UNIVERSIDAD POLITÉCNICA DE VALENCIA

Escuela Técnica Superior de Ingenieros Agrónomos

Departamento de Ecosistemas Agroforestales

Black scale Saissetia oleae (Hemiptera: Coccidae) on citrus and olives: population structure and biological control

DOCTORAL THESIS Presented by: Alejandro Tena Barreda Directed by: Dr. Ferran Garcia Marí

Valencia 2007

DEPARTAMENTO DE ECOSISTEMAS AGROFORESTALES E.T.S. INGENIEROS AGRÓNOMOS Universidad Politécnica Valencia Camino de Vera, s/n Telf.96-3879260 -Fax96-3879269 46022 VALÈNCIA

El Dr. Ferran Garcia Marí, Catedrático de la Universidad Politécnica de Valencia.

CERTIFICA:

Que la presente memoria titulada: “Black scale Saissetia oleae (Hemiptera: Coccidae) on citrus and olives: population structure and biological control”, realizado bajo mi dirección por D. Alejandro Tena Barreda, durante el periodo comprendido entre 2003 a 2007, constituye su Memoria de Tesis para optar al grado de Doctor, en el Departamento de Ecosistemas Agroforestales de la Universidad Politécnica de Valencia.

Para que así conste a todos los efectos oportunos, firma el presente certificado

Fdo: Dr. Ferran Garcia Marí

Valencia, Mayo 2007

Agradecimientos

Nombrar a cuantos han hecho posible la realización de esta memoria es cuanto menos una pretensión descabellada, así como injusto es pretender resumir en una simple frase toda su aportación. Así pués, y sabiendo de antemano que pecaré por omisión, quisiera expresar mi más sincero agradeciemiento

al Dr. Ferran Garcia Marí, el meu mestre, per qui sent admiració i respecte, per haver- me donat la seua confiança i l’oportunitat de realitzar la tesis baix la seua supervisió, per haver-me guiat, aconsellat, dedicat el seu temps i sobretot per haver-me transmès la riquesa de la seua experiència respectant sempre les meues idees.

to Dr. Robert F. Luck for giving me the opportunity of learning and working in his lab. For the long conversations about parasitoids and citrus pests.

a la Dra. Antonia Soto porque sin su esfuerzo nada hubiera sido posible. Al Dr. Francisco Ferragut por su ayuda y por transmitirme su pasión por los “bichos”. A Carmen por su infinita paciencia y por sus consejos (y por las recetas), a Marta y Lupita por haberme introducido en la taxonomía y por haberme guiado cuando andaba muy perdido, a Miguel Angel por todo, a Tolis por descubrir el comportamiento de los parasitoides y hormigas juntos, a Cristina por transmitir mucha alegría (y organizar las comidas). A todos mis compañeros y amigos del Departamento, a los que siempre estuvieron ahí (Toni, Juanjo), a los que pasaron (Nabor, Juan, Laura, Pablo, María, Teresa,…) y a los recien llegados (Amparo, Alex, Amador, Raquel) por haberme ayudado siempre, por las risas, por los buenos momentos vividos y por haber aprendido juntos.

to Dr. Apostolos Kapranas for transmitting all his knowledge about parasitoids, for teaching and guiding me in Riverside and for the drinks after work. To Porfirio Pacheco because “Metaphycus world” would not be possible without him, for teaching me English and for the “Mexican food”. To Bob’s lab people for helping me. To Dr. Gregory Walker for helping me with the montage and pictures of Metaphycus larvae. To Dr. Morse for his corrections in the second chapter and interesting conversations about thrips. To all my friends and colleagues in Riverside, Vicent and Aroa, Rory, Robbie, Rodrigo and Mariana, Diego, Jardel, Laura Simpson, Paul Flores, Laura,… for their friendship and support in the difficult moments.

a mis compañeros y amigos de la ETSIA por todo lo vivido y por haberme ayudado en lo que hiciera falta. En especial a David y Juan por cuidarnos tanto.

a les meues germanetes, a Vio y Carla, por haber crecido, aprendido y jugado juntos y sobretodo por todo el cariño y fuerza que me transmitis.

a mis padres por habérmelo dado todo, por todos los esfuerzos que han tenido que realizar a lo largo de su vida y por haberme comprendido y apoyado en este camino.

a Vero, por todo.

A mi familia

Table of contents

Resumen ...... i Resum ...... iii Summary ...... v

Chapter 1. Introduction ...... 1 1.1- Systematic classification of Saissetia oleae 1.2. Importance of Saissetia oleae 1.2.1. Importance on citrus 1.2.2. Importance on olives 1.3. Biology of Saissetia oleae 1.3.1. Morphology 1.3.2. Life cycle, dispersal and migration 1.3.3. Seasonal history 1.3.4. Influence of abiotic factors 1.4. Damages 1.5. Biological control 1.5.1. Natural enemy complex 1.5.2. Augmentative releases 1.6. Metaphycus as parasitoids of soft scales 1.6.1. Host range and specificity 1.6.2. Immature development 1.6.3. Adult 1.7. Superparasitism 1.7.1. Host discrimination 1.7.2. Consequences of superparasitism 1.7.3. Ovicide

Chapter 2. Justification and objectives ...... 37

Chapter 3. Density and structure of Saissetia oleae populations on citrus and olives: relative importance of the two annual generations...... 41

Chapter 4. Parasitoid complex of black scale Saissetia oleae on Citrus and Olives: seasonal trend and impact on host population...... 57

Chapter 5. Host discrimination, superparasitism and ovicide by Metaphycus flavus an endoparasitoid of Coccus hesperidum...... 79

Chapter 6. Conclusions ...... 109

Resumen

La caparreta negra es la principal plaga de cítricos y olivos del género Coccidae en todo el Mundo. Su incidencia es especialmente importante en la cuenca del Mediterráneo donde ambos cultivos se encuentran ampliamente distribuidos y coexisten a nivel local. Diversos enemigos naturales, principalmente parasitoides, han sido introducidos con el fin de controlar las poblaciones del cóccido. Sin embargo, su incidencia parece ser limitada y la caparreta negra continua siendo una plaga ocasional en muchos países Europeos, incluido España. Para mejorar el control de esta especie bajo los principios del Manejo Integrado de Plagas en ambos cultivos hemos estudiado el número y la importancia de cada una de sus generaciones, sus parasitoides a lo largo de los año 2003-05, así como algunos aspectos de la biología del parasitoide Metaphycus flavus.

Las poblaciones de caparreta negra mostraron una tendencia similar en ambos cultivos, presentando un máximo en julio, cuando las larvas móviles emergieron tras el periodo de puesta de las hembras adultas. A continuación se produjo una disminución poblacional debida a la alta mortalidad de las larvas de primera edad durante los meses de verano. Se observó una segunda salida de larvas móviles parcial, heterogénea y variable en otoño-invierno. Sin embargo, las poblaciones no aumentaron durante este periodo debido al efecto de las bajas temperaturas en la supervivencia de las larvas de primera edad y a la menor fertilidad de las hembras adultas, las cuales fueron la mitad de grandes y fecundas que las de primavera. No se encontraron diferencias entre el tamaño de las hembras adultas desarrolladas sobre cítricos y olivos. Si se realizan tratamientos para controlar posibles explosiones de la plaga recomendamos su aplicación a finales de julio, cuando las poblaciones son homogéneas, todas las larvas han emergido y las larvas de primera edad predominan en las poblaciones.

Los parasitoides más abundante y ampliamente distribuidos de la caparreta negra en cítricos y olivos del este de España fueron Scutellista caerulea, Metaphycus flavus y Metaphycus lounsburyi. Scutellista caerulea fue encontrada bajo el 35.4 ± 7.5% y el 22.4 ± 3.5% del cuerpo de las hembras fijadas en cítricos y olivos respectivamente. Sin embargo, su eficacia parece limitada porque las poblaciones de los dos parasitoides aumentan demasiado tarde para poder prevenir explosiones poblacionales del cóccido.

i

Metaphycus flavus fue el parasitoide de estadios inmaduros más abundante. Metaphycus helvolus, introducido hace 30 años, no ha desplazado a M. flavus como en otras regiones del Mediterráneo.

Metaphycus flavus es un endoparasitoide gregario facultativo que tiende a superparasitar cuando se cría sobre Coccus hesperidum, por lo que se llevó a cabo un estudio del comportamiento del parasitoide cuando este se encuentra con huéspedes previamente parasitados, así como las consecuencias de su comportamiento. Las hembras de M. flavus con y sin experiencia previa fueron capaces de distinguir entre los cóccidos sanos y parasitados, pero no pudieron distinguir entre aquellos parasitados por ellas mismas o por otras hembras de su misma especie. Las hembras no disminuyeron el tamaño de la puesta al superparasitar. Metaphycus flavus destruye los huevos previamente depositados en el cóccido antes de depositar los suyos, es decir, practica el ovicidio. Las hembras detectaron los huevos por la presencia del pedúnculo de estos, utilizando posteriormente el ovipositor para destruirlos. El número final de parasitoides que emergió de los huéspedes superparasitados se vió afectado por tres factores, encapsulación, competición entre larvas y ovicidio. El porcentaje de huevos encapsulados por el cóccido disminuyó desde alrededor del 65% para los huevos puestos en huéspedes sanos a menos del 25% en huéspedes previamente parasitados. Cuando la segunda puesta se retrasó dos o más días la descendencia de la segunda hembra estuvo en desventaja frente a la primera. Así, el ovicidio puede haber evolucionado en este sistema huésped-parasitoide porque la descendencia de la hembra que superparasita está en desventaja frente a la descendencia de la primera hembra y porque el sistema de defensa del huésped es menos eficiente al superparasitar y consecuentemente el huésped parasitado podría ser una mejor opción reproductiva que un huésped sano.

ii

Resum

La caparreta negra és la principal plaga de cítrics i olius del gènere Coccidae en tot el Món. La seua incidència és especialment important en la conca del Mediterrani on els dos cultius es troben àmpliament distribuïts i coexisteixen a nivell local. Diverses enemics naturals, principalment parasitoides, han segut introduïts per a controlar les poblacions del còccid. Però la seua incidència pareix ser limitada y la caparreta negra continua sent una plaga ocasional en molts països Europeus, inclòs Espanya. Per a millorar el control d’aquesta espècie sota els principis del Maneig Integrat de Plagues en els dos cultius hem estudiat el nombre i la importància de cadascuna de les seues generacions, els seus parasitoides al llarg del anys 2003-05, així com alguns dels aspectes de la biologia del parasitoide Metaphycus flavus.

Les poblacions de caparreta negra mostraren una tendència similar en els dos cultius, presentant un màxim en juliol, quan les larves mòbils emergiren seguint el període de posta de les femelles adultes. A continuació es va produir una disminució poblacional deguda a la alta mortalitat de les larves de primera edat durant els mesos d’estiu. Es va observar una segona eixida de larves mòbils parcial, heterogènia i variable durant la tardor-hivern. Tanmateix, les poblacions no augmentaren durant aquest període degut al efecte de les baixes temperatures en la supervivència de les larves de primera edat i a la menor fertilitat de les femelles adultes, les quals van ser la meitat de grans i fèrtils que les de primavera. No es varen trobar diferencies entre la grandària de les femelles desenvolupades sobre cítrics i olius. Si es realitzen tractaments per a controlar possibles explosions de la plaga recomanem la seua aplicació a finals de juliol, quan les poblacions son homogènies, totes les larves han emergit i les larves de primera edat predominen en les poblacions.

Els parasitoides més abundants i àmpliament distribuïts de la caparreta negra en cítrics i olius del est d’Espanya van ser Scutellista caerulea, Metaphycus flavus i Metaphycus lounsburyi. Scutellista caerulea va ser trobat baix el 35.4 ± 7.5% i el 22.4 ± 3.5% de les femelles adultes fixades en cítrics i olius respectivament. Tanmateix, la seua eficàcia pareix limitada perquè les poblacions dels dos parasitoides augmenten massa tard per a poder prevenir explosions poblacionals dels còccid. Metaphycus flavus va ser

iii

el parasitoide de estadies immadurs més abundant. Metaphycus helvolus, introduït ara fa 30 anys, no ha desplaçat M. flavus com ha passat en altres regions del Mediterrani.

Metaphycus flavus és un endoparasitoide gregari facultatiu que tendís a superparasitar quan es cria sobre Coccus hesperidum, per aquesta raó es va realitzar un estudi del comportament del parasitoide quan aquest es troba amb hostes prèviament parasitats, així com les conseqüències del seu comportament per a la descendència. Les femelles de M. flavus amb i sense experiència prèvia van distingir entre els còccids sans i parasitats però no distingiren entre aquells parasitats per elles mateixes o per altres femelles de la seua mateixa espècie. Les femelles no disminuïren la grandària de la posta quan van superparasitar. Metaphycus flavus destrueix els ous prèviament dipositats en el còccid abans de dipositar els seus, es a dir, practica l’ovicidi. Les femelles detectaren els ous per la presencia del peduncle d’aquests, utilitzant el ovipositor per a destruir-los. El nombre de parasitoids que va emergir dels hostes superparasitats es va veure afectat per tres factors, encapsulació, competició entre larves i ovicidi. El percentatge d’ous encapsulats per el còccid va disminuir des d’aproximadament el 65% per al ous dipositats en hostes sans a menys del 25% en hostes prèviament parasitats. Quan la segona posta es va retardar dos o més dies la descendència de la segona femella va estar en desavantatge front a la primera. L’ovicidi pot haver evolucionat en aquest sistema hoste-parasitoide perquè la descendència de la femella que superparasita està en desavantatge front a la de la primera femella i perquè el sistema de defensa del hoste parasitat és menys eficient al superparasitar i conseqüentment el hoste parasitat podria ser una millor opció reproductiva que un hoste no parasitat.

iv

Summary

Black scale is the most serious soft scale pest of citrus and olives throughout the World. Its incidence is especially important in the Mediterranean basis where both crops are widely distributed and coexist locally. Several natural enemies, mainly parasitoids, have been introduced to control the scale populations. However, their incidence seems to be limited and black scale remains as an occasional pest in many European countries, included Spain. In order to improve the control of this species under Integrated Pest Management practices in both crops we studied the number and importance of black scale generations, its parasitoid complex during 2003-05 and some aspects of the biology of its parasitoid Metaphycus flavus.

Black scale populations showed a similar trend in both crops, presenting one important peak in July, when crawlers emerged after the egg-laying period. Populations decreased during several moths due to mortality of first instars in summer. A second partial, heterogeneous and variable crawler emergence was observed in fall-winter, but populations did not increase during this time of the year due to the effect of low temperatures on first instar survival and the lower fertility of adult females. Females that gave rise to this fall-winter generation were half as big and fecund as spring females. No differences were found between the size of mature females developed on citrus and olives during spring. If chemical sprays are applied to control population outbreaks, we recommend to apply them at the end of July, when populations are homogenous, all crawlers have already emerged and first instars predominate in populations.

The most abundant and widely distributed parasitoids of black scale in citrus and olive crops in eastern Spain were Scutellista caerulea, Metaphycus flavus and Metaphycus lounsburyi. Scutellista caerulea was found beneath 35.4 ± 7.5% and 22.4 ± 3.5% adult female scale’s body in citrus and olive groves, respectively. However, their effectiveness seems limited when the scale is univoltine because they build up its populations too late to prevent scale outbreaks. Metaphycus flavus was the most abundant parasitoid of young instars. Metaphycus helvolus, introduced 30 years ago, has not displaced M. flavus as in other Mediterranean areas.

v

Metaphycus flavus is a gregarious facultative endoparasitoids which tends to superparasitize brown soft scale Coccus hesperidum under the colony conditions. The behaviour of M. flavus when encountering a parasitized host and its consequences were analyzed. Naïve and experienced females were able to discriminate between healthy and parasitized scales, but they could not discriminate between hosts parasitized by them or other conspecific females. Metaphycus flavus did not reduce the clutch size allocated in parasitized hosts. Metaphycus flavus practised ovicide/larvicide. They used the presence of egg stalks to detect the eggs previously laid, using their ovipositor to destroy the eggs inside the scale. The final brood size of superparasitized hosts was affected by three factors: encapsulation, larval competition and ovicide. The percentage of encapsulation decreased from around 65% for the eggs allocated in healthy hosts to less than 25% in parasitized hosts. Larval competition benefited the first clutch when the second oviposition was delayed two or four days. Ovicide may have evolved in this parasitoid- host system because the offspring of the superparasitizing female is in competitive disadvantage versus the offspring of the first female, and because the host defences has been already overcome when superparasitizing and, consequently, a parasitized host can be a better resource than a healthy host.

vi INTRODUCTION Chapter

1

Introduction

- 1 - CHAPTER 1

- 2 - INTRODUCTION

1.1. Systematic classification of Saissetia oleae

Black scale Saissetia oleae (Olivier) belongs to the order Hemiptera. Its classification is at follows: Order Hemiptera Suborder Homoptera Series Sternorrhyncha Superfamily Coccoidea Family Coccidae Genus Saissetia Species Saissetia oleae (Olivier 1791)

Prior to 1971, the facies of “S. oleae” was poorly understood, particularly in Africa and America, but De Lotto (1971, 1976) clarified the morphology of this complex of species, showing that it included Saissetia miranda (Cockerell and Parrott), S. neglecta De Lotto, S. oleae (Olivier) and S. privigna De Lotto. In Spain, only S. oleae is present on citrus and olives (Morillo 1977).

Saissetia oleae is world wide known as “black scale” (Ebeling 1959, Bedford et al.1998), “olive soft scale” (Bodenheimer 1951), “cochenille noire” (Panis 1974), “cochinilla de la tizne” (Gomez-Menor Ortega 1937, Ripollés 1990), “caparreta” (Llorens 1984), “caparreta negra” (Ripollés 1990, García Marí et al. 1994) or “cochonilha negra” (Passos de Carvalho et al. 2003).

1.2. Importance of Saissetia oleae

Black scale is a cosmopolitan and polyphagous insect which is considered one of the most important pests of olive and citrus trees (Morillo 1977, Ben-Dov and Hodgson 1997, Passos de Carvalho et al. 2003, Franco et al. 2006). Black scale is widely distributed in many parts of the world, in particular the tropics and subtropics, but it has only attained pest status in olive and citrus cultivated in temperate areas (Ben- Dov and Hodgson 1997), while in colder areas it thrives in greenhouses (Avidov and Harpaz 1969).

- 3 - CHAPTER 1

Black scale was originally described from specimens in olives trees on the Mediterranean coast of France in 1791 (De Lotto 1971). It is generally assumed that it originated in South Africa, where it is not a pest and is kept at low density by its natural enemies (Smith and Compere 1928, De Lotto 1976). However, some authors consider that it is native to Mediterranean coast, from where it expanded to the rest of the world altogether with the olive trees (Morillo 1977).

In Spain, black scale was firstly recorded at the beginning of the 19th century (Ruiz Castro 1951). It can be find on the Mediterranean and Atlantic coasts and on the main basin rivers (Morillo 1977) and it is considered an important pest of citrus (Limón et al.1976, Morillo 1977, Llorens 1984, Ripolles, 1990), olives (Morillo 1977, Fernandez et al. 1979, Llorens 1984, Montiel and Santaella 1995) and some ornamental plants (Morillo 1977, Llorens 1984).

1.2.1. Importance on citrus

All citrus varieties are attacked by the scale (Smith et al.1997) and, although actual assessment of losses caused by black scale on citrus is not available on a world wide basis nowadays, according to Talhouk (1975) it was the fourth citrus pest causing damages in the world. The damage black scale causes varies because of variation in the combined effect of mortality factors such as those arising from natural enemies and climatic effects (Panis 1977, Mendel et al. 1984). Thus, it is not considered a problem in hot and dry areas as the interior of California and Australia or in humid areas as Florida and South Africa (Reuther et al.1989).

Black scale was a pest of major importance on citrus in California until 1940 (Ebeling 1959, Reuther et al.1989), when the introduction of some parasitoids and the use of pesticides against California red scale Aonidiella aurantii Maskell (Hemiptera: Diaspididae) decreased considerably its importance (Reuther et al.1989). Nowadays, it is a cyclical pest only in southern California where it requires intervention every 5-10 years (Flint 1991). In Western Australia, it was also considered a major pest at the beginning of 20th century, until several natural enemies were introduced and controlled the pest (Waterhouse and Sands 2001).

- 4 - INTRODUCTION

A recent survey on the actual situation of citrus pest management in Mediterranean countries show that black scale is considered a key pest in Corsica (France); an occasional pest in Algeria, Italy, Montenegro, Morocco, Portugal, Spain and Turkey; and a potential pest in Greece and Georgia (Franco et al. 2006). In Israel, the importance of black scale increased considerably in the 1970s (Blumberg and Swirski 1988) but the introduction and management of several parasitoids controlled it (Argov and Rössler 1993) and nowadays the scale is not considered a pest (Franco et al. 2006).

In Spanish citriculture the importance of black scale increased during the 1980- 90’s because the use of petroleum oil sprays decreased during the summer, when the pest is more susceptible to treatments (García Marí et al.1994). In Comunidad Valenciana, the scale is found mainly in La Plana where the climate is more temperate and humid during the summer (Morillo 1977, www.agricultura.gva.es/rvfc/index.htm 2007), although its importance varies annually (Ripollés 1990).

1.2.2. Importance on olives

Of the soft scales living on olives only black scale has attained permanent pest status everywhere. Some olive varieties appear to be more susceptible than others (Rosen et al. 1971, Paraskakis et al. 1980) but olive trees of whatever variety are relatively free of black scale if planted singly or in single rows (Rosen et al. 1971). On the other hand, where branches of different trees touch and form canopy, as in olive groves, favourable microhabitats for the survival of scale populations occur (Ben-Dov and Hodgson 1997).

In California, black scale is spread throughout the olive groves of the state, causing economic damages since the 1890’s. During the 1940’s the use of DDT and parathion to control the new and more damaging olive scale, Parlatoria oleae Colvée (Hemiptera: Diaspididae) also suppressed black scale. Successful biological control of olive scale made insecticide use unnecessary and black scale again rose to prominence. Nowadays, black scale is a sporadic and explosive olive pest with wide fluctuations between years, regions and groves. Natural regulation is better in northern groves (Sacramento Valley) while San Joaquin groves have more frequent and damaging scale

- 5 - CHAPTER 1 outbreaks. These fluctuations are influenced mainly by pruning (Daane and Caltagirone 1989).

In the Mediterranean region, where 95% of the nearly 800 million olive trees of the world are grown, olive fruit fly Bactrocera oleae (Gmelin) (Diptera: Tephritidae), olive moth Prays oleae (Bern.) (Lepidoptera: Yponomeutidae) and black scale are the three most important olive pests. According to Haniotakis (2005) black scale is considered a major secondary pest, meaning that it occurs throughout the region, causing damages of major economic importance locally or ocasionally. Black scale was previosuly categorized as a major or key pest, but its status as an olive pest was reduced due to the advances in pest management, mainly pruning.

In Spain, blak scale was the second most important pest of olive trees during the 1970’s. It caused over 98.000 Tm of olives loss anually, that represented 3.45% of the total production (de Andrés 1991). Nowadays, it still remains as a main olive pest (Alvarado et al. 1997, Civantos 1999). In the interior north of Comunidad Valenciana the importance of black scale has increased during last years, and chemical sprays have been applied to control population outbreaks (Noguera et al. 2003).

1.3. Biology of Saissetia oleae

1.3.1. Morphology

Morillo (1977) gave a detailed description of the morphology of black scale and most of the following observations are based on his work.

Eggs: are oval, one of the extremes is more pointed than the other. Adult females secrete wax particles, which cover the eggs and prevent their sticking together (Bodenheimer, 1951). Eggs change from white color to pink, increasing the intensity as they develop. The eyes can be observed as two red points before hatching. Eggs are 0.26-0.32 mm long and 0.13-0.22 mm wide.

- 6 - INTRODUCTION

First instar: the body is flat, oval and lightly convex dorsally. They are light pink-orange with black eyes and six-segmented antennae. There are three long terminal setae at the anal plates. The central is longer and they can be used to distinguish this instar stage. Scales are 0.3-0.4 mm long and 0.18-0.20 mm wide before settling and 0.58-0.75 mm long and 0.20-0.35 mm wide at the end of this stage.

Second instar: a longitudinal ridge begins to take shape along the median line of the dorsum. A gradual obliteration of the two ends of this ridge leaves a central portion which later forms the bar of the letter H seen on older individuals. The body is pale brown in colour and four purple spots appear at the dorsum. They have also six-jointed antennae, which lack the long terminal setae, characteristic of the antennae of the first instar. The long setae of the anal plates of the first instar also disappear. They are 0.62- 0.80 mm long and 0.32-0.40 mm wide.

Fig. 1. Different developmental stages of black scale.

Third instar: they are less oval and more convex that the previous stages and the dorsum ridges are clearly present. The body is white-light brown coloured and darkens with age, the purple spots are bigger and darker. They have seven-jointed antennae. Scales are 1-1.2 mm long and 0.5-0.6 mm wide at the beginning of the stage, and 1.4- 1.6 mm long and 0.6-0.8 mm wide.

- 7 - CHAPTER 1

Adult female: after the third molt, the female increases rapidly in size and changes its shape, becoming nearly circular and hemispherical. The ridges of the letter H becomes distinctly outlined on the dorsum. As the egg-laying stage is approached, the scales become dark, mottled gray. At this moment the scales are known as “rubber stage”. When the egg laying begins, the scales became more leathery and acquire a smoother surface. They also become much darker in colour, finally becoming brilliant and black. They have eight-jointed antennae, the third joint being the longest. The size of the adults is quite variable, 1.9-5 mm long, 1-4 mm wide and 1.2-2.5 mm high.

Adult male: are usually very scarce. They have the usual two wings that occur on the coccid males, are about 1 mm long, and honey-yellow in colour (Quayle 1911).

1.3.2. Life cycle, dispersal and migration

The life cycle is similar on citrus and olive. Black scale is an oviparous and generally parthenogenetic species. Unmated females give rise to female progeny. Males are very rare and have been only reported in California (Quayle 1911), Australia (Simmonds 1951) and Chile (Gonzalez and Lamborot 1989). Black scale develops through three instars before reaching adult stage. The adult female lays the eggs under her body. Black scale is known by its high reproductive capacity, the number of eggs laid by the scale range from a few hundreds to 4000 depending on authors (Bodenheimer 1951, Babolini 1958, Ebeling 1959, Morillo 1977, Brailes and Campos 1986, Reuther et al. 1989, Smith et al. 1997, Passos de Carvalho et al. 2003). After hatching, the crawlers move out of the egg chamber beneath the mother’s body and they wander over the host plant searching for a suitable place to settle. Although they can wander for up to 36 hours they usually settle within 2-3 hours (Bibolini 1958). Generally the crawlers prefer to colonize the nearest suitable place to the mother scale, consequently, they tend to form groups and their distribution on the host plant is highly aggregated (Briales and Campos 1988). Most of the crawlers settle along the midrib on the undersurface of the leaves (Bibolini 1958, Ebeling 1959, Argyriou 1963, Morillo 1977).

The main instar dispersive stage is the crawler and dispersion may be active or passive. The passive has been explained above and the crawlers move as maximum from one tree to another searching for a suitable place to settle. The passive dispersal

- 8 - INTRODUCTION plays a fundamental role in the spread of the soft scale. The most important means of passive dispersal is the wind and this may easily transfer crawlers over large distances (Bibolini 1958, Argyriou 1963, Mendel et al. 1984).

Once they are settled, mainly in the leaves, they remain stationary until some stimulus induces them to migrate to twigs (Bibolini 1958, Ebeling 1959, Argyriou 1963, Morillo 1977). This migration appears to be stimulated by the search for nutritionally and climatically suitable niches (Briales and Campos 1986) or as an instinct still prevails, as consequence of feeding previously on deciduous trees (Ebeling 1959). The majority migrate during the third instar (Ebeling 1959, Santaballa 1972, Llorens 1984, Smith et al.1997). The percentage of the population that migrates to twigs varies depending authors. Thus, some earlier authors considered that only a small percentage moves to the twigs (Bibolini 1958, Argyriou 1963), whereas more recent observations suggest that the number of scales moving can be so high that, during the oviposition period, most adult females are on the twigs (De Freitas 1972, Briales and Campos 1986).

After the third molt and once in the twigs, the adult females increase rapidly (Morillo 1977), and as they develop the amount of honeydew secreted increases until it stops just before the oviposition (Bodenheimer 1951). The adult females die after the oviposition, remaining the body in the twig (Morillo 1977).

1.3.3. Seasonal history

The number of generations of black scale per year varies from one to four depending on the ecological conditions (Bodenheimer 1951, Panis 1977, Ben-Dov and Hodgson 1997). Thus, in citrus cultivated in humid and temperate areas such as Florida black scale has three generations, and even four in the subtropical areas of Australia (Rosen et al.1994, Panis 1977, Smith et al.1997, Waterhouse and Sands 2001). Whereas in hotter and drier areas such as California and the Mediterranean basin black scale has one or two generations on citrus (see Panis 1977) and olives (see Ben-Dov and Hodgson 1997, Passos de Carvalho 2003).

There are numerous studies about the history life of black scale on citrus and olives in the Mediterranean countries. The number of generations varies depending on

- 9 - CHAPTER 1 crop, area and author. Most of the authors find only one generation on citrus: Panis (1977) in France; Jarraya (1974) in Tunisia; Tuncyüreck (1975) in Turkey; Argyriou (1963) in the interior of Grecee; De Freitas (1977) in Portugal; Bodenheimer (1951) and Avidov and Harpaz (1969) in Israel; Barbagallo et al. (1992) in Italy; Panis (1977) in Spain. However, other authors find a second partial or complete generation: Santaballa (1972) and Llorens (1984) in Spain; Argyriou (1963) in the coast of Greece; Bodenheimer (1951) in Italy; Blumberg et al. (1975) in Israel.

Something similar occurs in the Mediterranean olives where black scale presents one complete generation per year according to: Morillo (1977), Briales and Campos (1986); Montiel and Santaella (1995) and Noguera et al. (2004) in Spain; Paraskakis et al. (1980) in Crete (Grecee); Argyriou (1963) in Grecee; Bibolini (1958) and Pucci (1986) in Italy. Other authors found that the scale completes a second partial or complete generation: Morillo (1977), Briales and Campos (1986) in Spain; Bibolini (1958) and Nuzzaci (1969) in Italy; Argyriou (1963), Canard and Laudeho (1977) in Grecee; De Freitas (1972) in Portugal.

On citrus cultivated in Comunidad Valenciana, the number of generations varies depending on authors, presenting one sole generation, one and a second partial or two complete (Ripollés 1990). Panis (1977) observed only one generation in Castellón. Santaballa (1972) described two generations in Valencia, emerging the crawlers in June and October. Llorens (1984) also found two generations, but according to this author the crawlers emerged in February-March and August-September. In olives cultivated in the province Alto Palancia (Castellón) black scale presented one sole generation and the crawlers emerge in June-July (Noguera et al. 2003).

Several theories have been proposed to explain the different number of generations. In general, it is considered that the number of generations depend mainly of the ecological conditions and the agronomic management (Panis 1977, Ben-Dov and Hodgson 1997). Thus, in groves irrigated (Peleg 1965) and well fertilized (De Freitas 1972), and in coastal areas (i.e. high summer humidity and mild winters) black scale may give rise to a second generation. Whereas there is usually only one annual generation in inland regions (i. e. hot and dry summers and cold winters) (Bodenheimer 1951, Argyriou 1963, Bartlett 1978). Other authors suggest that the plant physiology and the availability of nutrients are essential for the scale development (Ishaaya and

- 10 - INTRODUCTION

Swirski 1976, Bartlett 1960). Finally, Blumberg et al. (1975) suspect that the difference in the number of generations could be due to the presence of different black scale strains.

1.3.4. Influence of abiotic factors

Black scale populations suffer intense abundance oscillations not only along the year but also between years as consequence of biotic and abiotic factors (Panis 1977, Mendel et al. 1984). Orphanides and Kalmoukos (1970) summarized the main mortality factors; climate: mainly temperature, humidity, wind and rain; cultural practices: irrigation, fertilization, pruning and plant density; and biological: represented by the action of the natural enemies, these will be explained below.

Climate factors have a fundamental effect on black scale populations, mainly in the first instars. Mortality due to these factors can reach 99% in the active crawler stage. Such high mortality is caused by wind, heavy rain solar radiation and extreme temperatures (Bibolini 1958, Argyriou 1963, Mendel et al. 1984). In summer, once settled, temperatures over 30 ºC associated with relative humidity below 30% can cause a mortality rate over 80% (De Freitas 1972, Pucci et al. 1982). Low winter temperatures can also affect them. Pucci et al. (1982) recorded a 90% mortality of this stage at 3 ºC, while Canard and Laudeho (1977) considered that almost all eggs and crawlers that hatch during the winter perish. Mortality due to abiotic factors decreases with the age of the scales. At temperatures below 0 ºC, the mortality of second and third instars usually varies between 10 and 50% depending on authors. Adult females have the greatest resistance to low temperatures (Argyriou 1963, De Freitas 1972, Paparatti 1986, Pucci et al.1982).

Heavy rain, over 35 mm, may cause 50% of mortality when the temperature is 22-29 ºC and 80% when the temperature is unfavourable to the soft scale (Pucci et al. 1982).

Cultural factors: excessive plant density, lack of pruning, excessive irrigation and application of nitrogen fertilizer may improve the development of the scale(Panis 1977, Ben-Dov and Hodgson 1997).

- 11 - CHAPTER 1

Other factors: excess of sooty mould may impede the settlement and feeding of crawlers, increasing the mortiity by starving; some eggs form a melt mass under the scale impeding the free movement of the crawlers, which may die under the scale before leaving it (Morillo 1975, Katsoyannos 1996).

1.4. Damage

Black scale infests leaves and twigs of citrus and olives producing two different kinds of damages: direct and indirect. Direct damages are produced when large populations are present, their feeding can cause physiological damage to the host plant through an increase in the transpiration rate and by depletion of nutrients. Severe infestations cause premature leaf-fall, die-back of the branches and reduction in yield or even the absence of fruit of years (Ebeling 1959, Flint 1991, Ben-Dov and Hodgson 1997, de Andrés 1991), although Morillo (1977) asserts that the direct damages are scarce, since the insect feeds very small quantities of sap. A great deal of damage is caused indirectly by the large amount of honeydew excreted and by the subsequent development of sooty mould fungi belonging to the genera Capnodium, Cladosporium, Alternaria, etc. The sooty mould covers the leaves, reducing photosynthesis and transpiration, branches and fruits. The trees take on an unsightly, black appearance and the yields decrease in quantity and quality (Bodenheimer 1951, Ebeling 1959, Llorens 1984, Ben-Dov and Hodgson 1997, Agustí 2000, de Andrés 1991). Moreover, in olive groves the olive fly Bactrocera oleae, another important olive pest, is attracted by the honeydew secreted by the scale (de Andrés 1991).

Fig. 2. Indirect damages produced by Saissetia oleae on citrus

- 12 - INTRODUCTION

1.5. Biological control

Since the introduction of Rhizobious ventralis Erich (Coleoptera: Coccinalidae) in Califonria (EEUU) at the end of the 19th century (Bartlett 1978), extensive efforts have been made to control biologically black scale on citrus and olive around the world. So far different strategies have been carried out with different success. Summarizing two strategies have been the most successful for the satisfactory biological control. The commonest has been the introduction of a complex of parasitoids and predators rather than just a single species (Mendel et al. 1984, Gonzalez and Lamborot 1989, Orphanides 1993, Waterhouse and Sands 2001), which establishment is ensured by the presence of alternate hosts (Viggiani 1978). The other strategy has been the rearing and augmentative release of parasitoids of genus Metaphycus (Graebner et al. 1984, Daane et al. 1991).

1.5.1. Natural enemy complex

The commonest natural enemies associated with black scale are similar on citrus and olive, though their relative abundance and action on the soft scale may vary depending on the crop.

Some species of genus Metaphycus, which have been imported into Europe or exported from Europe for the control of black scale, had been incorrectly named until the revision of the European species of this genus by Guerrieri and Noyes (2000). Thus, the authors consider that the name Metaphycus lounsburyi (sensu Compere [1940] and Annecke and Mynhardt [1971]) is based on a misidentification of the type material described by Howard (1989). Metaphycus lounsburyi (Howard) is a valid name for the species up to now called Metaphycus bartletti Annecke and Mynhardt (M. bartletti is no longer a valid name). And what had been previously thought to be a single species (and referred to as M. lounsburyi), is a mix of two species: M. hageni Daane and Caltigirone and M. anneckei Guerrieri and Noyes. In the next revision we refer to them with the correct name according to Guerrieri and Noyes (2000).

Since 1902 until 1947, 22 species of parasitoids and two species of predators were introduced into Australia to control black scale. Nowadays, 13 of these parasitoids are regarded as permanently established and they control satisfactorily the pest. The

- 13 - CHAPTER 1 main parasitoids are considered M. anneckei and the pteromalid Scutellista caerulea (= S. cynea Motschulsky) (Fonscolombe), which larvae feed on the eggs laid by the scale. They are complemented by the native pteromalid Moranila californica (Howard) (Waterhouse and Sands 2001). Moreover, the entomopathogenic fungus Verticillium lecanii (Zimm.) may control high population densities of black scale in conditions of high relative humidity (Smith et al.1997).

In California (EEUU), over 50 natural enemies have been introduced from many regions of the world, approximately 20 of which are established (Kennet 1986, Lampson and Morse 1992). In the citrus groves located in the coastal area, where overlapping scale generations provide susceptible stages for a long time, the parasitoids Metaphycus helvolus Compere, M. lounsburyi and S. caerulea may control the pest if they are not disrupted by dust, pesticides, severe weather or the presence of Argentine ants (Linepithema humile [Mayr]) (Flint et al. 1991, Lampson and Morse 1992). However, in interior regions biological control is often ineffective in both crops because black scale's populations are strongly univoltine and synchronous, making it difficult for the parasitoids to establish (Kennett 1986).

Fig. 3. Larvae of Scutellista caerulea feeding on the eggs of Saissetia oleae.

Prior to the 1960’s, the natural enemies of black scale in the Mediterranean basin were mostly represented by predators, Chilocorus bipustulatus L., Exochomus quadripustulatus L. (Coleoptera: Coccinellidae), Eublemma scitula Rambur (Lepidoptera: Noctuidae), Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae), and the pteromalid S. caerulea. And by scarce general parasitoids, such as Metaphycus flavus (Howard) and Coccophagus lycimnia (Walker) (Hymenoptera: Aphelinidae). However, the mortality caused by these beneficial insects was considered too low to be

- 14 - INTRODUCTION effective and several natural enemies, mainly parasitoids from South Africa, where the pest is considered under natural control, were imported (Bedson and Hodgon 1997).

Several parasitoids of mature female scale were introduced, Moranila californica (Howard) (Hymenoptera: Pteromalidae), Diversinervus elegans Silvestri (Hymenoptera: Encyrtidae) and Metaphycus lounsburyi. The gregarious parasitoid M. lounsburyi was initially introduced into Israel from South Africa, and this has since become established in many Mediterranean countries. Nowadays, it is considered one of the most successful natural enemies (Viggiani and Mazzone 1980, Stratopoulou and Kapatos 1984, Argov and Rössler 1993, Orphanides 1993, Noguera et al. 2003, Pereira 2004). Moranila californica, considered a Chinese species, displaced Scutellista caerulea in Corfu, Greece (Stratopoulou et al. 1981), and it has been recovered in Israel and Italy (Mendev et al. 1984, Raspi and Brevin 1991). Diversinervus elegans was introduced and became established in Italy, France and Israel, where it was subsequently displaced by several species of genus Metaphycus (Viggiani and Mazzone 1971, Panis 1979, Mendel et al. 1984).

To improve the biological control of black scale, specific parasitoids, mainly of the second and third instars were introduced. Metaphycus helvolus, from South Africa, was the first and most successful parasitoid introduced in the Mediterranean basin (Argov and Rössler 1993). It was initially introduced in Greece by Argyriou and DeBach (1968) and subsequently was introduced in the rest of the countries (Viggiani 1978, Carrero 1981, Orpahnides 1993). Metaphycus helvolus has displaced the native parasitoid M. flavus in Crete, Greece (Argyriou and Michelakis 1975). Other parasitoids of young instars of black scale were reared and released but their real impact on black scale populations is poor or unknown (Argov and Rössler 1993, Guerrieri and Noyes 2000).

In Spain, the natural enemy complex of black scale has been studied mainly in olive groves in Andalucía, but also in citrus and olives from Comunidad Valenciana. In the late 1970’s M. lounsburyi and M. helvolus were introduced in eastern Spain to improve the biological control of the scale (Carrero 1981, Meliá and Blasco 1981, Ripollés 1986). Metaphycus lounsburyi became established (Noguera et al. 2003, Montiel and Santaella 1995), whereas the establishment of M. helvolus remains uncertain (Ripollés 1990). Summarizing, the most abundant and widely distributed

- 15 - CHAPTER 1 natural enemies are the parasitoids S. caerulea, Coccophagus lycimnia, C. semicircularis (Förster) (=Scutellaris) and M. flavus which have been documented in both areas and crops (Table 1). The real impact of these parasitoids on black scale populations is difficult to assess, since most of the studies, especially in citrus, are qualitative but not quantitative. Thus, S. caerulea is the most abundant on olive in Córdoba (Fernández et al. 1979), whereas, in Altura (Castellón) and Jaén are C. lycimnia and C. semicircularis respectively (Montiel and Santaella 1995, Noguera et al. 2003).

Fig. 4. Saissetia oleae infested by the entomopathogenic fungae Verticillium lecanii.

Finally, on citrus, the entomopathogenic fungus Verticillium lecanii may affect the surviving populations of black scale under certain conditions (Tuset 1992).

Table 1. Parasitoid species of Saissetia oleae reported in Spain.

Family Species Biology Crop Referenceb Aphelinidae Coccopahgus lycimnia (Walker) Facultative autoparasitoid Citrus, olive 1,3,4,7,8 Coccopahgus semicircularis (Förster) (=scutellaris) Obligate autoparasitoid Citrus, olive 2,3,6,8 Coccophagus cowperi Girault Facultative autoparasitoid Olive 8 Marietta picta Andre Secondary parasitoid Olive 4,8 Encyritidae Diversinervus elegans Silvestri Primary parasitoid Olive 6 Metaphycus anneckei Guerrieri y Noyes (=lounsburyi) Primary parasitoid Olive 9 Metaphycus flavus (Howard) Primary parasitoid Citrus, olive 2,4,7,8 Metaphycus hageni (Daane y Caltagirone) (=lounsburyi) Primary parasitoid Olive 9 Metaphycus hageni o Metaphycus anneckei (=lounsburyi) Primary parasitoid Citrus, olive 3,8 Metaphycus helvolus Compere Primary parasitoid Citrus, olive 3,6,8 Metaphycus lounsburyi (Howard) (=bartletti) Primary parasitoid Olive 4,6 Metaphycus zebratus Mercet Primary parasitoid Citrus 2 Eulophidae Baryscapus sp Secondary parasitoid? Olive 4 Pteromalidae Conomorium patulum (Walker) Secondary parasitoid Olive 8 Pachyneuron concolor Förster Secondary parasitoid Olive 8 Pachyneuron sp Secondary parasitoid? Olive 4 Scutellista caerulea (Fonscolombe) (=cynea) Egg predator Citrus, olive 1,2,3,4,5,6,7,8 Scutellista nigra Mercet Egg predator Citrus 8 b 1, Morillo (1977); 2, Limón et al (1976); 3, Panis (1977); 4, Nogera et al. (2003); 5, Fernández et al. (1979); 6, Montiel and Santaella (1995); 7, Briales and Campos (1985); 8, Carrero et al. (1977); 9, Daane et al. (2000).

- 16 - INTRODUCTION

1.5.2. Augmentative releases

As explained above, one of the main problems in improving the biological control of black scale is the fact that its populations can be strongly univoltine and synchronous. Consequently, there are long periods during the year when suitable scales are not available for parasitism. This causes a delay in the build up of parasitoid populations or even more, makes it difficult for them to establish (Flanders 1942, Panis 1977, Kennett 1986, Lampson and Morse 1992). One solution to this problem is the periodic augmentative releases.

Augmentative releases of M. helvolus have shown encouraging results in central California olive orchards (Daane et al. 1991) and in coastal southern California citrus (Graebner et al. 1984). Metaphycus helvolus was mass-reared in California by the grower-owned Fillmore Insectary (Fillmore, California) since 1937. The parasitoid was reared on black scale grown on 2 to 3-yr old oleander bushes, Nerium oleander L. (Rose and Stauffer 1997). Due to the huge amount of parasitoids recommended and to the large demand that exists to suppress black scale on citrus and olive, several studies on rearing M. helvolus were carried out to develop an economically efficient method of mass-producing (Lampson et al. 1996, Weppler et al. 2003). Unfortunately, the introduction of the glassy-winged sharp-shooter Homoladisca coagulata (Say) (Hemiptera: Cicadellidae), which transfer Xylella fastidiosa pathovar that kills oleander plants, increased the cost of this rearing system (Schweizer 2003).

More recently, the potential of M. flavus as an augmentative agent for black scale control has been also evaluated by Scheweizer et al. (2003). Metaphycus flavus resulted to be as effective as M. helvolus in controlling black scale, when retained inside sleeves cages, and they decreased black scale populations in an open-field situation. The release of M. flavus might be advantageous because the mass production of this species is less costly than that of M. helvolus, since it can be reared on brown soft scale Coccus hesperidum L. (Hemiptera: Coccidae).

Brown soft scale is grown on excised leaves of Yucca recurvifolia Salisbury (Agavaceae) maintained hydroponically in the University of California, Riverside. Brown soft scale-Yucca system is a method under development for rearing different species of Metaphycus. Among them, M. flavus has been successfully reared. However,

- 17 - CHAPTER 1 recent observations show that M. flavus tends to superparasitize under the colony conditions (L. D. Forster, P. Pacheco and R. F. Luck, unpublished data). Superparasitism in parasitoids may lead to very expensive rearing procedures in order to prevent high parasitoid mortalities, development of small and weak adults, as well as strongly male based sex ratios (van Lenteren 1981).

1.6. Metaphycus as parasitoids of soft scales

Metaphycus were firstly described by Mercet (1917) as subgenus of Aphycus Mayr. Species of Metaphycus play a crucial role in the natural regulation of their hosts and many of them have been used successfully in biological control programmes against agricultural pests, mainly soft scales (Guerrieri and Noyes 2000). In the next lines some aspects of their biology and behaviour are described.

Metaphycus flavus Metaphycus hhelvoluselvolus

Metaphycus luteolus Metaphycus angustifrons

Metaphycus lounsburyi Metaphycus stanleyi

Fig. 5. Different species of genus Metaphycus reported as parasitoids of Saissetia oleae.

- 18 - INTRODUCTION

1.6.1. Host range and specificity

Where their biology is known, all species of Metaphycus are primary endoparasitoids. In terms of host range, all levels of host specificity can be found in the genus. Strict specialists are M. deluchii Viggiani and several South African species that are reported from a single host (see Annecke and Mynhart 1971, 1972, 1981), while M. helvolus and M. insidious Mercet can be considered generalists. These species have been reported as parasitoids of a large number of soft scales. The degree of specificity seems to vary in terms of host species rather than host families. However, M. flavus has been recorded from many soft scales, a few Diaspididae and Kerriidae (Guerrieri and Noyes 2000).

1.6.2. Immature development

Maple (1947) was the first to give a detailed account of the morphology and physiology of some Metaphycus species eggs. When the egg is inside the female parasitoid it consists of two ovoid bulbous bodies connected by a narrow tube. The anterior bulb serves as a reservoir for the contents of the posterior body (the egg proper). When the egg passes down the ovipositor, the anterior bulb collapses and its contents are forced into the swollen posterior bulb, which contains the embryo. When the ovipositor is withdrawn, it frequently leaves a portion of the neck protruding through the host’s integument, which appears as an externally visible stalk.

The pre-imaginal development of Metaphycus follows the usual encyrtid pattern with tree to five larval instars and a pupal phase, although some variation has been observed (Guerrieri and Noyes 2000). Saakyan-Baranova (1966) gave detailed observations of the characteristics of each larval stage of M. luteolus (Timberlake). During the last larval development, septal walls are formed within the host that isolates each brood member in individual “cells” whenever a gregarious brood ensues (Bartlett and Ball 1964, Kapranas 2002). Pupation takes place inside these cells, after the last larval instar has voided its meconil pellets. Adults emerge via a hole chewed through the host’s dorsum, and only one exit hole is present per septal cell (Bartlett and Ball 1964, Kapranas 2002).

- 19 - CHAPTER 1

Fig. 6. Pre-imaginal development of Metaphycus flavus (L1, L2, L3 and L4).

Some of the eggs allocated can be encapsulated. Encapsulation of parasitoid’s eggs is the commonest physiological host defence against endoparasitoids (Quicke 1997). This mechanism involves the adhesion of host haemocytes to the surface of the parasitoid eggs/larvae that may produce the dead of these, preventing successful parasitism (Salt 1961, 1968). The capsule isolates the encapsulated parasitoid egg/larvae in the host’s haemocoel, causing its death by suffocation, starvation, or by physically preventing it from emergence. This explains why the capsule must be complete to be effective. Partially encapsulated parasitoids can survive and may continue to develop normally. Blumberg (1997) summarized the information of encapsulation by soft scales in response to Metaphycus parasitoids. The author identified seven factors as influencing Encyrtidae egg encapsulation rates by soft scales: 1) host and parasitoid species, eggs of a parasitoid species may be differently encapsulated in two species of hosts and vice versa; 2) physiological age of the host, larger individuals have larger number of hemocytes available for reaction; 3) host origin (or strain), geographic races of a given insect host may react differently to the same parasitoid; 4) physiological condition of the host, weak soft scales present lower encapsulation capacity; 5) superparasitism, this has been attributed to the host weakening by excessive parasitism, which lessens its ability to produce the complete reaction; 6) temperature, different effects on the incidence have been reported; 7) host plant, the host plant may sometimes determine the degree of immunity conferred on the host insect. Kapranas (2002)

- 20 - INTRODUCTION observed that the physiological conditions of the parasitoid may also influence the encapsulation rates.

A. Kapranas

Fig. 7. Encapsulated eggs of Metaphycus spp. by Coccus hesperidum.

Metaphycus reduce the supernumerary number of larvae developing inside their host, soft scales, through physical conflicts that result in the consumption of the loser. The larvae have mandibles that can be used against competitors (Bartlett and Ball 1964, Kapranas 2002). Such larval aggression might be adaptive under conditions of multiparasitism, if it occurs frequently, or if high rates of superparasitism occur, since it allows post oviposition regulation of brood size (Pexton and Mayhew 2001).

1.6.3. Adult

Kapranas (2006) reviewed the biology, ecology and behaviour of these parasitoids. Generally, Metaphycus are synovigenic, females emerge with at most a small fraction of their egg complement, (Flanders 1942, Lampson et al. 1996, Bernal et al. 1999). Although some species as M. insidiosus, M. anneckei, and M. stanleyi Compere emerge with mature eggs and start oviposition within a few hours after emergence (Bernal et al. 1999).

Host locating has been investigated in M. hageni and M. anneckei attacking S. oleae on different plants (Panis and Marro 1978, as M. lounsburyi). The female parasitoid usually walks back and forth along shoots and leaves infested by its host while tapping the substrate with the apices of both antenna. Once located, the host is similarly examined before assuming the oviposition posture. Lo Bue et al. (2004) found that M. sp. nr. flavus responded to chemical cues emitted directly by brown soft scale and not by the host plant from which the scale had been recently removed. Also,

- 21 - CHAPTER 1 preliminary studies showed that both M. sp. nr. flavus and M. luteolus recognize brown soft scale using a polar contact kairomone associated with the scale’s epidermis, which also stimulated oviposition behaviour (Kapranas et al. 2004).

Oviposition has been described for several species. Metaphycus flavus, M. hageni, M. helvolus, M. lounsburyi and M. luteolus insert the ovipositor through the dorsum of their soft scale host (Flanders 1942, Bartlett and Ball 1964, Lampson et al. 1996, Daane et al. 2000, Bartzman and Daane 2001, Kapranas 2002). While M. anneckei shows the unusual habit of inserting the ovipositor beneath the body of S. oleae (Panis and Marro 1978, Daane 2000). Oviposition durations range from 7 second in M. stanleyi to 189 seconds in M. luteolus (Bartlett 1961).

Metaphycus are arrhenotokous, unfertilized eggs produce only males. Sex ratio is usually female based when they develop gregariously (Kapranas 2006). Moreover, in the case of M. flavus and M. luteolus the sex of each egg is precisely ordered as the eggs are laid within a clutch. The wasp allocates several female eggs first, followed by one or more male eggs (Kapranas 2002; Kapranas 2006).

Metaphycus develop as solitary or gregarious parasitoids of soft scales. The size of the scale influences the number of developing offspring (Avidov and Podoler 1968, Bartllet and Ball 1964, Bernal et al. 1999, Kapranas 2006). When Metaphycus develop solitary in a host they usually follow the model host size-dependent sex allocation (Charnov et al. 1981), males emerge from smaller hosts, whereas females emerge from larger (Lampson et al. 1996, Bernal et al. 1999).

Some Metaphycus females feed on scale’s haemolymph to mature their eggs and to increase their longevity; however, to be effective, a carbohydrate source (honeydew or nectar) must be provided concurrently (Lampson et al. 1996). They insert their ovipositor into the host, and when the haemolymph oozes out, they feed on it. Host- feeding has been described in M. flavus, M. hageni, M. helvolus, M. lounsburyi and M. luteolus, whereas it has not been found in M. anneckei and M. stanleyi (Flanders 1942, Lampson 1996, Bernal 1999, Daane et al. 2000, Kapranas 2002). High mortality caused by Metaphycus may arise from host-feeding (Flanders 1942, Schweizer 2003).

- 22 - INTRODUCTION

1.7. Superparasitism

Superparasitism is defined as the deposition of a clutch of eggs on a host that has already been parasitized by members of the same species (Dijken and Waage 1987). Superparasitism is divided into self- and conspecific-superparasitism when the second clutch is laid by the same or other female respectively. (Waage 1986, Dijken and Waage 1987, van Alphen and Visser 1990). Long considered the result of mistake made by imperfect animals, superparasitism is now considered to be advantageous and adaptive under determinate characteristics (see van Alphen and Visser 1990). In the next lines some circumstances under which superparasitism in gregarious parasitoids is favourable are described. Conspecific-superparasitism can be advantageous when the survival rate of the second clutch is higher than zero and female parasitoids face a limited availability of hosts (Stephens and Krebs 1986). In this case the models predict that the superparasitizing female should lay a smaller clutch of eggs than the first female (Godfray 1994). Self-superparasitism can be beneficial when the total fitness performance of all the immatures in self-superparasitized hosts is higher than the fitness performance of the immatures in host attacked once only, or when conspecific are present (Vet et al. 1994, Gu et al. 2003, Yamada and Sugara 2003, Ito and Yamada 2005).

1.7.1. Host discrimination

A female parasitoid can only avoid superparasitism if it can recognize that a host has been previously parasitized, ability called “host discrimination”. This ability occurs in the major of families of parasitoid Hymenoptera (van Lenteren 1981).

The decision of a parasitoid to avoid a parasitized host may be influenced by 1) host availability, the probability of superparasitize is higher when unparasitized host are scarce (van Alphen and Visser 1990); 2) host quality, parasitoids are more willing to superparasitize recently parasitized hosts where their larvae have the greatest probability of survival (van Lenteren 1981, Mackauer 1990), they can detect it through the physiological changes in the host produced by the presence of developing parasitoids (Strand 1986); 3) external and internal markers, the use of marking chemicals in host

- 23 - CHAPTER 1 discrimination is known in many parasitoids and its recognition often depends on the time elapsed since the first oviposition (Chow and Mackauer 1986), in addition to chemicals cues, some parasitoids may use other mechanisms of host discrimination, as the presence of ectoparasitoid eggs or larvae (Godfray 1994), or the protruding egg stalk of some encyrtids (Takasu and Hirose 1988); 4) presence of adult conspecific, some parasitoids are more willing to superparasitize in the presence of conspecific than when they are searching alone (Bakker et al. 1985, van Alphen 1988, Visser et al. 1990, Yamada and Sugaura 2003); 6) physiological condition of the parasitoids, the supply of mature eggs influence the final decision (Rosenheim and Rosen 1991, Minkenberg et al. 1992); and 8) parasitoid previous experience, generally naïve parasitoids (i.e. those that have never oviposited before) are more likely to superparasitize than experienced ones (Van Alphen and Nell 1982, Van Alphen et al. 1987), whether a female has to learn to distinguish between a parasitized or unparasitized host or whether the female is adopting a conditional strategy based on the perceived density of unparasitized hosts remains a matter of debate (for recent discussion see: Henneman et al. 1995). There is a scarcity on data whether truly naïve wasps have an innate ability to discriminate (Henneman et al. 1995).

Van Dijken et al. (1992) reviewed if a parasitoid may distinguish between host attacked by itself or by others. Some of these studies failed to demonstrate the detection of self-parasitized hosts. The avoidance of self-superparasitism could work in two ways: the wasp might recognize the host itself, or might recognize the patch.

1.7.2. Consequences of superparasitism

The progeny of a superparasitizing female are normally at a competitive disadvantage in comparison with the progeny of the first parasitoid. In solitary species, the secondary larvae are often more likely to be eliminated, while in gregarious competition between individuals is normally restricted to exploitation competition for host resources (Godfray 1994). However, in parasitoids from genus Metaphycus supernumerary larvae may be eliminated by either physiological suppression (van Baaren and Nenon 1996) or by physical conflicts that result in the consumption of the loser, when developing gregariously inside their host (Bartlett and Ball 1964, van Baaren and Nenon 1996). The competitive disadvantage of the progeny of the

- 24 - INTRODUCTION superparasitizing female depend on the time elapsed since the first oviposition (Bartlett and Ball 1964, van Baaren and Nenon 1996).

Other results suggest that superparasitism not only increases the mortality of the second progeny but also delays the development of the progeny (Wylie 1983, Eller et al. 1990, Harvey et al. 1993, Potting et al.1997), results in smaller offspring (Harvey et al. 1993, Vet et al. 1994, Potting et al.1997) and/or produces a more male-biased sex ratio (Werren 1980).

Importantly, a parasitized host may not always be a lower quality resource than unparasitized host, since the host has already been overcome and prepared (Takasu and Hirose 1991). Thus, superparasitism could be considered as a mechanism by which parasitoids avoid encapsulation since the eggs allocated by the first female overwhelm the host immune system (Salt 1968, Blumberg and Luck 1990, van Alphen and Visser 1990).

Fig. 8. Coccus hesperidum superparasitized by Metaphycus flavus.

1.7.3. Ovicide

Ovicide may be considered a special case of superparasitism in which the female increases the probability of her offspring’s survival by killing previous female’s eggs (Strand and Godfray 1989, Mayhew 1997, Netting and Hunter 2000). Killing eggs laid previously may shift the competitive balance in favour of the ovicidal female.

Perhaps due to constrains on the ability of parasitoids to find previously laid eggs, the occurrence of ovicide is far less well documented than superparasitism and has been mentioned as an ovipositional strategy in only few parasitoids (Netting and Hunter 2000). Of these, all but one are ectoparasitoids (see Godfray 1994). Ovicide by

- 25 - CHAPTER 1 ectoparasitoids has been documented mainly in bethylids, which combine a precise oviposition, few large externally-laid eggs, and an asymmetrical larval competition that have made them to evolve ovicidal tactics (Mayhew 1997). In endoparasitoids, ovicide has been cited only for the solitary whitefly parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae), which uses its ovipositor to find and kill the egg of the first visitor inside the host (Arakawa 1987, Netting and Hunter 2000). Netting and Hunter (2000) suggested that ovicide is more probable to occur in endoparasitoids when an external cue identifies the location of the egg(s) within the host, as in the case of some Encyrtidae, where egg stalks protrude from the host cuticle.

The conditions under which adaptive ovicide might evolve in parasitoids has been modelled by Strand and Godfray (1989) using a game theoretical approach. Whether ovicide evolves depends on the relative pay-offs of ovicide, superparasitism and host rejection. Thus, according to Strand and Godfray’s model ovicide is more likely to evolve when: i) the time necessary to kill the eggs is short; ii) the proportion of parasitized hosts increases; iii) travel time between host increases; iv) the advantage of the first clutch is large. Smith and Lessells (1985) considered also that ovicide is more prone to evolve when there is little risk of killing one’s own eggs. Finally, some assumptions of the Strand and Godfray (1989) model may not hold for all parasitoids. Importantly, they predicted that a parasitized host could be a much worse resource than an healthy host, if ovicide takes time or the host deteriorates. Alternatively, since the host has already been overcome and prepared, a parasitized host could be a better resource than an unparasitized host (e.i. Takasu and Hirose 1988, 1991) (Mayhew 1997).

- 26 - INTRODUCTION

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Press, Princeton, UK. Strand, M. R. 1986. The physiological interactions of parasitoids with their hosts and their influence on reproductive strategies. In: Waage J. and Geathead D. (eds) Insects Parasitoids. Academic, London, UK. Strand, M. R., and H. C. J. Godfray. 1989. Superparasitism and ovicide in parasitic Hymenoptera: theory and a case study of the ectoparasitoid Bracon hebetor. Behavioral Ecology and Sociobiology 24: 421-432. Stratopoulou, E. T., and E. T. Kapatos. 1984. Prelimenary results for the evaluation of the action of Saissetia oleae parasites in Corfu. Entomologia Hellenica 2: 3-9. Stratopoulou, E. T., E. T. Kapatos, and G. Viggiani. 1981. Preliminary observations on the distribution and the action of Moranila californica (How.) in Corfu, a possible case of competitive displacement. Bollettino del Laboratorio di Entomologia Agraria Filippo Silvestre 38: 139-142. Takasu, K., and Y. Hirose. 1988. Host discrimination in the parasitoid Ooencyrtus nezarae: the role of the egg stalk as an external marker. Entomologia Experimentalis et Applicata 47: 45-48. Takasu, K., and Y. Hirose. 1991. The parasitoid Ooencyrtus nezarae (Hymenoptera: Encyrtidae) refers hosts parasitized by conspecific over unparasitized hosts. Oecologia 87: 319-323 Talhouk, A. S. 1975. Citrus pests throughout the world, pp. 21-23. In: Hafliger E. (ed), Citrus. Ciba-Geigy Agroquimicals. Basle, Swiss. Tuncyüreck, M. 1975. Observations sur la bio-ecologie de Saissetia oleae Bern. dans les vergers de la region egeene. Fruits 30: 163-165. Tuset, J. J. 1992. El hongo entomopatógeno Verticillium lecanii en el control de cóccidos en los agrios. Phytoma 40: 105-108. Vet, L. E. M., A. Datema, A. Janssen, and H. Snellen. 1994. Clutch size in a larval- pupa endoparasitoid: consequences for fitness. Journal of Animal Ecology 63: 807- 815 Viggiani, G. 1978. Current state of biological control of olive scales. Bollettino del Laboratorio di Entomologia Agraria Filippo Silvestre 35: 30-38. Viggiani, G., and P. Mazzone. 1980. Metaphycus bartletti Annecke et Mynhardt (1972), (Hym. Encyrtidae), nuovo parassita introdotto in Italia per la lotta biologica alla Saissetia oleae (Oliv.). Bollettino del Laboratorio di Entomologia Agraria Filippo silvestre 37: 171-176. Waage, J. K. 1986. Family planning in parasitoids: adaptive patterns of progeny and sex allocation. In: Waage J. and Geathead D. (eds). Insects Parasitoids. Academic, London, UK. Weppler, R. A., R. F. Luck, and J. G. Morse. 2003. Studies on rearing Metaphycus helvolus (Hymenoptera: Encyrtidae) for augmentative release against black scale (Homoptera: Coccidae) on citrus in California. Biological Control. 28: 118-128. Waterhouse, D. F. and D. P. A. Sands. 2001. Classical biological control of arthropods in Australia. ACIAR Monograph 67, ACIAR (Australian Center for International Agricultural Research). Canberra, Australia.

- 34 - INTRODUCTION

Werren, J. H. 1980. Sex ratio adaptations to local mate competition in a parasitic wasp. Science 208: 1157-1159. Wylie, H. G. 1965. Discrimination between parasitized and unparasitized housefly pupae by females of Nasonia vitripennis (Walk.) (Hym.: Pteromalidae). Canadian Entomologist 97: 279-286. Yamada, Y. Y., and K. Ikawa. 2005. Superparasitism strategy in a semisolitary parasitoid with imperfect self/non-self recognition, Echthodelphax fairchildii. Entomologia Experimentalis et Applicata 114: 143-152. Yamada, Y. Y., and K. Sugaura. 2003. Evidence for adaptive self-superparasitism in the drynid parasitoid Haplogonatopus atratus when conspecifics are present. Oikos 103: 175-181.

- 35 - CHAPTER 1

- 36 - JUSTIFICATION AND OBJECTIVES Chapter

2

Justification and objectives

- 37 - CHAPTER 2

- 38 - JUSTIFICATION AND OBJECTIVES

Justification and objectives

Black scale is considered an occasional pest of citrus and olives all along the Mediterranean basin, where these two crops coexist locally. Economic injuries caused by black scales vary because their populations suffer intense oscillations in abundance along the year and between years due to biotic and abiotic factors, making occasionally necessary the use of insecticides to maintain its populations under established thresholds. Thus, we studied and compared the density oscillations and structure changes of S. oleae populations on citrus and olives along the year to determine the best moment to apply chemicals for controlling occasional population outbreaks under Integrated Pest Management practices.

So far two different strategies have been the most successful for the biological control of black scale, introduction of exotic parasitoids and augmentative releases. In countries such as Australia and Israel, several parasitoids were introduced and nowadays they are regarded as permanently established and control satisfactorily the pest. Similarly, in eastern Spain M. helvolus and M. lounsburyi were introduced more than 30 years ago. However, their establishment and impact on the host and native parasitoids is unclear and black scale remains as an occasional pest. Thus, we initiated a study of the parasitoid complex of black scale to determine the main species present in citrus and olives, their geographical distribution, their seasonal abundance, and their incidence on the host population, as a first step in improving the biological control of black scale in Spain.

In California, augmentative releases of M. helvolus showed encouraging results in controlling black scale. However, this successful method, used during more than 40 years in southern California citrus, had to be abandoned. Recently, Metaphycus flavus showed promising results controlling black scale. Metaphycus flavus tends to superparasitize when it is reared on C. hesperidum. This behaviour might lead to very expensive rearing procedures in order to prevent high parasitoid mortalities, development of small and weak adults, as well as strongly male based sex ratios. Thus, we started a project to study the behaviour of M. flavus females when confronting with C. hesperidum previously parasitized and to examine the mortality causes of the first and second clutches in superparasitized hosts. Moreover, we describe ovicide by M.

- 39 - CHAPTER 2 flavus, reproductive strategy which had not been previously documented in gregarious endoparasitoids.

- 40 - SAISSETIA OLEAE POPULATIONS

Chapter

3

Density and structure of Saissetia oleae populations on citrus and olives: relative importance of the two annual generations

- 41 - CHAPTER 3

- 42 - SAISSETIA OLEAE POPULATIONS

Density and Structure of Saissetia oleae (Hemiptera: Coccidae) Populations on Citrus and Olives: Relative Importance of the Two Annual Generations

Abstract: Saissetia oleae (Olivier) (Hemiptera: Coccidae) populations were studied and compared in citrus (Citrus spp.) and olive (Olea europaea L.) groves to determine the number of generations, crawler emergence periods and changes in population density along the year. Ten citrus and four olive groves were sampled regularly between March 2003 and December 2005 in eastern Spain, covering an area of 10,000 km2. Each sample consisted of 16 branches and 64 leaves. Saissetia oleae populations presented a similar trend in both crops during the three years of study. Populations peaked in July, when crawlers emerged after the egg-laying period, and decreased during several moths due to mortality of first instars in summer. A second crawler emergence period less abundant, more dispersed, and variable depending on years, appeared between September and March. Populations did not increase during this period as expected, probably because almost all eggs and crawlers perished during the winter and also females that gave rise to this fall-winter generation were half as big and fecund as spring females. No differences were found between the size of mature females developed on citrus and olives during spring. The best moment to apply chemicals for controlling occasional population outbreaks would be at the end of July, when populations are synchronous, all crawlers have already emerged and first instars predominate.

- 43 - CHAPTER 3

3.1. Introduction

Black scale Saissetia oleae (Olivier) (Hemiptera: Coccidae) is one of the most widely distributed pests of citrus (Citrus spp.) and olive (Olea europaea L.) (Jeppson 1989, Ben-Dov and Hodgson 1997, Passos de Carvalho et al. 2003). Saissetia oleae populations suffer intense oscillations in abundance along the year and between years due to biotic and abiotic factors such as the action of natural enemies, the impact of broad-spectrum insecticides and environmental conditions (Panis 1977, Mendel et al. 1984). Low temperatures in winter and high temperatures coupled with low humidity in summer produce intense mortalities (Fernández et al. 1979, Pucci et al. 1982, Katsoyannos 1996). Economic injuries caused by S. oleae vary among years and groves, making occasionally necessary the use of insecticides to maintain its populations under established thresholds (Flint et al. 1991, Shoemaker et al. 1979). The main damage is caused by the large amount of honeydew excreted by the scale and the subsequent development of sooty mould fungi (Bodenheimer 1951, Ben-Dov and Hodgson 1997).

Saissetia oleae develops through three nymphal instars which migrate gradually from leaves to twigs. Once on twigs the parthenogenetic adult females lay from a few hundred to more than 2500 eggs (Morillo 1977, Briales and Campos 1986, Pereira 2004). High fecundity of S. oleae contributes to the outbreaks of the pest when environmental conditions are favorable. Determining the seasonal trend of S. oleae along the year is critical, since chemical sprays must be used against the first and second instars to ensure successful control (Flint et al. 1991). Else, the predominant stage and size of soft scales in the population is an important factor to carry out augmentative releases of parasitoids to control soft scales (Schweizer et al. 2003).

Several studies have been conducted in order to determine the number of generations per year of S. oleae and the main crawler emergence periods (Argyriou 1963, Blumberg et al. 1975, Briales and Campos 1986, Lampson and Morse 1992, Montiel and Santaella 1995). In Mediterranean countries, the number of generations per year varies between one, one and a partial second, or two, depending on the ecological conditions: crop species, nutrition of the tree, agricultural practices, possible different strains of S. oleae and, most importantly, climate (Bodenheimer 1951, Blumberg et al. 1975, Panis 1977, Passos de Carvalho et al. 2003). Usually, the second generation has

- 44 - SAISSETIA OLEAE POPULATIONS been observed in coastal areas, with higher humidity and less extreme temperatures compared with continental areas (Argyriou 1963, Flint et al. 1991). However, the relative contribution of both generations to the overall population abundance has not been clarified. In eastern-Spain, the life cycle of S. oleae remains unclear since the reported number of generations varies between one and two, being March, June and October the possible moments of crawler emergence along the year (Santaballa 1972, Panis 1977, Llorens 1984, Noguera, 2004). Moreover, no long-term and area-wide studies have been conducted to determine and compare its life cycle in both crops at the same time.

The aim of this research was to study and compare the density oscillations and structure changes of S. oleae populations on citrus and olives along the year. We also sought to determine the number and importance of S. oleae generations, crawler emergence periods and fecundity of the females, in order to improve the management of this species under Integrated Pest Management practices in two crops which are widely distributed and coexist locally all along the Mediterranean basin.

3.2. Materials and methods

3.2.1. Groves

Ten citrus and four olive groves selected for presenting medium to heavy infestations of S. oleae were sampled in different localities of eastern Spain, from March 2003 to December 2005. Groves were included in an area 200 Km long (north- south) and 50 km wide (east-west). The citrus groves were located in the following localities: Albal, Alcora, Altura, Castellón, Moncófar, Museros, Onda, Real de Montroy, Ribarroja and Xilxes. The olive groves included Altura, Castellón, Planes and Villar del Arzobispo. Each grove was sampled for a period ranging from 6 to 18 mo. Two to four groves were always sampled simultaneously on each crop and sampling date. The groves were sampled twice a moth during periods of rapid scale growth (April-October) and monthly during winter moths (November-March). No insecticide sprays were applied to the groves during the sampling period. Sampling was

- 45 - CHAPTER 3 discontinued when groves were treated with insecticides or when they had such light infestations that not enough S. oleae could be found.

3.2.2. Seasonal trend and mortality

For each sampling date, sixteen 15-cm long twigs with leaves were collected from a minimum of four different trees. The twigs were selected among the most heavily infested. The infested twigs were enclosed in plastic bags and transported to the laboratory. There, the twigs and four of their leaves (both sides) were examined under a stereomicroscope. The total number of alive and dead scales was segregated into the three nymphal instars (according to the description of Morillo 1977) and three types of adult females: young females (before oviposition), females with eggs, and females with crawlers (or eggs and crawlers), in order to determine the phenology of S. oleae populations and the mortality of different developmental stages. Mortality rates were calculated as number of dead scales divided by total number of scales either alive or dead. It was calculated separately for each developmental stage of S. oleae and only when number of scales alive and dead in that stage was higher than 20.

3.2.3. Fecundity and volume of ovipositing females

The fecundity and volume of S. oleae females on citrus and olives were analyzed between May 2004 and August 2005. In the laboratory, 775 egg-laying females, sampled in different dates and groves, were turned over carefully and the eggs were deposited into a marked Petri dish of 20 mm in diameter and counted under a stereomicroscope. The length, height and width of the females were measured under the stereomicroscope with the help of a micrometer. The volume of the females was calculated considering the shape of the female body as a half ellipsoid (Pereira 2004). Females whose eggs had already started to hatch were measured but their eggs were not counted.

3.2.4. Data analysis

Mortality rates were analyzed with a multifactorial ANOVA (Statgraphics 1994) considering as main factors, crop (citrus and olive), developmental stage (1st instar = L1; 2nd instar = L2; 3rd instar = L3) and plant substrate where nymphs settled (leaves

- 46 - SAISSETIA OLEAE POPULATIONS and twigs). To approximate a normal distribution, mortality rates were arcsine-square- root transformed (z = arcsine (x0,5)) prior to analysis.

The volume of mature females developed in spring was compared with a multifactorial ANOVA (Statgraphics 1994), with crop (citrus and olive) and year (2004 and 2005) as main factors. One-way ANOVAs (Statgraphics 1994) were also used to compare mature female volume between spring and fall on citrus; between leaves and twigs on olives; and among groves of the same crop. A two-way ANOVA was applied to compare the percent of S. oleae population settled on leaves and twigs, with developmental stage (1st instar = L1; 2nd instar = L2; 3rd instar = L3; adult female) and crop (citrus and olive) as main factors. Means were always compared with a protected LSD test at a 5% significance level.

3.3. Results

3.3.1. Seasonal trend, mortality and crawler emergence

Saissetia oleae populations showed a similar trend on citrus and olives during the three years of the study (Figs. 1 and 2), suffering intense fluctuations in abundance along the year. Populations peaked in summer (July), when crawlers emerged after the egg-laying period at the end of the spring (May and June). Subsequently, population density decreased during several moths, especially in summer, due to mortality of the nymphal instars.

No significant interactions were found between the three factors analyzed for mortality, crop, plant substrate and developmental stage (crop x plant substrate: ANOVA: F = 1.18; df = 2, 96; P = 0.3130; crop x developmental stage: ANOVA: F = 0.45; df = 2, 96; P = 0.6391; plant substrate x developmental stage: ANOVA: F = 0.45; df = 4, 96; P = 0.7717). Mortality rates decreased significantly as S. oleae went through the different developmental stages (L1 = 35.8 ± 2.7%, L2 = 22.4 ± 2.3% and L3 = 9.9 ± 2.2%) (ANOVA: F = 28.04; df = 2, 38; P < 0.0001). Saissetia oleae showed higher mortality rates on citrus compared with olives (ANOVA: F = 14.93; df = 1, 77; P < 0.0002). Finally, no significant differences were found between scales settled on leaves or twigs (ANOVA: F = 0.57; df = 1, 38; P = 0.56).

- 47 - CHAPTER 3 2000 2003

1500 Females with crawlers Females with eggs Young females Third instar 1000 Second instar First instar

500

0 JFMAMJJASOND 400 400 2004 2004 300 300 Females with crawlers Females with eggs Young females 200 200 Third instar Second instar First instar 100 100

No. insects per sample sample insects per No. 0 0 JFMAMJJASOND JFMAMJJASOND 4000 1200 2005 2005 900

3000 No. sample insects per

2000 600

1000 300

0 0 JFMAMJJASOND JFMAMJJASOND

Fig. 1. Seasonal trend of Saissetia oleae on citrus in eastern Spain from March 2003 to Fig. 2. Seasonal trend of Saissetia oleae on olives in eastern Spain from May 2004 to December 2005. The figure shows the average of the total number of insects alive per December 2005. The figure shows the average of the total number of insects alive per sample in 2-4 groves. Different colors and textures show different developmental stages. sample in 2-4 groves. Different colors and textures show different developmental stages.

- 48 - SAISSETIA OLEAE POPULATIONS

Figure 3 shows a homogeneous and concentrated crawler emergence in July, and a second extended crawler emergence which started between September and October and lasted until March. This second emergence period was variable depending on years. Saissetia oleae overwintered mainly as second and third instars (Figs. 1 and 2).

Figure 4 compares the accumulated percentage of first instars emerged in spring- summer. Differences were lower than 20 days when comparing different groves and crops sampled the same year (except for citrus in 2004 due to the low population levels observed).

Citrus 100%

80%

60%

40%

20% % stages (log(n+1)) (log(n+1)) stages %

0% JFMAMJJASONDJFMAMJJASONDJFMAMJJASOND

Olives 100%

Females with crawlers 80% Females with eggs Young females Third instar 60% Second instar First instar 40%

% stages (log(n+1)) (log(n+1)) stages % 20%

0% J FMAMJ J ASONDJ FMAMJ J ASOND Fig. 3. Relative seasonal trend of different developmental stages of Saissetia oleae on citrus and olives in eastern Spain from March 2003 to December 2005. Number of insects alive was logarithm transformed before calculating the percentage of each developmental stage. Average values of 2-4 groves.

- 49 - CHAPTER 3

Citrus 2003 Olives 2004 100 100

75 75

Moncofar Xilxes 50 50 Albal Planes Museros Villar 25 25

0 0 MJ J AS O MJ J ASO

Citrus 2005 Olives 2005

100 100

75 75 Percent of 1st instar accumulated 1stof Percent instar accumulated Percent of 1st of Percent instar accumulated Villar Castellon Castellon 50 Altura 50 Altura Onda-Alcora Planes 25 25

0 0 MJ J ASO MJ J ASO Fig. 4. Cumulative percentage of first instar Saissetia oleae in summer generation, in different citrus and olive groves sampled in eastern Spain between 2003 and 2005.

3.3.2. Fecundity and volume of ovipositing females

Adult females developed along the spring laid a maximum of 2603 eggs in citrus and 2675 in olives, whereas females developed in autumn laid a maximum of 774 eggs. There was a significant relationship between ovipositing female volumes and number of eggs found beneath females (Fig. 5). Thus, the volume of female represents accurately its potential fecundity. Female volume was variable along the year (Fig. 6) depending on season and location on the tree. On citrus, the average volume of spring females (12.07 ± 0.47 mm3) was much higher than the average volume of fall-winter females (4.52 ± 0.29 mm3) (ANOVA: F = 215.57; df = 1, 227; P < 0.0001) in 2004. Moreover, females settled on olive twigs (12.79 ± 0.33 mm3) were twice as big as females on olive leaves (6.08 ± 1.33 mm3) (ANOVA: F = 24.04; df = 1, 261; P < 0.0001).

When comparing only spring females, differences in female body volume were found between years in olives (2004 = 12.41 ± 0.32 mm3; 2005 = 8.73 ± 0.5 mm3) but not in citrus (2004 = 12.24 ± 0.61 mm3; 2005 = 10.96 ± 0.56 mm3) (ANOVA: F = 6.34; df = 1, 527; P = 0.0121). Significant differences in female volumes were found between years (2004 = 12.24 ± 0.34 mm3; 2005 = 9.85 ± 0.37 mm3) (ANOVA: F = 22.2; df = 1,

- 50 - SAISSETIA OLEAE POPULATIONS

527; P < 0.0001), but not between crops (citrus = 11.52 ± 0.41 mm3; olives = 10.57 ± 0.3 mm3) (ANOVA: F = 3.44; df = 1, 527; P = 0.0642).

3000

2500

2000

1500

1000

500 No.of per eggs female

0 0 5 10 15 20 25 30 Volume of ovipositing females

Fig. 5. Relationship between the volume (mm3) of Saissetia oleae ovipositing females and number of eggs found beneath their body. Data from citrus and olive groves in eastern Spain from May 2004 to December 2005 (R2 = 0.64; P < 0.0001; Y = -102.59 + 84.44X).

30

2004 25 2005 20

15

10

5 Volume of ovipositing females females ovipositing of Volume

0 MJ J ASOND

Fig. 6. Volume (mm3) variability of Saissetia oleae ovipositing females on citrus and olives groves in eastern Spain from May 2004 to December 2005.

3.3.3. Migration

Saissetia oleae migrated progressively from leaves to twigs (Fig. 7). At the beginning of their development, around 75% of first instar scales were settled on leaves, whereas only 5% of adult females appeared on this substrate. Settlement preference for leaves or twigs differed significantly among developmental stages of S. oleae in both citrus (ANOVA: F = 45.17; df = 3, 35; P < 0.0001) and olives (ANOVA: F = 14.82; df = 3, 15; P = 0.0002), but migration started earlier in scales developed in citrus than in olives (Fig. 7).

- 51 - CHAPTER 3

100

Olives 75 Citrus

50

25

insects on leaves%

0 st nd Adult 1 instar 2 instar 3rd instar female Developmental stages Fig. 7. Mean (± SE) percentage of Saissetia oleae on leaves during different developmental stages. Data from ten citrus and four olive groves sampled in eastern Spain from 2003 to 2005.

3.4. Discussion

This study presents the density and structure changes of S. oleae populations, the number and importance of its generations and the crawler emergence periods between 2003 and 2005 in eastern Spain citrus and olive groves. This research was carried out to improve the management of this species under Integrated Pest Management practices in two important Mediterranean crops.

Saissetia oleae populations showed a similar trend in both crops during the three years of the study. Populations reached a maximum in summer (July) due to a homogeneous and concentrated crawler emergence, following the egg-laying period at the end of the spring (May-June). First instars were present at the same time in all groves and crops sampled (Fig. 4). Thus, the crawler emergence of July here described might be generalized for all eastern Spain. Our observations are similar to other western Mediterranean authors, since most of them found a crawler emergence in June-July (Santaballa 1972, Briales and Campos 1986, Noguera 2004, Pereira 2004). Some of these authors observed also a second generation at least partial in autumn, which was not found by others. In our study a second partial crawler emergence appeared from September to March, although it was heterogeneous and variable depending on years, crops, and groves (Figs. 1 and 2). This second crawler emergence is apparently not important to the overall population, since S. oleae populations did not increase during this period (Fig 3). In citrus groves in 2004, when the populations were very low, the

- 52 - SAISSETIA OLEAE POPULATIONS increase during fall was due to the sampling of new groves with higher population levels.

Saissetia oleae populations suffered intense abundance fluctuations along the year. After peaking in July, population density decreased during several moths, especially in summer, due to high mortality of first instar scales. Temperatures over 30ºC associated with a relative humidity below 30% can cause mortality rates over 80% in first instar scales (De Freitas 1972, Pucci 1982). During fall-winter, when some egg- laying females were present, S. oleae population did not decreased as severely as in summer, but they neither increase, as expected. Likely, all eggs and crawlers that hatched during the winter perished (Canard and Laudeho 1977, Pucci et al. 1982). Saissetia oleae overwintered as second and third instars, which apparently have greater resistance to low temperatures (Argyriou 1963, De Freitas 1972, Pucci 1982). Thus, low temperatures in winter did not reduce the populations but homogenized them, and consequently populations were strongly synchronous after the winter.

In the study of female fecundity, we found a significant relationship between female volume and number of eggs that they contained beneath their body. This relation was also observed by Pereira (2004) in olive groves. We counted the number of eggs in a particular moment of the egg-laying period but not the total number of eggs laid by each female along its life. Saissetia oleae eggs hatched before females had finished the egg-laying period. For this reason, we consider that in many cases the number of eggs counted does not match the numbers expected considering the volume of the female, since many females observed may be in the early stages of their oviposition period. Consequently, the slope relating female volume and number of eggs appears lower than it would be if the relationship had been established with the total number of eggs laid. Thus, we consider that the volume of the female represents its potential fecundity more accurately than the number of eggs found in a particular moment.

Female volumes measured in fall-winter were lower than those measured at the end of spring. Duration of S. oleae stadia decreased with increasing temperatures in summer and some individuals became adults and gave rise to a second generation after summer, but high temperatures also reduced the volume of scales developed during summer. A decrease in body size with increasing temperature is well documented in insects in general (Ray 1960). The lower fertility of fall-winter females helps to explain

- 53 - CHAPTER 3 the poor contribution of the second generation to the overall population abundance of S. oleae.

Overall, our results show that S. oleae presents one important and concentrated crawler emergence in July in citrus and olive crops in eastern Spain. A second partial crawler emergence was also observed in fall-winter but populations did not increase during this time of the year due to the effect of low temperatures on first instars and the lower fertility of mature females. Thus, when chemical sprays should be applied to control population outbreaks, we recommend to apply them at the end of July, when populations are homogenous, all crawlers have already emerged and first instars predominate in populations. If parasitoids are augmentatively released to improve the biological control of S. oleae, the seasonal trend described above should be considered, since the predominant size and stage of the host scale are important factors in the success of augmentative releases.

Acknowledgements

We are grateful to Alejandro Alicart, Andrés Alonso, Ana Cano, Salut Cuñat, Miriam García, Francisco Girona, José Miguel Martinez, Cristina Mases, Juan Carlos Meliá, Vicente Mestre, Santiago Mompó, José Enrique Sanz, Mª Carmen Torralba and Manuel Viciedo for assistance in locating suitable groves for sampling. We also thank the following staff at the Entomology Department of the Universidad Politécnica de Valencia for their assistance: Carmen Marzal, Paco Ferragut, Lupita Alvis, Marta Martinez, Miguel Angel Martinez, Antonio Muñoz, Laura Bargues and Cristina Navarro. This study was supported by Ministerio de Ciencia y Tecnología into the Project AGL2002-00725.

- 54 - SAISSETIA OLEAE POPULATIONS

References Argyriou, L. 1963. Studies on the morphology and biology of the black scale (Saissetia oleae (Bernard)) in Greece. Annales de l’Institut Phytopathologique Benaki n.s. 5: 353- 377. Ben-Dov, Y., and C. Hodgson. 1997. Soft scale insects: their biology, natural enemies and control. World Crop Pest, Vol. 7b. Elsevier Science B. V., Amsterdam, Holland. Blumberg, D., E. Swirski, and S. Greenberg. 1975. Evidence for bivoltine populations of the Mediterranean Black Scale Saissetia oleae (Olivier) on citrus in Israel. Israel Journal of Entomology 10: 19-24. Bodenheimer, F. 1951. Citrus Entomology in the Middle East. Hoitsema Brothers- Groningen Holland. Briales, M. J., and M. Campos. 1986. Estudio de la biología de Saissetia oleae (Olivier, 1791) (Hom.: Coccidae) en Granada (España). Boletín Asociación Española Entomología 10: 249-256. Canard, M., and Y. Laudeho. 1977. Etude d’une deuxième géneration d’hiver de Saissetia oleae Oliv. (Hom., Coccidae) en Attique (Grèce) et de sa réduction par Metaphycus lounsburyi How. (Hym., Encyrtidae) et Scutellista cynea Motsch. (Hym., Pteromalidae). Fruits 32: 554-561. De Freitas, A. 1972. A cochonilha-negra (Saissetia oleae (Oliv.)) em Oliveira. Bio- ecologia e influência dos tratamentos antidácicos. Agronomia Lusitana 33: 349-390. Fernández, J. M., Z. Mendivil, and S. Almagro. 1979. Estudio de Saissetia oleae en Córdoba. Boletín Servicio de Plagas 5: 149-156. Flint, M. L., B. Kobbe, J. K. Clark, S. H. Dreistadt, J. E. Pehrson, D. L. Flaherty, N. V. O’Connell, P. A. Phillips, and J. G. Morse. 1991. Integrated pest management for citrus, 2nd ed. University of California. Oakland, CA. Jeppson, L. R. 1989. Biology of the Citrus Insects, Mites, and Molluscs, pp. 1-49. In the Citrus Industry. Division of Agriculture and Natural Resources, University of California. Oakland, CA. Katsoyannos, P. 1996. Integrated insect pest management for citrus in northern Mediterranean countries. Benaki Phytopatological Institute (BPI). Athens. Lampson, L. J., and J. G. Morse. 1992. A survey of Black Scale, Saissetia oleae (Homoptera: Coccidae) Parasitoids (Hym.: Chalcidoidea) in Southern California. Entomophaga 37: 373-390. Llorens, J. M. 1984. Las cochinillas de los agrios. Consellería de Agricultura, Pesca y Alimentación. Valencia, Spain. Mendel. Z., H. Podoler, and D. Rosen. 1984. Population dynamics of the Mediterranean black scale, Saissetia oleae (Olivier), on the citrus in Israel. 4. The natural enemies. Journal of the Entomological Society of South Africa 47: 1-21. Montiel, A., and S. Santaella. 1995. Evolución de la población de Saissetia oleae OLIV en condiciones naturales. Periodos susceptibles de control biológico. Boletín Sanidad Vegetal de Plagas 21: 445-455. Morillo, C. 1977. Morfología y biología de Saissetia oleae (Homoptera Coccidae). Boletín de la Real Sociedad Española de Historia Natural Sección Biológica 75: 87-108.

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Noguera, V., M. J. Verdú, A. Gómez-Cadenas, and J. A. Jacas. 2003. Ciclo biológico, dinámica poblacional y enemigos naturales de Saissetia oleae Olivier (Homoptera: Coccidae), en olivares del Alto Palencia (Castellón). Boletín Sanidad Vegetal de Plagas 29: 495-504. Panis, A. 1977. Contribución al conocimiento de la biología de la “cochinilla negra de los agrios” (Saissetia oleae Olivier). Boletín Servicio de Plagas 3: 199-205. Passos de Carvalho, J., L. M. Torres, J. A. Pereira, and A.A. Bento. 2003. A cochonilha-negra Saissetia oleae (Olivier, 1791) (Homoptera - Coccidae). Ed. Instituto Nacional de Investigaçao Agraria. Universidade de Tras-os-Montes e alto Douro. Escola Superior Agraria de Bragança. Bragança, Portugal. Pereira, J. A. C. 2004. Bioecologia da cochonilha negra Saissetia oleae (Olivier), na oliveira, em Trás-os-Montes. PhD disertation. Unversidade de Trás-os-Montes e alto Douro. Vila Real. Pucci, C., D. Salmistraro, A. Forcina, and G. Montanari. 1982. Incidenza dei fattori abiotici sulla mortalità della Saissetia oleae (Oliv.). Redia 65: 355-366. Ray, C. 1960. The application of Bergmann’s and Allen’s rules to the poikilotherms. Journal of Morphology. 106: 85-108. Santaballa, E. 1972. La Caparreta (Saissetia oleae) en agrios. Levante Agrícola. 22: 20- 25. Schweizer, H., J. G. Morse, and R. F. Luck. 2003. Evaluation of Metaphycus spp. for suppression of black scale (Homoptera: Coccidae) on southern California citrus. Biological Control 32: 377-386. Shoemaker, C. A., C. B. Huffaker, and C. E. Kennet. 1979. A systems approach to the integrated management of a complex of olive pests. Environmetal Entomology 8: 182-189. Statgraphics. 1994. Version 4.0 Plus. Statistical graphics system by Statistical Graphics Corporation. Manugistics, Rockville, MD.

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Chapter

4

Parasitoid complex of black scale Saissetia oleae on Citrus and Olives: seasonal trend and impact on host population

- 57 - CHAPTER 4

- 58 - PARASITOID COMPLEX

Parasitoid complex of black scale Saissetia oleae on Citrus and Olives: seasonal trend and impact on host population

Abstract: The parasitoid complex of black scale Saissetia oleae (Olivier) (Hemiptera: Coccidae) was studied on citrus and olives to determine their relative abundance, seasonal trend, geographical distribution, and their incidence on black scale populations. Branches and leaves of ten citrus and four olive groves infested with black scale were periodically collected over the period March 2003-December 2005 in eastern Spain, covering an area of 10,000 km2. Adult parasitoids were also sampled with a portable engine-powered suction device. Black scale females were often attacked by Scutellista caerulea (Fonscolombe) (Hymenoptera: Pteromalidae), which was found beneath 35.4 ± 7.5% and 22.4 ± 3.5% female scale’s body in citrus and olive groves, respectively. However, it attacked the scales when most of their eggs had already hatched. The parasitic mite Pyemotes herfsi (Oudemans) (Prostigmata: Pyemotidae) fed on all development stages of S. caerulea. The gregarious female’s endoparasitoid Metaphycus lounsburyi (Howard) (Hymenoptera: Encyrtidae) was common in citrus and olive trees, but the parasitism rates it reached was low. Second and third instars of black scale were mainly parasitized by the solitary endoparasitoid Metaphycus flavus (Howard), and secondarily by Metaphycus helvolus (Compere) which was much less abundant and limited in distribution. Thus, M. helvolus, introduced 30 years ago, has not displaced M. flavus as in other Mediterranean areas. According to their abundance, distribution and incidence, M. flavus and S. caerulea appeared as the main parasitoids of black scale in eastern Spain, whereas M. helvolus and M. lounsburyi, considered the main parasitoids in other citrus and olive areas of the world, had a limited incidence. Recommendations for improving the level of biological control are discussed.

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4.1. Introduction

Black scale Saissetia oleae (Olivier) (Hemiptera: Coccidae) is a cosmopolitan and polyphagous soft scale pest of more than 60 plant species in the Mediterranean region, including citrus and olives (Morillo 1977; Passos de Carvalho et al. 2003). The damage black scale causes to these plants varies annually because of variation in the combined effect of mortality factors such as those arising from natural enemies, climatic effects, and/or broad-spectrum insecticide applications (Panis 1977, Mendel et al. 1984). Satisfactory biological control of black scale has been achieved through the releases of parasitoids reared in the laboratory (Graebner et al. 1984) or through the introduction of a complex of parasitoids and predators (Mendel et al. 1984, Waterhouse and Sands 2001). In eastern Spain, different black scale parasitoids were introduced (Carrero 1981, Melia and Blasco 1981) but their establishment and incidence on black scale remain unclear.

Previous studies of the parasitoid complex of black scale around the world have identified four dominant parasitoid species associated with black scale. These include Metaphycus lounsburyi (Howard) (= M. bartletti Annecke & Mynhard), M. helvolus (Compere) (Hymenoptera: Encyrtidae), Coccophagus lycimnia (Walker) (Hymenoptera: Aphelinidae) and Scutellista caerulea (Fonscolombe) (= S. cynea Motschulsky) (Hymenoptera: Pteromalidae) (Mendel et al.1984, Kennett 1986, Lampson and Morse 1992, Pereira 2004).

Metaphycus helvolus and M. lounsburyi are parasitoids of black scale in South Africa where they and the scale are endemic. These parasitoids have been introduced into citrus and olive growing regions world wide (including Australia, California (USA), Cyprus, France, Greece, Israel, Italy and Spain), to reduce black scale populations to non economic densities (Argov and Rössler 1993, Guerrieri and Noyes 2000, Malipatil et al. 2000). Metaphycus helvolus is a solitary, primary endoparasitoid that attacks 2nd and 3rd instar black scale (Lampson et al. 1996). It has been augmentatively released against black scale in an inland coastal valley of southern California where it effectively suppressed the scale as part of an integrated citrus pest management program (Graebner et al. 1984). Such releases have also shown promise in California olive groves (Daane et al. 1991). In Crete (Greece) M. helvolus displaced the native parasitoid M. flavus

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(Howard) (Hymenoptera: Encyrtidae) (Argyriou and Michelakis 1975). In 1976, M. helvolus was successfully introduced into eastern Spain (Carrero 1981); however, ten years later, Ripollés (1990) reported that it was not well established in this region.

Metaphycus lounsburyi is a gregarious endoparasitoid that attacks 3rd instars, mature, and ovipositing female black scale (Barzman and Daane 2001). In Israel, M. lounsburyi is considered the principal parasitoid responsible for suppressing black scale (Argov and Rössler 1993). This species was introduced into eastern Spain in 1979 (Meliá and Blasco 1981) but its establishment and efficacy are unknown. A recent local study of black scale parasitoids in olive groves in eastern Spain by Noguera et al. (2003) found M. lounsburyi, but not M. helvolus.

A second genus of parasitoids associated with soft scales is Coccophagus (Hymenoptera: Aphelinidae). These are heteronomous hyperparasitoids in which the females are primary endoparasitoids of soft scales whereas the males are hyperparasitoids of their females (Walter 1983) or of other primary parasitoids including species of Metaphycus (Bernal et al. 2001). Coccophagus lycimnia has been collected from black scale infesting citrus and olives in Valencia (Carrero et al. 1977, Noguera et al. 2003). If C. lycimnia is abundant, it may be hyperparasitizing soft scales previously parasitized by Metaphycus and thereby reducing the potential of Metaphycus to suppress these soft scales (Bernal et al. 2001).

Scutellista caerulea is a well known cosmopolitan parasitoid of black scale, which larvae usually develop as an egg-predator feeding on the eggs beneath the scale body (Ehler 1989). However, it seldom consumes all the eggs and some of them may survive. The percentage of surviving eggs depends on the total number of eggs laid by the host female (Mendel et al.1984). Although S. caerulea is relatively well-known, its incidence on black scale populations appears unclear since it varies depending on authors. Thus, on olives in southern Spain, Montiel and Santaella (1995) detected S. caerulea in 5.9% of the black scale females, whereas on citrus in Israel, Mendel et al. (1984) found it in as many as 80% of the scales they assessed.

Despite being the most serious soft scale pest of citrus and olives in the Mediterranean region, no long-term, area-wide studies of the parasitoid complex of black scale have been conducted recently. Comparing the parasitoid complex on both

- 61 - CHAPTER 4 crops would be especially interesting due to their coexistence all around the Mediterranean Basin (Passos de Carvalho et al. 2003). Moreover, after the introduction of M. helvolus and M. lounsburyi more than 30 years ago, their establishments and impact on native parasitoids remain unclear. Thus, we initiated a study of the parasitoid complex of black scale to determine the main species present in citrus and olives, their geographical distribution, their seasonal abundance to ensure correct timing for possible augmentative releases, and their incidence on the host population, as a first step in improving the biological control of black scale in Spain.

4.2. Materials and Methods

4.2.1. Groves

We selected ten citrus and four olive groves in eastern Spain with sparse to dense black scale populations and sampled them between March 2003 and December 2005 (citrus) and May 2004 to December 2005 (olives). Groves were included in an area 200 Km long (north-south) and 50 km wide (east-west). The citrus groves were located in Albal, Alcora, Altura, Castellón, Moncófar, Museros, Onda, Real de Montroy, Ribarroja and Xilxes; and the olive groves in Altura, Castellón, Planes, and Villar del Arzobispo. The groves were sampled twice a month during periods of rapid scale growth (April- October) and monthly during the cooler, winter months. Each grove was sampled for different periods of time, ranging from 6 months to 1.5 years. Sampling was discontinued in a grove when it was treated with insecticides or when it was so lightly infested that no black scales could be found. Samples were collected from 2-4 citrus groves and from 2-4 olive groves on each date.

4.2.2. Black scale phenology and parasitoid incidence

Sixteen, 15-cm long twigs with green-wood and leaves were collected from a minimum of four trees. We only selected trees and twigs within trees that were heavily infested with black scale since we wanted to determine whether parasitoids were present in these groves. The infested twigs were placed in plastic bags and transported to the laboratory for processing. In the laboratory, we processed each twig and four leaves

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(both sides) from each twig using stereomicroscope. We sought to determine the phenology of the black scale population and the identity and incidence of its parasitoids. To determine the age structure of black scale that was present on a sample date, we counted the total number of live scales and categorized them into the three immature stages (i.e., see Morillo 1977) and into three adult female stages: young females, females with eggs, and females with crawlers (or eggs and crawlers). Simultaneously, we examined the black scale instars for signs of parasitism (i.e., parasitoid larvae or pupae). Each female was turned over and carefully examined for signs of parasitism. These assessments allowed us to calculate parasitism rates as the number of parasitized scales/number of live and parasitized scales. Parasitism rates were calculated for each black scale stage when the number of live and parasitized scales were > than 20. Parasitism rates for M. flavus and M. helvolus was a combined estimation since we could not differentiate the larval stages of these two species. We referred to this parasitism as Metaphycus spp. Similarly, we also combined the parasitism by C. lycimnia and C. semicircularis (Förster) (=C. scutellaris (Dalman)) and referred to as parasitism by Coccophagus spp. Scutellista caerulea is usually referred as a parasitoid, even though its larvae are predators of black scale eggs beneath the scale’s body. After examining them, twigs and leaves from each sample were placed in a ventilated transparent 25 x 8 cm plastic cage for parasitoid emergence. Previously, all other soft scale species were removed from the plant material. The cages were held at 23-27ºC, 16:8 (L:D) and 60- 80% RH for 25-30 days and then held at -20ºC for at least one day to kill surviving parasitoids. Finally, dried twigs, leaves and debris were brushed, sieved, and examined under stereomicroscope to count and identify the adult parasitoids that had emerged. This sampling method is referred in the text as “emergence cages”.

4.2.3. Flight period of parasitoids

On each sampling date, adult parasitoids were collected from the tree canopy with a portable, engine-powered, suction device. The device was constructed by modifying a commercial vacuum-blower (McCulloch, model Mac 320 BV, Tucson, AZ) and adapting it to collect insects from the foliage. We modified it by adding a cylindrical plastic tube 30-cm long with a 30-cm diameter opening. The sampling was standardized by placing the opening of the cylindrical tube a total 70 times on the foliage of citrus or olive trees per sample. We ensured that the samples were obtained around the tree

- 63 - CHAPTER 4 canopy and up to 2 m. high, on eight different trees selected for their high levels of black scale infestation. The collected material from each grove was bagged and transported to the laboratory, where it was held at -20ºC for one day to kill the insects. The black scale parasitoids were then counted under a stereomicroscope and labeled by date and grove. These data are referred in the text as “suction samples of parasitoids”.

We used the keys of Guerrieri and Noyes (2000) and Malipatil et al. (2000) to identify the Hymenoptera in the collected material and our identifications were confirmed by M. J. Verdú (Instituto Valenciano de Investigaciones Agrarias, Valencia, Spain). The parasitic mite was identified by A. Baker and the cecidomyiid egg predator was indentified by N. Wyatt (Natural History Museum, London, UK).

4.2.4. Data analysis

We compared the percent parasitism between years, host plants (citrus vs olive), host stages, and parasitoid species using a multifactorial analysis of variance (ANOVA) (Statgraphics 1994). We arcsine-square-root transformed (z = arcsine (x0.5)) percentage parasitism to approximate a normal distribution before subjecting the data to analyses. Means were compared using an LSD test at a 5% significance level.

4.3. Results

4.3.1. Parasitoids species

During the three year study, 6,864 parasitoid specimens were obtained using two sampling methods. These parasitoids belonged to nine species, with the genus Metaphycus, Scutellista and Coccophagus comprising 99% of the parasitoid fauna. The most abundant and widely distributed parasitoids in both olive and citrus groves were Metaphycus flavus (Howard) (44.5% of 6,864), Scutellista caerulea (26.6%), M. lounsburyi (17.4%) and Coccophagus lycimnia (6.5%) (Table 1). Their abundance depended on the crop and the sampling methodology used.

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Table 1. Relative abundance of the main Saissetia oleae natural enemies, observed using three sampling methods in citrus (March 2003-December 2005) and olive groves (May 2004-December 2005) in eastern Spain.

Citrus Olives Combined crops

% Abundancea % Abundancea Species Grove Grove Total Grove Emerging Suction- presence Emerging Suction- presence % presence

parasitoids sampled parasitoids sampled Aphelinidae Coccophagus lycimnia 4,0 6,2 7/10 10,8 13,4 3/4 6,5 10/14

C. semicircularis 0,8 0,7 3/10 0,3 0,4 1/4 0,7 4/14 Encyrtidae Metaphycus flavus 22,3 69,7 10/10 2,6 26,1 4/4 44,5 14/14

M. helvolus 6,1 3,2 5/10 5,6 8,2 3/4 4,7 8/14

M. lounsburyi 19,1 7,1 9/10 51,0 28,5 4/4 17,4 13/14 Pteromalidae Scutellista caerulea 47,7 13,2 10/10 29,7 23,3 4/4 26,6 14/14

TOTAL NUMBER 2174 3460 10 694 536 4 6864 14 a Parasitoid species percentage in each sampling method.

Among the instars parasitoids, M. flavus was the most abundant (44.5%) and widely distributed, especially on citrus. It was present in all the groves sampled, whereas M. helvolus was much less abundant (4.7%) and was present only in eight of the 14 groves sampled. Parasitoids of genus Coccophagus were more abundant on olives than on citrus. On olives, Coccophagus lycimnia was the most abundant parasitoid in the emergence cages (10.8%), where instars parasitoids of genus Metaphycus (M. helvolus (5.6%), M. flavus (2.6%)) were less abundant.

The female parasitoids S. caerulea and M. lounsburyi were widely distributed, since they were collected in almost all the citrus and olive groves sampled. The pteromalid S. caerulea was the most abundant if considering both crops, though the gregarious endoparasitoid M. lounsburyi was more abundant on olives than the pteromalid. The mite Pyemotes herfsi (Oudemans) (Prostigmata: Pyemotidae) was observed feeding on the larvae, pupae and adults of Scutellista caerulea. Thus, P. herfsi acts as a secondary parasite of black scale since it attacked its parasitoids. Pyemotes herfsi was found in three citrus and all four olive groves.

Two specimens of a male Pteromalinae (Pteromalidae: Hymenoptera), three female specimens of Mycroteris nietneri (Motschulsky) (Hymenoptera: Encyrtidae) and

- 65 - CHAPTER 4 one specimen of the hyperparasitoid Marietta picta (André) (Aphelinidae: Hymenoptera) were also obtained in the emergence cages.

During the observations to calculate the parasitism rates, 23 predacious cecidomyiid larvae were observed feeding on black scale eggs under the female’s body. It was found only in the citrus groves. The cecidomyiid was identified as Lestodiplosis sp. or another closely related genus.

4.3.2. Black scale phenology and seasonal trend of its parasitoids

In Fig. 1 and 2 the three years of data have been combined to compare the total number black scale and its age structure along with the total number of parasitoids recovered from the emergence cages and the parasitoid’s relative abundance per crop and date. The number of parasitoids recovered from the emergence cages peaked in both crops at the beginning of summer (June-July), just at the end of the female black scale’s development. The main species recovered during these dates were the female parasitoids M. lounsburyi and S. caerulea. This peak did not occur during the summer of 2004 in citrus groves, due to the sparse number of black scale and parasitoids present that year.

When black scale occurred as 2nd and 3rd instars (between September and May each year) the number of parasitoids collected was lower (Figs. 1 and 2). Metaphycus flavus was the commonest species recovered in citrus, although other young instar parasitoids as M. helvolus, C. lycimnia and C. semicircularis were also present. Similarly, low numbers of C. lycimnia, M. helvolus and M. flavus were also collected in the olive groves during this period.

The suction samples of the adult parasitoid in the groves confirmed the seasonal trend observed in the emergence cages (fig. 3). Adult specimens of Metaphycus lounsburyi and S. caerulea were captured in the groves in summer (June-September), after female black scales were abundant. These parasitoids were low or absent during the rest of the year. In contrast, the parasitoid M. flavus was present throughout the year, but it was scarce in winter. Metaphycus flavus numbers peaked at the end of the spring (June) and during autumn (October-November), during or shortly after 2nd and 3rd instar black scale occurred in the groves. These instars are the preferred stages for oviposition

- 66 - PARASITOID15000 COMPLEX 300 1250 300

250 12000 250 1000

200 Saissetia oleae Saissetia oleae 200 9000 Parasitoids Parasitoids 750 150 150 6000

S. oleae 500

100 oleaeS. Nº 100 Nº 3000 50 ParasitoidsNº reovered 250 50

0 0 M-03 M-03 J-03 A-03 O-03 J-04 M-04 J-04 S-04 N-04 J-05 A-05 J-05 A-05 O-05 0 0 May-04 Jun-04 Aug-04 Oct-04 Dec-04 Apr-05 Jun-05 Aug-05 Nov-05 100 1st instar 100 75 75 1st instar 50 50 25 25 0 100 0 100 75 2nd instar 2nd instar 75 50 50 25 25 0 100 0 3rd instar 100 75 3rd instar 75 50 50 25 25 0 100 0 75 inmature females 100 50 75 inmature females 25 50

Developmental stages of S. oleae 0 25 100 ovipositing females 0 75 100 Developmental stages of S. oleae stages Developmental 50 75 ovipositing females 25 50 1000 25 75 females with crawlers 0 100 50 75 females with crawlers 25 50 0 25 M-03 M-03 J-03 A-03 O-03 J-04 M-04 J-04 S-04 N-04 J-05 A-05 J-05 A-05 O-05 0 1,0 Parasitoid M-04 J-04 A-04 O-04 D-04 A-05 J-05 A-05 N-05 species Parasitoid 1,0 spe ci e s 0,5 Scutellista caerulea Scutellista 0,5 0,0 caerule a 1,0 0,0 0,5 Metaphycus 1,0 lounsbur yi Metaphycus 0,0 0,5 lounsbur yi 1,0 0,0 0,5 Metaphycus 1,0 flavus Metaphycus 0,0 0,5 1,0 flavus Metaphycus 0,0 0,5 helvolus 1,0 proportionSample Sample proportionSample Metaphycus 0,0 0,5 1,0 helvolus 0,0 Coccophagus 0,5 1,0 lycimni a Coccophagus 0,0 0,5 a 1,0 lycimni Coccophagus 0,0 0,5 semicircularis M-04 J-04 A-04 O-04 D-04 A-05 J-05 A-05 N-05 0,0 M-04 M-03 J-03 A-03 O-03 J-04 M-04 J-04 S-04 N-04 J-05 A-05 J-05 A-05 O-05 Fig. 1. Saissetia oleae phenology and the relative abundance of its main parasitoids on citrus Fig. 2. Saissetia oleae phenology and the relative abundance of its main parasitoids on olives in eastern Spain from March 2003 to December 2005. (a) Seasonal abundance of Saissetia in eastern Spain from May 2004 to December 2005. (a) Seasonal abundance of Saissetia oleae oleae and total number of parasitoids recovered in the “emerging cages”. (b) Relative and total number of parasitoids recovered in the “emerging cages”. (b) Relative abundance of abundance of S. oleae developmental stages. (c) Relative abundance of the main parasitoids S. oleae developmental stages. (c) Relative abundance of the main parasitoids recovered in the recovered in the “emerging cages”. “emerging cages”.

67 CHAPTER 4

by M. flavus. Interestingly, a second M. flavus peak occurred in summer when suitable black scale stages for oviposition by M. flavus were absent. Coccophagus lycimnia also parasitizes the immature stages of black scale and was usually abundant during the spring (May-June). It was also abundant in one olive grove in autumn 2005.

Scutellista caerulea Metaphycus flavus 120 250

100 2003 200 2003 80 2004 2004 2005 150 2005 60 100 parasitoids 40 º N 20 50

0 0 JFMAMJJASOND JFMAMJJASOND

Metaphycus lounsburyi Coccophagus lycimnia

120 50 100 2003 40 2003 80 2004 2004 2005 30 2005 60

parasitoids 20

º 40 N 20 10 0 0 JFMAMJJASOND JFMAMJJASOND Fig 3. Seasonal abundance of the main parasitoids of Saissetia oleae collected with a suction engine- powered device on citrus and olive groves in eastern Spain from February 2003 to December 2005

4.3.3. Parasitism rates

Parasitism of black scale by Metaphycus spp. (M. flavus and M. helvolus) was greater than by Coccophagus spp. (C. lycimnia and C. semicircularis) (ANOVA: F = 32.52; df = 1, 33; P < 0.0001) (Table 2). Metaphycus spp. were found parasitizing all immature stages, although 1st instar was rarely parasitized. Third instar black scale suffered higher rates of parasitism than 2nd instar (ANOVA: F = 9.95; df = 1, 16; P = 0.0036). Moreover, Metaphycus spp. parasitized significantly more black scale in citrus than in olives groves (ANOVA: F = 7.41; df = 1, 17; P = 0.0107). Finally, Metaphycus spp. parasitized significantly more immature black scale in 2003 (8.96 ± 1.01%) than in 2004 (3.03 ± 0.63%) or 2005 (2.04 ± 0.57%) (ANOVA: F = 17.2; df = 2, 13; P < 0.0001).

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Table 2. Mean parasitism rates by the natural enemies of Saissetia oleae, in citrus (March 2003-December 2005) and olive groves (May 2004-December 2005) in eastern Spain. Citrus Olives Natural enemies Host % ± SE (n*) % ± SE (n*)

Metaphycus spp. L2 3.6 ± 1.2 (10) 0.8 ± 0.6 (4)

L3 7.6 ± 1.6 (9) 1.3 ± 0.4 (4)

Coccophagus spp. L2 0.1 ± 0.1 (10) 0.1 ± 0.0 (4)

L3 0.4 ± 0.2 (10) 1.1 ± 0.7 (4)

Metaphycus lounsburyi ♀ with eggs 3.0 ± 1.4 (9) 4.8 ± 3.6 (4)

Scutellista caerulea ♀ with eggs 35.4 ± 7.5 (10) 22.4 ± 3.5 (4)

Pyemotes herfsi** S.caerulea 15.7 ± 7.1 (7) 2.5 ± 1.4 (3)

*Number of groves **Parasite of any postembrionic Scutellista caerulea stage present beneath the body of Saissetia oleae.

Coccophagus spp. parasitized both 2nd and 3rd instar black scale but it parasitized a significantly higher percentage of the 3rd instars (ANOVA: F = 6.2; df = 1, 16; P = 0.0186). Parasitism rates by this parasitoid were similar in citrus and olive groves (ANOVA: F = 0.98; df = 1, 17; P = 0.3311) and in different years (ANOVA: F = 0.49; df = 2, 13; P = 0.6181).

Scutellista caerulea larvae were always found developing as egg predators. On citrus, its larvae and pupae were found beneath 35.4 ± 7.5% of the female black scale with eggs, or with eggs and crawlers, whereas on olives its larvae or pupae were found beneath 22.4 ± 3.5% of the females with eggs, or with eggs and crawlers. These represent the highest percentages of parasitism encountered in this study. Parasitism rates were similar between crops (ANOVA: F =1.42; df = 1, 7; P = 0.2559) and years (ANOVA: F = 2.69; df = 2, 5; P = 0.1080). The parasitism rates of S. caerulea reached high values (>80%) at the end of the development of the black scale females (end of June-beginning of July) (fig. 3), being lower during the maximum of black scale females (May-beginning of June). Similarly, the incidence of Pyemotes herfsi, a parasite of S. caerulea larvae, pupae and adults, increased when the pteromalid was most abundant beneath the female scale body. In late July almost 100% of S. caerulea were found attacked by P. herfsi.

- 69 - CHAPTER 4

The percentage of gravid females parasitized by M. lounsburyi was much lower than by S. caerulea in both citrus and olive groves (Table 2). Parasitism by M. lounsburyi was similar in the citrus and olive groves (ANOVA: F = 0.01; df = 1, 7; P = 0.9632) and in both years (ANOVA: F = 0.05; df = 2, 5; P = 0.9497). Metaphycus lounsburyi rarely parasitized the 3rd instars of the black scale.

Citrus 2003 500 Females with crawlers Females w ith eggs 250 Young females S.oleae

Nº 0

100

75 S. caerulea M. lounsburyi S. caerulea +P. hersfi 50 % Parasitsm 25

0 AMJ J A

250 Citrus 2005

125 S.oleae Nº

0 amj j a 100

75

50

% Parasitsm 25

0 AMJ JA

Olives 2005 100 50 S.oleae Nº

0 amj j a 100

75 50

% Parasitsm 25

0 AMJ J A Fig. 4. Changes in parasitism rates by Metaphycus lounsburyi and Scutellista caerulea (either alone or with its parasite Pyemotes herfsi) related with the phenology of their host, Saissetia oleae. Each crop and year includes data collected from four different groves.

- 70 - PARASITOID COMPLEX

4.4. Discussion

Six species predominate in the parasitoid complex associated with black scale on citrus and olives in eastern Spain. According to their preferred host stage, they can be divided into those associated with instar stages and those associated with the female stage. The instar parasitoids were Coccophagus lycimnia¸ C. semicircularis, Metaphycus flavus and M. helvolus; while the female parasitoids were M. lounsburyi and S. caerulea. All of them had already been reported in eastern Spain (Limón et al. 1976, Carrero et al. 1977, Ripollés 1986, Noguera et al. 2003), but their relative abundance, geographical and crop distribution, flight period and incidence were not previously assessed.

Metaphycus flavus was the most abundant and widely distributed instar parasitoid in citrus groves and, consequently, it has not been displaced by the introduced parasitoid M. helvolus as it happened in Crete, Greece (Argyriou and Michelakis 1975). Metaphycus flavus parasitized black scale mainly in spring and fall, according to data obtained from emergence cages, but it was present all along the year in the groves, even when its black scale preferred stages were not present (Fig. 3). During that time, M. flavus might have emerged from alternate hosts present in spanish citrus groves, as brown soft scale Coccus hesperidum L. (Hemiptera: Coccidae) (Llorens 1984). The availability of using alternate host species could explain, at least in part, the superiority of M. flavus over M. helvolus observed in our study, since M. helvolus suffers high encapsulation rates when developing in brown soft scale (Blumberg 1977). The encapsulation rates of M. helvolus when developing in C. hesperidum decrease at low temperatures (Blumberg and DeBach 1981), and in our observations this parasitoid was found at high levels just in the most interior and, consequently, the most continental citrus grove sampled, being scarce or absent in other nine groves.

On olives, where black scale instars appeared poorly parasitized, C. lycimnia predominate in the instar parasitoid complex. Coccophagus lycimnia is a facultative autoparasitoid (Walter 1983), with females acting as primary endoparasitoids and males as hyperparasitoids of females of other parasitoid species. In this study, C. lycimnia reached the highest population levels coinciding with the maximum of M. flavus in May- June. This result suggests that C. lycimnia could reduce the potential of M. flavus and M. helvolus to suppress soft scales in spring, as observed by Bernal et al. (2001). More

- 71 - CHAPTER 4 studies are needed to clarify the role of C. lycimnia in the parasitoid complex of black scale.

The parasitoids of black scale females, S. caerulea and M. lounsburyi, play apparently a significant role in the biological control of black scale since they were encountered abundantly in almost all citrus and olive groves. Scutellista caerulea presented the highest parasitism rates in both crops, reaching almost 80% at the end of the scale development in 2003 and 2005 (fig. 4). However, an important part of the eggs laid by black scale females usually escape predation by S. caerulea larvae (Mendel et al. 1984). We found S. caerulea beneath scale’s body when most of the eggs had already hatched and crawlers had gone away. Thus, the real impact of S. caerulea on black scale populations is lower than our parasitism rates suggest. Although Pyemotes herfsi had not been previously cited in Spain as a parasite of S. caerulea, we have found that it is a common natural enemy regulating the pteromalid populations and, consequently, decreasing the efficacy of S. caerulea as a biological control agent. The parasitism levels reached by P. herfsi might be overestimated in our study, since S. caerulea parasitized by P. herfsi remain for longer beneath black scale body than the unparasitized, which emerge and migrate.

Metaphycus lounsburyi appeared as one of the most abundant parasitoids, like in other studies in which the emergence cages have been used as sampling method (Lampson and Morse 1992, Mendel et al. 1984). However, the incidence of M. lounsburyi on black scale populations may be overestimated using this method because it is a gregarious parasitoid (Barzman and Daane 2001), emerging up to 12 adults per parasitized scale (personal observations). Thus, the incidence of M. lounsburyi on black scale populations is considerably lower when considering its parasitism rates (3 and 4.8% on citrus and olives respectively). Further, M. lounsburyi might build up the populations too late to prevent scale outbreaks, because the host scale is univoltine and no alternate hosts were present. Only one flight peak of M. lounsburyi was observed along the year from our suction samples.

The real impact of the parasitoids on black scale populations is difficult to assess, since its populations are affected not only by biotic but also by abiotic factors, mainly the climate (Bodenheimer 1951, Panis 1977, Montiel and Santaella 1995). During spring and autumn, M. flavus in the coast and M. helvolus in the interior may be responsible, at

- 72 - PARASITOID COMPLEX least in part, for the second and third instar mortality observed in citrus groves. Development of black scale females appears strongly synchronized during a short summer-time period, being highly attacked by S. caerulea and M. lounsburyi when the scales have already laid the eggs and many crawlers have hatched. Consequently, the effectiveness of S. caerulea and M. lounsburyi seems to be limited as biological control agents of black scale when the scale is univoltine, unless they were augmentatively released just before the female reach the optimum stage to be parasitized.

Overall, our results show that the most abundant and widely distributed parasitoids of black scale in citrus and olive crops in eastern Spain are nowadays S. caerulea, M. flavus and M. lounsburyi. These parasitoids should be considered when determining the side-effects of pesticides on beneficials, as an important component of Integrated Pest Management strategies. We also recommend the rearing and augmentative release of M. flavus instead of M. helvolus for black scale outbreaks in citrus, since the native parasitoid appears to be better adapted and, moreover, mass- production of M. flavus is less costly than that of M. helvolus (Scheweizer et al. 2003). Finally, more studies should be carried out to determine: (i) the effectiveness of augmentative release of M. flavus; (ii) the scarce distribution of M. helvolus in eastern Spain, especially in coastal areas; and (iii) the relationship between C. lycimnia and the abundance of Metaphycus females.

Acknowledgements

We wish to thank Dr. Robert F. Luck for their advice and careful review of our manuscript. We are grateful to Alejandro Alicart, Andrés Alonso, Ana Cano, Salut Cuñat, Miriam García, Francisco Girona, José Miguel Martinez, Cristina Mases, Juan Carlos Meliá, Vicente Mestre, Santiago Mompó, José Enrique Sanz, Mª Carmen Torralba and Manuel Viciedo for assistance in locating suitable groves for sampling. We also thank to the following staff at Entomology Department of the Universidad Politecnica de Valencia for their technical and taxonomic assistance: Carmen Marzal, Paco Ferragut, Lupita Alvis, Marta Martinez, Miguel Angel Martinez, Laura Bargues and Cristina Navarro. This study was supported by Ministerio de Ciencia y Tecnología into the Project AGL2002-00725.

- 73 - CHAPTER 4

References Argyriou, L. C., and S. Michelakis. 1975. Metaphycus lounsburyi Howard (Hym. Encyrtidae), parasite nouveau de Saissetia oleae Bern. in Crète, Grèce. Fruits 30: 251-254. Argov, Y., and Y. Rössler. 1993. Biological Control of the Mediterranean Black Scale, Saissetia oleae (Hom: Coccidae) in Israel. Entomophaga 38: 89-100. Barzman, M. S., and D. M. Daane. 2001. Host-handling behaviours in parasitoids of the black scale: a case for ant-mediated evolution. Journal of Animal Ecology 70: 237- 244 Bernal, J. S., R. L. Luck, J. G. Morse, and M. S. Drury. 2001. Seasonal and scale size relationships between Citricola Scale (Homoptera: Coccidae) and its parasitoid complex (Hymenoptera: Chalcidoidea) on San Joaquin Valley Citrus. Biological Control 20: 210- 221. Blumberg, D. 1977. Encapsulation of parasitoid eggs in soft scales (Homoptera: Coccidae). Ecological Entomology 2: 185-192. Blumberg, D., and P. DeBach. 1981. Effects of temperature and host age upon the encapsulation of Metaphycus stanleyi and Metaphycus helvolus eggs by brown soft scale Coccus hesperidum. Journal of invertebrate pathology 37: 73-79. Blumberg, D., and E. Swirski. 1988. Colonization of Metaphycus spp. (Hymenoptera: Encyrtidae) for Control of the Mediterranean Black Scale, Saissetia oleae (Olivier) (Homoptera: Coccidae) in Israel. In: R. Goren and K. Mendel (eds.), Proceedings of the Sixth International Citrus Congress, March 6-11 1988,Tel Aviv, Israel, pp. 1209-1214. Bodenheimer, F. 1951.Citrus Entomology in the Middle East. Dr. W. Hunk, The Hague, Netherlands. Carrero, J. M. 1981. Etat actuel de la lutte biologique contre les cochenilles des agrumes a Valence. IOBC/WPRS Bulletin 42: 25-31. Carrero, J. M., F. Limon, and A. Panis. 1977. Note biologique sur quelques insectes entomophages vivant sur olivier et sur agrumes en Espagne. Fruits 32: 548-551. Daane, K. M., M. S. Barzman, and L. E. Caltagirone. 1991. Augmentative release of Metaphycus helvolus for control of black scale, Saissetia oleae, in olives. KAC Plant Protection Quarterly 2: 7-9. Ehler, L. E. 1989. Observations on Scutellista cynea Motsch. (Hymenoptera: Pteromalidae). Pan-Pacific Entomology 65: 151-155. Graebner, L., D. S. Moreno, and J. L Baritelle. 1984. The Fillmore citrus protective district: a success story in integrated pest management. Bulletin of the Entomological Society of America 30: 27-33. Guerrieri, E., and J. S. Noyes. 2000. Revision of European species of genus Metaphycus Mercet (Hymenoptera: Chalcidoidea: Encyrtidae), parasitoids of scale insects (Homoptera: Coccoidea). Systematic Entomology 25: 147-222. Kennett, C. E. 1986. A survey of the parasitoid complex attacking black scale, Saissetia oleae (Olivier) in central and northern California (Hymenoptera: Chalcidoidea; Homotera: Coccidae). Pan-Pacific Entomology 62: 363-369.

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Lampson, L. J., and J. G Morse. 1992. A survey of Black Scale, Saissetia oleae (Homoptera: Coccidae) Parasitoids (Hym.: Chalcidoidea) in Southern California. Entomophaga 37: 373-390. Lampson, L. J., J. G.Morse, and R. F. Luck. 1996. Host selection, sex allocation, and host feeding by Metaphycus helvolus (Hymenoptera: Encyrtidae) on Saissetia oleae (Homoptera: Coccidae) and its effect on parasitoid size, sex and quality. Environmental Entomology 25: 283-294. Limón, F., A. Meliá, J. Blasco, and P. Moner. 1976. Estudio de la distribución, nivel de ataque, parásitos y predadores de las cochinillas lecaninas (Saissetia oleae Bern y Ceroplastes sinensis Del Guercio) en los cítricos de la provincia de Castellón. Boletín de Sanidad Vegetal y Plagas 2: 263-276. Llorens, J. M. 1984. Las cochinillas de los agrios. Consellería de Agricultura, Pesca y Alimentación. Valencia, España. Malipatil, M. B., K. L. Dunn, and D. Smith. 2000. An Illustrated Guide to the Parasitic Wasps Associated with Citrus Scale Insects and Mealbugs in Australia. Knoxfield, Victoria. Meliá, A., and J. Blasco. 1981. Cochenilles nuisibles aux citrus de la region de Castellon et leurs parasites. IOBC/WPRS Bulletin 4: 5-11. Mendel, Z., H. Podoler, and D. Rosen. 1984. Population dynamics of the Mediterranean black scale, Saissetia oleae (Olivier), on the citrus in Israel. 4. The natural enemies. Journal of the Entomological Society of South Africa 47: 1-21. Montiel, A., and S. Santaella. 1995. Evolución de la población de Saissetia oleae Oliv. en condiciones naturales. Periodos susceptibles de control biológico. Boletín de Sanidad Vegetal y Plagas 21: 445-455. Morillo, C. 1977. Morfología y biología de Saissetia oleae (Homoptera Coccidae). Boletín de la Real Sociedad Española de Historia Natural Sección Biológica 75: 87-108. Noguera, V., M. J. Verdú, A. Gómez-Cadenas, and J. A. Jacas. 2003. Ciclo biológico, dinámica poblacional y enemigos naturales de Saissetia oleae Olivier (Homoptera: Coccidae), en olivares del Alto Palencia (Castellón). Boletín de Sanidad Vegetal y Plagas 29: 495-504. Orphanides, G. M. 1993. Control of Saissetia oleae (Hom.: Coccidae) in Cyprus through establishment of Metaphycus barletti and M. helvolus (Hym.: Encyrtidae). Entomophaga 38: 235-239. Panis, A. 1977. Contribución al conocimiento de la biología de la “cochinilla negra de los agrios” (Saissetia oleae Olivier). Boletín de Sanidad Vegetal y Plagas 3: 199-205. Passos de Carvalho, J., L. M. Torres, J. A. Pereira, and A. A. Bento. 2003. A cochonilha-negra Saissetia oleae (Olivier, 1791) (Homoptera - Coccidae). Instituto Nacional de Investigaçao Agraria, Universidade de Tras-os-Montes e alto Douro, Escola Superior Agraria de Bragança. Pereira, J. A. C. 2004. Bioecologia da cochonilha negra Saissetia oleae (Olivier), na oliveira, em Trás-os-Montes. PhD disertation. Universidade de Trás-os-Montes e alto Douro, Vila Real. Ripollés, J. L. 1986. Integrated Pest Management in citrus. Symposium Parasitis, Geneva, 9-13 December.

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Ripollés, J. L. 1990. Las cochinillas de los agrios. Levante Agrícola 297: 37-45. Schweizer, H., J. G. Morse, and R. F. Luck. 2003. Evaluation of Metaphycus spp. for suppression of black scale (Homoptera: Coccidae) on southern California citrus. Biological Control 32: 377-386. Statgraphics, 1994. Version 4.0 Plus. Statistical graphics system by Statistical Graphics Corporation, Manugistics, Rockville, MD. Waterhouse, D. F., and D. P. A. Sands. 2001. Classical biological control of arthropods in Australia. ACIAR Monograph 67, ACIAR (Australian Center for International Agricultural Research). Canberra, Australia. Walter, G. H. 1983. Divergent male ontogenies in Aphelinidae (Hymenoptera: Chalcidoidea): A simplified classification and a suggested evolutionary sequence. Biological Journal of the Linnean Society (London) 19: 63-82.

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Chapter

5

Host discrimination, superparasitism and ovicide by Metaphycus flavus

- 77 - CHAPTER 5

- 78 - SUPERPARASITISM BY METAPHYCUS FLAVUS

Host discrimination, superparasitism and ovicide by Metaphycus flavus, a gregarious endoparasitoid of Coccus hesperidum

Abstract: Metaphycus flavus (Howard) (Hymenoptera: Encytidae) is a facultative gregarious endoparasitoid of different soft scales (Hemiptera: Coccidae) including brown soft scales, Coccus hesperidum L. Host discrimination, superparasitism and ovicide by M. flavus and how it is affected by factors such as experience and time interval between ovipositions were studied. Naïve and experienced females discriminated healthy from parasitized scales, but they could not discriminate between hosts parasitized by them or other conspecific females. Metaphycus flavus practised ovicide/larvicide. They used the presence of egg stalks to detect the eggs previously laid, using their ovipositor to destroy the eggs inside the scale. The final brood size of superparasitized hosts was affected mainly by three factors: encapsulation, larval competition and ovicide. Ovicide may have evolved in this parasitoid-host system because the offspring of the superparasitizing female was in competitive disadvantage when the second oviposition was delayed, and because the host defences had been already suppressed when superparasitizing.

- 79 - CHAPTER 5

5.1. Introduction

The decision a parasitoid makes when it encounters a previously parasitized host has been thoroughly explored to understand the reproductive behaviour of insect parasitoids (van Alphen and Visser 1990, Godfray 1994). A parasitized host is generally considered a host of low quality relative to a healthy host. The female parasitoids may reject the parasitized host and search for other healthy ones, feed on the haemolymph of the host to gain nutrients for future egg maturation, superparasitize the host, or kill the eggs/larvae of the first clutch (ovicide) and lay her own eggs (Strand and Godfray 1989, Mayhew 1997, Netting and Hunter 2000). To decide its reproductive strategy the parasitoid has to discriminate between parasitized and unparasitized hosts, and if the host has been parasitized by it-self or by other conspecific female. The ability of host discrimination occurs in the major of families of parasitoid Hymenoptera (van Lenteren 1981). However, some studies have failed to demonstrate the detection of self- parasitized hosts (van Dijken et al. 1992).

Superparasitism is defined as the deposition of a clutch of eggs on a host that has already been parasitized by members of the same species (Dijken and Waage 1987). Long considered the result of mistake made by imperfect animals, superparasitism is now considered to be adaptive under certain situations (see van Alphen and Visser, 1990). Superparasitism is categorized as self- or conspecific-superparasitism when the second clutch is laid by the same or other female respectively. (Waage 1986, Dijken and Waage 1987, van Alphen and Visser 1990). Conspecific-superparasitism can be advantageous when the survival rate of the second clutch is higher than zero and female parasitoids face a limited availability of hosts (Stephens and Krebs 1986). Self- superparasitism can be beneficial when the total fitness performance of all the immatures in self-superparasitized hosts is higher than the fitness performance of the immatures in host attacked once only, or when conspecific are present (Vet et al. 1994, Gu et al. 2003, Yamada and Sugaura 2003, Ito and Yamada 2005).

Encapsulation of parasitoid’s eggs is the commonest physiological host defence against endoparasitoids (Quicke 1997). The intensity and incidence of encapsulation decrease as the number of parasitoid eggs laid increase (see Blumberg 1997). Thus, superparasitism has been considered as a mechanism by which parasitoids avoid

- 80 - SUPERPARASITISM BY METAPHYCUS FLAVUS encapsulation (Blumberg and Luck 1990, Van Alphen and Visser 1990). However, these studies are based on solitary parasitoids.

The offspring of superparasitizing females are at disadvantage because they suffer from competition with the immature parasitoids already present in the host. The disadvantage depends on the time that elapses between the ovipositions (Strand and Godfray 1989, van Baaren and Nénon 1996). Gregarious larvae usually do not have fighting mandibles and competition between individuals is normally restricted to exploitation competition for host resources (Godfray 1994). Parasitoids from genus Metaphycus (Hymenoptera: Encyrtidae) reduce the supernumerary number of larvae developing inside their host, soft scales (Hemiptera: Coccidae), through physical conflicts that result in the consumption of the loser (Bartlett and Ball 1964). The advantage depends also on the time between ovipositions since the one-day-older larvae present advantage over the younger (Bartlett and Ball 1964). Thus parasitoids should be more willing to attack recently parasitized hosts where their larvae have the greatest probability of survival (van Lenteren 1981, Strand 1986, Mackauer 1990).

Ovicide may be considered a special case of superparasitism in which the female increases the probability of her offspring’s survival by killing previous female’s eggs (Strand and Godfray 1989, Mayhew 1997, Netting and Hunter 2000). Ovicide as an ovipositional strategy has been well documented in ectoparasitoids, mainly bethylids, which combine a precise oviposition, few large externally-laid eggs, and an asymmetrical larval competition that have made them to evolve ovicidal tactics (Mayhew 1997). In endoparasitoids, ovicide has been reported only for the solitary whitefly parasitoid Encarsia formosa Gahan (Hymenoptera: Aphelinidae), which uses its ovipositor to find and kill the eggs that have been laid by conspecifics (Arakawa 1987, Netting and Hunter 2000). Netting and Hunter (2000) suggested that ovicide is more probable to occur in endoparasitoids when an external cue identifies the location of the egg(s) within the host, as in the case of some Encyrtidae, where egg stalks protrude from the host cuticle.

Metaphycus flavus (Howard) (Hymenoptera: Encyrtidae) is a synovigenic, idiobiont, endoparasitoid of several soft scale insects (Hemiptera: Coccidae), including brown soft scale Coccus hesperidum L (Guerrieri and Noyes 2000). Oviposition of M. flavus has been previously studied (Kapranas 2002). Metaphycus flavus eggs are of

- 81 - CHAPTER 5 typical encyrtiform type egg, and part of them appears as a stalk protruding from the host cuticle (Maple 1947). The stalks and the transparent dorsum of C. hesperidum permit continuous observation of the development of its parasitoids without their removal (Bartlett and Ball 1964). That makes M. flavus and C. hesperidum an interesting parasitoid-host complex to study the reproductive behaviour of the parasitoid and the consequences for its offspring.

In this study we i) document host discrimination and superparasitism behaviour by M. flavus and how it is affected by factors such as experience and time interval between ovipositions ii) analyze the mortality of immature M. flavus offspring when developing in superparasitized hosts, iii) we describe ovicide by M. flavus and give an adaptive explanation for such behaviour based on the life-history of this species.

5.2. Materials and methods

Scale and parasitoid cultures. A brown soft scale colony was established with scales obtained from a guava plant, Feijoa sellowiana O. Berg (Myrtiaceae), located on the University of California, Riverside, CA campus. The scales were reared on excised leaves of Yucca recurvifolia Salisbury (Agavaceae) maintained hydroponically in the University of California, Riverside, insectary at 27-28º C, 60 % R.H. and a 21L: 3D photoperiod. The excised leaves were obtained from yucca plants grown on the Agricultural Experiment Station, University of California, Riverside.

Metaphycus flavus was collected from Citricola scale Coccus pseudomagnoliarum Kuwana (Hemiptera: Coccidae) infesting citrus near Kozan, in south central Turkey. The parasitoid colony was maintained by introducing mated females (ca. one per 10 scales) into plastic tubes (7.5 cm diam × 50 cm long) containing 1-2 scales-infested yucca leaves (ca. 300 brown soft scales/leaf) and honey as a carbohydrate source for the parasitoids. Tubes were maintained at 25 ± 1º C and 50-70 % R.H. under continuous light. Adult parasitoids for the experiments were obtained from sections of the yucca leaves with parasitized scales. Scales containing pupal parasitoids were isolated and placed into glass vials (2.5 cm diam. × 9.5 cm long) to ensure that they had no previous encounters with healthy scales. The emerging wasps

- 82 - SUPERPARASITISM BY METAPHYCUS FLAVUS were maintained as mixed-sexed groups during three days for mating and egg maturation. Each vial was sealed with a cap that had a hole covered with fine nylon mesh for ventilation. All the vials were stored at 25 ± 1ºC and 50-70 % R. H. at 14L: 10D photoperiod for 24 h and contained honey.

Parasitoids and hosts used. One day before the experiments, three day-old female were isolated in a 1 cm diam. glass vial with a drop of honey and stored with a cotton plug and kept at 25 ± 1ºC and 50-70 % R. H. at 14L: 10D photoperiod. Female parasitoids with different previous experienced were used in the experiments. “Naïve females” are considered those that had never been in previous contact with any healthy scale. “Experienced females” are considered those that had parasitized one scale four hours, two days or four days before, depending on the treatment.

All the scales used were 23-28 days old at the beginning of the experiment and their size ranged between 1.8-1.9 mm wide by 2.5-2.7 mm long. They were measured using an ocular micrometer mounted in the eyepiece of a dissecting-microscope. Different types of hosts (regarding parasitism) such as unparasitized, self- and conspecific-parasitized, were offered to the wasps at different time intervals between ovipositions. The time intervals between ovipositions for superparasitism were four hours, two days and four days. Parasitized hosts were obtained by exposing an individual scale to a four day-old naïve parasitoid female. Only scales in which the female laid two-three eggs were used for the experiment (92.5 % of the naïve females laid 2-3 eggs).

Behavioural observations. Each observation consisted of a single wasp foraging for a scale on a yucca leaf. The experimental arena, onto the yucca leaf, was delimitated by a glass Petri dish (4 cm diam × 1.5 cm high). The wasp was observed using a dissecting microscope at 10× to 50× magnification and fibre cool light. An observation began when the wasp was placed onto the leaf arena. Our observations of M. flavus foraging and its oviposition behaviour on an unparasitized host were generally in agreement with Kapranas (2002). Wasps examined the leaf by drumming their antenna while walking. Upon encountering a host, the female examined the host with her antenna while remaining on the yucca substrate. Once the female mounted the scale, she continued to examine it with her antenna. She then usually jabbed once the host with her ovipositor along the median axis and in some hosts oviposited there. Then she walked to

- 83 - CHAPTER 5 the periphery of the host and laid several eggs there. If they rejected the scale they abandoned it immediately after jabbing it with the ovipositor.

In parasitized host, the wasps behaved as in unparasitized hosts until they jabbed the scale with their ovipositor. After that, if a wasp rejected the scale for oviposition, either she abandoned the scale immediately after jabbing it or she fed on the haemolymph as it oozed from the puncture they had made. In this study we refer “probe only” when the wasps rejected the scale just after jabbing it once and “host-feeding” when the wasps fed on the scale haemolymph. After jabbing once the scale, if the wasp decided to use this scale for oviposition, she continued examining the scale with her antenna and jabbing with the ovipositor several times in different points of the dorsum’s scale before laying their own eggs in the periphery of the host (this behaviour is referred as “superparasitism”). Whenever, during this examination the female found a stalk of an egg previously laid (physical contact of antenna with the stalk of the deposited egg) she jabbed with her ovipositor close to the remaining stalk and consequently pierced the egg or young larva that was still connected to the stalk. Our results indicated that jabbing behaviour after encountering the eggs stalks led to the death of the egg/larvae and this behaviour is referred to as “ovicide/larvicide” in this paper (only two eggs survived to the female attack out of 114). Each female always laid at least one egg after oviciding. We use the term “jabbing” for any behaviour that involved the ovipositor piercing the scale dorsum without laying any egg. “Rejected hosts” were considered those in which the wasps did not lay any egg (probed only or host-feed). “Accepted hosts” were those in which the wasps laid at least one egg.

The time between the wasp encountering the host and leaving it for more than two minutes was used as an index of host handling time. During oviposition, wasps occasionally fed on the honeydew secreted by the scale or dismounted the host and wandered a short distance only to quickly return and continue the oviposition sequence. The time spent during these behaviours was not considered.

Successful ovipositions were recognized by the egg stalks protruding externally from each host which was visible using a dissecting microscope and fibre cool light. The number of eggs laid by each parasitizing and superparasiting female, and the number of jabs and eggs/larvae killed were counted and their position was mapped using a

- 84 - SUPERPARASITISM BY METAPHYCUS FLAVUS schematic drawing of the scale. The experimental design (treatments and number of observations/treatment) is detailed in the table 1.

Table 1. Experimental design (treatments) and number of observations per treatment. Each treatment is characterized by the type of host offered (parasitized or superparasitized and the interval between ovipositions) and the previous experience and age of the wasps encountering the scales. Time between Parasitoid Number Host offered ovipositions Age (days) Experience observations Healthy - 4 Naïve 40 Healthy - 4 Experienced (4 h earlier) 32 Parasitized * 4 hours 4 Naïve 40 Parasitized * 4 hours 4 Experienced (4 h earlier) 33 Self-superparasitized** 4 hours 4 Experienced (4 h earlier) 33 Healthy - 6 Naïve 40 Healthy - 6 Experienced (2 d earlier) 29 Parasitized * 2 days 6 Naïve 40 Parasitized * 2 days 6 Experienced (2 d earlier) 33 Self-superparasitized** 2 days 6 Experienced (2 d earlier) 31 Healthy - 8 Naïve 38 Healthy - 8 Experienced (4 d earlier) 30 Parasitized * 4 days 8 Naïve 38 Parasitized * 4 days 8 Experienced (4 d earlier) 32 Self-superparasitized** 4 days 8 Experienced (4 d earlier) 28 * Scales had been previously parasitized by a naïve female which laid 2-3 eggs. ** Scales had been previously parasitized by a naïve female which laid 2-3 eggs and the same wasps was used to superparasitize the scale.

5.2.1. Host discrimination and host use

Host rejection and ovicide rates, and number of jabs were analyzed using ANOVAs (number of jabs) and Yates' chi-square tests (rejection and ovicide rates) to study: i. Whether naïve females are able to discriminate between parasitized vs unparasitized hosts. To this aim rejection rates and number of jabs by naïve females encountering unparasitized and hosts parasitized four hours, two days and four days earlier were compared. ii. Whether experience affected host rejection and ovicide. To this aim rejection and ovicide rates and number of jabs by naïve and experienced (both self- and conspecific-superparasitism) females encountering hosts parasitized four hours, two and four days earlier were compared.

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iii. Whether females discriminate between scales parasitized by themselves and those parasitized by conspecific females. To this aim rejection and ovicide rates and number of jabs by females encountering scales parasitized by themselves and by others four hours, two and four days earlier were compared. iv. The effect of the interval between ovipositions. To this aim rejection and ovicide rates by naïve females encountering scales parasitized four hours, two days and four days before were compared. The same analyses were carried out for experienced females encountering scales parasitized by themselves and by others.

5.2.2. Clutch size

The possibility that M. flavus adjusted the second clutch size allocated in parasitized hosts was investigated. Clutch size allocated by naïve and experienced females on unparasitized and parasitized hosts (self-parasitized; or conspecific- parasitized) four hours, two and four days after the first encounter were analyzed using an ANOVA. The clutch size allocated by wasps that performed ovicide was compared with those that did not perform using also ANOVA.

5.2.3. Ovicide

The time spent during host encounters that led M. flavus to parasitize, superparasitize or ovicide and the number of parasitoid eggs pierced by the superparasitizing female at different oviposition intervals (four hours, two days and four days after) were analyzed using ANOVA and the means were compared using an LSD test at 5 % significance level. The data from these observations were used to test if: (i) ovicide confers a time cost on ovipositing wasps that engage in this behaviour and; (ii) the handling time and the number of eggs killed depends on the time last since the first oviposition.

5.2.4. Mortality of immatures

Parasitized and superparasitized scales, along with the leaf (the bottom part of which was in constant contact with water), were maintained in a state that allowed parasitoids to develop normally (at 25 ± 1ºC and 50-70 % R. H. during 14 hours of light and 18 ± 1ºC and 50-70 % R. H. during 10 hours of darkness). The incubated scales

- 86 - SUPERPARASITISM BY METAPHYCUS FLAVUS were examined daily until the parasitoids had pupated. The translucent body of the host, the light yellowish colour of the larvae altogether with the previous observations of the positions of the eggs allowed to track of each immature M. flavus within the host using dissecting microscope at 10× to 50× magnification and fibre cool light. These observations allowed also to assess different causes of mortality of each immature of M. flavus within parasitized and superparasitized scales.

Three different causes of mortality were observed and measured by this method: egg encapsulation, larval competition and mortality of eggs or larvae as a consequence of a scale dying in response to parasitism. Encapsulated parasitoid eggs and/or larvae were detected between the second and fourth day after oviposition. We considered that the eggs and larvae had been encapsulated when they and the surrounding tissue become melanised and no development was observed. Two kinds of larval competition were observed: elimination through “physical combat” and “physiological suppression”. The outcome of the “physical combat” was the consumption of the loser by the winner larvae. Moreover, some eggs and young larvae were neither encapsulated nor consumed by older larvae but they did not develop. In this study we could not clarify if the cause of mortality was asphyxiation or starvation; thus it is referred as “physiological suppression”. Mapping of the position of each deposited egg during each observation and subsequent observation of larval development within the host allowed us to determine which larvae died as consequence of physical combats or physiological suppression, which was the winner of the combats, and its age and sex as well. The sex of the winner was determined once it pupated. Male pupae have darker abdomen than females.

Mortality, encapsulation and larval competition (physical combats and physiological suppression) rates of the two broods present in superparasitized and in parasitized scales were compared using ANOVA. When ANOVA indicated significant main effects LSD test at 5 % significance level were used to compare means. Mortality and encapsulation rates were arcsine transformed before being analyzed. Data of these observations were used to test if: (i) the rates of encapsulation decrease in superparasitized versus parasitized scales and if specifically rates of survival of both clutches are higher in superparasitized hosts (ii) the second brood is in disadvantage relative to the first in superparasitized scales because of larval competition. A binary

- 87 - CHAPTER 5 logistic regression was used to assess the incidence of physical attack within the host depending on the number of larvae presented. For this study the larvae developing in parasitized and superparasitized (four hours between ovipositions) hosts were considered.

5.2.5. Influence of superparasitism on offspring

Two to three days after pupation, mummified scales were removed from the yucca leaf and isolated in 1 cm diam. glass vial with a drop of honey and stored with a cotton plug. The vials were inspected daily for emergence of adult wasps and, upon emergence, the wasps were killed within the vials by freezing. The date of emergence and the number, sex and size of offspring was recorded. When the interval between ovipositions differed two days and both broods developed together they could put apart and measured separately because of the daily examination and their different size. The pupae and consequently the parasitoids emerging from the second brood were much smaller than those from the first. Wasp’s hind tibia lengths (= HTL) were used as an index of host size. The wasps were temporarily slide-mounted using distilled water and their HTL measured to the nearest 0.0025 mm. Measurements were made using an ocular micrometer mounted in the eyepiece of a compound-microscope. The sex ratios of the offspring emerging from each scale were represented as the proportion of male wasps, and were arc-sine transformed before analysis.

Data were used to analyze and compare the influence of superparasitism on several characteristics of the offspring, under different oviposition time intervals (four hours, two days and four days). Brood size, egg-adult development time, sex ratio (% males), hind tibiae length of females and males (× 0.1 mm) of M. flavus emerged from parasitized and superparasitized scales were compared using ANOVA and means were compared using an LSD test at 5% significance level.

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5.3. Results

5.3.1. Host discrimination and host use

Figure 1 represents the proportion of naïve and experienced females that accepted and rejected unparasitized scales and scales parasitized four hours, two and four days before. Figure 2 represents the number of times that females jabbed the scales. Table 2 shows the analysis of the different results represented in figures 1 and 2. i. Are naïve females able to discriminate between parasitized vs unparasitized hosts? No significant differences were found between the rejection rates of unparasitized and parasitized scales by naïve females. However, the numbers of jabs in parasitized scales were significantly different than in the unparasitized scales (Table 2, treatment a). Naïve females killed the eggs/larvae of the first wasp. ii. Does experience affect host rejection and ovicide? Naïve females rejected significantly less and ovicided more in parasitized scales than experienced females when the time between ovipositions was four hours. However, after two days the percentage of rejection and ovicide became similar. No significant differences were found between the numbers of jabs (Table 2, treatment b). iii. Do females discriminate between scales parasitized by themselves and by conspecific females? There were no significant differences in the rejection rates and in the number of jabs of experienced females when they encountered a scale parasitized by themselves or by a conspecific (Table 2, treatment c). Experienced females killed their own eggs when they encountered scales already parasitized by themselves. Ovicide rates were significantly higher in self parasitized scales than in those parasitized by others four days after the first oviposition. iv. Effect of interval between ovipositions on rejection and ovicide rates. Rejection rates depended on the intervals between oviposition for naïve females and for selfsuperparasitism. Rejection rates increased with the time between ovipositions for naïve females and decreased for females that oviposited in scales parasitized by their self (Figure 1, Table 2). The ovicide rate was not affected by the interval between ovipositions.

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4 hours 2 days 4 days Was p Host N N N U Naive 40 40 38

P 32 29 30 Oviposition U 40 40 38 Ovicide + Oviposition Experienced Probe only SP 33 31 28 Host-feeding

CP 33 33 32

-100 -50 0 50 100 -100 -50 0 50 100 -100 -50 0 50 100 Rejection rates Acceptance rates Rejection rates Acceptance rates Rejection rates Acceptance rates

Fig. 1. Rejection (host feeding and probing only) and acceptance rates (oviposition and ovicide plus oviposition ) of unparasitized (U) and parasitized hosts (P) [self-parasitized (SP); or conspecific-parasitized (CP)] by naïve and experienced females four hours, two and four days after the first encounter. The numbers in the right are the number of observations. See table 2 for statistical analysis.

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4 hours 2 days 4 days 7 7 7 6 6 6 s 5 5 5 4 4 4 3 3 3 2 2 2 Number of jab of Number 1 1 1

0 0 0 UPUSPCP UPUSPCP UPUSPCP Naive Experienced Naive Experienced Naive Experienced

Fig. 2. Number of times that naïve and experienced Metaphycus flavus females jabbed in unparasitized (U) and parasitized hosts (P) (self-parasitized (SP); or conspecific-parasitized (CP)) four hours, two and four days after the first encounter.

Table 2. Analysis of superparasitism-rejection, ovicide rates and number of jabs by Metaphycus flavus (see also figure 1). Significance differences were tested using Yates' chi-square test of independence for acceptance and ovicide rates and one way-ANOVA for the number of jabs.

Time RejectionOvicide Number of jabsstings Treatment between ovipositions χ2dfP value χ2dfP value F df P value a 4 hours 0,013 1 0,910 39,48 1, 70 0,0001 2 days 0,000 1 1,000 38,12 1, 67 0,0001 4 days 2,546 1 0,111 33,38 1, 66 0,0001 b 4 hours 5,311 1 0,021 5,479 1 0,019 2,84 1, 96 0,0954 2 days 3,050 1 0,081 1,064 1 0,302 2,37 1, 91 0,1269 4 days 0,009 1 0,924 0,318 1 0,573 0,39 1, 58 0,5356 c 4 hours 2,063 1 0,151 0,142 1 0,706 0,21 1, 64 0,6485 2 days 0,000 1 1,000 0,189 1 0,664 2,21 1, 62 0,1423 4 days 1,846 1 0,174 4,195 1 0,041 2,76 1, 88 0,1004 Naïve 4h, 2d, 4d 9,527 2 0,0085 0,04 2 0,9801 Conspecific superparasitism 4h, 2d, 4d 2,32 2 0,3135 1,183 2 0,5534 Selfsuperparasitism 4h, 2d, 4d 6,24 2 0,0442 5,92 2 0,0518 Treatment a: Compares naïve females when they encountered parasitized and unparasitized scales. Treatment b: Compares naïve and experienced females (conspecific and self-parasitism) when they encountered parasitized scales. Treatment c: Compares experienced females when they encountered a scale parasitized by themselves or by a conspecific. Naïve: compares naïve females when they encountered parasitized hosts at different interval times between ovipositions. Conspecific superparasitism: compares experienced females when they encountered parasitized hosts at different interval times between ovipositions. Selfsuperparasitism: compares experienced females when they encountered hosts parasitized by themselves at different interval times between.

5.3.2. Clutch size

No significant differences were found between clutch sizes allocated at different times between ovipositions or treatments (ANOVA: Time between ovipositions: F = 2.29; df = 2, 388; P = 0.1024; Treatment: F = 0.68; df = 4; P = 0.6062; Interaction: F =

- 91 - CHAPTER 5

1.28; df 8; P = 0.2520) (Table 3). According to these results M. flavus do not adjust their clutch size to the quality of the host. Moreover, no significant differences were found when analyzing the clutch sizes in unparasitized scales (2.65 ± 0.07, n = 167) and in parasitized scales after killing the first clutch (2.69 ± 0.1, n = 68) and when the wasps just did superparasitize (2.47 ± 0.07, n = 167) (ANOVA: F = 2.51; df = 2, 400; P = 0.0829).

Table 3. Mean clutch sizes (mean ± SE (n)) laid by naïve and experienced females on unparasitized and parasitized hosts (self-parasitized; or conspecific-parasitized) four hours, two and four days after the first encounter. Scales had the same size and parasitized scales had two-three eggs (or larvae four days post- oviposition). Treatment Time between ovipositions

Wasp Host 4 Hours 2 Days 4 Days Naïve female Unparasitized 2,75 ± 0,13 (40) 2,52 ± 0,16 (27) 2,53 ± 0,21 (17) Parasitized 2,55 ± 0,15 (31) 2,66 ± 0,16 (29) 2,32 ± 0,16 (28) Experienced Unparasitized 2,59 ± 0,14 (39) 2,83 ± 0,16 (29) 2,67 ± 0,22 (15) female Self-parasitized 2,43 ± 0,19 (21) 2,65 ± 0,17 (26) 2,71 ± 0,17 (24) Conspecific-parasitized 2,33 ± 0,16 (27) 2,89 ± 0,16 (27) 2,26 ± 0,18 (23)

5.3.3. Ovicide

Metaphycus flavus females killed a total of 144 eggs/larvae present in 84 different parasitized scales when they ovicided. In 19 of these scales the wasps killed all the eggs/larvae laid by the first wasp. Only two eggs survived the attack of the females. The number of eggs/larvae killed in one scale was significantly higher when the time elapsed between ovipositions was four days (2 ± 0.15 n = 24) than when it was four hours (1.58 ± 0.14 n = 30) or two days (1.58 ± 0.14 n = 30) (ANOVA: F = 3.44; df = 2, 81; P = 0.037). The host handling time depended on the behaviour adopted by the wasp (parasitize, superparasitize or ovicide) and the time between ovipositions (Fig. 3). Thus, handling time was significantly longer whenever ovicide/larvicide ensued. The wasps spent double time oviciding and laying their own eggs than just ovipositing in unparasitized hosts. Moreover, when oviciding the parasitoids spent more time handling the scales parasitized four days before than those parasitized four hours or two days before.

- 92 - SUPERPARASITISM BY METAPHYCUS FLAVUS

900 15d

9c

15c 600 33b 21b

33a 39a 39a 42a Time (seg) (seg) Time 300

0 Ovicide Ovicide Ovicide Parasitism Parasitism Parasitism Superparasitism Superparasitism Superparasitism 4 hours 2 days 4 days

Fig. 3. Time (mean ± SD, n) spent during host encounters that led Metaphycus flavus to parasitize, superparasitize or ovicide 4 hours, 2 days and 4 days after the first oviposition. Numbers above the columns show the number of replications. Different letters denote significant differences at 5 % level in handling time. Means were compared using an LSD test. ANOVA: Time between ovipositions: F = 9.09; df = 2, 237; P = 0.0002; Behaviour: F = 62.36; df = 2, 237; P < 0.0001; Interaction: F = 2.69; df = 4, 237; P = 0.0318).

5.3.4. Mortality of immatures

Figure 4 illustrates the mortality rates of immature parasitoids and its different causes in parasitized and in superparasitized scales when the second oviposition is delayed four hours, two and four days. In hosts that were supeparasitized within four hours, the mortality rates for the immature offspring of each parental female were significantly lower than those in parasitized hosts (control) (In superparasitized hosts: 1st brood mortality: 53 ± 5%; 2nd brood mortality: 36 ± 5%, in parasitized hosts 68 ± 5%) (ANOVA: F = 11.8; df = 2, 197; P < 0.0001). The main cause of mortality was encapsulation, which was significantly lower for the clutches laid in superparasitized (1st clutch: 25.87 ± 4.78 %; 2nd clutch: 22.38 ± 4.74 %) than in parasitized hosts (control: 64.29 ± 4.78 %) (ANOVA: F = 26.80; df = 2, 197; P < 0.0001). Larval mortality because of competition between larvae was never observed in singly parasitized hosts, but only in superparasitized ones. Larvae of both broods died as consequence of physical combat. There were no significant differences between the number of larvae consumed of the first (6.71 ± 2.08 %) and the second brood (7.84 ± 2.08 %) (ANOVA: F = 0.28; df = 1, 133; P = 0.5954). Thus, the larvae of the second brood were not in disadvantage when the second oviposition was delayed 4 hours. In addition, the clutch of eggs laid by the

- 93 - CHAPTER 5 first female suffered significantly higher mortality rates than the clutch laid by the second female because of ovicide (15.67 ± 3.7 % eggs were killed).

When the second oviposition was delayed two days no significant differences were found between the mortality rates of the immature parasitoids developed in superparasitized (1st brood: 66.92 ± 4.98 %; 2nd brood: 64.55 ± 5.09 %) and in parasitized hosts (53.45 ± 6.71 %) (ANOVA: F = 1.06; df = 2, 187; P = 0.3490). The main mortality causes were different between the broods. The encapsulation rate of the second clutch in superparasitized hosts (14.05 ± 4.97 %) was significantly lower than either the first clutch (49.25 ± 4.97 %) or those clutches allocated in single parasitized hosts (49.83 ± 5.7 %) (ANOVA: F = 14.45; df = 2, 187; P < 0.0001). The second brood was the only that suffered mortality due to competition between larvae (40.05 ± 5.55 %). Specifically, 30.10 ± 4.91 % of the larvae of the second brood were eliminated by physical attacks and 9.95 ± 3.23 % by physiological suppression. Moreover, the survival rate of the larvae of the second brood was 65 ± 7% when none larvae of the first survived, whereas it was lower than 20% when at least one of them survived (Table 4). Finally, 14.18 ± 3.48 % eggs of the first clutch were killed by the superparasitizing female.

Table 4. Survival rates (mean ± SE, n) of second broods when developing in scales where none larvae of the first brood survived and when at least one of them survived at different time intervals between ovipositions.

Number of Time Survival rates larvae of 1st between of the 2nd brood ovipositions brood developing 4 hours 0 0.41 ± 0.09 (23) ≥ 1 0.75 ± 0.04 (54)

2 days 0 0.65 ± 0.07 (47) ≥ 1 0.18 ± 0.05 (35)

4days 0 0.62 ± 0.07 (35) ≥ 1 0.00 ± 0.00 (23)

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4 hours 2 days N N N 4 days Control

Control a Control a a 66 51 30 Encapsulation Encapsulation Encapsulation Wasp 1 Ovicide Ovicide Ovicide Wasp 1 b Wasp 1 a b Larval competition Larval competition Larval competition 67 67 67 Dead scale Dead scale Dead scale Wasp 2 Survival Wasp 2 c Survival Wasp 2 a Survival ab 67 67 67 -1,0 -0,5 0,0 0,5 1,0 -1,0 -0,5 0,0 0,5 1,0 -1,0 -0,5 0,0 0,5 1,0 Survival Mortality Survival Mortality Survival Mortality

Fig. 4. Survival and mortality rates (mean ± SE, n) of the first and second clutches (Wasp 1, Wasp 2) when developing in superparasitized at different time intervals between ovipositions (4 hours, 2 and 4 days) and of the first clutch in parasitized hosts (control). Different colours and textures represent different causes of mortality. Different letters denote significant differences at 5 % level in mortality rates. Means were compared using an LSD test at a 5% significance level.

95 CHAPTER 5

When the second oviposition was delayed four days no significant differences were found between the mortality rates of the immature parasitoids developed in superparasitized (1st brood: 83.33 ± 3.43 %; 2nd brood: 73.61 ± 5.56 %) and in parasitized hosts (62.18 ± 8.7 %) (ANOVA: F = 2.30; df = 2, 147; P = 0.1039). The encapsulation rate of the second clutch in superparasitized hosts (13.61 ± 5.26 %) was again significantly lower than either the first clutch (53.05 ± 5.26 %) or those clutches allocated in single parasitized hosts (61.78 ± 7.56 %) (ANOVA: F = 17.78; df = 2, 147; P < 0.0001). The second clutch was again the only that suffered mortality due to competition between larvae (33.33 ± 6.14 %). Specifically, 13.05 ± 4.08 % of the larvae were eliminated by physical attack between larvae and 20.27 ± 4.99 % by physiological suppression. Any larvae of the second brood survived when one of larvae of the first clutch did (Table 4). Finally, 21.39 ± 4.41 % eggs of the first clutch were killed by the superparasiting female.

Physical combats. Sex and age of the winner. In superparasitized hosts a total of 94 instances of physical attacks were observed. When the interval between ovipositions was four hours, 30 instances were observed; in 27 attacks the winner was a female and in the other three we could not identify the sex. When the interval between ovipositions was two days, 48 attacks were observed; 41 of the winners were females, five were males and two could not be recognized. Finally, 16 larvae of the second brood were consumed when the second wasp oviposited four days later. Fourteen of the consumers observed were females and the other two were males.

The probability of physical attack increased with the number of larvae developing in a host (Binary logistic regression: χ2 = 36.08; df =1, 200; P < 0.0001). All the wining fighters were between 6 to 9 days old.

5.3.5. Influence of superparasitism on offspring

The number of offspring emerging from superparasitized hosts was significantly higher than in just parasitized scales when the second oviposition was delayed four hours, but no such difference was observed when the second oviposition was delayed two and four days (ANOVA: F = 25.9; df = 3, 219; P < 0.0001) (Table 5). The size of females and males (hind tibiae length) emerging from hosts superparasitized within four

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hours were significantly shorter than in those from parasitized or superparasitized after two or four days (Size females; ANOVA: F = 13.11; df = 3, 190; P < 0.0001), (Size males; ANOVA: F = 10.18; df = 3, 157; P < 0.0001), as well as their immature developmental time (ANOVA: F = 15.30; df = 3, 202; P < 0.0001). The secondary sex ratios did not vary significantly between treatments (ANOVA: F = 1.73; df = 3, 212; P = 0.1617).

Table 5. Brood size (when at least one larvae emerged), mean egg-adult development time (days), sex ratio (% males), hind tibiae length of females and males (× 0.1 mm) of Metahycus flavus emerged from parasitized and superparasitized scales. Time intervals between ovipositions in superparasitized hosts varied between 4 hours, two and four days. Different letters denote significant differences in between columns. Means were compared using an LSD test at a 5% significance level.

Parasitized scales Superparasitized scales 4 hours 2 days 4 days Brood size 2.58 ± 0.12 (66) b 3.39 ± 0.12 (62) c 2.44 ± 0.14 (52) b 1.7 ± 0.12 (43) a Developmental time 17.66 ± 0.11 (63) b 17.07 ± 0.12 (58) a 17.59 ± 0.13 (46) b 18.31 ± 0.14 (39) c Sex ratio 0.37 ± 0.035 (65) a 0.39 ± 0.037 (60) a 0.32 ± 0.04 (51) a 0.42 ± 0.045 (40) a Female size (0.1 mm) 2.85 ± 0.028 (61) b 2.64 ± 0.029 (55) a 2.84 ± 0.033 (45) b 2.89 ± 0.038 (33) b Male size (0.1 mm) 2.25 ± 0.037 (51) b 2.02 ± 0.037 (51) a 2.21 ± 0.045 (34) b 2.33 ± 0.052 (25) b

Due to the competition between larvae, broods of the first and second wasp rarely developed together when the second oviposition was delayed two days, and never when delayed four days (see table 4). In the few cases that offspring of both parents developed together, when the second oviposition was delayed two days, emerging parasitoids of the second female were significantly smaller than those of the first female (Size of females of the 1st brood: 2.99 ± 0.074; Size of females of the 2nd brood: 2.41 ± 0.074; ANOVA: F = 31.83; df = 1, 16; P < 0.0001) (Size of males of the 1st brood: 2.43 ± 0.14; Size of males of the 2nd brood: 1.78 ± 0.11; ANOVA: F = 12.84; df = 1, 8; P = 0.007).

5.4. Discussion

Host discrimination and host use. The present study has demonstrated that M. flavus females are able to discriminate parasitized from unparasitized brown soft scales for oviposition purposes. This is supported by the fact that the wasps jabbed more times in parasitized scales and they ovicided. Although naïve females were willing to

- 97 - CHAPTER 5 superparasitize, their behaviour on encountering unparasitized and parasitized hosts were not identical since they jabbed more times in parasitized hosts. Hence, our results show that host discrimination by M. flavus is innate, but the tendency to superparasitize is influenced by experience. Van Alphen et al. (1987) also showed that although naïve females of Leptopilina heterotoma (Thompson) (Hymenoptera: Figitidae) and Trichogramma evanescens Westwood (Hymenoptera: Trichogrammatidae) were willing to superparasitize, their behaviour handling parasitized and unparasitized hosts was not identical.

The fact that M. flavus females rejected at equal rates parasitized hosts by themselves or conspecific females and the fact that the wasps killed their own eggs/larvae show that M. flavus females cannot discriminate between hosts parasitized by them or other conspecific females. However, recognition of hosts parasitized by a female (self-) may depend on the time elapsed since the first ovipositions. This is because rejection rates of selfsuperparasitism decreased when the time between ovipositions was increased. Other parasitoids appear to develop similar superparasitism strategies in which imperfect self/non-self recognition is involved (van Dijken et al. 1992, Godfray 1994, Yamada and Ikawa 2005), and/or this capability may decrease with the time elapsed since the first oviposition (Ueno 1994).

The handling time of parasitized scales depended on the time interval between ovipositions. Metaphycus flavus females spent more time and consequently killed more egg/larvae in scales parasitized four days before than two days and four hours. This result may suggests that M. flavus also discriminate against hosts containing parasitoid larvae, since the larvae of the first female hatched after three-four days. It is unclear whether females discriminate because of the presence of the larvae or because of the lower quality of the hosts.

Metaphycus flavus did not adjust the clutch size allocated in parasitized hosts, independently of the behaviour manifested by the superparasitizing female. Models predict that gregarious parasitoids should lay a reduced clutch size when they superparasitize (Strand and Godfray 1989, Charnov and Skinner 1985). This prediction has been demonstrated for other gregarious parasitoids (Wylie 1965, Van Dijken and Waage 1987). However, M. flavus did not reduce the clutch size when they only superparasitized. Charnov and Skinner (1985) model also predicts that when the

- 98 - SUPERPARASITISM BY METAPHYCUS FLAVUS probability of superparasitizing is high, the first female should lay a smaller clutch. Although superparasitism by M. flavus is frequent in the laboratory i.e. in mass rearing conditions of these wasps (A.T, personal observations), it is unknown whether superparasitism is frequent in the field. Suzuki et al. (1984) also found no difference in the number of eggs laid on parasitized and unparasitized host by Trichogramma evanescens, a gregarious endoparasitoid. Metaphycus flavus did not reduce the clutch size after oviciding either. Similarly, the clutch size laid by Laelius pedatus (Say) (Hymenoptera: Bethylidae) did not differ between the first (sole) parasitoid and the second (ovicidal) parasitoid (Mayhew 1997). This author suggested that parasitized and unparasitized host could be resources of similar quality when ovicide is performed.

Ovicide in a gregarious endoparasitoid. Metaphycus flavus practise ovicide/larvicide. Females examine the hosts externally by drumming the antenna on the dorsum and internally by ovipositor contact (Kapranas 2002). Other encyrtids previously studied use only the antenna or both to reject parasitized hosts (Takasu and Hirose 1988, Islam and Copland 2000). In this study M. flavus always rejected the scales immediately after jabbing them with the ovipositor once and never before, indicating that they might use the ovipositor to detect either the presence of cues/markers, the host’s physiological condition or both (Wylie 1965, Fisher 1971). After jabbing the scale, if the females decided to get involved in an ovicide/larvicide strategy they drummed the scale dorsum searching for the location of the protruding egg stalks. Once the females found the stalks (physical contact of the antenna with the stalks) they jabbed the ovipositor close to the stalks to damage the eggs/larvae, which are attached to the stalk, and consequently kill them. The females also jabbed close to stigmatic spines of the scale after their antenna contacted with them indicating that the location of the eggs was due to a physical contact with the protruding egg stalks. This represents the first case of ovicide/larvicide in Encyrtidae and in gregarious endoparasitoids.

Ovicide/larvicide is an alternative tactic to superparasitism or host rejection. The conditions under which ovicide/larvicide might be adaptive for parasitoids have been modelled by Strand and Godfray (1989). Generally their model predict that ovicide would evolve when i) the advantage of the first female’s offspring is great, ii) when unparasitized host are rare, iii) when the time needed to ovicide/larvicide is short, and iv) when there is little risk of killing one’s own eggs (Strand and Godfray 1989, Smith and

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Lessells 1985). The first condition is met for M. flavus, because the second clutch will be in competitive disadvantage relative to the first clutch, especially when the time between ovipositions is long. On the other hand, the total time involved in ovipositions wherein females also killed the eggs previously laid by another female was increased. Moreover, our results show that superparasitizing females could not recognize their own eggs.

However, there are several reasons for the evolution of ovicide/larvicide in the endoparasitoid-host system of M. flavus and its scale host. Mayhew (1997) speculated that the “precise oviposition” (laying eggs at specific parts of the host) and few large external-laid eggs should favour ovicide in bethylids (ectoparasitoids). Metaphycus flavus do not manifest such “precise oviposition”, but they may use the protruding egg stalks as external cues to identify preciselly the location of the eggs within the host (Netting and Hunter 2000). Some assumptions of the Strand and Godfray model may not apply necessarily to all parasitoid-host associations. More importantly, a parasitized host may not be considered always a host of lower quality than an unparasitized one, because the host defences have been already suppresed. For example, oviposition in a parasitized host could be adaptative when encapsulation of the first eggs laid in the host have exhausted or depleted the host’s supply of haemocytes (Salt 1968, Blumberg and Luck 1990, van Alphen and Visser 1990). In our experiments with M. flavus, the mortality rates of the second clutch caused by encapsulation are much lower in parasitized than unparasitized hosts, indicating that parasitized hosts could represent better oviposition options than unparasitized. Additionally, the fact that the second brood is at a disadvantage relative to the first one when increasing time delayed between ovipositions may have made M. flavus particularly prone to evolve ovicidal tactics.

Partial destruction of the first clutch occurred when M. flavus ovicided. Females killed all egg/larvae present in the scale in 19 of 84 cases that ovicide was observed. According to Strand and Godfray (1989) “all or none” ovicide shall occur if the first clutch is readily apparent to superparasitizing female while partial destruction might occur if the first clutch must be searched for. This occurs in the system M. flavus-brown soft scale where the females have to search for the stalks of the eggs distributed along the periphery of the scale dorsum.

Mortality of immatures. Encapsulation was the major cause of mortality (50-65 %) in just parasitized hosts. Our results show that M. flavus is able to reduce the

- 100 - SUPERPARASITISM BY METAPHYCUS FLAVUS percentage of encapsulation by saturation of the immune system through superparasitism. Superparasitism benefited both clutches when the interval between ovipositions was four hours and only the second clutch when the second oviposition was delayed more than two days.

Elimination through physical combat and physiological suppression regulated supernumerary individuals of M. flavus within the scale. Their importance varied depending on the time elapsed between the two ovipositions (Fisher 1963, 1971). Physiological suppression has also been reported in other parasitoids of the family Encyrtidae (Laraichi 1978, van Baaren and Nénon 1996). In our study physiological suppression occurred mainly when the second oviposition was delayed four days, since the older larvae had consumed most of the scale when the second clutch hatched. The other mechanism by which supernumerary larvae were eliminated was physical combat between larvae which resulted in the death and consumption of one or several brood mates. This aggressive behaviour has been previously documented for M. flavus and also for ther encyrtids Diversinervus elegans Silvestri, M. luteolus and Microterys nietneri (Motschulsky), which are facultatively gregarious endoparasitoids of soft scales (Bartlett and Ball 1964, Bartlett and Medved 1966, Kapranas 2002, 2006). High rates of multiparastism and/or superparasitism favour this aggressive larval behaviour, which allows post-oviposition regulation of the brood size (Pexton and Mayhew 2001). Second oviposition was regulated by older larvae when the oviposition was delayed 2 or 4 days. However, there were no significant differences between the number of larvae consumed of the first and the second brood when the oviposition was delayed four hours. Thus, if the second female does not destroy the older larvae these will control the final clutch size when the second oviposition is delayed two or more days. Bartlett and Ball (1964) also found a definite superiority among larvae with 1-day age advantage for M. luteolus and M. nietneri.

In solitary endoparasitoids the first instars, which are usually equipped with robust, often sickle-shaped mandibles (Fisher 1961, Salt 1961), attack and kill later instars larvae that either have reduced mandibles or lack mandibles altogether (Chow and Mackauer 1986). Gregarious parasitoid larvae usually do not posses fighting mandibles, and competition between individuals is normally restricted to exploitation competition for host resources (Godfray 1994). In encyrtid parasitoids, in which the four

- 101 - CHAPTER 5 larval instars are equipped with mandibles, fights have been reported at the second stage (Bartlett and Ball 1964, van Baaren and Nénon 1996). Considering other studies of Metaphycus immature stages (Saakyan-Baranova 1966), all the fights observed in our study occurred after the first stage since they were at least five days old. In some gregarious parasitoid species, death of immature females in superparasitized hosts was responsible for an increase in the proportion of males produced (Salt 1936, Kuno 1962, Suzuki et al. 1984). According to Waage (1986) males could be better competitors than females because of their more rapid development and lower nutritional requirements. However, Pickering (1980) showed that in the gregarious Pachysomoides stupidus (Cresson) (Hymenoptera: Ichneumonidae), females were better competitors than their brothers because they were more selfish. According to inclusive fitness theory, the selection threshold for a male helping his sister is lower than the selection threshold for a female helping her brother. When the available food is limited, a female should therefore limit the access of her brothers to the food more often than the reverse (van Baaren et al. 1999). In M. flavus, females won all the fights when the time between ovipositions was four hours (no significant differences between the number of larvae consumed of the first and the second brood were found for this time interval). However, more detailed studies would be necessary to understand the reasons of these differences on the competitive abilities between sexes.

Overall, in our study, which was designed to create the conditions promoting superparasitism, the final brood size of superparasitized hosts was affected mainly by three factors: encapsulation, larval competition and ovicide/larvicide. Their importance varied according to the time elapsed between ovipositions. Metaphycus flavus overwhelmed the host immune system when superparasitizing the host. The percentage of encapsulation decreased from around 65% in parasitized hosts to less than 25% for the clutch allocated by the superparasitizing female. Consequently, the number of larvae presented in the scale and the probability of physical combats between brood mates increased. This aggressive behaviour and the physiological suppression regulated the final brood size and they benefited the first clutch when the second oviposition was delayed. But, the second female might also influences the survival rate of its progeny by destroying the first parasitoid eggs/larvae and laying their eggs in a host that has already been overwhelmed (Arakawa 1987, Strand and Godfray 1989, van Baaren et al. 1995, Mayhew 1997). Metaphycus flavus females should pierce all the eggs/larvae present in

- 102 - SUPERPARASITISM BY METAPHYCUS FLAVUS the scale when encountering scales parasitized two or more days before. Otherwise its progeny will be consumed or will be much smaller.

When may superparasitism and ovicide be advantageous for M. flavus? Superparasitism in gregarious parasitoids leads generally to smaller offspring because there is a trade-off between the number of offspring reared from a host and their size. Although the fitness per offspring declines with increasing clutch size, total fitness gained from a host may still increase until a maximum is reached (van Alphen and Visser 1990). Our results indicate that when M. flavus superparasitize within four hours there is an increase of the survival probability of both progenies that consequently leads to emerged adults of smaller size (Figure 4, Table 6). Under this circumstance, and considering that emerging wasp cannot be considered as size marginal when comparing with those of the study of Bernal et al. (1999), superparasitism could be advantageous, especially if considering the highest mortality of the offspring in healthy hosts due to encapsulation. However, Metaphycus spp. are idiobionts; they arrest their host’s development following oviposition and the parasitoid larvae develop rapidly within the host (Lampson et al. 1996, Bernal et al. 1999). Thus, a narrow window of opportunity exists in which to allocate additional eggs to a previously parasitized host, since the older larvae are in advantage respect the younger larvae. After two days most of the larvae of the superparasitizing female died because of physical combat or physiological suppression whenever any larvae of the first female developed. Under these circumstances superparasitism is not advantageous and the females should leave the scale, host feed or kill the eggs/ young larvae already in the hosts and then allocate a clutch of eggs.

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References van Alphen, J. J., M. J. Dijken, and , J. K. Waage. 1987. A functional approach to superparasitism, host discrimination need to be learnt. Netherlands Journal of Zoology 37: 167-179. van Alphen, J. J., and M. E. Visser. 1990. Superparasitism as an adaptive strategy for insect parasitoids. Annual Review of Entomology 35: 59-79. Arakawa, R. 1987. Attack on the parasitized host by a primary solitary parasitoid, Encarsia Formosa (Hymenoptera: Aphelinidae): the second female pierces with her ovipositor, the egg laid by the first one. Applied Entomology and Zoology 22: 644-645. van Baaren, J., and J. P. Nénon. 1996. Intraspecific larval competition in two solitary parasitoids, Apoanagyrus (Epidinocarsis) lopezi and Leptomastix dactylopii. . Entomologia Experimentalis et Applicata 81: 325-333. van Baaren, J., B. L. Landry, and G. Boivin. 1999. Sex allocation and larval cometition in a superparasitizing solitary egg parasitoid: competing strategies for an optimal sex ratio. Functional Ecology, 13: 66-71. Bartlett, B. R., and J. C. Ball. 1964. The developmental biologies of two encyrtid parasites of Coccus hesperidum and their intrinsic competition. Annals of the Entomological Society of America 57: 496-503. Bartlett B. R., and R. A. Medved. 1966. The biology and effectiveness of Diversinervus elegans (Encyrtidae: Hymenoptera), an imported parasite of lecaniine scale insects in California. Annals of the Entomological Society of America 59: 974- 976. Bernal, J. S., R. F. Luck, and J. G. Morse. 1999. Host influences on sex ratio, longevity, and egg load of two Metaphycus species parasitic on soft scales: implications for insectary rearing. Entomologia Experimentalis et Applicata 92: 191-204. Blumberg, D. 1997. Parasitoid encapsulation as a defense mechanism in the Coccoidea (Homoptera) and its importance in biological control. Biological control 8: 225-236. Blumberg, D., and R. F. Luck. 1990. Differences in the rates of superparasitism between two strains of Comperiella bifasciata (Howard) (Hymenoptera: Encyrtidae) parasitizing California red scale (Homoptera: Diaspididae): an adaptation to circumvent encapsulation? Annals of the Entomological Society of America 83: 591-597. Charnov, E. L., and S. W. Skinner. 1985. Complementary approaches to the understanding of parasitoid oviposition decisions. Environmental Entomology 14: 383- 391. Chow, F. J., and M. Mackauer. 1986. Host discrimination and larval competition in the aphid parasite Ephedrus californicus. Entomologia Experimentalis et applicata 41: 243-254. van Dijken, M. J., P. Neuenschwander, J. J. M. van Alphen, and W. N. D. Hammond. 1992. Recognition of individual-specific marked parasitized hosts by the solitary parasitoid Epidinocarsis lopezi. Behavioural Ecology and Sociobiology 30: 77- 82

- 104 - SUPERPARASITISM BY METAPHYCUS FLAVUS van Dijken, M. J., and J. K. Waage. 1987. Self and conspecific superparasitism by the egg parasitoid Trichogramma evanescens. Entomologia Experimentalis et Applicata 43: 183-192. Fisher, R. A. 1961. A study in insect multiparasitism. II. The mechanism and control of competition for the host. Journal of Experimental Biology 38: 605-628. Fisher, R. A. 1963. Oxygen requirements and the physiological suppression of supernumerary insect parasitoids. Journal of Experimental Biology 40: 531-540. Fisher, R. A. 1971. Aspects of the physiology of endoparasitic Hymenoptera. Biological Reviews 46: 243-278. Godfray, H. C. J. 1994. Parasitoids: Behavioral and Evolutionary Ecology. Princeton University Press, Princeton, New Jersey. Gu, H., Q. Wang, and S. Dorn. 2003. Superparasitism in Cotesia glomerata: response of hosts and consequences for parasitoids. Ecological Entomology 28: 422-431. Islam, K. S., and M. J. W. Copland. 2000. Influence of egg load and oviposition time interval on the host discrimination and offspring survival of Anagyrus pseudococci (Hymenoptera: Encyrtidae), a solitary endoparasitoid of citrus mealybug, Planococcus citri (Hemiptera: Pseudoccidae). Bulletin of Entomological Research 90: 69-75. Ito, E., and Y. Y. Yamada. 2005. Profitable self-superparasism in an infanticidal parasitoid when conspecifics are present: self-superparasitism deters later attackers from probing for infanticide. Ecological Entomology 30: 714-723. Kapranas, A. 2002. Clutch size, pattern of sex allocation and encapsulation of Metaphycus sp. nr flavus (Hymenoptera: Encyrtidae) eggs, a facultative gregarious endoparasitoid of brown soft scale Coccus hesperidum L. (Homoptera: Coccidae), M. S. thesis, University of California, Riverside. Kapranas, A. 2006. Parasitoids of Brown Soft Scale Coccus hesperidum L. in Southern California and Their Reproductive Strategies. Ph. D. dissertation. Department of Entomology, University of California, Riverside. Kuno, E. 1962. The effect of population density on the reproduction of Trichogramma japonicum Ashmead (Hymenoptera: Tricogrammatidae). Research in Population Ecology 4: 47-59. Lampson L. J., J. G. Morse, and R. F. Luck. 1996. Host selection, sex allocation, and host feeding by Metaphycus helvolus (Hymenoptera: Encyrtidae) on Saissetia oleae (Homoptera: Coccidae) and its effect on parasitoid size, sex and quality. Environmental Entomology 25: 283-294. Laraichi, M. 1978. Etude de la compétition intra et interspécifique chez les parasites oophages des punaises des blés. Entomophaga 23: 115-120. van Lenteren, J. C., 1981. Host discrimination by parasitoids. In: Nordlund D. A. et al. (eds), Semiochemicals: their role in pest control. John Wiley, New York. Mackauer, M. 1990. Host discrimination and larval competition in solitary endoparasitoids. In: Mackauer, M. et al. (eds), Critical issues in biological control. Intercept, Andover, U.K. Maple, J. D. 1954. The eggs and first instar larvae of Encyrtidae and their morphological adaptations for respiration. University of California, publications in Entomology 8: 25-122.

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Mayhew, P. J. 1997. Fitness consequences of ovicide in a parasitoid wasp. Entomologia Experimentalis et Applicata 84: 115-126. Netting, J. F., and M. S. Hunter. 2000. Ovicide in the whitefly parasitoid, Encarsia Formosa. Animal behaviour 60: 217-226. Pexton, J. J., and P. J. Mayhew. 2001. Immobility: the key to family harmony? Trends in Ecology and Evolution 16: 7-9. Pickering, J. 1980. Larval competition and brood sex ratios in the gregarious parasitoid Pachysomoides stupidus. Nature 283: 291-292. Quicke, D. L. J. 1997. Parasitoc Wasps. Chapman & Hall, London, UK. Saakyan-Baranova, A. A. 1966. The life cycle of Metaphycus luteolus Timb. (Hymenoptera: Encyrtidae), parasite of Coccus hesperidum L. Homoptera: Coccidae), and the attempt of its introduction into the USSR. Entomological Review 45: 414-423. Salt, G. 1961. Competition among insect parasitoids. Mechanisms in biological competition. Symposium of the Society for Experiemental Biology 15: 96-119. Salt, G. 1968. The resistance of insect parasitoids to the defence reactions of their hosts. Biological Reviews of the Cambridge Philosophical Society, 43: 200-232. Smith, R. H., and C. M. Lessells. 1985. Oviposition, ovicide and larval competition in granivorous insects. In: Sibly, R. M. and Smith, R. H. (eds), Behavioral Ecology. Blackwell Scientific, Oxford, U.K. Stephens, D. W., and J. R. Krebs. 1986. Foraging theory. Princeton University Press, Princeton, U.K. Strand, M. R. 1986. The physiological interactions of parasitoids with their hosts and their influence on reproductive strategies. In: Waage, J. and Geathead, D (eds), Insects Parasitoids. Academic, London, U.K. Strand, M. R., and H. C. J. Godfray. 1989. Superparasitism and ovicide in parasitic Hymenoptera: theory and a case study of the ectoparasitoid Bracon hebetor. Behavioral Ecology and Sociobiology 24: 421-432. Suzuki, Y., H. Tsuji, and M. Sasakawa. 1984. Sex allocation and effects of superparasitism on secondary sex ratios in the gregarious parasitoid, Trichogramma chilonis (Hymenoptera: Tricogrammatidae). Animal Behaviour 32: 478-484. Takasu, K., and Y. Hirose. 1988. Host discrimination in the parasitoid Ooencyrtus nezarae: the role of the egg stalk as an external marker. Entomologia Experimentalis et Applicata 47: 45-48. Ueno, T. 1994. Self-recognition by the parasitic wasp Itoplectis naranyae (Hymenoptera: Ichneumonidae). Oikos 70: 333-339. Vet, L. E. M., A. Datema, A. Janssen, and H. Snellen. 1994. Clutch size in a larval- pupa endoparasitoid: consequences for fitness. Journal of Animal Ecology 63: 807-815. Waage, J. K. 1986. Family planning in parasitoids: adaptive patterns of progeny and sex allocation. In: Waage, J. and Geathead, D (eds), Insects Parasitoids. Academic, London, U.K. Wylie, H. G. 1965. Discrimination between parasitized and unparasitized housefly pupae by females of Nasonia vitripennis (Walk.) (Hym.: Pteromalidae). Canadian Entomologist 97: 279-286.

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Yamada, Y. Y., and K. Sugaura. 2003. Evidence for adaptive self-superparasitism in the drynid parasitoid Haplogonatopus atratus when conspecifics are present. Oikos 103: 175-181. Yamada, Y. Y., and K. Ikawa. 2005. Superparasitism strategy in a semisolitary parasitoid with imperfect self/non-self recognition, Echthodelphax fairchildii. Entomologia Experimentalis et Applicata 114: 143-152.

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- 108 - CONCLUSIONS

Chapter

6

Conclusions

- 109 - CHAPTER 6

- 110 - CONCLUSIONS

Density and structure of Saissetia oleae populations on citrus and olives: relative importance of the two annual generations.

i. Black scale populations show a similar trend in both crops, presenting one important and concentrated crawler emergence in July

ii. A second partial, heterogeneous and variable crawler emergence was observed in fall-winter but populations did not increase during this time of the year due to the effect of low temperatures on first instars and the lower fertility of mature females.

iii. If chemical sprays should be applied to control population outbreaks, we recommend to apply them at the end of July, when populations are homogenous, all crawlers have already emerged and first instars predominate in populations.

Parasitoid complex of Saissetia oleae on citrus and olives: seasonal trend and impact on host population.

i. The most abundant and widely distributed parasitoids of black scale in citrus and olive crops in eastern Spain are nowadays Scutellista caerulea, Metaphycus flavus and Metaphycus lounsburyi. Thus, M. flavus has not been displaced by the introduced parasitoid M. helvolus.

ii. The effectiveness of the adult female parasitoid, S. caerulea and M. lounsburyi, seems to be limited as biological control agents of black scale when the scale is univoltine because they build up the populations too late to prevent scale outbreaks, unless they were augmentatively released just before the female reached the optimum stage to be parasitized.

iii. If outbreaks occur in citrus we recommend the augmentative release of M. flavus instead of M. helvolus because the native parasitoid appears to be better adapted and, moreover, mass-production of M. flavus is less costly than that of M. helvolus.

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Host discrimination, superparasitism and ovicide by Metaphycus flavus a gregarious endoparasitoid of Coccus hesperidum.

i. Metaphycus flavus females are able to discriminate healthy from parasitized brown soft scales although they have no ability for self/ non-self recognition.

ii.The final brood size of superparasitized hosts was affected mainly by three factors: encapsulation, larval competition and ovicide/larvicide. Their importance varied according to the time elapsed between ovipositions.

iii. Metaphycus flavus females practised ovicide/larvicide. This reproductive strategy had not been previously documented in gregarious endoparasitoids. It might have evolved because: a. The larvae of the second female are in disadvantage relative to the larvae of the first female. b. The eggs allocated in parasitized hosts are encapsulated less frequently than those allocated in unparasitized ones.

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