UNIVERSIDAD DE GUADALAJARA · 2018-04-23 · Agradecimientos A Dios por la oportunidad diaria de aprender y a mi familia por el apoyo continuo. A la Universidad de Guadalajara por

UNIVERSIDAD DE GUADALAJARA

CENTRO UNIVERSITARIO DE CIENCIAS

BIOLÓGICAS Y AGROPECUARIAS

DIVISION DE CIENCIAS AGROPECUARIAS

DEPARTAMENTO DE PRODUCCIÓN AGRÍCOLA

Dinámica del comportamiento de selección: especies de

parasitoides limitados en carga ovárica vs especies limitadas en

tiempo

Tesis Presentada por el Sustentante:

M. en C. María Concepción Velasco Hernández

Como Requisito Parcial para Obtener el Grado de:

DOCTORA EN CIENCIAS EN BIOSISTEMÁTICA,

ECOLOGÍA Y MANEJO DE RECURSOS NATURALES Y

AGRÍCOLAS

ZAPOPAN, JALISCO

OCTUBRE 2017

DECLARATORIA DE ORIGINALIDAD A quien corresponda: Por este conducto el abajo firmante, autor del Trabajo Recepcional (Tesis) titulado: “Dinámica del comportamiento de selección: especies de parasitoides limitados en carga ovárica vs especies limitadas en tiempo”, declaro que el contenido del mismo constituye un documento inédito y original por lo que cumple con los términos de originalidad a los que se hace mención en el Artículo 73 del Reglamento General de Posgrado y el Artículo 14 Fracción I del Reglamento General de Titulación de la Universidad de Guadalajara.

ATENTAMENTE

Zapopan, Jalisco, 6 de octubre de 2017

__________________________ M. en C. María Concepción Velasco Hernández

Código 213503303

Agradecimientos

A Dios por la oportunidad diaria de aprender y a mi familia por el apoyo continuo.

A la Universidad de Guadalajara por permitir el desarrollo de mi investigación dentro de sus

instalaciones.

A mi director de tesis Dr. Ricardo Ramírez Romero por dirigirme en este proyecto.

Al Dr. Nicolas Desneux por la confianza de otorgarme este proyecto, la guía y consejos

durante la estancia y a lo largo de este proceso.

A mi asesora interna Dra. María Magdalena Martínez Ramírez, no solo por el tiempo

proporcionado en las evaluaciones, sino por el compromiso mostrado en cada momento.

Dr. Gustavo Moya Raygoza por sus valiosos comentarios, consejos y aportes durante las

reuniones anuales y durante el examen de candidatura.

Dra. Carla V. Sánchez Hernández por su apoyo en el examen de candidatura y en la

colaboración de la revisión final de la tesis.

Dra. Rosalba Mireya Hernández Herrera y Dr. Alejandro Urias por su participación en la

revisión final de la tesis.

Dr. Fernando Santacruz Ruvalcaba por el apoyo durante mis estudios.

A Luis y Héctor, gracias por la ayuda durante mis experimentos y además por alegrar mis

momentos de mayor trabajo.

A mis compañeros de laboratorio: Adal, Estefania, Lulu, Delia, Luis, Héctor, me llevo grandes

alegrías y anécdotas divertidas gracias a ustedes, pero sobre todo gracias por su amistad

sincera.

A los chicos nuevos con los cuales conviví muy poco tiempo pero que de no ser por ellos no

hubiese encontrado abierto el lab: Pau, Walter, Pit, Vale, Caro, Kike.

A la nueva familia alrededor del mundo: Ha, Lucie, Philip, Awawing, Senegal, Christine, Dr.

Abid Alí, Hang, Wan-Yen, Dani, Kercya, Paty, Vanessa, Vitor, Van, Francois, Victor,

Madalina, Lionel, Sylvan, Christiane, Christian, Yu-sha, Yen-yen, Chen y Alex.

A los amigos de siempre Gaby y Juve, gracias por no dejarme sola y seguir forjando esta

amistad de años y “sigamos yendo a esos tacos”

A los amigos lejanos que siempre me han brindado su apoyo.

A Jury y Emilia, por el apoyo en los tramites.

A la Universidad de Guadalajara por permitir el desarrollo de mi investigación dentro de sus

instalaciones.

A Koppert-Mexico por el apoyo parcial en la adquisición de las avispas parasitoides.

A CONACyT y al pueblo mexicano por el apoyo de beca durante los estudios para la

obtención del grado de Doctor de MCVH (CONACyT ID:367437).

A CONACyT por el apoyo financiero del proyecto No 0157259 de Ciencia Básica otorgado a

RRR y a UC-Mexus-CONACyT (número CN12608) por el apoyo otorgado a RRR y el apoyo

de FP7-PEOPLE-2013-IRSES (proyecto de APHIWEB, número 611810) otorgado a ND y

MCVH.

Dedicado a mi familia

“El hombre nunca sabe de lo que es capaz, hasta que lo intenta” Charles Dickens

i

Dinámica del comportamiento de selección: especies de parasitoides limitadas en carga

ovárica vs especies limitadas en tiempo

María Concepción Velasco Hernández

Resumen

Es aceptado que parasitoides limitados en tiempo son no-selectivos y tienden a aceptar a hospederos de baja calidad cuando los hospederos de alta calidad no están disponibles. Del mismo modo se acepta que parasitoides limitados en carga ovárica son selectivos tendiendo a aceptar solo a hospederos de alta calidad. Sin embargo, también se postula que la selección de hospederos en parasitoides es un proceso dinámico afectado por su estado fisiológico y el tipo de hospedero presente. Por lo que el objetivo de este estudio fue determinar cómo el estado fisiológico de dos parasitoides de áfidos (Aphidius ervi Haliday and Aphelinus abdominalis Dalman) con diferentes historias de vida (parasitoide limitado en tiempo y parasitoide limitado en carga ovárica) influye en su proceso de selección de hospederos. En un bioensayo con opción en laboratorio se observaron a parasitoides bajo los siguientes tratamientos: 1) hembras parasitoides alimentadas-jóvenes, 2) hembras parasitoides en ayuno-jóvenes, 3) hembras parasitoides alimentadas-viejos y 4) hembras parasitoides en ayuno-viejos. Una hembra parasitoide de cada tratamiento fue expuesta a su hospedero de alta y baja calidad de manera simultánea, observando y registrando su comportamiento. Los resultados muestran que A. ervi (parasitoide limitado en tiempo) oviposita similarmente en hospederos de alta y baja calidad en todos los tratamientos. Mientras que el parasitoide A. abdominalis (especie limitada en carga ovárica) no oviposita en ningún hospedero expuesto en todos los tratamientos. En un bioensayo sin opción y en condiciones de laboratorio los parasitoides en los

tratamientos antes mencionados fueron expuestos a sus hospederos de alta y baja calidad

de manera separada, observando y registrando su comportamiento. En condiciones de

invernadero se expuso a los parasitoides de tratamientos alimentados a sus hospederos de

alta y baja calidad durante 24 horas y recapturados al término del tiempo. Los resultados de

laboratorio indicaron que ambos parasitoides ovipositan más en hospederos de alta calidad y

menos en hospederos de baja calidad en todos los tratamientos. Pero el parasitoide limitado

en carga ovárica (A. abdominalis) solo lo hace bajo ciertas condiciones fisiológicas. Los

resultados de invernadero muestran que ambas especies provenientes de tratamientos

alimentadas-jóvenes ovipositan más en hospederos de alta calidad y ovipositan menos en

hospederos de baja calidad y, que cuando son parasitoides alimentados-viejos ambas

especies no muestran preferencia por hospederos de alta o baja calidad. Los resultados

confirman que el estado fisiológico afecta el comportamiento de selección de hospederos

contribuyendo a aceptar que el proceso de selección es dinámico afectado por el estado

fisiológico del parasitoide.

ii

Abstract

Time-limited parasitoids are accepted to be non-selective, accepting low -quality hosts when high-quality hosts are not available. And egg limited parasitoids are considered selective, accepting only high-quality hosts. However, is accepted that host selection is a Dynamic process affected for physiological status and the sort of host available. Then the objective in this study was determine how the physiological status of the aphid parasitoids (Aphidius ervi Haliday and Aphelinus abdominalis Dalman) with different life histories (time-limited and egg-limited parasitoids) influence the host selection process. In a choice bioassay under laboratory conditions we observed to parasitoids under the following treatments: 1) fed-young female parasitoid, 2) starved-young female parasitoid, 3) fed-old female parasitoid and 4) starved-old female parasitoid. Each parasitoid from each treatment was exposed to its high and low-quality hosts in a simultaneously way, observing and recording its behavior. Results showed that A. ervi (time-limited parasitoid) oviposited in a similar way in high and low-quality hosts in all treatments. While A. abdominalis (egg-limited parasitoid) does not oviposit in any exposed host in all treatments. In a choice bioassay and under laboratory conditions, parasitoids under the treatments aforementioned were exposed to high and low-quality hosts in a separated way, observing and recording its behavior. Under greenhouse conditions, parasitoids from fed treatments were exposed to high and low-quality hosts for 24 hours and recaptured to the end of this time. Results from laboratory conditions showed that both species of parasitoids oviposited more in high quality hosts tan low quality hosts in all treatments. But the egg-limited parasitoid (A. abdominalis) only made it under certains physiological conditions. Greenhouse results showed that both species oviposited more in high quality hosts than in low-quality hosts. Besides fed-old parasitoids did not showed differences in oviposition in high or low-quality hosts.ad Results confirmed that physiological status of parasitoids affects the host selection behavior to reaffirm that host selection process is dynamic and it is affected for parasitoid physiological status.

iii

TABLA DE CONTENIDO

Resumen………………………………………………..………………………………….……... i

Abstract………………………………………………………..………………………….………. ii

Lista de figuras …….…..…….………………………….…………………………………….… v

Lista de cuadros ………………………………………….……………………………… …..... vi

CAPÍTULO 1. INTRODUCCIÓN GENERAL................................................................................... 1

1.1 Hipótesis General ....................................................................................................................... 3

1.1.1 Hipótesis específicas .......................................................................................................... 3

1.2 Objetivo general .......................................................................................................................... 4

1.2.1 Objetivos particulares ......................................................................................................... 4

CAPÍTULO 2. IDONEIDAD DE ESPECIES HOSPEDERAS Y PREFERENCIA DE INSTAR

DE APHIDIUS ERVI Y APHELINUS ABDOMINALIS ......................................................... 5

2.1 Abstract. ....................................................................................................................................... 6

2.2 Introduction .................................................................................................................................. 7

2.3 Materials and Methods ............................................................................................................. 11

2.3.1 Biological material .............................................................................................................. 11

2.3.2 Bioassay 1: Host species suitability ................................................................................ 12

2.3.3 Bioassay 2: Instar preference .......................................................................................... 14

2.4 Results ........................................................................................................................................ 15

2.4.1 Bioassay 1: Host species ................................................................................................. 15

2.4.2 Bioassay 2: Instar preference .......................................................................................... 17

2.5 Discussion .................................................................................................................................. 18

2.5.1 Host species suitability ..................................................................................................... 18

2.5.2 Instar preference ................................................................................................................ 20

2.6 Conclusion.................................................................................................................................. 21

2.7 References ................................................................................................................................. 22

CAPÍTULO 3. ESTADO FISIOLÓGICO DE PARASITOIDES AFECTANDO EL PROCESO

DE SELECCIÓN DE HOSPEDEROS ................................................................................. 27

3.1 Resumen .................................................................................................................................... 28

3.2 Introducción ............................................................................................................................... 29

iv

3.3 Materiales y Métodos ........................................................................................................... ….31

3.3.1 Material Biológico .............................................................................................................. 31

3.3.2 Bioensayo de Laboratorio ................................................................................................ 33

3.4 Resultados ................................................................................................................................. 35

3.4.1 Observaciones de comportamiento ........................................................................ ……..35

3.4.2 Análisis Bioquímicos ......................................................................................................... 39

3.5 Discusión .................................................................................................................................... 41

3.5.1 Observaciones de comportamiento ................................................................................ 41

3.5.2 Análisis Bioquímicos ......................................................................................................... 44

3.6 Conclusiones ............................................................................................................................. 45

3.7 Referencias ................................................................................................................................ 46

CAPÍTULO 4. EFECTOS DEL ESTADO FISIOLÓGICO EN EL RANGO DE HOSPEDERO

Y COMPORTAMIENTO DE OVIPOSICIÓN ...................................................................... 51

4.1 Abstract ...................................................................................................................................... 52

4.2 Introduction ................................................................................................................................ 53

4.3 Materials and Methods ............................................................................................................. 55

4.3.1 Biological Material ............................................................................................................. 55

4.3.2 Selection behavior observations ..................................................................................... 56

4.3.3 Greenhouse bioassay ....................................................................................................... 57

4.3.4 Data analysis ...................................................................................................................... 57

4.4 Results ........................................................................................................................................ 58

4.4.1 Laboratory Bioessay ......................................................................................................... 58


4.5 Discussion .................................................................................................................................. 63

4.5.1 Laboratory bioessay .......................................................................................................... 63


4.6 Conclusions ............................................................................................................................... 67

4.7 References ................................................................................................................................. 69

CAPÍTULO 5. DISCUSIÓN GENERAL ......................................................................................... 75

CAPÍTULO 6. CONCLUSIONES GENERALES .......................................................................... 78

Referencias ........................................................................................................................................ 80

v

Lista de figuras

CAPITULO 2

Figure 2.1 Variables analyzed for the species A. ervi. A) number of parasitized aphids, B)

percentage of parasitism, C) percentage of emergence, D) proportion of females, E)

development time (in days), F) size of the progeny tibia (in

millimeters).…………………………………………………………………..…………………… 15

Figure. 2.2 Variables analyzed for the species A. abdominalis. A) number of parasitized

aphids, B) percentage of parasitism, C) percent-age of emergence, D) proportion of females,

E) development time (in days), F) size of the progeny tibia (in millimeters)

……………………………………………………………………………………………..……..…16

Figure 2.3 Instar preference bioassay. A) Mean (± SE) number of mummies produced by A.

ervi in each nymphal stage of the host aphid species M. persicae, B) Mean (± SE) number of

mummies produced by A. abdominalis in each nymphal stage of the host aphid species R.

padi……………………………………..…………………………………………….…………… 17

CAPITULO 3

Figura 3.1 Selección de hospedero para el parasitoide limitado en tiempo Aphidius ervi A) y

selección de hospedero para el parasitoide limitado en carga ovárica A. abdominalis B)

……………………………………………………………………………………………………… 36

Figura 3.2 Parametros de comportamiento del parasitoide limitado en tiempo Aphidius ervi

en A) Tiempo de detección, B) Ataque, C) Tiempo de aceptación, D) Numero de huevos

ovipositados en los áfidos y E) Carga ovárica.

…………………………………………………………….……………………………………..… 37

Figura 3.3 Parametros de comportamiento del parasitoide limitado en carga ovárica

Aphelinus abdominalis en A) Tiempo de detección, B) Ataque, C) Tiempo de aceptación y D)

Carga ovárica……………………………………………………………………………………... 39

Figura 3.4 Aphidius ervi A) Contenido de fructosa, B) Contenido de glicógeno y C) Contenido

de sacarosa en µg……………………………………………………………………………...… 40

Figura 3.5 Aphelinus abdominalis A) Contenido de fructosa, B) Contenido de glicógeno y C)

Contenido de sacarosa en µg ………………………………………………………….…..…... 41

vi

CAPITULO 4

Figure 4.1 Behavioral parameters of Aphidius ervi (time-limited parasitoid) in A) Time of host

detection, B) Attacks, C) Time of host acceptance, D) Number of eggs oviposited in aphids

and E) Egg load……………………….………………………………………………….………. 59

Figure 4.2. Behavioral parameters of Aphelinus abdominalis (egg-limited parasitoid) in A)

Time of host detection, B) Attacks, C) Time of host acceptance, D) Number of eggs

oviposited in aphids and E) Egg

load…………………………………………………………………………………………….……. 61

Figure 4.3 Aphidius ervi A) Egg load and B) Number of aphids

parasitized…………………………………………………………………………………………. 62

Figure 4.4 Aphelinus abdominalis A) Egg load and B) Number of aphids

parasitized……………………………………………………………………………………….... 63

Lista de cuadros

CAPITULO 2

Table 2.1 Detailed information on the contributions of the present study. For each parasitoid

species, we formed 18 cells by crossing 6 biological parameters studied and 3 hosts species

analyzed. For each cell it is indicated: ‘Not reported previously’ (when we found no previous

published literature reporting that information) or ‘Reported previously’ (when previous

literature reports information for that specific cell). In the last case, we provide also the

reference that reports such information. ‘Reported here’ indicates that we generated data in

this study for that specific cell……………………………………………………………………. 9

1

CAPÍTULO 1. INTRODUCCIÓN GENERAL

Los áfidos son un importante grupo plaga alrededor del mundo por el daño que causan en

cultivos (Blackman & Eastop 2007). El daño es notorio no solo por la extracción de savia sino

porque se convierten en importantes vectores de numerosos virus de plantas, además de

contribuir en la proliferación de hongos en plantas (Boquel et al. 2014, Xiao et al. 2015).

Los parasitoides son enemigos naturales de muchos insectos y pueden mantener bajo

control muchas plagas potenciales, entre ellos los áfidos, (Godfray 2004). Un parasitoide es

un insecto del cual su larva se desarrolla alimentándose de los cuerpos de otros artrópodos,

como parásito requieren de un solo hospedero para completar su desarrollo, pero como

depredadores matan a su hospedero (Godfray 2004). Los parasitoides por su reproducción

son clasificados en dos grupos, el primer grupo son aquellos que nacen con su complemento

de huevos completo (número de huevos maduros que tiene el parasitoide a determinado

tiempo), y el segundo grupo son aquellos parasitoides que nacen con su complemento de

huevos incompleto pero que pueden madurar más huevos a lo largo de su vida (Mangel

1989). Sin embargo, los primeros se consideran limitados en tiempo desde que su

complemento de huevos es numeroso y su longevidad es corta. Y los segundos se

consideran limitados en carga ovárica (carga ovárica= número de ovocitos maduros que una

hembra parasitoide tiene en sus ovariolas a cierto tiempo) desde que su complemento de

huevos es pequeño, pero tienen una longevidad larga (Mangel 1989, Godfray 1994, Heimpel

& Rosenheim 1998, Van Baalen 2000). Los parasitoides con longevidad corta llegan a vivir

de una a dos semanas de edad (Hofsvang & Hagvar 1975, Malina & Praslicka 2008),

mientras que los parasitoides de longevidad larga llegan a vivir varios meses (Wahab 1985,

Holler & Haardt 1993).

El proceso de localizar y aceptar un hospedero o presa es llamado selección de hospedero y

todos los organismos en un hábitat son potenciales hospederos o presas, los cuales varían

en calidad (Strand & Obrycki 1996). El grupo de especies donde el parasitoide se puede

desarrollar es referido con el término de rango de hospederos (Strand & Obrycki 1996). Y

cada especie de enemigo natural es capaz de atacar un rango de hospederos que va desde

los mejores para el desarrollo de su progenie (alta calidad), a aquellos que solo les permite

un desarrollo marginal (baja calidad) (Strand & Obrycki 1996).

Se han establecido paradigmas sobre la selección de hospedero por los parasitoides, con

base en el tipo de historia de vida que presentan, los cuales dicen que:

2

1.- Parasitoides limitados en carga ovárica tienden a aceptar a hospederos de alta calidad

sobre los de baja calidad (Heimpel & Rosenheim 1998)

2.- Parasitoides limitados en tiempo tienden a aceptar a hospederos de baja calidad sobre

los de alta calidad cuando no hay hospederos de alta calidad disponibles (Heimpel et al.

1998, Papaj et al. 2000).

Sin embargo, se sugiere que algunos modelos de selección de hospedero en parasitoides

son dinámicos (Rosenheim 1999), y dependen fuertemente del estado fisiológico de los

parasitoides (Rivero 2000, Wackers 1994). Ya que se ha observado que especies de baja

calidad para los parasitoides han sido aceptadas bajo diferentes condiciones fisiológicas

(Jenner et al. 2012). Por ejemplo, Jenner et al. (2014) encontraron que parasitoides viejos de

la especie Diadromus pulchellus Wesmael (Hymenoptera:Ichneumonidae), aceptan a

hospederos de baja calidad. También se ha observado en especies de Aphelinus que los

parasitoides bajo condiciones de hambre o viejos disminuyen su reproducción en especies

de baja calidad (Hopper et al. 2013).

La mayoría de los estudios que prueban factores fisiológicos en parasitoides, lo hacen de

manera separada. Este estudio propone combinar los factores fisiológicos del parasitoide

(alimentación y edad) de manera que se tengan más posibilidades de diferentes estados

fisiológicos de parasitoide, esperando encontrar en qué condiciones el parasitoide acepta a

sus hospederos de baja o alta calidad. Ya que los paradigmas solo hacen alusión al tipo de

historia de vida del parasitoide.

3

1.1 Hipótesis General

El estado fisiológico, definido por el estado nutricional y la edad del parasitoide, modificará el

comportamiento de selección de los parasitoides en las dos historias de vida analizadas

(limitados en tiempo y limitados en carga ovárica).

1.1.1 Hipótesis específicas

1) El parasitoide A. ervi, al ser una especie limitada en tiempo parasitará y se comportará de

manera similar frente a hospederos de baja y/o alta calidad cuando el estado fisiológico sea

óptimo (i.e. alimentados y jóvenes) tal como lo indica el paradigma de selección. Es decir, no

será selectivo, ovipositando en todo aquel hospedero que se le exponga.

2) Al variar el estado fisiológico del parasitoide limitado en tiempo A. ervi (i.e. cuando las

hembras estén viejas o en ayuno), la no-selectividad esperada de acuerdo con el paradigma,

cambiará y el parasitoide parasitará de manera diferencial en hospederos de baja y alta

calidad.

3) El parasitoide A. abdominalis, al ser una especie limitada en carga ovárica parasitará y se

comportará de manera diferencial frente a hospederos de baja o alta calidad cuando el

estado fisiológico sea óptimo (i.e. alimentados y jóvenes) tal como indica el paradigma de

selección. Es decir, será selectivo, prefiriendo a hospederos de alta calidad sobre los de baja

calidad.

4) Al variar el estado fisiológico del parasitoide A. abdominalis (i.e. cuando las hembras estén

más viejas o en ayuno), la selectividad esperada de acuerdo con el paradigma cambiará y el

parasitoide parasitará de manera similar a hospederos de baja y alta calidad.

4

1.2 Objetivo General

Determinar cómo el estado fisiológico (estado nutricional y edad) de los parasitoides

modifican la selección del hospedero en estas dos historias de vida diferentes (limitados en

tiempo y limitados en carga ovárica) bajo condiciones de laboratorio e invernadero.

1.2.1 Objetivos particulares

1) Determinar si el parasitoide A. ervi, al ser una especie limitada en tiempo, parasitará y se

comportará de manera similar frente a hospederos de baja o alta calidad cuando el estado

fisiológico sea óptimo (i.e. alimentados y jóvenes) tal como lo indica el paradigma de

selección.

2) Determinar si el estado fisiológico del parasitoide A. ervi (i.e. cuando las hembras estén

viejas o en ayuno) hace parasitar de manera diferencial en hospederos de baja y alta

calidad.

3) Determinar si el parasitoide A. abdominalis, al ser una especie limitada en carga ovárica

parasitará y se comportará de manera diferencial frente a hospederos de baja o alta calidad

cuando el estado fisiológico sea óptimo (i.e. alimentados y jóvenes) tal como indica el

paradigma de selección.

4) Determinar si el estado fisiológico del parasitoide A. abdominalis (i.e. cuando las hembras

estén más viejas o en ayuno) hace aceptar de manera similar a hospederos de baja y alta

calidad.

5

CAPÍTULO 2. IDONEIDAD DE ESPECIES HOSPEDERAS Y PREFERENCIA DE INSTAR DE APHIDIUS ERVI Y APHELINUS ABDOMINALIS

Artículo publicado en prensa, cuya referencia bibliográfica es:

Velasco-Hernández MC, Desneux N, Ramírez-Martínez MM, Cicero L, Ramírez-Romero R

(2017). Host species suitability and instar preference of Aphidius ervi and Aphelinus

abdominalis. Entomologia Generalis 36(4), DOI:10.1127/entomología/2017/0500. En prensa

6

Entomologia Generalis, Vol. 36 (2017), Issue 4, XXX–XXX Article

Published in print XXX 2017

Host species suitability and instar preference

of Aphidius ervi and Aphelinus abdominalis

M.C. Velasco-Hernández1, N. Desneux2, M.M. Ramírez-Martínez3,

L. Cicero4 and R. Ramirez-Romero1*

1 Biological Control Laboratory, Department of Agricultural Production,

CUCBA, University of Guadalajara, Zapopan, Jalisco, México

2 French National Institute for Agricultural Research (INRA), UMR1355, 400 Route

des Chappes, 06903, Sophia-Antipolis, France

3 Department of Ecology and Natural Resources, CUCSUR, University of

Guadalajara, Autlán de Navarro, Jalisco, México

4 Forest Health and Agriculture Program, C.E. Mocochá – CIRSE, National Institute for

Research in Forestry, Agriculture, and Livestock (INIFAP), Mocochá 97454,

Yucatán, México

* Corresponding author: [email protected]

With 3 figures and 1 table

2.1 Abstract: Parasitism rates and parasitoid development can be influenced by the species and

developmental stage of the host; both factors can influence parasitoid performance and fitness. In

this study, parasitism rates and developmental parameters were assessed for two widely

distributed and commercially available species of aphid parasitoid: Aphidius ervi (Hymenoptera:

Braconidae) and Aphelinus abdominalis (Hymenoptera: Aphelinidae). In a first bioassay,

parasitism rates and parasitoid development were investigated in different host species. The wasp

A. ervi was tested on Macrosiphum euphorbiae, Rhopalosiphum padi, and Myzus persicae

(Hemiptera: Aphididae) and A. abdominalis was tested on M. persicae, R. padi, and

Rhopalosiphum maidis (Hemiptera: Aphididae). The results indicated that A. ervi had a greater

percentage of emergence, higher percentage of parasitized aphids, longer developmental time,

and higher proportion of females in M. persicae than in the other hosts. A. abdominalis had a

greater percentage of emergence, larger progeny, and shorter developmental time in R. padi than

in the other hosts. A second bioassay evaluated the preference of the parasitoids for different

instars of their respective optimum host aphids in terms of parasitism and development, as

determined in the first bioassay. The results showed that A. ervi produced a greater number of

mummies in the fourth instar and in adults of M. persicae. In contrast, A. abdominalis preferred

the first instar of R. padi. In conclusion, our results indicate that both parasitoid species exhibit

different parasitism parameters depending upon the host species and the host stage. This suggests

that these parasitoid species could be potentially complementary on multiple or combined

releases of biological control programs.

© 2017 E. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart, Germany www.schweizerbart.de

DOI: 10.1127/entomologia/2017/0500 0171-8177/17/0500 $ 0.00

7

2 M.C Velasco Hernández et al.

Keywords: host preference, biological control, natural enemies, parasitoids, aphids

2.2 Introduction

Biological control is a pest control strategy that has been effective in several cases (Van

Driesche & Belows 1996). Besides, it can be an alternative or complementary strategy to

the traditional use of synthetic pesticides for pest control (Van Driesche et al. 2008).

However, its use involves knowledge on the implication of several factors (e.g. inter-

actions among organisms), which in some cases are not fully known or understood. For

example, if the control of an aphid pest is intended using a parasitoid species, once

released the parasitoid it could attack other aphid species, producing unexpected results

on the efficiency of our pest control strategy. Then, previous knowledge on the host

species suitability or instar preference of natural enemies on different hosts will be

helpful to better predict or understand pest control results (Godfray 1994, Brodeur & Vet

1995, Desneux et al. 2009a, 2009b, 2009c, Desneux et al. 2012).

Aphids are an important pest group throughout the world. Because of the damage

they cause to vegetable, fruit, and grain crops, a few species stand out as particularly

important: Macrosiphum euphorbiae Thomas, Myzus persicae Sulzer, Rhopalosiphum

padi Linnaeus and Rhopalosiphum maidis Fitch (Hemiptera: Aphididae) (Blackman &

Eastop 2007, Gavkare et al. 2014). The main damage they cause is due to extraction of

sap and fungal proliferation due to honeydew, but above all, they are vectors of a number

of viral diseases (Boquel et al. 2014, Xiao et al. 2015). One of the methods for their

control has been the use of natural enemies such as predators and parasitoids. For

example, Aphidius ervi Haliday (Hymenoptera: Braconidae) and Aphelinus abdominalis

Dalman (Hymenoptera: Aphelinidae) are two species of solitary parasitoids increasingly

being used in America and Europe for biological control of aphids (Daza-Bustamante et

al. 2003, Ceballos & Duarte 2012, Legarrea et al. 2014, Shrestha et al. 2015). They are

commercially available and can share some hosts attacking common crops such as

eggplant, tomato, pepper and beans. The wasp A. ervi can be used to control species of

the tribe Macrosiphini such as M. euphorbiae, M. persicae, and Acyrthosiphon pisum

Harris (Takada & Tada 2000, Acheampong et al. 2012, Daza-Bustamante et al. 2003).

For its part, A. abdominalis is a wasp that attack species of aphids including M. persicae,

R. padi, R. maidis, and M. euphorbiae, among others (Duarte et al. 2012, Acheampong et

al. 2012, Ceballos & Duarte 2012, Japoshvili & Abrantes 2006, Botto 1981). The quality

of the host, from the perspective of the parasitoids, can be related to its nutritional

content (Mackauer et al. 1996). At the same time, the nutritional content of the host will

be influenced by its species (Sequeira & Mackauer 1993). Thus, the host species can

determine its quality and whether or not it would be chosen by a parasitoid. In an

ecosystem, it is common for different host species to coexist (Van Alphen & Jervis

1996), and it is predicted that the host of the best quality for parasitoid progeny

development will be chosen for reproduction (Godfray 1994, Heimpel et al. 1996,

Cingolani et al. 2014). The mechanisms used by female parasitoids to locate and

discriminate among hosts are widely described by Isidoro

8

Host suitability and instar preference 3

et al. (2001) and van Baaren et al. (2007). In this process, antennae play a key role for the

recognition of cues associated to hosts, both, volatile and contact semiochemicals

(Romano et al. 2016). Thus, faced with a host of good quality, it is expected that a

parasitoid will choose this host over others and will have high rates of parasitism and

survival to the adult stage, as well as progeny with greater size and fertility, shorter

development time, and a biased female sex-ratio (at least a 1:1 female:male proportion

(i.e. 50% females) is expected) (Mackauer 1996, Van Driesche et al. 2008, Teppa-Yotto

et al. 2013, Ghimire & Phillips 2014).

In order to effectively employ parasitoids in biological control programs, it is

important to know about their host selection behavior and host suitability in different host

species (Rakhshani et al. 2004). Indeed, during their life cycle, one of the important

decisions facing female parasitoids is whether or not to parasitize certain host species and

developmental stages (Barrete et al. 2009). These decisions will determine whether or not

her progeny will survive and develop adequately.

Previous studies have approached this subject of host suitability and selection of

parasitoids in different species and stages of aphids (Rakhshani et al. 2004, Tomanovick

et al. 2009, Sídney et al. 2010, Silva et al. 2011, Monje et al. 1999). Among the principal

biological parameters measuring host suitability are rates of parasitism, survival to the

adult stage, sex-ratio, immature developmental times and offspring size (Sidney et al.

2010, Gavkare et al. 2014, Duarte et al. 2012, Gavkare et al. 2013, Stilmant et al. 2008).

For the specific case of A. ervi and A. abdominalis, some of these parameters have been

previously studied using some aphid hosts (Table 1). However, information taking into

account other biological parameters or host species is lacking (see details in Table 1). For

example, for A. ervi, Sidney et al. (2010) studied developmental parameters using the

host M. euphorbiae. Percentage of parasitism has been assessed on M. persicae (Gavkare

et al. 2014) and R. padi (Stilmant et al. 2008). However, important parameters such as

mean number of parasitized hosts, sex-ratio and offspring size have not still been

assessed for M. persicae and R. padi (Table 1). Information on A. abdominalis is less

available. For example, Duarte et al. (2012) evaluated some biological parameters (e.g.,

percentage of emergence and developmental time) in M. persicae. However, the sex-ratio

and offspring size of A. abdominalis developing in M. persicae was not reported.

Furthermore, all these developmental parameters have not been assessed using R. maidis

or R. padi as hosts (Table 1).

In another hand, it has been observed that some species of parasitoids are able to

oviposit and develop in various developmental stages of a host (Rakhshani et al. 2004,

López et al. 2009, Latham & Mills 2012). Nevertheless, some species of parasitoids show

a preference for parasitism of certain stages of development of the host (Weisser 1994,

Rakhshani et al. 2004, He et al. 2005, Talebi et al. 2006). In the case of A. ervi, its

preference for specific developmental stages has been assessed in species such as M.

persicae, Acyrthosiphon kondoi, and Aulacorthum solani (Hemiptera: Aphidoidea)

(Colinet et al. 2005, He & Wang 2006, He et al. 2011). These studies found that A. ervi

has a preference for intermediate stages of development. As a possible explanation, these

authors proposed defensive behavior and nutritional value of the host. Instar preference of

A. abdominalis has been studied in Nasonovia ribisnigri

9

4 M.C. Velasco-Hernández et al.

and Myzus ascalonicus (Hemiptera: Aphidoidea) (Shrestha et al. 2015, Wahab 1985).

These studies report that for N. ribisnigri, the preference is for first, second, and third

stages, while in M. ascalonicus, the preference is for third and fourth stages, as well as

for adults. The authors relate this preference to the presence of effective defensive

mechanisms in the other stages.

Table 2.1. Detailed information on the contributions of the present study. For

each parasitoid species, we formed 18 cells by crossing 6 biological parameters

studied and 3 hosts species analyzed. For each cell it is indicated: ‘Not reported

previously’ (when we found no previous published literature reporting that

information) or ‘Reported previously’ (when previous literature reports information

for that specific cell). In the last case, we provide also the reference that reports

such information. ‘Reported here’ indicates that we generated data in this study

for that specific cell.

Aphidius ervi

Hosts

M. persicae R. padi M. euphorbiae

Mean of Not reported Not reported Not reported

parasitized aphids previously previously previously

(Reported here) (Reported here) (Reported here)

Percentage of Reported previ- Reported previ- Reported previ-

parasitism ously: Gavkare ously: Stilman ously: Sidney

et al. 2014 et al. 2008 et al. 2010


Percentage of Reported previ- Not reported Reported previ-

emergence ously: Hofsvang & previously ously: Sidney

Hagvar 1975 (Reported here) et al. 2010

(Reported here) (Reported here)

Sex-ratio Not reported Not reported Reported previ-

previously previously ously: Sidney

(Reported here) (Reported here) et al. 2010

(Reported here)

Development time Reported Not reported Reported previ-

previously: previously ously: Sidney

Hofsvang & (Reported here) et al. 2010

Hagvar 1975 (Reported here)

(Reported here)

Offspring size Not reported Not reported Reported previ-

previously previously ously: Sidney

(Reported here) (Reported here) et al. 2010

(Reported here)

10

Host species suitability and instar preference 5

Aphelinus abdominalis

Hosts

M. persicae R. padi R. maidis

Mean of Not reported Not reported Not reported

parasitized aphids previously previously previously


Percentage of Reported Not reported Not reported

parasitism previously: previously previously

Duarte et al. 2012 (Reported here) (Reported here)

(Reported here)

Percentage of Reported Not reported Not reported

emergence previously: previously previously


(Reported here)

Sex-ratio Not reported Not reported Not reported

previously previously previously


Development time Reported Not reported Not reported

previously: previously previously


(Reported here)

Offspring size Not reported Not reported Not reported

previously previously previously


An understanding of the preference of parasitoids for certain developmental stages is

important because it forms a part of the life history of these organisms (Trotta et al.

2014), and can contribute to the understanding of foraging and host-selection strategies

(Henry et al. 2009), as well as their population dynamics (Lin & Ives 2003, Sengonca et

al. 2008). From an applied point of view, this information can be valuable during mass

rearing optimization (He & Wang 2006), and release of these parasitoid species (Shrestha

et al. 2015).

For the above reasons, the aims of the present study were: 1) to assess the suitability

of different aphid species for A. ervi and A. abdominalis and, 2) to determine which host

developmental stage is preferred by the parasitoids A. ervi and A. abdominalis, when

faced with different developmental stages of M. persicae and R. padi, respectively. We

choose these host species for the second bioassay on the basis of the results of the first

bioassay that indicated that these hosts were the best for parasitoid development.

11


2.3 Materials and Methods

2.3.1 Biological material

Plants

To maintain the different species of aphids, the plants used were chili (Capsicum annuum

L. var. Serrano), oat (Avena sativa L. var. Avemex), potato (Solanum tuberosum L. var.

Blanca), and maize (Zea mays L. var. Tuxpeño). The chili plants were grown from

commercial seeds acquired at La Casa del Hortelano SA de CV (Guadalajara, Jalisco,

México). Seeds for the oat plants were acquired at Grupo Industrial Vida SA de CV

(Venta del Astillero, Jalisco, México). Potato plants were grown from tubers acquired

from a local commercial market (Zapopan, Jalisco, México). The seeds for the maize,

were provided by CIMMyT (Centro Internacional de Mejoramiento de Maíz y Trigo,

Estado de México, México).

All of the seeds and tubers were planted in square pots (8.5 cm × 8.5 cm × 9 cm)

containing Nutrigarden® soil: 50% black soil and 50% compost (Sulfatos y Derivados,

S.A. de C.V., México) and perlite (Agrolita de México, S.A. de C.V.) mixed in a 1:1

ratio. The plants were fertilized with “triple 18” fertilizer (SQM Comercial de México

S.A. de C.V.) every two days. All of the plants were used at 1–2 months of age and were

maintained free of herbivores until the experiment. The chili plants were maintained in

an acclimatized room with 24 ± 3 °C, relative humidity (RH) of 50 ± 10% and a

light:dark (L:D) photoperiod of 12:12 hours in the Laboratorio de Control Biológico de

la Universidad de Guadalajara, México (LCB-UdG). The maize plants and oat were

maintained under greenhouse conditions (17 ± 6.5 °C, RH: 68 ± 26.5%) in cages covered

with anti-aphid mesh to preclude the entrance of herbivores. The maize and oat plants

were grown in the greenhouse because, during previous growing attempts, we observed

that development proceeded better under greenhouse conditions than under laboratory

conditions. In contrast, the chili and potato plants developed well for our purposes in the

laboratory.

The plants used to maintain the aphid colonies were: chili plants for M. persicae, oat

plants for R. padi, potato plants for M. euphorbiae, and maize plants for R. maidis.

Insects

All insects were maintained under laboratory conditions, at 24 ± 3 °C, RH of 50 ± 10%

and a photoperiod of 12:12 (L:D) hours.

Aphid colonies were established in the LCB-UdG. The colonies of R. padi, M.

euphorbiae, and R. maidis were initiated with individuals collected in crops adjacent to

the LCB-UdG campus (Latitude: North 20° 44’ 47”, Longitude: West 103° 30’ 43”)

between August and November of 2013. R. padi was collected on oat plants, R. maidis

on maize plants, and M. euphorbiae on tomatillo plants (Physalis philadelphica Lam.).

To collect aphids, we carefully sampled about 20 individuals per aphid species using a

fine paintbrush. These individuals were left on its corresponding host plant placed in an

acrylic cage (45 cm high × 38 cm long × 30 cm wide) covered

12


with anti-aphid mesh. After sampling, cages with plants and aphids were transferred to a

rearing room and provided with new host plants as required. These cages were supervised

daily and newborn aphids were carefully transferred to a different cage containing new

host plants. The newborn aphids were used to increase populations and perform

experiments. The colony of M. persicae was established from individuals provided by

Mary Carmen Torres Quintero (Laboratorio de control Biológico del Centro de

Investigaciones en Biotecnología de la Universidad Autónoma del Estado de Morelos,

México) in February of 2012. All the aphid species were taxonomically verified by the

Aphidoidea specialist MSc. Rebeca Peña Martínez (Escuela Nacional de Ciencias

Biológicas, Instituto Politécnico Nacional IPN, México) (Blackman & Eastop 2000,

Blackman & Eastop 2007).

Parasitoids of both species, A. ervi and A. abdominalis, were provided as mummies by

the company Koppert México SA de CV (Querétaro, México). After their arrival at the

laboratory, they were maintained in acrylic cages (45 cm high × 38 cm long × 30 cm

wide) and fed ad libitum with a honey solution (Carlota, McCormick de México SA de

CV, Morelos, México) diluted to a honey:water ratio of 7:3, v/v, and drinking water in

folded absorbent paper (7 cm × 7 cm) placed in the base of a petri dish (8.5 cm in

diameter) (Bao-Fundora et al. 2016). After adult emergence of these mummies, pairs of

parasitoids were randomly selected and using an insect aspirator were placed together in

petri dishes (8.5 cm in diameter) to increase the probability of mating by the females. The

pairs were fed with a honey solution (7:3 ml honey:water) and drinking water in 1 cm3 of

cotton. For the experiments, 24- to 48-hour old females of both species (A. ervi and A.

abdominalis) were used. From previous studies, it is known that both parasitoids are able

to oviposit 1 day after their emergence (Wahab 1985, He et al. 2004).

2.3.2 Bioassay 1: Host species suitability

All bioassays were performed at 24 ± 3 °C and a RH of 50 ± 10%, an L12:D12

photoperiod and 1600 lux. We followed a randomized block design (with days as the

block factor) in which the different aphid species were tested (no-choice tests) with the

two species of parasitoids. For A. ervi, the aphid species M. persicae, R. padi, and M.

euphorbiae were tested. For A. abdominalis, the aphid species M. persicae, R. padi, and

R. maidis were tested. These aphid species were chosen taking into account that they can

share some host plants and be present simultaneously in the field. For example, R. padi

and R. maidis can be found simultaneously on maize (Zea mays) and grasses (SAGARPA

2013). Myzus persicae and M. euphorbiae can be simultaneously present on plants of the

Solanacea family (Srinivasan & Alvarez 2011, Davis et al. 2006).

We tested host species suitability based on the methodology described by Desneux et

al. (2009b). In this methodology, each host plant corresponding to a specific species of

aphid was covered with a cylinder of transparent plastic (11 cm in diameter, 21 cm high).

Each cylinder had 12 holes (3.5 cm in diameter) and an additional hole at the upper end,

these were all covered with anti-aphid mesh for ventilation. Fifty aphids

13


of the corresponding species were placed on each host plant (i.e. M. persicae on chili, R.

padi on oat, R. maidis on maize, and M. euphorbiae on potato). These 50 aphids were

taken at random from the general colony of each species and included individuals from

different developmental stages following Desneux et al. (2009b) and with the aim of

simulating what can be found in the field (i.e. several aphid instars present

concomitantly). After this time, a female parasitoid was introduced into the cylinder. For

this, we used an insect aspirator to take the parasitoid from the petri dish and release it

over the leaves of the plant. Afterwards, the plant was immediately covered with the

plastic cylinder. Once released, the parasitoid was allowed to forage for 24 hours. After

this period of time, the female parasitoid was withdrawn and the plants were checked

daily for the presence of mummies (Capinera 2008). When mummies were detected, they

were separated into individual gelatin capsules (Capsugel qualityMR, Envases Velasco,

Guadalajara, Jalisco, México) and checked every 24 hours in order to register the day of

emergence and the sex of the emerging wasps.

For this bioassay, the response variables were the mean number of aphids parasitized

(i.e., number of mummies formed), the sex of the emergent wasps, and the development

time in days (time after oviposition until emergence of the parasitoid adult), for each

wasp. Using these variables, three values were calculated: the percentage of parasitism

(number of parasitized aphids*100/total exposed aphids), the percentage of emergence

(number of emerged wasps*100/number of parasitized aphids), and sex ratio (number of

female emerged wasps/total number of emerged wasps). In addition, the posterior right

tibia of emerged wasps was measured as an estimation of size. The tibias were measured

with a stereoscope (Stemi 2000-C Carl Zeiss) connected to an AxioCam ICc1 camera,

using AxioVision 4.8 software (Carl Zeiss, Guadalajara, Jalisco, México). Fifteen

replicates were performed for A. ervi (in total 45 A. ervi wasps were tested) and 20

replicates for A. abdominalis (in total 60 A. abdominalis wasps were tested).

For A. ervi, the response variables (mean number of parasitized aphids, percentage of

parasitism, percentage of emergence, sex ratio, and development time) were compared

among aphid species using linear mixed models (LMM) with the host species as the fixed

factor and the block (days) as the random factor (Crawley 2007). Size was analyzed

using a linear model with the host species as the main factor and the block integrated into

the model (Crawley 2007), but after sqrt (x + 0.5) transformation of data (Zar 1998) to fit

normality and homoscedasticity requirements.

For A. abdominalis, the four response variables, mean number of parasitized aphids,

percentage of parasitism, percentage of emergence, and development time, were

compared among host species using an LMM with the host species as the fixed factor

and the block as the random factor. The sex ratio was not compared because only females

emerged in the three species of aphids evaluated. The size of the progeny was compared

among aphid host species in the same way as for A. ervi. All analyses were performed

using R, version 3.3.1 R Core Team (2013).

14


2.3.3 Bioassay 2: Instar preference

For this bioassay, we also followed a randomized block design, with time (days) as the

blocking factor. Ten individuals from each nymphal developmental stage, first to fourth

instars (Perdikis et al. 2004) as well as wingless adults (Tahriri et al. 2007), from each

aphid species, were selected. With these 50 aphids in total, we sought to provide the

wasps with a sufficient number of aphids to parasitize (see Results, Fig. 1). For this

bioassay, for A. ervi we used M. persicae as host species and for A. abdominalis we used

R. padi, since these were the species with the highest rates of parasitism in the first

bioassay (see Results, Figs. 1 and 2). The aphids were placed on a leaf of their

corresponding host plant that was inserted into a piece (1 cm2) of floral foam (OASIS ®,

Smithers-Oasis, Zapopan, Jalisco, México) to maintain hydration, afterwards they were

transferred to a petri dish (8.5 cm in diameter). The leaves were changed every three days

to ensure their turgidity. Damp filter paper (ISOLAB average porosity, 8.3 cm in

diameter, Prolab, Guadalajara, Jalisco, México) was placed in the base of each petri dish

and to provide food to wasps, a drop of undiluted honey was placed on the inside of the

lid. One leaf of the host plant for each species of aphid was placed on the damp filter

paper, and different stages of aphids were randomly placed on the leaf and allowed to

establish themselves for one hour. After this period, one female parasitoid was taken

using an insect aspirator and then released into each petri dish and allowed to forage there

for 24 hours. At the end of this time period, the parasitoid was withdrawn and the aphids

were separated according to their developmental stage. The different stages of aphids

were placed on a leaf of their corresponding host plant in a petri dish (described earlier)

in order to allow development of the aphids and parasitoids to the mummy phase

(Capinera 2008). The leaf was changed every three days in order to maintain nourishment

for the aphids. After the day of exposure to the parasitoid, the aphids were observed every

24 hours and the number of mummies produced for each stage was recorded. For each

parasitoid species, this procedure was replicated fifteen times (i.e. the total number of

tested individuals was: A. ervi, 750 M. persicae, 15 A. abdominalis and 750 R. padi). For

A. ervi, the mean number of mummies was compared among stages using a linear model

with the host instar as the main factor and the block integrated into the model (Crawley

2007), after sqrt (x + 0.5) transformation of data (Zar 1998) to fit normality and

homoscedasticity requirements.

For A. abdominalis, the mean number of mummies produced was compared among

the different stages using a linear mixed model (LMM) with the stage as the fixed factor

and the block as the random factor.

15


2.4 Results

2.4.1 Bioassay 1: Host species

For A. ervi, we found significant differences in the mean number of parasitized aphids

(F2,28 = 75.554, P < 0.0001) (Fig. 1A), percentage of parasitism (F2,28 = 61.987, P <

0.001) (Fig. 1B), percentage of emergence (F2,28 = 26.256, P < 0.001) (Fig. 1C),

Figure 2.1. Variables analyzed for the species A. ervi. A) Mean (± SE) number of

parasitized aphids, B) Mean (± SE) percentage of parasitism, C) Mean (± SE)

percentage of emergence, D) Mean (± SE) proportion of females, E) Mean (± SE)

development time (in days), F) Mean (± SE) size of the progeny tibia (in millimeters).

Different letters indicate significant differences among the host species (P < 0.05).

16


Figure 2.2. Variables analyzed for the species A. abdominalis. A) Mean (± SE)

number of parasitized aphids, B) Mean (± SE) percentage of parasitism, C) Mean (±

SE) percentage of emergence, D) Mean (± SE) proportion of females, E) Mean (±

SE) development time (in days), F) Mean (± SE) size of the progeny tibia (in

millimeters). Different letters indicate significant differences between the host species

(p < 0.05).

sex ratio (F2,28 = 9.822, P < 0.001) (Fig. 1D), and development time (F2,108 = 13.098, P <

0.001) (Fig. 1E). However, no significant differences were found in the size of the

emerged wasps from different hosts (F2,108 = 2.665, P = 0.074) (Fig. 1F). In terms of mean

number of parasitized aphids, percentage of parasitism, percentage of emergence, and

female:male sex ratio, the parasitoid A. ervi had significantly higher levels in M. persicae

than in R. padi and M. euphorbiae (Figs. 1A, B, C and D). Furthermore, development

time of A. ervi was significantly longer in M. persicae than in the other

17


Figure 2.3. Instar preference bioassay. A) Mean (± SE) number of mummies

produced by A. ervi in each nymphal stage of the host aphid species M. persicae, B)

Mean (± SE) number of mummies produced by A. abdominalis in each nymphal stage

of the host aphid species R. padi. Different letters indicate significant differences

between the host species (p < 0.05).

two aphid species (Fig. 1E). In contrast, the size of the A. ervi offspring emerging from

the three aphid host species was not significantly different (Fig. 1F).

For the parasitoid A. abdominalis, significant differences were found for the mean

number of parasitized aphids (F2,38 = 16.550, P < 0.0001) (Fig. 2A), percentages of

parasitism (F2,38 = 18.765, P < 0.0001) (Fig. 2B), percentages of emergence (F2,38 =

6.439, P = 0.003) (Fig. 2C), development time (F2,204 = 10.610, P < 0.001) (Fig. 2E), and

size of the progeny (F2,202 = 121.270, P < 0.001) (Fig. 2F). The highest percentage of

parasitism was obtained in R. padi, and the lowest was obtained in R. maidis (Fig. 2B).

We did not find significant differences in sex ratio because in all three aphid species,

only female wasps were obtained (Fig. 2D). Regarding the percentage of emergence,

more A. abdominalis individuals were obtained from M. persicae and R. padi compared

with R. maidis (Fig. 2C). Development time for A. abdominalis was shorter in R. padi

than in M. persicae and R. maidis (Fig. 2E). When the size of the offspring was analyzed,

A. abdominalis was found to produce significantly larger daughters in R. padi compared

with those obtained from M. persicae and R. maidis (Fig. 3D).

2.4.2 Bioassay 2: Instar preference

Each species of parasitoid showed a preference for parasitism of some host instars over

others (A. ervi: F4,56 = 5.813, P < 0.001, A. abdominalis: F4,56 = 15.598, P < 0.001). A.

ervi produced a significant larger number of mummies in the fourth nymphal stage and in

adult aphids (Fig. 3A). In the third instar, the number of produced mummies was

marginally larger relative to those produced in the second instar (Fig. 3A). This indicates

that A. ervi can produce more offspring in more advanced stages of develop-ment in the

aphid M. persicae.

In contrast to A. ervi, the parasitoid A. abdominalis produced more mummies in early

stage nymphs (F4,56 = 15.598, P < 0.001) than in more advanced developmental stages

(Fig. 3B). In particular, the number of mummies produced in first

18


instar nymphs was significantly higher compared with other stages of development

(Fig. 3B). Mummy production followed in descending order from the second to the

third nymphal stage, with the fourth nymphal stage and the adults producing the

smallest number of mummies (Fig. 3B).

2.5 Discussion

2.5.1 Host species suitability

Parasitoids can attack, reproduce, and develop in different host species. However,

they must choose the most optimal species for their development (Ghimire &

Phillips 2014). Because of this, it is expected that female parasitoids would prefer to

select hosts with high nutritional quality to ensure development of their offspring

(Wang & Liu 2002). In the present study, the suitability of three aphid host species

for two species of widely employed aphid parasitoids was tested. In addition, it was

assessed whether some developmental stages of some host species were preferred

over others. We found that each species of parasitoid developed better in different

host species, as well as in distinct developmental stages. That is to say, they can

exploit distinct niches, which could prove useful in aphid control programs.

Studies on host preference and quality have been conducted for the parasitoid A.

ervi (Sidney et al. 2010, Bueno et al. 1993). Our work brings new information on the

development of this parasitoid species in different host species and the way that the

host species can affect some developmental traits of the parasitoid. Previous studies

have reported parasitism rates for A. ervi as 74% in M. euphorbiae (Sidney et al.

2010), 16.9% or 4.1% in M. persicae (Kavallieratos et al. 2004, Gavkare et al. 2014),

14.3% or no parasitism in R. padi (Rakhshani et al. 2008, Stilmant et al. 2008,

Tomanović et al. 2008). These results differ from our findings in the present study,

as we found parasitism rates of 2%, 19.8%, and 4% in M. euphorbiae, M. persicae,

and R. padi, respectively. For other species of aphids such as A. pisum and A. kondoi,

reported percentages of parasitism are of 34% and 32%, respectively (Bueno et al.

1993). Considering a related measurement, we found that the greatest percentage of

emergence of A. ervi (70%) was from the host aphid M. persicae, a higher rate than

the 50% reported by Hofsvang & Hagvar (1975). The percentage of emergence we

found in the potato aphid, M. euphorbiae, was 25%, in contrast to the 92% found by

Sidney et al. (2010).

Regarding sex ratio of A. ervi progeny emerged from M. euphorbiae, we found a

lower percentage of females (20%), compared with that reported by Sidney et al.

(2010) (68%). Further, despite the assertion in the literature that sex ratio will be

biased toward females in large hosts (Spitzen & van Huis 2005), we found a lower

quantity of females in the largest host (M. euphorbiae: 1.7–3.6 mm > M. persicae:

1.2 a 2.5 mm and R. padi: 1.2–2.4 mm (Enciclop’Aphid 2016)).

The mean development time of A. ervi in M. persicae in our study was 12.6 days

on average, in contrast to the 19.9 days reported by Hofsvang & Hagvar (1975).

19


In our analysis of the size of the emerged wasps of A. ervi, our results coincided with

those of Sidney et al. (2010) in that M. euphorbiae produced wasps of the greatest size,

although the size we observed (0.55 mm) was lower than that reported by them (0.73

mm). For the most part, parasitoids evaluate the size of the host as an indication of

quality when choosing hosts of greater size (Sequeira & Mackauer 1992). Nevertheless,

our results contradict this trend since, although M. euphorbiae is the largest host (1.7–3.6

mm), it produces fewer parasitoids, and, within these, few females, whereas M. persicae

produces more parasitoids and, within these, the most females. In other words, if the

production of adults and especially females is important for the fitness of the parasitoid,

M. persicae represents a good quality host for A. ervi, in fact, the best among the studied

species. Likewise, M. persicae has been reported as the most optimal host for the

development of other species of parasitoids, such as Aphidius colemani Viereck,

Diaeretiella rapae Stary, Praon volucre (Haliday) (Hymenoptera: Braconidae)

(Kavallieratos et al. 2004, Silva et al. 2011).

A preference for parasitizing some species over others has also been found in the

genus Aphelinus (Kang et al. 2012). For example, Prinsloo (2000) found that Aphelinus

sp. nr. varipes (Hymenoptera: Aphelinidae) oviposited in Diuraphis noxia Kurdjumov,

Schizaphis graminum Rondani, Sitobion avenae Fabricius, R. padi, and Metopolophium

dirhodum Walker, but development was not achieved in R. padi and S. avenae.

Moreover, a greater number of eggs were laid in D. noxia. Their results suggest that

some hosts are more adequate for the parasitoid.

The percentage of parasitism of A. abdominalis in the hosts evaluated in the present

study differed from the results found in other studies (Duarte et al. 2012), supporting the

hypothesis of specialization of populations of parasitoids in accordance with their

origins.

In this way, differences among populations could explain the variations of host

acceptance and parasitism rates. For example, differences among mechanoreceptor

sensilla (i.e. number and distribution) of female antennae could perhaps result on

different rates of host location and acceptance (Isidoro et al. 2001, van Baaren, et al.

2007, Romano et al. 2016). In general, the percentage of emergence of A. abdominalis in

M. persicae was high (70%), coinciding with that reported by Duarte et al. (2012) on the

same species.

It should be noted that in this study, A. abdominalis only produced female progeny in

all three host species, in contrast to the findings of Honek et al. (1998), who observed the

emergence of both sexes. If the models of sex ratio presuming that female parasitoids

utilize hosts of the best quality (Godfray 1994) are correct, our results would indicate that

the hosts utilized were perceived by the females as being of high quality. Nonetheless, it

is important to consider that A. abdominalis has been reported as a species that exhibits

deuterotoky, and that for the most part, its progeny are females (Wahab 1985).

In general, it was observed that the host conferring substantial adaptive advantages on

A. abdominalis was R. padi, indicating that it can be considered to be of higher quality

than the other two species evaluated.

In general, we found that the results obtained in this study, together with those of

the cited previous studies, differed in developmental and experimental conditions,

and

20


therefore, differences in results are not surprising. However, some of these differences

may be due to the parasitoids belonging to different biotypes and/or populations. With

respect to this, Takada & Tada (2000) reported that they found differences in variables

such as percentage of parasitism when comparing different “lines” (strains) of A. ervi.

Likewise, it has been previously reported that A. ervi is a species with differences

between populations, due principally to geographic isolation and their adaptation to

specific hosts (Atanassova et al. 1998, Kavallieratos et al. 2004).

2.5.2 Instar preference

Our work confirms that both species of parasitoids are capable of parasitizing all of the

stages of their aphid hosts, but have a preference for some of these stages over others

(Lopez et al. 2009, Latham & Mills 2012). In general, it can be said that this study

confirms previous findings of instar preference in these parasitoids, which is an important

point for their optimization for practical use.

We found that A. ervi more frequently parasitized the last two nymphal stages and

adults of M. persicae, a similar finding to that reported by Colinet et al. (2005), who

observed a majority of parasitism in intermediate stages (corresponding to instars 3 and

4) of M. persicae. Likewise, this behavior of choosing intermediate stages has been

observed in other hosts, such as Acyrthosiphon kondoi Shinji (He & Wang 2006, He et al.

2011). The preference of A. ervi for more advanced instars in M. persicae is related to

improved quality. This has also been observed by He et al. (2011), who reported that A.

ervi prefers to parasitize more advanced instars of Acyrthosiphon pisum (Harris). This is

similar to the behavior of the parasitoid Lysiphlebus testaceipes Cresson (Hymenoptera:

Braconidae), which prefers larger stages of Aphis gossypii Glover (Hopkinson et al.

2013). In addition, the females of A. ervi have the advantage of an absence of defensive,

aggressive behavior in more advanced instars, in contrast to what has been observed in

other species of aphids like Aphis fabae Scopoli, Aphis glycines Matsumura, and A. pisum

(Ameri et al. 2014, Wyckhuys et al. 2008).

Similarly to findings in other species of parasitoids, such as Aphelinus spiraecolae

Evans & Schauff and Monoctonus paulensis (Ashmead) (Tang & Yokomi 1996, Chau &

Mackauer 2000), A. abdominalis prefers to oviposit in the smaller developmental stages

(instars 1 and 2, principally). Although in general, hosts of larger size (in this case, more

advanced instars) are preferred by female parasitoids, the preference for small instars

could be related to the capacity of larger aphids to escape or damage the parasitoid as a

part of a defensive strategy while the parasitoids are ovipositing (Wyckhuys et al. 2008).

Along these lines, it has been reported that in various species of aphids, defensive

behavior is more pronounced in larger stages in comparison with smaller ones (Kouamé

& Mackauer 1991). In addition, and in concordance with our results, Shrestha et al.

(2015) observed that A. abdominalis displays greater parasitism in the first stages (instars

1, 2, and 3) in Nasonovia ribisnigri Mosley (Hemiptera: Aphididae), in comparison with

fourth instar nymphs and adults. Although it was found that A. abdominalis is capable of

developing in all stages of N. ribisnigri, significantly more successful parasitism was

reported in the second instar than in the fourth. In addition, it has been

21


reported that in other species of Aphelinus, the preference for parasitizing the first stages

is frequently seen (Gerling et al. 1990, Sengonca et al. 2008, Rohne 2002). In this sense,

an advantage of parasitizing younger instars stems from the fact that the immune system

of the host is less developed and tends to be more susceptible to wasp venom, which is

convenient for endoparasitoids like A. abdominalis (Stoepler et al. 2013, Brodeur & Vet

1995, Chau & Mackauer 2000). In our observations, we could see that in the first instar,

the parasitoid approaches the host, becoming sufficiently close for insertion of the

ovipositor while remaining out of range of the defensive movements of the aphid.

Nevertheless, in the case of the third and fourth instars, the parasitoid is forced to get as

close as possible while still staying out of reach of the defense systems of the aphids, this

has also been seen in other species (Gerling et al. 1990). Thus, there may exist certain

tendencies of small parasitoids like A. abdominalis, which can more easily parasitize

early stages because the defenses of the aphids do not affect the parasitoid. On the other

hand, Wahab (1985) reported that A. abdominalis oviposited more in the third stage in

Myzus ascalonicus van der Goot (Hemiptera: Aphididae) in comparison with the other

stages (first, second, fourth, and adult). However, they did not account for the quantity of

mummies produced, that is to say, of effective ovipositions. In addition, the parasitoids

stayed for three days in the presence of their host, so that possibly, and in the absence of

more individuals of other instars, the females may have opted to oviposit in the next most

numerous host stages.

2.6 Conclusion

In general, we can conclude that A. ervi developed better on the host species M. persicae,

over the hosts R. padi and M. euphorbidae. As for A. abdominalis, we found that R. padi

is a better host for its development than the hosts M. persicae and R. maidis. Regarding

instar preference, we observed that A. ervi prefers to parasitize larger/older instars over

smaller/younger ones. In contrast, A. abdominalis prefers to parasitize smaller/younger

stages. Considering the potential of these parasitoids in biological control programs, we

estimate that they could be complementary to each other, since with their differences in

preferred host species and instar, they will not be competing for the same targets.

Acknowledgments: We acknowledge CONACyT (Grant Number 157259) and the UC-

Mexus-Conacyt Program (Grant Number CN12608) which provided grant support to RRR,

and support from the FP7-PEOPLE-2013-IRSES fund (project APHIWEB, grant no. 611810)

to ND and MCVH. This work formed a part of the requirements for the degree of Doctor of

MCVH (CONACyT ID: 367437), supervised by RRR. We thank Koppert-México SA de CV

for the provision of the wasps.

22


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CAPÍTULO 3. ESTADO FISIOLÓGICO DE PARASITOIDES AFECTANDO EL PROCESO DE SELECCIÓN DE HOSPEDEROS

Artículo en preparación (70% avance) para Oecologia cuya referencia bibliográfica es:

Velasco-Hernandez MC, Zañudo-Hernandez J, Desneux N, Ramírez-Romero R.

Physiological status of parasitoids affecting host selection process. En preparación

28

Parasitoid physiological state affecting host selection process

Velasco-Hernández, M.C.1, Zañudo-Hernández J.1, Desneux, N2 and Ramírez-Romero R.1

1Departamento de Producción Agrícola, CUCBA, Universidad de Guadalajara, Zapopan,

Jalisco, México.

2French National Institute for Agricultural Research (INRA), UMR1355, 400 Route des

Chappes, 06903, Sophia-Antipolis, France.

3.1 Resumen

La selección de hospederos por parasitoides es dinámica afectada por el estado fisiológico

de los mismos al momento del encuentro con sus hospederos. Este estudio investigó las

relaciones entre el estado fisiológico de dos parasitoides (Aphidius ervi Haliday and

Aphelinus abdominalis Dalman) con diferentes historias de vida y su proceso de selección.

Probamos los tratamientos: 1) hembras parasitoides jóvenes-alimentados, 2) hembras

parasitoides jóvenes-en ayuno, 3) hembras parasitoides viejos-alimentados y 4) hembras

parasitoides viejos-en ayuno. Una hembra parasitoide de cada tratamiento fue expuesta a su

hospedero de baja y alta calidad de manera simultánea. Se observó el comportamiento de

selección del parasitoide y se registraron como variables de respuesta la selección del

hospedero, el tiempo de detección, el resultado de la selección (ataque o no) y el tiempo de

aceptación. Después de las observaciones los áfidos utilizados en las observaciones fueron

disecados para contar el número de huevos ovipositados por el parasitoide. La mitad de los

parasitoides utilizados en las observaciones se disecaron para conocer su carga ovárica y la

otra mitad para conocer su contenido de fructosa, glicógeno y sacarosa. Los resultados

muestran que el proceso de selección de hospedero no se ve afectado por el estado

fisiológico del parasitoide y que los contenidos de carbohidratos no difieren en cantidad en

los tratamientos al escoger hospederos de alta o baja calidad. El contenido de carbohidratos

debe ser punto clave en las modificaciones del comportamiento de selección y debe tomarse

en cuenta en cuestiones de control biológico de áfidos.

Palabras clave: estado fisiológico, fructosa, glicógeno, sacarosa, selección de hospedero,

parasitoide limitado en tiempo, parasitoide limitado en carga ovárica.

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3.2 Introducción

La selección de hospederos de parasitoides ha sido ampliamente estudiada no solo por su

importancia aplicada en control biológico sino por su importancia ecológica y evolutiva

(Vinson 1976, Charnov & Stephens 1988, Hagvar & Hofsvang 1991, Rehman & Powell

2010). Tanto la selección de hospedero como la subsecuente oviposición por parte de los

parasitoides son decisiones en donde un hospedero encontrado puede ser aceptado o

rechazado (Chau & Mackauer 2001). Se dice que la aceptación de los hospederos va a

variar dependiendo del estado fisiológico del parasitoide, la idoneidad, la calidad y la

disponibilidad de los tipos de hospederos presentes (Charnov & Skinner 1984, Mackauer et

al. 1996, Chau & Mackauer 2001), así como de factores ambientales (Powell & Wright 1992,

Heimpel et al. 1996, Rehman & Powell 2010). Sin embargo, se ha establecido que el proceso

de selección se ve afectado por el tipo de historia de vida que presenta la especie

parasitoide, estas son principalmente dos, especies de parasitoides limitados en tiempo (pro-

ovigénicos con longevidad corta) y especies de parasitoides limitados en carga ovárica (sin-

ovigenicos con longevidad larga) (Mangel 1989, Godfray 1994, Van Baalen 2000). Se han

establecido paradigmas en cuanto al comportamiento de selección de parasitoides

dependiendo de sus historias de vida, los cuales mencionan que 1) parasitoides limitados en

tiempo tienden a aceptar a hospederos de baja calidad cuando los de alta no están

presentes, es decir no son selectivos (Heimpel et al. 1998; Papaj 2000). Mangel (1989)

concuerda al asegurar que es óptimo aceptar hospederos inferiores cuando el complemento

de huevos es alto, desde que los hospederos inferiores proveen al menos algo de

adecuación para el parasitoide. Y 2) parasitoides limitados en carga ovárica serán más

selectivos aceptando solo aquellos hospederos de alta calidad, pues tienen tiempo suficiente

para seguir madurando más huevos (Heimpel & Rosenheim 1998).

El comportamiento de parasitoides hacia sus presas es modulado por un gran número de

factores (Wajnberg et al. 2016). Y las características fisiológicas principalmente estudiadas

han sido la edad (Hopper et al. 2013), alimentación (Lewis et al. 1998, Collier 1995), carga

ovárica (Deas & Hunter 2014, Dieckhoff et al. 2014), estado de apareamiento, (Zhang et al.

2015, Fletcher et al. 1994), experiencia de oviposición (Nurindah et al. 1999). Siendo la edad

y la alimentación los factores más importantes afectando el proceso de selección (Jenner et

al. 2012 y 2014). Pero otros autores han mostrado que ciertos contenidos nutricionales en

parasitoides podrían ser un indicador de la fecundidad y por ende de la selección de sus

especies hospederas (Heimpel & Rosenheim 1995). Aquí, la hembra parasitoide tendrá que

evaluar si los hospederos son adecuados para el desarrollo de su progenie, así como la

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cantidad de tiempo y energía necesaria invertida para la oviposición (Chow & Mackauer

1991). Dado que la calidad de un hospedero está determinada por el tipo y la cantidad de

nutrientes disponibles para un parasitoide (Sequeira & Mackauer 1992). Cada especie de

enemigo natural será capaz de atacar un rango de hospederos que va desde aquellos que

permitirán el desarrollo óptimo de su progenie (de alta calidad), hasta aquellos que solo

permitirán un desarrollo marginal (de baja calidad) (Strand & Obrycki 1996).

La nutrición es el principal factor limitando la reproducción (Wheeler 1996), debido a que

ciertos recursos como azúcares son utilizados en el proceso de producción de huevos (Luo

et al. 2010). Entonces la información acerca de la dinámica energética en las decisiones del

parasitoide puede tener influencia o no en las tasas de oviposición de la hembra parasitoide

(Casas et al. 2003, Lee & Heimpel 2007). Es sabido que los insectos almacenan sus

reservas de energía como carbohidratos y/o grasas, y los parasitoides dependen de estos

como el recurso energético (Akman et al. 2010). Además se ha encontrado que los

carbohidratos son un indicador confiable del estado nutricional del parasitoide (Steppuhn y

Wackers 2004). Y se sabe que parasitoides adultos son especialmente sensitivos a la falta

de estos (Olson et al. 2000); por lo que el análisis de carbohidratos es un punto clave para

conocer su contenido nutricional y probablemente un indicador de su respuesta a la

selección de hospederos. Poco se sabe de cómo estos indicadores del estado nutricional,

entendidos como niveles de carbohidratos, pueden afectar las decisiones de selección de

hospederos de los parasitoides y semejantes atributos podrían cambiar los paradigmas antes

descritos.

Aphidius ervi Haliday (Hymenoptera: Braconidae) y Aphelinus abdominalis Dalman

(Hymenoptera: Aphelinidae) son dos especies generalistas de parasitoides de áfidos

(Acheampong et al. 2012, Duarte et al. 2012). A. ervi es considerado una especie limitada en

tiempo, mientras que A. abdominalis es considerado una especie limitada en carga ovárica

(Couty & Poppy 2001, Molck et al. 2000). Nuestro estudio analiza la selección del hospedero

dependiente del estado fisiológico de estos parasitoides. Analizando no solo el

comportamiento de selección desde un punto de vista comportamental, sino desde el punto

de vista de la nutrición de los parasitoides, refiriéndonos al contenido de carbohidratos

(fructosa, sacarosa y glicógeno) como condición importante en la selección de sus

hospederos en un rango altamente variable; desde los hospederos idóneos para la

reproducción del parasitoide hasta los hospederos que le permiten una reproducción

marginal. Por lo tanto, nuestra hipótesis es que el estado fisiológico definido por la edad

(jóvenes y viejos) y el estado nutricional (alimentados y en ayuno) de los parasitoides con

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dos historias de vida distintas (limitados en tiempo y limitados en carga ovárica), va a

influenciar las decisiones de selección de sus hospederos con variación simultánea de la

calidad del hospedero (baja y alta). Y el objetivo de este estudio fue determinar si el estado

fisiológico (definido por el estado nutricional y la edad) de los parasitoides A. ervi (limitado en

tiempo) y A. abdominalis (limitado en carga ovárica) influencia el comportamiento de

selección de los parasitoides frente a hospederos de alta y baja calidad en un ensayo con

opción en condiciones de laboratorio.

3.3 Materiales y Métodos

3.3.1 Material Biológico

3.3.1.1 Plantas

Se utilizaron semillas comerciales de plantas de chile (Capsicum annum L.) variedad

Serrano, plantas de avena (Avena sativa L.) variedad Avemex y plantas de maíz (Zea mays

L.) variedad Tuxpeño. Las plantas de chile se obtuvieron de semillas comerciales adquiridas

en la Casa del Hortelano SA de CV (Guadalajara, Jalisco, México). Las semillas de avena

fueron adquiridas al grupo industrial Vida SA de CV (Venta del Astillero, Jalisco, México). Las

semillas de maíz fueron donadas por el Centro Internacional de Mejoramiento de Maíz y

Trigo (CIMMyT, Estado de México, México). Todas las semillas fueron cultivadas en macetas

cuadradas (8.5 cm × 8.5 cm × 9cm) conteniendo tierra Nutrigarden ®, 50% tierra de hoja

(Sulfatos y Derivados, SA de CV, México) y 50% perlita (Agrolita de México, SA de CV) en

una proporción 1:1). Las plantas se fertilizaron con fertilizante “triple 18” (SQM Comercial de

México SA de CV) cada dos días. Todas las plantas se utilizaron cuando tenían de uno a dos

meses de edad y se mantuvieron libres de herbívoros hasta su uso en el experimento. Las

plantas de chile serrano se mantuvieron en la sala de cría del Laboratorio de Control

Biológico de la Universidad de Guadalajara, campus CUCBA. Estas plantas fueron

mantenidas para su desarrollo en condiciones controladas con temperatura de 24±3°C,

humedad relativa de 50±10% y fotoperiodo de 12:12 horas (Luz:Oscuridad). Las plantas de

maíz y avena se mantuvieron dentro de jaulas cubiertas con malla tipo organdí para evitar la

entrada de herbívoros y bajo condiciones de invernadero con una temperatura de 17°C

(±6.5), y una humedad relativa de 68%(±26.5), (Estación Meteorológica de la Universidad de

Guadalajara del Centro Universitario de Ciencias Biológicas y Agropecuarias-CUCBA-). Las

plantas de maíz y avena se desarrollaron en invernadero porque en intentos previos, se

observó que su desarrollo era mejor que en condiciones de laboratorio. Por el contrario, las

plantas de chile se desarrollaron adecuadamente en laboratorio. Las plantas de chile fueron

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utilizadas para mantener la colonia de pulgón Myzus persicae, las de avena para el

mantenimiento de la colonia de Rhopalosiphum padi y las de maíz para la colonia de

Rhopalosiphum maidis (todos Hemíptera: Aphidoidea).

3.3.1.2 Áfidos

Las colonias de áfidos fueron establecidas en el laboratorio de Control Biológico de la

Universidad de Guadalajara, campus CUCBA. Las especies R. padi y R. maidis se iniciaron

con individuos colectados en cultivos aledaños al CUCBA (LN 20°44’47”, LO 103°30’43”)

durante los meses de agosto a noviembre del 2013. Rhopalosiphum padi fue colectada sobre

plantas de avena y R. maidis fue encontrada sobre plántulas de maíz. La especie M.

persicae fue establecida con una donación de la Biol. Mary Carmen Torres Quintero del

Laboratorio de Control Biológico del Centro de Investigaciones en Biotecnología de la

Universidad Autónoma del Estado de Morelos, en febrero del 2012. Todas las especies

fueron confirmadas por la especialista en taxonomía Hemíptera: Aphidoidea, M. en C.

Rebeca Peña Martínez (Escuela Nacional de Ciencia Biológicas, Instituto Politécnico

Nacional IPN, México) y con la ayuda de las claves de identificación de Blackman y Eastop

(2007). Todas las colonias y bioensayos se desarrollaron bajo condiciones de laboratorio con

temperatura de 24±3°C, humedad relativa de 50±10% y fotoperiodo de 12:12 horas

(Luz:Oscuridad).

3.3.1.3 Parasitoides

Los parasitoides de ambas especies A. ervi y A. abdominalis fueron provistos como momias

por la compañía Koppert México SA de CV (Querétaro, México). Las especies de

parasitoides fueron confirmadas con la ayuda de las claves de identificación de Powell

(1982), Zumoffen et al. (2015) y la enciclopedia de áfidos (Encyclop’Aphid 2014). Desde su

llegada al laboratorio fueron mantenidos en jaulas de acrílico (45 cm de alto × 38 cm de largo

× 30 cm de ancho) y alimentados ad libitum con una solución de miel (Carlota, McCormick de

México SA de CV, Morelos, México) diluida en agua (proporción 7:3 ml, solución de

miel:agua) y agua potable en papel absorbente doblado (7 cm × 7 cm) sobre la base de una

caja petri (8.5 cm) (Bao-Fundora et al. 2016). Desde su emergencia se tomaron parejas de

parasitoides (para incrementar la probabilidad de apareamiento de las hembras) que

emergieron por la mañana (de 9:00 a 12:00 del día) y se colocaron en una caja petri (8.5 cm

de diámetro) provista con miel diluida en agua (proporción 7:3 ml, solución de miel:agua)

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sobre un pedazo de papel absorbente doblado (1cm2) y agua potable en algodón (1cm de

diámetro, Protec MR, Walmart, Jalisco, México). Para los experimentos se utilizaron hembras

de 24 a 48 horas de edad para ambas especies de parasitoide (A. ervi y A. abdominalis). A

partir de estudios previos, se sabe que ambos parasitoides pueden ovipositar desde el

primer día de emergidos (Wahab 1985, He et al. 2004).

3.3.2 Bioensayo de Laboratorio

Basándonos en la metodología de Desneux et al. (2009) y en los resultados de Velasco

Hernández et al. (2017) para A. ervi se tomó como hospedero de alta calidad a M. persicae y

como hospedero de baja calidad a R. padi, mientras que para A. abdominalis se utilizó como

hospedero de alta calidad a R. padi y como hospedero de baja calidad a R. maidis.

3.3.2.1 Observaciones de comportamiento de selección

Se realizaron las observaciones en bloques completamente al azar de la selección de

hospedero de los siguientes tratamientos. Se probaron cuatro tratamientos para cada

especie parasitoide. 1) Una hembra parasitoide alimentada-joven, 2) una hembra parasitoide

alimentada-vieja, 3) una hembra parasitoide en ayuno-joven y 4) una hembra parasitoide en

ayuno-vieja. Cada uno de los parasitoides con su respectivo tratamiento fueron expuestos a

un hospedero de alta y baja calidad de manera simultánea.

Para la especie A. ervi y A. abdominalis las hembras alimentadas-jóvenes y en ayuno-

jóvenes fueron parasitoides de 24 horas de emergencia, para las hembras alimentadas-

viejas en la especie A. ervi fueron de 9 a 10 días mientras que para A. abdominalis fueron de

34-36 días. Las hembras en ayuno-viejas para A. ervi fueron de 38 horas y para A.

abdominalis fueron de dos días de edad, esto porque al día siguiente las hembras

parasitoides morían. Para hembras alimentadas se utilizaron a hembras recién emergidas

puestas en cajas petri (8.5 cm de diámetro) y provistas con miel diluida en agua (proporción

7:3 ml, solución de miel:agua) sobre un pedazo de papel absorbente doblado (1cm2) y agua

potable en algodón (1cm de diámetro, Protec MR, Walmart, Jalisco, México). Para las

hembras en ayuno se utilizaron hembras parasitoides puestas en cajas petri (8.5 cm de

diámetro) y provistas únicamente con agua potable en algodón (1cm de diámetro). Cada

hembra parasitoide fue dejada con un macho para permitir la cópula entre ellos durante 24

horas después de la emergencia. Después de este tiempo los machos fueron retirados

dejando solo a las hembras bajo cada tratamiento y para su uso en experimentos.

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Entonces se observó individualmente el comportamiento de los parasitoides bajo el

estereoscopio (Stemi 2000C, Carl Zeiss, Guadalajara, Jalisco, México) a un aumento de 6x

hasta la oviposición del huevo por parte del parasitoide o máximo diez minutos para A. ervi y

20 minutos para A. abdominalis. Se registraron como variables de respuesta el tipo de

hospedero seleccionado (hospedero de alta o baja calidad), el tiempo de detección con el

hospedero seleccionado, el tiempo de aceptación y el resultado del contacto (oviposición o

no). Después de estas observaciones se tomaron los áfidos utilizados en las observaciones y

se disecaron con una gota de solución de Ringer en un portaobjetos y cubierto con un

cubreobjetos bajo microscopio (Stemi DV4 Zeiss, Guadalajara, Jalisco, México) conectado a

una cámara AxioCam ICc1 y haciendo uso del software Axiovisión Rel 4.8 (Carl Zeiss,

Guadalajara, Jalisco, México) y se registró el número de huevos dejados por el parasitoide

dentro del áfido.

Se realizaron 20 réplicas para las observaciones de cada tratamiento y para cada especie

parasitoide. Inmediatamente después de las observaciones se disecó a los parasitoides para

realizar el conteo de huevos y la otra mitad fue almacenada en el congelador a -80°C para su

posterior evaluación bioquímica de contenido de fructosa, glicógeno y sacarosa.

Las disecciones de los parasitoides se llevaron a cabo sobre un portaobjetos conteniendo

una gota de solución de Ringer bajo microscopio (Stemi 2000 C Zeiss, Guadalajara, Jalisco,

México), donde se extrajeron las ovariolas de cada hembra. El revestimiento de las ovariolas

fue cortado para liberar los huevos y una vez liberados fueron cubiertos con un cubreobjetos

y se procedió a la observación y conteo del número de huevos en cada ovariola bajo

microscopio compuesto (PrimoStar, Zeiss, Guadalajara, Jalisco). Para A. ervi los huevos

fueron observados a un aumento de 20X y para A. abdominalis el aumento utilizado fue de

10X.

3.3.2.2 Análisis bioquímicos

Los análisis bioquímicos donde se analizaron los contenidos de fructosa, sacarosa y

glicógeno se realizaron con una adaptación a las técnicas de Olson et al. (2000) y Wyckhuys

et al. (2008). Donde a las hembras parasitoides almacenadas a -80°C fueron descongeladas

y pesadas previo a los análisis. Los parasitoides fueron macerados en 5µl de sulfato de sodio

al 2% y se enjuagaron con 45 µl de cloroformo:metanol y centrifugados por 2 min a 13000

rpm. El sobrante en el fondo se utilizó para el análisis de glicógeno y el líquido para el

análisis de contenido de fructosa y sacarosa usando el método colorimétrico de Antrona.

Todos los nutrientes fueron determinados con el uso de espectrofotómetro (X-Mark, Bio-Rad,

35

México) formato de placa de 96 pozos y su correspondiente software (Microplate Manager 6,

Bio-Rad, México) para realizar las lecturas de absorbancia. Los cálculos se realizaron a partir

de la absorbancia presente para cada individuo y deducido de la curva de regresión de

absorbancia. Este resultado expresado en µg/µl fue dividido al peso del insecto (mg) entre el

volumen (µl) analizado en la muestra, obteniendo así el contenido total de los carbohidratos

analizados expresando los resultados en unidades de µg/mg.

3.3.2.3 Análisis de datos

Los análisis estadísticos se realizaron de la manera siguiente: para la variable de selección

del hospedero se analizó mediante el modelo de análisis de proporciones de Chi cuadrada.

Las demás variables fueron analizadas mediante ANOVA-anidada cuando las distribuciones

cumplieron con los supuestos de normalidad y homocedasticidad. Cuando los supuestos no

se cumplieron los datos fueron transformados, y si estos no se ajustaban a los supuestos de

normalidad y homocedasticidad, se analizaron con Modelos Lineales Generalizados (GLM)

con las distribuciones apropiadas (e.g. Poisson, Gaussian o binomial) y así saber si existían

diferencias significativas entre los hospederos de alta o baja calidad seleccionados. Se

hicieron las correspondientes pruebas de contraste entre los tratamientos para evaluar si

hubo diferencias significativas en los tratamientos que seleccionaron a hospederos de alta

calidad comparados con los de baja calidad. Todos los datos fueron analizados con el

software gratuito R versión 3.1.3.

3.4 Resultados

3.4.1 Observaciones de comportamiento

3.4.1.1 Para A. ervi

Para la variable selección de hospedero se encontraron diferencias significativas solo para el

tratamiento de los parasitoides en ayuno-viejos (χ 2=5, gl=1, P=0.0126) seleccionando mayor

número de hospederos de alta calidad (80%) sobre los de baja calidad (20%); mientras que

en los tres tratamientos restantes no mostraron diferencias significativas, es decir,

seleccionaron de manera similar a hospederos de alta como de baja calidad (Figura 3.1-A).

36

Figura 3.1. Selección de hospedero para el parasitoide limitado en tiempo Aphidius ervi A) y selección de hospedero para el parasitoide limitado en carga ovárica A. abdominalis B). Las frecuencias son mostradas, letras diferentes significan diferencias significativas (P=<0.05).

En el tiempo de detección de los hospederos no hubo diferencias significativas entre

hospederos de baja y alta calidad en la totalidad de los tratamientos (F=1.031, gl=4,

P=0.397). En promedio (± error estándar) los parasitoides alimentados-jóvenes tardaron en

detectar a su hospedero de alta calidad en 88.2s (± 27.8s), mientras que la detección de los

hospederos de baja calidad fue de 104.5s (±38.0 s). Los parasitoides en ayuno-jóvenes

tardaron en promedio (± error estándar) en detectar al hospedero de alta calidad a los 173.3s

(±50.5s) y en los hospederos de baja calidad tardaron 55.2s (±20.5s). Los parasitoides

alimentados-viejos tardaron 96.8s (±35.9s) en detectar a sus hospederos de alta calidad,

mientras que a los de baja calidad tardaron 156.0s (±44.2s). Los parasitoides en ayuno-

viejos tardaron 99.0s (±25.1s) en detectar a su hospedero de alta calidad y tardaron 35.9s

(±15.5s) en detectar a su hospedero de baja calidad (Figura 3.2-A).

Para ataque de los hospederos seleccionados no encontramos diferencias significativas

cuando los parasitoides de los cuatro tratamientos están en contacto con los hospederos de

alta calidad en comparación con los de baja calidad (χ=70.151, gl=4, P=>0.05) (Figura 3.2-

B).

Cuando observamos el tiempo de aceptación no se encontraron diferencias significativas en

hospederos de alta calidad comparados con los de baja calidad en todos los tratamientos

analizados (F=0.834, gl=4, P=>0.05). En promedio (± error estándar) los parasitoides

alimentados-jóvenes aceptaron a sus hospederos de alta calidad a los 95.7s (±30.0s) y a los

hospederos de baja calidad a los 114.6s (±57.8s). Parasitoides en ayuno-jóvenes tardaron en

aceptar 204.1s (±53.9s) a sus hospederos de alta calidad, y a sus hospederos de baja

calidad les tomo 98.6s (±39.5s). Parasitoides alimentados-viejos aceptaron a los 88.6s

(±43.8s) a sus hospederos de alta calidad y 168s (±35.9s) a sus hospederos de baja calidad.

37

Parasitoides en ayuno-viejos aceptan a hospederos de alta calidad a los 232.0s (±44.2s) y

aceptan a hospederos de baja calidad a los 49.3s (Figura 3.2-C).

Para el número de huevos dejados dentro de los áfidos, no hubo diferencias significativas en

hospederos de alta o baja calidad en algún tratamiento (χ=82.979, gl=4, P=>0.05) (Fig. 3.2-

D).

Cuando observamos la carga ovárica de los parasitoides, no se encontraron diferencias

cuando estos fueron expuestos en hospederos de alta calidad en comparación con los de

baja calidad en todos los tratamientos (F=0.142, gl=4, P=>0.05) (Figura 3.2-E).

Figura 3.2. Parametros de comportamiento del parasitoide limitado en tiempo Aphidius ervi en A) Tiempo de detección, B) Ataque, C) Tiempo de aceptación, D) Numero de huevos ovipositados en los áfidos y E) Carga ovárica. Promedio ± EE son mostrados, diferentes letras muestran diferencias significativas (P=<0.05).

3.4.1.2 Para A. abdominalis

No se encontraron diferencias significativas al analizar el hospedero seleccionado por los

parasitoides, de alta o baja calidad para los distintos tratamientos de A. abdominalis

(χ2=5.318, gl=1.77, P=>0.1149) (Figura 3.1-B).

Con respecto al tiempo de detección con el hospedero seleccionado no hubo diferencias

significativas en los tratamientos entre hospederos de alta o baja calidad (F=0.179, gl=4,

P=>0.05). En promedio (± error estándar) parasitoides alimentados-jóvenes detectan a sus

hospederos de alta calidad en 236.5s (±73.4s) y a los hospederos de baja calidad los

detectan a los 95.4s (±24.0s). Parasitoides en ayuno-jóvenes detectan a los 261.4s (±90.7s)

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a sus hospederos de alta calidad, mientras que detectar a sus hospederos de baja calidad

les toma 316.1s (±98.7s). Parasitoides alimentados-viejos tardan 281.3s (±71.0s) en detectar

a sus hospederos de alta calidad y tardan 328.2s (±262.9s) en detectar a sus hospederos de

baja calidad. Parasitoides en ayuno-viejos tardan en detectar a sus hospederos de alta

calidad 239.6s (±72.6s) y tardan 214.1s (±137.0s) en detectar a sus hospederos de baja

calidad (Figura 3.3-A).

Para la variable ataque no encontramos diferencias significativas en todos los tratamientos

en parasitoides atacando hospederos de alta calidad en comparación con los hospederos de

baja calidad (χ=61.867, gl=4, P=>0.05) (Figura 3.3-B).

Cuando observamos el tiempo de aceptación no encontramos diferencias significativas en

algún tratamiento cuando los parasitoides seleccionaron a hospederos de alta calidad

comparados con los de baja calidad (F=0.773, gl=4, P=>0.05). En promedio (± error

estándar) parasitoides alimentados jóvenes aceptan a sus hospederos de alta calidad a los

321.9s (±104.2) y los hospederos de baja calidad no fueron aceptados. Parasitoides en

ayuno-jóvenes aceptan a los 404.3s (±116.1s) a sus hospederos de alta calidad mientras que

a los 443.2s (±100.9s) aceptan a sus hospederos de baja calidad. Parasitoides alimentados-

viejos aceptaron a sus hospederos de alta calidad a los 429.7s (±104.7s) mientras que los

hospederos de baja calidad no fueron aceptados. Parasitoides en ayuno-viejos aceptaron a

sus hospederos de alta calidad a los 312.3s (±58.9s) y aceptaron a sus hospederos de baja

calidad a los 575.0s (±174.1s) (Figura 3.3-C).

El número de huevos ovipositados dentro de los áfidos no pudo analizarse ya que no se

encontraron huevos del parasitoide en ninguno de los hospederos.

Al analizar la carga ovárica de los parasitoides, no se encontraron diferencias en todos los

tratamientos cuando estos fueron expuestos en hospederos de alta calidad en comparación

con los de baja calidad (F=0.318, gl=4, P=>0.05) (Figura 3.3-D).

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Figura 3.3. Parámetros de comportamiento del parasitoide limitado en carga ovárica Aphelinus abdominalis en A) Tiempo de detección, B) Ataque, C) Tiempo de aceptación y D) Carga ovárica. Promedio ± EE son mostrados, diferentes letras muestran diferencias significativas (P=<0.05).

3.4.2 Análisis Bioquímicos

3.4.2.1 Para A. ervi

Cuando se analiza el contenido de fructosa en los parasitoides, no se observan diferencias

significativas en la totalidad de tratamientos cuando los parasitoides aceptan hospederos de

alta calidad comparados con los hospederos de baja calidad (F=0.546, gl=4, P=>0.05)

(Figura 3.4-A).

Al analizar el contenido de glicógeno, no se encontraron diferencias significativas en todos

los tratamientos cuando los parasitoides son expuestos a hospederos de alta calidad en

comparación con los de baja calidad (F=1.3315, gl=4, P=>0.05) (Figura 3.4-B).

Cuando analizamos el contenido de sacarosa no se encontraron diferencias significativas en

ningún tratamiento, teniendo similar cantidad de sacarosa en parasitoides expuestos a

hospederos de alta calidad en comparación con los de baja calidad (F=0.553, gl=4, P=>0.05)

(Figura 3.4-C).

40

Figura 3.4. Aphidius ervi A) Contenido de fructosa, B) Contenido de glicógeno y C) Contenido de sacarosa en µg. Promedio ± EE son mostrados, diferentes letras significan diferencias significativas (P=<0.05).

3.4.2.2 Para A. abdominalis

Cuando se analiza el contenido de fructosa en los parasitoides, no se observan diferencias

significativas en los tratamientos cuando los parasitoides son expuestos a hospederos de

alta calidad comparados con los hospederos de baja calidad (F=0.785, gl=3, P=>0.05)

(Figura 3.5-A).

Al analizar el contenido de glicógeno no se encontraron diferencias significativas para todos

los tratamientos en parasitoides expuestos a hospederos de alta calidad en comparación con

los de baja calidad (F=1.408, gl=3, P=>0.05) (Figura 3.5-B).

Cuando analizamos el contenido de sacarosa no se encontraron diferencias significativas en

todos los tratamientos, teniendo similar cantidad de sacarosa en parasitoides expuestos a

hospederos de alta calidad en comparación con los de baja calidad (F=0.255, gl=3, P=>0.05)

(Figura 3.5-C).

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Figura 3.5. Aphelinus abdominalis A) Contenido de fructosa, B) Contenido de glicógeno y C) Contenido de sacarosa en µg. Promedio ± EE son mostrados, diferentes letras significan diferencias significativas (P=<0.05). NA significa no analizado.

3.5 Discusión

3.5.1 Observaciones de comportamiento

Se sostiene ampliamente que hembras parasitoides frecuentemente rechazan hospederos

de calidades pobres, donde la sobrevivencia y la adecuación de su progenie podrían ser

bajos (Segoli et al. 2010). Además, el comportamiento de forrajeo y por ende la selección de

sus hospederos se verá afectado por el estado fisiológico de los parasitoides (Lewis et al.

1990). Nuestros resultados muestran que A. ervi no muestra una discriminación entra

hospederos de alta y baja calidad cuando las hembras son alimentadas-jóvenes, en ayuno-

jóvenes y alimentadas-viejas. Solo se observó que A. ervi selecciona más a hospederos de

alta calidad sobre los de baja calidad cuando los parasitoides están en ayuno-viejos,

contradiciendo a Vinson et al. (1998) quien afirma que las hembras hambrientas reducen sus

esfuerzos dirigidos a la búsqueda de hospederos.

Para A. abdominalis, no se vieron diferencias de selección entre hospederos de alta y baja

calidad en los tratamientos analizados, sugiriendo que el parasitoide no es selectivo como lo

sugiere el paradigma. A corto alcance se espera que los parasitoides seleccionen al mejor de

sus hospederos con base en las sustancias que detectan (Hagvar & Hofsvang 1991). Ya que

los hospederos pueden generar sustancias que permiten la identificación por parte de los

42

parasitoides la detección de las señales de un hospedero puede ser modificada por el

trasfondo de una red de volátiles presentes en el hábitat (Hilker & McNeil 2008). Desde que

no es solo influenciado por los químicos emanados, el color o forma del hospedero sino

también por la planta de la que se alimenta el hospedero (Vinson et al. 1998). Los volátiles

de insectos herbívoros y sus plantas hospederas sirven como señales confiables para la

búsqueda de hospederos idóneos de parasitoides (Hilker & McNeil 2008). Entonces, si al

menos dos especies hospederas se encuentran juntas, como lo fue en nuestro bioensayo,

tales sustancias pueden mezclarse haciendo que el parasitoide se confunda en el proceso

de selección. Es decir, estas señales asociadas a una especie podrían influenciar la

respuesta de la otra especie (Vinson 1976). Entonces la presencia de una especie

hospedera podría estimular un ataque falso-positivo en la especie no-objetivo (de baja

calidad) a través de una excitación central (Barton & Withers 2002). O puede ser que los

encuentros con hospederos no idóneos pueden hacer disminuir las respuestas a ciertas

señales (Turlings et al. 1993). Aunque hay estudios que señalan que la presencia del

hospedero preferido por parasitoides no incrementa el ataque en los hospederos de menor

rango o menor preferidos (Murray et al. 2010).

Los parasitoides gastan segundos en distinguir los tipos de hospederos cada vez que estos

se encuentran con un hospedero potencial, independientemente si acepta o no (Charnov &

Stephens 1988), ya que los hospederos encontrados al azar difieren en la contribución a la

adecuación de los parasitoides y requieren diferentes tiempos de manipulación (Mangel

1989). Sin embargo, nuestros resultados de tiempo de detección y aceptación no mostraron

diferencias en hospederos de alta y baja calidad en todos los tratamientos para ambas

especies parasitoides. Sugiriendo que ambas especies podrían manifestar una influencia o

confusión en la detección y aceptación por parte del parasitoide al tener presente a las dos

especies hospederas sin importar el estado fisiológico en el que se encontraban los

parasitoides. En un estudio previo sin opción el tiempo de detección y aceptación observado

para A. ervi fue menor en hospederos de alta calidad en comparación con los hospederos de

baja calidad en la mayoría de tratamientos analizados, mientras que A. abdominalis no

muestra ninguna diferencia entre hospederos de alta o baja calidad. Por lo que mientras el

comportamiento de A. ervi se ve modificado según el estado fisiológico del parasitoide, para

A. abdominalis confirmamos que toma tiempos similares en la de detección y aceptación de

sus hospederos de alta y baja calidad sin importar el estado fisiológico de los parasitoides.

Contradiciendo estudios como el de Takasu y Lewis (1993) quienes observaron que hembras

parasitoides de Microplitis croceipes Cresson (Hymenoptera:Braconidae) en estado de ayuno

43

son menos eficientes en la búsqueda de hospederos en comparación con las alimentadas

sugiriendo a las hembras hambrientas las cuales se sugiere son menos sensitivas a olores

asociados a sus hospederos.

Para la variable ataque, los resultados muestran que fue similar en hospederos de alta y baja

calidad en todos los tratamientos para ambas especies. En A. abdominalis se observó que la

mayoría de los individuos atacados se encontraban muertos y este comportamiento se ha

observado en hembras viejas de Diadromus pulchellus Wesmael

(Hymenoptera:Ichneumonidae), las cuales en pruebas de selección mataron a sus

hospederos de Plutella xylostella L. (Lepidoptera:Noctuidea) que era el hospedero no-

objetivo o de baja calidad (Jenner et al. 2012).

Para el número de huevos ovipositados en los áfidos, en A. ervi no se mostraron diferencias

en todos los tratamientos, la oviposición fue similar sobre hospederos de alta calidad en

comparación con los de baja calidad. Sin embargo, Pennacchio et al. (1994) observaron que

A. ervi atacó y ovipositó más en su hospedero natural Acyrthosiphon pisum (Harris) que en

su hospedero no idóneo (o de baja calidad) Microlophium carnosum Buckton (Homoptera:

Aphididae) en condiciones de opción. Otros estudios han mostrado que las hembras

parasitoides modifican la oviposición dependiendo del estado fisiológico presente, por

ejemplo; Microplitis croceipes Cresson (Hymenoptera:Braconidae) en ayuno son menos

selectivas que hembras alimentadas atribuyendo la respuesta a que las hembras tienen

muchos huevos y poco tiempo de vida (Takasu & Lewis 1995). Otro estudio con

Glyptapanteles militaris Ashmead (Hymenoptera: Braconidae) muestra un comportamiento

selectivo de oviposición cuando los parasitoides son alimentados con miel en comparación

con los que están en ayuno, los cuales muestran una acelerada oviposición (Costamagna et

al. 2004). La oviposición de A. abdominalis en nuestros resultados fue nula en todos los

tratamientos sugiriendo que probablemente hay caracteres de los hospederos afectando el

proceso de selección del parasitoide. Hilker & McNeil (2008) mencionan que la falta de

discriminación o respuesta a señales de los hospederos (infoquímicos) puede ser causada

por factores fenotípicos o genotípicos. Nosotros sugerimos que la respuesta podría ser

influenciada por los semioquímicos que despide el hospedero; por ejemplo, puede ser que el

hospedero de baja calidad despida semioquímicos que haga que se rechace el hospedero y

al estar los dos hospederos juntos, el parasitoide puede detectar el semioquímico del

hospedero de alta calidad y atacar al hospedero de baja calidad pensando que era el idóneo

para el desarrollo de su progenie. O podría ser que la defensa de los áfidos afectó en la

44

oviposición desde que se observó que los áfidos se defendían levantando las patas traseras

y golpeando a los parasitoides.

En cuanto a la carga ovárica, en ambas especies no se mostró una diferencia de número de

huevos maduros en parasitoides expuestos a hospederos de alta calidad en comparación a

los de baja calidad de todos los tratamientos. Mientras que en un estudio previo (Velasco-

Hernández et al., en preparación) se observó que al menos en A. ervi, la carga ovárica

incrementa en parasitoides que son expuestos a hospederos de alta calidad y la carga

ovárica es menor en parasitoides expuestos a hospederos de baja calidad sin importar el

estado fisiológico del parasitoide. Tal y como se ha observado en otras especies de

Braconidos (Casas et al. 2009). Para A. abdominalis en el estudio previo (Velasco-

Hernández et al., en preparación) se obtuvieron resultados similares confirmando que no

existe una diferencia en la producción de huevos cuando los parasitoides se presentan frente

a hospederos de alta o baja calidad, sugiriendo que la carga ovárica no se ve afectada por el

tipo de hospedero a parasitar.

3.5.2 Análisis Bioquímicos

Se sabe que parasitoides adultos dependen de los carbohidratos como recurso energético

(Rivero & West 2002, Casas et al. 2003, Steppuhn & Wackers 2004). Estos se ven afectados

por el estado fisiológico observándose menores cantidades en parasitoides viejos o sin

alimento (Olson 2000, Rivero y Casas 1999, Fadamiro & Heimpel 2001, Giron & Casas 2003,

Wyckhuys et al 2008, Akman et al. 2010). Esta afectación en contenidos se ve reflejada en la

modificación de su comportamiento durante el forrajeo de hospederos (Lee & Heimpel 2007).

Nosotros esperábamos un cambio en el contenido de los carbohidratos no sólo por el estado

fisiológico del parasitoide sino por el forrajeo presentado en sus hospederos, pero cuando los

parasitoides son expuestos a hospederos de alta o baja calidad no muestran una diferencia

en el contenido de fructosa, glicógeno o sacarosa. Lo que sugiere que estas características

no se ven afectadas en el parasitoide al estar frente diferentes tipos de hospederos. Sin

embargo, al igual que en los resultados de comportamiento podría estar en una percepción

confusa al estar frente a sus dos hospederos que emiten diferentes señales y no saber a qué

estímulos responder y por lo tanto el contenido de carbohidratos no se ve afectado. Aunque

faltan por hacer más estudios desde que algunos tratamientos fueron analizados con un bajo

número de muestras resultado de la selección de los hospederos.

45

3.6 Conclusiones

Se esperaba que el comportamiento de los parasitoides A. ervi fuese selectivo, mientras que

el parasitoide A. abdominalis por su condición de ser limitado en carga ovárica y poder

producir más huevos a lo largo de su vida pudiese ser no selectivo en condiciones donde su

estado fisiológico disminuyera su esperanza de vida (viejos y/o en ayuno) y no como es

sugerido por los paradigmas. Y que estos comportamientos se verían reflejados en

contenidos energéticos de fructosa, glicógeno y sacarosa de los parasitoides. Sin embargo,

A. ervi no mostró selectividad y A. abdominalis ni siquiera ovipositó en los hospederos

presentes, además de que ambas especies no mostraron diferencias en los contenidos de

carbohidratos. Se sugiere que los resultados se debieron a una mezcla de señales

producidas por los hospederos y por ende el no discernimiento específico del parasitoide

hacia los hospederos presentes.

Agradecimientos

A Luis Angel Godoy Tapia y Héctor Pelayo Uribe por su asistencia técnica durante los

experimentos. A CONACyT Ciencia-Básica (No. 157259) otorgado a RRR para la compra de

materiales, a el apoyo del proyecto de FP7-PEOPLE-2013-IRSES fondos de APHIWEB grant

no. 611810 para ND y MCVH. A CONACyT por la beca de doctorado de MCVH (ID: 367437)

y al CINVESTAV- Irapuato especialmente al Dr. John Délano por el apoyo otorgado para

realizar los análisis bioquímicos en su laboratorio. A Koppert-México SA de CV por el apoyo

parcial de las avispas.

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3.7 Referencias

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Akman GE, Gulel A, Varer IO, Boz A, Ceur O (2010). Effects of sugar feeding on lipid, glycogen, and total sugar levels of a female parasitoid, Bracon hebetor (Say) (Hymenoptera: Braconidae). Turk J Agric For 34:343-347

Bao-Fundora L, Ramírez-Romero R, Sánchez-Hernández CV, Sánchez-Martínez J, Desneux N (2015). Intraguild predation of Geocoris punctipes on Eretmocerus eremicus and its influence on the control of the whitefly Trialeurodes vaporariorum. Pest Manag Sci 72:1110-1116

Barton BL, Withers TM (2002). Time-dependent changes in the host-acceptance threshold of insects: implications for host specificity testing of candidate biological control agents. Biocontrol Sci Technology 12(6):677-693

Blackman RL, Eastop VF (2007). Aphids on the world’s herbaceous plants and shrubs: an identification and information guide, John Wiley Sons Inc, United States.

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Casas J, Vannier F, Mandon N, Delbecque JP, Giron D, Monge JP (2009). Mitigation of egg limitation in parasitoids: immediate hormonal response and enhanced oogenesis after host use. Ecology 90(2): 537-545

Charnov EL, Skinner SW (1984). Evolution of host selection and clutch size in parasitoid wasps. Florida Entomol 67(1):5-21

Charnov EL, Stephens DW (1988). On the evolution of host selection in solitary parasitoids. American Naturalist 132(5):707-722

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CAPÍTULO 4. EFECTOS DEL ESTADO FISIOLÓGICO EN EL RANGO DE HOSPEDERO Y COMPORTAMIENTO DE OVIPOSICIÓN

Artículo en preparación, cuya referencia bibliográfica es:

Velasco-Hernandez MC, Ramirez-Romero R, Desneux N. Effects of physiological status on

parasitoid host range and oviposition behavior. En preparación para Oecologia

52

Effects of physiological status on parasitoid host range and oviposition

behavior

Velasco-Hernández, M.C.1, Ramírez-Romero, R.1, Desneux, N.2,

1Biological Control Laboratory, Department of Agricultural Production, CUCBA, University of

Guadalajara, Zapopan, Jalisco, México.

2French National Institute for Agricultural Research (INRA), UMR1355, 400 Route des

Chappes, 06903, Sophia-Antipolis, France.

4.1 Abstract

Parasitoids' host range is usually seen as a static feature; however, the physiological state of

an individual may modify established behavioral patterns. This study investigated

relationships between the physiological status of two parasitoids with different life histories

(Aphidius ervi and Aphelinus abdominalis) and their host selection for parasitism under

laboratory and greenhouse conditions. In laboratory, we tested the following treatments: 1)

fed, young parasitoids, 2) starved, young parasitoids, 3) fed, old parasitoids and 4) starved,

old parasitoids. Each treatment group was exposed to its high and low-quality hosts

separately. We observed behavior under a microscope and recorded time of detection,

acceptation time and time to complete oviposition. After these observations, the aphid hosts

were dissected to check if the parasitoid laid egg(s) in the hosts and recovered parasitoid

adults were also dissected to assess the egg load. Later, young and old fed parasitoids were

tested under greenhouse conditions, where they were exposed to their high and low-quality

hosts for 24h before being recaptured. We counted the number of mummies produced under

each treatment; the parasitoids used were dissected and their eggs counted. Our results in

laboratory conditions indicate that time-limited parasitoids prefer to oviposit more in high

quality host than in low quality host in all treatments. Egg-limited parasitoids prefer to oviposit

more in high quality hosts than in low quality hosts when parasitoids are fed-young and

starved-old. In greenhouse conditions, our results indicate that both parasitoid species prefer

to oviposit in high quality hosts than in low quality host when parasitoids are young. No

differences were found when they are fed and old over high and low-quality hosts. Then

different physiological conditions can modify parasitoid host selection behavior. These

behavior modifications may be adaptive for parasitoid species and may have consequences

for their use in aphid pest management.

Keywords: Host selection, physiological status, aphids, parasitoid

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4.2 Introduction

Parasitoids have been used as models of many ecological theories and behavioral studies

(Rosenheim 1999, Jervis et al. 2001, Jenner et al. 2014, Montovan et al. 2015) and is one of

the main biological control agents used in the management of crop pest around the world

(Schmidt et al. 2003, Wajnberg et al. 2008, Van Lenteren 2012). The selective behavior of

parasitoids has been widely studied, such behavior allows them to choose the best host for

oviposition and subsequent development of its progeny (Vinson 1998). In field, parasitoids

could find diverse kinds of hosts; these range from suitable ones to those that allow them

marginal conditions for their development (Heimpel et al. 2003, Sidney et al. 2010, Bueno et

al. 1993, Bryden 2003). In order to increase fitness, it is imperative for parasitoids to locate

suitable hosts (Salt 1935; Rivero 2000). Therefore, females have to evaluate the energy

needed to invest in oviposition and if potential hosts are suitable for the development of its

progeny (Chow & Mackauer 1991). Hosts with low survival potentials should be eventually

rejected (Jansen 1989).

Parasitoids have been classified into two categories with respect to their life histories. This

includes, the time-limited parasitoids and the egg-limited parasitoids (Godfray 1994, Van

Baalen 2000, Jervis et al. 2008). Time-limited parasitoids are pro-ovigenic species (females

that emerge with their full or nearly full complement of mature eggs) but its longevity is short

(Jervis et al. 2001, Deas & Hunter 2014). Egg-limited parasitoids are synovigenic species

(females born without or with a few complement of mature eggs) but they can mature more

eggs along their life since they have longer life spans (Mangel 1989, Heimpel & Rosenheim

1998). In this case, the question is how these features affect host selection behavior when

host range is wide? Differences in their life history have been associated with host selection

behavior. It is widely accepted that time-limited parasitoids tend to choose low quality hosts

when high quality host are no present, then they are considered non-selective species

(Heimpel & Rosenheim 1998; Papaj et al. 2000; Withers & Browne 2004). An egg-limited

parasitoid prefers a high-quality host over a low-quality host because they are highly selective

species (Drost & Cardé 1992; Heimpel et al 1996).

Host selection models for time-limited and egg-limited parasitoids suggest a dynamic

behavior (Rosenheim 1999). This behavior depends on multiple factors including

physiological status, oviposition experience and environmental characteristics (Roitberg

1993, Heimpel et al. 1996, Mackauer et al. 1996, Jacob & Evans 2001, Ueno & Ueno 2005,

Jenner et al. 2012). Some authors have reported that host selection depends strongly on the

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parasitoid’s physiological status (Rivero 2000, Wackers 1994) since non-target hosts have

been accepted under different physiological conditions (Jenner et al 2012, 2014). Several

studies have focused on the physiological factors affecting host selection in parasitoids. Their

results reveal that factors as age (Hopper et al 2013; Jenner et al 2012), nutritional state (fed

or starved) (Lewis et al 1998; Jenner et al 2012), egg load (Ueno & Ueno 2005, Deas &

Hunter 2014, Diekkof et al 2014), mating (Zhang et al. 2015, Fletcher et al. 1994), previous

ovipositional experience (Nurindah et al. 1999) and host-quality influence (Silva et al 2011)

are important on host selection. However, after testing these factors independently, it was

revealed that, hunger and age are the main factors affecting host selection behavior (Jacob &

Evans 2001, Jenner et al 2012). Scarce are the studies focused on the effects of age and

nutritional status on host selection (all together) affecting parasitoid host selection on high

suitable hosts and low suitable hosts (Jenner et al 2012; Hopper et al. 2013).

This study focuses on two major families of aphid parasitoids which have time-limitation and

egg-limitation behaviors (Hagvar & Hofsvang 1991). Aphidius ervi Haliday (Hymenoptera:

Braconidae) and Aphelinus abdominalis Dalman (Hymenoptera: Aphelinidae) are two solitary

endo-parasitoids species of aphids (Acheampong et al. 2012, Duarte et al. 2012, Haardt &

Holler 1992) with different life history. Both are considered generalist (Henry et al. 2008;

Mölck et al. 2000) and commercialized for control of aphids. Aphidius ervi has a short life

span of 8-15 days (Hofsvang & Hagvar 1975, Malina & Praslicka 2008) and a high fecundity

rate (He & Wang 2006). On the other hand, A. abdominalis is considered synovigenc

because this species mature eggs along its lifetime (Couty & Poppy 2001, Viggiani 1984) and

has a long life span around 85 days (Wahab 1985, Holler & Haardt 1993).

In this study we hypothesized that, the parasitoid’s physiological status defined by age (young

and old) and nutritional status (fed and starved) could influence the hots selection behavior of

parasitoids with two life histories (time-limited and egg-limited). This decision will be affected

in the choice of a wide host range (high and low-quality hosts). For example, for Lysiphlebus

cardui (Hymenoptera:Braconidae) showed differences between young and old parasitoids

during the time on the host patch, where residence time increased with parasitoid age

(Weisser 1994). Then behavior was affected by parasitoids age. In Aphelinus species Hopper

et al. (2013) tested factors such as starvation and old age in oviposition behavior on a low-

suitable host. However, parasitoids did not were influenced to accept low-suitable hosts

under these conditions, they accept more high-suitable hosts in these conditions. In different

species of parasitoids such as Diadromus Pulchellus (Hymenoptera: Ichneumonidae) Jenner

55

et al. (2014) found that young and old females attacked the same number of hosts from

different non-target species and parasitoid offspring were not affected.

The main objective of this study was therefore to determine if the physiological state (defined

by age and nutritional state together) of the parasitoids A. ervi (time-limited) and A.

abdominalis (egg-limited) have an effect on their host selection behavior under laboratory and

greenhouse conditions.

4.3 Materials and Methods

4.3.1 Biological Material

4.3.1.1 Plants

Commercial tomatoes seeds (Solanum lycopersicum L.) variety Nano, wheat (Triticum

aestivum L.) and broad beans (Vicia faba L.) were used as plant host. Plants were grown in

plastic pots (7cm x7cm x10cm) with 50% organic soil (TONUSOL®, Agriver, Saint Papoul,

France) and 50% perlite (Perlite Italiana ®, AGRILIT, Corsico, Italy). Fertilization was carried

out using nutrient solution composed of mineral salts, trace elements, micro and macro

nutrients. Three weeks old tomato plant and one-week old broad bean and wheat plants were

used for experimental trials. Prior to experimental trials. Plants were carefully checked for any

herbivoral infestation. Experiments were carried out under controlled conditions

(temperature: 23±3°C, relative humidity 70±5% and a photoperiod 16:8 light: darkness).

4.3.1.2 Aphids:

For the time-limited species (A. ervi) we used Macrosiphum euphorbiae Thomas aphid as its

high-quality host and Rhopalosiphum padi L. aphid was used as its low-quality host. For egg-

limited parasitoid (A. abdominalis) we used Metopolophium dirhodum Walker as its high-

quality host and Aphis gossypii Glover was used as the low-quality host (all aphid species

Hemiptera:Aphididae). First and second instar of aphids were used for A. ervi. Third and

fourth instar and adults of aphids were used for A. abdominalis. This was determined in a

preliminary study following the protocol of Desneux et al 2009, Perdikis et al. (2004) and

Tahriri et al. (2007).

Macrosiphum euphorbiae were maintained on tomato plants; R. padi, A. gossypii and M.

dirhodum were maintained on wheat plants. And Acyrthosiphon pisum Harris were

maintained on broad beans and it was used for maintenance of parasitoid colonies. Aphid

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colonies were established in the Mixed Unit Research (UMR) Laboratory-Sophia-Agrobiotech

at the National Institute of Agricultural Research (INRA) in Sophia Antipolis, France. All aphid

species were confirmed for specialists. Colonies were reared under laboratory conditions at a

temperature of 24±3°C, relative humidity of 60±5% and a photoperiod of 10:14 hours (light:

darkness).

4.3.1.3 Parasitoids:

Aphidius ervi used as time-limited species were established in laboratory with individuals

collected from field collects made on February 2015 on Provence of Alpes-Cote d’Azur

(PACA), France. Samples collected from Koppert France in March 2015 were used to

establish a laboratory colony of the egg-limited species (A. abdominalis). Both colonies were

maintained in plastic boxes (55cm x 55cm x 55cm) covered by fine mesh under the following

conditions; temperature of 23±3°C, relative humidity 70±5 and a photoperiod 8:16 light:

darkness. Both species were reared on the host aphid A. pisum Harris (Hemiptera:

Aphididae) under conditions before described. New emerged female parasitoids were used

for experiments and they were paired with males in couples of 10 (10:10, male:female) to

increase chances of mating, every 24 hours we took females parasitoids for using them in

experiments.

4.3.2 Selection behavior observations

Host selection experiments were carried out in completely randomized block design.

Parasitoids of the two species used for the experiments were of the following conditions; 1)

young-fed female parasitoid, 2) young-starved female parasitoid, 3) old-fed female parasitoid,

4) old-starved female parasitoid. Each female with the previous treatments were exposed to

its high-quality host and low-quality host separately, we used a new parasitoid for every host.

For A. ervi y A. abdominalis young females were 24 hours old after emergence. For older

females of A. ervi were females of 9-10 days old and for older female parasitoids of A.

abdominalis were of 34-36 days old. For starved-old individuals of A. abdominalis we used

females of 4 days old, A. ervi old-starved was not used since parasitoids died before time for

experiments (two days old). Newly emerged fed females were obtained by allowing females

to emerge in plastic cylinders (6 cm high × 3 cm diameter). Small drops of honey were placed

on the wall of the plastic container and water was provided using a cotton ball. Insects were

allowed to fed ad libitum. Honey and water were replaced every 2 days. Only water was

provided to starved females. In order to improve on the mating chances in the first 24 hours

57

after emergence, equal number of females and males were allowed to emerge in the same

plastic container. Prior to experimental trails, all males were removed and dead females also

were removed daily.

Parasitoid behavior was recorded after observing a single parasitoid under a stereoscope

microscope (LEICA EZ24, Carl Zeiss, France). Observation time was maximum five minutes

for A. ervi and 10 minutes for A. abdominalis. Parameters recoded included: 1) the time lapse

before the parasitoid made contact with the aphis was recorded, 2) if parasitoids stunk the

aphids or not and 3) time interval before ovipositor insertion. After observations, aphids were

dissected with a drop of Ringer solution on a microscope slide under a microscope

(Discovery V12, Carl Zeiss, France). After dissection, a coverslip was over the dissected

aphids and the number of eggs oviposited in the aphids were counted under a microscope

(Olympus CX40, Olympus, France).

Similarly, the parasitoids used in each experiment were also dissected to know their egg-load.

Each observation was replicated 30 times.

4.3.3 Greenhouse bioassay

In this trial, only fed parasitoids were used: 1) young and fed female parasitoid, 2) old and fed

female parasitoid. These two treatments were exposed to high-quality hosts and low-quality

hosts separately.

A total of 24 cages (125cm x 65cm x 95 cm) covered with a fine mesh and containing five

host plants per cage were used for green house experiments. An hour prior to experimental

trials, 50 aphids were placed on each plant and allowed to establish. Two parasitoids were

released into each cage for 24 hours and each plant was covered with a plastic cylinder

(47cm high x 15 cm diameter). After this time, parasitoids were recaptured and half of the

recaptured parasitoids were used for dissections and the number of parasitoid eggs (egg-

load) in the females were counted and registered as describes above. Also, the numbers of

aphids parasitized for parasitoids of each treatment was registered. Three repetitions were

made for this bioassay.

4.3.4 Data analysis

Data were first tested for normality using the Shapiro-test, if data were not normal, we

transformed data when it was possible. When data distributions were normal, Nested-ANOVA

was used to analyze the variables. On the other hand, abnormal distributions were analyzed

using the Generalized Linear Models (GLM) with their correspondent distributions (e.g.

58

Poisson, Gaussian or binomial and link function appropriated). Host quality (low and high)

was nested in physiological status (age and feeding factors). Appropriated contrast tests were

carried out between treatments exposed to high and low quality. All tests were carried out

using free software R version 3.1.3 (2017/03/09).

4.4 Results

4.4.1 Laboratory Bioassay

4.4.1.2 Aphidius ervi

In general, the detection time for each treatment varied significantly (F=7.81, P=<0.0005)

(Fig. 4.1-A). Young-fed parasitoids took a longer time to detect low-quality aphid host (64.5s

±10.5s; Mean±SE) and a relative shorter time to detect the high-quality host (23.7s ± 5.8s),

time is significantly minor (t1,58= 3.29, P= <0.001). Parasitoids young-starved did not show

significant differences (t=0.47, P=>0.05) when they were exposed to high or low-quality hosts,

they took a similar time to arrive at detection on high and low-quality hosts (46.6s ±10.1s vs

44.7s ± 9.3s respectively). Parasitoids of the old and fed group took a significantly longer time

(t=2.86, P= 0.004) to make contact with a low-quality host (77.6s ± 14.5s) and relatively less

time (37.0s ± 8.2s) to make contact with a high-quality host.

In the variable attack, treatments differed strongly (X2= 24.19, P=<0.0005) (Fig. 4.1-B). The

young-fed parasitoids significantly attacked more high-quality host than the low-quality host

(t=3.61, P= <0.0005) (Fig. 4.1-B). Parasitoids young-starved did not show any significant

differences (t= 1.63, P= 0.103) in attacking the high-quality host and the low-quality host. The

fed-old parasitoids significantly attacked more high-quality hosts compared with the low-

quality hosts (t=2.02, P= 0.044).

With respect to the time of acceptance, the response time of aphids to the different

treatments were significantly different (F=8.998, P= <0.0005) (Fig. 4.1-C). Fed-young

parasitoids attacked high-quality host significantly faster (21.1s ± 3.4s) and low-quality host

relatively slower (77.6s ± 14.8s) (t=2.952, P= 0.003), and same behavior was observed with

young-starved parasitoids (t=2.509, P= 0.013), fed-old (t=3.462, P= <0.0005).

The parasitoid eggs oviposited in the aphids in the different treatments were significantly

different (X2= 42.58 P=<0.0005) (Fig. 4.1-D). Young-fed (t=2.62, gl=44, P=0.011), young-

starved (t= 2.18, gl=44, P= 0.034) and old-fed (t=2.97, gl= 44, P= 0.004) (Fig.4.1-D)

oviposited significantly more eggs in high-quality hosts when compared with low quality hosts.

At least an egg was oviposited in each aphid, no more than an egg was found inside the

aphids. The egg-load was significantly different across the treatments of parasitoids

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(F=11.444, P= <0.0005) (Fig. 4.1-E). Young-fed parasitoids had significantly (t=4.256,

P=<0.0005), more eggs when they were exposed to high-quality hosts (223.8 ± 10.6, mean ±

standard error) meanwhile the number of eggs is minor (150.3 ± 12.7) when parasitoids were

exposed to low-quality hosts. Parasitoids young-starved also had significantly (t=2.869,

P=0.005) bigger egg load when exposed to high-quality hosts (226.6 ± 14.5) than exposed to

low quality hosts (177.0±12.8). And parasitoids old-fed had significantly (t=2.827, P=0.005)

higher egg number when exposed to high-quality hosts (171.6 ±10.2) vs low quality hosts

(122.8 ±11.6). Most old-starved parasitoids died before the experimental trials, therefore their

data was not included in the analysis.

Figure 4.1. Behavioral parameters of Aphidius ervi (time-limited parasitoid) in A) Time of host detection, B) Attacks, C) Time of host acceptance, D) Number of eggs oviposited in aphids and E) Egg load. Mean ± SE are showed, different letters mean significative differences (P=<0.05).

4.4.1.2 Aphelinus abdominalis

In general, the parasitoid detection time was not significantly different across the treatments

(F=1.411, P=0.230) (Fig. 4.2-A). The young-fed parasitoids took an average of 77.7 seconds

(±16.1, SE) to detect the high-quality host and it took 68.2 seconds for the low-quality host (±

19.3) (t= 1.663, P=<0.097). On the other hand, young-starved parasitoids it took 78.4

seconds (±16.7) to detect high-quality host and an average of 57.5 seconds (±12.9) to detect

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low-quality host it took. In the case of old-fed parasitoids, it took an average of 49.9 seconds

(±12.1) to detect a high-quality host and 69.8 seconds (±14.3) to detect a low-quality host.

Old-starved parasitoids to only 51.4 seconds (±8.1) to detect a high-quality host and up to

82.2 seconds to detect a low-quality host (±13.8) (t=1.206, P=0.229).

Though the attacks were significantly different across the treatments (X2= 6.86, P=<0.0005)

(Fig. 4.2-B). The young-fed (t=0.667, P= 0.498), young-starved (t=0.355, p= 0.723), old-fed

(t=0.764, P= 0.445) parasitoids made their attacks in an analogous pattern when they were

exposed to high and low-quality host. However, old-starved parasitoids attacked more high-

quality hosts and less low-quality hosts (t= 2.205, P= 0.028).

The acceptation time were not significantly different between high and low-quality hosts

(F=0.358, P=0.837) (Fig. 4.2-C). Young-fed, young-starved, old-fed, old -starved parasitoids

took approximately the same amount of time to accept the high and low-quality hosts.

The numbers of parasitoid eggs oviposited in the aphids were significantly different across

treatments (X2= 13.5 P=<0.004) (Fig. 4.2-D). Young-fed (t=2.260, P=0.030) and old-starved

(t=2.260, P= 0.024) parasitoids oviposited significantly more in high-quality host than in low-

quality host. However, the number of eggs oviposited in by young-starved (t= 1.387, P=

0.166) and old-fed (t=0.013, gl= 44, P= 0.989) parasitoids were not significantly different in

high and low-quality host. Finally, the parasitoid egg load was not significantly different in all

treatments (F=0.996, P= 0.412) (Fig. 4.2-E).

61

Figure 4.2. Behavioral parameters of Aphelinus abdominalis (egg-limited parasitoid) in A) Time of host detection, B) Attacks, C) Time of host acceptance, D) Number of eggs oviposited in aphids and E) Egg load. Mean ± SE are showed, different letters mean significative differences (P=<0.05).


4.4.2.1 For A. ervi

The egg load of young-old parasitoids exposed to high-quality hosts were not significantly

different from to those exposed to low-quality hosts (F=1.895, P=0.161) (Fig. 4.3-A)

However, after analyzing the egg load in young and old parasitoids, it was realized that

young parasitoids had significantly more number of eggs compared with older parasitoids

(t=8.284, P=<0.0005). Parasitism were significantly (t=3.83, P=0.0003) higher in high-quality

hosts (9.06±1.29) than in low-quality hosts (3.7±0.96) when parasitoids are younger (Fig. 4.3-

B). But after analyzing this variable in older parasitoids, it was realized that, this difference

was not significant (t=0.910, P=0.336).

62

Figure 4.3. Aphidius ervi A) Egg load and B) Number of aphids parasitized. Mean ± SE are showed, different letters mean significative differences (P=<0.05).

4.4.2.2 For A. abdominalis

Egg load were not significantly higher (F88,3=0.22, P=0.896) in young or old parasitoids

exposed to high and low-quality hosts (Fig. 4.4-A). However, the younger parasitoids had

significantly more number of eggs than the older ones (t=5.650, P=<0.005).

Parasitism rates of young parasitoids were significantly (t=17.64, P=<0.005) higher in high

quality hosts (33.2 ± 2.54) than in low-quality hosts (3.2 ± 0.70) (Fig. 4.4-B). However, for old

parasitoids no significant difference was recorded when parasitoids were exposed high or

low-quality hosts (t=1.066, P=0.291). Sometimes parasitism was almost zero.

63

Figure 4.4. Aphelinus abdominalis A) Egg load and B) Number of aphids parasitized. Mean ± SE are showed, different letters mean significative differences (P=<0.05).

4.5 Discussion

4.5.1 Laboratory bioassay

4.5.1.1 Aphidius ervi

From our results, it was observed that A. ervi young and old-fed parasitoids spent less time to

detect a high-quality host when compared with a low-quality host. Wajnberg et al. (2006)

suggests that old parasitoids are less efficient in finding host and exploiting resources.

Contrarily, our results suggest that old parasitoids responded faster in high quality hosts.

Maybe when they find a good host for oviposition in its last days of life, they want to take

advantages and oviposit. On the other hand, young-starved parasitoids did not show a

noticeable change in behavior when exposed to high and low-quality hosts. This result agrees

that of Vinson et al. (1998) which suggested that, starving females reduces their foraging

efforts. Since our results showed no differences in time acceptance for high and low-quality

hosts. Rosenheim & Rosen (1991) attribute the intensity of behaviors of handling times to the

egg-load, since it decreases when egg load decreases and increases when the egg load

increases.

64

Parasitoids young-fed and old-fed tend to be more selective since they prefer high-quality

hosts over low-quality hosts. This reaction could be possible since they have egg to be

oviposited. Only young-starved parasitoids did not show any significant differences in attack

between high and low-quality hosts.

The acceptance times in all treatments were lower in high-quality hosts and higher in low

quality hosts. This result contrast that of Stilmant et al (2008), who reported that A. ervi is

able to accept non-suitable hosts slower. This difference may be as a result of the

physiological state of parasitoids, stressors factors (age and hunger) producing delays in the

female’s response.

Almost all high-quality aphids host received a parasitoid egg and the low-quality hosts were

less accepted for parasitoid oviposition in all treatments. This result is contradicts that of

Drost & Cardé (1992) who said that a pro-ovigenic species deprived from its hosts is able to

increase its acceptance of a non-suitable hosts. Jenner et al. (2012) also suggest that young-

fed parasitoids may have a reduced acceptance threshold compared with old-starved

parasitoids. However, the results from the pro-ovigenic species treatment suggest that even

under difficult physiological state all parasitoids have some preferences for the most suitable

host. Contrarily, Heimpel et al. (2003) suggested that, time-limited parasitoids are used to

utilizing low quality host when the best ones are not present. Similarly, Weisser (1994) also

suggested that, time-limited parasitoids are more selective when they are younger and when

they are older will not show preferences among hosts.

After dissecting the different parasitoids, it was observed that all parasitoids exposed to high-

quality host had higher number of eggs and parasitoids exposed to low-quality hosts had

lower number of eggs. Mangel (1989) suggest that eggs are only produced when they are

needed. Therefore, it could be suggested that, parasitoids on high quality hosts detect it as a

suitable host for its offspring and start to produce more eggs. Some other studies have

showed that parasitoids increase their eggs production when they have contact with its

suitable hosts and, it happened a few minutes after the contact with hosts (Casas et al.

2009). However, it is noticeable that even in low quality hosts the number of eggs is higher

however, the number of ovipositions made on low quality hosts were lower. In other

parasitoids such as Monoctonus pseudoplatanis Marsshall (Hymenoptera: Braconidae), it has

been observed that females with a high number of mature eggs stopped foraging for suitable

hosts (Collins & Dixon 1986). Therefore, parasitoids try to maintain their fitness for survival

over oviposition and reproduction. However, the number of eggs is higher in all parasitoids

65

(more than 70 eggs) as it has been seen in other Braconidae species which had around 40

eggs at emergence and around 200 eggs at one day old after emergence (Dieckhoff &

Heimpel 2010).

4.5.1.2 Aphelinus abdominalis

It has been mentioned that parasitoids with a small egg load need more time for foraging to

discover its hosts (Rosenheim & Rosen 1991). This idea is coherent with our results since

hosts detection time for A. abdominalis showed that parasitoids took approximately the same

amount of time to locate different hosts (high and low-quality hosts) in all treatments. Vinson

et al. (1998) suggested that that starved female parasitoids could reduce their efforts in hosts

searching. Here our results showed that starved condition is not a factor affecting host

handling times, since in all treatments were not differences in amount of time at acceptance.

The recorded attacks in the present study were very selective. It was higher in high-quality

hosts when compared with low-quality hosts in old-starved treatment. The higher number of

attacks recorded in old-starved parasitoids to high-quality hosts was probably due to host-

feeding start, because old-starved insects were not really old females, but they were hungry

enough to attack and fed from their hosts thereby increasing its life expectancy as in other

Aphelinus species (Azzous et al. 2005). In the rest of our treatments, the attack was similar

on its high and low-quality hosts. It contrasts Hopper et al. (2013) who found out that,

stressed (starved and older ages) Aphelinus females could always accept high-suitable hosts

over low-suitable host, showing a high capacity for discriminate among hosts.

The results from the present study suggest that, the acceptation time in all treatments were

similar when parasitoids were exposed to high quality host and low-quality hosts. Aphelinus

species usually takes a longer hosts handling time, since it has to assess the host’s quality

with its ovipositor (De Farias & Hopper 1999). Longer periods of host handling in Aphelinus

species are taken in comparison with Braconidae species.

Eggs oviposited on aphids when female parasitoids are young-fed and old-starved were

higher on all the high-quality hosts and they laid fewer eggs in low quality hosts. In the

present study, old-starved parasitoids were four days old, these insects were not really very

old. Probably that is why they displayed similar behavior. This result supports that of Hopper

et al. (2013) who reported that, Aphelinus rhamni did not increase its ovipositions on low-

suitability hosts when the parasitoid’s life expectancy was decreased by stressors (age and

66

starvation). Also, our results partially support that of Jenner et al. (2012) who found out that,

younger and sugar-fed individuals showed higher host exploitation than older and sugar-

starved parasitoids. However young-starved and old-fed parasitoids had similar oviposition on

high and low-quality host. Heimpel et al. (1996) observed that, old parasitoids have a reduced

egg load and it probably makes the parasitoids more selective. However, oviposition of old-

fed parasitoids was almost zero. Importance of trade-offs between survival and reproduction

in parasitoids have been widely studied since one of these features might be more important

for parasitoids fitness (Ellers 1996, Ellers & Alphen 1997, Sevenster et al 1998, Ellers et al.

2000). In young-starved parasitoids exposed to high quality host and low-quality hosts similar

oviposition was showed. Maybe these parasitoids perceive a low life expectancy Whiters &

Browne (2004). Parasitoids can express a wide range of host acceptance (Stary 1975, Pike

et al 1999, Ideo et al. 2008). Parasitoids such as Venturia canescens Gravenhorst

(Hymenoptera: Ichneumonidae) often uses low-quality hosts when its life expectancy is

reduced (Fletcher et al. 1994).

The egg load was similar in female parasitoids exposed to aphids of high quality and of low

quality in all treatments. For young-fed, young-starved and old-starved, the egg load was

around 20 eggs for parasitoids exposed to high and low-quality hosts. It is known Aphelinus

species have few eggs in ovaries at emergence but after 24 hours they produce around 20

eggs more (Wu & Heimpel 2007). Only old-fed parasitoids in both high and low-quality hosts

the number of eggs was almost zero. Some studies showed that parasitoids have no reaction

of egg production after the contact with its hosts as in Braconidae species happened (Mangel

1989. Casas et al. 2009). And other suggest egg resorption when parasitoids have a long

time without hosts even when they are feed (Collier 1995).


Time-limited and egg-limited parasitoids have effect on behavioral decisions since they have

to look for the reproductive success and it is difficult extrapolate studies made in laboratory

and field conditions (Heimpel & Casas 2007).

The number of available eggs for oviposition is crucial in foraging behavior (Collins & Dixon

1986). Both species when they were young and fed, had a high number of eggs in

greenhouse trials. A. ervi had about 220 eggs and A. abdominalis 20. Previously, Dieckhoff et

al (2014) reported that the egg load for Braconidae species was about 77 eggs in field

conditions. Aphelinus species in field conditions has been reported to have around 15 eggs

(Hopper & Diers 2014). A. ervi and A. abdominalis old-fed parasitoids had about 100 and five

67

eggs respectively. The number of eggs these species these species could have under

greenhouse conditions has not been reported before. Although the number of eggs in

parasitoids exposed to high quality and low-quality hosts were not different in both

treatments. The age strongly affected parasitoids since the old ones had a few eggs for

oviposition.

Young-fed parasitoids oviposited more eggs in high-quality hosts compared with low quality

hosts in both species. The rejection of three from thirty-three low quality hosts under field

conditions has been observed in Asobara tabida Nees (Hymenoptera: Braconidae) (Janssen

et al. 1989). This contradicts the findings of Barrat et al. (1997) who reported that, parasitism

in non-target species was around 70% in field conditions. When the parasitoids were old-fed,

parasitism were lower in high and low-quality hosts in both species.

4.6 Conclusions

We had hypothesized that, time-limited parasitoid was non-selective and the behavior will be

lightly modified when the physiological status of the two parasitoids were influenced.

However, our results revealed that, parasitoids were selective. They preferred to oviposit in

high quality hosts to low quality hosts under laboratory conditions. Although under

greenhouse conditions young-fed parasitoids continued with this behavior, old-fed parasitoids

did not show preference for either of two host qualities even when they had a high egg load

so, old-fed parasitoids were non-selective.

The egg-limited parasitoid which we expected to be selective all the time had a little modified

behavior in the laboratory. However, their ovipositional behavior showed that they were

always selective except when they were old in which case their egg load had been decreased

to almost zero. In the green house, these insects also exhibited a similar behavior.

Species showing other behaviors are possible and more studies in this topic are necessary to

be developed however here we show a different result to those assumed by some theories.

68

Acknowledgments

To Philipe Bearez, Ha Nguyen and Lucie Monticelli for technical assistance. To Andongma

Awawing for English review. To A CONACyT Ciencia-Básica (No. 157259) to RRR for

materials, to project FP7-PEOPLE-2013-IRSES founding from APHIWEB grant no. 611810

para ND y MCVH. To CONACyT for the PhD fellowship of MCVH (ID: 367437). To Koppert-

France for partial support on wasp obtaining. To INRA for the support to realize the academic

stay.

69

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75

CAPÍTULO 5. DISCUSIÓN GENERAL

El parasitoide A. ervi, al ser una especie limitada y según el paradigma debió parasitar de

manera similar en hospederos de alta y baja calidad cuando se encontraba en un estado

fisiológico óptimo (i.e. alimentados y jóvenes). Esto se vio corroborado en el bioensayo con

opción, reafirmando el paradigma predicho (Heimpel & Rosenheim 1998, Papaj 2000)

afirmando que los parasitoides no son selectivos desde que tienen muchos huevos y poco

tiempo para la oviposición. Sin embargo, al observar los bioensayos sin opción en laboratorio

e invernadero, estos refutan fuertemente el paradigma desde que el parasitoide prefirió a

hospederos de alta calidad sobre los de baja calidad. Apoyando lo dicho por Rosenheim

(1996) que parasitoides jóvenes no deben desperdiciar huevos en hospederos de baja

calidad. Puesto que los parasitoides jóvenes tienen una carga ovárica alta, y que esta

influencia la oviposición de los parasitoides (Mangel 1989). Además, estos resultados

contradicen a Papaj (2000) quien menciona que el incremento en la carga ovárica reduce la

selectividad.

El parasitoide A. ervi (limitado en tiempo) bajo condiciones de ayuno y edad avanzada se

esperaba mostrara un comportamiento diferencial en la selección de hospederos de alta y

baja calidad y no como como lo menciona el paradigma. Sin embargo, nuestros resultados

del bioensayo con opción y los resultados de invernadero sin opción confirman el paradigma

establecido, ya que no hubo selectividad por parte del parasitoide, aceptando número similar

de hospederos de alta y baja calidad. Al respecto Drost & Carde (1992) mencionan que la

tasa de aceptación de hospederos de baja calidad podría incrementar si una especie es

privada de hospederos. Lo cual fue hecho al tener los parasitoides privados de hospederos

bajo los tratamientos establecidos y esto también pudo afectar en la respuesta de los

parasitoides. Pero los resultados del bioensayo sin opción muestran una alta selectividad por

hospederos de alta calidad coincidiendo con Wackers (1994) y Roitberg et al. (1993) quienes

mencionan que los parasitoides poseen diferentes preferencias y priorizan alguna

dependiendo de sus necesidades fisiológicas. Ya que, aunque el estado fisiológico era

distinto y aun así los parasitoides decidieron ovipositar en hospederos de alta calidad y no en

los de baja calidad.

Se esperaba que el parasitoide A. abdominalis, al ser una especie limitada en carga ovárica

parasitará y se comportará de manera diferencial frente a hospederos de baja o alta calidad

76

cuando el estado fisiológico fuera óptimo (i.e. alimentados y jóvenes) tal como indica el

paradigma de selección. Los resultados en el bioensayo con opción muestran que la

respuesta no es la esperada por el paradigma desde que los parasitoides no ovipositaron en

ninguno de los dos hospederos presentados (de alta y baja calidad). Respuesta que puede

atribuirse a los volátiles generados por los insectos. Dado que los volátiles de insectos

herbívoros y sus plantas hospederas sirven como señales confiables para la búsqueda de

hospederos de parasitoides (Hilker & McNeil 2008). Lo que pudo haber ocurrido al estar las

dos especies hospederas juntas, las sustancias que los identifican pudieron haberse

mezclado haciendo que el parasitoide se confundiera en el proceso de selección y tal vez al

detectar más señales del hospedero de baja calidad, decidió no ovipositar ninguno de los

hospederos suponiendo que ambos no eran de buena calidad. Puesto que como menciona

Vinson (1976), ciertas señales asociadas a una especie podrían influenciar la respuesta de la

otra especie cuando comparten un mismo sitio. Sin embargo, los resultados del bioensayo

sin opción en condiciones de laboratorio e invernadero muestran que existe una alta

selectividad hacia hospederos de alta calidad, esto apoya el paradigma de selección en

parasitoides limitados en carga ovárica (Heimpel & Rosenheim 1998) al asegurar que estos

van a aceptar solo hospederos de alta calidad. Y coincidiendo con Heimpel et al. (1996) al

decir que parasitoides con baja carga ovárica exhiben una alta selectividad de hospederos.

Para el parasitoide limitado en carga ovárica (A. abdominalis) en condiciones de ayuno y

edad avanzada (viejos) se esperaba que aceptarían de manera similar a hospederos de baja

y alta calidad, puesto que el estado fisiológico afectaría su comportamiento. Dado que se ha

observado que parasitoides en ayuno ovipositan más frecuentemente en hospederos de baja

calidad por su reducida esperanza de vida (Fletcher et al 1994). Sin embargo, los resultados

del bioensayo con opción muestran que los parasitoides no ovipositan en ningún hospedero

expuesto. Sugiriendo que los parasitoides detectan un ambiente con hospederos de baja

calidad y como se mencionó anteriormente señales asociadas a una especie podrían

influenciar la respuesta de otra especie cuando las especies hospederas distintas comparten

el mismo sitio (Vinson 1976). Los resultados en el bioensayo sin opción en laboratorio

mostraron que cuando los parasitoides estuvieron en ayuno-jóvenes y alimentados-viejos, no

mostraron la selectividad esperada por el paradigma. Resultados similares observados por

Hopper et al. (2013) muestran que factores como edad y ayuno, que hacen que la esperanza

de vida disminuya, no incrementaron la oviposición de Aphelinus rhamni Hopper & Woolley y

Aphelinus jurdjumovi Mercet (Hymenoptera:Aphelinidae) en hospederos de baja calidad.

77

Nuestros resultados para el tratamiento de parasitoides en ayuno-viejos del mismo

bioensayo sin opción en laboratorio mostró una alta selectividad, ovipositando más huevos

en hospederos de alta calidad y menos en hospederos de baja calidad. Este resultado podría

apoyar al modelo realizado por Mc Gregor (1997), el cual predice que la oviposición en

hospederos de baja calidad disminuye a cero en la etapa del parasitoide cercana al fin de su

vida. Confirmando que el estado fisiológico afecta fuertemente la habilidad de responder a

señales en el proceso de selección de hospedero por parte del parasitoide como Lewis et al.

(1990) mencionan. En los resultados del bioensayo sin opción en invernadero la selectividad

desapareció, ya que mostró número similar de oviposiciones en hospederos de alta y baja

calidad. Jenner et al. (2012) sugiere que parasitoides sinovigénicos al tener mayor edad y

por lo tanto menor esperanza de vida deben incrementar la oviposicion. Además, es

importante mencionar que la oviposición en tratamiento alimentados-viejos, en el bioensayo

sin opción en laboratorio e invernadero fue casi nula, lo que hace sugerir que en esta etapa

el parasitoide prefiere mantenerse vivo que reproducirse, tal y como Denis et al. (2013)

señalan.

78

CAPÍTULO 6. CONCLUSIONES GENERALES

Los resultados obtenidos permiten formular las siguientes conclusiones:

1. Se esperaba que el parasitoide A. ervi, al ser una especie limitada en tiempo parasitará y

se comportará de manera similar frente a hospederos de baja o alta calidad cuando el estado

fisiológico es óptimo (i.e. alimentados y jóvenes) tal como lo indica el paradigma de

selección. Los resultados en bioensayos con opción mostraron apoyan el paradigma desde

que el parasitoide se muestra como no-selectivo. Mientras que, en el bioensayo sin opción

en condiciones de laboratorio e invernadero, los parasitoides presentan un comportamiento

altamente selectivo, ovipositando más en hospederos de alta calidad.

2. Se esperaba que estado fisiológico del parasitoide A. ervi cuando fuesen hembras viejas o

en ayuno continuarían parasitando de manera similar a hospederos de baja y alta calidad.

Los resultados en bioensayos con opción mostraron que el parasitoide no es selectivo, tal

como el paradigma lo señala. Sin embargo, en los bioensayos sin opción en laboratorio, los

parasitoides muestran un comportamiento selectivo, ovipositando más en hospederos de alta

calidad que en hospederos de baja calidad. Finalmente, en condiciones de invernadero la

oviposición es similar en ambos hospederos, aunque en menor frecuencia que en su

condición óptima.

3. Se esperaba que el parasitoide A. abdominalis, al ser una especie limitada en carga

ovárica parasitará más en hospederos de alta calidad en comparación con los hospederos

de baja calidad cuando el estado fisiológico fuese óptimo (i.e. alimentados y jóvenes) tal

como indica el paradigma de selección. Los resultados en el bioensayo con opción muestran

que el parasitoide no oviposita en ningún hospedero, este resultado pudo deberse a la

confusión en la percepción de las señales por parte del parasitoide. Sin embargo, los

resultados del bioensayo sin opción concuerdan con el paradigma desde que el parasitoide

muestra un comportamiento selectivo hacia los hospederos de alta calidad. Confirmado así

por los resultados de invernadero donde la oviposición es más alta en hospederos de alta

calidad.

79

4. Se esperaba que el estado fisiológico del parasitoide A. abdominalis cuando fuesen

hembras viejas y/o en ayuno, aceptarían de manera similar a hospederos de baja y alta

calidad. Los resultados del bioensayo con opción no muestran una respuesta clara desde

que el parasitoide no ovipositó en ninguno de los dos hospederos. Sin embargo, en el

bioensayo sin opción en laboratorio se observa que los parasitoides cuando fueron en

ayuno-viejos prefirieron a hospederos de alta calidad sobre los de baja calidad. Y la

oviposición fue similar en hospederos de alta y baja calidad para parasitoides en ayuno-

jóvenes y alimentados-viejos, confirmando que el estado fisiológico modifica el

comportamiento de selección. Y en condiciones de invernadero se confirma el resultado que

muestra un comportamiento de oviposición similar en hospederos de alta y baja calidad,

aunque esta oviposición es casi nula.

80

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