agallas): Meloidogyne, Pratylenchus… Not important Phanerogam Mistletoe, Jopo, Cuscuta Not important Abiotic diseases Lack of essential nutrients : boron, iron, potassium Moderate‐ Low Other damages: frost, drought, flooding… High ‐ Low 1The table has been organised according to the different pathogen agents or the causes of abiotic diseases. Their economic importance, according to the damage caused, has also been pointed out as high, moderate, low or unimportant. 2The Spanish name of the pests is indicated in brackets.

Sources: Iannotta, 2003; Trapero and Blanco, 2008; Trapero et al., 2009

Introduction

In intensive farming systems, the high‐density plantations and the continuous presence of fresh shoots during the whole vegetative cycle allow phytophagous species to have a constant availability of food. Apart from traditional damaging organisms, others which had never caused significant damage on the crop could be now categorized as potential pests or disease‐causing agents (Torrell and Celada, 1998).

11.6. 1 The olive fruit fly (Bactrocera oleae )

Bactrocera oleae distribution is primarily limited to the regions where cultivated and wild olive trees are found. Today, the olive fruit fly is reported throughout the Mediterranean basin, Africa and from Middle East to India (Guerrero, 2003; Alvarado et al.,

2008). It is also found in all the countries Figure 2: Female of B. oleae (Anonymous, 2009c) where olive crop has been introduced during recent years, as USA, China, but it has not been observed in South America and Australia (Civantos, 1999; Iannotta, 2003; Rotundo and De Cristofaro, 2003; Daane and Johnson, 2010).

It was recorded attacking olives in biblical times and has long been a major pest in the Mediterranean basin. Larvae are monophagous on olive fruits in the genus Olea, including O. europaea (cultivated and wild), O. verrucosa and O. chrysophylla (Daane and Johnson, 2010). However, it has also been reared in laboratory on Ligustrum and Jasminum berries (Civantos, 1999; Alvarado et al., 2008) and on tomatoes (Navrozidis and Tzanakakis, 2005).

As soon as adults emerge, they look for the sweetened and nitrogenous substances they need as nutritional requirements. They feed on a variety of organic sources including insect honeydews (for example, black scale honeydew), plant nectar, plant

10 Introduction pollens and fruits exudates (De Andrés, 1991), and also bird dung, bacteria and yeasts. Females lay their eggs in ripening but also in green fruits, in which the newly hatched larvae feed upon the pulp. They pupate inside the olive or exit to do it on the ground (Daane and Johnson, 2010). Larval development is largely temperature dependent. It has been reported that the lower temperature threshold is 6ºC and the upper one is 35ºC, while the optimal temperature ranges from 20º to 25ºC. Relative humidity is only important if its value is low and temperatures are high during a long period (De Andrés, 1991; Civantos, 1999; Rotundo and De Cristofaro, 2003; Alvarado et al., 2008).

The number of annual generations depends not only on the temperature, but also on the relative humidity, on the microclimate within the olive canopy and on the availability and quality of olive fruits. This results in variation in the reported number of generations per season within the fly’s endemic range, which encompasses a variety of climatic regions. Two to three generations have been reported in continental climate areas, while three generations are always common in coastal regions (Civantos, 1999), or even four (De Andrés, 1999; Alvarado et al., 2008). Some authors have also reported up to six or seven generations (De Andrés, 1991).

Several studies have shown that olive cultivars vary in their susceptibility to the olive fruit fly. Some of the factors that possibly play a role include fruit size and weight, colour, fruit epicarp hardness, surface covering (mainly aliphatic waxes), phenological stage of the crop, and chemical factors (Daane and Johnson, 2010). Oil destined cultivars are less susceptible to olive fly attack than table olive cultivars (Civantos, 1999; Rotundo and De Cristofaro, 2003; Alvarado et al., 2008). It has also been demonstrated that the most susceptible fruits in a tree are the biggest and those which are in the outer part of the tree’s crown (Alvarado et al., 2008).

The relative importance of the economic damage provoked depends either on the olive fly population density or on the period of the year considered (De Andrés, 1991; Civantos, 1999; Alvarado et al., 2008; Daane and Johnson, 2010). In areas of the world where the olive fruit fly is established, it has been reported as responsible for losses of up to 80% of oil value and 100% of some table cultivars. It has been estimated to

Introduction damage 5% of total olive production, resulting in economic losses of approximately USD 800 million per year (Haniotakis, 2005; Daane and Johnson, 2010). In table olives damage is more important because oviposition stings on fruits totally reduce their value (Alvarado et al., 2008). Therefore, the tolerable infestation level is near zero larvae per fruit (Daane and Johnson, 2010). Economic damage can be classified as direct

Figure 3: Detail of a B. oleae larva in an olive and indirect: fruit. Microorganism growth can be observed in the feeding gallery

Direct damage: premature fruit dropping or loosing of fruit weight resulting from larvae feeding the pulp. The production rates can decrease between 5 and 10 %

Indirect damage: they are referred to the lowered quality and value of pressed oil due to increased acidity as a result of microorganism growth inside olive tree fruits (bacteria, yeasts and mould).

In the Mediterranean area, it seems that none of the olive fruit fly’s predators or parasitoids is able to totally control the pest (González‐Núñez, 2008). According to the earlier surveys, it appeared that sub‐Saharan Africa might provide a rich source of natural enemies of B. oleae. The most recent surveys suggest that a smaller group best represents the primary parasitoids attacking olive fruit fly in its native range. Most of these wasps are synovigenic, koinobiont, larval‐pupal or larval‐prepupal parasitoids in the Opiinae subfamily. The exception is the idiobiont larval ectoparasitoid Bracon celer Szépligeti. (Hymenoptera, Braconidae). Some chalcid parasitoids have also been reared from olive fruit fly, although many of the species are polyphagous parasitoids that may opportunistically attack the olive fruit fly (Daane and Johnson, 2010). In the Mediterranean basin the most common parasitoids found are the Hymenoptera Eupelmus urozonus Dalman (Eupelmidae), Pnigalio mediterraneus (Ferriere and Delucchi) (Eulophidae), Eurytoma martellii Domenichini (Eurytomidae), Psyttalia concolor (Szèpligeti) (Braconidae) and Cyrtoptyx latipes (Rondani) (Pteromalidae)

12 Introduction

(Iannotta, 2003; Rotundo and De Cristofaro, 2003). The most commonly found are E. urozonus and P. mediterraneus, but they are not effective enough to control B. oleae populations (Civantos, 1999). Neonate larvae of the olive fruit midge, Lasioptera berlesiana Paoli (Diptera, Cecydomiidae), feed on the eggs of B. oleae. The problem is that females, at the time of egg‐laying, also introduce the fungus S. dalmatica (C. dalmaticum), which causes Dalmatian disease in olive fruits. There seems to be a large number of B. oleae larvae and pupae predators too. Predators attack B. oleae when third instar larvae drop to the ground to pupate beneath the trees. This group of predators includes ants, Carabidae, Staphyllinidae, spiders and earwigs (González‐ Núñez, 2008; Daane and Johnson, 2010). Some authors also take into account the role of insectivorous birds and birds which feed on olive fruits because they can decrease B. oleae populations of wild, overgrown and ornamental olive trees (González‐Núñez, 2008).

Over the last four decades B. oleae management has been based on the use of different insecticides (such as organophosphates, pyrethroids and spinosad). However, the fly has already evolved resistance to dimethoate (Daane and Johnson, 2010) and spinosad (Kakani et al. 2010). Furthermore, the continued use of such products has been questioned in recent years, especially by environmentalists. For example, in the case of dimethoate, which is used to control both B. oleae and the anthophagus generation of the olive moth, a strong and dramatic effect on the abundance of different trophic groups has been reported (Santos et al., 2010). In addition, residues of pesticides have been detected both in olive oil and in the environment where olives are grown. These facts have caused concern in most olive growing countries and have lead to a concerted effort to reduce the amount of pesticides used in this crop (Montiel‐Bueno and Jones, 2002).

Introduction

1.1.6.2 The olive moth (Prays oleae )

Prays oleae (Bernard) (Lepidoptera: Yponomeutidae) is considered the second important pest in olive groves. It is found throughout the Mediterranean basin. Although its main host plant is the olive tree, it can also Figure 4: Adult of P. oleae feed on other Oleaceae species (Rotundo and De (Anonymous, 2009d) Cristofaro, 2003; Alvarado et al., 2008).

It has three generations per year, synchronized with the olive tree phenology. The first one infests the leaves (phyllophagous), the second one the flowers (antophagous) and the third one the fruits (carpophagous). The most harmful is the third one (Iannotta, 2003) because feeding damage causes premature fruit dropping. Chemical treatments against the first or the second generation are only justified when trees are young or when moth population is high and the number of flowers is low (Rotundo and De Cristofaro, 2003; Alvarado et al., 2008).

Climatic conditions are very important to the olive moth development and determine its presence in the different geographical regions (Civantos, 1998a). Cold weather during the winter (<10ºC) or hot weather (>30ºC) and a high relative humidity percentage (> 70%) during the summer control the populations. A relative humidity percentage below 50% makes survival difficult for the eggs (Alvarado et al., 2008;

Civantos, 1998a; Rotundo and De Cristofaro, 2003).

Parasitism rate is high and varies among generations, years and geographical areas. It is responsible for between 10 and 50% of moth population mortalities. Amongst their parasitoids, the Hymenoptera Ageniaspis fuscicollis var. praynsicola (Silvestri) (Encyrtidae), Chelonus eleaphilus Silvestri (Braconidae) (both of them specific to prays), Diadegma armillata (Gravenhorst) (Ichneumonidae), Apanteles xanthostigmus (Haliday) (Braconidae), the Eulophidae Pnigalio mediterraneus (Ferriere and Delucchi)

14 Introduction and P. pectenicornis (L.), and other Trichogrammatidae species stand out. They parasitize larvae, pupae or eggs (Civantos, 1998a; González‐Núñez, 2008). Among their predators, the chrysopids Chrysoperla carnea (Stephens) and Dichochrysa flavifrons (Brauer) (Neuroptera, Chrysopidae) are very important (González‐Núñez, 2008). They feed on the eggs (BOJA, 2002; Iannotta, 2003; Rotundo and De Cristofaro, 2003; Alvarado et al, 2008), although they can also feed on larvae of the phyllophagous and the anthophagous generations (González‐Núñez, 2008). Also different spider species, especially mites which feed on eggs and larvae (Civantos, 1998a), ants, Heteroptera and Coleoptera are important predators of the olive moth (González‐Núñez, 2008). Bacteria, fungi, protozoa and virus can also affect the pest, but they are not usually efficient enough (Civantos, 1998a).

1.1.6.3 The black scale (Saissetia oleae )

The black scale, Saisettia oleae (Olivier) (Homoptera, Coccidae), is spread all over the world, but mainly in the Mediterranean basin. It is found in olive groves and citrus, but also in some other trees and bushes. It prefers shady areas and humid environments (Rotundo and De Cristofaro, 2003; Figure 5: S. oleae adult females Alvarado et al., 2008).

They feed on the trees by piercing of host tissues and sucking the sap. Although direct damage is not really significant, the black fungi developing on the honeydew they deposit on trees (sooty mould) are responsible for reducing photosynthesis and can be referred to as contamination, often rendering plants or fruits unmarketable (Iannotta, 2003; Alvarado et al., 2008). Furthermore, honeydew is one the of B. oleae adults’ favourite sweet foodstuff, so it can also attract them (Guerrero, 2003).

Saissetia oleae holds a high number of natural enemies which parasitize different nymphal stages and even preovipositional females. They are mainly Hymenoptera

Introduction parasitoids of the genus Metaphycus (M. helvolus (Compere) and M. lounsburyi (Howard); Encyrtidae) and other native Aphelinidae, such as Coccophagus lycimnia (Walker), C. semicircularis (Föster) and C. scutellaris (Dalman), which also parasitize other Coccidae species (González‐Núñez, 2008). Among predators, the most important are the Hymenoptera Scutellista cyanea (=S. caerulea) (Mostchulsky) (Pteromalidae), whose larvae feed on the eggs under the female scale, and some Coleoptera Coccinellidae, such as Chilocorus bipustulatus (L.) (Iannotta, 2003; Rotundo and De Cristofaro, 2003; Alvarado et al., 2008), Brumus quadripustulatus (L.), Rhyzobius spp., Scymnus spp. The Lepidoptera Eublemma scitula Rambur (Noctuidae) and the Neuroptera Coniopteryx spp. (Coniopterygidae) are important as well (González‐ Núñez, 2008).

1.1.6.4 The olive leaf spot (Spilocaea oleagina )

The olive leaf spot, caused by the fungus Spilocaea oleagina Cast., is the most common disease in Spanish olive orchards (Trapero et al., 2009). Its importance is due both because of the

extensive areas where it can be found Figure 6: Olive leaf spot and because of the damage caused when development conditions are in its favour (rainy years, high density and poorly aerated plantations and olive groves irrigated or close to wet areas) (Guerrero, 2003; Pajarón, 2007; Trapero and Blanco, 2008). Despite the fact that the fungus is only pathogenic on olive trees, morphologically similar fungus have been described as disease‐causing on Ligustrum, Phillyrea and Quercus species (Trapero et al., 2009).

Typical symptoms of the disease are the black circular spots on the adaxial surface of the leaves, often surrounded by a yellowish hallow. Leaves fall prematurely and death of twigs may ensue. Conidia are spread out mainly by the rain. Once they are on the susceptible tissues of the plant, they germinate only if there is water available or

16 Introduction the relative humidity is higher than 98% and temperature swung by around 0º and 27ºC, being 15ºC the optimum. Consequently, cultural practices helping aeration of the trees are very important (Trapero et al., 2009). Premature defoliation has serious consequences on the plant vegetative activity and yield. It could reduce either the differentiation rate of auxiliary buds into flower‐bearing shoots or the productivity of the trees. Sometimes, it can also infect fruit peduncles and provoke their premature fall, which decreases their quality and fatty yield. Olive oil quality from these fruits, however, remains unaffected (Guerrero, 2003; Pajarón, 2007; Trapero and Blanco, 2008).

1.2 Control of pests and diseases

The use of different substances with insecticide properties dates from Roman and Greek times. During the 19th Century, artificial fertilizers were developed. They were cheap, powerful and easy to transport in bulk. Similarly, it also occurred in the 1940’s with chemical pesticides, leading to the decade being referred to as the “Pesticide era” (MARM, 2006). Indeed, the synthesis of the DDT in 1939 seemed to have solved all pest problems (Dent, 1991; Casida and Quistad, 1998).

Traditional agricultural systems are based on the use of different chemical products, without taking into account the possible negative impacts caused by their widely and uncontrolled use and abuse. For example, environmental contamination, intoxications or the appearance of resistances to insecticides among the pests have already been reported in different crops, including olive groves (Cirio, 1997; De Ricke, 1998; Chamorro and Sánchez, 2003; Iannotta, 2003; Alvarado et al., 2008).

In contrast to these chemical‐based traditional pest management practices, once farmers and researchers realized it was necessary to rationalise the use of pesticides, the concept of “Integrated Pest Management” (IPM) appeared. Furthermore, as a reaction to agriculture growing reliance on synthetic fertilizers, the organic movement had already started between 1930 and 1940 (MARM, 2006).

Introduction

1.2.1 Integrated Pest Management

In 1967, the FAO (Food and Agriculture Organization of the United Nations) defined IPM as “the careful consideration of all available pest control techniques and subsequent integration of appropriate measures that discourage the development of pest populations and keep pesticides and other interventions to levels that are economically justified and reduce or minimize risks to human health and the environment. IPM emphasizes the growth of a healthy crop with the least possible disruption to agro‐ecosystems and encourages natural pest control mechanisms”

(FAO, 2011a).

Later on, in 1976, the International Organisation for Biological and Integrated Control of Noxious Animals and Plants (IOBC) defined Integrated Production: “Integrated Production is a concept of sustainable agriculture based on the use of natural resources and regulating mechanisms to replace potentially polluting inputs. The agronomic preventive measures and biological/physical/chemical methods are carefully selected and balanced taking into account the protection of health of both farmers and consumers and of the environment”. The aim of the IOBC is the promotion of biological and integrated methods to fight against pest, diseases and weeds (IOBC, 2011).

Integrated Protection (IP) fits among the different Integrated Production measures. The aims of IP are both to protect the environment and to be profitable for farmers, although both situations are not always compatible. IP is based on three practices (Cirio, 1997):

‐ Crop monitoring: that is, a routinely checking process against defined objectives and targets. It takes place periodically and evaluates and verifies standards across a range of plantation activities. It is a rigorously documented process that will normally result in a programme of improvements.

18 Introduction

‐ Application of the Economic Injury Level (EIL), which is the point when economic damage that occurs from insect injury equals the cost of managing insect populations; it is the breakeven point (Alvarado et al., 2008). Damage that occurs below that point is not worth the cost of preventing it. Because these insect or injury levels are not wanted to be reached, a point that is set well below the EIL is used, usually meaning when an insecticide can be applied. This “take action” level is known as the Economic Threshold (ET). ‐ Evaluation of the proper control methods both for effectiveness and risk. Good practices related to crop protection include those that use resistant cultivars and varieties, crop sequences, associations, and cultural practices that maximize biological prevention of pests and diseases; maintaining regular and quantitative assessment of the balance status between pests and diseases and beneficial organisms of all crops; adopt organic control practices where and when applicable; applying pest and disease forecasting techniques where available; determining interventions following consideration of all possible methods and their short‐ and long‐term effects on farm productivity and environmental implications in order to minimize the use of agrochemicals

(FAO, 2011b).

Biological control will always try to exploit agrosystem trophic food chains (Urbaneja and Jacas, 2008). Therefore, all the measures which protect auxiliars should be favoured. Special attention should be paid to cultural practices and pesticide applications, which can control pests but also have a negative impact on natural enemies (Jiménez et al., 2002). For that reason, it is very important to know the biological and phenological cycles of auxiliary fauna, their role in pest control and the side effects of pesticides on them (Civantos, 1998b). Furthermore, regional regulations of integrated production have established at least two natural enemies whose protection and increase are important (González‐Núñez, 2008).

Pesticides should only be applied as a last resort when there are no adequate non‐ chemical alternatives and their use is economically justified. They should be as specific as possible for the target and have the least side effects on human health, non‐target

Introduction organisms and the environment. Their use should be kept at minimum levels, e.g., by partial applications (Civantos, 1998b; Hassan, 1998; BOJA, 2002; Malavolta et al., 2002;

FAO, 2011a).

In 2009, the European Parliament approved the new legislation about the sustainable use of pesticides and their trade (Regulation EC 1107/2009 of the European Parliament and the Council of 21 October 2009 concerning the placing of plant protection products on the market. It repeals the Council Directives 97/117/EEC and 91/414/EEC and the Directive 2009/128/EC of the European Parliament. It establishes a framework for Community action to achieve the sustainable use of pesticides). The compromise deal on the proposed regulation will put in place a system where a positive list of approved active substances in pesticides will be drawn up. Pesticides will then be licensed at the national level based on this list. The deal allows exemptions for banned active ingredients to be used in pesticides for up to five years, if they are proven essential for crop survival. Certain types of banned active ingredients (candidates for substitution) have to be replaced within three years, if safer alternatives exist. The compromise deal on the framework Directive requires Member States to adopt National Action Plans with quantitative targets, measures and timetables. The deal prohibits pesticide use, or at least requires it to be kept to a minimum, in specific areas used by the general public or by vulnerable groups (IEEP,

2009; OJEU, 2009a,b; Palomar, 2009; Ruiz‐Torres, 2009).

In Spain, IP practices are carried out under the supervision of public regulated organisms, the ATRIAS (“Agricultural Integrated Treatment Groups”: “Agrupaciones de Tratamientos Integrados en Agricultura”, in Spanish), since 1979. They control the crops and decide the proper treatments according to the methodology tuned up by Plant Health Services (Chamorro and Sánchez, 2003).

20 Introduction

There is a national logo which identifies products produced under IP techniques, as well as some other different Autonomous Communities’ logos (BOE, 2004; MARM,

2004a,b).

Figure 7: National and Autonomous Integrated Protection logos (BOE, 2004; MARM, 2004b)

1.2.2 Organic farming

Despite having started at the beginning of the XXth century, organic farming was not recognized as a feasible agricultural method until the beginning of the seventies (MARM, 2006).

There is a worldwide umbrella organization for the organic movement which unites more than 750 member organizations in 116 countries, the International Federation of Organic Agriculture Movement (IFOAM). It actively participates in international agricultural and environmental negotiations with the United Nations and multilateral institutions to further the interests of the organic agricultural movement worldwide. According to IFOAM, organic agriculture can be defined as “A production system that sustains the health of soils, ecosystems and people. It relies on ecological processes, biodiversity and cycles adapted to local conditions, rather than the use of inputs with adverse effects. Organic agriculture combines tradition, innovation and science to

Introduction benefit shared environment and promote fair relationships and good quality of life for all involved” (IFOAM, 2011).

Just as in IPM, plant health should be achieved through the maintenance of preventive measures. For example, the choice of appropriate species and varieties resistant to pest and diseases, suitable crop rotations, mechanical and physical methods and the protection of natural enemies. In case of needing a treatment to a crop, plant protection products may only be used if they have been authorised for its use in organic production (there is a restricted list of products and substances that can be used). All products and substances should have plant, animal, microbial or mineral origin, except if products are not available in sufficient quantities or if alternatives are not possible. If they are not identical to their natural form, they may only be authorised whether their conditions for use preclude any direct contact with the edible part of the crop. Each European Union Member State regulates the use of these products within their territory (OJEU, 2007).

The council Regulation (CE) 834/2007 regulates on organic production and labelling of organic products. It repeals the previous Regulation (EEC) 2092/91. In order to ensure that organic products are produced in accordance with requirements laid down under the Community legal framework of organic production, activities performed by operators at all stages of production, preparation and distribution of organic products should be submitted to a control system set up and managed on official controls (OJEU, 2007). In Spain, the MARM has a specific program whose main aims are to promote organic farming, to improve the trade, the consumption and the knowledge about organic products, as well as to get better collaboration among institutions and farmers. Autonomous Communities are directly responsible for regulations (MARM, 2007; OJEU, 2007).

22 Introduction

There is a European Union logo, a national one and some others from some Autonomous Communities. There is also a logo which certifies Spanish organic products for export (OJEU, 2007; Anonymous, 2008; MARM,

2011c).

Figure 8: Spanish, Autonomous Communities and European Union logos. Certification for European organic products (ECO CERT, SHC) (Anonymous 2008, MARM, 2011c)

1.2.3 Integrated Protection in olive groves

Integrated Olive Production in Spain has specific regulations in six Autonomous Communities (Andalusia, Balearic Islands, Catalonia, Extremadura, Murcia and Valencian Community). The number of hectares growing under this production system is 290,505 ha, which accounts for11% of the total olive crop hectares in our country

(MARM, 2011e,f).

Within the IOBC, there is a specific Working Group (WG) focus on olive crops, the “Integrated Protection of Olive Crops”, which was initiated in 1991. The main goal of the group is to promote collaboration in multidisciplinary research on the development, evaluation and implementation of integrated control strategies for olive pests and diseases. An important priority is the exchange of knowledge and the main ultimate targets are to minimize the impacts of olive crop protection on the environment, to increase sustainability and also to support the production of higher quality products (IOBC, 2011). IOBC has also published specific guidelines for

Introduction integration production of olives. The purpose of these guidelines is to define the basic requirements of integrated production in olives in such a generalised way that these rules can be applied in all geographical regions (Malavolta et al., 2002).

Table 3 summarizes some of the specific integrated production practices of olive groves.

Table 3: Integrated pest management in olive crops

Crop monitoring

As olive grove areas tend to be big, in order to simplify pest and diseases monitoring they are divided into homogeneous smaller areas. Control plots, as more representative of the whole area as possible, are then the monitoring units. Different traps are placed in the control plot and vegetative parts of the trees or fruits, depending on the phenological stage and the pest or the disease evaluated, are periodically sampled

Economic threshold levels

Tolerable thresholds are somewhat debatable because several factors influence them (the region, the variety, the destination of the harvest, etc.). That is the reason why the specific pest or disease, their secondary effects and the particular conditions of each farm should be taken into consideration when evaluating. Data from similar areas can be extrapolated

Sources: (Cirio, 1997; Civantos, 1998b; Civantos, 1999; BOJA, 2002; Chamorro and Sánchez, 2003; Iannotta, 2003; Rotundo and De Cristofaro, 2003; Romero et al., 2006; Moretti et al., 2007; Pajarón, 2007; Alvarado et al., 2008; González‐Núñez, 2008; Trapero and Blanco, 2008; IAEA, 2009; Trapero et al., 2009; Bento et al., 2010; Delrio, 2010).

24 Introduction

Table 3: Integrated pest management in olive crops (continuation)

Control methods Agricultural practice Pest or disease controlled New plantations Tuberculosis, verticillium wilt, olive leaf spot, Selecting best varieties for local growing anthracnose, black scale, olive fruit fly and conditions olive moth Verifying that both the soil and the new Verticillium wilt plants are pathogen‐free Planting trees in a density that provides Olive leaf spot, anthracnose a good aeration Cultural techniques Avoiding nitrogen over‐fertilisation Verticillium wilt, black scale, olive leaf spot Fruit fly and olive moth pupae could be Limiting heavy tillage practices destroyed by tillage; however, limiting tillage practices increases populations of beneficials. Olive fly populations increase while beneficial Herbicide applications populations decrease It avoids nitrogen run off and increases Maintaining permanent soil covering biodiversity Limiting water inputs in irrigated Verticillium wilt, root fungus, white grubs, plantations black scale, olive leaf spot Olive pruning Black scale, bark beetles, Euzophera pingüis, Achieving a good aeration tuberculosis, fungal diseases Disinfecting pruning tools and ensuring Olive knot disease that there are not damage Removing the pruned branches and Zeuzera pyrina and Cossus cossus larvae and destroyed them Phloeotribus scarabaeoides adults Pruning during the dormant season Different pests Harvesting Early harvest B. oleae Do not mixing fruits collected from olive B. oleae trees with those lying on the ground

Introduction

Table 3: Integrated pest management in olive crops (continuation)

Control methods Agricultural practice Pest or disease controlled Biological control Releases of the Hymenoptera Psyttalia concolor, B. oleae Fopius arisanus, P. lounsburyi, Eupelmus urozonus Releases of the parasitoids Methaphycus swirskii, Diversinervus elegans, M. barletti, M. helvolus, M. S. oleae lounsburyi and the predators Rhyzobius forestieri, Brumus quadripustulatus Releases of the fungus Bauveria bassiana Balsamo B. oleae and Metarhizium anisopliae Metchnikoff Releases of different nematode species Z. pyrina Applications of the fungus Talaromyces flavus Verticillium wilt Klöcker Inoculative releases of the fungus Fomes spp. and Vegetable parasites Agrobacterium tumefaciens Releases of the parasitoid Trichogramma spp., the predator Chrysoperla carnea and the parasporal P. oleae crystals of the bacterium Bacillus thuringiensis var. kurstaki Releases of the parasporal crystals of the Palpita unionalis bacterium Bacillus thuringiensis var. kurstaki Management of the agroecosystem to maximize the effect of native or introduced biological control agents (conservative or natural control): sowing Different olive pests plants that attract natural enemies, offering honey or other sugar sources… Biotechnical methods Mass trapping (traps baited with pheromones) Z. pyrina, C. cossus Mass trapping (traps baited with food lures); “Attract and kill” (integrating the sexual pheromone and ammonium bicarbonate as lures), sexual confusion B. oleae and releases of sterile males (Sterile Insect Technique)

26 Introduction

Fruit flies, such as B. oleae, are not attractive targets for classical biological control. This is partly because of several features in their life histories which make conditions very difficult for parasitoids. Adults of many fruit fly species disperse widely when they emerge and leave their parasitoids behind. This also happens when fruits disappear from crops and fruit flies disperse widely to other areas. Some examples of failures and successes in efforts to establish parasitoids in countries have been demonstrated by many years of extensive biological control programs conducted in the Pacific region and other countries outside it (Peters, 1996).

Conservative biological control programs are effective against some olive grove pests. For example, a study carried out by Boccaccio and Petacchi (2009) showed that landscape structure and natural or semi‐natural woodland play a role in enhancing B. oleae parasitoid activity. Some other experiments have studied the effect of different attractive sources (sugars, yeasts, etc.) on the abundance of olive pests predatory arthropods and the possible enhancement of their activity (Bento et al., 2004), or the effect of the establishment of vegetation patches which produce flowers (Jorge et al., 2005).

Pesticide applications in olive crops are sometimes necessary against B. oleae, P. oleae, olive leaf spot and anthracnose, less frequently with S. oleae and rarely with the rest of the pests (Iannotta, 2003). The most used products in these agrosystems are syntheticc insecticides, like organophosphates and pyrethroids against pests, and copper‐based compounds against diseases (MARM, 2011c). However, it should be pointed out that their use is lower compared to other crop systems (Cirio, 1997).

Spanish regulation has established the predator C. carnea as one of the two natural enemies whose protection and increase is important in olive groves. The other natural enemy should be chosen among the most important natural enemies in each region (González‐Núñez, 2008).

Introduction

1.2.4 Organic olive farming

Organic olive groves occupy a surface of 126,328.26 ha (4.9% of the total), chiefly in Cordoba (Andalusia), where a 53.30% of organic crop is located (according to the

MARM, in 2010 there were 1,650,866 ha of organic farming in Spain) (MARM, 2011d).

Agricultural practices to fight against olive pests and diseases are similar to those previously described for IP systems. However, in organic olive systems there is a lack of a wide range of effective products to control some of them, as it occurs with B. oleae. In this case, the interest on using repellent and antiovipositional products, as well as products able to kill both their larvae and eggs, has increased in the last years (Caleca and Rizzo, 2006; Caleca et al., 2008).

1.3 Side-effects of pesticides on non-target organisms

The effects of chemical pesticides on predators and parasites are much less known than on herbivorous. However, literature on natural enemy/pesticide research has increased at an exponential rate since the late 1950s.

It is necessary to test the possible negative effects of pesticides on non‐target arthropods, not only for regulatory requirements before a product is able to be registered, but also for knowing whether a plant protection product is suitable for using in IPM programs. In Europe, according to the Council Regulation (EC 1107/2009), the objective of protecting human and animal health and the environment should take priority over the objective of improving plant production. Therefore, it should be demonstrated, before plant protection products are placed on the market, that they present a clear benefit for plant production and do not have any harmful effect on human or animal health, including that of vulnerable groups, or any unacceptable effects on the environment. Regarding the effects on the environment, the following requirements have to be considered: the fate and distribution of the products in the

28 Introduction environment, their impact on non‐target species, including the ongoing behaviour of those species, and their impact on biodiversity and the ecosystem (OJEU, 2009a). With the aim of developing and validating test methods to assess the side‐effects of plant protection products to non‐target arthropods, the IOBC, the BART (Beneficial Arthropod Testing Group) and the EPPO (European and Mediterranean Plant Protection Organisation) in collaboration with the Council of Europe), decided in 1994 to set up a Joint Initiative (JI) (Barret et al., 1994). JI activities started in 1995 and different reports and conferences resulted in the publication of a guidance document for regulatory testing and interpretation of semi‐field and field studies with non‐target arthropods. They describe test systems, treatments, validity criteria of the studies, information on test organisms, test procedures, test conditions, biological observations, data analyses and reporting for selected terrestrial non‐target arthropods (Candolfi et al., 2000).

In 1974, the working group of the OILB/SROP, “Pesticides and Beneficial Organisms” was founded. Their main objective was to coordinate the developing of standard methodologies to evaluate side‐effects on the most important natural enemies and to choose selective pesticides to be used in IPM programs (Hassan, 1998). Pesticides are selected according to a sequential process which assumes that harmless pesticides in laboratory tests will be also harmless in semi‐field and field, and they do not need additional studies. Since 1980, standard guidelines to test side‐effects of pesticides on natural enemies; rearing methods for beneficial arthropods; comparison of results of laboratory, semi‐field, field experiments, and results of the joint programs to test the side‐effects of pesticides on beneficial organisms have been published (IOBC, 2011).

The measurement of the acute toxicity of pesticides to beneficial arthropods has traditionally relied on the determination of an acute median lethal dose or concentration. However, these tests can only be a partial measure of their deleterious effects. In addition to direct mortality induced by pesticides, their sublethal effects on arthropod physiology and behaviour must be considered for a complete analysis of their impact (Desneux et al., 2007). In extended laboratory and semi‐field experiments, treated vegetal material is used. In the case of extended laboratory assays, plants are

Introduction treated and carried to the laboratory, where experiments are performed. When fresh residues of the pesticides caused negative effects, possible effects due to the persistence of the products on the plants are also evaluated. Treated plants are maintained in greenhouses or outdoors up to the desired‐age residue is available. Then, they are carried to the laboratory to continue the experiment. Semi‐field tests are carried out with fresh applied products or residues on the plants, under similar field conditions, i.e. in greenhouses. Whether field studies are necessary, they are designed taking into account the habitat of the natural enemy and the good agricultural practices procedure, which involves respecting both the number of applications of a product and the minimum time period between treatments.

1.4 Natural enemies used in the experiments

1.4.1 Psyttalia concolor

Psyttalia (Opius) concolor (Szèpligeti) (Hymenoptera, Braconidae) is a koinobiont parasitoid of second‐ and third‐instar larvae of tephritids. Its host records are available for about 24 species. Originally described from material reared from olive fruit fly (B. oleae) infested olives in Tunisia (Szèpligueti, 1911), it Figure 9: P. concolor female has also been reared in Kenya from olive fly collected from Olea europaea var. cuspidata and from medfly (Ceratitis capitata (Wiedemann); Diptera, Tephritidae) in arabica coffee also in Kenya and in argan trees (Sapotaceae) in Morocco (Kimani‐Njogu et al,. 2001; Anonymous, 2011).

It belongs to the P. concolor species complex, which also includes P. humilis and P. perproximus (which have been treated as synonyms of one another), amongst others (Kimani‐Njogu et al., 2001; Rugman‐Jones et al., 2009). These species have been

30 Introduction distinguished by subtle differences in the length of the ovipositor and the size of the eye (Billah et al., 2008).

The adult body colour varies between light brown and yellowish, and measures in the region of 3.5 cm. Antennae are darker than the body. The female is able to parasitize different host’s larval instars although it prefers the third one, when larvae are close to pupation and are located nearer the surface of the fruits (Arambourg, 1986; Jiménez et al., 2002; Canale and Loni, 2006; Sime et al., 2006). The relative short ovipositor of females makes them unable to reach larvae that have burrowed deep into the olive, as commonly happens with second instars, which feed near the pit (Sime et al., 2006). Under laboratory conditions, the relative failure of P. concolor to locate the second larval stage it is likely to be related to the fewer vibrations produced by second instar hosts during feeding and/or movement (Canale and Loni, 2006). Sometimes there is superparasitism and more than one egg is laid inside the same larva (normally by different females). It is quite likely that the female paralyses its hosts to oviposit successfully, but by definition this paralysis must disappear fairly rapidly in order to allow the host to continue to fend for it and pupate. Parasitoid larvae have four instars (Cals‐Usciati, 1972) during which they consume the host up to its own pupation. The development of the parasitoid occurs in the range between 15ºC and 30ºC. Males’ development is shorter compared to females’ (Loni, 1997). Under laboratory conditions (25 ± 2ºC and 75 ± 5%HR), male developmental time is 17‐18 days and female’s is 21‐22 days. In the field, they overwinter as adults or immature stages inside the pupae of B. oleae found in soil of olive groves. Their survival is conditioned on winter temperatures, which can reduce populations or even make them disappear (Jiménez et al., 2002; Liaropoulos et al., 2005).

Members of P. concolor complex have been extensively used in both classical and augmentative biological control programs directed against tephritid pests. In fact, shortly after being described in Tunisia, it was introduced to olive‐growing regions of Italy (1914 and 1917‐1918) and France (1919 and 1931) (Rugman‐Jones et al., 2009) for controlling both B. oleae and C. capitata. In 1912 it was introduced in Hawaii from South Africa with the aim of controlling C. capitata (Daane and Johnson, 2010).

Introduction

However, despite the high percentage of parasitation reached at the beginning, this species was finally replaced by Diachasmimorpha tryoni Cameron (Hymenoptera, Braconidae) (Peters, 1996). P. concolor was the only olive fruit fly parasitoid found in Morocco and the Canary Island. However, few olive fruit flies were collected and parasitism rates were limited to 14.6 and 2.3% respectively. Similarly, less than 7% of the parasitism was recorded in the Republic of South Africa. On the contrary, it was the dominant parasitoid in Namibia (18 to 35% parasitism rates) (Daane and Johnson, 2010). Parasitism rates of P. concolor between 22.4% and 23.4% have also been detected in Spain in organic orchards in the Balearic Islands (Miranda et al., 2008).

After the development of its efficient mass‐rearing method using medfly in artificial diet in the 1950s, most European programs for olive fruit fly focused on P. concolor (Jiménez et al., 1990; Daane and Johnson, 2010; Anonymous, 2011). It has been routinely used in the Mediterranean Region (Spain, France, Italy, Greece, Portugal and former Yugoslavia) (EPPO, 2011a) for augmentative releases against B. oleae (Kimani‐ Njogu et al., 2001; Jiménez et al., 2002; Rugman‐Jones et al., 2009). To date, it has been the only imported species widely released and established in olive‐growing regions (Daane and Johnson, 2010).

Over the years, different releases of the parasitoid have been made in olive groves in order to evaluate the parasitism rates, their adaptation to the environment and the survival capacity either of the released individuals or their progeny. Most experiments have shown that releases should be made when fruit fly population levels are low, at the beginning of the summer, to control the first fruit fly generations. Otherwise, when B. oleae populations are high the parasitoid is not able to maintain them under the economic threshold levels (Jiménez et al., 1990; Civantos, 1999):

‐ In Italy, it is recommended to release twice a year: one inoculative release in spring, to control the first generation of the pest (which is inside the olive fruits from last year or as pupae in the soil), and a second inundative one to control summer‐ autumn generations (Delrio, 1995; Rotundo and De Cristofaro, 2003). P. concolor releases are only recommended when olive grove yield is high and as a part of an

32 Introduction integrated pest management program, being combined with other methods (Delrio et al., 2005). In Sardinia, parasitism rates between 60% and 100% were reached even if a low amount of parasitoids released. However, if climatic conditions are not suitable, there is not parasitism despite releasing a high amount of individuals (Delrio et al., 2005). ‐In Spain, Jiménez et al. (1990) carried out some experiments in Jaén, where parasitoids were released inundatively in order to test whether they were able to hibernate there or not. They demonstrated that progeny of P. concolor could be obtained from parasitized B. oleae the year after releases were done, however, the number of parasitoids collected was low compared to the number of them released. Other experiments carried out between 1997 and 1999 evaluated the parasitic activity of P. concolor in some different regions, demonstrating the efficacy of the parasitoid when is released during the summer (Jiménez et al., 2002). ‐In Corfu, Greece, P. concolor and P. concolor var. siculus Mon. were released during spring to determine whether they could be used to control olive fly infestations at that period. At an initial density of 300‐400 parasites per tree, the mean parasitism rates of 3rd stage larvae ranged from 30% to 50% in the first week following the release, indicating that P. concolor could work well in the spring in tall trees with large numbers of ripe and heavily infested fruits (Kapatos et al., 1977). ‐In California, where olive fruit fly arrived in 1998 and it is considered an invasive pest (Yokoyama et al., 2008), a classical biological control program was initiated in 2002 (Sime et al., 2006). Results from several experiments suggest that high summer temperatures limit olive fly abundance in California’s Central Valley (Wang et al., 2009) and it seems that nowadays biological control is effective enough to control pest populations (Yokoyama et al., 2008).

Parasitism rate differences among experiments can be due to the low quality of laboratory mass‐rearing parasitoids, climatic conditions and the abundance of fruit flies at the beginning of the summer or the olive grove yield (Delrio et al., 1995). Adult foodstuff availability and the possibility that parasitoids attack other non‐target fruit flies could also cause those different results (Yokoyama et al., 2008). Furthermore, mass‐reared parasitoid’s flight ability has been demonstrated to be lower than that of

Introduction wild parasitoids. This can be caused either by the mass‐rearing technique employed or by the genetic population composition (Delrio et al., 1995). Because mass‐rearing conditions can have an influence on host localization and parasitic capacity of some braconids used in biological control programmes, it could be interesting to periodically renew laboratory populations with wild individuals (Loni and Canale, 2005/2006; Estes et al., 2012). Psyttalia species are known to have specific host fruit and/or host fly preferences and despite the fact that they can successfully produce viable offspring in the laboratory, it is not known whether this populations/species interbreed in the field. When releasing these species, potential effects of interbreeding must be taken into account, especially in environments where other closed related species are presented or in situations where multiple introductions are intended. (Billah et al., 2008).

In Spain, the company Econex marketed P. concolor up to 2006 (De Liñán, 2007). They recommended releases of 100 to 500 adults per olive tree, choosing the most appropriate time depending on the state of development of the pest: in spring, against the first generation; from the end of June up to the end of August, against the summer generations; and during September and October against the overwintering generation, which will become the fruit fly adults of the next spring.

34 Introduction

1.4.2 Chilocorus nigritus

Amongst predaceous ladybeetles (Coleoptera, Coccinellidae), species of the genus Chilocorus (65 known species) are important scale‐predators, capable of removing heavy infestations and thereby increasing crop yields (Hattingh and Samways, 1994; Omkar and Pervez, 2003).

In olive groves, one of the most common species within the Coccinellid community is C. bipustulatus (Santos et al., 2010), which is indigenous to the Mediterranean region (Smith, 1915). It is a polyphagous predator of scale insects, such as the California red scale (Aonidiella aurantii (Maskell) (Homoptera, Diaspididae), the Egyptian black scale (Florida red scale), Chrysophalmus aonidium L. (Homoptera, Diaspididae), the Florida wax scale, Ceroplastes floridensis Comstock (Homoptera, Coccidae), and the black scale, S. oleae (Nadel and Biron, 1964). However, due to the difficulties to have enough individuals to carry on laboratory experiments, the coccinellid C.nigritus F., which is sold by different European companies, has been used as representative species of it in this thesis.

C. nigritus is native to the Indian subcontinent (Samways and Tate, 1984; 1986; Hattingh and Samways, 1994; Omkar and Pervez, 2003) and South‐east Asia (Ponsonby and Copland, 1998; 2000). After its establishment in South Africa it soon spread naturally or was artificially introduced in other regions. Evidence Figure 10: C. nigritus adults suggests that once established in an area, the beetle becomes a permanent member of the local fauna. Amongst other Chilocorus species, it is maybe one of the most tolerant to extreme temperatures, although their theoretical lower thermal development threshold is 16.6ºC (Samways and Tate, 1984; Ponsoby and Copland, 1998; Omkar and Pervez, 2003). This adaptive

Introduction strategy has probably helped it in its establishment in the tropical and sub‐tropical regions. It prefers hot and humid summers and cold dry winters (Omkar and Pervez, 2003), although is well adapted to the drier savannah areas too (Greathead and Pope, 1977). It has not yet established in high altitudes (Samways and Tate, 1986). Because of its tolerance to a wide range of temperature and humidity, it is suggested that perhaps the reasons for the failure to the species in apparently favourable climates may be due to other factors. For example, prey suitability and/or the presence of natural enemies or pathogens to which the species has no resistance (Ponsonby and Copland, 1998), although it seems to be rarely attacked by natural enemies (Samways and Tate, 1986; Ponsonby, 2009). It is an active predator of many insect species, such as different aphid species or the spiralling whitefly Aleurodicus disperses Russel (Hemiptera, Aleyrodidae). However, it prefers scales, especially Diaspididae, such as A. aurantii Maskell, Asterolecanium miliaris Boisduval or Aspidiotus nerii Bouché (Greathead and Pope, 1977; Samways, 1984; Samways and Tate, 1984; 1986; Ponsonby and Copland, 2000; Omkar and Pervez, 2003), when they are on citrus, sugar cane, coconut and other tropical and subtropical crops (Ponsonby and Copland, 1996). It feed on all sessile stages of the scales and it is the most important predator of gravid adult females (Samways, 1984; Samways and Tate, 1986). It is more efficient in removing medium and medium‐high densities of scale (15.8‐23.5 scales/cm2) than at very high ones (60 scales/cm2) (Omkar and Pervez, 2003).

Adults are easily recognizable due to their almost semi‐spherical shape, with sizes ranging from 3 to 5.5 mm. Their elytra are shiny black with sparse simple punctures. They have dull orange areas between the eyes and on the antero‐lateral tips of the pronotum. Females lay bright yellow spindle‐shaped eggs individually on the substrata, to which they are attached by one of their ends (in the shields of death scales, on Figure 11: C. nigritus larva the silk of spider webs or any other substrata composed of loose fibres, and on scale‐infested leaves). Early first instars hatch out and they usually moult thrice to undergo four larval instars. Greyish yellow larvae have the

36 Introduction dorsal surface covered with spiny hairs. They also have characteristic dark patches on the second, the third and from the seventh to the ninth segments, which impart a banded appearance. Mature larvae congregate inside the concavity of crumpled leaves to pupate (Samways, 1984; Omkar and Pervez, 2003). First instars chewed through the newly formed scale cover and sucked out the body juices, leaving the cuticle behind. The other three instars completely removed the scale cover and sucked out the body contents, leaving the cuticle behind or partially eaten (insects are rarely completely eaten). Piercings or puncturings on the scales lead to their rapid dehydration (Ponsonby and Copland, 2000; Omkar and Pervez, 2003). Larvae and adults are positively phototactic and negatively geotactic. They use the prominent features of the plant, such as leaf veins and margins, to guide their searching behaviour. Since prey species shows similar photo‐ and geotaxis and feed largely from the veins, such behaviour tends to concentrate predators at sites of high prey density (Ponsonby and Copland, 1995). Temperature varies developmental times, the pre‐oviposition period and the oviposition rate (Samways and Tate, 1984; Ponsonby and Copland, 1998; Omakar and Pervez, 2003). At 26ºC, mean development period days are 6.6 ± 0.6 for the eggs, 7.5 ± 0.8 for the first instar, 5.2 ± 0.6 for the second one, 5.4 ± 2.5 for the third one, 9.9 ± 1.6 for the forth one and 6.1 ± 0.2 for the pupa. This means 34.0 ± 3.7 days from egg to adult (Ponsonby and Copland, 1996). The influence of biotic and abiotic factors of prey development can reduce predator fitness, i.e., their immature development and adult reproduction. Immature stages are always more affected than adults (Hattingh and Samways, 1994). The reproductive activity of the females can also be inhibited, delaying oviposition for several days (Omkar and Pervez, 2003).

It has been successfully utilized as a natural enemy in many biological control programs, and classically introduced in scale prevalent regions time and again (Samways, 1984; Omkar and Pervez, 2003; Ponsonby, 2009). Success of C. nigritus and other coccidophagous beetles has been attributed to their high searching efficiency and quick response to the changes in prey density (Omkar and Pervez, 2003). According to the data available on the EPPO website, both C. nigritus and C. bipustulatus are commercially used as biological control agents (EPPO, 2011b). There have been many experiences in releasing C. nigritus over the years:

Introduction

‐ In the EPPO countries, data of first use of C. nigritus is 1985 and it is currently used in Belgium, Denmark, France, Germany, the Netherlands and United Kingdom. It is used indoors, mainly against Diaspididae and Asterolecaniidae,

but it has not been successfully established in this area (EPPO, 2011b). ‐ In South Africa and Swaziland, it has been reared in several insectaries primarily against California red scale, (Samways and Tate, 1984), which became resistant to organophosphate insecticides (Samways, 1984). The predator has been found to be the most effective natural enemy, along with Aphytis spp. (Hymenoptera, Aphelinidae), against A. aurantii (Uygun and Elekçioglu, 1998). ‐ In the coconuts industries of the Seychelles and Mauritius it has been used against A. aurantii and other scales on various plants, including citrus. ‐ In India, it has been released to control Coccus viridis Green (Homoptera, Coccidae) on coffee (Samways, 1984). ‐ In Pakistan, against the diaspidids Aspidiotus destructor Signoret (coconut scale), Aspidiotus orientalis Newstead, Pinnaspis strachani Cooley and Quadraspidiotus perniciosus Comstock (Samways, 1984). ‐ In the New Hebrides, against the coconut scale (Samways, 1984). ‐ In UK glasshouses it has been considered as a potential natural enemy of different scales (Ponsonby and Copland, 1996).

Chilocorus nigritus (F.) mass rearing can be done using several diaspids and/or certain artificial diets (Omkar and Pervez, 2003). Nutritional requirements of coccinellids, similarly to other predatory groups, are very specific. Thus, artificial diets that support normal rates of coccinellid egg production are not commercially available. Honeybee products or brood have been used for semi‐artificial diets (Obrycki and Kring, 1998). However, artificial diets are still inferior to natural prey and not adequate as the sole food source for rearing consecutive generations. They are just valuable as substitute food in the insectary during shortages of natural prey (Hattingh and Samways, 1993). Cannibalism by larvae and adults is another persistent problem in mass rearing of many coccinellid species (Obrycki and Kring, 1998).

38 Introduction

According to some authors, the introduction of predator eggs in the scale‐infested areas is not advisable, since most of the hatched larvae starve when scale population is composed mostly of adult females (Omkar and Pervez, 2003). Therefore, adult releases are better, followed by older larvae (Hattingh and Samways, 1991). However, successful experiences in South Africa to control A. aurantii were done distributing predator eggs in polyester fibre pads (Samways and Tate, 1984; Obrycki and Kring, 1998). It enabled far more material to be introduced into the field, encouraged establishment of the species throughout larvae developing in situ (Samways and Tate, 1986), and it also partially overcome the problem of dispersal away from specific release sites (Samways, 1984).

Coccinellidae activity in suppressing pest populations is significant. However, it is poorly documented in many pest management programs that expect preserving natural enemies. They are often less susceptible than their prey to treatments, but they are highly affected by several insecticides. Toxicities vary widely among and within classes of insecticides and coccinellid species. Coccinellids efficacy in natural or managed systems is difficult to determine given their mobility and typically polyphagous nature. Adults may disperse from treated areas in response to severe prey reductions or because of insecticide repellence (Obrycki and Kring, 1998).

Introduction

40 Objectives

Chapter 2

OBJECTIVES

Chemical control is still the most common strategy applied against B. oleae. However, there is a shortage of a wide range of effective products to control it, especially in organic olive systems.

The objective of chapter number four of this thesis is to study the ecotoxicology of kaolin and two copper‐based products (Bordeaux mixture and copper oxychloride) on the two natural enemies P. concolor and C. nigritus. The use of kaolin and copper is allowed both in integrated management systems in organic farming. They act as deterrents of oviposition (kaolin) or bactericidal (coppers), rather than having insecticidal activity. Hence, seven different experiments both at laboratory and semi‐ field level using different routes of uptake have been designed to be able to better understand their activity. Several studies have demonstrated some negative effects of kaolin on auxiliary fauna at field level, which cannot be explained through classical laboratory assays. Therefore, specific experiments to study kaolin effects on the behaviour of insects, rather than the direct effects of the product on them, have also been designed.

In the fifth chapter of this thesis, the potential of three different insect growth regulators for controlling B. oleae is studied. The ecdysone agonists, methoxyfenozide, tebufenozide and RH‐5849 have been chosen. Furthermore, the ecotoxicology of the three products on P. concolor females is also considered. For both insects, the pest and the beneficial, not only of biological assays are performed, but also molecular and docking experiments.

Objectives

General material and methods

Chapter 3

GENERAL MATERIAL AND METHODS

In the current chapter, general material and methods of the experiments on side‐ effects and efficacy carried out in this study are explained. The following items can be found:

‐ Environmental conditions of the different insect rearing and laboratory experiments. ‐ Insect rearing. ‐ Common characteristics of the experiments. ‐ Parameters evaluated. ‐ Statistical analysis.

3.1 Environmental conditions of insect rearing and laboratory experiments

Both insect rearing and laboratory experiments, unless otherwise specified, were performed in a controlled environmental cabinet (4.25 x 2 x 2.5 m3) in the laboratory of “Protección Vegetal” (Polytechnic University of Madrid) with the following conditions:

‐ Temperature: 25 ± 2º C ‐ Relative humidity: 75 ± 5 % ‐ Photoperiod: 16 : 8 (L : D)

In order to keep the adequate temperature, there is an air‐conditioner‐heat pump (Interclisa®, CUCVO26M3 + CXE 26M3), regulated by a thermostat (Sunvic®).

Relative humidity percentage is controlled with a humidistat connected to a humidifier (Defensor®, model 505).

General material and methods

To control either temperature or humidity regulation systems, there are a thermo‐ hygrograph (model Salmoiraghi® 1750) and a digital thermo‐hygrometer with memory for maximum and minimum absolute values.

Photoperiod is achieved with two strip lighting (Sylvanya Gro‐Lux®) placed above each shelf, which give a luminance in the region of 2.500 lux at 20 cm distance. Switching on and off of lights is controlled with a switch clock (Orbis®).

3.2 Insect rearing

3.2.1 Psyttalia concolor

The parasitoid P. concolor is reared in the laboratory on the alternative host C. capitata (medfly) following the methodology proposed by González‐Núñez (1998), which is slightly modified from the one described by Jacas and Viñuela (1994). Neither C. capitata nor P. concolor have ever been exposed to insecticides and both rearings have been renewed from time to time with field individuals.

Psyttalia concolor population in our laboratory comes from the original one that the “Instituto Nacional de Investigaciones Agrarias” had in “El Encín”, a researching centre which was located en Alcalá de Henares, Madrid.

General material and methods

3.2.1.1 Mass rearing of Ceratitis capitata

3.2.1.1.1 Adults’ cage

Medflies are reared in methacrylate cages (40 x 30 x 30 cm) with around 3,000 flies per cage. The front side of the cages is covered with mesh, which is used for females to oviposite. Mesh also allows aeration inside cages. Eggs are laid through the mesh after a pre‐oviposition period of Figure 12: Cage of C. capitata adults’ rearing 4‐5 days and they fall into a plastic tray containing water, from which they are collected daily by filtering.

Cages have two round holes (8 cm diameter) on the upper side covered with mesh. The diet, a mixture of hydrolyzed protein and sugar (1:4; MP Biomedicals Inc: Azucarera Ebro S.A.), is offered through the holes. Water is supplied ad libitum in a plastic pot with a piece of Spontex® wiper, placed inside the cage.

Adults are usually used for obtaining eggs during a week, although they can survive longer.

3.2.1.1.2 Eggs handling

Eggs (less than 1 day old) are collected daily from the plastic trays (39.5 x 30.5 x 4.5 cm) with water in which they have been conserved since they were laid. A thin mesh (for example, tights) fixed to a funnel is used to filter the eggs. About 2,000 eggs are placed on each larval diet tray (25 x 15 x 4 cm; 3 eggs/g diet). They should be previously mixed with a small amount of water to be able to scatter them evenly.

General material and methods

3.2.1.1.3 Larvae rearing

Larvae are reared in plastic trays containing a specific diet consisted of:

 Wheat bran (Harinas Polo S.A.) 400 g  Sugar (Azucarera Ebro S.A.) 112 g  Brewer’s yeast (Vigor®, Santiveri S.A.) 58 g  Methylparaben (Nipagin®, Central Ibérica de Drogas S.A.) 4.5 g  Propylparaben (Nipasol®, Central Ibérica de Drogas S.A.) 4.5 g  Benzoic acid (Panreac ®, Montplet & Esteban S.A.) 4 g  Water 900 ml

All ingredients, except water and benzoic acid, are mixed in a two‐liter container with a mechanical mixer (Turbula®, WAB) during an hour to ensure a homogenous mixture.

Water is heated up and when it boils, benzoic acid is added and dissolved with the help of a magnetic mixer. Then, they cool down up to 40ºC and they are mixed in a tray with the rest of the ingredients, using a spatula. Diet is then distributed into two plastic trays (25 x 15 x 4 cm; 750 g diet/tray). It is squashed and covered with aluminium foil. It can be conserved in the fridge up to two weeks, being careful to let it get warmer up to room temperature before placing eggs on it.

Trays with the diet are placed inside a methacrylate cage. Eggs hatch two days after placing them on the diet. 8‐9 days later, third instar larvae (already fully developed) search for a dry place to pupate. Thus, larvae jump out from the diet and pupate on the floor of the cages. Once pupated, pupae are collected and stored in small plastic cages (12 cm diameter, 5 cm high).

Some days before adult emergence, new adult cages are built up. Pupae are placed inside them and mass‐rearing continues.

General material and methods

3.2.1.2 Mass-rearing of Psyttalia concolor

Psyttalia concolor adults are kept in methacrylate cages (40 x 30 x 30 cm) containing around 500 wasps per cage. Lids of the cages consist of a mesh which provides both ventilation and the physical support for the parasitization process. Water is supplied ad libitum in a glass pot with a piece of Spontex® cloth protruding out of it. Food, consisted of a milled mixture of brewer’s yeast and icing sugar (1:4), is also provided ad libitum in a plastic stopper. Cages are renewed weekly. Figure 13: P. concolor adults’ cage

3.2.1.2.1 Parasitization

About 500 fully grown larvae of C. capitata are offered to P. concolor adults by sandwiching them between two pieces of mesh hold together with a wooden frame (14 cm diameter). Frame is placed on the roof of the parasitoid rearing cage and females sting medfly larvae through the mesh. A bag with sand is put on top of the frame to prevent larvae from jumping (otherwise females are not able to parasitize them). After one hour of exposure, larvae are transferred to a plastic cage (12 cm diameter, 5 cm high).

Adult flies emerge from the non parasitized pupae after 7‐8 days. Parasitoid males do it after 17‐18 days approximately and females after 21‐22 days, at 25±2 ºC. Parasitoid adults are collected and transferred to the rearing cages for renewing the population or are used in the experiments.

General material and methods

3.2.2 Chilocorus nigritus

The availability of C. nigritus adults and larvae was limited. Only a few companies in Europe sell them. Adults used in the current experiments were normally supplied by the company Entocare Biological Crop Protection (Wageningen, The Netherlands). When it was possible, adults were reared in the laboratory, but Figure 14: Temptative C. nigritus rearing established in the laboratory their number was usually too low to perform assays.

A mass rearing of the predator was tried to be established in the laboratory. Hattingh and Samways (1993) screened promising diets for C. nigritus based on different artificial diets for other entomophagous insects. Two suitable diets, one for adults and one for larvae, were obtained. However, they were still inferior to natural prey and not adequate as the sole food source for rearing consecutive generations (Ponsoby, 2009). Other coccinellid mass rearing experiences had also demonstrated that predators acquired significantly higher survival and faster development when feeding on live prey, as it occurred with Menochilus sexmaculatus (F.) (Coleoptera, Coccinellidae) when compared the parameters above after feeding on Myzus persicae (Sulz.) (Homoptera, Aphidae) or artificial diets (Khan and Khan, 2002). Females also showed more preference for laying eggs on scale‐infested butternuts than on an artificial substrate (Murali‐Baskaran and Suresh, 2007).

In order to have an available source of live prey for C. nigritus, a mass‐rearing of scales was established in the laboratory. In absence of living prey, C. nigritus adults were fed with eggs of Ephestia kuehniella Zeller (Lepidoptera, Pyralidae). Distilled water was also provided ad libitum in glass vials similar to those described for P. concolor.

General material and methods

3.2.2.1 Mass-rearing of scales

The literature on host relations of C. nigritus is extremely ambiguous and appears to suggest the presence of different ecotypes and/or biotypes. For example, first instar larvae of South Africa origin were only capable of feeding on first instar A. nerii, while larvae of a similar stage from Pakistan were able to feed on first and second instar Aspidiotus cyanophylli Signoret (Homoptera, Diaspididae). However, prey species may not be as important as prey population structure. Indeed, highest levels of beetle reproduction occur when there are high densities of overlapping generations of scale that include all stages of development (Ponsoby, 2009).

Best survival rates and adult fecundity appeared to be from beetles cultured on biparental A. nerii reared on potato tubers (Solanum tuberosum L.). Butternuts (Cucurbita moschata Duch. ex Lam.) have also demonstrated to be a good alternative (Samways and Tate, 1986; Hattingh and Samways, 1993; Ponsoby, 2009). Thus, both butternuts Figure 15: A. nerii rearing. Infested and uninfested butternuts and potatoes are placed on wire baskets and potatoes were used in A. nerii rearing.

Butternuts are more convenient to handle and provide a large surface area on which beetles can feed. In contrast, potatoes are used principally as mother stocks. They are able to support heavier scale infestations than butternuts and crawlers can also readily leave potatoes to move onto new material rather than settling on an open area, as they are inclined to do on butternuts. Scale‐infested vegetal should be removed from the culture from time to time because fungal contamination of the butternuts occurred.

General material and methods

Both fresh butternuts and potatoes were bought in the supermarket, because picking them direct from the field was not available. They were carefully washed in a 0.025% solution of sodium hypochlorite and dried before offering to scales.

Rearing of scales was started with A. nerii‐infested butternuts from a public insectary in Silla, Valencia (Spain). It was carried out slightly modifying the methodologies proposed by Samways and Tate (1986) and the public insectary mentioned above.

The culture of the scales was maintained in plastic cages (500 x 400 x 250 mm) in the previously described climatic chamber. Vegetal material was placed on wire baskets to improve aeration and avoid fungal contamination. Cages had two holes on both sides and one on the front part (30 x 10 cm) covered with mesh to allow aeration and to prevent crawlers from escaping. Cages were painted black colour. Because crawlers are attracted upwards to the light (Samways and Tate, 1986), they were supposed to walk up to the front part of the cages, so it would be easier to collect them. This mass‐rearing procedure was followed in the insectary of Silla, where scales were reared in big dark chambers with a light point in the middle. However, the methodology did not work in the laboratory and infestation of new butternuts by crawlers was decided to be by contact. Thus, uninfested potatoes and butternuts were placed among the infested ones. Within the hours, crawlers infested the new material. This made it easier to get homogeneously infested vegetal material because crawlers did not need to walk long distances to find non infested surfaces. There was not a strict routine to this procedure.

When scales on butternuts had developed up to a level in which they formed a white layer on the surface of the vegetable, they were then ready to be transferred to beetle cages or used in the experiments. However, despite the efforts to rear the scales, their development was not fast enough, as it could take several months to have butternuts with an appropriate scale‐infestation level. Infested material could only be used in the dual choice and no‐choice experiments (explained later in Chapter 4).

General material and methods

3.2.3 Bactrocera oleae

Mass‐rearing of B. oleae on an artificial diet has difficulties and the procedure still needs different improvements. Issues include: the desing of the cages and oviposition substrated, the cost and quality of artificial diets, the maintenance of endosymbiotic microbiota, the control of pathogenic microbes, the collection of pupae and the fitness of adults (Estes et al., 2012).

Although we attempted to rear B. oleae in our laboratory with individuals sent from the FAO/IAEA (International Atomic Energy Agency) Agriculture and Biotechnology Laboratory in Seibersdorf (Austria), different problems forced us to use wild olive fruit flies for the experiments

We were able to obtain eggs using a paraffined gauze as artificial substrate, but fungal contamination on the artificial diets prevented the larvae from developing. This fungal contamination affected either some of the ingredients used in the diet or the laboratory installations, and it was not possible to decontaminate none of them. Figure 16: Fungal contamination of B. oleae artificial diet

Bactrocera oleae adults were finally obtained from infested olive fruits collected from different olive orchards in Spain: Villarejo de Salvanés in Madrid (cv. Manzanilla and Cornicabra); Jaén and Alcalá la Real in Jaén (cv. Picual); Alía and Guadalupe in Cáceres (cv. Verdial, Manzanilla Figure 17: Methacrylate cages where third‐instar larvae of B. cacereña and Cornicabra). oleae were collected when they jumped from the olive fruits Infested fruits were taken to the

General material and methods laboratory and placed on plastic grilles situated on the top of methacrylate cages. When larvae were close to pupate, they jumped out from the fruits into the cages and pupae were collected. Adults emerged from these pupae were used in the experiments. They were fed with a mixture of icing sugar: hydrolyzed protein (Azucarera Ebro S.A.: MP Biomedicals Inc; 4:1). Distilled water was provided ad libitum as previously described.

3.3 Common characteristics of the experiments

Unless otherwise specified, in the case of P. concolor ten individuals per replicate were used to perform the different experiments. Eight or nine adults per replicate in the case of C. nigritus were used, depending on their availability. Five replicates per treatments were always used if possible.

Unfed, mated females, less than 48‐h old of P. concolor, were always used. Females were used instead of males because they live longer. Furthermore, females, which only need mating once, are the ones which control pest populations (Ragusa, 1974). Newly emerged individuals were used because they use to be the most sensitive to pesticides (Croft, 1990). They were not fed because it has been demonstrated that pesticide sensitivity of some insect is modified when they are fed (Viñuela and Arroyo, 1983).

In the case of C. nigritus, adults (unknown age or sex) were always used in the experiments. Experiments with larvae were not performed because it was not possible to achieve a homogeneous‐age population of larvae.

In the case of B. oleae, ten less than 48‐h old adults were used.

General material and methods

Unless otherwise specified, individuals were placed in ventilated round plastic cages (12 cm in diameter by 5 cm, with a 5.5 cm‐diameter ventilation hole covered with mesh on the top). Distilled water was provided ad libitum in small glass vials (30 x 35 mm) covered with Parafilm® and a piece of Spontex® wiper leaking out of it. No water was offered to C. nigritus. Diet was supplied in small plastic stoppers (24 x 6 mm) (diets for each insect were the same as the diets described for their mass rearing: brewer’s yeast and icing sugar (1:4) for P. concolor, E. khueniella eggs for C. nigritus and sugar and hydrolysed protein (4:1) for B. oleae).

Both the glass vials and the plastic stoppers were fixed to the floor with Plasticine®. Water and diet were renewed when necessary.

Figure 18: Round plastic cages used in the experiments

General material and methods

3.4 Parameters evaluated

3.4.1 Mortality

Mortality was always scored at 24, 48 and 72 hours. Depending on the test, a longer period could also be evaluated. Normally, 72h after the treatments, survivors were moved to non‐treated cages to study the effects of the products on reproduction or life span.

3.4.2 Life span

Life span was measured as the average of days that each insect survived in each replicate after the treatments. It was evaluated daily (or twice a week when more than two months had been lasted since the experiment started) using similar cages and conditions than the ones described above.

3.4.3 Effects on reproductive parameters

Reproductive parameters in the case of parasitoids are also known as “beneficial capacity”. It is measured as the percentage of attacked hosts (percentage of puparia without medfly emergence) and the percentage of progeny size (percentage of parasitoids emerged from parasitized puparia). To evaluate these two parameters on P. concolor, 72h after the treatments five females per replicate, when possible, were transferred from the plastic cages described above to a Figure 19: Cages used to evaluate beneficial capacity of P. concolor parasitization cage.

General material and methods

Parasitization cages consist on untreated round cages similar to those described above but with a hole (5.5 cm‐diameter) covered with mesh on the bottom, through which females parasitized C. capitata larvae. Distilled water and diet are provided ad libitum as described above. When possible, five replicates per treatment were also performed to evaluate beneficial capacity.

During the following five days, 30 fully‐grown C. capitata larvae are offered to females in each cage replicate. The larvae of the medfly are previously collected from their diet and placed in water before offering them to female wasps to avoid pupation. Larvae are immobilized by sandwiching them between the mesh of the cage’s floor and a piece of Parafilm® placed on the bottom of a glass pot, all held together with a rubber band. After one hour of exposure, C. capitata larvae are transferred to Petri dishes to allow them Figure 20: C. capitata larvae transferred into pupate (effects of the products on beneficial Petri dishes after 1hour of exposure to P. concolor females capacity can approximately be evaluated one month after parasitization).

Data from the first day of parasitization are rejected because previous experiments had shown that females needed one day before getting used to parasitizing in their new cages.

Effects on reproductive parameters were only possible to be measured in the case of P. concolor. In the case of C. nigritus, the difficulty of determining the number of eggs laid by females on the butternut surface, together with the difficulty of sexing individuals to make pairs, made it not possible to determine fecundity and fertility rates during the experiments using butternuts as oviposition substrata. Surgical gauzes were provided as artificial substrata, as it was demonstrated that it was highly suitable as substrate for oviposition and thus for augmentative releases in glasshouses environments (Ponsoby, 2009). Synthetic cotton was also provided as oviposition

General material and methods substrata, but a low amount of eggs were laid on them and the larvae hatched died always before pupating. Furthermore, C. nigritus preferably needs prey to reproduce and our scale mass‐rearing was not big enough to continuously provide A. nerii.

These parameters were not evaluated in the case of B. oleae either because we did not have enough non damaged olive fruits to offer to females to oviposit.

3.5 Statistical analysis

The data were subjected to a one‐way analysis of variance (ANOVA). Fisher’s least significant difference (LSD) test was used to compare the responses to the insecticides. All statistical analyses were performed using Statgraphics® version 5.1 (STSC 1987). If necessary, the data were transformed using arcsin (x/100) for percentages and log (x+1) otherwise. The untransformed data (mean values and standard errors (S.E.)) were shown in the tables. If any of the assumptions of the analysis of variance were violated after appropriate transformations, the non‐parametric Kruskal‐Wallis test was applied. When P < 0.05, the idea that the differences are all a coincidence can be rejected. This doesn´t mean that every group differs from every other group, only that at least one group differs from one of the others. In this case, the Dunn’s post‐test was applied. This test compares the difference in the sum of ranks between two columns with the expected average difference (based on the number of groups and their size). Median values are considered significantly different if the 95% confidence intervals of the medians did not overlap.

The mean values of each parameter studied were corrected using the Schneider‐

Orelli formula [M (%) = [(Mtreated ‐ Mcontrol)/ (100 ‐ Mcontrol)] x 100] for mortality or the

Abbot formula [P (%) = [1‐ (Ptreated/ Pcontrol)] x 100] for reproductive parameters and life span. These corrected values were used to rank the products according to the IOBC. Pesticides were classified into the following four toxicity categories: for laboratory experiments the categories are: 1, harmless (<30%); 2, slightly harmful (30‐79%); 3, moderately harmful (80‐99%); 4, harmful (>99%). For extended laboratory and semi‐

General material and methods field tests the categories are: 1, harmless (<25%); 2, slightly harmful (25‐50%); 3, moderately harmful (51‐75%); 4, toxic (>75%) (Hassan 1998).

In the dual choice and no‐choice assays different statistical analyses were done.

For P. concolor, significant differences between treatment means were detected using the two sample t‐tests. If any of the assumptions of the analysis are violated, the non‐parametric Mann‐Whitney U test is applied.

For C. nigritus, data were analysed by multifactorial ANOVA because more than one factor was taken into account (A. nerii infestation and treatment).

Different models were used to fit survival of C. nigritus with regard to treatments in the two experiments performed with kaolin and coppers. Survivorship curves are a graophical expression of the probability of surviving to age t as a function of t (Southwood, 1976). Survivorship data can be effectively summarized and compared using the shape and the scale parameters of the Weibull frequency distribution (Pinder et al., 1978). TableCurve 2D (Jandel Scientific, 1994) was used to models fitting and parameter estimation.

F (t) = 1 –exp [‐(t/b)c] t,c,b > 0

in which b and c are respectively the scale and shape of the Weibull frequency distribution. The c parameter controls the rate of change of the age‐specific mortality rate and, therefore, the general form of the survivroship curve (Southwood, 1976).This function shows four basic types of curve (Slobodkin, 1962; Southwood, 1976); in type I mortality acts most heavily on the old individuals, in type II a constant number die per unit of time; in type III the mortality rate is constant and in type IV mortality acts most heavily on the young stages. In the Weibull frequency distribution, high values of c determine a type I curve, while low values determine type IV values (Pinder et al., 1978).

General material and methods

Kaolin and copper-based products

Chapter 4

LETHAL AND SUBLETHAL EFFECTS OF KAOLIN PARTICLE FILMS AND COPPER- BASED COMPOUNDS ON THE NATURAL ENEMIES PSYTTALIA CONCOLOR AND CHILOCORUS NIGRITUS1

4.1 Introduction and objectives

As previously mentioned in the introduction, B. oleae has been proved to be able to develop resistance to some of the products commonly applied against it. Therefore, a suitable pest management program should include different control measures that prevent resistance development. In this context, kaolin particle films and copper‐based compounds might be considered as an alternative. Furthermore, both products could be also applied in organic olive groves.

Up to nowadays, side‐effects of kaolin and copper‐based products on natural enemies found in olive orchards have been mainly tested at field level. Most of these studies showed results concerning the presence/absence or mortality of the non‐ target organisms after the application of the products. However, much less is known about which the causes of those results are. Whether a minor number of insects are due to a direct mortality of them after the treatments or due to other sublethal effects is what the current study will try to evaluate.

1 BENGOCHEA P, HERNANDO S, SAELICES R, ADÁN A, BUDIA F, GONZÁLEZ‐NÚÑEZ M, VIÑUELA E, MEDINA P, 2010. Side effects of kaolin on natural enemies found on olive crops. IOBC/wprs Bull 55:61‐67. BENGOCHEA P, SAELICES R, AMOR A, ADÁN A, BUDIA F, MEDINA P, Effects of kaolin particle film and copper‐based compounds on the endoparasitoid Psyttalia concolor (Szépligetti) and the predator Chrysoperla carnea (Stephens) in olives. Sent to be published BENGOCHEA P, AMOR F, SAELICES R, HERNANDO S, BUDIA F, ADÁN A, MEDINA P, The lethal and sublethal effects of kaolin particle films and two copper‐based products on six natural enemies: laboratory assays. Sent to be published

Kaolin and copper-based products

Hence, the aim of the following experiments will be to better understand the possible sublethal effects of these products on the two natural enemies chosen, P. concolor and C. nigritus. The ecotoxicology of the products on P. concolor female adults has been carried out throughout different laboratory, extended laboratory and semi‐field experiments. In contrast, in the case of the predator, and due to the difficulty of having enough C. nigritus adults available, only a residual contact experiment and an extended laboratory assay have been carried out. When it was possible, additional experiments to test behavioural changes when insects are in contact with kaolin have also been performed.

4.2 Material and methods

All the experiments were carried out with P. concolor adult females or pupae, C. capitata larvae and C. nigritus adults obtained as it was specified in chapter 3: “General material and methods”. Unless otherwise specified, environmental conditions were the same as mentioned in that chapter.

Diet and distilled water were supplied ad libitum as it was described in chapter 3, although in the laboratory test in which insects were exposed to a treated inert surface, glass vials were a little bit smaller (15 x 22 mm) because the bigger ones did not fit inside the cages.

4.2.1 Chemicals

Active ingredients tested, their trade names and their formulations are listed in Table 4. A systemic insecticide, dimethoate, was used as a commercial standard because it is the most commonly applied insecticide in Spanish olive groves (Alvarado et al., 2008), even in integrated production systems (Civantos, 1999). It is utilized against the main olive pests in bait treatments either in terrestrial applications or in aerial treatments, being the last ones more specific against the olive fruit fly (Ruiz‐

Kaolin and copper-based products

Torres and Montiel‐Bueno, 2007). Solutions of product were prepared freshly in distilled water prior to the assays, based on their respective maximum field recommended concentrations (MFRC) in accordance with the Spanish registration, with a delivery rate of 1000 liter water ha‐1.

In the laboratory assays in which products were applied on an inert surface, the amount of insecticide applied per hectare was corrected by using the following formula: PIEC = (dose rate x fd)/100, where PIEC in the Predicted Initial Environmental Concentration (formulated product in µg/cm2); dose rate (formulated product in g/ha); and fd is the correction factor representing deposits under field conditions (0.4 for foliage dwelling predators), according to Barret et al. (1994) (see Table 4).

Table 4: Chemicals evaluated in the experiments

Active ingredient %a.i; PIEC2 Trade name Conc1 Trade Company (a.i.) form (µg/cm2) Surround BASF Española S.L., Kaolin 95 WP 5,000 g/hl 200 WP® Barcelona (Spain) Bordeaux Poltiglia 20 Manica SPA, 20 WP 1,000 g/hl 40 mixture WP® Trento (Italy) Copper Syngenta Agro S.A., ZZ‐Cuprocol® 70 SC 250 cc/hl 10 oxychloride Madrid (Spain) Danadim Cheminova Agro Dimethoate 40 EC 150 cc/hl 6 Progress® S.A. Madrid (Spain) 1Formulated product (concentration) 2Used only in some laboratory tests, in which an inert surface is treated

Figure 21: Chemicals used in the experiments

Kaolin and copper-based products

4.2.1.1 Kaolin

Kaolin is a white, non‐abrasive, fine‐grained aluminosilicate [Al4Si4O10(OH)8] mineral clay. Kaolin‐based particle film was originally employed in fruit production because of its agronomical benefits. It reduces heat stress by reflecting sunlight with its bright colour. It does not affect plant photosynthesis or productivity due to the porous nature of the film (Glenn et al., 1999; Glenn and Puterka, 2005). Indeed, crop yield and flower production can be increased by decreasing transpiration (Sisterson et al., 2003). It has also been hypothesized to control fungal and bacterial plant pathogens by preventing disease inoculum or water from directly contacting the leaf surface too (Glenn et al., 1999).

When sprayed on crops, researchers observed that this protective barrier creates a hostile environment for insects (Bürgel et al., 2005), making the host plant visually or tactually unrecognizable for pests (Glenn et al., 1999; Showler, 2003; Saour and Makee, 2004). Furthermore, it obstructs insect movements and feeding when particles attach to the insect’s body (Showler, 2003; Daniel et al., 2005). Additionally, it prevents egg‐laying (Bürgel et al., 2005) and it also impedes insect’s ability to grasp the plant (Markó et al., 2008), resulting effective against a range of pest insects such as psyllas (Puterka et al., 2000; Pasqualini et al., 2003; Vincent et al., 2003; Daniel et al., 2005; Gobin et al., 2005; Saour, 2005; Laffranque et al., 2009), tephritids (Caleca et al., 2008; Mazor and Erez, 2004; Braham et al., 2007), the Hymenoptera Ophelimus maskelli (Ashmead) (Eulophidae), which is a pest of Eucaliptus spp (Lo Verde et al., 2011), and some aphids, mites, leafhoppers, scales, Lepidoptera and Figure 22: Kaolin‐coated olive tree Coleoptera species (Knigth et al. 2000; Puterka

Kaolin and copper-based products et al., 2000; Phillips and De la Roca, 2003; Showler and Sétamou, 2004; Bürgel et al., 2005; Daniel et al., 2005; Glenn and Puterka, 2005; Kourdoumbalos et al., 2006; Markó et al., 2006; Laffranque et al., 2009).

In the last few years it has been tested against olive pests with good results on B. oleae (Phillips and De la Roca, 2003; Saour and Makee, 2004; Perri et al., 2007; Caleca and Rizzo, 2006; Iannotta et al. 2006; Pennino et al., 2006; Romero et al., 2006; Caleca and Rizzo, 2007; Iannotta et al., 2007b; Caleca et al., 2008; González‐Núñez et al., 2008; Laffranque et al., 2009). Visual and chemical stimuli lead the female olive fruit fly to oviposit into fruits, so the clay, especially white clays as kaolin, disrupts ovipositing females (Saour and Makee, 2004; Caleca and Rizzo, 2006; Iannotta et al., 2008). De la Roca (2003) also reported a good control of the carpophagous generation of P. oleae, as well as a minor presence of S. oleae, the second and third important pests in olive groves, respectively.

Other advantages of kaolin particle films are that they are not‐toxic to humans and they are relatively safe to natural enemies. They have no phytotoxic effects either. They last longer than most insecticides on the plants when it does not rain or there is not excessive dew formation. Additionally, they are washable and form a suspension in water, so they can be easily applied using conventional spray equipment. Furthermore it seems that pests are unlikely to develop resistance, because they are not an insecticide and, therefore, they do not have a specific target site (Peng et al., 2011). However, possible changes of insects’ behavior after several kaolin applications should be evaluated because they could get used to these coated surfaces.

However, the effectiveness of kaolin particle film as a control method is reduced by two factors: the frequent rainfalls, which washed off the particles, and the reduction of the number of predators and parasitoids (Markó et al., 2006). Beneficials can be affected both due to a direct effect on them and because of a reduction on their prey number, as it occurs with some coccinellid species (Pascual et al., 2010a). Nevertheless, the specific mechanisms that produce the decrease in certain arthropod taxa at the field level remain unclear. The reduction of some beneficials due to kaolin treatments

Kaolin and copper-based products could have strong negative effects and provoke an indirect increase of the populations of several pests, as it has been already reported for some apple pests and cotton aphids (Markó et al., 2006; Showler and Sétamou, 2004; Ulmer et al., 2006; Showler and Amstrong, 2007).

In Spain the use of kaolin is authorised in orange, clementine and pear orchards. In olive groves it can be used to control B. oleae and P. oleae. It should be applied before egg‐laying on fruits (MARM, 2011c)

4.2.1.2 Copper

Copper is nowadays the only fungicide allowed in organic agriculture, together with sulphur. Although it has been widely used in olive crops against fungal diseases, farmers noticed a positive effect on the control of B. oleae (Belcari and Bobbio, 1999), increasing the interest in the possible application of this product against this pest (Belcari et al., 2005; Rosi et al., 2007). It is known that copper products can play an important role as an oviposition deterrent, but researches have more recently been focused on its effectiveness as a bactericide. The fitness of the olive fly

Figure 23: Olive tree leaves and fruits covered by copper is greatly assisted by the presence of associated bacteria living on the phylloplane and in specialised parts of the gut. These microorganisms play an important role as a proteinaceous source for the adults and as elicitors of protein hydrolysis in the blind sac of the midgut of the larvae (Belcari et al, 2005; Estes et al., 2012). The symbiotic bacterium, named “Candidatus Erwinia dacicola”, is always associated to B. oleae and mothers, endowed with contractile perianal glands that become filled with bacteria, transmit symbionts to their offspring (Capuzzo et al.,

Kaolin and copper-based products

2005). Whether the bacterium colonizes the egg via the micropyle, or the larva consumes the bacterium during the eclosion, is unknown (Estes et al., 2012). Different studies have shown a high percentage of mortality on young larvae of the fly (first and second instar) in the absence of the bacteria (Belcari and Bobbio, 1999; Rosi et al., 2007). It seems to be important for both adult and larval nutrition (Estes et al., 2012).

It is the decrease of fruit fly populations after copper applications that could also favour the subsequent action of their natural enemies, which are only efficient when B. oleae population levels are low (Belcari and Bobbio, 1999).

Two copper formulations, Bordeaux mixture (CuSO4 + CaOH) and copper oxychloride have been tested in this study because the formulation of products can influence their action. Both of them are authorised in Spain to control different diseases in several crops. In olive orchards they can be applied to control olive knot disease and olive leaf spot (MARM, 2011c).

4.2.2 Laboratory tests

4.2.2.1 Residual contact on glass surfaces

A standard methodology to evaluate the residual contact activity of pesticides on non target arthropods in laboratory experiments was developed by Jacas and Viñuela (1994), according to the IOBC criteria.

Glass plates (12 x 12 x 0.5 cm in thickness) are treated with the chemicals under a Potter Precision Spray Tower (Burkard Manufacturing Co., UK) with 1 ml of each test solution at a pressure of 55 kPa to obtain a homogenous deposit of 1.5‐2 mg fluid per cm‐2. This deposit is within the interval recommended by the IOBC’s validity criteria for running ecotoxicological experiments on beneficial arthropods (Hassan, 1998).

Kaolin and copper-based products

Adults are exposed to dry residues of insecticides after spraying them on glass surfaces, using the slightly modified test cages designed for P. concolor tests by Jacas and Viñuela (1994). Test units consist of a round methacrylate frame (10‐cm diameter, 3‐cm high) and the two square glass plates described above. The plastic frame has six holes: two small (0.5‐cm diameter) and four bigger than them (0.7‐cm diameter). The smallest ones are covered by a mesh (for aeration), three of the biggest with tape (to prevent in sects from escaping), and the last one hold a hypodermic needle connected to a rubber tube which provides a continuous flow of air produced by an aquarium pump (to assure forced ventilation). As soon as the plates are dry (about half an hour after the application of the products, depending on the compound), adults are introduced to each test unit, which are then mounted and holding together with two crossed rubber bands.

The glass vials use in the glass cages are smaller than those described in Chapter 3 (15 x 22 mm) because the others do not fit in the cages.

Figure 24: Residual contact on glass surfaces test. Cages (which contain C. nigritus adults) are in the climatic chamber. The forced ventilation system is also observed

Kaolin and copper-based products

Psyttalia concolor experiment: each treatment consisted of five replicates and ten females per replicate. Cumulative mortality was recorded 24, 48 and 72 h after treatment. Subsequently, five females per replicate, when possible, were transferred to non‐treated cages to measure beneficial capacity as described in chapter 3.

Chilocorus nigritus experiment: each treatment consisted of five replicates. 9 adults per replicate were used. 72 h later, survivors were transferred to untreated round plastic cages to measure effects on life span after exposure to pesticides.

4.2.2.2 Oral toxicity

To evaluate the oral toxicity of the pesticides, they are offered via the drinking water. In this case, the experiment was done with P. concolor females and it consisted of five replicates per treatment; fifteen females per replicate were used.

After three days of exposure, five females per replicate were transferred to untreated parasitization cages and beneficial capacity was measured. Apart from the effects of kaolin and copper on direct mortality and beneficial capacity, their effects on life span were also measured (the experiment continued with the females that were not used to evaluate beneficial capacity). Pesticides were offered during the whole experiment (also during beneficial capacity measurement) until the last female died. Pesticide solutions were prepared at the beginning of the experiment in big quantities. Thus, if it was necessary to refill Figure 25: Pesticide solutions in the glass vials and any of the glass‐vials, the “age” of the plastic stoppers with the diet

Kaolin and copper-based products insecticide was similar to those in the other vials and we assured that no effects on insects were caused by freshly‐prepared solutions.

Figure 26: Testing of the effects when products are ingested

4.2.2.3 Treatment of parasitized pupae

The purpose of this test is to analyse the possible side effects of the products on the most protected stage of P. concolor. According to Jacas (1992), the eleventh day after parasitization is considered the moment from which parasitoids are more protected. The colour of pupae is used to distinguish the parasitized pupae from the non‐ parasitized. Although it was considered that the darkest pupae were the ones which should be used in the experiments, we observed that P. concolor emergence from them was not always as high as expected. In contrast, the pupae whose colour was between light brown and dark brown were the ones which shown a higher parasitoid emergence percentage. Thus, these pupae were chosen for the experiments. Light colour pupae were considered as not parasitized, while the darkest ones were probably superparasitized.

30 pupae per replicate (5 replicate per treatment) are first placed in Petri dishes and then treated using hand sprayers. After that, they are transferred to new Petri dishes, which have a filter paper on the bottom glass, to better absorb the excess of insecticide. Once they are dried (1 hour later), they are transferred to the round plastic cages described above.

Kaolin and copper-based products

One or two days before adult emergence (5‐6 days after the treatment), distilled water in glass vials and diet are placed in the cages. Emergence is evaluated daily. Immediately after emerging, adults are transferred to untreated cages to evaluate mortality and life span. When no more adults are emerged, beneficial capacity is evaluated, using five females per replicate. Females used for parasitization are between 3 and 5 days old. As we decided to transfer newly emerged adults to the same cage, we could not exactly know how old females were. Nevertheless, we made sure they were more than 72‐h‐old and had previously been in contact with males.

Figure 27: Treatment of pupae using hand sprayers

4.2.2.4 Treatment of the parasitization surface

The aim of this experiment is to evaluate whether kaolin and copper salts may modify beneficial capacity when P. concolor females parasitized through a treated surface. This experiment is an attempt to simulate which occurs in the field, when olive trees are treated and the surface of the fruits is covered with the products.

The bottom mesh of the parasitization cages (which is the mesh with which P. concolor females are in contact to reach L3‐larvae of C. capitata) is treated with the products using a hand sprayer. Five females per replicate and five replicates per treatment were performed. Females are transferred to treated cages 24 h after the

Kaolin and copper-based products treatment, when meshes are totally dried. However, the previous day they are placed in untreated cages with the aim of getting them to parasitize. Beneficial capacity is then measured during four more days as previously described in chapter 3. However, because at least 30 days after the treatments are needed to obtain data of attacked hosts and progeny size, it is not possible to know whether any deleterious effect on this parameter might be caused by the products after the four days of parasitization. Therefore, we decided to continue the experiment for three more days. Hence, it can be evaluated whether females, in case of previous negative effects, could reach their normal beneficial capacity rates when a non‐treated surface is offered. Thus, surviving females are transferred to untreated parasitization cages and 72 hours later beneficial capacity is measured again during four more days.

Figure 28: Treatment of the meshes through which P. concolor females parasitize

Kaolin and copper-based products

4.2.3 Extended-laboratory experiments

4.2.3.1 Treatment of olive tree leaves

Leaves were collected from small two‐year‐old olive trees (cv. Picual) grown in a greenhouse in the Experimental Fields of the ETSI Agrónomos (Polytechnic University of Madrid) and taken to the laboratory.

The products are applied using hand sprayers until the liquid ran off the leaves. Once the leaves are dried (1 hour later, approximately), they are transferred in groups of 18 (3 small branches with 6 leaves per branch) to the previously described plastic cages (12‐cm in diameter and 5‐cm high). Drinking water is offered in the small glass vials described in the residual contact activity assay.

Figure 29: Treatment of olive tree leaves

Kaolin and copper-based products

Psyttalia concolor experiment: each treatment consists of five replicates and ten females per replicate. Cumulative mortality is recorded 24, 48 and 72 h after treatment. Subsequently, five females per replicate, when possible, are transferred to non‐ treated cages to measure beneficial capacity as described in chapter 3. Figure 30: Detail of kaolin‐treated leaves in the plastic cages

Chilocorus nigritus assay: each treatment consists of 4 replicates and 8 adults per replicate are used. 72 h later, survivors are transferred to untreated plastic cages to measure effects on life span.

4.2.3.2 Treatment of the parasitization surface and olive tree leaves

This experiment also aims to evaluate whether kaolin and copper salts could modify beneficial capacity when P. concolor females parasitized through a treated surface. However, it tries to more accurately simulate field conditions than the previous one, in which only the surface was treated. Therefore, olive tree leaves are also treated and females are in contact with them during the first part of the assay (the four first parasitization days during which females parasitized through the treated surfaces).

The methodology followed is similar to the

Figure 31: Olive tree leaves and previously described. Both the parasitization surface parasitization surface treated and the leaves are treated using had sprayers. 3 small branches (18 leaves) are introduced in each cage. Drinking water is offered in the small glass vials described in the residual contact activity assay. 72

Kaolin and copper-based products

Similarly, after four days of parasitization, females are transferred to untreated parasitisation cages. No olive tree leaves are placed in untreated cages.

4.2.4 Semi-field experiment

Effects of kaolin and copper salts were evaluated under semifield conditions on P. concolor females.

Small‐55‐cm cv. Picual olive trees were grown in a greenhouse in Madrid (in the experimental fields of the ETSI Agrónomos). The average environmental conditions in the greenhouse during the experiment were 14.06 ± 2ºC, 65 ± 10 % r.h.

Trees are treated with hand sprayers with the corresponding compound at their maximum field recommended concentration until the liquid ran off. As soon as trees dried, they are covered with a wooden cage with a slight modification of the design by González‐Núñez (1998) to conduct semifield assays with the parasitoid P. concolor. Each cage consists of a wooden base and a wooden, plastic and gauze frame. The wooden square bases are 25 cm long and are painted in white. In one side there is a rectangular hole (13 cm x 2 cm) for the trunk of the tree. To contain insects in the cage, Plasticine® is used to seal the space between the trunk and the floor. The frames Figure 32: Olive tree in the wooden are 60 cm high. The top and three of the sides are cage. Glass vials the stoppers can also be observed covered with mesh. The fourth side consists of a methacrylate sheet to easily monitor the experiment. Each tree is considered an experimental unit. Each cage contains two small glass vials to supply water, similar to 73

Kaolin and copper-based products those already described. The diet is supplied using two plastic stoppers. Both water and diet containers are hung with a wire to the branches.

A total of 30 P. concolor females (less than 24 h old) were introduced per cage and exposed to the insecticides for a week. Three replicates per compound and control were performed. The beneficial capacity was measured directly in the greenhouse to avoid carrying the females to the laboratory; thus, measurements were carried out in the treated cages. Because the mortality was no high after the three

Figure 33: Semi field experiment in the greenhouse. days of exposure to the compounds, with In the top of the wooden frames, the sand bags used to prevent C. capitata larvae from jumping when P. the exception of dimethoate, the concolor females are parasitizing can be observed measurement of the beneficial capacity was made using six times more C. capitata larvae than in the other experiments. Therefore, 180 larvae were offered to the females, using two mesh pieces hold together with a wooden frame (15‐cm diameter). A bag with sand was placed on the top of the frames to prevent larvae from jumping (otherwise, the females would not be able to parasitize them, as it occurred in the mass rearing of the parasitoids). After one hour, larvae were placed onto Petri dishes and transferred to the climatic chamber in the laboratory to allow them pupate. Percentage of attacked host and progeny size were recorded as previously described.

Kaolin and copper-based products

4.2.5 Dual choice and no-choice experiments

4.2.5.1 Psyttalia concolor

This experiment was designed with the aim of evaluating the possible behavioural effects of kaolin when P. concolor females have the possibility of choosing between parasitizing through a treated surface and an untreated one. Both a dual choice test and a no‐choice test were carried out. In both cases, six replicates per treatment performed. Five females per replicate (72‐h‐old) were used. Every 15 minutes, during the hour of parasitization, the number of females Figure 34: Dual choice and no‐choice experiments. C. capitata larvae were searching for larvae or parasitizing both in the offered either on the top and the floor of the parasitization cages. The upper mesh and the bottom mesh was counted. small plastic stopper placed in the top of the cages to prevent larvae from Distilled water and diet were provided ad libitum jumping and escaping is apparent as previously described.

In this case, the two pieces of mesh of the parasitization cage are treated (the one usually used for ventilation and the one used for parasitize in the previous experiments, because it is not possible to treat only half of the parasitization mesh). 15 C. capitata larvae are offered on the top of the cages, and other 15 on the bottom. Larvae placed on the top mesh are covered with piece of Parafilm® and a small plastic stopper (4‐cm diameter) to prevent them from being mashed. Larvae of the bottom side are immobilized by sandwiching them between the mesh of the cage’s floor and a piece of Parafilm®. Parafilm® is placed on the floor of a glass pot put upside down. All the structure (the cage, the plastic stopper and the glass pot) is held together with a rubber band.

Kaolin and copper-based products

In the dual choice test, one of the meshes of the cages is sprayed with kaolin. To avoid possible effects on parasitization when females sting through the upper or the bottom mesh, two different treatments should be performed. Thus, the upper mesh of the cages is treated in 6 replicates, and the bottom one in the other 6 replicates.

In the no‐choice test, both the top and the bottom meshes are sprayed with kaolin. Distilled water was used to spraying the mesh in control units. 6 replicates for

Figure 35: Detail of P. concolor females parasitizing the kaolin treatment and 6 for the controls through the bottom mesh of the cages were done.

4.2.5.2 Chilocorus nigritus

The main objective of this experiment was to evaluate the possible repellence caused by kaolin treated surfaces on C. nigritus adults. The assay was carried out offering vegetal material to adults and observing whether they were found on the treated material or, by contrast, they preferred untreated parts. The plant material used was butternuts (C. moschata). Three replicates per treatment were done.

Both infested and uninfested butternuts were used in the experiment. Non infested butternuts were washed according to the procedure described in Chapter 3 for butternuts and potatoes. Infested butternuts were obtained from the vegetal material used for A. nerii rearing (see Chapter 3). Hand sprayers were used to treat the butternuts. When they dried, they were placed in the cages on an egg box to prevent them rolling in the cage.

Kaolin and copper-based products

Each experimental unit consists of a plastic cage (40 x 30 x 21 cm) covered with a piece of mesh to allow aeration. The mesh is held to the cage with a rubber band and some binder clips to prevent adults from escaping. Each cage contained the following materials: two butternuts, one infested with A. nerii and other one uninfested, placed on egg cardboards; a glass vial with distilled water (similar to those previously described); a plastic stopper with E. kuehniella eggs; and a piece of a semi‐solid diet (34‐mm diameter, 1‐cm high). The semisolid diet is elaborated with 66.67 g of honey, 33.33 ml of distilled water and 0.5 g of agar. Water and honey are heated up and when they boil, agar is carefully added and dissolved with the help of a magnetic mixer. They have to boil all together for three or four more minutes. Then, the mixture is let warmer up and store in the fridge until it is used.

Figure 36: Experimental units: plastic cages covered with a piece of mesh held with a rubber band and binder clips

In the dual choice experiment, just half of the butternut surface is covered with kaolin. In the no‐choice test, the surface of treated butternuts is completely kaolin‐ covered.

Adults were previously sexed according to the methodology proposed by Samways and Tate (1984). However, due to the difficulty of sexing the adults when they are alive, they were sexed again by dissection under a stereoscopic microscope when the experiment finished. Six pairs of adults were introduced in each cage with the help of a brush. Because it was not easy to distinguish between males and females at a glance, it was not possible to detect behavioural differences between sexes.

Kaolin and copper-based products

Figure 37: No‐choice experiment: controls. The non‐ Figure 38: No‐choice experiments: kaolin infested butternut is on the left of the picture and the replicates (non‐infested butternut on the left infested one is on the right. Butternuts are placed on and the infested one on the right). egg boxes. In the middle of the cage there is a glass vial with distilled water, a plastic stopper with E. kuehniella eggs and a piece of the semi‐solid diet

Figure 39: Dual choice experiment (on the left, the Figure 40: Detail of a kaolin‐treated infested butternut; on the right the non‐infested one). butternut. C. nigritus adults can be Half of the butternut was treated with kaolin and the observed on the treated surface other half with distilled water

The experiment lasted four days. Cages were transferred to the laboratory for the daily measurements. The rest of the time, they were maintained in the previously described climatic chamber. During the four days, daily measurements were done every half an hour during three hours. The number of adults on the butternuts or on the cages was counted. Butternuts were gently turning and then placed again on the egg cardboards. It was not possible to evaluate either fertility or fecundity because C. nigritus eggs were not easily visible on the butternuts. Nevertheless, the experiment was maintained 15 more days to observe the possible presence of larvae. Adults and

Kaolin and copper-based products unifested butternuts were removed from the cages. Because we observed that females tend to oviposit under the scales, only the A. nerii‐infested material was maintained.

Both in the case of P. concolor and C. nigritus, when the dual choice and no‐choice tests were finished every adult was observed under stereoscopic microscope, in order to find out whether kaolin particles were attached to their bodies or not.

4.3 Results

Results of the experiments carried out are stated below. Instead of giving the results of each experiment separately, they are grouped depending on the parameter evaluated. Different figures have been built up with the results obtained.

As an appendix at the end of the chapter, mean data and standard errors of each experiment are shown in different tables (Tables 6, 7, 8, 9 10 and 11). Furthermore, an additional table with the classification of the products according to the IOBC criteria for each parameter and has also been done (Table 12).

4.3.1 Direct mortality

Kaolin, Bordeaux mixture and copper oxychloride did not caused any deleterious effect on the percentage of mortality 72 h after the treatments, either for P. concolor or C. nigritus in most of the experiments performed (percentages of mortality < 10%). The sole exception was the evaluation of the oral toxicity of these pesticides on P. concolor females. In this case, when females ingested kaolin via their drinking water, an increase in the percentage of mortality was observed (36.0% of mortality). None of the two copper‐based products provoked a high mortality, compared to the controls. In great contrast, dimethoate killed 100% of insects within the first 24 h of all the treatments, except in the semifield experiment, in which 100% of P. concolor adults were killed 4 days after the treatment (Figure 41; Tables 6, 7 and 8).

Kaolin and copper-based products

Different experiments: % mortality

* * * * ** 100 90 80 70 60 Control 50 Mortality

Kaolin 40 % Bordeaux mixture 30 20 Copper oxychloride 10 Dimethoate 0 Residual Extended Semi field Oral toxicity Residual Extended contact laboratory contact laboratory (glass) (olive tree (glass) (olive tree leaves) leaves) Psyttalia concolor Chilocorus nigritus

Figure 41: Percentage of P. concolor and C. nigritus mortality 72 hours after different treatments. Asterisks indicate statistical differences between the treatments and the control (P<0.05)

4.3.2 Life span

No deleterious effects on the life span of P. concolor females emerged from treated pupae were found for none of the tested products, including dimethoate (F4,20 = 1.14, P = 0.3678). However, although the average number of days females lived after the pupae treatments was between 46.3 (dimethoate) and 55.3 (control), some females died 20 days after emerging, while others survived for 91 days. In contrast, when females ingested the products via their drinking water, statistical differences amongst all the treatments were found (F4,20 = 78.01, P < 0.0001). After dimethoate ingestion, the life span was reduced to less than 24 h. Furthermore, a clearly reduction of this parameter was caused by kaolin. The average number of days females survived in this experiment was lower than in the previous one. Females survived 53 days, as maximum, in the case of the controls; between 15 and 20 days when they ingested coppers, and less than 8 days if they ingested kaolin or dimethoate (Figure 42; Table 5).

Kaolin and copper-based products

Psyttalia concolor life span (number of days)

60 50 Control 40 Kaolin days 30 Bordeaux mixture No. 20 ** Copper oxychloride 10 * * Dimethoate 0 Oral toxicity Treatment of pupae

In the case of C. nigritus, either kaolin or copper salts did not provoke a reduction on the life span, both in the residual contact activity and in the extended laboratory experiments. When exposed to dimethoate, however, 100% of adults died after 24 h in both experiments (F4,20 = 26.58, P < 0.0001 and F4,15 = 14.80, P < 0.0001, respectively). Although the average number of days C. nigritus adults exposed to water, kaolin or copper treated surfaces (glass or leaves) were able to survive around 120‐140 days, some of them could survive longer (up to 357 days) (Figure 43; Table 8).

Chilocorus nigritus life span (number of days)

160 140 120 Control 100

days Kaolin 80

No. 60 Bordeaux mixture 40 Copper oxychloride 20 * * 0 Dimethoate Residual contact Extended (glass) laboratory (olive tree leaves)

Figure 43: C. nigritus life span (number of days) during the residual contact on a glass surface and the extended laboratory experiments. Asterisks indicate statistical differences between the treatments and the control (P<0.05)

Kaolin and copper-based products

The Weibull distribution fitted well the population survivorship data obtained for the four treatments considered, with R2 values greater than 0.92 in most of the cases (Table 5). Similar patterns were obtained for controls, kaolin and Bordeaux mixture both in the residual contact on glass surfaces and the extended laboratory experiments. For copper oxychloride, however, different patters compared to the rest of the treatments were obtained (Figures 44 and 45). In the residual contact on glass surfaces, the obtained curves correspond to a curve type I (taking into account the different survivorship curve data described by Slobodkin (1962) for control, kaolin and Bordeaux mixture (mortality acts most heavily on the old individuals) and type II‐III for copper oxychloride (the mortality rate is more or less constant). In the extended laboratory experiment, copper oxychloride curve correspond to a curve type II‐III, while a curve type III‐IV fits better with the rest of the treatments (although the mortality rate is more or less constant, it acts most heavily on the young stages). Because no statistical differences were found on life span amongst the treatments, the different curves mean that although the final effect of products id not different from one to each other, copper oxychloride acts different than the rest of the treatments. The differences on formulation between the two copper‐based products used in the experiments could explain why Bordeaux mixture and copper oxychloride patterns are different.

Table 5: Parameters estimated for the Weibull function describing the survivorship of C. nigritus adults at different treatments in two experiments: residual contact on glass surfaces and an extended laboratory experiments in which olive tree leaves were treated (mean data ± standard error)

b ±SEa c ± SEb R2c Residual contact on glass surfaces Control 155.9231 ± 0.8887 13.2611 ± 1.2251 0.6444 Kaolin 143.7869 ± 0.8641 5.3357 ± 0.2207 0.9498 Bordeaux mixture 153.9784 ± 1.1663 4.4493 ± 0.1963 0.9362 Copper oxychloride 142.3211 ± 1.4290 1.5776 ± 0.0386 0.9432 Extended laboratory Control 120.8383 ± 1.5806 1.2885 ± 0.0347 0.9309 Kaolin 119.1735 ± 1.2598 1.3707 ± 0.0310 0.9531 Bordeaux mixture 147.0034 ± 1.8742 1.3683 ± 0.0392 0.9221 Copper oxychloride 144.7783 ± 2.0494 0.7818 ± 0.0189 0.9208 aScale of the Weibull distribution bShape of the Weibull distribution cCorrelation coefficient

Kaolin and copper-based products

Figure 44: Survival probability of C. nigritus adults (Series 1) and line of best fit by Weibull function (Series 2) at the different treatments on the residual contact on glass surfaces experiment

Figure 45: Survival probability of C. nigritus adults (Series 1) and line of best fit by Weibull function (Series 2) at the different treatments on the extended laboratory experiment

Kaolin and copper-based products

4.3.3 Emergence

When C. capitata pupae previously parasitized by P. concolor, were treated with kaolin, copper formulations and dimethoate, statistical differences were found between dimethoate and the rest of the treatments (F4,15 = 7.16, P = 0.0010). A reduction of 32.9% emergence was found for dimethoate compared to the controls, while no differences were found when results of kaolin and coppers were compared to the controls (Figure 46; Table 7).

Treatment of pupae: % emergence

100

80 Control Kaolin 60 Bordeaux mixture * Copper oxychloride Emergence

40 % Dimethoate 20

Figure 46: Percentages of P. concolor emergence from treated pupae. Asterisks indicate statistical differences between the treatments and the control (P<0.05)

Kaolin and copper-based products

4.3.4 Beneficial capacity

Beneficial capacity (i.e. percentage of attacked hosts and progeny size) of P. concolor females during or after the treatments remained unaffected in all the experiments performed (P ≥ 0.05). Percentage of attacked hosts was higher than 95% in most of the cases. When females ingested kaolin, however, statistical differences were found (F = 5.99, P = 0.0098) and the percentage of attacked hosts was reduced 13.5 compared to the controls (Figure 47; Tables 6, 7 and 8).

Different experiments: % attacked hosts

100

90 * 80

60 hosts Control 50 Kaolin

Attacked 40 Bordeaux mixture

% 30 Copper oxychloride

20 Dimethoate

0 Residual Extended Semi field Oral Treatment contact laboratory toxicity of pupae (glass) (olive tree leaves)

Figure 47: Percentage of P. concolor attacked host in different experiments. Asterisks indicate statistical differences between the treatments and the control (P<0.05)

Values of the percentage of progeny size were much more changeable. Depending on the experiment, they ranked from the 30% up to the 77%, although the average was around 45‐50%. However, no statistical differences were found among the treatments in none of the experiments. Thus, differences could be related to the specific characteristics and fitness of the females and the medfly larvae used in each experiment (Figure 48; Tables 6, 7 and 8).

Kaolin and copper-based products

Beneficial capacity of females treated with dimethoate was not evaluated because they did not survive long enough to do it, with the exception of the experiment in which pupae were treated with the products. Reproductive parameters of females emerged from dimethoate‐treated pupae did not present any statistical differences when compared to controls (F4,15 = 1.51, P = 0.2479 for attacked hosts and F4,15 = 0.18, P = 0.9429 for progeny size) (Figures 47 and 48).

Different experiments: % progeny size

100

60 size Control 50 Kaolin Progeny 40 Bordeaux mixture % Copper oxychloride 30 Dimethoate 20

0 Residual Extended Semi field Oral Treatment contact laboratory toxicity of pupae (glass) (olive tree leaves)

Figure 48: Percentage of P. concolor progeny size in different experiments

In the experiments in which the parasitization mesh was treated, a slight reduction of the percentage of attacked host was found for kaolin (83%, compared to the values higher than 99% for the control and coppers), although these differences disappeared in the second part of the experiment, when parasitization meshes were not treated

(F3,12= 22.89, P < 0.0001 and F3,12= 1.52, P = 0.2654, respectively). In contrast, when treated olive tree leaves were added, no statistical differences in this parameter were observed during the whole experiment (F3,12= 2.82, P = 0.0840 for the first part of the assay and F3,12= 1.77, P = 0.2070 for the second one). No statistical differences for progeny size were found in any of the experiments (P ≥ 0.05) (Figures 49 and 50).

Kaolin and copper-based products

Treatment of the parasitization surface and olive tree leaves: % attacked hosts

100

90 *

hosts 60 Control 50 Kaolin 40 Attacked Bordeaux mixture % 30 Copper oxychloride 20 10 0 Only mesh Mesh + olive Only mesh Mesh + olive tree leaves tree leaves Treated material After the treatment

Figure 49: Percentage of P. concolor attacked hosts in the experiment in which females had to parasitize throughout a treated surface and in the experiment in which also treated olive tree leaves are placed into the parasitization cages. The differences between the beginning of the two experiments (treated materials) and the end (untreated meshes and no olive trees, in the second case) have also been compared. Asterisks indicate statistical differences between the treatments and the control (P<0.05)

Treatment of the parasitization surface and olive tree leaves: % progeny size

100 90 80 70 size 60 Control 50 40 Kaolin Progeny

% 30 Bordeaux mixture 20 Copper oxychloride 10 0 Only mesh Mesh + olive Only mesh Mesh + olive tree leaves tree leaves Treated material After the treatment

Figure 50: Percentage of P. concolor progeny size in the experiment in which females had to parasitize throughout a treated surface and in the experiment in which also treated olive tree leaves are placed into the parasitization cages. The differences between the beginning of the two experiments (treated materials) and the end (untreated meshes and no olive trees, in the second case) have also been compared

Kaolin and copper-based products

4.3.5 Dual choice and no-choice experiments

4.3.5.1 Psyttalia concolor

No statistical differences were found when compared kaolin with controls in the no‐ choice experiment (t = 2.13365, P = 0.076821 for the percentage of attacked hosts and W = 9.0, P = 0.885229 for the percentage of progeny size). Slight statistical differences were found, however, for the percentage of attacked hosts in the dual choice experiment. This percentage was 81.7 for the controls, while it was 63.8 for the kaolin replicates (W = 593.5, P = 0.03489). There were no differences for the percentage of progeny size (t = 1.60231, P = 0.113129). In this dual choice assay, when the percentages of attacked hosts and progeny size between kaolin and controls were compared depending on which the treated mesh was, i.e., depending on the position in which C. capitata larvae were offered to females, curious results were observed. There were no statistical differences between kaolin and controls when the bottom mesh was treated with kaolin (W = 15.0, P = 0.0590715 for attacked hosts and t = ‐ 0.901544, P = 0.402037 for progeny size). In contrast, when the upper mesh was treated, statistical differences were observed (W = 16.0, P = 0.0294009 for attacked hosts and W= 16.0, P = 0.0303826 for progeny size). However, both in the dual choice and the no‐choice experiments, the percentage of attacked hosts and progeny size were always higher when females parasitized through the mesh in the bottom than through the upper mesh, even if no statistical differences were observed (Figures 51, 52, 53 and 54).

Kaolin and copper-based products

Dual choice experiment: % attacked hosts

100 90 80 70 hosts 60 50 * 40

Attacked 30

% 20 10 0 Kaolin Water Kaolin Water Upper mesh treated Bottom mesh treated Dual choice

Figure 51: Percentage of P. concolor attacked hosts in the dual choice experiment. Asterisks indicate statistical differences between the kaolin and the control

Dual choice and no‐choice experiments: % attacked hosts

100

80 * hosts 60

40 Attacked

% 20

0 Kaolin Water Kaolin Control Dual choice No choice

Figure 52: Percentage of P. concolor attacked hosts in the dual choice and no‐choice experiments. With the aim of comparing experiments, in the dual choice assay all the kaolin treated and water treated surfaces have been grouped together. Asterisks indicate statistical differences between the kaolin and the control

Kaolin and copper-based products

Dual choice experiment: % progeny size

100

size 80

60 40 * Progeny

20 % 0 Kaolin Water Kaolin Water Upper mesh treated Bottom mesh treated Dual choice

Figure 53: Percentage of P. concolor progeny size in the dual choice experiment. Asterisks indicate statistical differences between the kaolin and the control

Dual choice and no‐choice experiments: % progeny size

100

size 80

60 40 Progeny 20 % 0 Kaolin Water Kaolin Control Dual choice No choice

Figure 54: Percentage of P. concolor progeny size in the dual choice and no‐choice experiments. With the aim of comparing experiments, in the dual choice assay all the kaolin treated and water treated surfaces have been grouped together

Although daily observations of females while they were parasitizing showed that they were able to parasitize through both surfaces, a detail study of the percentage of attacked hosts demonstrated that females were more likely to do it through the bottom mesh. Figures 55 and 56 showed that both in the dual choice and no‐choice

Kaolin and copper-based products experiments, the percentage of attacked hosts remained more or less constant during the four days of the experiments when females parasitize through the bottom mesh. In contrast, these percentages strongly fluctuate during the experiment when the parasitisation in the upper mesh was evaluated.

No choice experiment: daily fluctuation of % attacked hosts

100 90 80 70

Control Upper 60 hosts Control Bottom 50 Kaolin Upper Attacked

% 40 Kaolin Bottom

20 10

0 Day 1Day 2Day 3Day 4

Figure 55: Daily fluctuation in the percentage of P. concolor attacked hosts in the no‐choice experiment

Dual choice experiment: daily fluctuation of % attacked hosts

100

90 80 70 Kaolin top 60 hosts Water bottom 50 Kaolin bottom

Water top Attacked

% 30 20

0 Day 1Day 2Day 3Day 4

Figure 56: Daily fluctuation in the percentage of P. concolor attacked hosts in the dual choice experiment

Kaolin and copper-based products

4.3.5.2 Chilocorus nigritus

Most of C. nigritus adults were found on the egg card boxes or other parts of the cages, rather than on the butternuts in all the observations done during the four days the experiment lasted (in the no‐choice experiment, 13.9 % of the adults were found on the butternuts and 85.5% in different parts of the cages in the controls. Similar percentages, 12.4% and 87.6%, respectively, were observed in the kaolin replicates). In the dual choice assay, results were similar (12.1% of adults were on the butternuts, mostly on the non‐treated infested butternuts, and 87.9% on other parts of the cages) (Figures 57 and 58; Table 11).

In the no‐choice experiment, statistical differences were found when the level of A. nerii infestation was compared, and more adults were found on the infested butternuts than on the uninfested ones (F = 165.42, P < 0.0001). In contrast, no differences were found when the treatment was compared (F = 1.24, P = 0.2668). These results suggest than C. nigritus adults are more likely to be on the infested butternuts, no matter whether they are treated or not.

In the dual choice assay, statistical differences were found either when the treatment or the infestation level were compared (F = 87.78, P < 0.0001 and F = 14.72, P = 0.0001, respectively). These results show that C. nigritus are found mainly on the infested butternuts and that once on these infested surfaces, if they can choose between being on a treated surface or not, they prefer to be on the untreated parts. The results also suggested that there is an interaction among the two considered parameters (i.e. infestation level and treatment) (P = 0.001).

Kaolin and copper-based products

Dual‐choice experiment: % Chilocorus nigritus adults on the butternuts or other different parts

100 90 80 70 Other

adults 60 Non infested butternut‐ kaolin 50 Non infested butternut‐ water

nigritus Infested butternut‐ kaolin

40 C. 30 Infested butternut‐ water % 20 10 0 Day 1Day 2Day 3Day 4

Figure 57: Dual choice experiment: percentages of C. nigritus adults placed in the infested butternuts, the non‐infested ones or other parts of the experimental cages. Percentages were recorded during 4 days. “Water” means the half of the butternut treated with distilled water and “Kaolin” the other half, treated with kaolin

No choice experiment: % Chilocorus nigritus adults on the butternuts or other different parts

100 80 adults 60 Other 40 nigritus Non infested butternut C. 20 % Infested butternut 0 Kaolin Kaolin Kaolin Kaolin Control Control Control Control Day 1Day 2Day 3Day 4

Figure 58: Percentages of C. nigritus adults placed in the infested butternuts, the non‐infested ones or other parts of the experimental cages in the no‐choice experiments. Percentages were recorded during 4 days. “Control” means the replicates in which both butternuts were treated with distilled water and “Kaolin” indicates the replicates in which both were treated with kaolin

Kaolin and copper-based products

Despite not evaluating fecundity and fertility, the presence/absence of C. nigritus larvae was recorded at the end of the experiment. Larvae were found both in the cages and on the butternuts, especially close to the places were first instar scales (crawlers) were, as for example the butternuts (where most of the larvae were observed) and the corners of the cages (maybe crawlers walked to the corners of the cages because there was more light there than in other parts of the cages and they were attracted to it). Although the number of larvae was very changeable: from 4 up to 35, depending more on the replicate than on the treatment), in most of the cages this number was related to the level of A. nerii infestation of the butternuts and the abundance of A. nerii N1, which are the food of newly emerged predator’s larvae (they were more abundant in the cages in which the number of scales, including first instars, was higher). It should be also remarked that at the time of checking the presence of larvae, a high percentage of them had already died, which could be due either to the treatment with kaolin or a limited number of crawlers (Figure 59).

Number of C. nigritus larvae found at the end of the experiments

35 30

larvae 25 20 15

nigritus Death 10 C. Infested butternuts 5 No. 0 Cage (egg box, walls, etc.) Cage Cage Cage Cage Cage Cage Cage Cage Cage 1 2 3 1 2 3 1 2 3 Controls Kaolin Control + kaolin No‐choice Dual choice

Figure 59: Number of C. nigritus larvae found in the different replicates of each treatment. In the dual choice experiment, larvae on the butternuts were always observed on the non‐treated parts of the butternuts. It can be observed the high percentage of dead larvae, especially on the kaolin treated butternuts

Kaolin and copper-based products

4.4 Discussion

Based on the results obtained in the different experiments performed, both kaolin and copper salts seem to be not toxic to the natural enemies tested. The sole exception was the oral toxicity assay, in which a reduction of life span of P. concolor females was observed for the three products. In the case of C. nigritus, although no effects have been proved on the parameters studied, possible negative effects on fecundity and fertility still remained unknown, and they should be determined.

According to the IOBC categories, kaolin and copper salts were classified as harmless (1), or slightly toxic (2), depending on the parameter studied. Only when kaolin was ingested by P. concolor females, it was classified as moderately toxic (3). However, this product did not cause as high deleterious effect as in this case in a similar experiment performed with kaolin solved into the drinking water or sprayed on the females’ food (data non shown).

Dimethoate was classified as toxic, except when parasitized pupae were treated with the product. In this last case, negative effects of this product were lower. This result can be explained taking into account that the intrinsic toxicity of an insecticide is determined by the rate of arrival at the site of action. Pupae are the most protected stage both for C. capitata and P. concolor. When dimethoate was sprayed on parasitized pupae, P. concolor individuals were at pupal stage. Thus, a lower amount of the insecticide arrived to the target site; which explained the reduction of the percentage of emergence. No negative effects were reported for emerged adults, compared to the other treatments. Youssef et al. (2004) observed similar results when Sitotroga cerealella (Olivier) eggs including mature pupae of Trichogramma spp. were directly sprayed with dimethoate.

Kaolin and copper-based products

4.4.1 Lethal and sublethal effects of kaolin, Bordeaux mixture and copper oxychloride

At field level, results of the different studies performed all over the world, showed contradictory results. This could be due to a variety of factors, such as the pest or beneficial studied, the methodology of the experiment, the particular climatic conditions during the assay, etc. Kaolin has been reported to have a strong effect on the olive grove’s arthropod community at the canopy level, in which the abundance of arthropods is usually reduced after the treatments. Similarly, in a study conducted in an olive grove in Madrid (Spain), Pascual et al. (2010a) showed that kaolin reduced the abundance and diversity of arthropods after three consecutive years of treatment (the treatment covered the entire foliage, and two treatments were applied per year). The most affected taxa were certain coccinellids, mirids, different species of Orius and the families Philodromidae, Scelionidae, Pteromalidae, Chrysopidae and Aphelinidae. At soil level, however, the abundance did not decrease but the structure of the community was modified (i.e. the overall number of auxiliars remained unaffected, but the relative number within the same taxa changed). This effect can be the result of the interference between the kaolin particle film and the feeding strategies utilised by pollinators, phytophagous insects and predators (Iannotta et al., 2007a; 2008). Studies carried out in pear orchards also reported a reduction on the reproduction of the pear psyllas predator Anthocoris nemoralis (F.) (Heteroptera, Antocoridae) (Pasqualini et al., 2003; Gobin et al., 2005). Showler and Sétamou (2004) also observed a reduction on dipterans, Orius spp. and wasps after kaolin sprayed in cotton, although no adverse effects were reported for other arthropod populations, including different natural enemies. In contrast, Porcel et al. (2011) did not find any difference in the abundance of adult C. carnea after kaolin applications in olive orchards and Kourdoumbalos et al. (2006) showed no adverse effects on coccinellid populations after kaolin applications in pear orchards either.

Fewer references can be found about the effects of copper application on non‐ target arthropods. Studies of the application of copper to olive orchards apparently showed that the product seemed to be harmless to the taxa at the canopy level, 96

Kaolin and copper-based products whereas it had a stronger negative effect on the arthropod communities at the soil level (Iannota et al., 2007a; Scalercio et al., 2009). This effect could be due to the lower dispersal ability of above‐ground arthropods (primarily walkers) compared with canopy arthropods (primarily fliers). However, a reduction of Chrysopids in olive groves after copper applications was also observed by González‐Núñez et al. (2008). Nevertheless, long term effects of copper salts should be also taken into account because copper has a high persistence in the soil. Thus, copper residues can be accumulated in the environment (Scalercio et al., 2009), which could explain the results showed above.

To the best of my knowledge, few references are available on the effects of kaolin and copper on beneficial insects of olive orchards in the laboratory.

No effects on mortality have been found on P. concolor and C. nigritus, except when the products were ingested. Adán et al. (2007) also reported no effects of kaolin on P. concolor mortality when females were exposed to kaolin‐treated vegetation. Similarly, no effects on mortality were observed on the natural enemies C. carnea, Chelonus inanitus L. (Hymenoptera, Braconidae) and Scutellysta cyanea Motschulsky (= S. caerulea; Hymenoptera, Pteromalidae), in residual contact and extended laboratory experiments (Bengochea et al., 2010 and Bengochea et al., 2012‐sent). Porcel et al. (2011) reported no effects on the mortality of third‐ instar C. carnea sprayed directly with kaolin. However, larvae coated with the product showed a slightly hampered movement capacity and a preference for clean surfaces. The authors have also observed that if younger larvae were sprayed with kaolin, they removed the particle layer deposited on their cuticle when they moulted.

Boyce (1932) suggested that when adults of the fruit fly Rhagoletis completa Cresson (Diptera, Tephritidae) ingested different undissolved dust particles, they died because particles abraded their alimentary canal tissues. This could explained the higher mortality rates observed when P. concolor females ingested kaolin solutions, compared to the rest of the experiments carried out in this work. A higher mortality of insect after kaolin ingestion was also observed on Rhagoletis indifferens Curran

Kaolin and copper-based products

(Diptera, Tephritidae) (Yee, 2007), and when larvae of Choristoneura rosaceana (Harris) (Lepidoptera, Tortricidae) were fed on kaolin‐sprayed apple leaves (Sackett et al., 2005). Similarly, when second instar larvae of Trichoplusia ni (Hübner) (Lepidoptera, Noctuidae) were fed on cabbage leaves, a 60% of mortality was registered (Díaz et al., 2002). It has been reported that Hymenopteran parasitoids are especially sensitive to dust due to their specialized structures for removing dust particles. Dust is trained from liquid food and placed in a “gnathal pouch” just beneath the mouth. It can be observed a lack of coordination in their movements, and they wallow helplessly too (Quarles, 1992). In the case of predators, examinations of the digestive tracts of dusted beetles before or after death showed that most of the beetles which succumbed to toxic substances on dusted foliage were killed by particles which were removed from their body and apendages by the mandibles rather than by particles ingested with the food. The excitation resulting from particles which have fallen upon the bodies, or which adhere to the appendages of the beetles as they walk over dusted plant surfaces, is so great that the process of removal and ingestion is begun at once (Richardson and Glover, 1932). However, no kaolin particles were found attached to P. concolor or C. nigritus bodies after the treatments.

Effects of copper salts on natural enemies have been reported in several previous studies. Copper oxychloride did not affect the mortality of the predators Orius laevigatus Fieber (Hemiptera, Anthocoridae) if fourth‐instar larvae and young adults were exposed to the product by contact or by ingestion, respectively (Angeli et al. 2005). Silva et al. (2005) also reported no negative effects of this product when larvae of C. carnea were directly sprayed using the Potter´s Tower. Similarly, Ventura et al. (2009) found basic copper sulphate to be harmless when the product was sprayed on pupae parasitised by Trichogramma cordubense Vargas & Cabello (Hymenoptera, Trichogrammatidae). When first and third instar larvae of three species of ladybeetle (Coleoptera, Coccinellidae: Curinus coeruleus Mulsant, Harmonia axyridis Pallas, and Olla v‐nigrum Mulsant) were exposed during 24 h to field rates of copper sulphate in combination with petroleum oil, all larvae of all three species survived to adulthood at the same rate as control larvae, but larvae of O. v‐nigrum experienced a significant increase in developmental time (Michaud and Grant, 2003). In contrast, a slightly

Kaolin and copper-based products higher mortality 72 h after the treatments was reported for A. nemoralis after kaolin and copper applications (Bengochea et al., 2010 and Bengochea et al., 2012‐sent).

Effects on longevity after exposure to lethal or sublethal doses of pesticides have been described mostly for parasitoids species and to lesser extent for predators. Depending on the study, reduced longevity may be considered a sublethal effect or latent mortality. Extrapolation of these effects to the population level is difficult because, depending on the biology of the particular natural enemy (proovigenic or synovigenic, parasitod or predator), they may be more or less likely to reproduce and/or kill pests before their premature death. From a practical perspective, it is the resulting amount of feeding and reproduction that occurs between exposure and death that is important (Desneux et al., 2007). Previous studies of the effects of kaolin and copper on longevity have obtained different results, depending on the insect, the product tested and the experiment performed. In this work, no effects of the three tested products have been observed in any of the experiments (except oral toxicity). In agreement, Ventura et al. (2009) did not found a reduction on the life span of the parasitoid T. cordubensis after copper applications. In contrast to my results, Villanueva‐Jiménez and Hoy (1998), observed a 39.2% reduction of the survival of the parasitoid Ageniaspis citricola Logvinovskaya (Hymenoptera, Encyrtidae) after copper hydroxide applications on grapefruit leaves. Bengochea et al. (2012‐sent) also reported a reduction of S. cyanea and C. inanitus life span when adults were exposed to kaolin‐ and copper‐treated olive tree leaves. Similarly, Knight et al. (2000) observed a reduction of C. rosaceana female’s longevity after their exposure to kaolin‐treated apple leaves. The reduction of life span when kaolin is ingested could be explained from autointoxication as a result of failure to eliminate excrement or starvation (Boyce, 1932).

Reductions in the reproductive parameters associated with pesticides may be due to both physiological and behavioral effects (Desneux et al., 2007). The effects of the products on reproduction also depend on the insect tested. In the case of parasitoids, the differences in host location strategies, as well as larval habits (degree of larvae enclosure inside fruits, leaf miners or leaf folds), may affect the interactions between

Kaolin and copper-based products kaolin and parasitism and explain why some parasitoids are not affected by treatments (Sackett et al., 2005; 2007).

Kaolin had no effects on the beneficial capacity of P. concolor, in any of the experiments performed. Similarly, Adán et al. (2007) found no effects of kaolin exposure on the beneficial capacity of P. concolor. Porcel et al. (2011) observed no sublethal deleterious effects on C. carnea adults emerged from treated larvae or on the early survival of newly emerged first instars after spraying the eggs with kaolin. In agreement, no effects of kaolin on the fecundity or fertility of the predator C. carnea have been also reported by Bengochea et al. (sent to Journal of Economic Entomology.). However, deleterious effects on these parameters after kaolin treatments have been reported. A reduction of A. nemoralis fecundity was observed when adults were in contact with kaolin‐treated olive tree leaves, although no effects were detected on the fertility of the eggs laid (Bengochea et al., 2012‐sent). Kahn et al. (2001) also reported negative effects on the reproductive parameters of the parasitoid Pnigalio flavipes Erdos (Hymenoptera, Eulophidae). The females were not able to recognise their hosts, the western tentiform leafminer, if they were covered by kaolin particles. This was the same effect as Grafton‐Cardwell and Reagan (2003) reported on the citrus Diaspididae parasitoids, Aphytis melinus DeBach (Hymenoptera, Aphelinidae) and Comperiella bifasciata Howard (Hymenoptera: Encyrtidae). Laboratory and greenhouse assays carried out by Sisterson et al. (2003) and Showler (2003) on different pests are consistent with the results of this study. The two assays cited showed that kaolin was a partial deterrent. The kaolin treatment reduced the number of eggs laid by the pink bollworm, Pectinophora gossypiella (Saunders) (Lepidoptera, Gelechiidae) and the beet armyworm, Spodoptera exigua (Hübner) (Lepidoptera,

Noctuidae) on cotton bolls or plants (Cadogan and Sabaranch 2005a,b). However, Porcel et al. (2011) observed in a laboratory study that C. carnea females preferred to oviposit on the treated surface rather than on the controls. The explanation for this finding may be that the treated surface was more suitable for anchoring the eggs. However, the particle film attraction effect on adults was not observed at field level.

100

Kaolin and copper-based products

Copper‐based products did not affect the reproductive parameters of P. concolor either. In agreement with our results, Angeli et al. (2005) observed that the fertility and fecundity of the predator O. laevigatus were unaffected when adults were exposed to copper residues through contact or ingestion. Little or no adverse effects on emergency rates, longevity or fecundity of T. cordubense have been reported either after basic copper sulphate applications (Ventura et al., 2009). Bengochea et al.a,b (sent to Journal of Economic Entomology and 2012‐sent) observed similar results in treated C. carnea larvae. In contrast, laboratory experiments with adults of C. carnea showed a decrease of fecundity in individuals exposed to Bordeaux mixture. No negative effects on fertility were reported, however. Similar effects were observed in this study for A. nemoralis, although in this case the effects of copper oxychloride were more severe than the effects of Bordeaux mixture. Female adults of C. coeruleus and H. axyridis receiving copper sulphate exposures as larvae did not differ from control adults in pre‐ reproductive period, fecundity or fertility over ten days of reproduction. Treated O. v‐ nigrum females, however, had significantly longer pre‐reproductive periods than control females and laid significantly fewer eggs, although egg fertility was equivalent (Michaud and Grant, 2003). Laboratory tests carried out by Ye et al. (2009) showed that copper oxychloride could be transferred along food chains to secondary consumers (parasitoids) in small amounts, resulting in negative effects on parasitoid growth and development (body weight and developmental duration), as well as fecundity (number of offspring per female). Copper exposure also inhibited vitellogenesis of parasitoids from Cu‐contaminated host pupae. When the effect of fresh copper hydroxide residue was tested on Phyllocnistis citrella Stainton (Lepidoptera, Noctuidae) and its parasitoid A. citricola, it allowed high survival of leaf miner adults, but reduced survival of A. citricola adults, and it was ranked as moderately selective to the parasitoid (Villanueva‐Jiménez and Hoy, 1998).

101

Kaolin and copper-based products

4.4.2 Effects of kaolin treated surfaces in dual choice and no-choice experiments

Different laboratory dual choice experiments showed a reduction of oviposition or feeding damage on kaolin treated surfaces in the case of several pests, such as B. oleae, C. rosaceana, P. gossypiella, Lymantria dispar (L.) (Lepidoptera, Lymantriidae), Choristoneura fumiferana (Clemens) (Lepidoptera, Tortricidae), Thrips tabaci Lindeman (Thysanoptera, Thripidae), S. exigua or R. indifferens (Knight et al., 2000; Sisterson et al., 2003; Showler, 2003; Cadogan and Sabaranch, 2005a,b; Yee, 2007; Larentzaki et al.,

2008; Pascual et al., 2010b; Yee, 2010). These results are in agreement with those obtained in the experiment with C. nigritus and P. concolor. Adults of the predator seemed to prefer non kaolin‐treated surfaces when they were able to choose between a treated and an untreated one. Similarly, the percentage of attacked hosts by P. concolor females was higher in the controls than in the kaolin. In an experiment carried out with the potato psylla, Bactericera cockerelli (Sulc) (Homoptera, Psyllidae), the number of adults on the leaves depended on the leaf surface treated: when kaolin was applied on the upper surface of the leaves, adults were found in a higher number in the controls. In contrast, when the lower or both surfaces were treated, there were not differences between the number of adults found in the controls and in the kaolin‐ treated leaves. However, 72‐h after the treatments, the number of psyllids on the controls was significantly greater. It was also observed that the adults landed on the treated leaves, apparently tried to escape from them by moving around. They tested the leaf surface because their mouthparts could penetrate through the kaolin particle film barrier on the surface into leaf tissue. Even though potato psyllid adults could land on the kaolin‐treated plants, when given a choice, the psyllids avoided plants treated with kaolin particle film under laboratory and field conditions. When no choice was given, a fewer number of eggs was observed (Peng et al., 2011). Liang and Liu (2002) reported a similar effect for the adults of the silverleaf whitefly, Bemisia argentifolii (Bellows & Perring) (Homoptera, Alerodidae). They also observed that whitefly adults after landing on the treated leaves quickly move forward to the uncoated areas, forming many clusters of adults and eggs on the green spots on the leaves. These results are in agreement with the results obtained in the C. nigritus

102

Kaolin and copper-based products

In the no‐choice tests, when insects cannot choose between a kaolin‐coated surface and a clean one, the product did not have the same deleterious effects as those reported in the dual choice experiments. Although Pascual et al. (2010b), found that both the percentage of attacked olives and the number of oviposition stings per olive were reduced by kaolin treatment, the product did neither completely inhibited oviposition nor did negatively influence the percentage of egg hatching or larval feeding, in the case of C. fumiferana, L. dispar, R. indifferens (Cadogan and Scharbach,

2005a,b; Yee, 2007). In agreement with the last results, there were not differences between the percentage of C. nigritus adults found on the treated and the untreated butternuts, and no differences on beneficial capacity of P. concolor were detected either.

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Kaolin and copper-based products

4.5 Appendix (tables of results)

104

Table 6: Percentages of mortality 72 hours after exposure, attacked hosts and progeny size of P. concolor after kaolin, Bordeaux mixture, copper oxychloride and dimethoate applications on an inert surface, an extended laboratory and a semifield experiments (mean data ± standard error) Reduction1 (%) Reduction2 (%) Reduction2 (%) % Mortality 72h % Attacked hosts % Progeny size IOBC3 IOBC3 IOBC3 Residual contact on glass surfaces Control 0.0a ± 0.0 ‐ 83.0a ± 6,6 ‐ 54.8a ± 7.2 ‐ Kaolin 0.0a ± 0.0 0/1 97.7a ± 1.3 ‐17.6/1 65.0a ± 3.9 ‐18.6/1 Bordeaux mixture 0.0a ± 0.0 0/1 91.8a ± 3.7 ‐10.5/1 60.2a ± 4.8 ‐9.9/1 Copper oxychloride 0.0a ± 0.0 0/1 92.2a ± 5.8 ‐11.0/1 52.1a ± 4.4 4.8/1 Dimethoate 100.0b ± 0.0 100/4 ‐ ‐ ‐ ‐ 4 K=24.0 F3,12=1.57 F3,12=1.21 P<0.0001 P=0.2484 P=0.3497 Extended laboratory

Control 3.8a ± 2.3 ‐ 96.3a ± 1.6 ‐ 54.8a ± 10.4 Kaolin 0.0a ± 0.0 ‐4.0/1 97.4a ± 0.6 ‐1.2/1 65.1a ± 9.5 ‐18.7/1 Bordeaux mixture 1.8a ± 1.8 ‐2.1/1 95.8a ± 1.7 0.5/1 77.4a ± 4.4 ‐41.2/1 Copper oxychloride 5.3a ± 3.4 1.6/1 96.9a ± 1.7 ‐0.7/1 64.3a ± 12.1 ‐17.2/1 Dimethoate 100.0b ± 0.0 100/4 ‐‐ ‐ ‐

F4,20= 460.75 F3,12=0.24 F3,12= 0.94 P<0.0001 P=0.8676 P=0.4522 Semi‐field Control 1.1a ± 1.1 ‐ 92.7a ± 3.3 ‐ 49.2a ± 4.8 ‐ Kaolin 1.1 ± 1.1 0.0/1 94.3 ± 2.7 ‐1.22/1 45.2 ± 4.3 ‐18.7/1 a a a Bordeaux mixture 3.3a ± 1.9 2.24/1 90.2a ± 6.5 0.5/1 54.0a ± 3.5 ‐41.2/1 Copper oxychloride 1.1a ± 1.1 0.0/1 93.3a ± 3.9 0.7/1 45.3a ± 4.1 ‐17.2/1 Dimethoate 98.9b ± 1.1 98.9/4 ‐‐ ‐‐ F4,10=1094.77 F3,12=0.16 F3,12= 0.99 P<0.0001 P= 0.9196 P= 0.4314

Data followed by the same letter are not significantly different (ANOVA, LSD; P≥0.05) 1 Mortality corrected following the Schneider‐Orelli formula: M (%) = [(Mtreated – Mcontrol)/(100 – Mcontrol)]*100 2 Attacked hosts and progeny size corrected following the Abbot formula: P (%) = [1‐ (Ptreated/ Pcontrol)] x 100 3IOBC toxicity rating: Laboratory: 1 (harmless) < 30%; 2 (slightly harmful) 30‐79%; 3 (moderately harmful) 80‐99%; 4 (harmful) >99%; Extended laboratory and semifield: 1 <25%; 2 25‐50%; 3 51‐75%; 4 >75% 4Data analyzed using Kruskal‐Wallis test.

Table 7: Percentages of mortality 72 hours after exposure, life span, emergence, attacked hosts and progeny size of P. concolor after kaolin, Bordeaux mixture, copper oxychloride and dimethoate applications on parasitized pupae or ingested via their drinking water (mean data ± standard error) Oral toxicity % Mortality Reduction1(%) Life span Reduction2(%) % Attacked Reduction2(%) % Progeny Reduction2(%)

72h IOBC3 (days) IOBC 3 hosts IOBC 3 size IOBC 3

Control 0.0a ± 0.0 ‐ 20.1a ± 2.1 ‐ 94.4a ± 4.0 ‐ 49.8a ± 3.0 Kaolin 36.0ab ± 21.1 36.0/2 3.9c ± 0.6 80.5/3 81.6b ± 6.9 13.5/1 46.4a ± 4.8 6.9/1 Bordeaux mixture 4.0ab ± 2.7 4/1 13.8b ± 0.7 31.1/2 97.2a ± 1.6 ‐2.9/1 61.5a ± 6.4 ‐23.4/1 Copper oxychloride 0.0a ± 0.0 0/1 13.3b ± 1.0 33.6/2 99.8a ± 0.2 ‐5.7/1 51.7a ± 6.9 ‐3.8/1 Dimethoate 100.0b ± 0.0 100/4 1.38d ± 0.13 93.1/3 ‐ ‐ ‐ ‐ 4 K= 17.3868 F4,20= 78.01 F3,12=5.99 F3,12=1.38 P= 0.0016255 P < 0.0001 P=0.0098 P=0.2950 Treatment of pupae % Reduction2(%) Life span Reduction2(%) % Attacked Reduction2(%) % Progeny Reduction2(%) Emergence IOBC 3 (♀) IOBC3 hosts IOBC3 size IOBC3

Control 55.7a ± 4.3 ‐ 55.3a ± 2.5 ‐ 99.1a ± 0.5 ‐ 31.6a ± 9.8 ‐ Kaolin 66.5a ± 6.6 ‐19.4/1 49.6a ± 2.8 10.3/1 96.4a ± 1.5 2.8/1 38.6a ± 8.3 ‐22.1/1 Bordeaux mixture 64.8a ± 4.3 ‐16.2/1 48.2a ± 3.8 12.8/1 98.7a ± 0.9 0.4/1 37.9a ± 10.4 ‐19.9/1 Copper oxychloride 63.6a ± 3.6 ‐14.2/1 48.6a ± 2.7 12.0/1 98.8a ± 0.3 0.3/1 30.0a ± 13.9 4.9/1 Dimethoate 37.4b ± 2.7 32.9/2 46.3a ± 3.8 16.2/1 89.5a ± 7.1 9.6/1 29.6a ± 6.7 6.2/1

F4,20=7.16 F4,20=1.14 F4,15=1.51 F4,15= 0.18 P=0.0010 P=0.3678 P= 0.2479 P= 0.9429 Data followed by the same letter are not significantly different (ANOVA, LSD; P≥0.05) 1 Mortality corrected following the Schneider‐Orelli formula: M (%) = [(Mtreated – Mcontrol)/(100 – Mcontrol)]*100 2 Life span, emergence and attacked hosts and progeny size corrected following the Abbot formula: P (%) = [1‐ (Ptreated/ Pcontrol)] x 100 3IOBC toxicity rating: Laboratory: 1 (harmless) < 30%; 2 (slightly harmful) 30‐79%; 3 (moderately harmful) 80‐99%; 4 (harmful) >99% 4Data analyzed using Kruskal‐Wallis test.

Kaolin and copper-based products

Table 8: Percentages of mortality 72h after exposure and life span C. nigritus adults after kaolin, Bordeaux mixture, copper oxychloride and dimethoate applications on an inert surface and in an extended laboratory experiment (mean data ± standard error)

% Mortality Reduction1 (%) Life span Reduction2(%) 72h IOBC3 (days) IOBC3 Residual contact on glass surfaces Control 0.0a ± 0.0 ‐ 140.7a ± 8.3 ‐ Kaolin 2.2a ± 2.2 2.2/1 126.9a ± 7.7 9.8/1 Bordeaux mixture 0.0a ± 0.0 0.0/1 140.9a ± 7.5 ‐0.1/1 Copper oxychloride 4.4a ± 2.7 4.4/1 119.6a ± 21.8 ‐15.0/1 Dimethoate 100.0b ± 0.0 100.0/4 1.0b ± 0.0 99.3/4 F4,20= 784.60 F4,20=26.58 P<0.0001 P<0.0001 Extended laboratory Control 3.1a ± 3.1 ‐ 117.8a ± 7.2 ‐ Kaolin 3.1a ± 3.1 0.0/1 108.7a ± 11.2 7.7/1 Bordeaux mixture 3.1a ± 3.1 0.0/1 134.9a ± 11.3 ‐14.6/1 Copper oxychloride 3.1a ± 3.1 0.0/1 126.9a ± 26.8 ‐7.71 Dimethoate 100.0b ± 0.0 100/4 1.0b ± 0.0 99.1/4

F4,15= 240.25 F4,15=14.80 P<0.0001 P<0.0001

Data followed by the same letter are not significantly different (ANOVA, LSD; P≥0.05) 1 Mortality corrected following the Schneider‐Orelli formula: M (%) = [(Mtreated – Mcontrol)/(100 – Mcontrol)]*100 2 Life span corrected following the Abbot formula: P (%) = [1‐ (Ptreated/ Pcontrol)] x 100 3IOBC toxicity rating: Laboratory: 1 (harmless) < 30%; 2 (slightly harmful) 30‐79%; 3 (moderately harmful) 80‐99%; 4 (harmful) >99%

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Treated surface After treatment (non‐treated surface) % Attacked Reduction1(%) % Progeny Reduction1(%) % Attacked Reduction1(%) % Progeny Reduction1(%) hosts IOBC2 size IOBC2 hosts IOBC2 size IOBC2 Treatment of parasitization surface

Control 99.5a ± 0.3 ‐ 66.0a ± 2.9 ‐ 98.7a ± 0.7 ‐ 52.0a ± 4.7 ‐ Kaolin 83.1b ± 4.7 16.4/1 70.4a ± 2.7 ‐6.6/1 97.3a ± 1.2 1.4/1 69.3a ± 4.5 ‐33.4/1 Bordeaux mixture 99.3a ± 0.3 0.1/1 69.5a ± 3.5 ‐5.2/1 99.2a ± 0.3 ‐0.0/1 53.2a ± 5.4 ‐2.3/1 Copper oxychloride 99.5a ± 0.2 0.0/1 65.8a ± 4.5 0.3/1 99.2a ± 0.5 0.5/1 58.3a ± 4.2 ‐12.2/1

F3,12=22.89 F3,12=0.45 F3,12=1.52 F3,12=2.77 P<0.0001 P=0.7219 P=0.2604 P=0.0872 Treatment of parasitization surface and olive tree leaves

Control 92.1a ± 4.7 ‐ 73.4a ± 3.5 ‐ 94.5a ± 1.3 ‐ 76.0a ± 5.8 ‐ Kaolin 89.4a ± 2.2 2.9/1 70.8a ± 1.4 3.6/1 94.2a ± 2.4 0.3/1 71.2a ± 4.5 6.2/1 Bordeaux mixture 94.5a ± 1.1 ‐2.6/1 72.3a ± 4.9 1.5/1 89.7a ± 4.4 5.1/1 72.8a ± 4.3 4.2/1 Copper oxychloride 98.3a ± 0.8 ‐6.8/1 78.6a ± 3.6 ‐7.1/1 98.2a ± 0.8 ‐3.9/1 75.6a ± 4.1 0.4/1 F3,12=2.82 F3,12=0.89 F3,12=1.77 F3,12=0.23 P=0.0840 P=0.4761 P=0.2070 P=0.8720 Data followed by the same letter are not significantly different (ANOVA, LSD; P≥0.05) 2 Attacked hosts and progeny size corrected following the Abbot formula: P (%) = [1‐ (Ptreated/ Pcontrol)] x 100 3IOBC toxicity rating: Laboratory: 1 (harmless) < 30%; 2 (slightly harmful) 30‐79%; 3 (moderately harmful) 80‐99%; 4 (harmful) >99%; Extended laboratory: 1 <25%; 2 25‐50%; 3 51‐75%; 4 >75%

Kaolin and copper-based products

Table 10: Percentages of attacked hosts and progeny size in the dual choice and the no‐ choice experiments when P. concolor females parasitize through a kaolin‐treated surface (mean data ± standard error)

% Attacked hosts % Progeny size No‐choice Control 74.0a ± 3.2 43.8a ± 1.8 Kaolin 59.1a ± 6.2 48.7a ± 8.0 t = 2.13365 W = 9.0 P = 0.076821 P = 0.885229 Dual choice (all together) Control 81.7a ± 5.7 48.3a ± 4.0 Kaolin 63.8b ± 7.0 38.0a ± 5.0 W = 593.5 t = 1.60231 P = 0.03489 P = 0.113129 Dual choice: upper mesh treated Control 98.3a ± 0.6 52.9a ± 5.0 Kaolin 30.55b ± 5.8 22.8b ± 5.5 W = 16.0 W = 16.0

P = 0.0294009 P = 0.0303826 Dual choice: bottom mesh treated

Control 65.1a ± 16.9 43.7a ± 7.6

Kaolin 97.0a ± 0.8 53.2a ± 7.3 W = 15.0 t = ‐0.901544 P = 0.0590715 P = 0.402037

Significant differences between treatment means were detected using the two sample t‐tests for the study of the beneficial capacity. If any of the assumptions of the analysis of were violated, the non‐parametric Mann‐ Whitney U test was applied. Data followed by the same letter are not significantly different (P≥0.05)

Table 11: C. nigritus: dual choice and no‐choice experiments. Percentage of adults found on the butternuts (mean data ± standard error) Infested Uninfested Marginal mean butternut butternut No choice

Control 14.4 ± 1.3 0.1 ± 0.1 7.25a ± 0.66 Kaolin 11.1 ± 1.2 1.3 ± 0.5 6.21a ± 0.66 Marginal mean 12.76a ± 0.66 0.70b ± 0.66 Dual choice

Control 8.5 ± 1.2 0.0 ± 0.0 4.25a ± 0.46 Kaolin 3.6 ± 0.5 0.0 ± 0.0 1.79b ± 0.46 Marginal mean 6.05a ± 0.46 0.0b ± 0.46

Data followed by the same letter are not significantly different (Multifactorial ANOVA; P≥0.05)

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Table 12: Classification of the products according to the IOBC criteria

Bordeaux Copper Kaolin Dimethoate mixture oxychloride Mortality 72h after the treatments Residual contact (glass) P. concolor 1 1 1 4 Residual contact (glass) C. nigritus 1 1 1 4 Extended laboratory P. concolor 1 1 1 4 Extended laboratory C. nigritus 1 1 1 4 Semifield* 1 1 1 4 Ingestion* 2 1 1 4 Treatment of pupae* 1 1 1 4 Life span Residual contact (glass) C. nigritus 1 1 1 4 Extended laboratory C. nigritus 1 1 1 4 Ingestion* 3 2 2 3 Treatment of pupae (of surviving 1 1 1 1 females)* Emergence Treatment of pupae* 1 1 1 2 Reproductive parameters Attacked hosts Residual contact (glass) P. concolor 1 1 1 ‐ Extended laboratory P. concolor 1 1 1 ‐ Semifield* 1 1 1 ‐ Ingestion* 1 1 1 ‐ Treatment of pupae* 1 1 1 1 Treatment of surface 1 1 1 ‐ Treatment of surface (after treatment) 1 1 1 ‐ Tr. Surface + olive tree leaves 1 1 1 ‐ Tr. Surface + leaves (after treatment) 1 1 1 ‐ Progeny size Residual contact (glass) P. concolor 1 1 1 ‐ Extended laboratory P. concolor 1 1 1 ‐ Semi‐field* 1 1 1 ‐ Ingestion* 1 1 1 ‐ Treatment of pupae* 1 1 1 1 Treatment of surface 1 1 1 ‐ Treatment of surface (after treatment) 1 1 1 ‐ Tr. Surface + olive tree leaves 1 1 1 ‐ Tr. Surface + leaves (after treatment) 1 1 1 ‐

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Chapter 5

ECDYSONE AGONISTS: EFFICACY AND ECOTOXICOLOGY ON BACTROCERA OLEAE AND PSYTTALIA CONCOLOR. INSECT TOXICITY BIOASSAYS AND MOLECULAR DOCKING APPROACHES1

5.1 Introduction

As it was already explained in Chapter 1, control methods against B. oleae include bait sprays, cover sprays and mass trapping (Haniotakis, 2005). Both traditional insecticides, such as organophosphates, and other more recently developed compounds, like spinosad, are commonly applied in bait sprays. However, B. oleae can develop resistance to some of these insecticides, as it has already been demonstrated for dimethoate (Skouras et al., 2007; Daane and Johnson, 2010) and spinosad (Kakani et al., 2010). Therefore, searching alternative treatments against this pest is necessary if an accurate resistance management program is likely to be applied. Insect growth regulators (IGRs) such as ecdysone agonists could be an alternative.

The ecdysteroid agonists or moulting accelerating compounds (MACs) act upon binding specifically with the ecdysone receptor (EcR) of susceptible insects. They are chemically described as substituted dibenzoylhydrazines (DBHs) and mimic the natural function of the endogenous insect moulting hormone 20‐hydroxyecdysone (20E). They induce a premature lethal molting in larval stages of different insect orders, and they affect reproduction reducing egg production, producing ovicidal activity and disrupting

1 BENGOCHEA P, CHRISTIAENS O, AMOR F, VIÑUELA E, ROUGÉ P, MEDINA P, SMAGGHE G. 2012. Ecdysteroid receptor docking suggests that dibenzoylhydrazine‐based insecticides are devoid to any deleterious effect on the parasitic wasp Psyttalia concolor (Hym. Braconidae). Pest Management Science 68 (DOI 10.1002/ps.3274) BENGOCHEA P, CHRISTIAENS O, AMOR F, VIÑUELA E, ROUGÉ P, MEDINA P, SMAGGHE G. Insect growth regulators as potential insecticides to control olive fruit fly (Bactrocera oleae (Rossi)): insect toxicity bioassays and molecular docking approach. Pest Management Science (In Press) 111

Ecdysone agonists normal spermatogenesis processes (Dhadialla et al., 1998; 2005; Nakagawa, 2005).

5.1.1 The ecdysone receptor

The morphogenetic events associated with insect development are largely triggered by the action of a single class of steroid hormones, the ecdysteroids. Among all insect orders it has been established that the ecdysteroid‐induced orchestration of molting and metamorphosis is mediated by a heterodimer comprised of the ecdysone receptor (EcR) and Ultraspiracle (USP, also named RXR) (i. e. this protein is composed of two similar but not identical subunits; polypeptide chains differ in composition, order, number, or kind of their amino acid residues). The heterodimer is stabilized by 20E and recognizes specific promoter elements in the insect genome to regulate transcription (Henrich, 2005).

Both proteins (EcR and USP) belong to the superfamily of nuclear hormone receptors, which consists of ligand‐dependent transcription factors that share two conserved domains: the DNA‐binding domain (DBD) and the ligand‐binding domain (LBD). They play a central role in controlling gene expression during the development of arthropods. Their general modular structure commonly has four domains, namely the A/B, C, D and E domains, although some receptors also contain an F domain at the carboxy‐terminal end of the protein. Individual domains are at least partially autonomous in their function (Henrich, 2005) (Figure 60). The DBD and the LBD are the most conserved across all taxa for both receptors. The LBD is composed of 12 α‐helices that form a ligand‐binding pocket which holds the cognate ligand. The binding between the receptor and a ligand starts the hormone signalling cascade regulating important physiological events in an insect’s life, such as growth, metamorphosis and reproduction (Bonnetton et al., 2003; Henrich, 2005; Nakagawa, 2005; Tohidi‐Esfahani et al., 2011; Fahrbach et al., 2012). Crystal structures of the LBD provide important information on the recognition of the ligands and the mechanisms of activation of nuclear receptors (Kasuya et al., 2003).

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A/B C D E F

Figure 60: Modular structure (domains) of the insects’ ecdysone receptors

5.2 Objectives and procedures

In the current study, the efficacy of three MACs (tebufenozide (RH‐5992), methoxyfenozide (RH‐2485), and RH‐5849) on B. oleae has been tested and compared to dimethoate and spinosad. Furthermore, with the aim of evaluating their possible compatibility in IPM programs, the toxic effects on P. concolor have also been tested.

In the first part, two biological experiments have been carried out using B. oleae adults and P. concolor females: exposition to treated glass surfaces and oral toxicity of the products.

In a second part, the LBD of the EcR of both insects have been cloned and sequenced. Then, a three dimensional (3D) modelling of the LBD of the EcR of B. oleae (BoEcR‐LBD) and P. concolor EcR (PcEcR‐LBD) have been constructed to evaluate if they exhibit the typical canonical structure with 12 α‐helices.

Finally, a ligand docking was performed to support the hypothesis that DBH‐based insecticides could fit in the ligand binding pocket of susceptible insects. This should happen in the case of the pest, while it should be devoid of any deleterious effect on the parasitic wasp.

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5.3 Materials and methods

5.3.1 Insect bioassays

The residual contact activity on glass surfaces and the oral toxicity test procedures, as well as, the statistical analysis, were similar to those described in Chapters 3 and 4. However, in the case of the residual contact activity experiment the PIEC was not applied. Chemicals used in these experiments are listed in Table 13. Solutions of the products were prepared freshly in distilled water prior to the assays, based on their respective MFRC in accordance with the Spanish registration, with a delivery rate of 1000 litre water ha‐1. In the case of RH‐5849 the dose applied was the same as the dose of tebufenozide. 5 ml of acetone were used for solving for this technical product. The activity of the three IGRs was compared to spinosad and dimethoate. Both dimethoate and spinosad were chosen as commercial standards.

P. concolor females and B. oleae adults were obtained as described in chapter 3. Diets and distilled water were supplied as described in chapter 3.

Experiments consisted of five replicates per treatment. Per replicate, 10 unfed adults (<48h‐old) of B. oleae and 10 unfed, mated females (<48h‐old) of P. concolor were used. Mortality 72 hours after the treatments was measured both for B. oleae and P. concolor. Life span, in the case of the pest, and beneficial capacity, in the case of the parasitoid, were evaluated following the procedures described in Chapter 3.

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Table 13: Chemicals tested in the experiments

Active ingredient %a.i; Trade name Conc1 Trade Company (a.i.) form Dow Agrosciences Iberica Methoxyfenozide Runner® 24 SC 75 cc/hl2 S.A., Madrid (Spain) Dow Agrosciences Iberica Tebufenozide Mimic 2F® 24 SC 75 cc/hl3 S.A., Madrid (Spain) Technical Rohm and Hass, Spring RH‐5849 > 95% 75 cc/hl3 Product House (PA) Spintor 480 Dow Agrosciences Iberica 48 SC 25 cc/hl4 SC® S.A., Madrid (Spain) Spinosad Spintor‐ Dow Agrosciences Iberica 0.024CB 1 l/ha5 Cebo® S.A., Madrid (Spain) Danadim Cheminova Agro S.A. Madrid Dimethoate 40 EC 150 cc/hl Progress® (Spain) 1Formulated product 2Dose applied in citrus orchards against Phyllocnistis citrella Stainton (Lepidoptera: 3Dose applied in tangerine tree orchards against P. citrella (for RH‐5849, the same dose as for tebufenozide was tested). 4Dose applied in horticulture crops against different insect pests. The product was used only when the oral toxicity of the insecticides was evaluated in both insects. 5Rate applied in olive orchards against B. oleae. It was only used to evaluate the residual contact activity on a glass surface because it cannot be solved (therefore, the other spinosad formulation was decided to be used in the oral toxicity experiments). Because the product is too dense, instead of applying it with the Tower of Potter, a pipette was used to place small drops on the glass. The dose at field level and the surface of the test cages were taking into account to calculate the amount of product to be applied (1l/ha is the rate for B. oleae; 219.7 cm2 is the area of the cage; thus, 2.19 µl/per replicate should be applied).

Figure 61: Insecticides tested in the experiments

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5.3.2 EcR-LBD sequence

Total RNA was extracted from B. oleae and P. concolor adults using TRI Reagent (Sigma‐Aldrich, Bornem, Belgium), based on a single‐step liquid phase separation method (Chomeczynski and Sacchi, 1987). The product is a mixture of guanidine, thiocyanate and phenol in a monophase solution which effectively dissolves RNA, DNA and protein on homogenization or lysis of tissue samples. The resulting RNA is intact with Figure 62: PCR machine used in the experiments little or no contaminating DNA and protein. The quality and quantity of the extracted RNA were examined by gel electrophoresis and spectrophotometry using a NanodropTM ND‐1000 (Thermo Fisher Scientific, Asse, Belgium). Subsequently, first strand cDNA synthesis was performed using SuperScriptTM II reverse transcriptase (Invitrogen, Merelbeke, Belgium) with the oligo(dT)12‐18 primers according to the manufacturer’s protocol.

The complete BoEcR‐LBD and PcEcR‐LBD coding sequences were then determined through a number of Polymerase Chain Reaction (PCR) reaction steps (SensoQuest labcycler, SensoQuest GmbH Göttingen, Germany). The specific conditions of PCR reaction steps are specified in Table 14. Partial sequences of the LBD were obtained using degenerate and specific primers (Table 15) located in the coding sequence of the LBD and the DBD of the receptor and designed using Primer3 software (Rozen and Skaletsky, 2000). Design of degenerate primers was based on Figure 63: Agarose gel with different PCR known EcR sequences from different Mecoptera, Trichoptera, products loaded Strepsiptera, Coleoptera, Hymenoptera, Lepidoptera and Diptera insect species. Gene specific primers were designed in the partial sequence obtained with the degenerate primers. PCR products were purified using the Cycle Pure kit (Eppendorf centrifuge 5424; Omega Bio‐Tek, Beaver Ridge Circle, Norcross,

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GA) to eliminate the DNA of interest from soluble proteins and other nucleic acids. After purification, samples were sent for sequencing (AGOWA, Berlin, Germany). The first fragment of both BoEcR‐LBD and PcEcR‐LBD was amplified using two degenerate primers within the LBD, located at helices 5 and 12, respectively. In a second step, a degenerate primer in the DBD was used in combination with a gene‐specific primer at helix 9‐10 in order to amplify and sequence the LBD (fragment 2). Then, the 3’ end of the transcripts were eventually also amplified by RACE‐PCR (Rapid amplification of cDNA Ends‐PCR; i.e. a PCR technique which facilitates the cloning of full‐length cDNA sequences when only a partial cDNA sequence is available) using the 3’RACE System for RACE (Invitrogen). A specific primer in helix 11 together with the Abridged Universal Amplification Primer (AUAP) that is delivered with the kit were used in the reaction. In the case of P. concolor, we also amplified and sequenced a small missing part between helices 10‐12 and afterwards (Bridge fragment), the whole fragment, starting in the DBD and ending in the 3’ UTR was cloned and sequenced for confirmation. In the case of B. oleae, cDNA for RACE‐PRCs was obtained from B. olae adults stored in RNAlater (Sigma‐Aldrich, Bornem, Belgium) because there were no adults alive available at the time of isolating RNA.

Figure 64: Gel electrophoresis apparatus (an Figure 65: Bio‐Rad. Once the electrophoresis is agarose gel is placed in the buffer‐filled box and completed, the molecules in the gel can be electrical field is applied via the power supply to stained to make them visible. DNA can be the rear. The negative terminal is at the side of visualized using ethidium bromide which, the apparatus closest to the tip box (colour fluoresces under ultraviolet light, when blue), so DNA migrates toward it intercalated into DNA. This apparatus is used to visualize DNA. Photographs of the gels can be taken using Gel Doc

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Same B. oleae and P. concolor cDNAs as used in the identification of EcR‐LBD were used for the initial PCR reactions of the cloning. After purification, the PCR products were ligated into a pGEM‐T vector (Promega, Madison, WI) according to the manufacturer’s instructions. Afterwards, plasmids were transformed in competent Escherichia coli XL‐1 Blue Cells by heat stock and then plated out on an ampicilin‐ containing LB (Lysogeny broth) agar plate. After 16 h incubation, formed colonies were checked by colony PCR and several of these positive colonies were then purified using a Plasmid mini prep kit (Omega Bio‐Tek) and sent for sequencing (AGOWA) (Figures 66,67 and 68).

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Table 14: Specific conditions of PCR reaction steps for determining BoEcR‐LBD and PcEcR‐LBD coding sequences

µl primer/ 10 µl Denaturation Annealing Extension Phases Fragment Primers PCR reaction (T/t) (T/t) (T/t) total Bactrocera oleae Degen F2‐ 1 0.4‐0.4 94º/30’’ 51º/30’’ 72º/45’’ 32 degen R3 Degen F3‐ 2 0.4‐0.4 94º/30’’ 53º/30’’ 72º/1’0’’ 32 spec R1 Bridge 3 0.4‐0.4 94º/30’’ 55º/30’’ 72º/30’’ 32 fragment Cloning F‐ Cloning 1‐1 94º/30’’ 60º/30’’ 72º/1’0’’ 32 cloningR Psyttalia concolor Degen F2‐ 1 0.4‐0.4 94º/30’’ 49º/30’’ 72º/45’’ 32 degen R3 Bridge 2 0.3‐0.3 94º/30’’ 60º/30’’ 72º/45’’ 32 fragment Degen F3‐ 3 0.4‐0.4 94º/30’’ 53º/30’’ 72º/1’30’’ 32 specific R1 Race F5‐ 4 0.6‐0.2 94º/30’’ 60º/30’’ 72º/45’’ 32 AUAP* Cloning F‐ Cloning 1‐1 94º/30’’ 60º/30’’ 72º/30’’ 32 cloningR Cloning F‐ Ovaries 1‐1 94º/30’’ 60º/30’’ 72º/30’’ 32 cloningR Table 15: Degenerate and specific primers using for obtaining the partial sequences of the LBD

Bactrocera oleae Forward primer GAAGTVATGATGYTNMGNATG Fragment 1 Reversed primer ACGTCCCAKATYTCWKCNARVAA Forward primer GVCGVAARTGYCARGAGTG Fragment 2 Reversed primer GCAGTGAGGAGAGCGTATT Forward primer CCACAAGAGGATCAAATCAC Bridge fragment Reversed primer CGCAGTTCAGTTAGTATGGA Forward primer TGTCCGTTGCTACCTGATGA Cloning Reversed primer CGGCAGTTTACGATTCTTCA Psyttalia concolor Forward primer GAAGTVATGATGYTNMGNATG Fragment 1 Reversed primer ACGTCCCAKATYTCWKCNARVAA Forward primer GVCGVAARTGYCARGAGTG Fragment 2 Reversed primer ATTTGTGTTTACGGCGACTG Forward primer GAACGGCTCACCTGGAAGTA Bridge fragment Reversed primer AGTGGGCGTCGTTATTGAAA Forward primer CGAGGCACTCAGAACATACG RACE fragment Reversed primer GGCCACGCGTCGACTAGTAC Forward primer CTGGCAGCACTGACTCGTTA Cloning Reversed primer CCACTGGGGCAATTACACTG

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Figure 66: Ethidium bromide‐stained PCR products of the cloning (before and after purification) after gel electrophoresis (P. concolor)

Figure 67: Formed bacteria colonies on an ampicilin‐ Figure 68: Ethidium bromide‐stained containing LB agar plate (B. oleae) plasmids after gel electrophoresis (P. concolor)

The EcR‐LBD sequences of several arthropods and two human orthologs of EcR were retrieved by Blast searches against the Genbank database. The chosen sequences were then aligned by CLUSTALW2/CLUSTALX2 and the phylogenetic trees were made using MEGA4 software (Larkin et al. 2007; Tamura et al., 2007). Bootstrap analysis with 1000 replicates for each branch position was used to assess support for nodes in the tree (Felsenstein, 1985).

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Figure 69: nucleotide and amino acid sequences of B. oleae and P. concolor

Bactrocera oleae (nucleotides): CTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTGTCCGTTGCTACCTGATGATATTGTG GCCAAGTGCAAGGCGAGCAACATTCCGCCGCTCACGCGTAACCAGTTGGCGGTCATATACAAATTGAT CTGGTATCAGGATGGCTATGAACAGCCATCGGAGGAAGATCTGAAGCGTATTATGAGCACCCCCGATG AAAACGAAAGCCCGAATGATATCAGCTTTCGGCATATAACCGAAATTACCATTTTGACAGTACAACTTA TTGTGGAGTTTGCAAAAGGTTTACCGGCATTTACAAAAATTCCACAAGAGGATCAAATCACGTTGCTGA AGGCCTGCTCATCGGAAGTGATGATGTTGCGTATGGCCCGACGTTACGATCACAATTCGGATTCCATAT TCTTCGCCAACAATCGTTCATATACGCGTGATGCGTACAAAATGGCCGGTGTGGCCGATAATATTGAGG ATCTATTGCATTTTTGTCGGCAGATGTACTCGATGAAGGTCGACAACGTCGAATACGCTCTCCTCACTGC CATTGTGATCTTCTCCGATCGGCCGGGACTTGAAAAGGCCGAACTAGTCGAAGCGATACAAAGTTACTA CATCAATACGCTGCGCGTATATATAATTAATCGACATTGCGGCGATACAAAGAGTCTGGTCTTCTTCGC GAAATTACTCTCCATACTAACTGAACTGCGCACGCTTGGCAATCAGAATGCCGAGATGTGTTAATCCCG CGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGCCCTATAGTGAGTCGTATTACAATTC ACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGC ACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCC

Bactrocera oleae (amino acids): QLAVIYKLIWYQDGYEQPSEEDLKRIMSTPDENESPNDISFRHITEITILTVQLIVEFAKGLPAFTKIPQEDQITLL KACSSEVMMLRMARRYDHNSDSIFFANNRSYTRDAYKMAGVADNIEDLLHFCRQMYSMKVDNVEYALLT AIVIFSDRPGLEKAELVEAIQSYYINTLRVYIINRHCGDTKSLVFFAKLLSILTELRTLGNQNAEMCSRGHGGRE HATSGPIRPIVSRITIHWPSFYNVVTGKTLALPNLIALQHIPLSPAGVIAKRPAPIA

Psyttalia concolor (nucleotides): CTCTCCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTCTGGCAGCACTGACTCGTTATTAGAAT TTAAGAACGGATTGTCAATTGTCAGTCCTGAACAAGCTGAGCTCATTGAGAGACTGGTTTATTTTCAAG GACTCTATGAGAATCCCAGTCCCGAAGATCTTGAAAGAATTACGTATCGACCAGTCGAGGGTGAAGAT CCTGTTGACGTTAGATTCAGGCATATGGCGGAAATAACGATACTGACTGTCCAGCTTATTGTTGAATTT GCCAAAAACTTATCGGGTTTCGATAAATTGCTGAGGGAGGATCAAATTGCATTGCTAAAGGCATGCTCC AGTGAAGTCATGATGCTGAGAATGGCAAGAAAGTATGACGCTAGGACAGACAGTATCCTATTTGCTGA TAATCAGCCGTACACGAGAGACAGCTACAGTTTGGCTGGAATGGGTGATACAATTGAGGATTTGCTGC GTTTTTGCCGGCACATGTTCAATATGAAAGTCAACAATGCTGAGTATGCGTTATTAACGGCTATCGTCAT TTTCTCAGAACGGCCGATGCTCTTGGAGGGCTGGAAAGTCGAAAAAATCCAAGAAATATACCTCGAGG CACTCAGAACATACGTGGACAGTCGCCGTAAACACAAATCAGGAACAATATTCGCTAAACTACTCTCCG TATTGACGGAATTACGAACACTCGGCAATCAAAACAGCGAAATTTGCTTCAGTTTGAAGCTCAAAAACA AGAAGCTACCTCCATTTCTTGCCGAGATCTGGGATGTCATGCCC

Psyttalia concolor (amino acids): QAELIERLVYFQGLYENPSPEDLERITYRPVEGEDPVDVRFRHMAEITILTVQLIVEFAKNLSGFDKLLREDQIA LLKACSSEVMMLRMARKYDARTDSILFADNQPYTRDSYSLAGMGDTIEDLLRFCRHMFNMKVNNAEYALL TAIVIFSERPMLLEGWKVEKIQEIYLEALRTYVDSRRKHKSGTIFAKLLSVLTELRTLGNQNSEICFSLKLKNKKLP PFLAEIWDVMP

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5.3.3 Confirmation of EcR expression in Psyttalia. concolor ovaries

The expression of EcR in the ovaries was confirmed on the parasitoid. As it occurred in the biological experiments, in which effects on reproduction of B. oleae were not evaluated, there were not olive fly females available at the moment of the experiments to confirm the expression of this gene in their ovaries.

Ovaries were carefully dissected form P. concolor female adults under stereoscopic microscope with the help of entomological needles (using a buffer solution to prevent desiccation of the ovaries). Newly dissected ovaries were stored in TRI Reagent. 43 pairs of ovaries were used to extract the RNA. RNA was extracted and subsequently cDNA was synthesized following the same procedure described before for total RNA. The expression of the EcR in the ovaries was investigated by PCR using the specific primers designed for the cloning process.

Figure 70: P. concolor ovaries

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5.3.4 Modeling of PcEcR-LBD and docking studies

Homology modeling of the BoEcR‐LBD and the PcEcR‐LBD were performed with the YASARA structure program running on a 2.53 GHz Intel core duo Macintosh computer (Krieger et al., 2002). Different models were built from the X‐ray coordinates of the EcR of the lepidopteran Heliothis virescens F. (Lepidoptera, Noctuidae) in complex with synthetic ligand as BYI‐06830 (PDB code 3IXP), the RXR‐USP receptor of the coleopteran Tribolium castaneum Herbst. (Coleoptera, Tenebrionidae) bound to ponasterone A (PoA; PDB Code 2NXX) (Iwema et al., 2007), the EcR‐LBD of the hemipteran Bemisia tabaci Gennadius (Homoptera, Aleyrodidae) complexed to PoA (PDB code 1Z5X) (Carmichael et al., 2005), the EcR‐USP of H. virescens in complex with 20E (PDB code 2R40) (Browning et al., 2007), and the human RXRα (PDB code 3FC6) used as templates (Washburn et al., 2009), respectively. Finally, a hybrid model built up from the five previous models. PROCHECK was used to assess the geometric quality of the 3D‐model (Laskowski et al, 1993). In this respect, about all of the residues of BoEcR‐LBD and 87% of PcEcR‐LBD were correctly assigned on the best allowed regions of the Ramachandran plot. For BoEcR‐LBD the sole exception is the residue Ala75, which occurs in the non allowed region of the plot (results not shown). For PcEcR‐LBD, the remaining residues were located in the generously allowed regions of the plot (result not shown). Using ANOLEA to evaluate the models (Melo and Feytmans, 1998), a single residue of BoEcR‐LBD over 235 and 5 residues of PcEcR‐LBD over 246 exhibit and an energy level over the threshold value. All of these residues are located in the loop regions connecting α‐helices. Molecular cartoons were drawn with YASARA and PyMol (W.L. DeLano, http://pymol.sourceforge.net). Docking of 20E, PoA, tebufenozide, methoxyfenozide and RH‐5849 to BoEcR‐LBD and PcEcR‐LBD was performed with the YASARA structure program. Clipping planes of BoEcR‐LBD and PcEcR‐LBD complexed to 20E, PoA, tebufenozide, methoxyfenozide and RH‐5849 were rendered with PyMol. Complexed to halofenozide has also been performer for PcEcR‐ LBD.

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5.4 Results

5.4.1 Efficacy and toxicological effects of methoxyfenozide, tebufenozide and RH-5849

Either residual contact or ingestion exposure to methoxyfenozide, tebufenozide and RH‐5849 did not cause any deleterious effect on B. oleae adults 24 h after exposition. Then, in the continuation of the experiment, methoxyfenozide and tebufenozide were not toxic to B. oleae. In great contrast, RH‐5849 provoked a significant higher mortality. 48 h after being in contact with the products 26% of adults were killed, 86% on day 7 and 100% on day 15. Results were a little bit different when oral toxicity of the product was evaluated. After 72 h ingesting RH‐5849, only 10% of adults have died. 31.8% of adults were killed at 7 days after the treatments and the percentage increased to 98.2% on day 15. When exposed to dimethoate, however, 100% of B. oleae adults died after 24 h in both experiments. In the case of spinosad, mortality 24 h after the treatments was 62% when adults were exposed to the fresh residue of the insecticides and 50% when they ingested the products. 24 h later these percentages increased up to 88% and 98%, respectively (Figure 71).

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Different experiments: % mortality of Bactrocera oleae ******** * * ******* 100 * * 90 * *

70 * Control 60 * * Methoxyfenozide 50 Tebufenozide Mortality

% 40 RH‐5849 * 30 * Spinosad * Dimethoate 20

0 24h 48h 72h 7d 15d 24h 48h 72h 7d 15d Residual contact (glass) Oral toxicity

Figure 71: Percentage of mortality of B. oleae adults during the two performed experiments. Spintor‐Cebo® was applied in the residual contact on glass surfaces experiment. Spintor 480 SC® was used for evaluating the oral toxicity. Asterisks indicate statistical differences between the treatments and the controls within the same parameter evaluated (P < 0.05)

In the case of P. concolor, the three tested MACs did not cause any mortality on females. No effects were detected either when females were exposed to a treated glass surface or when they ingested the products. When exposed to dimethoate, however, females died 24 after exposure to this insecticide. Residual contact with spinosad caused a 68.0% of mortality 72h after exposure, while the percentage increased to 99.0% when females ingested the insecticide (mean data ± standard errors are given in supplementary tables in an appendix at the end of this chapter; Tables 17 and 18). Furthermore, no sublethal effects of the three MACs on reproductive parameters, namely percentage of attacked hosts and progeny size, were observed in both assays (P>0.05) and no change in behaviour of treated wasps was seen (Figures 72 and 73).

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Different experiments: % mortality of Psyttalia concolor **** * * ** 100 * 90

80 * * 70 Control 60 Methoxyfenozide Tebufenozide 50

Mortality RH‐5849

% 40 * Spinosad 30 * Dimethoate

20 * 10

0 24h 48h 72h 7d 24h 48h 72h Residual contact (glass) Oral toxicity

Figure 72: Percentage of mortality of P. concolor females during the two performed experiments. Spintor‐Cebo® was applied in the residual contact on glass surfaces experiment. Spintor 480 SC® was used for evaluating the oral toxicity. Asterisks indicate statistical differences between the treatments and the controls within the same parameter evaluated (P < 0.05)

Different experiments: % attacked hosts and progeny size

100 90 80 70 60 Control

% 50 Methoxyfenozide 40 Tebufenozide 30 RH‐5849 20 10 0 % Attacked % Progeny size % Attacked % Progeny size hosts hosts Residual contact (glass) Oral toxicity

Figure 73: Effects of methoxyfenozide, tebufenozide and RH‐5849 on P. concolor beneficial capacity

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5.4.2 Bactrocera oleae EcR-LBD sequence

The cDNA encoding the BoEcR‐LBD was cloned in order to obtain its sequence. However, it was not possible to amplify the 3’ end of the transcript. RACE‐PCR using the 3’RACE System for Rapid Amplification of cDNA Ends (Invitrogen) did not work, despite the multiple tentative to sequence it. Thus, a consensus sequence of other dipteran species, such as the medfly C. capitata, and the Culicidae Anopheles gambiae Giles, Aedes aegypti (L.) and Aedes albopictus (Skuse), were used to complete the helix 12 of BoEcR‐LBD. A multiple alignment with the amino acid sequences of BoEcR‐LBD together with the EcR‐LBD from other dipteran species, and insects from other different orders is shown in Figure 74. The alignment includes ecdysone receptors from Lepidoptera (Heliothis, Chilo, Bombyx), Coleoptera (Tribolium, Tenebrio, Leptinotarsa), Hymenoptera (Apis, Bombus, Psyttalia), Hemiptera (Nilaparvata, Nezara, Bemisia) and Diptera (Drosophila, Calliphora, Aedes, Chironomus, Culex, Bradisia, Anopheles, Ceratitis, Bactrocera). It has been constructed using CINEMA (Colour INteractive Editor for Multiple Alignments, Utopia, http://utopia.cs.man.ac.uk/utopia/). Amino acid colors indicate similar structure. Sequence analysis showed that the EcR‐LBD of B. oleae exhibits a strong sequence identity towards the insect order Diptera.

In Figure 74, red dots indicate amino acids that are similar towards dipterans but different from the other insect orders (residues at positions 5, 6 and 11 in helix 1; positions 75, 80 and 117; positions 171 and 179 in helix 9; and 236 in helix 12). Other residues are similar in Lepidoptera and Diptera but differ from the other orders. These residues are marked in the figure with green dots (position 19 in helix 1; 71 and 77; 81 and 86 in helix 4; 126; 130 in helix 7; 167 and 169; 173, 180, 184 and 190 in helix 9; 201; 239 in helix 12). In addition, amino acids involved in the ligand binding according to Kasuya et al. (2003), are marked in the figure by blue dots (positions 49, 51, 52, 53, 55, 56, 58, 59 and 62 in helix 3; 93, 96, 97, 99, 100 and 101 in helix 5; positions 103, 111, 113; positions 129, 132, 133 and 136 in helix 7; positions 216, 220, 223, 224, 225, 227, 228, 229, 230, 231, 232 in helix 10‐11 and positions 238 and 242 in helix 12).

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Figure 74: Sequence alignment of ecdysone receptor ligand‐binding domains (LBD), including BoEcR‐LBD (Helix 1 to 8). In the following order: Heliothis, Chilo, Bombyx, Tribolium, Tenebrio, Leptinotarsa, Apis, Bombus, Psyttalia, Nilaparvata, Nezara, Bemisia, Drosophila, Calliphora, Aedes, Aedes, Chironomus, Culex, Bradisia, Anopheles, Ceratitis, Bactrocera

128

Figure 74 (continuation): Sequence alignment of ecdysone receptor ligand‐binding domains (LBD), including BoEcR‐LBD (Helix 9 to 12). In the following order: Heliothis, Chilo, Bombyx, Tribolium, Tenebrio, Leptinotarsa, Apis, Bombus, Psyttalia, Nilaparvata, Nezara, Bemisia, Drosophila, Calliphora, Aedes, Aedes, Chironomus, Culex, Bradisia, Anopheles, Ceratitis, Bactrocera

Ecdysone agonists

5.4.3. Psyttalia concolor EcR-LBD sequence, phylogenetic tree and expression in the ovaries

The cDNA encoding the PcEcR‐LBD fragment was also cloned in order to obtain the amino acid sequence. Figure 75 shows a multiple alignment with the amino acid sequence of PcEcR‐LBD, together with the EcR‐LBD from most other known hymenopteran species, and several members from other insect orders. It has also been constructed using CINEMA. The alignment includes ecdysone receptors from Lepidoptera (Heliothis, Chilo, Bombyx), Coleoptera (Tribolium, Tenebrio, Leptinotarsa), Hymenoptera (Apis, Bombus, Psyttalia), Hemiptera (Nilaparvata, Nezara, Bemisia) and Diptera (Drosophila, Calliphora, Aedes, Chironomus, Culex, Bradisia, Anopheles, Ceratitis, Bactrocera).

PcEcR‐LBD exhibits some amino acid substitutions in positions where conservation is usually very high throughout the class of Insecta. These residues are marked in Figure 75 with red dots: residues at positions 2 and 16 in helix 1; 26 in helix 2; 54 and 70 in helix 3, 72, 113 and 195. Blue dots indicate amino acid substitutions in PcEcR‐LBD that are similar to those in hemipteran species and different form the hymenopteran. Yellow dots indicate the amino acids involved in the ligand binding in the EcR‐LBD (Kasuya et al., 2003): positions 47, 49, 50, 51, 53, 54, 56, 57 and 60 in helix 3; 91, 94, 95, 97, 98 and 99 in helix 5; positions 101, 109, 111; positions 127, 130, 131 and 134 in helix 7; positions 214, 218, 221, 222, 223, 225, 226, 227, 228, 229, 230 in helix 10‐11 and positions 236 and 240 in helix 12).

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Figure 75: Sequence alignment of ecdysone receptor ligand‐binding domains (LBD), including PcEcR‐LBD (Helix 1 to 5). In the following order: Drosophila, Aedes, Ceratitis, Bombyx, Junonia, Bicyclus, Tribolium, Tenebrio, Leptinotarsa, Apis, Polistes, Nasonia, Acromyrmex, Camponotus, Bombus, Solenopsis, Pheidole, Psyttalia, Nezara, Bemisia, Nilaparvata.

Figure 75 (continuation): Sequence alignment of ecdysone receptor ligand‐binding domains (LBD), including PcEcR‐LBD (Helix 6 to 12). In the following order: Drosophila, Aedes, Ceratitis, Bombyx, Junonia, Bicyclus, Tribolium, Tenebrio, Leptinotarsa, Apis, Polistes, Nasonia, Acromyrmex, Camponotus, Bombus, Solenopsis, Pheidole, Psyttalia, Nezara, Bemisia, Nilaparvata

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RT‐PCR from RNA extracted from dissected ovaries of female adults of P. concolor confirmed the expression of the EcR gene in the female tissues (Figure 76).

Figure 76: Confirmation of the expression

of the EcR in the ovaries of P. concolor

Sequence identity analysis was been performed for P. concolor. It shows that the LBD of P. concolor exhibits a stronger sequence identity towards the insect order of Hemiptera and also to the Coleoptera than to the Hymenoptera (Table 16).

Table 16: Sequence identity between PcEcR‐LBD and the EcR‐LBD in other insect orders (%) Diptera Lepidoptera Coleoptera Hymenoptera Hemiptera 65.3 (64‐67) 58.7 (57‐60) 81.0 (79‐82) 77.9 (74‐81) 79.7 (77‐84)

Data are given as average. Data in brackets refer to the range. Species used are Drosophila melanogaster, Aedes aegypti, Ceratitis capitata, Bombyx mori, Junonia coenia, Bicyclus anynana, Tribolium castaneum, Tenebrio molitor, Leptinotarsa decemlineata, Apis mellifera, Polistes dominulus, Nasonia vitripennis, Acromyrmex echinatior, Camponotus japonicus, Bombus terrestris, Solenopsis invicta, Pheidole megacephala, Bemisia tabaci, Nilaparvata lugens, Nezara viridula.

Phylogenetic trees of the EcR‐LBD, including various insect species from several orders such as Diptera, Lepidoptera, Hymenoptera, Hemiptera, Orthoptera, Coleoptera together with some Crustacea and Chelicerata, group PcEcR‐LBD together with the Hemiptera, close to Nezara viridula L. (Pentatomidae), instead of the Hymenoptera clade (Figure 77). Maximum parsimony trees also confirmed this result (data not shown).

This tree was constructed with the neighbour‐joining method using the amino acid sequences of the LBD of the selected sequences. Bootstrap values as percentage of a 1000 replicates, >50 are indicated on the tree.

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Figure 77: Phylogenetic trees of the EcR‐LBD, including various insect species from several orders.

Ecdysone agonists

5.4.4 Modeling of BoEcR-LBD and PcEcR-LBD and docking studies

BoEcR‐LBD and PcEcR‐LBD modeled from the X‐ray coordinates of different insect LBD, exhibited the canonical 3D‐conformation of the LBD of arthropod EcR made of twelve α‐helices (labeled H1‐H12). These twelve α‐helices are distributed along the polypeptide chain and are associated to a hairpin of two short β‐strands, β1 and β2 (Figure 78A and B, respectively). They form a protruding hairpin motif A very similar model was obtained for the fruit fly Drosophila melanogaster Meigen (Diptera, Drosophilidae) DmEcR‐LBD (Koelle et al. 1991) (Figure 78C). Both dipteran models readily resemble the tobacco budworm H. virescens HvEcR‐LBD three‐dimensional (3D) structure (PDB code 3IXP) used as a template, even though α‐helix H2 was not correctly X‐ray solved and is absent from the 3D structure of the HvEcR‐LBD (Figure 78D). The 3D conformation of the coleopteran T. castaneum TcEcR‐LBD has also been modeled (Figure78E).

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A B

C D E

Figure 78: Overall 3D conformation of the modeled LBD domain of the EcR receptors from B. oleae (A), P. concolor (B), D. melanogaster (C), H. viscerens (D) and T. castaneum (E), all in complex with ponasterona A (PoA) (colored stick). The twelve α‐helices and the two‐β strands are indicated. N and C consist of the N‐terminal and C‐ terminal ends of the polypeptide chain, respectively

Helices H2, H3, H5, H8 and H11 delineate a ligand‐binding cavity which usually accommodates the natural insect ecdysteroids 20E (Figure 79) and also the ponasterone A (PoA) molecule (i.e. an insect steroid hormone involved in regulating methamorphosis) (Figure80). Docking experiments performed with these two ecdysteroids yielded a typical H‐bonding scheme the BoEcR‐LBD and the PcEcR‐LBD share with other arthropod EcR‐LBD. Both ecdysteroids interacted with the BoEcR‐LBD pocket via a network of 6 hydrogen bonds involving the hydrophilic residues Glu16, Thr48, Thr51, Ala103 and Tyr113 (Figure 79‐2A and 82‐ 2A) (Glu21, Thr59, Trh62, Ala114 and Tyr124 in Figure 74). The PcEcR‐LBD pocket interacted via a network of 7

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hydrogen bonds involving the hydrophilic residues Glu73, Thr105, Thr108, Arg145, Ala160 and Tyr10 (Figure 79‐2B and 80‐ 2B).

1A 1B

2A 2B

3A 4A

3B 4B

Figure 79: Clip view (dashed yellow line) of the ligand‐binding pocket of the BoEcR‐LBD (1A), PcEcR‐LBD (1B), HvEcR‐LBD (1C) and TcEcR‐LBD (1D) harboring 20‐hydroxyecdysone (20E) (pink stick). (E) Network of hydrogen bonds (dashed dark lines) anchoring 20E to the BoEcR‐LBD (2A), PcEcR‐LBD (2B), HvEcR‐LBD (2C) and TcEcR‐LBD (2D). Aromatic residues interacting with the ligand by stacking interactions are colored orange. In the figures A, residues are labeled according to the three‐dimensional model built for the BoEcR‐LBD. In figures B, C and D, residues are labeled according to the three‐dimensional model built for the PcEcR‐LBD

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1A 2A

2B 1B

3A 4A

3B 4B

Figure 80: Clip view (dashed yellow line) of the ligand‐binding pocket of the BoEcR‐LBD (1A), PcEcR‐LBD (2A), HvEcR‐LBD (3A) and TcEcR‐LBD (4A), harboring ponasterone A (PoA) (pink stick). Network of hydrogen bonds (dashed dark lines) anchoring PoA to the BoEcR‐LBD (1B), PcEcR‐LBD (2B), HvEcR‐LBD (3B) and TcEcR‐LBD (4B). Aromatic residues interacting with the ligand by hydrophobic interactions are colored orange. In the figures A, residues are labeled according to the three‐dimensional model built for the BoEcR‐LBD. In figures B, C and D, residues are labeled according to the three‐dimensional model built for the PcEcR‐LBD

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In addition, stacking interactions occurring with various aromatic residues located at the periphery of the ligand‐binding cavity complete the ligand anchorage into the pocket.

For the DBH‐based compounds, however, due to the restricted extent of the ligand binding cavity, a steric clash occurred with the methoxy‐phenyl ring of tebufenozide and methoxyfenozide, upon docking of these two agonists to the BoEcR‐LBD and PcEcR‐LBD, respectively. The same results are obtained with the chloride‐phenyl ring of halofenozide and PcEcR‐LBD (data not performed for BoEcR‐LBD). In the case of PcEcR‐LBD, another steric clash occurred with the unsubstituted phenyl ring RH‐5849. Although much less severe, a very light steric hindrance still occurred upon docking of RH‐5849 to the BoEcR‐LBD (Figures 81 and 82).

A B C

Figure 81: Clip view of the ligand‐binding pocket of the B. oleae BoEcR‐LBD harboring tebufenozide (A), methoxyfenozide (B) and RH‐5849 (C) (blue sticks). Note the steric clash () of tebufenozide and methoxyfenozide with the wall of the ligand‐binding pocket (A and B). Note the very light steric hindrance () of the B‐phenyl ring of RH‐5849 with the wall of the ligand‐binding pocket (C)

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A B C

Figure 82: Clip view (dashed yellow line) of the ligand‐binding pocket of the P. concolor PcEcR‐LBD domain harboring tebufenozide (A), methoxyfenozide (B), RH‐5849 (C) and halofenocide (D) (blue sticks). Note the steric conflicts (and ) of the four compounds with the wall of the ligand‐binding pocket of PcEcR‐LBD. Network of amino acid residues of PcEcR‐LBD (E) interacting with tebufenozide by hydrogen bond (dashed blue line), and hydrophobic interactions. Hydrophobic and aromatic residues are colored orange

In contrast there was no steric clash for the four DbH‐based agonists in the TcEcR‐ LBD and HvEcR‐ LBD (Figure 83).

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C DD

H E F G

I J

Figure 83: Clip view (dashed yellow line) of the ligand‐binding pocket of the T. castaneum TcEcR‐LBD domain (A) and the H. virescens HvEcR‐LBD domain (B) harboring tebufenozide. Network of amino acid residues of TcEcR‐LBD (C) and HvEcR‐LBD (D) interacting with tebufenozide by hydrogen bond (dashed blue line) and hydrophobic interactions. Hydrophobic and aromatic residues are colored orange. E, F, G, H, I and J, clip views (dashed yellow line) of the ligand‐binding pocket of the TcEcR‐LBD domain (E, G and I) and the HvEcR‐LBD domain (F, H and J) harboring methoxyfenozide (METHO), RH‐5849 (BH) and halofenozide (HALO)

These docking experiments suggest that DBH‐based insecticides like tebufenozide and methoxyfenozide readily differ from the agonist RH‐5849 by their effects on the

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Ecdysone agonists insect pest B. oleae, which is in agreement with the previous reported experimental data showing a rather different biological effect of the insecticides in this insect.

5.5 Discussion

5.5.1 Efficacy and toxicology of methoxyfenozide, tebufenozide and RH-5849 on Bactrocera oleae and Psyttalia concolor, respectively

In the present study, tebufenozide and methoxyfenozide did not shown any deleterious effect on direct mortality or life span of B. oleae adults, while RH‐5849 killed (nearly) all treated olive fruit flies (98‐100%), although 7 days after the treatments. Deleterious effects of RH‐5849 were higher when adults were exposed to treated surfaces than when they ingested the insecticide solution. The last result was not expected because although MACs have some topical activity, they primarily act by ingestion (Carlson et al., 2001).

Studies on larval stages of dipteran species proved that tebufenozide, methoxyfenozide and RH‐5849 were effective against larvae of the mosquitoes A. aegypti, Culex quinquefasciatus (Say) (Culicidae), A. gambiae and midges Chironomus tentans F. (Diptera, Chironomidae) (Smagghe et al., 2002; Beckage et al., 2004). In contrast, Paul et al. (2006) found a lack of activity of tebufenozide on A. aegypti larvae, while pyriproxyfen resulted effective against them. Studies carried on with tephritid fruit flies larvae shown that the IGR lufenuron had negative effects on different parameters when larvae of the tephritids C. capitata and Bactrocera. dorsalis (Hendel) were fed with the product, but no effects were reported for Bactrocera cucurbitae 142

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(Coquillett) (Chang et al., 2012). Similar experiments with azadirachtin and tebufenozide added to the larval C. capitata medium shown a reduction on adult emergence for the first compound, while no effects were detected for the MAC (González‐Núñez and Viñuela, 1997).

Deleterious effects on adults have also been demonstrated on different Tephritidae species. After the ingestion of neem leaf dust and a commercial formulation of neem, a reduction of life span of B. cucurbitae and B. dorsalis was reported (Khan et al., 2007). Lawrence (1993) also observed high mortality percentages (>75%) after 12‐14 days when females of Anastrepha suspensa (Loew) ingested diet that was treated with RH‐5849, which is in agreement with our results. In contrast, no effects were observed when B. oleae were fed with artificial diet treated with the IGRs azadirachtin, cyromazine, flufenoxuron and pyriproxyfen, although a slight negative effect on life span of adults was reported for lufenuron (Sánchez‐Ramos et al., 2011). The last compound also caused a significant mortality on adults of Bactrocera latifrons (Hendel), but had no effects on C. capitata, B. dorsalis and B. cucurbitae (Chang et al., 2012).

Although as explained before, it was not possible to evaluate the effects of the three products on the reproductive parameters of B. oleae, different studies shown deleterious effects on different dipteran species caused by IGRs on these parameters. Ecdysone has a regulatory role in yolk protein synthesis and ecdysone agonists would act on this parameter, suppressing egg development (Lawrence 1993). This author reported a suppression of the level of A. suspense egg development and maturation and a reduction of ovaries size when RH‐5849 was topically applied on females. When RH‐5849 was ingested, females were able to oviposit, but on the days 8‐10, 60‐75% of the eggs were not viable. It has been observed that neem decreased fecundity of B. cucurbitae and B. dorsalis due to the block of ovarian development. Similarly, diets

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Ecdysone agonists treated with neem caused a reduction on fertility of B. cucurbitae and B. dorsalis (Singh 2003; Khan et al., 2007). Complete egg mortality was also observed when B. oleae and C. capitata females were fed with diet treated with lufenuron (Casaña et al., 1999; Sánchez‐Ramos et al., 2011). The product also affected fertility of B. dorsalis, B. latifrons (Chang et al., 2012), Anastrepha ludens (Loew), Anastrepha obliqua Mcquart, Anastrepha serpentina Wied. and Anastrepha striata Schiner (Moya et al., 2010). However, it had no effects on B. cucurbitae fertility or on C. capitata fecundity (Chang et al., 2012). A lower activity on B. oleae and C. capitata reproductive parameters was observed for the IGRs cyromazine, azadirachtin, flufenoxuron, triflumuron, diflubenzuron, methoprene, fenoxycarb, buprofezin, benzylphenol J2644 and pyriproxyfen (Casaña et al., 1999; Sánchez‐Ramos et al., 2011), although pyriproxyfen had no effects on B. oleae (Sánchez‐Ramos et al., 2011).

Evidence collected to date indicates that the MAC insecticides such as methoxyfenozide, tebufenozide and RH‐5849 have an excellent margin of safety to non‐target organisms, including a wide range of non‐target and beneficial insects as well as mammals, birds and fishes (Dhadialla et al., 1998; 2005; Aller and Ramsay, 1988; Carlson et al., 2001; Mommaerts et al., 2006). The latter is in agreement with the results obtained in this study for the parasitic wasp P. concolor. These products also resulted harmless when their residual contact activity on an inert surface was tested on the nymphs or adults of the predators of O. laevigatus, Macrolophus pygmaeus (Rambur) (=M. caliginosus) (Hemiptera, Miridae) and Amblyseius californicus (McGregor) (= Neoseiulus. Acari, Phytoseiidae) (Van de Veire and Tirry, 2003). No deleterious effects were detected when the hemipterans Orius insidiosus Say, Podisus maculiventris (Say) and Podisius sagitta (F.) (Pentatomidae) were exposed to relatively high doses of RH‐5849 and tebufenozide either (Smagghe and Degheele,

1994a,b, 1995; Schneider et al, 2003). Similarly, tebufenozide was harmless for C. carnea adults and pupae and P. maculiventris (Viñuela et al., 2001). Amongst parasitoids, they proved also to be safe for the parasitic wasps Encarsia formosa (Gahan) (Aphelinidae) (Van de Veire and Tirry, 2003), Hyposoter didymator (Thunberg) (Ichneymonidae) (Schneider et al., 2003; 2008; Viñuela et al., 2001), Telenomus remus

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(Nixon) (Scelionidae) (Carmo et al., 2010), Trichogramma cacoeciae (Marchal) (Grützmacher et al., 2004), Trichogramma pretiosum (Riley) (Trichogrammatidae ) and Allorhogas pyralophagus (Marsh) (Braconidae) (Bueno et al., 2008; Legaspi et al., 1999). In agreement with our results, several studies with tebufenozide have shown no deleterious effect of the product for P. concolor females (Jacas et al., 1995; González‐ Núñez and Viñuela, 1997; Viñuela et al., 2001), except a reduction of life span when females ingested the product via the drinking water (Jacas et al., 1995; González‐ Núñez and Viñuela, 1997).

In the present, no sublethal effects on the beneficial capacity of the parasitic wasps of P. concolor, namely their reproductive parameters, have been reported.

Ecdysteroids, together with the juvenile hormones (JH), are the two most important groups responsible for regulating insect growth, development, metamorphosis, and reproduction. Insect ecdysteroids are potent regulatory molecules, perhaps best known for their ability to trigger a molt. Other functions associated with ecdysteroid action include metamorphosis, egg and sperm production, chitin synthesis, and the inhibition of eclosion. Inscet host hormones have been demonstrated to influence endoparasites and endosymbionts in some host‐parasite systems. Bodin et al. (2007) identified ecdysone as the main ecdysteroid found in females and produced by the ovaries. This consistent with many studies showing that ecdysone stimulates vitellogenesis in female insects. Furthermore, the wasp parasitoid itself, typically by means of its secretory products, alters the biochemistry and physiology of its host by a variety of different mechanisms. Parasitoids have been reported to release ecdysteroids and JH into their host’s hemolymph, presumably to fine‐tune the levels of these hormones to meet their own developmental needs. Parasitized hosts typically exhibit abnormal patterns of hemolymph ecdysteroid fluctuation, especially in the last instar. The mechanisms responsible for these anomalies vary with the host‐parasite

145

Ecdysone agonists system under investigation and for most systems are not well understood. The parasitoid as well as its venom, calyx fluid, and teratocytes have been shown to play a role in altering host ecdysteroid levels. Their actions could serve as useful models for developing insect control strategies, for when the ecdysteroid that triggers the molt (typically 20E) does not reach threshold levels, host molting is prevented (Becakge and Gelman, 2004).

Synovigenic insects (i.e., insects emerging with few ripe eggs and maturing more eggs during the course of their lifetime) may suffer from transient egg limitation due to the stochastic nature of encounters with patchy hosts and the low availability of ripe eggs at any given time point. Egg limitation also affects the stability of host–parasitoid models. Ecdysone levels increased within two minutes of contact with a host, the fastest hormonal response reported for any insect. Even simple contact with a host, without further host use, triggered an increase in hormone levels, leading to the maturation of a single egg, using body reserves only. Feeding on the host caused a much larger increase in ecdysone levels and was followed by a marked increase in oogenesis. Oviposition had a weak effect on hormone levels, but increased oogenesis (Casas et al., 2009). In a study carried out by Bodin et al. (2007) with the parasitoid Eupelmus vuilleti (Hymenoptera, Eupelmidae), it was observed that a larger secretion of ecdysone was found in female during their reproductive period compared with inactive females. Furthermore, the presence of the host, which represents both the oviposition site and the nutritional source, induced an active biosynthesis of ecdysone. When hosts were available, this synthesis was cyclic and continuous during the entire female lifetime. These results showed that host presence triggered ovarian synthesis of ecdysteroids, which are involved in a stop‐and‐go regulation of egg production linked to host availability.

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Ecdysone agonists

Members of the genus Psyttalia are synovigenic parasitoids (Pemberton and Willard, 1918). Therefore, we can suppose that ecdysone plays an important role on reproduction, although there are not specific studies on this topic for P. concolor.

In agreement to our results no adverse effects were detected on reproduction (production of males) nor on the development of the larvae in the treated nests when MACs were applied to bumblebees, Bombus terrestris L. (Hymenoptera, Apidae) (Mommaerts et al., 2006). Experiments carried out by Jacas et al. (1995), González‐ Núñez and Viñuela (1997) and Viñuela et al. (2001) also shown no effects on P. concolor reproduction after the topical application or the ingestion of tebufenozide. In contrast, MACs strongly affect reproduction in sensitive insect species as Lepidoptera and Coleoptera which in many cases resulted in sterile female adults and/or abnormal genitalia, which hinder the mating process or the capacity to produce fertile offspring (Dhadialla et al., 1998; 2005; Tunaz and Uygun, 2004). The prevention or cessation of the oviposition of some Coleoptera, Lepidoptera and Diptera was also observed, and similar effects were reported for Hemiptera (Aller and Ramsay, 1988; Lawrence, 1993). Dissection of lepidopteran and coleopteran females treated with MACs and which had stopped oviposition, showed that the formation of new ovarioles seemed inhibited and they already showed signs of degeneration, resulting in very frail ovarioles. However, all the eggs that had been deposited by the treated females were equally viable (Smagghe and Degheele, 1994a,b, 1995; Farinós et al., 1999). In contrast, no alterations on the oocyte growth or on the ovulation process were detected in tebufenozide‐treated lacewing predatory adults (Medina et al., 2002).

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5.5.2. Molecular docking studies

Although sequence conservation for the LBD of NRs, including the EcR, is high, small amino acid substitutions in this domain can have a major impact on the 3D‐structure of the protein, and in particular on the size and shape of the ligand‐binding pocket

(Kasuya et al., 2003 According to Smagghe and Degheele (1994a), the wide range of susceptibilities to the ecdysteroid‐mimicking compounds in the different insect species may be explained by differences in the structure of the EcR and their binding affinity for the ecdysteroid agonist ligand molecules.

BoEcR‐LBD and PcEcR‐LBD exhibit a high conservation among the ligand binding‐ involved residues (indicated with blue dots in Figures 74 and 75). In Lepidoptera, which show a high sensitivity for tebufenozide and methoxyfenozide, the residues methionine58 and the valines100 and 111 (Figure 74) (corresponded to methionine56 and the valines 98 and 109 in Figure 75) are the divergent residues lining the binding pocket. In the case of other insect and non‐insect arthropods that show no or low susceptibility for tebufenozide and methoxyfenozide, these residues are substituted by isoleucine, methionine and isoleucine, respectively (Wurtz et al., 2000). These substitutions are also observed in BoEcR‐LBD and PcEcR‐LBD, which supports the results obtained in the current biological experiments with these compounds. The latter authors also reported that especially the isoleucine58 generates steric clashes between the γ‐methyl group of the isoleucine and the C5‐methyl group at the A‐ring or the C4‐ethyl group at the B‐ring of the tebufenozide molecule, depending on the orientation of the insecticide molecule.

As previously mentioned, in the case of BoEcR‐LBD, a steric clash also occurred with the methoxy‐phenyl ring of methoxyfenozide and tebufenozide. However, in the case of RH‐5849, which contains no substitutions on the two benzoyl rings, the steric 148

Ecdysone agonists

hindrance occurring upon docking of the products is much less severe (only light) as compared to the other two DBH‐based products. Thus, in this case, the differences on insect susceptibility might be due to the size and shape of the insecticide molecule. In the case of PcEcR‐LBD, two of the amino acid residues are completely different from the other EcR‐LBD sequences, namely threonine54 and methionine221, which are substituted by alanine and isoleucine in P. concolor, respectively.

Similar results than those obtained for P. concolor have been reported for bees (Mommaerts et al., 2006). Indeed the current data are strong indications that target site differences in the molting hormone reception play an important role. The latter hypothesis is consistent with the concept that structure and biochemical properties of EcR may differ among insect species. However, it needs also to be mentioned here that, next to the structure of the EcR‐LBD pocket, other factors as pharmacokinetics and metabolic detoxification additionally play an important role in determining the biological spectrum of the MAC insecticides (Wurtz et al., 2000). For instance, the penetration of tebufenozide in non‐sensitive C. carnea female adults was relatively slow and low, while the absorption in sensitive Lepidoptera was much more rapid (Medina et al., 2002). The latter results demonstrated that the low penetration and absorption patterns of tebufenozide also help to explain its non‐toxicity towards C. carnea larvae.

To date different sequences for the EcR‐LBD in Hymenoptera are already available. For a number of social insects belonging to the Formicidae, Vespidae and Apoidae families, the sequence is known, but these three families comprise only a small part of the large order of Hymenoptera. Apart from these social insects, only one EcR sequence is known for another hymenopteran, namely the Pteromalidae parasitoid wasp Nasonia vitripennis (Walker), of which the genome has recently been sequenced (Werren et al., 2010). Sequence alignment analysis with PcEcR‐LBD indicated a number

149

Ecdysone agonists of substitutions in regions of the LBD that are usually strongly conserved. It also indicated a higher sequence identity towards the Hemiptera than to the Hymenoptera. Phylogenetic analysis confirmed this, showing that PcEcR‐LBD grouped together with the Hemiptera rather than the Hymenoptera. Indeed the EcR‐LBD of P. concolor exhibits higher sequence identity on amino acid level towards most Hemiptera orthologs such as N. viridula, B. tabaci and Nilaparvata lugens (Stål.) (Delphacidae) (77‐ 84%, average 79.7%), than to the other Hymenoptera orthologs (74‐81%, average 77.9%). However, these differences could also be partly caused by the limited amount of data that is available for non‐social Hymenoptera. It is clear from the sequence data that in some conserved regions, social hymenopteran insects, especially ants, have the same amino acid substitutions, while these are not shared by the EcR of the two parasitic wasps (N. vitripennis and P. concolor) or other insect species. Other examples of nuclear receptors not following the normal phylogeny have also been described before. For instance, the Mecopterida EcR proteins failed to cluster together with the rest of the Holometabola group, despite this being considered a monophyletic group (Bonnetton et al., 2003). A similar phenomenon was found in Hemiptera where members of the Sternorrhyncha suborder did not group together with the Heteroptera or Auchenorrhyncha suborders to form a hemipteran clade. The phylogenetic distance of PcEcR‐LBD from that of Lepidoptera can explain the negative correlation with the high affinity of MAC for Lepidoptera. This negative correlation was also found for RH‐ 5849 in N. viridula (Tohidi‐Esfahani et al., 2011), whose EcR‐LBD exhibits a high sequence identity with PcEcR‐LBD.

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5.6 Appendix (tables of results)

151

Table 17: Efficacy of methoxyfenozide, tebufenozide, RH‐5849, dimethoate and spinosad on B. oleae (mean data ± standard error)

Mortality 24h Mortality 48h % Mortality 72h % Mortality 7d % Mortality 15d Residual contact on glass surfaces Control 2.0a ± 2.0 4.0a ± 4.0 4.0a ± 4.0 4.0a ± 4.0 45.8a ± 16.2 Methoxyfenozide 0.0a ± 0.0 0.0a ± 0.0 4.0a ± 4.0 4.0a ± 4.0 49.1a ± 14.4 Tebufenozide 0.0a ± 0.0 2.0a ± 2.0 2.0a ± 2.0 18.0b ± 5.8 58.0a ± 11.6 RH‐5849 0.0a ± 0.0 26.0b ± 8.7 54.0b ± 13.3 86.0c ± 7.5 100.0b ± 0.0

Spinosad 62.0b ± 6.6 88.0c ± 3.7 96.0c ± 4.0 100.0d ± 0.0 100.0b ± 0.0 Dimethoate 100.0c ± 0.0 100.0c ± 0.0 100.0c ± 0.0 100.0d ± 0.0 100.0b ± 0.0 F5,24= 295.87 F5,24= 113.16 F5,24= 73.35 F5,24= 135.0 F5,24= 7.33 P<0.0001 P<0.0001 P<0.0001 P<0.0001 P = 0.0003 Oral toxicity

Control 0.0a ± 0.0 0.0a ± 0.0 2.5a ± 2.5 2.5a ± 2.5 2.5a ± 2.5 Methoxyfenozide 2.5a ± 2.5 2.5a ± 2.5 2.5a ± 2.5 2.5a ± 2.5 2.5a ± 2.5 Tebufenozide 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 RH‐5849 0.0 ± 0.0 2.0 ± 2.0 10.0 ± 7.7 31.8 ± 8.1 98.2 ± 1.8 a a a b b Spinosad 50.0b ± 6.3 98.0b ± 2.0 100.0b ± 0.0 100.0c ± 0.0 100.0b ± 0.0 Dimethoate 85.2c ± 14.8 100.0b ± 0.0 100.0b ± 0.0 100.0c ± 0.0 100.0b ± 0.0

F5,22= 48.51 F5,22= 841.31 F5,22= 96.52 F5,22= 139.64 F5,22= 924.4 P<0.0001 P<0.0001 P<0.0001 P<0.0001 P<0.0001

Data followed by the same letter are not significantly different (ANOVA, LSD; P≥0.05)

Table 18: Toxicological effects of methoxyfenozide, tebufenozide, RH‐5849, dimethoate and spinosad on P. concolor females (mean data ± standard error)

Mortality Mortality % Mortality % Mortality % Attacked % Progeny 24h 48h 72h 7 days hosts size Residual contact on glass surfaces

Control 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 93.4a ± 2.3 57.6a ± 13.2 Methoxyfenozide 2.0a ± 2.0 2.0a ± 2.0 2.0a ± 2.0 4.0a ± 4.0 96.7a ± 1.4 55.3a ± 9.3 Tebufenozide 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 86.2a ± 8.1 59.4a ± 11.0 RH‐5849 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 4.0a ± 4.0 89.8a ± 7.5 53.6a ± 7.9 Spinosad 8.0b ± 3.7 25.3b ± 4.4 35.3b ± 7.9 68.0b ± 13.6 ‐ ‐ Dimethoate 100.0c ± 0.0 100.0c ± 0.0 100.0c ± 0.0 100.0c ± 0.0 ‐ ‐

F5,24= 536.76 F5,24= 399.61 F5,24= 207.71 F5,24= 54.52 F3,12= 0.42 F3,12= 0.06 P<0.0001 P<0.0001 P<0.0001 P<0.0001 P = 0.7450 P = 0.9804 Oral toxicity

Control 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 ‐ 89.9a ± 0.7 53.9a ± 7.4 Methoxyfenozide 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 ‐ 98.3a ± 0.6 58.9a ± 7.2

Tebufenozide 0.0a ± 0.0 0.0a ± 0.0 0.0a ± 0.0 ‐ 99.1a ± 0.2 52.8a ± 4.9 RH‐5849 0.0a ± 0.0 2.0a ± 2.0 4.0a ± 4.0 ‐ 99.0a ± 0.2 40.6a ± 7.7 Spinosad 92.0c ± 3.7 98.0b ± 2.0 100.0b ± 0.0 ‐ ‐ ‐ Dimethoate 73.8b ± 12.0 100.0b ± 0.0 100.0b ± 0.0 ‐ ‐ ‐ 1 F5,24= 69.61 F5,24= 1941.20 K = 27.5455 ‐ F3,12= 0.68 F3,12= 1.27 P<0.0001 P<0.0001 P<0.0001 ‐ P = 0.5834 P = 0.3289

Data followed by the same letter are not significantly different (ANOVA, LSD; P≥0.05) Effects of oral toxicity of the products 7 days after the treatment were not measured 1Data analysed using Kruskal‐Wallis test

Conclusions

Chapter 6

CONCLUSIONS

After evaluating the ecotoxicological effects of kaolin, Bordeaux mixture and cooper oxychloride on the parasitic wasp P. concolor and the scale predator C. nigritus it can be concluded:

Neither direct mortality nor high negative sublethal effects of the products have been recorded on adults of P. concolor and C. nigritus through the different experiments performed. However, when P. concolor females ingested the pesticides via the drinking water some deleterious effects on mortality, life span and attacked hosts could be observed, especially in the case of kaolin. Progeny size was not affected by the products.

Kaolin seems to be a promising compound to be used in olive crops, taking into account that it affects beneficial arthropods to a lesser extent than compounds commonly used, such as dimethoate. However, because of its uncommon mode of action, special attention should be paid to its sublethal effects, such as reproduction and behaviour.

The two copper‐based products evaluated in these experiments can also be considered as alternatives against B. oleae. Furthermore, as they washed away easier than kaolin, they can be a good alternative for table olives (especially in organic farming). Nevertheless, the possible negative effects that copper residues could provoke on the ecosystems if they accumulated in the soil should also be taken into account.

From a practical point of view, they might be considered together with other insecticides, such as dimethoate or spinosad, if an accurate resistance management program is likely to be applied, both for integrated pest management programs and

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organic oliviculture. However, because their effectiveness is conditioned by the pest level, they should be used in those years in which populations are low.

The study of the efficacy of methoxyfenozide, tebufenozide and RH‐5849 on B. oleae and their ecotoxicological effects on P. concolor through biological assays and molecular and docking experiments has reported the following conclusions:

For B. oleae adults, data showed no biological activity of methoxyfenozide and tebufenozide, while insecticidal effects were found for RH‐5849. Modeling and docking experiments also suggest that tebufenozide and methoxyfenozide are not effective against the pest. In contrast, RH‐5849 demonstrated a promising activity against it. More experiments for testing the product on other pest developmental stages pest are needed to confirm this issue. Evaluation of effects on olive fruit fly reproduction should also be performed.

No biological activity of methoxyfenozide, tebufenozide and RH‐5849 on P. concolor was found. Modeling of the PcEcR‐LBD and docking experiments also indicate that DBH‐based insecticides are devoid of any deleterious effect on the wasp.

Searching and developing of new insecticides to control B. oleae could be based on the basic lead structure of RH‐5849 molecule (1, 2‐dibenzoyl‐1‐tert‐butylhydrazine).

These products could be safely applied in IPM programs in which the parasitic wasp is present. However, it is recommended to test MACs also on other species to prevent undesirable effects on the auxiliary fauna.

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References

Chapter 7

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182

Appendix

INDEX OF FIGURES

Chapter 1:

Figure 1: An olive grove in Castile-La Mancha 1 Figure 2: Female of B. oleae 10 Figure 3: Detail of a B. oleae larva in an olive fruit. Microorganism growth can be observed in the feeding gallery 12 Figure 4: Adult of P. oleae 14 Figure 5: S. oleae adult females 15 Figure 6: Olive leaf spot 16 Figure 7: National and Autonomous Integrated Protection logos 21 Figure 8: Spanish, Autonomous Communities and European Union logos. Certification for European organic products (ECO CERT, SHC) 23 Figure 9: P. concolor female 30 Figure 10: C. nigritus adults 35 Figure 11: C. nigritus larva 36

Chapter 3:

Figure 12: Cage of C. capitata adults’ rearing 45 Figure 13: P. concolor adults’ cage 47 Figure 14: Temptative C. nigritus rearing established in the laboratory 48 Figure 15: A. nerii rearing. Infested and uninfested butternuts and potatoes are placed on wire baskets 49 Figure 16: Fungal contamination of B. oleae artificial diet 51 Figure 17: Methacrylate cages where third-instar larvae of B. oleae were collected when they jumped from the olive fruits 51 Figure 18: Round plastic cages used in the experiments 53 Figure 19: Cages used to evaluate beneficial capacity of P. concolor 54 Figure 20: C. capitata larvae transferred into Petri dishes after 1hour of exposure to P. concolor females 55

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Appendix

Chapter 4:

Figure 21: Chemicals used in the experiments 61 Figure 22: Kaolin-coated olive tree 62 Figure 23: Olive tree leaves and fruits covered by copper 64 Figure 24: Residual contact on glass surfaces test. C. nigritus cages have been mounted in the climatic chamber. The forced ventilation system is also observed 66 Figure 25: Pesticide solutions in the glass vials and plastic stoppers with the diet 67 Figure 26: Testing of the effects when products are ingested 68 Figure 27: Treatment of pupae using hand sprayers 69 Figure 28: Treatment of the meshes through which P. concolor females parasitize 70 Figure 29: Treatment of olive tree leaves 71 Figure 30: Detail of kaolin-treated leaves in the plastic cages 72 Figure 31: Olive tree leaves and parasitization surface treated 72 Figure 32: Olive tree in the wooden cage. Glass vials and stoppers can also be observed 73 Figure 33: Semi field experiment in the greenhouse. In the top of the wooden frames, the sand bags used to prevent C. capitata larvae from jumping when P. concolor females are parasitizing can be observed 74 Figure 34: Dual choice and no-choice experiments. C. capitata larvae were offered either on the top and the floor of the parasitization cages. The small plastic stopper placed on the top of the cages to prevent larvae from jumping and escaping is apparent 75 Figure 35: Detail of P. concolor females parasitizing through the bottom mesh of the cages 76 Figure 36: Experimental units: plastic cages covered with a piece of mesh held with a rubber band and binder clips 77 Figure 37: No-choice experiment: controls. The non-infested butternut is on the left of the picture and the infested one is on the right. Butternuts are placed on egg boxes. In the middle of the cage there is a glass vial with distilled water, a plastic stopper with E. kuehniella eggs and a piece of the semi-solid diet 78 Figure 38: No-choice experiments: kaolin replicates (non-infested butternut on the left and the infested one on the right) 78 Figure 39: Dual choice experiment (on the left, the infested butternut; on the right the non-infested one). Half of the butternut was treated with kaolin and the other half with distilled water 78 Figure 40: Detail of a kaolin-treated butternut. C. nigritus adults can be observed on the treated surface 78 Figure 41: Percentage of P. concolor and C. nigritus mortality 72 hours after different treatments. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 80

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Figure 42: Life span (number of days) of P. concolor when oral toxicity and treatment of pupae were evaluated. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 81 Figure 43: C. nigritus life span (number of days) during the residual contact on a glass surface and the extended laboratory experiments. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 81 Figure 44: Survival probability of C. nigritus adults (Series 1) and line of best fit by Weibull function (Series 2) at the different treatments on the residual contact on glass surfaces experiment 83 Figure 45: Survival probability of C. nigritus adults (Series 1) and line of best fit by Weibull function (Series 2) at the different treatments on the extended laboratory experiment 83 Figure 46: Percentages of P. concolor emergence from treated pupae. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 84 Figure 47: Percentage of P. concolor attacked host in different experiments. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 85 Figure 48: Percentage of P. concolor progeny size in different experiments 86 Figure 49: Percentage of P. concolor attacked hosts in the experiment in which females had to parasitize throughout a treated surface and in the experiment in which also treated olive tree leaves are placed into the parasitization cages. The differences between the beginning of the two experiments (treated materials) and the end (untreated meshes and no olive trees, in the second case) have also been compared. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 87 Figure 50: Percentage of P. concolor progeny size in the experiment in which females had to parasitize throughout a treated surface and in the experiment in which also treated olive tree leaves are placed into the parasitization cages. The differences between the beginning of the two experiments (treated materials) and the end (untreated meshes and no olive trees, in the second case) have also been compared 87 Figure 51: Percentage of P. concolor attacked hosts in the dual choice experiment. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 89 Figure 52: Percentage of P. concolor attacked hosts in the dual choice and no-choice experiments. With the aim of comparing experiments, in the dual choice assay all the kaolin treated and water treated surfaces have been grouped together. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 89 Figure 53: Percentage of P. concolor progeny size in the dual choice experiment. Asterisks indicate statistical differences between the treatments and the control (P < 0.05) 90

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Figure 54: Percentage of P. concolor progeny size in the dual choice and no- choice experiments. A With the aim of comparing experiments, in the dual choice assay all the kaolin treated and water treated surfaces have been grouped together 90 Figure 55: Daily fluctuation in the percentage of P. concolor attacked hosts in the no-choice experiment 91 Figure 56: Daily fluctuation in the percentage of P. concolor attacked hosts in the dual choice experiment 91 Figure 57: Dual choice experiment: percentages of C. nigritus adults placed in the infested butternuts, the non-infested ones or other parts of the experimental cages. Percentages were recorded during 4 days. “Water” means the half of the butternut treated with distilled water and “Kaolin” the other half, treated with kaolin 93 Figure 58: Percentages of C. nigritus adults placed in the infested butternuts, the non-infested ones or other parts of the experimental cages in the no-choice experiments. Percentages were recorded during 4 days. “Control” means the replicates in which both butternuts were treated with distilled water and “Kaolin” indicates the replicates in which both were treated with kaolin 93 Figure 59: Number of C. nigritus larvae found in the different replicates of each treatment. In the dual choice experiment, larvae on the butternuts were always observed on the non-treated parts of the butternuts. It can be observed the high percentage of dead larvae, especially on the kaolin treated butternuts 94

Chapter 5:

Figure 60: Modular structure (domains) of the insects’ ecdysone receptors 113 Figure 61: Insecticides tested in the experiments 115 Figure 62: PCR machine used in the experiments 116 Figure 63: Agarose gel with different PCR products loaded 116 Figure 64: Gel electrophoresis apparatus (an agarose gel is placed in the buffer-filled box and electrical field is applied via the power supply to the rear. The negative terminal is at the side of the apparatus closest to the tip box (colour blue), so DNA migrates toward it 117 Figure 65: Bio-Rad. Once the electrophoresis is completed, the molecules in the gel can be stained to make them visible. DNA can be visualized using ethidium bromide which, fluoresces under ultraviolet light, when intercalated into DNA. This apparatus is used to visualize DNA. Photographs of the gels can be taken using Gel Doc 117 Figure 66: Ethidium bromide-stained PCR products of the cloning (before and after purification) after gel electrophoresis (P. concolor) 120 Figure 67: Formed bacteria colonies on an ampicilin-containing LB agar plate (B. oleae) 120

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Figure 68: Ethidium bromide-stained plasmids after gel electrophoresis (P. concolor) 120 Figure 69: nucleotide and amino acid sequences of B. oleae and P. concolor 121 Figure 70: P. concolor ovaries 122 Figure 71: Percentage of mortality of B. oleae adults during the two performed experiments. Spintor-Cebo® was applied in the residual contact on glass surfaces experiment. Spintor 480 SC® was used for evaluating the oral toxicity. Asterisks indicate statistical differences between the treatments and the control within the same parameter evaluated (P<0.05) 125 Figure 72: Percentage of mortality of P. concolor females during the two performed experiments. Spintor-Cebo® was applied in the residual contact on glass surfaces experiment. Spintor 480 SC® was used for evaluating the oral toxicity. Asterisks indicate statistical differences between the treatments and the control within the same parameter evaluated (P<0.05) 126 Figure 73: Effects of methoxyfenozide, tebufenozide and RH-5849 on P. concolor beneficial capacity 126 Figure 74: Sequence alignment of ecdysone receptor ligand-binding domains (LBD), including BoEcR-LBD (Helix 1 to 8). In the following order: Heliothis, Chilo, Bombyx, Tribolium, Tenebrio, Leptinotarsa, Apis, Bombus, Psyttalia, Nilaparvata, Nezara, Bemisia, Drosophila, Calliphora, Aedes, Aedes, Chironomus, Culex, Bradisia, Anopheles, Ceratitis, Bactrocera 128 Figure 74 (continuation): Sequence alignment of ecdysone receptor ligand- binding domains (LBD), including BoEcR-LBD (Helix 9 to 12). In the following order: Heliothis, Chilo, Bombyx, Tribolium, Tenebrio, Leptinotarsa, Apis, Bombus, Psyttalia, Nilaparvata, Nezara, Bemisia, Drosophila, Calliphora, Aedes, Aedes, Chironomus, Culex, Bradisia, Anopheles, Ceratitis, Bactrocera 129 Figure 75: Sequence alignment of ecdysone receptor ligand-binding domains (LBD), including PcEcR-LBD (Helix 1 to 5). In the following order: Drosophila, Aedes, Ceratitis, Bombyx, Junonia, Bicyclus, Tribolium, Tenebrio, Leptinotarsa, Apis, Polistes, Nasonia, Acromyrmex, Camponotus, Bombus, Solenopsis, Pheidole, Psyttalia, Nezara, Bemisia, Nilaparvata 131 Figure 75 (continuation): Sequence alignment of ecdysone receptor ligand- binding domains (LBD), including PcEcR-LBD (Helix 6 to 12). In the following order: Drosophila, Aedes, Ceratitis, Bombyx, Junonia, Bicyclus, Tribolium, Tenebrio, Leptinotarsa, Apis, Polistes, Nasonia, Acromyrmex, Camponotus, Bombus, Solenopsis, Pheidole, Psyttalia, Nezara, Bemisia, Nilaparvata 132 Figure 76: Confirmation of the expression of the EcR in the ovaries of P.concolor 133 Figure 77: Phylogenetic trees of the EcR-LBD, including various insect species from several orders 134

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Figure 78: Overall 3D conformation of the modeled LBD domain of the EcR receptors from B. oleae (A), P. concolor (B), Drosophila melanogaster (C), H. viscerens (D) and T. castaneum (E), all in complex with ponasterona A (P1A) (colored stick). The twelve α-helices and the two-β strands are indicated. N and C consist of the N-terminal and C-terminal ends of the polypeptide chain, respectively 136 Figure 79: Clip view (dashed yellow line) of the ligand-binding pocket of the BoEcR-LBD (1A), PcEcR-LBD (1B), HvEcR-LBD (1C) and TcEcR-LBD (1D) harboring 20-hydroxyecdysone (20E) (pink stick). (E) Network of hydrogen bonds (dashed dark lines) anchoring 20E to the BoEcR-LBD (2A), PcEcR-LBD (2B), HvEcR-LBD (2C) and TcEcR-LBD (2D). Aromatic residues interacting with the ligand by stacking interactions are colored orange. In the figures A, residues are labeled according to the three- dimensional model built for the BoEcR-LBD. In figures B, C and D, residues are labeled according to the three-dimensional model built for the PcEcR-LBD 137 Figure 80: Clip view (dashed yellow line) of the ligand-binding pocket of the BoEcR-LBD (1A), PcEcR-LBD (1B), HvEcR-LBD (1C) and TcEcR-LBD (1D), harboring ponasterone A (PA1) (pink stick). Network of hydrogen bonds (dashed dark lines) anchoring P1A to the BoEcR-LBD (2A), PcEcR-LBD (2B), HvEcR-LBD (2C) and TcEcR-LBD (2D). Aromatic residues interacting with the ligand by hydrophobic interactions are colored orange. In the figures A, residues are labeled according to the three-dimensional model built for the BoEcR-LBD. In figures B, C and D, residues are labeled according to the three-dimensional model built for the PcEcR- LBD 138 Figure 81: Clip view of the ligand-binding pocket of the B. oleae BoEcR-LBD harboring tebufenozide (A), methoxyfenozide (B) and RH-5849 (C) (blue sticks). Note the steric clash () of tebufenozide and methoxyfenozide with the wall of the ligand-binding pocket (A and B). Note the very light steric hindrance () of the B-phenyl ring of RH-5849 with the wall of the ligand-binding pocket (C) 139 Figure 82: Clip view (dashed yellow line) of the ligand-binding pocket of the P. concolor PcEcR-LBD domain harboring tebufenozide (A), methoxyfenozide (B), RH-5849 (C) and halofenocide (D) (blue sticks). Note the steric conflicts (and ) of the four compounds with the wall of the ligand-binding pocket of PcEcR-LBD. Network of amino acid residues of PcEcR-LBD (E) interacting with tebufenozide by hydrogen bond (dashed blue line), and hydrophobic interactions. Hydrophobic and aromatic residues are colored orange 140

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Figure 83: Clip view (dashed yellow line) of the ligand-binding pocket of the T. castaneum TcEcR-LBD domain (A) and the H. virescens HvEcR-LBD domain (B) harboring tebufenozide. Network of amino acid residues of TcEcR-LBD (C) and HvEcR-LBD (D) interacting with tebufenozide by hydrogen bond (dashed blue line) and hydrophobic interactions. Hydrophobic and aromatic residues are colored orange. E, F, G, H, I and J, clip views (dashed yellow line) of the ligand-binding pocket of the TcEcR-LBD domain (E, G and I) and the HvEcR-LBD domain (f, H and J) harboring methoxyfenozide (METHO), RH-5849 (BH) and halofenozide (HALO) 141

INDEX OF TABLES

Chapter 1:

Table 1: Main olive grove phytophagous and their eating habits 8 Table 2: Olive grovepathogenic agents and abiotic diseases. Significance of the damage caused by them 9 Table 3: Integrated pest management in olive crops 24, 25, 26

Chapter 4:

Table 4: Chemicals evaluated in the experiments 61 Table 5: Parameters estimated for the Weibull function describing the survivorship of C. nigritus adults at different treatments in two experiments: residual contact on glass surfaces and an extended laboratory experiments in which olive tree leaves were treated (mean data ± standard error) 82 Table 6: Percentages of mortality 72 hours after exposure, attacked hosts and progeny size of P. concolor after kaolin, Bordeaux mixture, copper oxychloride and dimethoate applications on an inert surface, an extended laboratory and a semi-field experiments (mean data ± standard error) 105 Table 7: Percentages of mortality 72 hours after exposure, life span, emergence, attacked hosts and progeny size of P. concolor after kaolin, Bordeaux mixture, copper oxychloride and dimethoate applications on parasitized pupae or ingested via their drinking water (mean data ± standard error) 105 Table 8: Percentages of mortality 72h after exposure and life span C. nigritus adults after kaolin, Bordeaux mixture, copper oxychloride and dimethoate applications on an inert surface and in an extended laboratory experiment (mean data ± standard error) 107

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Table 9: Percentages of attacked hosts and progeny size when P. concolor parasitize through a kaolin, Bordeaux mixture or copper oxychloride treated surface with or without olive tree treated leaves. Percentages have been recorded when the surfaces were treated and when females were transferred into non treated cages (mean data ± standard error) 108 Table 10: Percentages of attacked hosts and progeny size in the dual choice and the no-choice experiments when P. concolor females parasitize through a kaolin-treated surface (mean data ± standard error) 109 Table 11: C. nigritus: dual choice and no choice experiments. Percentage of adults found on the butternuts and other parts of the experimental cages (mean data ± standard error) 109 Table 12: Classification of the products according to the IOBC criteria 110

Chapter 5:

Table 13: Chemicals tested in the experiments 115 Table 14: Specific conditions of PCR reaction steps for determining BoEcR- LBD and PcEcR-LBD coding sequences 119 Table 15: Degenerate and specific primers using for obtaining the partial sequences of the LBD 119 Table 16: Sequence identity between PcEcR-LBD and the EcR-LBD in other insect orders (%) 133 Table 17: Efficacy of methoxyfenozide, tebufenozide, RH-5849, dimethoate and spinosad on B. oleae (mean data ± standard error) 152 Table 18: Toxicological effects of methoxyfenozide, tebufenozide, RH-5849, dimethoate and spinosad on P. concolor females (mean data ± standard error) 153

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ACRONYMS

20E: the endogenous insect moulting hormone 20-hydroxyecdysone 3D: three dimensional modelling ATRIAS: Agrupaciones de Tratamientos Integrados en Agricultura (Agricultural Integrated Treatment Groups) AUAP: Abridged Universal Amplification Primer BART: Beneficial Arthropod Testing Group Bo-EcR-LBD: LBD of the EcR of B. oleae DBD: DNA-binding domain DBHs: Dibenzoylhydrazines EcR: Ecdysone receptor EIL: Economic Injury Level EPPO: European and Mediterranean Plant Protection Organisation in collaboration with the Council of Europe ET: Economic Threshold FAO: Food and Agriculture Organization of the United Nations IAEA: International Atomic Energy agency IFOAM: International Federation of Organic Agriculture Movement IGR: Insect Growth Regulators IP: Integrated Protection IPM: Integrated Pest Management JH: Juvenile Hormones JI: Joint Initiative LB: Lysogeny broth LBD: Ligand-binding domain MACs: Moulting Accelerating Compounds MARM: Ministerio de Medio Ambiente y Medio Rural y Marino (Ministry of the Environmental, Rural and Marine Environs). Now, MAGRAMA (Ministerio de Agricultura, Alimentación y Medio Ambiente) MFRC: Maximum Field Recommended Concentrations OIBC: Organisation for Biological and Integrated Control of Noxious Animals and Plants PcEcR-LBD: LBD of the P. concolor EcR PCR: Polymerase Chain Reaction PIEC: Predicted Initial Environmental Concentration PoA: Ponasterone A RACE-PCR: Rapid amplification of cDNA Ends-PCR SIT: Sterile Insect Technique USP: Ultraspiracle gen

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