Interaction entre la teigne du chou Plutella xylostella

(L.) et ses principaux parasitoïdes en conditions

tropicales : approche éthologique, écologique et évolutive
Laurence Arvanitakis

To cite this version:

Laurence Arvanitakis. Interaction entre la teigne du chou Plutella xylostella (L.) et ses principaux
parasitoïdes en conditions tropicales : approche éthologique, écologique et évolutive. Sciences agricoles.
Université Paul Valéry - Montpellier III, 2013. Français. �NNT : 2013MON30067�. �tel-00984578�

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 Délivré par l’Université Paul-Valéry-Montpellier 3

Préparée au sein de l’école doctorale Territoires, Temps,

Sociétés et Développement - ED 60
Et de l’unité de recherche UMR 5175 CEFE-CNRS

Spécialité : Écologie et Biologie des Populations

Présentée par ARVANITAKIS Laurence

INTERACTION ENTRE LA TEIGNE DU CHOU

PLUTELLA XYLOSTELLA (L.) ET SES PRINCIPAUX
PARASITOIDES EN CONDITIONS TROPICALES :
APPROCHE ETHOLOGIQUE, ECOLOGIQUE
ET EVOLUTIVE.

Soutenue le 19 décembre 2013 devant le jury composé de

Mme Marie-Stéphane TIXIER, Professeur, SupAgro Montpellier Rapporteur

M. Olivier BONATO, CR1 (HDR), IRD Rapporteur

Mme Anne-Nathalie VOLKOFF, DR, UMR 1333 INRA, UM2 Examinateur

Mme Marie-Claude BON, Chercheur, USDA-ARS-EBCL Examinateur

M. Jean-Pierre LUMARET, Professeur, UM3, UMR 5175 CEFE Co-Directeur de thèse

M. Dominique BORDAT, Chercheur (HDR), CIRAD UR HortSyst Co-Directeur de thèse

Résumé

L'espèce Plutella xylostella (L.) (Lepidoptera : Plutellidae) défoliatrice des choux constitue
surtout un problème dans les régions tropicales et subtropicales. La lutte chimique a
rapidement montré ses limites du fait de l'apparition de résistance dans les populations. Des
moyens de lutte alternatifs ont été mis en place, impliquant principalement des insectes
parasitoïdes, parmi lesquels Oomyzus sokolowskii (Kurdjumov) (Hymenoptera : Eulophidae)
et Cotesia vestalis (Haliday) (Hymenoptera : Braconidae) qui sont les plus couramment
utilisés en raison de leur spécificité envers P. xylostella. Afin de contribuer à une meilleure
connaissance du contrôle de la teigne en région tropicale, nous avons étudié les relations hôte-
parasitoïde entre P. xylostella et O. sokolowskii d’une part, et P. xylostella et C. vestalis
d’autre part, en conditions de laboratoire et sur le terrain au Sénégal et au Bénin. Au Sénégal,
quatre espèces de parasitoïdes sont présentes sur les chenilles : O. sokolowskii, Apanteles
litae, C. vestalis et Brachymeria citrae. Au Bénin, seule l'espèce C. vestalis est présente. Au
Sénégal comme au Bénin, les facteurs climatiques contribuent au développement de la teigne
et les précipitations ne régulent pas les populations du ravageur. Dans ces deux pays, la teigne
n’est pas contrôlée par ses ennemis naturels. La lutte biologique par conservation y est à
prendre en considération et l’utilisation de plantes compagnes cultivées en association avec le
chou peut être envisagée pour réduire les populations de la teigne. Les études en laboratoire
ont montré qu’Oomyzus sokolowskii est un parasitoïde larvo-nymphal performant. Concernant
C. vestalis, les femelles détectent et reconnaissent leur hôte grâce aux lipides cuticulaires émis
par les chenilles. Des marqueurs moléculaires (isozymes et ISSR) ont confirmé une forte
variabilité entre les populations de P. xylostella à l’échelle mondiale, les populations
d’Australie et du Japon étant très différentes des autres et formant deux groupes distincts. La
structuration des populations semble influencée par le type de climat : tropical et non tropical.

Mots-clés : Plutella xylostella, Oomyzus sokolowskii, Cotesia vestalis, interaction hôte-

parasitoïde, agroécosystème tropical, Brassicacées, lutte biologique.

I
Abstract

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is the most
destructive pest of Brassicaceae worldwide and poses particularly acute problems in tropical
areas. Chemical control is impaired by multiple-insecticide resistance in this species.
Alternative methods are based on biological control by parasitoids, such as Oomyzus
sokolowskii (Kurdjumov) (Hymenoptera: Eulophidae) and Cotesia vestalis (Haliday)
(Hymenoptera: Braconidae), which are commonly used due to their specificity towards DBM.
To help to improve the biocontrol of the moth in the tropics, we studied host-parasitoid
relationships between P. xylostella and these two parasitoids under both laboratory and field
conditions in Senegal and Benin. In both countries, climatic conditions are favourable for the
development of DBM and rainfall does not limit populations of this pest. In Senegal, four
parasitoid species are present on DBM larvae: O. sokolowskii, C. vestalis, Apanteles litae, and
Brachymeria citrae. In Benin, C. vestalis is largely dominant. In neither of these countries,
the moth is sufficiently controlled by natural enemies. Conservation biological control might
be combined with the use of companion plants in cabbage crops to reduce DBM populations.
Laboratory studies have shown that O. sokolowskii is an efficient larval-pupal parasitoid. In
C. vestalis, females detect and recognize their host using cuticular lipids produced by the
caterpillar. Studies of molecular markers (isozymes and ISSR) have confirmed high
variability among DBM populations around the world, those from Australia and Japan being
distinct and very different from any other population. Population structure seems to be
influenced by the type of climate (tropical vs. non-tropical).

Keywords: Plutella xylostella, Oomyzus sokolowskii, Cotesia vestalis, host-parasitoid

interactions, tropical agroecosystems, Brassicaceae, biological control.

II
Il est mon ami et mon maître,

Au-delà des apparences, il est profondément humain,

Au-delà de la passion, l’entomologie est sa vocation,

Sincère, curieux, obstiné, battant et sensible,

Il cache ses talents d’artiste peintre et de photographe,

Il a souvent été seul contre tous,

Il m’a tout appris de mon métier,

Je ne lui serais jamais assez reconnaissante.

Dominique

Je te dédie ce manuscrit

Avec toute ma gratitude et mon amitié

III
Ils ont aussi contribué à ce que je suis aujourd’hui…

Cette icône appartenait à mon arrière

grand-mère Parasquevi Arvanitakis. Je
tiens à lui rendre hommage, ainsi qu’à
tous mes aïeux. Depuis bien longtemps
déjà, ils ne sont plus de ce monde, mais je
sais, qu’ils seraient fiers de moi.

Cette icône représente la Présentation du

Christ au temple. Si j’en fais une
interprétation personnelle, ce manuscrit
représentera le jour de ma soutenance, la
présentation d’un travail de recherche de
plusieurs années, à mes pairs.
Icône grecque du début du XXe siècle

Je rends hommage à mon grand-

père Arthur-René Lauzière. Je lui
dois en partie ma passion pour
l’entomologie et ma fascination
depuis mon plus jeune âge pour les
cigales. Lou soulèu me fai canta.
La cigale Lyristes plebejus (Scopoli)
Aquarelle 1808

Je rends hommage à Maryse Fromen

avec qui j’entretiens depuis bientôt
trente ans une fidèle et sincère
amitié. Professeur de biologie, elle a
su me transmettre sa passion pour
la nature et son intérêt pour les
sciences naturelles.
Feuille de Ginkgo biloba récoltée au jardin des plantes
de Montpellier, 1984. Classe 2nde, Lycée La Frondaie,
Castelnau-le-lez

IV
 Remerciements

Cette thèse sur articles est la synthèse de plusieurs travaux effectués et co-publiés avec
plusieurs doctorants qui se sont succédé dans le laboratoire au sein duquel je travaille depuis
plus de 15 ans.

Dans ce contexte professionnel, j’ai choisi de rédiger un mémoire de thèse pour

valoriser une partie des travaux auxquels j’ai été passionnément impliquée durant plusieurs
années. Ce mémoire représente également un moyen d’obtenir la reconnaissance de mes pairs
dans ma discipline.

Je souhaite remercier en premier lieu mes deux codirecteurs de thèse : Jean-Pierre

LUMARET, Professeur à l’Université Paul-Valéry Montpellier III et Dominique BORDAT,
entomologiste (HDR) au CIRAD. Je tiens à les remercier tout particulièrement, car sans leur
confiance et leur soutien, ce mémoire n’aurait jamais eu le mérite d’exister. C’est donc à ce
titre, que je leurs exprime toute ma reconnaissance. Merci encore.

Je suis reconnaissante à Mme Marie-Stéphane TIXIER, Professeur à SupAgro

Montpellier et à M. Olivier BONATO, Chargé de Recherche (HDR) à l’IRD, pour avoir
accepté d’être les rapporteurs de cette thèse. Je remercie également les autres membres du
jury, Mme Marie-Claude BON, Chercheur à l’USDA-ARS-EBCL à Montpellier et Mme
Anne-Nathalie VOLKOFF, Directeur de Recherche à l’INRA.

Je remercie également les doctorants. Un grand merci à Thomas, à Apolline, à Olivier,

à Gallo et à Babacar. J’ai partagé d’agréables moments avec eux et appris beaucoup sur la
teigne des Brassicacées et sur ses parasitoïdes. Ce travail est un peu le votre.

Je tiens à exprimer toute mon amitié à Ernest GOUDEGNON, Professeur à

l’Université d’Abomey-Calavi au Bénin. Merci de m’avoir fait connaitre ton pays. C’était la
première fois que je posais le pied sur le continent africain. J’en garde un souvenir
inoubliable.

V
 Un grand merci à Jean-François DAVID, Chercheur au CNRS-CEFE à Montpellier.
Merci pour ta patience et ton investissement pour m’aider à achever l’article. J’espère que tu
ne détestes pas complètement Plutella !

Pour terminer, j’exprime toute mon affection et ma gratitude à ma famille. Je remercie

mes parents, pour avoir toujours été solidaires et avoir cru en ma détermination. Je vous dédie
également ce mémoire. Un grand merci également à ma sœurette pour son aide précieuse dans
la mise en page.

Pardonnes moi, Philippe, pour avoir perturbé tes nuits pendant que je rédigeais. Merci
pour ta compréhension et ton soutien sans limite. Merci pour tout ton amour.
J’embrasse Alexandre, notre petit garçon.

Et un grand merci à tous mes proches qui ont cru en moi. Merci Jojo.

A vous tous merci !!

VI
 La connaissance n’est que provisoire et jamais absolue.

Socrate

Le commencement de toutes les sciences, c’est l’étonnement

de ce que les choses sont ce qu’elles sont.

Aristote

Il n’est pas d’un homme raisonnable de blâmer

par caprice l’étude des insectes,

ni de s’en dégoûter par la considération des peines qu’elle donne.

La nature ne renferme rien de bas.

Tout y est sublime, tout y est digne d’admiration.

Aristote

VII
 TABLE DES MATIERES

Résumé I
Abstract II
Remerciements V
Liste des articles XII
Table des illustrations XIII
Liste des annexes XIV

INTRODUCTION GENERALE 1

CHAPITRE I : Présentation du modèle biologique étudié

1. Le ravageur : Plutella xylostella (L.) 13

1.1. Systématique 13
1.2. Origine, répartition géographique et migration 13
1.3. Spectre de plantes-hôtes 15
1.4. Morphologie, biologie et écologie 17
1.5. Les moyens de lutte contre Plutella xylostella 19
1.5.1. La lutte chimique 22
1.5.2. La lutte biologique 23
1.5.3. Les autres méthodes de lutte 29
1.5.4. La lutte intégrée 31

2. Les parasitoïdes 34

2.1. Oomyzus sokolowskii (Kurdjumov) 34

2.1.1. Systématique 34
2.1.2. Morphologie, biologie et écologie 34
2.1.3. Répartition géographique 36

2.2. Cotesia vestalis (Haliday) 38

2.2.1. Systématique 38
2.2.2. Morphologie, biologie et écologie 38
2.2.3. Répartition géographique 41

IX
 CHAPITRE II : Interaction entre Plutella xylostella et le parasitoïde
Oomyzus sokolowskii

1. Introduction 45
2. Etude de terrain : le Sénégal 46
2.1. Présentation générale 46
2.2. Statut de Plutella xylostella au Sénégal 47
2.3. Site d’expérimentation 48

3. Synthèse des résultats 50

4. Conclusion 51

Article 1 55
The relationship between the diamondback moth, climatic factors, cabbage crops and natural
enemies in a tropical area. Gallo SOW, Karamoko DIARRA, Laurence ARVANITAKIS and
Dominique BORDAT. Folia Horticulturae, 2013, 25: 3-12

Article 2 67
Life history traits of Oomyzus sokolowskii Kurdjumov (Hymenoptera: Eulophidae), a
parasitoid of the diamondback moth. Gallo SOW, Laurence ARVANITAKIS, Saliou
NIASSY, Karamoko DIARRA and Dominique BORDAT. African Entomology, 2013, 21:
231-238

Article 3 77
Performance of the parasitoid Oomyzus sokolowskii (Hymenoptera: Eulophidae) on its host
Plutella xylostella (Lepidoptera: Plutellidae) under laboratory conditions. Gallo SOW,
Laurence ARVANITAKIS, Saliou NIASSY, Karamoko DIARRA and Dominique BORDAT.
International Journal of Tropical Insect Science, 2013, 33: 38-45.

CHAPITRE III : Interaction entre Plutella xylostella et le parasitoïde

Cotesia vestalis

1. Introduction 87
2. Etude de terrain : le Bénin 89
2.1. Présentation générale 89
2.2. Statut de Plutella xylostella au Bénin 89
2.3. Site d’expérimentation 91

3. Synthèse des résultats 92

4. Conclusion 93

X
Article 4 97
Incomplete control of the diamondback moth, Plutella xylostella, by the parasitoid Cotesia
vestalis in a cabbage field under tropical conditions. Laurence ARVANITAKIS, Jean-
François DAVID and Dominique BORDAT. Soumis à BioControl.

Article 5 115
Antennal structure and oviposition behavior of the Plutella xylostella specialist parasitoid:
Cotesia plutellae. Olivier ROUX, Joan Van BAAREN, Charles GERS, Laurence
ARVANITAKIS and Luc LEGAL. Microscopy Research and Technique, 2005, 68:36-44.

Article 6 127
Chemical characterization of contact semiochemicals for host-recognition and host-
acceptance by the specialist parasitoid Cotesia plutellae (Kurdjumov). Olivier ROUX,
Charles GERS, Josephe Nathan TENE-GHOMSI, Laurence ARVANITAKIS, Dominique
BORDAT and Luc LEGAL. Chemoecology, 2007, 17: 13-18.

CHAPITRE IV : Structuration génétique de Plutella xylostella

1. Introduction 135
2. Synthèse des résultats 136
3. Conclusion 137

Article 7 141
Genetic differentiation among various populations of the diamondback moth, Plutella
xylostella (Lepidoptera : Yponomeutidae). Apolline PICHON, Laurence ARVANITAKIS,
Olivier ROUX, Alan KIRK, Dominique BORDAT and Luc LEGAL. Bulletin of
Entomological Research, 2006, 96: 137-144.

Article 8 151
ISSR-PCR: Tool for discrimination and genetic structure analysis of Plutella xylostella
populations native to different geographical areas. Olivier ROUX, M. GEVREY, Laurence
ARVANITAKIS, Charles GERS, Dominique BORDAT and Luc LEGAL. Molecular
phylogenetics and Evolution, 2007, 43:240-250

DISCUSSION GENERALE & PERSPECTIVES 165

REFERENCES BIBLIOGRAPHIQUES 175

ANNEXES 207

XI
 LISTES DES ARTICLES INCLUS DANS LA THESE

A1 : Sow G., Diarra K., Arvanitakis L., Bordat D. 2013. The relationship between the
diamondback moth, climatic factors, cabbage crops and natural enemies in a tropical
area. Folia Horticulturae, 25/1: 3-12.

A2 : Sow G., Arvanitakis L., Niassy S., Diarra K., Bordat D. 2013. Life history traits of
Oomyzus sokolowskii Kurdjumov (Hymenoptera: Eulophidae), a parasitoid of the
diamondback moth. African Entomology, 21 (2): 231-238.

A3 : Sow G., Arvanitakis L., Niassy S., Diarra K., Bordat D. 2013. Performance of the
parasitoid Oomyzus sokolowskii (Hymenoptera: Eulophidae) on its host Plutella xylostella
(Lepidoptera: Plutellidae) under laboratory conditions. International Journal of Tropical
Insect Science, 33 (1): 38-45.

A4 : Arvanitakis L., David J-F., Bordat D. Incomplete control of the diamondback moth,
Plutella xylostella, by the parasitoid Cotesia vestalis in a cabbage field under tropical
conditions. Soumis à BioControl.

A5 : Roux O., Van Baaren J., Gers C., Arvanitakis L., Legal L. 2005. Antennal structure and
oviposition behavior of the Plutella xylostella specialist parasitoid: Cotesia plutellae.
Microscopy Research and Technique, 68: 36-44.

A6 : Roux O., Gers C., Tene-Ghomsi J. N., Arvanitakis L., Bordat D., Legal L. 2007.
Chemical characterization of contact semiochemicals for host-recognition and host-
acceptance by the specialist parasitoid Cotesia plutellae (Kurdjumov). Chemoecology, 17: 13-
18.

A7 : Pichon A., Arvanitakis L., Roux O., Kirk A.A., Alauzet C., Bordat D., Legal L. 2006.
Genetic differentiation among various populations of the diamondback moth, Plutella
xylostella (Lepidoptera : Yponomeutidae). Bulletin of Entomological Research, 96: 137-144.

A8 : Roux O., Gevrey M., Arvanitakis L., Gers C., Bordat D., Legal L. 2007. ISSR-PCR:
Tool for discrimination and genetic structure analysis of Plutella xylostella populations native
to different geographical areas. Molecular phylogenetics and Evolution, 43: 240-250.

XII
 TABLE DES ILLUSTRATIONS (hors articles)

LISTE DES FIGURES

N°
1 Predicted worldwide distribution of diamondback moth (DBM) based on a
validated bioclimatic model (Zalucki & Furlong 2011) 14

2 Les différents stades pré-imaginaux de P. xylostella 18

3 Adultes de P. xylostella 19

4 Cycle de développement de P. xylostella 20

5 Culture de choux au Sénégal dont les feuilles sont entièrement perforées par
les chenilles de P. xylostella 21

6 Chou entièrement détruit par les chenilles de P. xylostella au Bénin. Les

feuilles sont réduites à de la dentelle 21

7 Principaux agents de lutte biologique utilisés contre les différents stades de

P. xylostella (Sarfraz et al. 2005) 27

8 Couple d’adultes d’O. sokolowskii (× 20) (femelle à gauche et mâle à droite) 35

9 Plusieurs adultes d’O. sokolowskii émergeant d’une nymphe de P. xylostella 37

10 Cycle de développement de C. vestalis à 20°C (Delucchi et al. 1954) 40

11 Les différents espaces agricoles du Sénégal 47

.
12 Localisation du site de Malika dans la zone périurbaine de Dakar 49

13 Parcelle de choux sur le site de Malika, dans la zone périurbaine de Dakar 49

14 Carte du Bénin avec localisation (en rouge) du site d’étude dans la zone
Périurbaine de Cotonou 90

15 Parcelle de choux sur le site de Kouhounou, dans la zone périurbaine de

Cotonou 91

XIII
LISTE DES TABLEAUX

N°
1 Production de Brassicacées dans le monde en 2011 (FAOSTAT 2013) 2

2 Espèces de Brassicacées les plus consommées 16

3 Principales espèces d’hyménoptères parasitoïdes de P. xylostella 29

4 Durée du cycle biologique d’O. sokolowskii à différentes températures

(Wang et al. 1999) 36

5 Liste des pays où la présence d’O. sokolowskii est confirmée (Delvare 2004 ;
Talekar 2004 ; Shelton et al. 2008) 37

6 Liste des pays où la présence de C. vestalis est confirmée (Delvare 2004 ;

Talekar 2004 ; Shelton et al. 2008) 41

LISTE DES ENCADRES

I Les Glucosinolates 16

II Integrated Pest Management (IPM) 32

LISTE DES ANNEXES

ANNEXE 1
Communication affichée, VII Conférence Internationale Francophone d'Entomologie,
Louvain la Neuve, Belgique, 2010. 207

ANNEXE 2
Communication affichée, Colloque National des Entomophagistes, Montpellier,
France, 2012. 209

XIV
INTRODUCTION GENERALE
 Introduction générale

La culture des Brassicacées est une des productions agricoles les plus importantes au
monde. D’après les données FAO (FAOSTAT 2013), 37 millions d’hectares on été cultivés
en 2011 avec une production annuelle globale de 152 millions de tonnes uniquement pour le
chou, le chou-fleur et le colza. Elles sont essentiellement produites en Asie, en Europe et en
Amérique du Nord (Tableau 1). Les deux tiers de cette production sont localisés en Asie, où
ces légumes représentent parfois la principale source alimentaire des populations (Grzywacz
et al. 2010). De nombreuses régions africaines et sud américaines en voie de développement
dépendent principalement de cette culture.
Cependant, la production de Brassicacées est sérieusement affectée par un nombre
important de ravageurs dont le principal est communément appelé « Teigne du chou »,
Plutella xylostella (Linné 1758) (Lepidoptera : Plutellidae). Les chenilles de ce micro-
lépidoptère sont défoliatrices et peuvent causer jusqu’à 90% de perte de production (Talekar
& Shelton 1993 ; Sarfraz et al. 2005). Cette espèce, originaire de la région Méditerranéenne
(Hardy 1938), est devenue cosmopolite et se rencontre partout où sont présentes les
Brassicacées cultivées et sauvages (Shelton 2004). Cette espèce envahissante est surtout un
problème dans les régions tropicales et subtropicales où la culture de chou se conduit toute
l’année. Les conditions climatiques très favorables à son développement, associées à un fort
potentiel reproductif, permettent à P. xylostella d’avoir plus de 20 générations par an (Vickers
et al. 2004).
La lutte engagée contre la teigne du chou fut d’abord axée sur l’utilisation de
traitements chimiques à base d’insecticides de synthèses qui, très vite, ont montré leurs
limites en se heurtant à l’étonnante capacité de résistance des populations de cette espèce à
toutes les familles de produits existants (Cheng 1988), y compris les biopesticides à base de
Bacillus thuringiensis (Berliner) (ou « Bt ») (Tabashnik et al. 1990 ; Sanchis et al. 1995).
Cette course entre les agriculteurs et le ravageur conduit à l’augmentation incessante des
doses de produits. L’estimation du coût annuel dans l’économie mondiale pour lutter contre la
teigne est estimée à plus de 4 milliards de dollars (Zalucki et al. 2012). Pour faire face à ce
problème, il a donc été nécessaire de trouver des méthodes alternatives permettant de
contrôler le niveau des populations de P. xylostella, tout en respectant l’environnement et la
santé des agriculteurs.
En région tempérée, la lutte intégrée (Integrated Pest Management ou IPM) a donné
des résultats encourageant en combinant l’utilisation de plusieurs pratiques compatibles entre
elles, telles que la rotation des cultures, les cultures intercalaires, la confusion ou le piégeage
sexuel, la sélection variétale et la lutte biologique à l’aide d’antagonistes.

1
 Introduction générale

Tableau 1: Production de Brassicacées dans le monde en 2011 (FAOSTAT 2013)

Production (en tonnes)

Monde Asie Europe Amérique du Nord

Chou 68 840 531 51 859 189 12 267 797 1 154 102

Chou-fleur et Brocoli 20 876 817 17 000 754 2 355 945 359 253
Colza 62 454 482 22 544 274 22 306 360 14 863 410

Surfaces cultivées (en hectares)

Monde Asie Europe Amérique du Nord

Chou 2 373 818 1 741 982 428 962 34 077

Chou-fleur et Brocoli 1 209 106 895 725 137 042 17 232
Colza 33 645 342 14 641 931 8 809 291 7 893 920

Parmi ces stratégies, la lutte biologique est de plus en plus utilisée et elle constitue le
moyen de lutte le plus efficace à grande échelle contre les populations de P. xylostella
résistantes aux insecticides (Lim 1992). Plusieurs agents de lutte, comme les virus, les
bactéries, les champignons, les prédateurs et les parasitoïdes, ont été recensés contre la teigne
(Delvare 2004), mais les parasitoïdes sont de loin les plus utilisés, les plus étudiés et les plus
efficaces.
Actuellement en Afrique, la teigne reste difficilement contrôlable, à l’exception de la
région du Cap en Afrique du sud où l’on trouve une grande diversité de parasitoïdes et un
taux de parasitisme proche de 90% (Smith & Villet 2004). Dans les autres régions,
notamment en Afrique de l’Ouest, la faune parasitaire associée à P. xylostella est relativement
pauvre. L’efficacité des parasitoïdes y est peu satisfaisante et les taux de parasitisme sont
faibles (Lörh & Kfir 2004).
Parmi ces parasitoïdes, Cotesia vestalis (Haliday) (Hymenoptera : Braconidae) et
Oomyzus sokolowskii (Kurdjumov) (Hymenoptera : Eulophidae) sont les plus couramment
rencontrés en zones tropicales, notamment en raison de leur tolérance aux températures
élevées, et aussi les plus utilisés en lutte biologique en raison de leur spécificité envers P.

2
 Introduction générale

xylostella. Pour autant, certaines de leurs introductions n’ont pas toujours été efficaces et les
échecs ne trouvent pas toujours d’explications (Cock 1985 ; Chaves et al. 1993). Dans la
pratique, le contrôle biologique est souvent aléatoire et les stratégies d’introductions sont
basées sur des critères peu pertinents. Néanmoins depuis de nombreuses années, ces deux
espèces sont présentes au Bénin et au Sénégal, avec des taux de parasitisme qui varient selon
les saisons, entre 5% et plus de 80% (Bordat & Goudegnon 1997 ; Sall-Sy et al. 2004 ;
Goudegnon et al. 2004). Cependant ces taux de parasitisme ne permettent pas pour autant un
contrôle suffisant de la teigne.

C’est à partir de ce constat que nous avons construit la problématique de notre travail.
Sans avoir la prétention de vouloir régler définitivement ce problème, nous voulons essayer
de contribuer à améliorer le contrôle biologique de la teigne du chou en région tropicale.
En effet, il s’agit d’essayer de comprendre pourquoi ce ravageur est aussi difficile à
contrôler dans ces conditions climatiques particulières et pourquoi ses parasitoïdes ne sont pas
suffisamment efficaces. D’une manière générale, on a tendance à faire reposer entièrement le
succès comme l’échec du contrôle biologique uniquement sur les caractéristiques biologiques
du parasitoïde, sans se préoccuper de la part du ravageur dans l’interaction hôte-parasitoïde.
Les hypothèses le plus souvent avancées mettent en cause des phénomènes de compétition
interspécifique, de mal-adaptation du parasitoïde à son environnement ou encore l’usage
d’insecticides par les agriculteurs. A ce jour, aucune réponse n’est satisfaisante et on ne s’était
pas réellement penché sur ce problème, ce que nous avons essayé de faire ici.
Mieux comprendre le fonctionnement de l’interaction hôte-parasitoïde peut représenter
à terme l’une des clés de la gestion raisonnée des introductions de parasitoïdes en lutte
biologique classique ou par le maintien des espèces déjà établies (lutte biologique par
conservation), et donc d’augmenter l’efficacité dans le contrôle du ravageur concerné.

Nous avons choisi d’étudier le modèle biologique Plutella xylostella - Oomyzus

sokolowskii et Cotesia vestalis. Dans le contexte de cette relation hôte-parasitoïde, nous nous
sommes intéressés d’une part à l’hôte et à ses deux parasitoïdes, et d’autre part à l’influence
de l’environnement en condition tropicale sur l’interaction entre ces trois espèces.
Dans la mesure où ce travail traite essentiellement des relations hôte-parasitoïde, il
nous a semblé important d’exposer d’abord les différentes caractéristiques qui régissent ce
type de relation.

3
 Introduction générale

Les insectes parasitoïdes

Le « parasitoïdisme » (Wajnberg & Ris 2007) est un mode de vie à l’interface entre la
prédation et le parasitisme. Comme les parasites, les insectes parasitoïdes dépendent aussi
d’un hôte (le plus souvent un autre insecte) dont ils vont tirer les ressources nécessaires à leur
développent (Godfray 1994). Cependant, seuls les stades pré-imaginaux nécessitent un tel
mode de vie (Askew 1971). Les adultes mènent en général une vie libre pendant laquelle les
femelles recherchent activement leurs hôtes pour y déposer leur progéniture. Les stades
immatures tirent leur substance de ces hôtes et les tuent directement ou indirectement à l’issue
de leur développement (Eggleton & Gaston 1990). Les parasitoïdes représenteraient entre 8 et
20 % des espèces d’insectes décrites à ce jour, la majorité appartenant soit à l’ordre des
hyménoptères (environ 50 000 espèces décrites), soit à celui des diptères (environ 16 000
espèces connues) (Feener & Brown 1997). Des parasitoïdes ont été également signalés chez
les coléoptères, les lépidoptères, les trichoptères, les neuroptères et les strepsiptères (Quicke
1997).

Quelque caractéristiques écologiques des parasitoïdes

Les parasitoïdes infestent principalement d’autres insectes. Cependant, certaines
espèces sont capables de parasiter d’autres arthropodes mais aussi des plathelminthes, des
annélides, des mollusques et même certains chordés (Feener & Brown 1997). Certains de ces
hôtes sont parfois eux-mêmes des parasitoïdes et on parle alors d’hyperparasitisme. Le stade
parasité est variable d’une espèce à l’autre, ce qui permet, lorsqu’il s’agit de parasitoïdes
d’insectes, de définir globalement des parasitoïdes d’œufs, de larves, de nymphes, voire dans
quelques cas d’adultes. Dans certains cas, le parasitisme peut avoir lieu au stade d’œuf, mais
avec un développement du parasitoïde suffisamment lent ou différé pour s’achever beaucoup
plus tardivement lorsque l’hôte a atteint un stade larvaire (parasitoïdes ovo-larvaires) ou le
stade nymphal (parasitoïdes ovo-nymphals). Le nombre d’espèces susceptibles d’être
infestées avec succès varie considérablement d’une espèce à l’autre. Par exemple, certains
Tachinides sont hautement généralistes (ou polyphages) et peuvent parasiter plusieurs
dizaines d’espèces hôtes dans des familles différentes (Stireman et al. 2006). De nombreuses
espèces sont en revanche spécialisées sur une ou quelques espèces hôtes seulement. Selon les
espèces, les stades immatures (œufs, larves) des parasitoïdes peuvent se développer soit à
l’intérieur, soit à l’extérieur de leurs hôtes : on parle respectivement d’endoparasitoïdes ou
d’ectoparasitoïdes. La possibilité de se développer ou non en présence d’un ou plusieurs
congénères aux dépens d’un même hôte conduit à faire la distinction entre les parasitoïdes

4
 Introduction générale

solitaires ou grégaires. Chez certains parasitoïdes solitaires, les femelles ont une fonction
discriminatoire qui leur permet de différencier les hôtes sains des hôtes déjà parasités par elle-
même, par une autre femelle de leur espèce ou par celle d’une autre espèce. Il en résulte le
développement d’une seule larve par hôte, sachant que la première pondue dévore ou élimine
par des moyens chimiques les larves les plus jeunes (Wajnberg & Ris 2007). Chez les espèces
grégaires, plusieurs adultes émergeront d’un seul hôte. Cela nécessite une évaluation par la
femelle de la taille de l’hôte pour que les ressources qu’il représente puissent suffire au
développement complet de tous les congénères (Wajnberg & Ris 2007). L’haplo-diploïdie est
le système qui prédomine chez la plupart des hyménoptères parasitoïdes. Les femelles
(diploïdes) sont issues d’ovocytes normalement fécondés tandis que les mâles fertiles
proviennent d’ovocytes non fécondés (parthénogenèse arrhénotoque) et sont par conséquent
haploïdes, avec un patrimoine génétique uniquement d’origine maternelle. Une conséquence
de ce mode de reproduction est la possibilité, pour les femelles fécondées, de « choisir » la
proportion des sexes dans leur descendance en fécondant ou non les œufs qu’elles pondent.
La durée relative de l’interaction entre l’hôte et le parasitoïde est également importante et l’on
distingue ainsi les espèces idiobiontes qui tuent et exploitent rapidement leurs hôtes, des
espèces koïnobiontes qui permettent à leur hôte de continuer plus ou moins normalement leur
développement avant de succomber sous l’effet du parasitisme (Askew & Shaw 1986). Chez
l’adulte parasitoïde, la disponibilité des œufs varie également de façon très nette, certaines
espèces disposant de la totalité de leurs œufs matures dès l’émergence de la femelle (espèces
pro-ovogéniques) tandis que d’autres produisent des nouveaux œufs tout au long de leur vie
(espèces syn-ovogéniques). En fait, la distinction est probablement beaucoup moins nette et il
semble exister en pratique un continuum entre ces deux stratégies extrêmes (Jervis et al.
2001).

Les étapes comportementales aboutissant au parasitisme

Pour qu’un parasitoïde réussisse son infestation et son développement, il est
communément admis que plusieurs étapes chronologiques doivent être franchies avec succès
(Doutt 1959 ; Vinson 1976). Elles correspondent à deux grandes parties du cycle de vie de ces
insectes. La première correspond à la perception, par une femelle, d’une série de stimuli qui
vont lui permettre de réduire progressivement son aire de recherche pour aboutir à la
découverte d’un hôte et à son acceptation en tant que site de ponte (on parle alors
d’oviposition). Les étapes de cette première phase sont qualifiées de pré-ovipositionnelles et
dépendent du comportement des femelles adultes (Vinson 1981). La seconde partie, qui

5
 Introduction générale

concerne les stades immatures se développant dans l’hôte, est qualifiée de post-
ovipositionnelle et met en œuvre des mécanismes liés à la physiologie de l’association entre
les deux partenaires (Vinson & Iwantsch 1980a, b).
Etant donné que nous nous sommes intéressés à l’une des étapes qui correspond à la
reconnaissance de l’hôte par le parasitoïde (Cf. Chapitre III), nous décrirons uniquement la
première phase (étapes pré-ovipositionnelles).

Les étapes pré-ovipositionnelles

C’est au cours de ces étapes qu’une femelle parasitoïde adulte partira à la recherche
d’hôtes pour y déposer une descendance. Les mécanismes impliqués dans ces étapes pré-
ovipositionnelles reposent sur les caractéristiques écologiques des niches occupées et les
caractéristiques comportementales des deux partenaires. Ces mécanismes vont déterminer la
capacité des femelles parasitoïdes à découvrir et à attaquer leurs hôtes et vont donc
conditionner l’impact parasitaire sur la dynamique des populations hôtes (Wajnberg & Ris
2007).

• Recherche de l’habitat de l’hôte

Une femelle parasitoïde adulte récemment émergée doit, dans un premier temps, partir
à la recherche d’habitats potentiellement colonisés par des hôtes. De nombreux travaux
démontrent le rôle important joué par les caractéristiques visuelles, acoustiques et surtout
olfactives de l’habitat des hôtes dans sa détection par les femelles parasitoïdes (Vinson 1976,
1981). Certaines plantes émettent, en cas d’attaque par des phytophages, des molécules
caractéristiques, appelées synomones, qui peuvent être utilisées par des parasitoïdes pour
trouver de façon précise un habitat infesté par leurs hôtes (Turlings et al. 1990).

• Recherche de l’hôte
Une fois un site potentiellement habitable trouvé, la femelle doit commencer à
rechercher un hôte proprement dit à partir de stimuli parfois acoustiques ou visuels, mais
généralement plutôt chimiques et olfactifs qui proviennent ici des hôtes eux-mêmes. Ces
signaux sont qualifiés de kairomones (Wajnberg & Ris 2007). Il s’agit, par exemple, de
phéromones sexuelles impliquées dans la recherche et la reconnaissance de partenaires pour la
reproduction. Dans ce cas, la femelle parasitoïde agit comme un véritable espion en
détournant à son avantage des signaux qui ne lui sont pas initialement destinés (Noldus 1989).

6
 Introduction générale

• Acceptation de l’hôte
Une fois l’hôte trouvé, la femelle doit encore s’assurer que celui-ci peut convenir au
développement de sa descendance. Autant de questions auxquelles la femelle va devoir
répondre grâce encore à des signaux physiques (taille, forme, couleur) ou chimiques
provenant de l’hôte découvert (Wajnberg & Ris 2007). Ces signaux peuvent être situés à
l’extérieur de l’hôte et sont perçus par le parasitoïde grâce à de nombreux récepteurs situés sur
ses antennes qui sont richement innervées (Wajnberg & Ris 2007). Ils peuvent aussi se situer
à l’intérieur de l’hôte et sont détectés grâce à des organes sensoriels situés sur l’ovipositeur
(organe de ponte) lors de son insertion dans l’hôte (Wajnberg & Ris 2007). De puissantes
interactions sélectives entre les deux partenaires ont probablement contribué à la riche
diversité des stratégies adoptées par les femelles parasitoïdes.
Chez de nombreuses espèces, les informations recueillies par les femelles parasitoïdes
permettent également de détecter des hôtes déjà parasités, soit par elles-mêmes
précédemment, soit par une ou plusieurs autres femelles conspécifiques ou d’une autre
espèce. La décision de pondre dans un hôte déjà parasité (on parle de superparasitisme) est
généralement risquée pour un parasitoïde solitaire puisqu’elle entraîne une compétition à
l’intérieur de l’hôte (van Alphen & Visser 1990 ; Plantegenest et al. 2004).

7
 Introduction générale

Présentation des chapitres de la thèse

Le mémoire présenté s’articule en quatre grands chapitres. Tous les résultats obtenus
sont présentés sous la forme d’articles scientifiques regroupés dans les trois derniers
chapitres.

Chapitre I : Présentation du modèle biologique étudié

Il s’agit d’un chapitre introductif dans lequel est présenté un bilan des connaissances acquises
sur les caractéristiques biologiques et écologiques des trois partenaires impliqués dans la
relation hôte-parasitoïde étudiée, ainsi que les moyens mis en œuvre pour combattre le
ravageur concerné.

Chapitre II : Interaction entre Plutella xylostella et Oomyzus sokolowskii

Ce chapitre est consacré à l’interaction entre P. xylostella et le parasitoïde O. sokolowskii.

Cette étude a été réalisée, dans un premier temps, en plein champ au Sénégal afin d’étudier
l’effet des facteurs environnementaux sur les populations de la teigne et de son parasitoïde
(article 1). Dans un second temps, les traits d’histoire de vie (article 2) ainsi que les
performances d’O. sokolowskii (article 3) comme éventuel agent de lutte contre la teigne, ont
été étudiés en conditions de laboratoire.

Chapitre III : Interaction entre Plutella xylostella et Cotesia vestalis

Ce chapitre est consacré à l’interaction entre P. xylostella et le parasitoïde C. vestalis. Cette

étude a été réalisée dans un premier temps en plein champ au Bénin afin d’essayer de
comprendre pourquoi, malgré un fort taux de parasitisme, ce parasitoïde ne contrôle pas pour
autant les populations de la teigne (article 4). Ensuite nous nous sommes intéressés au
système de reconnaissance qui lie C. vestalis à P. xylostella, en conditions de laboratoire.

8
 Introduction générale

Cette étude porte d’abord sur le mode de détection et d’identification de l’hôte par le
parasitoïde en recherchant chez C. vestalis quels sont les organes impliqués (article 5), puis
nous avons tenté de déterminer la nature exacte du stimulus permettant cette identification et
l’initiation de l’oviposition (article 6).

Chapitre IV : Structuration génétique de Plutella xylostella

Dans ce dernier chapitre, il est question d’explorer la variabilité génétique au sein de P.

xylostella à l’aide de deux marqueurs moléculaires (isoenzymes et ISSR), afin de différencier
des populations d’hôtes d’origines géographiques différentes à l’échelle mondiale. Il s’agit
également de voir si les populations sont structurées génétiquement et s’il existe une relation
entre les distances géographiques et les distances génétiques. Les résultats de cette étude
seront présentés dans les articles 7 et 8.

A l’issue des ces quatre chapitres, suivra ensuite une partie « discussion générale et
perspectives », où nous ferons le point sur l’apport de nos travaux tant sur le plan théorique
que sur le plan des applications.

9
 CHAPITRE I

Présentation du modèle biologique étudié

 Chapitre I : Présentation du modèle biologique étudié

1. Le ravageur : Plutella xylostella (L.)

1.1. Systématique
La teigne du chou (ou teigne des
Brassicacées) Plutella xylostella (Linné
1758) est un lépidoptère appartenant à la
superfamille des Yponomeutoidea et à la
famille des Plutellidae. Par le passé, cette
espèce a été appelée Plutella maculipennis
(Curtis) avant d’acquérir son nom actuel
Individu de collection monté sur paillette (×4)
(Moriuti 1986). C’est l’espèce la plus connue
de son genre, à cause de son importance économique et aussi la seule à être cosmopolite (Kfir
1998).

1.2. Origine, répartition géographique et migration

L’espèce P. xylostella est originaire de la région Est du bassin méditerranéen (Hardy
1938) où l’on trouve aussi le plus grand nombre d’espèces de parasitoïdes et d’où sont
originaires les principales Brassicacées cultivées (Tsunoda 1980). Cependant une origine en
Asie mineure a été suggérée (Chu 1986), voire même en Afrique du Sud, où l’on a recensé de
nombreuses espèces de Brassicacées sauvages et un important cortège de parasitoïdes et
d’hyperparasitoïdes des populations de P. xylostella, dont certains sont endémiques et
spécialistes de cette espèce (Kfir 1998).
Ce ravageur est cosmopolite et est l’une des espèces les plus répandues dans le monde.
L’étendue de sa propagation est due essentiellement à son importante capacité de déplacement
par migration passive et à l’extension des cultures de Brassicacées due à l’activité humaine.
Zalucki & Furlong (2011) ont élaboré et validé un modèle bioclimatique pour P. xylostella,
qui prévoit une distribution de base où elle persiste toute l'année, ainsi que les régions où elle
peut être un ravageur saisonnier (Fig. 1).
C’est en effet un grand migrateur, capable de franchir plus de 3000 km à l’aide des
vents (Chu 1986). Ceci explique qu’on la retrouve régulièrement au Canada ou au nord du
Japon (Hokkaido), où elle ne peut survivre en hiver, mais où les vents du sud la ramènent
chaque printemps (Honda et al. 1992). En Malaisie, la présence de ce ravageur est due à une
introduction de variétés de choux originaires de Chine, d’Inde et d’Europe (Ooi 1986). En
raison de ses grandes capacités d’adaptation à une large gamme de températures

13
14
Ecoclimatic index (EI)

Figure 1: Predicted worldwide distribution of diamondback moth (DBM) based on a

validated bioclimatic model (Zalucki & Furlong 2011). Areas shaded in red show regions of
0 ≤ 10 ≤ 20 ≤ 30 ≤ 40 ≤ 50 > 50
the world where the Ecoclimatic Index (EI) is positive and DBM can persist year-round; red
shading demarcates the core distribution of DBM. Areas shaded in blue show regions of the
world where the EI is zero but where the annual growth index (GI) is positive; in these
Growth index (GI)
regions DBM cannot persist year-round but it can become a seasonal pest following influxes
of moths from elsewhere.
Chapitre I : Présentation du modèle biologique étudié
 Chapitre I : Présentation du modèle biologique étudié

(développement de 5 à 35 °C et une survie à une exposition temporaire de - 9, - 14 et - 19 °C

respectivement pour les adultes, les larves et les nymphes) (Honda 1992), P. xylostella est
désormais présente partout où les Brassicacées sont susceptibles de pousser. Elle s’adapte
même au climat subantarctique (Crafford & Chown 1987) et elle aurait réussi à atteindre l’île
Marion (Chown & Avenant 1992). Dans certaines régions du monde, l’absence d’ennemis
naturels susceptibles d’avoir un impact suffisant pour assurer le contrôle de ses populations
lui permet de proliférer (Talekar & Shelton 1993). Seules les régions où la température ne
dépasse pas 0 °C pendant plus de 60 jours ne sont pas infestées (Honda 1992).

1.3. Spectre de plantes-hôtes

Plutella xylostella est une espèce spécialiste oligophage : elle se développe
exclusivement sur des plantes appartenant à la famille des Brassicacées. Elle est attirée par les
composés soufrés appelés glucosinolates (Cf. Encadré I) caractéristiques de cette famille
végétale. Elle vit essentiellement sur les choux et les autres Brassicacées cultivées (moutarde,
colza, navet, etc.), mais on la trouve aussi sur les Brassicacées sauvages (bourse à pasteur,
cardamine, ravenelle, etc.) qui peuvent servir de réservoir durant les périodes où les cultures
ne sont pas disponibles (Muhamad et al. 1994).
Les Brassicacées sont distribuées dans de nombreuses régions du monde sous les
climats tropicaux et tempérés selon les espèces. On les trouve principalement dans leur région
d’origine en Méditerranée, ainsi que dans les régions sud-est et centrale de l’Asie (Koch &
Kiefer 2006). Toutes ces plantes sont facilement cultivables quelque soient les différentes
conditions climatiques. Selon les variétés, les températures de culture sont comprises entre 4
et 30° C. Certaines ont un cycle de vie annuel (le brocoli), d’autres sont bisannuelles (le chou)
ou pérennes (Arabidopsis lyrata).
Anciennement nommées Crucifères, elles sont aujourd’hui divisées en 25 tribus et
comprennent 3709 espèces appartenant à 338 genres (Warwick et al. 2003). Elles représentent
une importante famille de plantes dicotylédones, essentiellement herbacées. C’est dans la
tribu des Brassiceae que l’on trouve le plus grand nombre d’espèces à haute valeur
économique. Ces espèces sont cultivées, entre autre, pour la production de légumes sous
forme de feuilles et de fleurs (chou-fleur, chou pommé, chou de Bruxelles, brocoli, cresson,
roquette, etc.) sous forme de racines (radis, navet, rutabaga), d’huiles à usage alimentaire et
industriel (le colza), de condiments (moutarde, raifort, wasabi), de fourrage ou de plantes
ornementales (Giroflée, Ibéris, etc.). Ce sont surtout les légumes du genre Brassica qui sont
les plus consommés et essentiellement l’espèce B. oleracea (L.) (Tableau 2).

15
 Chapitre I : Présentation du modèle biologique étudié

Encadré I : Les glucosinolates

Les glucosinolates sont des composés organiques dont la molécule de base comprend un élément dérivé du
glucose et une chaîne soufrée et azotée. Ce sont des métabolites secondaires présents dans toutes les parties de la
plante, que l’on trouve chez de nombreuses Brassicales et plus particulièrement chez les Brassicacées. De
nombreux dérivés des glucosinolates sont impliqués dans la défense des plantes contre les herbivores. Ces
glucosinolates sont toxiques pour la plupart des insectes. Cependant, certains d’entre eux, notamment P.
xylostella, sont capables de désactiver ces molécules grâce à une glucosinolate sulfatase, rendant ainsi la plante
comestible (Ratzka et al. 2002). Ils sont également responsable de goût âpre, amer ou piquant de certaines de ces
plantes (moutarde, radis, choux…). Ces molécules sont synthétisées à partir d’acides aminés (méthionine,
phénylalanine, tyrosine ou tryptophane). Les molécules actives (dérivées des glucosinolates) sont issues de
l’action de la myrosinase qui, en présence d’eau, supprime le groupement glucose. La molécule restante est
transformée en un thiocyanate, un isothiocyanate ou un nitrile qui sont les substances actives. Ces métabolites
secondaires initialement prévus pour dissuader les ravageurs peuvent également être attractifs pour les mâles et
les femelles de P. xylostella, être des stimulants de l’oviposition pour les femelles et des phagostimulants pour
les chenilles (Spencer et al. 1999). Ils vont également participer aux défenses indirectes de ces plantes puisqu’ils
sont également utilisés par les parasitoïdes spécialistes des phytophages inféodés aux Brassicacées pour localiser
leurs hôtes.

Tableau 2 : Espèces de Brassicacées les plus consommées

Genre Espèce et variété Nom commun

Brassica B. oleracea var. botrytis Chou-fleur
B. oleracea var. capitata Chou cabus ou pommé
B. oleracea var. gemmifera Chou de Bruxelles
B. oleracea var. gongyloides Chou rave
B. oleracea var. italica Brocoli
B. oleracea var. sabauda Chou de Milan
B. oleracea var. sabellica Chou frisé
B. rapa var. chinensis Chou chinois
B. rapa var. oleifera Navette
B. rapa var. rapa Navet
B. napus var. napus Colza
B. alba Moutarde blanche
B. juncea Moutarde chinoise
B. nigra Moutarde noire
Raphanus R. sativus Radis
R. niger Radis noir
Armoracia A. rusticana Raifort
Nasturtium N. officinalis Cresson de fontaine
Eruca E. sativa Roquette
Wasabia W. japonica Wasabi

16
 Chapitre I : Présentation du modèle biologique étudié

1.4. Morphologie, biologie et écologie

• Les stades pré-imaginaux
Les œufs sont jaunâtres, elliptiques et mesurent environ 500 µm. Ils sont pondus
isolement ou en petits groupes, le plus souvent sur la face inférieure des feuilles (Fig. 2). La
fécondité est élevée, la femelle pouvant pondre entre 150 et 300 œufs (Pichon 1999), avec
cependant d’importantes variations dépendant de multiples facteurs (température, qualité de la
nourriture de la femelle pendant les stades larvaires, densité des populations, etc.).
Les larves se développent en passant par quatre stades larvaires. Après éclosion, les
larves néonates puis de stade L1 se dispersent. Elles sont endophylles et creusent des galeries
ou « virgules » dans le parenchyme de la feuille (Fig. 2). Au stade L2, les larves sont de
couleur jaune ivoire avec une capsule céphalique noire et mesurent de 2 à 3 mm de long
(Fig. 2). Non mineuse, elles se nourrissent de l’épiderme des feuilles formant des « fenêtres »
caractéristiques de l’espèce (Chua & Lim 1979). Dès ce stade, les chenilles se suspendent à
un fil de soie au moindre danger. Au stade L3, elles sont de couleur jaune-brun, à pilosité plus
visible. La capsule céphalique est brun clair à brun foncé. Au stade L4, les chenilles sont d’un
vert vif et peuvent mesurer 8 mm de longueur. A ce stade, on observe un dimorphisme
sexuel : une tâche blanche sur le cinquième segment abdominal révèle la présence de gonades
visibles par transparence pour les chenilles qui donneront des mâles (Fig. 2) (Liu &
Tabashnik 1997).
Les nymphes, d’une longueur de 5 à 7 mm, sont entourées d’un cocon fusiforme et
étroit, constitué de mailles lâches (Fig. 2). Elles sont d’un vert pâle au début du stade
nymphal, puis brunissent progressivement à l’approche de la mue imaginale.

• L’adulte
L’adulte est un papillon de taille inférieure à 10 mm, pour une envergure d’environ15
mm. Les ailes antérieures sont étroites, allongées et finement frangées. Au repos, il conserve
les ailes plaquées contre le corps, en forme de toit. La frange claire le long des ailes
antérieures forme une série de 2-3 losanges alignés dorsalement rappelant la forme d’un
diamant, qui est à l’origine de son nom anglo-saxon de « Diamondback moth ». Ceci est plus
visible chez le mâle où les couleurs sont contrastées (Fig. 3). C’est le seul dimorphisme
sexuel visible. Pour différencier les sexes de façon certaine, une observation des valves
génitales est nécessaire. Les femelles attirent les mâles à l’aide d’une phéromone sexuelle

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 Chapitre I : Présentation du modèle biologique étudié

(Chow et al. 1974 ; Maa 1986). L’accouplement (Fig. 3) se fait dos à dos dès l’émergence. La
femelle ne s’accouple qu’une seule fois et le mâle de 1 à 3 fois (Talekar & Shelton 1993).

L2

Figure 2 : Les différents stades pré-imaginaux de P. xylostella. En haut à gauche

(× 2) et à droite (× 10), œufs disposés en petits groupes ou isolés autour des
nervures sous la face inférieure de la feuille. Au centre, à gauche, des galeries
creusées par des chenilles endophylles de stade L1, et à droite, des chenilles de
stade L2 (× 7) caractérisées par une capsule céphalique noire et une chenille de
stade L4. En bas à gauche, une chenille de stade L4 (× 6) avec une tâche blanche
qui révèle l’emplacement des gonades (G), et à droite, une nymphe (× 6) dans son
cocon.

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 Chapitre I : Présentation du modèle biologique étudié

Figure 3 : Adultes de P. xylostella.

En haut, à gauche un mâle (× 5)
et à droite une femelle (× 5).
En bas, accouplement (× 3,5)

La température optimale de développement du ravageur se situe entre 17°C et 25°C

(Atwal 1955), mais elle peut considérablement augmenter. Par exemple, des populations
tropicales peuvent effectuer leur cycle larvaire à 35°C (Observation personnelle). A 25°C, le
cycle complet peut durer 16 jours : trois jours pour l’incubation des œufs, neufs jours pour le
développement larvaire et quatre jours pour la nymphose (Fig. 4). Cette rapidité de
développement explique sa capacité à détruire rapidement les cultures attaquées sous les
climats chauds, même si le nombre d’individus est faible au départ (cas de migrants amenés
par le vent, par exemple). Le nombre de générations par an varie de trois dans les pays à
climat tempéré, à plus de 20 dans les pays possédant un climat tropical. Aucune période de
diapause n’a été mise en évidence chez P. xylostella (Harcourt & Cass 1966 ; Yamada &
Umeya 1972).

1.5. Les moyens de lutte contre Plutella xylostella

C’est le plus important ravageur des cultures de Brassicacées dans le monde et en
particulier dans les zones tropicales et subtropicales (Talekar & Shelton 1993 ; Furlong et al.
2013) (Fig. 5 et Fig. 6). L’estimation du coût annuel dans l’économie mondiale pour lutter
contre la teigne est supérieure à 4 milliards de dollars. Ceci peut représenter de 30 à 50% des
coûts de production, loin devant les coûts de fertilisation (Zalucki et al. 2012).

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 Chapitre I : Présentation du modèle biologique étudié

Figure 4 : Cycle de développement de P. xylostella à 20° C (à l’extérieur du cercle) et à 25 °C

(à l’intérieur du cercle) (Valeurs en jours, Salinas 1986) (Dessin : Carpenter 2005).
1.5. Les moens de lutte contre la teine du chou

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 Chapitre I : Présentation du modèle biologique étudié

Photographie : L. Arvanitakis (2012)

Figure 5 : Culture de choux au Sénégal dont les feuilles sont entièrement perforées
par les chenilles de P. xylostella

Photographie : L. Arvanitakis (2006)

Figure 6: Chou entièrement détruit par les chenilles de P. xylostella au Bénin.

Les feuilles sont réduites à de la dentelle

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 Chapitre I : Présentation du modèle biologique étudié

Dans le Sud-Est de l’Asie, des explosions démographiques de P. xylostella ont pu

provoquer jusqu’à 90% de perte de récolte (Verkerk & Wright 1996). Dans les pays
occidentaux, le seuil économique est atteint dès qu’il y a une seule chenille de stade L4 par
chou car les trous créés par celle-ci suffisent à altérer l’aspect de la plante et à la rendre
invendable (Shelton et al. 1983 ; Maltais et al. 1998). Dans certains pays où la culture du chou
représente près de 90% des cultures maraîchères, la lutte contre ce ravageur est plus qu’un
enjeu économique : c’est une condition de survie des traditions locales et cela concerne la
protection de la santé des populations humaines. Encore aujourd’hui, l’utilisation de
pesticides reste la méthode de lutte la plus répandue, bien que l’apparition de résistances à la
plupart des matières actives rend la lutte peu efficace. Dans certains cas, des solutions
alternatives sont conçues sous la forme de programmes de lutte intégrée ou Integrated Pest
Management (IPM), basés sur la combinaison de plusieurs méthodes afin de gérer les
résistances et de limiter la quantité de pesticides libérés dans l’environnement. Outre la lutte
chimique, nous passerons en revue les principales méthodes de lutte dites alternatives basées
sur différents critères : 1) la modification de la plante hôte (sélection variétale et
transgénèse) ; 2) la modification des pratiques culturales (rotation, plantes pièges, plantes
intercalaires, irrigation par aspersion) ; 3) le comportement du ravageur (phéromones
sexuelles) ; 4) l’utilisation d’autres organismes vivants (lutte biologique).

1.5.1. La lutte chimique

L’usage d’insecticides reste la méthode la plus répandue. Dans la première moitié du
XXe siècle, l’éventail des produits est resté limité : fumigants, composés minéraux (arsenic) et
végétaux (nicotine, roténone, pyréthrines) (Huckett 1934). Peu efficaces et trop toxiques, ils
sont supplantés à partir de 1945 par les insecticides de synthèse. Les organochlorés tels que le
Dichloro-diphényl-trichlore éthane (DDT) ont prouvé leur efficacité et leur utilisation s’est
alors largement répandue (Ankersmith 1953). Rapidement suivi du lindane, des
organophosphorés, des carbamates (carbofuran et méthomyl), des pyrèthrinoïdes
(cyperméthrine, deltaméthrine…), voire des cocktails de produits issus de plusieurs familles
chimiques (Sun et al. 1986). Au cours des années 70, l’efficacité des formulations contenant
les toxines de la bactérie Bacillus thuringiensis (Bt) (Berliner) a été démontrée et ces
biopesticides qui ont une action sélective sont devenus très populaires.
Cependant la limite des insecticides est vite apparue en donnant naissance à des
populations résistantes aux principales familles chimiques. Dès 1951, P. xylostella est
résistant au DDT en Indonésie (Ankersmith 1953). En 1980, on recense des résistances envers

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 Chapitre I : Présentation du modèle biologique étudié

36 produits différents dans 14 pays (Miyata et al. 1986) et envers 51 produits en 1989
(Shelton et al. 1993b) dont les formulations de biopesticides à base de Bt (Syed 1992). La
recherche de solutions alternatives a conduit à s’intéresser à des insecticides d’origine
biologique, extraits de végétaux. Seuls les extraits de graines de « neem » issus de
l’Azadirachta indica (Meliacea) (Goudegnon et al. 2000 ; Löhr & Kfir 2004) offrent la
possibilité d’une utilisation à grand échelle, peu coûteuse et efficace. Néanmoins, ils
présentent des effets phytotoxiques, modifiant la couleur des feuilles de choux (Schmutterer
1992 ; Leskovar & Boales 1996). Malgré tous les produits disponibles, des agriculteurs à
Hawaii ou au Japon, ont été amenés à abandonner leurs cultures de Brassicacées, car la teigne
était résistante à tous les produits autorisés aux doses sanitairement acceptables (Nakahara et
al. 1986 ; Tanaka 1992).

1.5.2. La lutte biologique

• Définition et généralités
La lutte biologique est définie comme « l’utilisation d’organismes vivants (appelés
auxiliaires) pour prévenir ou réduire les pertes ou dommages causés par des organismes
nuisibles » (OILB-SROP 1973). Elle est également définie comme la suppression, le contrôle
ou la régulation des populations de ravageurs à l’aide de prédateurs, de parasitoïdes, et de
pathogènes (DeBach & Rossen 1991 ; Hawkins & Cornell 1999). Parmi les moyens de
substitution aux insecticides chimiques, la lutte biologique est la plus employée et la seule qui
soit efficace à grande échelle (Mills & Gutierrez 1999). Les opérations de lutte biologique
réussies ont apporté des résultats significatifs pour l’agriculture et l’économie (Mills &
Gutierrez 1999). Cependant des estimations concernant le contrôle d’arthropodes indiquent
que seulement 34% des introductions ont abouti à l’installation de l’agent de lutte biologique
et seulement 47% d’entre elles ont réduit les populations de nuisibles (Hall & Ehler 1979).
Lynch et al. (2001) ont estimé à seulement 3 % le taux de succès des introductions. La lutte
biologique ne se limite pas à la régulation de populations d’insectes nuisibles, mais peut
également être utilisée contre les mauvaises herbes (Fraval & Silvy 1999) dont le contrôle
connait de meilleurs taux d’installation (Roderick & Navajas 2003). L’échec de nombreuses
introductions peut être en partie dû à la difficulté de prédiction des résultats potentiels des
introductions. Les critères de recherche et de sélection des agents sont restés empiriques.
Parmi ces critères, on peut citer celui qui prévoit qu’un agent de lutte biologique qui contrôle
moins de 30% de la population de son hôte dans la zone de provenance n’aura qu’un succès
limité, voire échouera dans la zone d’introduction (Hawkins & Cornell 1999). Les avantages

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 Chapitre I : Présentation du modèle biologique étudié

pratiques attribués à la lutte biologique par rapport à la lutte chimique sont : (1) les agents de
lutte biologique n’ont pas d’effets phytotoxiques et ont peu ou pas d’effets nocifs pour la
santé humaine et l’environnement ; (2) les applications d’agents sont plus simples et le travail
est moins pénible ; (3) la lutte biologique est appréciée du public ce qui constitue un avantage
pour la commercialisation des produits des cultures qui peuvent prétendre à des labels
valorisables en terme de prix ; (4) elle peut représenter une alternative à la pérennité des
traitements.

• Différentes stratégies de lutte biologique

La lutte biologique classique vise à introduire (on dit aussi acclimater) dans la
culture à protéger un (ou plusieurs) auxiliaire(s) exotique(s) pour un établissement permanent
et un contrôle durable des ravageurs. Cette stratégie fut la plus utilisées pendant plus d’un
siècle avec des succès notables. Le ravageur est dans la plupart des cas lui-même exotique et
pullule en l’absence de son cortège d’ennemis naturels, ou pour d’autres raisons écologiques
(Colautti et al. 2004). Il s’agit alors d’importer un ennemi naturel sympatrique efficace
(Eilenberg et al. 2001) et de recréer dans un nouveau contexte écologique l’équilibre
démographique existant entre l’hôte et l’auxiliaire dans leur aire d’origine. D’un point de vue
économique, cette stratégie par acclimatation est particulièrement intéressante puisque les
coûts liés à son développement sont relativement limités par rapport à la durabilité du contrôle
du ravageur et à la faible intervention humaine nécessaire.

La lutte par lâchers inoculatifs a pour objet d’établir une population d’auxiliaires
suffisante pour contrôler le ravageur durant une période limitée dans le temps. L’utilisation de
cette stratégie est particulièrement répandue pour les cultures sous serre puisque l’on estime
que 5 % des 300 000 ha de serres dans le monde sont gérées grâce à la Protection Biologique
Intégrée (PBI), notamment grâce à des lâchers inoculatifs de parasitoïdes ou d’autres
auxiliaires (van Lenteren 2000). Le succès de cette stratégie sous serre s’explique en partie
par la large gamme d’auxiliaires (plus d’une centaine d’espèces) disponibles et
commercialisés dans le monde. Contrairement à la lutte biologique classique, la lutte
biologique par lâchers inoculatifs nécessite un approvisionnement régulier et important
d’auxiliaires. Cette étape supplémentaire de production de masse soulève plusieurs
problèmes. En effet, les conditions de production et de stockage doivent préserver les qualités
des individus, notamment leur fécondité, leur longévité, leur capacité de dispersion, etc. Par
ailleurs, de nombreux auteurs soulignent l’importance d’évaluer l’impact des élevages de

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 Chapitre I : Présentation du modèle biologique étudié

masse au niveau génétique (van Lenteren 2003 ; Wajnberg 2004). En effet, les conditions
imposées lors de la production de masse sont souvent très différentes de celles rencontrées
dans l’agrosystème. Les pressions de sélection lors de la phase de production de masse
peuvent à long terme altérer les performances des auxiliaires initialement choisis. De même,
des phénomènes de dérive génétique ou de consanguinité importante pourraient également
conduire à une réduction de la variabilité génétique avec une modification des caractéristiques
initiales des souches utilisées.

La lutte par lâchers inondatifs vise à contrôler les populations de ravageurs par la
seule activité de destruction réalisée par les auxiliaires lâchés en grand nombre dans
l’agrosystème. Contrairement aux deux stratégies précédentes, l’action des auxiliaires sur la
population du ravageur se veut donc beaucoup plus brutale et limitée dans le temps. L’effet
des descendants des individus lâchés peut s’avérer également intéressant pour prolonger le
contrôle du ravageur, mais il constitue ici rarement un objectif en soi.

La lutte biologique par conservation est de modifier l’agrosystème ou les pratiques

culturales afin de protéger et de favoriser la présence d’ennemis naturels locaux, facilitant
ainsi leur capacité à contrôler les populations d’insectes nuisibles. À l’heure actuelle, cette
forme de lutte biologique est probablement la moins développée. Elle offre cependant des
solutions pratiques efficaces (Landis et al. 2000). Trois tactiques non exclusives peuvent
généralement être mises en œuvre dans cette stratégie. La première consiste à créer des abris
ou des microclimats susceptibles de favoriser l’installation et la pérennisation des auxiliaires.
La seconde stratégie consiste à mettre en place des sources de nourriture pour les auxiliaires
adultes. Par exemple, l’influence de différentes espèces de fleurs sauvages sur le parasitoïde
Diadegma insulare (Cresson) a été évaluée dans le cadre de la lutte biologique contre la
teigne Plutella xylostella (Idris & Grafius 1995). Une dernière tactique cherche à maintenir
des hôtes afin de maintenir la population d’auxiliaires sur la culture à protéger. D’un point de
vue écologique, l’ensemble de ces pratiques a tendance à augmenter la biodiversité de
l’agrosystème tant au niveau des espèces végétales qu’animales.

• Les différents auxiliaires utilisés en lutte biologique

La lutte biologique utilise des organismes vivants pour diminuer les niveaux de
population d'autres organismes, généralement nuisibles. Les agents de la lutte biologique
(appelés aussi auxiliaires) les plus souvent utilisés comprennent les microorganismes, les

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nématodes, les prédateurs et les parasitoïdes (Lim 1992). On considère que la mortalité causée
par les parasitoïdes est plus importante que celle attribuée aux prédateurs et aux
microorganismes combinés. Des efforts importants sont menés en faveur de la lutte
biologique contre P. xylostella, afin de ralentir l’apparition de résistances et de limiter la
quantité de pesticides libérés dans l’environnement. Plusieurs agents de lutte biologique ont
été étudiés, mais peu d’entre eux sont couramment utilisés contre P. xylostella (Fig. 7). Près
de 135 espèces de parasitoïdes (Talekar & Shelton 1993 ; Delvare 2004), 25 prédateurs, au
moins deux virus et deux bactéries, ainsi que sept champignons (Talekar 2004) ont été
recensés comme ennemis potentiels des différents stades de développement de P. xylostella.
Parmi eux, les parasitoïdes sont de loin les plus utilisés.

Les virus sont peu employés contre P. xylostella en raison d’un manque de virulence
(Kolodny-Hirsch & Van Beek 1997 ; Greval et al. 1998). Toutefois, certains granulovirus
semblent être très pathogènes, pouvant provoquer jusqu’à 90% de mortalité chez les larves de
premier et second stade (Asayama 1986). Toutes les souches de P. xylostella n’ont pas la
même sensibilité. Des épidémies apparaissent naturellement, mais elles restent limitées.

Les bactéries : la plus connue est Bacillus thuringiensis Berliner (Bacillales :

Bacillaceae) ou Bt. Sa grande spécificité et les faibles risques qu’elle présente pour la santé
humaine en ont fait un outil révolutionnaire dans la lutte contre les ravageurs de culture. Elle
est utilisée sous forme de spores contenant des δ-endotoxines protéiques « Cry ». Plusieurs
centaines de ces toxines sont connues, mais seules certaines classes sont actives contre les
Lépidoptères (d’autres n’agissent que sur les Coléoptères ou les Diptères) (Chaufaux 1995 ;
Monnerat 1995). Les variétés Kurstaki et aïsawaï (Eubactériales) sont utilisées pour le
contrôle des populations de chenilles de P. xylostella. Elles tuent les insectes en se fixant sur
la membrane des intestins et en créant des pores dans cette dernière (Gill et al. 1992).
Quoique considéré au début de son utilisation comme une « arme absolue », P. xylostella est
le premier insecte à avoir développé une résistance aux toxines de Bt en milieu naturel, avec
des résistances croisées aux diverses classes de toxines (Tabashnik et al. 1997). Ces
résistances sont la conséquence d’une utilisation intensive des traitements avec les
formulations à base de B. thuringiensis. Les toxines de Bt présentent l’avantage d’être non
toxiques vis-à-vis des parasitoïdes (Flexner et al. 1986 ; Lim 1992). En Malaisie, l’utilisation
de ces toxines, combinée à celle des parasitoïdes est la pierre angulaire des programmes de
lutte intégrée (Loke et al. 1992).

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Figure 7: Principaux agents de lutte biologique utilisés contre les différents

stades de P. xylostella (Sarfraz et al. 2005)

Les champignons entomopathogènes sont difficilement utilisables car ils ne

prolifèrent qu’en milieu très humide et se développent plus facilement dans les pays
tropicaux. Les deux champignons qui attaquent P. xylostella sont Beauveria bassiana
(Balsano) (Hyphomycètes : Moniliaceae) et Zoophtora radicans (Brefeld) (Zygomycètes :
Entomophthorales). Ils attaquent les larves et parfois les nymphes (Wilding 1986). Cependant,
ces champignons sont peu spécialistes et peuvent aussi infecter les espèces de parasitoïdes et
réduire de ce fait leur efficacité (Furlong & Pell 1996).

Les microsporidies appartenant au genre Nosema (Microspora : Nosematidae)

peuvent infecter les larves de P. xylostella au niveau du système digestif et des tissus adipeux.

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Cependant, les chenilles infectées peuvent nuire aux espèces de parasitoïdes vivant à leurs
dépens en réduisant considérablement leur efficacité (Bordat et al. 1994 ; Gruarin 1998).

Les nématodes entomopathogènes sont peu utilisés car ils sont coûteux à produire et
sensibles à la lumière et à la sécheresse (Baur et al. 1997 ; Lello et al. 1996). Au stade
infectieux, ils portent des bactéries du genre Xhenorhabdus (Steinernematidae) ou
Photorhabdus (Heterorhabitidae) qui infectent le ravageur en pénétrant dans l’hémocoele
(Ratnasinghe & Hague 1995). Les isolats de Steinernema carpocapsae (Weiser) sont les plus
efficaces contre les larves de P. xylostella (Baur et al. 1998).

Les prédateurs de P. xylostella peuvent être des Arachnides, des Insectes et des
Acariens (Alam 1992 ; Lim 1992). Malheureusement, ils ne sont pas spécifiques à P.
xylostella et leur taux de prédation est difficile à évaluer. D’après Goudegnon et al. (2004), les
fourmis Anomma nigricans (Illiger) (Hym. : Formicidae) peuvent être des agents de contrôle
de P. xylostella dans les zones périurbaines au Bénin.

Les parasitoïdes ont un mode de développement et des caractéristiques écologiques

qui conduisent, au niveau individuel et dans la plupart des cas, à la mort de leur hôte. Au
niveau populationnel, leurs caractéristiques contribuent également à la limitation des
populations hôtes. A ce titre, les parasitoïdes se révèlent intéressants pour réduire l’impact des
ravageurs des cultures. Ils sont responsables de nombreux succès en lutte biologique et
occupent dans les écosystèmes naturels une place importante.
Les parasitoïdes restent de loin le groupe d’organismes le plus étudié, le plus utilisé et
le plus efficace dans la lutte contre P. xylostella et souvent le moins coûteux (Waage &
Greathead 1986). Parmi les espèces de parasitoïdes recensées sur P. xylostella, on retient
principalement 6 parasites d’œufs, 38 de chenilles et 13 de nymphes (Lim 1986 ; Monnerat
1995 ; Delvare 2004). Ces espèces sont présentes un peu partout, mais individuellement
chacune possède une aire de répartition plus restreinte que celle de la teigne. La plus grande
diversité de parasitoïdes liés à P. xylostella se trouve dans la région méditerranéenne (Mustata
1992) mais également en Afrique du Sud (Kfir 1998). Dans son aire d’origine, l’espèce P.
xylostella est relativement bien contrôlée par les populations locales de parasitoïdes (70 à
80%) (Löhr & Kfir 2004). Par contre, en zone tropicale, les successions de générations du
ravageur sont loin d’être régulées par les parasitoïdes locaux souvent peu nombreux en termes
d’espèces et peu efficaces (15 à 20% de parasitisme) (Löhr & Kfir 2004). Pour renforcer les

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populations autochtones dans de nombreuses régions du monde, des introductions d’espèces

exotiques ont été réalisées (Hardy 1938 ; Talekar & Shelton 1993). Bien que les résultats
soient variables selon les régions, de nombreuses implantations ont réussi et la répartition
actuelle de ces espèces n’a plus rien à voir avec leur aire d’origine. Certains échecs observés
lors d’introductions sont liés à l’usage continu et abusif des traitements insecticides (Travis &
Rick 2000). Ceci conduit à la destruction des parasitoïdes qui sont plus sensibles que leur
hôte. L’utilisation d’insecticides reste donc incompatible avec la lutte biologique.
Les principaux parasitoïdes de P. xylostella appartiennent tous à l’ordre des
Hyménoptères. On trouve des parasitoïdes d’œufs qui appartiennent tous à la famille des
Trichogrammatidae. Ils ont fait l’objet d’études mais ne sont pas utilisés en lutte biologique
contre P. xylostella (Klemm et al. 1992 ; Pak 1992). Ils contribuent faiblement au contrôle
naturel et requièrent de fréquents lâchers de masse (Talekar & Shelton 1993). Par contre, les
espèces qui s’attaquent aux stades larvaires sont les plus importantes et les plus efficaces et
semblent donc offrir le meilleur potentiel de contrôle (Lim 1986). Elles appartiennent aux
familles des Braconidae, des Ichneumonidae et des Eulophidae. Plusieurs genres sont
particulièrement représentés : Cotesia et Apanteles (Braconidae), Diadegma (Ichneumonidae)
et Oomyzus (Eulophidae). L’espèce Diadromus collaris (Ichneumonidae) est une espèce qui
parasite les nymphes de P. xylostella (Tableau 3).

Tableau 3 : Principales espèces d’hyménoptères parasitoïdes de P. xylostella

Stade parasité Famille Espèce Caractéristiques

Oeuf Trichogrammatidae Trichogramma spp. Généralistes, solitaires
Chenille Braconidae Cotesia vestalis (Haliday) Spécialiste, solitaire
Apanteles spp. Spécialistes, solitaires
Microplitis plutellae Musebeck Spécialiste, solitaire
Ichneumonidae Diadegma spp. Spécialistes, solitaires
Eulophidae Oomyzus sokolowskii (Kurdjumov) Spécialiste, grégaire
Nymphe Ichneumonidae Diadromus collaris (Gravenhorst) Spécialiste, solitaire

1.5.3. Les autres méthodes de lutte

La sélection variétale : Il existe des variétés de choux moins sensibles aux attaques
des chenilles de P. xylostella, par la production de toxines responsables de mécanisme
d’antibiose (Eigenbrode et al. 1990 ; Eigenbrode & Shelton 1992) ou par des changements de
la structure des cires épicuticulaires qui diminuent l’appétence des feuilles (Dickson et al.

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 Chapitre I : Présentation du modèle biologique étudié

1990 ; Stoner 1992 ; Shimabuku et al. 1997). Ces variétés sont peu employées car les
modifications induites peuvent favoriser le développement d’autres ravageurs (tels que les
altises Phyllotreta spp. (Coleoptera : Chrysomelidae) (Bodnaryk 1997) et donnent un aspect
brillant aux feuilles (glossy leaves), peu apprécié des consommateurs (Lim 1992).

Les plantes transgéniques : La transgénèse « insecticide », avec intégration de gènes

codant pour la production de diverses toxines d’origine bactérienne, végétale ou
d’arthropodes venimeux, se développe de plus en plus (Schuler et al. 1998), surtout pour le
maïs ou le coton. Les tests de toxicité montrent généralement une grande efficacité de ces
plantes dans la suppression de leurs ravageurs (Stewart et al. 1996 ; Ramachandran et al.
1998). Cependant il existe déjà des cas où, des populations de P. xylostella résistantes au B.
thuringiensis, consomment des choux transgéniques sans problème (Tang et al. 1999).

Les phéromones sexuelles de synthèse de P. xylostella peuvent être utilisées en

pulvérisation pour désorienter les mâles qui recherchent les femelles (confusion sexuelle) ou
en association avec des pièges englués (piège sexuel). Cependant, les résultats sont peu
probants (Schroeder et al. 2000). Néanmoins, les pièges sexuels peuvent être utilisés pour
estimer le nombre d’individus présents sur le site afin de réaliser des traitements lorsque le
seuil économique est atteint ou dépassé (Reddy & Guerrero 2001 ; Lörh & Kfir 2004).

L’irrigation par aspersion réduit significativement les infestations de P. xylostella,

de 40 à 60% en une année d’irrigation aux Etats-Unis (McHugh & Foster 1995). Lorsque les
larves sont en présence d’une humidité relative très élevée (100%), le taux de mortalité est de
70%. Ces résultats sont confortés par la mise en évidence de la pluie en tant qu’un des
principaux facteurs de mortalité chez P. xylostella (Wakisaka et al. 1992). Cette méthode de
lutte est efficace mais coûteuse et favorise l’apparition de maladies cryptogamiques (Talekar
& Shelton 1993).

L’utilisation de plantes pièges consiste à introduire, dans la parcelle cultivée, des

plantes présentant une plus forte attractivité à la ponte pour les femelles. Dans le cas de P.
xylostella, il s’agit d’introduire des plants de moutarde sur lesquels les femelles vont pondre
préférentiellement (Charleston & Kfir 2000). Pour éliminer le ravageur, il suffit de détruire
les plants de moutarde. Simple et efficace, cette méthode est utilisée en Inde (Srinivasan &
Moorthy 1992 ; Talekar & Shelton 1993) et commence à se répandre en Afrique du Sud

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 Chapitre I : Présentation du modèle biologique étudié

(Charleston & Kfir 1999). Cependant certains agriculteurs sont réticents à utiliser cette
technique qui implique de détruire volontairement une partie des plantes cultivées
(observations personnelles.).

La rotation culturale consiste à ne pas laisser sur un cycle de culture se suivre deux
fois la même plante ou des plantes de la même famille sur une parcelle. En milieu tropical, il
est fréquent de trouver des cultures de choux toute l’année sur une même parcelle, ce qui
permet à P. xylostella de se maintenir à des densités élevées. La rotation permettrait de priver
temporairement le ravageur de sa plante hôte et donc de réduire ses populations (Talekar &
Shelton 1993). Mais cette pratique a un coût financier pour les producteurs. Il leur est difficile
d’arrêter de produire du chou qui constitue, dans certains cas, leur seule ressource.

Les cultures intercalaires (intercropping) : son principe consiste à cultiver des rangs
alternés de choux et d’une autre espèce végétale comme l’ail ou la tomate (Talekar et al.
1986), dont l’odeur inhibe l’oviposition des femelles (Srinivasan 1984). Certaines plantes de
grande taille peuvent agir comme une barrière physique limitant les déplacements du
ravageur, les contacts visuels avec ses congénères ou encore avec sa plante hôte (Talekar &
Shelton 1993). Morallo-Rejesus (1986) fait état de 88 plantes ayant un effet répulsif sur P.
xylostella. Les plantes intercalées entre les plants de choux peuvent également servir de
refuge pour les insectes parasitoïdes (Risch 1981 ; Sheehan 1986). Cette pratique s’avère peu
intéressante car la plante intercalaire n’est pas d’un aussi bon rapport économique que le chou
et ne convient pas aux agriculteurs spécialisés dans une seule culture. Il peut aussi y avoir des
problèmes de compétition entre les deux plantes, ce qui nuit au rendement (Shellhorn & Sork
1997).

1.5.4. La lutte intégrée

Les résistances à tous les insecticides, le coût élevé de certaines pratiques, le manque
d’efficacité de certaines autres et très souvent le manque de connaissance des populations
locales sur les pratiques à suivre, rendent la lutte contre P. xylostella extrêmement difficile.
Face à ces constats, l’importance d’une stratégie intégrée pour la gestion durable de P.
xylostella est au centre des préoccupations depuis plus de 20 ans (Grzywacz et al. 2010). La
lutte intégrée aborde la lutte contre les ravageurs d’une façon économiquement et
écologiquement saine, en utilisant un ensemble varié de techniques pour réduire et maintenir
les populations de ravageurs à des niveaux acceptables. La plupart des programmes IPM

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 Chapitre I : Présentation du modèle biologique étudié

comportent des éléments de prévention et de prédiction qui s’efforcent de réduire, sinon

d’éliminer, le besoin en mesures de lutte à grande échelle. Parmi les techniques utilisées dans
un programme IPM se trouvent la lutte biologique qui prend une place fondamentale, les
pesticides et les méthodes culturales de lutte. Le concept de lutte intégrée ou IPM (Integrated
Pest Management) (Encadré II) est apparu dans les années 1980 et repose sur cinq grands
points : 1) surveillance continue des parcelles par une présence constante sur le terrain et un
suivi des populations de ravageurs (monitoring, piégeage sexuel…) ; 2) établissement des
seuils économiques au-delà desquels un type bien déterminé d’intervention est préconisé ; 3)
utilisation conjointe de méthodes non chimiques (principalement auxiliaires de cultures
(parasitoïdes), moyens mécaniques, plantes pièges) ; 4) proscription des insecticides à large
spectre au profit de produits plus sélectifs (Bt) dont les quantités employées sont réduites afin
de faciliter l’implantation des auxiliaires utilisés ; 5) information et/ou participation des
acteurs locaux sur les programmes de lutte raisonnée.

Encadré II. Integrated Pest Management (IPM)

Dans le code international de conduite de la distribution et de l’utilisation des pesticides adopté par la FAO
en novembre 2002, la définition de l’IPM est la suivante :

Integrated Pest Management (IPM) means the careful consideration of all available pest control techniques
and subsequent integration of appropriate measures that discourage the development of pest populations and
keep pesticides and other interventions to levels that are economically justified and reduce or minimize risks to
human health and the environment. IPM emphasizes the growth of a healthy crop with the least possible
disruption to agro-ecosystems and encourages natural pest control mechanisms.

L’application de tout ou partie de ces diverses recommandations ont donné de bons

résultats. Par exemple, au Brésil, le seuil économique fixé à 6 trous sur la feuille centrale a
permis de diminuer de 50% les quantités de Bt utilisées (Branco et al. 2004). A Cuba, le
dépassement du seuil de 0,2 larves par plant préconise le lâcher de 50 000 Trichogramma
pintoi Voegele par hectare. Près de 85% des fermiers cubains respectent ces conseils (Branco
et al. 2004). L’application de tels programmes est bénéfique à de nombreux niveaux. En effet,
en plus de la sauvegarde de la qualité des sols, de l’eau, de la santé humaine et des agro-
écosystèmes, leur impact s’étend aux coûts de production et de manière concomitante aux
bénéfices. En Malaisie et en Thaïlande, plus de 80% des vaporisations de pesticides ont été
réduits et les profits ont été respectivement doublés et triplés (Lim 1992). Ces résultats sont

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 Chapitre I : Présentation du modèle biologique étudié

observés également à Hawaii et ont été obtenus en moins de sept années à Singapour (Ng et
al. 2004).

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2. Les parasitoïdes

2.1. Oomyzus sokolowskii (Kurdjumov)

2.1.1. Systématique
L’espèce a été décrite par Kurdjumov en 1912 et rattachée au genre Tetrastichus. En
1991, Graham l’a classée dans le genre Oomyzus.

Embranchement Arthropoda Famille Eulophidae

Classe Insecta Sous-famille Tetrastichinae
Ordre Hymenoptera Genre Oomyzus
Super-famille Chalcidoidea Espèce sokolowskii

2.1.2. Morphologie, biologie et écologie

L’œuf est de forme elliptique, transparent et mesure 0,3 × 0,06 mm. Les œufs sont
souvent regroupés en amas de 3 à 15 unités dans la partie postérieure interne de l’hôte bien
que la femelle n’ait pas de site de ponte préférentiel. Ces œufs se développent grâce aux
éléments nutritifs de la chenille puis de la nymphe d’où ils émergent sous forme d’adultes.
L’incubation des œufs dure en moyenne trois jours à 25°C (Saw et al. 2013).

La larve est de forme vermiforme, arrondie aux extrémités, quasi transparente et

possède un tube digestif sur toute sa longueur. Leur nombre est variable dans la chrysalide
parasitée. Au fur et à mesure de leur développement, les plus grosses larves colonisent la
totalité de la nymphe, tandis que la compétition qui s’instaure entre elles provoque une
diminution sensible de leur nombre au détriment des plus petites. En fin de développement,
les larves ont vidé la chrysalide de P. xylostella. La durée du stade larvaire varie de quatre à
sept jours à 25°C (Saw et al. 2013).

La nymphose se déroule en quatre étapes et dure en moyenne sept jours à 25°C. Une
pré-nymphe de couleur blanche précède la nymphe caractérisée par l’apparition et la
coloration des yeux et ocelles en rouges. Ensuite la nymphe se développe, grisaille puis prend
une coloration définitive noire (Saw et al. 2013).

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 Chapitre I : Présentation du modèle biologique étudié

L’adulte est de très petite taille entre 1 à 2 mm. Son corps est de couleur noir brillant
avec des reflets métalliques verts. La durée de vie moyenne d’un adulte est de sept jours
(Hirashima et al. 1990). Un dimorphisme sexuel assez marqué permet de distinguer le mâle de
la femelle à partir de la taille du corps (Fig. 8) et de la morphologie des antennes.
Le mâle a un abdomen cylindrique, de même diamètre que le thorax. Il possède des antennes
de grande taille pourvues de quatre articles portant des soies nombreuses et longues. La
femelle présente un abdomen plus renflé et anguleux en forme de losange. Elle est
reconnaissable grâce à son ovipositeur (tarière) disposé dans une gouttière visible sur la face
ventrale à l’extrémité de l’abdomen. Leurs antennes plus courtes possèdent des soies plus
courtes et moins nombreuses.

Figure 8 : Couple d’adultes d’O. sokolowskii (× 20) (femelle à gauche et mâle à droite)

L’accouplement, qui a lieu dès l’émergence, stimule fortement les capacités de

parasitisme de la femelle. Oomyzus sokolowskii est un endoparasitoïde dont la femelle pond à
l’aide de son ovipositeur dans les chenilles de P. xylostella. Tous les stades sont parasités,
avec une nette préférence pour les chenilles L3 et L4 où les taux de parasitisme sont en
moyenne respectivement de 54% et 76% (Saw et al. 2013). Bien qu’il soit généralement
considéré comme un parasitoïde larvaire (Ooi 1988 ; Talekar & Hu 1996 ; Kfir 1997), certains
auteurs le décrivent aussi comme un parasitoïde nymphal (Chelliah & Srinivasan 1986 ;
Waterhouse & Norris 1987 ; Wakisaka et al. 1992 ; Noyes 1994).

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 Chapitre I : Présentation du modèle biologique étudié

Les larves du parasitoïde continuent en effet leur développement dans la nymphe de P.

xylostella d’où émergeront les adultes. La durée du cycle biologique complet varie en
fonction de la température (Tableau 4) ; elle est en moyenne de 15 jours à 25°C (Wang et al.
1999). Ce parasitoïde comme beaucoup d’hyménoptères, présente à la fois une reproduction
sexuée et une reproduction asexuée par parthénogénèse arrhénotoque.

Tableau 4: Durée du cycle biologique d’O. sokolowskii à différentes

températures (Wang et al. 1999)

Température (°C) Durée du développement (jours)

35,0 13,4 ± 0,15

32,5 11,0 ± 0,13
30,0 12,7 ± 0,15
25,0 15,6 ± 0,18
22,5 20,9 ± 0,09
20,0 26,5 ± 0,71

Dans le premier cas la descendance, issue d’œufs fécondés, se compose de femelles

diploïdes. Dans le second cas, le développement embryonnaire des œufs non fécondés donne
une descendance uniquement composée de mâles haploïdes. La femelle gravide détermine le
sexe ratio de la descendance : elle pond en général plusieurs œufs fécondés, puis un petit
nombre d’œufs non fécondés d’où un sexe-ratio en faveur des femelles (Uraichuen 1999).
Oomyzus sokolowskii est une espèce grégaire (plusieurs congénères se développent aux
dépens d’un même hôte) (Fig. 9). L’espèce est oïoxène, son spectre d’hôte se limitant à une
seule espèce, mais cet hyménoptère peut aussi agir comme un hyperparasitoïde facultatif ou
parasitoïde secondaire de C. plutellae (Waterhouse & Norris 1987 ; Liu et al. 2000).

2.1.3. Répartition géographique

Cette espèce est répertoriée sur les cinq continents, faisant suite à un certain nombre
d’introductions, dont la première fut réalisée à Hawaii en 1953 avec une population originaire
du Kenya. Sa répartition à l’échelle mondiale, prouve sa grande capacité d’adaptation face à
des conditions climatiques variées, qualité nécessaire pour une lutte biologique efficace
(Gruarin 1998) (Tableau 5).

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 Chapitre I : Présentation du modèle biologique étudié

Figure 9 : Plusieurs adultes d’O. sokolowskii émergeant d’une nymphe de

P. xylostella

Tableau 5 : Liste des pays où la présence d’O. sokolowskii est confirmée

(Delvare 2004 ; Talekar 2004 ; Shelton et al. 2008)

Europe : France, Suisse, Italie, Hongrie, Roumanie, Russie

Asie : Pakistan, Inde, Sri Lanka, Japon, Chine, Corée du nord

Afrique : Egypte, Bénin, Sénégal, Kenya, Afrique du Sud,

Amérique du nord : Canada, USA

Amérique du sud : Brésil, Chili

Caraïbes : Martinique, Guadeloupe, Barbade, Jamaïque, République Dominicaine

Océanie : Australie, îles Fidji, Guam

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2.2. Cotesia vestalis (Haliday)

2.2.1. Systématique
Cotesia vestalis a subi quelques modifications taxonomiques : décrit au début du siècle
par Kurdjumov (1912), redécrit en détail par Wilkinson (1939), il a d’abord été nommé
Apanteles plutellae. Le genre Apanteles a éclaté et a vu une partie de ses espèces reclassées
vers de nouveaux genres au sein de la sous-famille des Microgastrinae, entièrement révisée
par Mason (1981). A. plutellae est devenu Cotesia plutellae. Fitton et Walker en 1992 ont
proposé une synonymie avec Cotesia vestalis (Haliday), récemment confirmée par Shaw
(2003).

Embranchement Arthropoda Famille Braconidae

Classe Insecta Sous-famille Microgastrinae
Ordre Hymenoptera Genre Cotesia
Super-famille Ichneumonoidea Espèce vestalis

2.2.2. Morphologie, biologie et écologie

Les stades pré-imaginaux sont décrits par Delucchi et al. (1954).
L’œuf est en forme de croissant, de couleur blanchâtre et mesure 0.3 mm de long. La
femelle pond ses œufs sans préférence de localisation dans la chenille hôte.

Le premier stade larvaire est caractérisé par une larve à grosse tête, munie d’un
prolongement caudiforme avec une vésicule caudale. La larve dispose de mandibules qui lui
permettent de lutter contre les autres larves en cas de super-parasitisme au sein de la chenille
hôte (Lloyd 1940).

Au second stade, la larve est vermiforme et possède une petite tête chitinisée avec des
mandibules légèrement dentées. Elle vit en se nourrissant de l’hémolymphe de son hôte en
évitant les organes vitaux pendant tout son développement.

La nymphose se réalise à l’extérieur de l’hôte. La larve émerge en perforant le

tégument de la chenille-hôte mourante et tisse son cocon juste à côté de la dépouille. Le cocon
long de 3 mm est de couleur blanc-jaune avec un aspect soyeux. A l’intérieur du cocon, la

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 Chapitre I : Présentation du modèle biologique étudié

nymphe se colore et se chitinise progressivement, avec séparation des appendices qui

deviennent visibles, plaqués contre le corps.

L’adule émerge en découpant l’extrémité de son cocon. Il mesure entre 3 et 5 mm, son
abdomen n’est pas pétiolé et son corps est de couleur marron-noir. Ses ailes sont transparentes
et la paire antérieure porte une tache le long de la nervure costale. Cette espèce présente un
dimorphisme sexuel. Le mâle, qui est haploïde, a une morphologie plus élancée et des
antennes plus longues que le corps. La femelle qui est diploïde, est plus massive avec des
antennes plus courtes ou égales à son corps. Son abdomen est terminé par un ovipositeur (ou
tarière). Les adultes vivent généralement une à deux semaines (Verkerk & Wright 1996).
La durée moyenne du développement complet de l’œuf à l’émergence de l’adulte est de 13
jours à 20°C (Delucchi et al. 1954) (Fig. 10).

La vie des adultes s’organise autour des Brassicacées. La femelle détecte son hôte par
l’intermédiaire de l’odeur caractéristique émise par le chou endommagé par les chenilles de P.
xylostella (Bogahawatte & van Emden 1996 ; Potting et al. 1999). Il se constitue ainsi un
complexe tri-trophique très spécialisé. Ceci est lié à la propre spécificité alimentaire de son
hôte, qui ne se nourrit que de Brassicacées et il a dû s’adapter à la phytochimie particulière de
cette famille végétale. Quant aux mâles, ils vont, outre les phéromones sexuelles, utiliser ce
même signal pour trouver les femelles afin de s’accoupler. Le mâle manifeste un
comportement de cour qui consiste en des vibrations des ailes. L’accouplement, très bref, peut
se réaliser dès l’émergence des adultes. La femelle commence à pondre au cours des 24
heures qui suivent (Saw et al. 2013).
Comme de nombreux endoparasitoïdes, C. vestalis injecte lors de l’oviposition un
polydnavirus qui permet d’éviter l’encapsulation de l’œuf et qui est donc chez cette espèce
indispensable à sa réussite parasitaire. Comme la plupart des Hyménoptères parasitoïdes, C.
vestalis a un mode de reproduction haplo-diploïde. La femelle a l’aptitude de pondre des œufs
fécondés ou non. Les œufs non fécondés (haploïdes) donnent des mâles par parthénogénèse
arrhénotoque et les œufs fécondés (diploïdes) donnent des femelles. En condition
expérimentale, la femelle est capable de s’attaquer à tous les stades larvaires de son hôte.
Cependant, elle préfère les L2 et L3 (Talekar & Yang 1991 ; Shi et al. 2002). Les L1 sont
endophylles et donc difficilement accessibles compte tenu de la taille réduite de l’ovipositeur
de la femelle. Les L4 au contraire sont plus grosses et plus agiles, et donc rarement parasitées

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 Chapitre I : Présentation du modèle biologique étudié

de P. xylostella

En fin de développement
larvaire, si la chenille de
stade L4 n’est pas
parasitée, elle peut alors
se nymphoser

Figure 10 : Cycle de développement de C. vestalis à 20°C (Delucchi et al. 1954).

(Lloyd 1940). Ce parasitoïde préfère donc pondre dans des hôtes plus petits que lui, ce qui en
fait obligatoirement un koïnobionte. En effet, une fois parasitée, la chenille continue son
développement jusqu’au dernier stade larvaire d’où sortira le parasitoïde ayant terminé son
développement larvaire.
Cotesia vestalis est un endoparasitoïde solitaire qui s’attaque à un microlépidoptère
qui ne fournit de ressources nutritives que pour une seule larve. La femelle ne possède pas de
capacités de discrimination. Il n’y a pas de phéromone de marquage comme on l’observe chez
d’autres Hyménoptères (Lloyd 1940). En effet, il n’est pas rare qu’une femelle ponde
plusieurs fois dans le même hôte. Dans le cas de super-parasitisme, quand plusieurs œufs sont

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 Chapitre I : Présentation du modèle biologique étudié

pondus par différentes femelles dans un même hôte, il y a compétition et élimination des
parasitoïdes surnuméraires (Mackauer 1990). Généralement, c’est la première larve à éclore
qui détruit les autres œufs (Vinson & Hegazi 1998).

2.2.3. Répartition géographique

Cotesia vestalis est probablement originaire de la région Méditerranéenne comme son
hôte. Sa répartition, bien que plus restreinte que celle de son hôte, reste relativement étendue.
L’aire géographique que l’espèce occupe actuellement (Tableau 6) n’a plus grand-chose à
voir avec sa répartition naturelle. De nombreux programmes de contrôle de P. xylostella ont
conduit à introduire de nombreux parasitoïdes dans des zones qui n’en comptaient pas ou très
peu. De ce fait, il est devenu l’un des parasitoïdes de P. xylostella le plus répandu au monde.
Très tolérante à la chaleur (30-35°C), c’est une espèce qui est préférentiellement relâchée
dans les zones tropicales. Introduite dans de nombreux pays depuis les années 70, elle a su
s’acclimater rapidement, bien que certaines introductions se soient soldées par des échecs.

Tableau 6 : Liste des pays où la présence de C. vestalis est confirmée (Delvare 2004 ;
Talekar 2004 ; Shelton et al. 2008)

Europe : Finlande, France, Autriche, Bulgarie, Serbie, Turquie, Russie,

Asie : Pakistan, Inde, Sri Lanka, Taiwan, Indonésie, Malaisie, Philippines,
Vietnam, Chine, Japon,
Afrique : Egypte, Sénégal, Bénin, Kenya, Afrique du Sud, Réunion
Amérique du Nord : USA
Amérique du Sud : Brésil, Venezuela
Caraïbes : Martinique, Guadeloupe, Jamaïque, Barbade, Ste Lucie,
République Dominicaine
Océanie : Australie, îles Fidji

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 CHAPITRE II

Interaction entre Plutella xylostella et le parasitoïde

Oomyzus sokolowskii
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

1. Introduction

Ce chapitre est consacré à l’interaction entre P. xylostella et ses auxiliaires naturels,

particulièrement O. sokolowskii. Les travaux correspondant ont été publiés et sont présentés
ici sous la forme de trois articles.

Nous avons choisi le Sénégal comme site d’étude pour étudier cette interaction, pour
plusieurs raisons : 1) ce pays présente des caractéristiques climatiques tropicales ; 2) le chou y
est cultivé toute l’année ; 3) Plutella xylostella en est le principal ravageur et ses dégâts sont
importants ; 4) la lutte chimique y est pratiquée mais elle s’avère inefficace ; 5) à ce jour,
aucune étude approfondie n’avait été réalisée sur la teigne.

Afin d’identifier les facteurs environnementaux (biotiques et abiotiques) pouvant

favoriser ou inhiber le développement de la teigne, nous avons étudié les relations croisées
entre les facteurs climatiques, P. xylostella, sa plante hôte et ses ennemis naturels. Cette étude
a été réalisée en plein champ à Malika, dans la zone périurbaine de Dakar (article 1).

Oomyzus sokolowskii est l’un des parasitoïdes de P. xylostella et on le rencontre

majoritairement au Sénégal (Delvare & Kirk 1999 ; Sall-Sy et al. 2004). Pour autant ce
parasitoïde a été très peu étudié. Afin de connaitre son potentiel comme éventuel agent de
lutte, nous avons étudié en conditions de laboratoire quelques traits de son histoire de vie
(article 2), ainsi que les facteurs qui peuvent contribuer à connaitre et augmenter ses
performances parasitaires vis-à-vis de son hôte (article 3).

Dans la mesure où une partie des travaux proposés dans ce chapitre a été réalisée en
condition de plein champ au Sénégal, il nous a semblé important d’exposer d’abord les
caractéristiques climatiques et agricoles de ce pays de l’Afrique de l’Ouest et de faire un point
sur le statut de la teigne du chou. Ensuite nous avons fait une synthèse des résultats obtenus à
partir des trois articles, puis une conclusion.

45
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

2. Etude de terrain : le Sénégal

2.1. Présentation générale

Situé dans la zone intertropicale (entre le tropique du cancer et l’équateur), le climat y
est de type sahélien au nord (région du fleuve Sénégal), voire semi-désertique, de type
subtropical (humide) au sud (Gambie et Casamance) et de type soudanien au centre. Il est
également caractérisé par l’alternance de deux saisons :

 Une saison des pluies, appelée « hivernage », qui dure de juin à octobre, avec
un pic de précipitations en août (250 mm). C’est la période des moussons. Les
précipitations s’échelonnent entre 1500 mm au sud et 200 mm par an au nord.
Les températures sont comprises entre 25°C et 35°C.

 Une saison sèche un peu plus fraîche, de novembre à juin avec des
températures comprises entre 17°C et 25°C.

L’agriculture sénégalaise est largement dominée par des exploitations de très petite
taille de type familial qui constituent la quasi-totalité des activités agricoles villageoises. Les
cultivateurs produisent des choux, essentiellement dans les Niayes, toute l’année pour
répondre à la demande nationale, mais aussi pour l’exportation dans la sous-région
(Mauritanie, Mali, Burkina Faso, etc.). Cette région fournit 80% de la production maraîchère
nationale. La région des Niayes (Fig. 11) couvre une bande dunaire littorale, d’une longueur
de 180 km et d’une largeur d’environ 30 km, s’étendant de la banlieue de Dakar jusqu’à celle
de Saint-Louis, au Nord. Appelées aussi "Poumons maraîchers du Sénégal", les Niayes
bénéficient de conditions hydriques (nappes phréatiques nombreuses et superficielles) et
pédoclimatiques (températures fraîches, faible amplitude thermique, dunes et dépressions)
exceptionnelles, propices aux cultures maraîchères.

Le chou est un produit de grande consommation au Sénégal puisqu’il fait partie du plat
national et il est consommé quotidiennement. C’est une culture importante parce que son
cycle est relativement court (60-90 jours après repiquage). La possibilité de le cultiver toute

46
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

l’année, tant en saison sèche qu’en hivernage, permet de financer d’autres activités et d’autres
cultures. En 2011, le Sénégal a produit 50 000 tonnes de choux sur une superficie de 2 444
hectares (FAOSTAT 2013). Ce pays est le second pays producteur de chou de l’Afrique de
l’ouest, après le Niger.

Figure 11 : Les différents espaces agricoles du Sénégal. La région des Niayes (colorée en
mauve) est une zone géographique du Nord-Ouest du Sénégal qui s’étend de Dakar jusqu’à
Saint-Louis. Elle représente la zone des cultures horticoles la plus importante.

2.2. Statut de Plutella xylostella au Sénégal

Bien que les tendances de consommation de chou soient à la hausse, le potentiel de
production est fortement freiné par l’impact négatif des ravageurs sur cette culture. D’après
l’ISRA (Institut Sénégalais de Recherches Agricoles), P. xylostella est le principal ravageur.
Il provoque d’important dégâts puisque les cultivateurs peuvent perdre jusqu’à 80% de leur
production.

47
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

Face à ce problème, les agriculteurs utilisent beaucoup de pesticides, mais peu formés
ils ne connaissent pas la réelle action des pesticides utilisés, ni leur mode d’utilisation, ce qui
se traduit par une utilisation abusive et/ou mal adaptée des produits phytosanitaires. De telles
pratiques ont pour conséquences d’affecter la santé des agriculteurs et des consommateurs, de
contaminer l’environnement et les nappes phréatiques, d’induire des phénomènes de
résistance chez les populations de la teigne tout en éliminant ses ennemis naturels.

Au Sénégal, les méthodes alternatives à la lutte chimique sont très peu développées.
Cependant, les produits à base de B. thuringiensis (Bt) et d’insecticides naturels, dont les
extraits de graines de « Neem » (Azadirachta indica), peuvent constituer une alternative
efficace et plus respectueuse de l’environnement (Grzywacz et al. 2010). Bien que ces
produits constituent des palliatifs aux pesticides chimiques, l’optimisation de leur application
et la prise en compte du cortège parasitaire de P. xylostella sont souvent négligées. La lutte
biologique est, de ce fait, très peu voire pas employée. L’insuffisance de données sur la
biologie et l’écologie de la teigne du chou et de ses ennemis naturels dans les conditions
tropicales que représente le Sénégal a ainsi justifié cette étude.

2.3. Site d’expérimentation

L’étude a été réalisée dans la zone périurbaine de Dakar, dans les Niayes, sur le site de
Malika (latitude: 14°47’38 N et longitude: 17°20’20 W) (Fig. 12 et Fig. 13). Cette localité
bénéficie d’un climat tropical de type côtier à deux saisons influencé par les alizés maritimes
et la mousson. Les précipitations y sont peu abondantes et dépassent rarement 500 mm par an.
Les températures moyennes varient entre 23°C et 30°C selon la saison. L’expérimentation a
été réalisée pendant deux ans, sur la parcelle d’un agriculteur qui cultive du chou (Brassica
oleracea L. var. capitata, cultivar « Marché de Copenhague ») toute l’année, sans utiliser
d’insecticide.

48
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

Figure 12 : Localisation du site de Malika dans la zone

périurbaine de Dakar

Figure 13 : Parcelle de choux sur le site de Malika, dans la zone périurbaine de Dakar

49
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

3. Synthèse des résultats

Parmi les facteurs climatiques étudiés, la saisonnalité influence particulièrement la

dynamique des populations de la teigne. En effet, l’abondance de P. xylostella est
significativement plus importante pendant la saison sèche quand les précipitations sont peu
importantes et les températures plus basses, comprises entre 18°C et 25°C. Les précipitations
n’ont pas été un facteur de régulation du ravageur, contrairement aux températures élevées
durant la saison chaude et humide (hivernage). La plante hôte influence également la
dynamique de la teigne. L’abondance des chenilles est plus importante en début de culture
quand les choux sont jeunes.
Quatre espèces d’hyménoptères ont été identifiées comme parasitoïdes de P.
xylostella. Il s’agit d’Oomyzus sokolowskii, Apanteles litae Nixon (Braconidae), Cotesia
vestalis et Brachymeria citrae Westwood (Chalcididae). Apanteles litae s’est révélée l’espèce
la plus abondante en saison sèche, par contre O. sokolowskii a été l’espèce rencontrée le plus
régulièrement pendant toute la durée de notre étude. Le taux de parasitisme total calculé a été
de l’ordre de 10% et aucun contrôle de la teigne n’a été observé durant toute la période
d’étude.
La durée totale du développement d’O. sokolowskii, entre l’oviposition et l’émergence
de l’adulte, est relativement rapide (15 jours à 25°C), avec trois jours pour l’incubation des
œufs, quatre jours pour le développement larvaire et huit jours pour la nymphose.
Cette espèce présente un dimorphisme sexuel basé sur la taille du corps et la longueur
des tibias des adultes. La femelle est plus grosse et ses tibias sont plus longs que ceux du
mâle. C’est une espèce synovogénique, puisque la femelle produit des œufs tout au long de sa
vie. Son mode de reproduction est basé sur un système haplo-diploïde avec une
parthénogénèse arrhénotoque.
La sex-ratio de la descendance est en faveur des femelles avec en moyenne huit
femelles pour deux mâles. La femelle peut parasiter tous les stades larvaires (L2 à L4) et le
stade pré-nymphal, avec une nette préférence pour le stade L4. Le taux de parasitisme est
quatre fois plus élevé quand la femelle a été accouplée.
Une femelle isolée peut parasiter 14% de chenilles L4 et engendrer une descendance
très faible. En présence de congénères, ce taux augmente significativement puisqu’elle peut
parasiter jusqu’à 78 % des chenilles L4 mises à sa disposition et sa descendance peut être
multipliée par cinq. La population du Sénégal a des nymphes significativement plus grosses

50
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

que celles de la population de Martinique qui nous a servi de référence. Les femelles d’O.
sokolowskii du Sénégal parasitent d’avantage les chenilles L4 martiniquaises que sénégalaises
(81% et 66%, respectivement). Le taux de parasitisme varie en fonction de l’âge de la femelle
parasitoïde. Dès les premiers jours de sa vie, le taux peut atteindre 82% puis décroître jusqu’à
31% au bout de 28 jours.

4. Conclusion

Les facteurs climatiques en conditions tropicales ont une influence sur la dynamique
des populations de la teigne du chou, en effet la saison sèche est plus favorable à son
développement et les précipitations ne sont pas un facteur de régulation.
Malgré la présence de quatre espèces d’hyménoptères parasitoïdes, le taux de
parasitisme est faible et le contrôle du ravageur reste insuffisant.
O. sokolowskii présente une durée de développement rapide (15 jours) et parasite
préférentiellement les chenilles de stades L4. Ses performances parasitaires dépendent du
nombre de congénères, de l’origine de l’hôte et de l’âge de la femelle et le taux de parasitisme
obtenu en conditions de laboratoire peut atteindre 80%.
Pour conclure, O. sokolowskii est un parasitoïde de P. xylostella qui présente des
performances parasitaires évidentes en conditions de laboratoire, ce qui n’est pas le cas en
conditions de terrain. Malgré la présence des trois autres espèces, le taux de parasitisme reste
faible et la teigne n’est pas contrôlée.

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 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

ARTICLE 1

The relationship between the diamondback moth, climatic factors,

cabbage crops and natural enemies in a tropical area

Gallo SOW, Karamoko DIARRA, Laurence ARVANITAKIS and Dominique BORDAT

Folia Horticulturae, 2013, 25: 3-12

 Folia
Folia Hort. 25/1 (2013): 3-12
Horticulturae
Published by the Polish Society
DOI: 10.2478/fhort-2013-0001
for Horticultural Science since 1989

ORIGINAL ARTICLE Open access http://www.foliahort.ogr.ur.krakow.pl

The relationship between the diamondback moth,

climatic factors, cabbage crops and natural enemies
in a tropical area
Gallo Sow1, Karamoko Diarra1*, Laurence Arvanitakis2, Dominique Bordat2
1
Département de Biologie Animale, Faculté des Sciences et Techniques
Equipe Production et Protection Intégrées en Agroécosystèmes Horticoles
UCAD, BP.5005 Dakar-Fann, Senegal
2
Laboratoire Biodiversité des Agroécosystèmes en Horticulture
UR-HortSys, TA B-103, Campus International de Baillarguet, CIRAD
34398 Montpellier cedex 5, France

ABSTRACT

The impact of abiotic and biotic factors (rainfall, temperature, host plant and natural enemies) on population
dynamics of the Plutella xylostella L. diamondback moth was investigated. The experiments were conducted
during the rainy and dry seasons for two years (June 2009-April 2011) on unsprayed cabbage plots in Malika
(Senegal). Every 10 days, 10 cabbages were randomly selected. Plutella xylostella larvae, pupae and parasitoid
cocoons were recorded on each plant. Before each sampling, the diameters and ages of plants were recorded.
Temperature and rainfall were also recorded during this study. Larvae and pupae of P. xylstella were higher for
the dry season than the rainy season. There was a negative correlation between temperature and P. xylostella
populations, and a strong relationship between P. xylostella populations and the age of cabbages. Females
oviposited on young cabbages where the presence of young larvae was important, whereas older immature
stages were mainly found in older cabbage plants. Parasitoid populations were higher for the dry season than
the rainy season. High temperatures did not increase the pest populations and parasitism rate. There was no
effect found on pest, plants and natural enemies due to rainfall. There was a positive correlation between
pest populations and parasitism. Four Hymenoptera species were found: Oomyzus sokolowskii, Apanteles litae,
Cotesia plutellae and Brachymeria citrae, but they were not eficient to control the P. xylostella populations.
These results are important for understanding the factors that promote or inhibit pest populations and their
natural enemies, and therefore essential for effective crop protection.

Key words: biological control, parasitoid, plant phenology, Plutella xylostella, rainfall, temperature

INTRODUCTION is oligophagous and considered to be the most

Cabbage, Brassica oleracea L., is an important important pest for the Brassicaceae family (Talekar
vegetable crop playing a key role in the economy and Shelton 1993, Sarfraz et al. 2006). The
of many countries, particularly in Asia and Africa proliferation of larvae pest populations is favoured
(Grzywacz et al. 2010). The diamondback moth by the short duration of the life cycle, with up to
Plutella xylostella L. (Lepidoptera: Plutellidae) 20 generations per year under tropical conditions

*Corresponding author.
Tel.: +221 774 50 27 54; fax: +221 338 24 63 18;
e-mail: [email protected] (K. Diarra).

55
4 Relationship between Plutella xylostella and its environment

(Vickers et al. 2004) and a high reproductive endemic lora, natural enemies and climatic factors
potential of the females (Justus et al. 2000). The (Regnault-Roger 2005). Temperature and humidity
damage caused by this pest has been estimated are among the most important climatic factors
globally to cost US$ 1 billion in direct losses and affecting the biology of the diamondback moth (Guo
control costs (Grzywacz et al. 2010). The use of and Qin 2010). According to Ansari et al. (2010),
synthetic insecticides is the main control strategy the development of P. xylostella depends on the
(Kibata 1996). This pest has developed resistance host plants and temperature. The development rate
against all major groups of pesticides, including in relation to temperature plays an essential role in
Bacillus thuringiensis bacterial based bio-pesticides pest management, especially in helping to predict
(Tabashnik et al. 1990, Zhou et al. 2011). the timing of the development of pests and natural
Several studies (Shelton et al. 1993, Hill and enemies in ield conditions (Roy et al. 2002).
Foster 2000, Liu et al. 2000) have shown that The biology and ecology of the pest population
the use of insecticides is not a sustainable pest and its relationship with the host plant and natural
management option for farmers, as it is fraught enemies must also be studied (Campos et al. 2003).
with problems such as the improper handling of Brassica IPM depends on a good understanding
pesticides, increased cost of pesticides, reduced of factors affecting P. xylostella population
control eficacy and contamination of the farming dynamics. In this study, the impact of abiotic and
environment (Dobson et al. 2002). A possible biotic factors (rainfall, temperature, host plant and
alternative to pesticides in the development of an natural enemies) on the population dynamics of
integrated management strategy against P. xylostella P. xylostella was investigated on cabbage plants in
is biological conservation control using endemic the ield.
parasitoids (Sarfraz et al. 2005). Parasitoids are
particularly susceptible to chemical insecticides MATERIAL AND METHODS
and understanding their role in the ecosystem is
important for the implementation of an integrated Study site
pest management strategy (Shepard et al. 1999). The study was conducted in Malika, a district in
More than 90 insect parasitoids have been the Niayes of Dakar, Senegal (N: 14°47’552, W:
recorded, but less than 10 have bio-control potential 17°19’818 and 189 m above sea level). The area is
for P. xylostella (Noyes 1994). Among these characterised by a long dry season from November
natural enemies, Cotesia plutellae Kurdjumov to June with a temperature range of 15-20°C and
(Hymenoptera: Braconidae) is the most abundant a short rainy season from July to October, with
larval parasitoid of P. xylostella in South Africa temperatures ranging between 25 to 35°C (Pereira
(Kir 1997, Mosiane et al. 2003). In Ethiopia, 1963). The yearly precipitations do not exceed
Oomyzus sokolowskii Kurdjumov (Hymenoptera: 500 mm between August and September. The
Eulophidae), Diadegma sp. (Hymenoptera: experiments were conducted during the rainy and
Ichneumonidae) and Cotesia plutellae are the dry seasons for two years from June 2009 to April
most important ones, accounting for more than 2011.
90% of the parasitoid complex (Ayalew et al.
Cabbage crops
2004). However, total parasitism of P. xylostella
rarely exceeds 15% in East Africa (Kir 2003). The host plants (Brassica oleracea L. var. capitata
According to Löhr and Kir (2004), the diversity 'Copenhagen Market' were grown in a small farmers’
of the parasitoid fauna associated with P. xylostella ield and no insecticide was used. Thirty-day old
in West Africa is relatively poor. Most common seedlings were transplanted to seven replicate
were C. plutellae and O. sokolowskii in Benin and plots. Plot size was six rows of 5 m length, each
Senegal (Goudegnon et al. 2004, Sall-Sy et al. with a spacing of 40 cm between plants and 60 cm
2004), while Apanteles litae Nixon (Hymenoptera: between rows. Spacing between plots was 1 m. In
Braconidae) was predominant in Ivory Coast (Löhr order to protect the plants from nematodes, Furadan
and Kir 2004). at 500 g was applied in the soil prior to planting.
The agro-ecological concept, which integrates Poultry manure at 50 kg was applied 10 days later
agriculture into the natural ecosystem, has been with intensive irrigation. Additional fertilizers NPK
found useful for population management of (10:10:20) at 5 kg and poultry manure at 75 kg
P. xylostella (Vandermeer 1995). Population were applied 15 days after planting. The crops were
management of pests integrates cultivated plants, watered daily using sprinkler irrigation.

56
Gallo Sow, Karamoko Diarra, Laurence Arvanitakis, Dominique Bordat 5

Sampling methods analysed using one-way ANOVA (XLSTAT).

The samplings started 10 days after transplanting Means were separated using the Student Newman
and were performed every 10 days on unsprayed Keuls test. Correlation analyses were performed
cabbage plots. Samples were collected randomly to determine relationships between the abundance
by selecting 10 cabbages in the central rows of of P. xylostella and rainfall, infestation levels
each plot. Each plant selected was examined and temperature, plant age and infestation levels,
and numbers of P. xylostella larvae (second to rainfall and parasitoid populations, temperature
and parasitoid populations, plant age and parasitoid
fourth instar), pupae and parasitoid cocoons were
populations, and abundance of P. xylostella and
recorded and left to develop in order to determine
parasitoid populations using XLSTAT version
parasitism levels in the ield (Nofemala and Kir
2012.1.01. In all statistical analyses, p = 0.05 was
2005). The eggs and larvae that were inside the
considered signiicant.
leaves were not considered. The samples were
taken to the laboratory where they were maintained
RESULTS
at 25°C, 60% relative humidity and 12 h light/
dark photoperiod. Emerging parasitoids were Relationships between P. xylostella populations
identiied (by the taxonomy Laboratory from Cirad, and climatic factors
Montpellier, France), and their incidence recorded. Effects of season
The diameters and ages of cabbage plants
The population density of the pest varied
collected during each sample were noted. The
signiicantly between the dry and rainy seasons
temperature of the air was recorded with the aid of (F = 11.17, p = 0.002, Tab. 1). The abundance of
an automatic tape recorder, “Tinytag”, programmed P. xylostella was higher in the dry season than in
via the software Tinytag Explorer 4.1 (Tinytag the rainy season. Infestation levels by larvae and
Explorer 2005). Parameters were recorded all pupae were high at the middle of the dry season
10 min and permitted to have a daily mean of (February-April), luctuating between 100 and 800
temperature. Rainfall was also recorded daily using larvae and pupae per plant. The immature stages of
a rain gauge. The effects of these factors on the P. xylostella were low in the rainy season (1 to 200
population dynamics of P. xylostella and parasitoid larvae and pupae per plant) and the beginning of the
populations were assessed. dry season (16 to 120 larvae and pupae per plant)
Statistical analysis (Fig. 1).
The data were normalised by logarithmic Effects of rainfall and temperature
transformation before being subjected to an No signiicant correlation was found between
analysis of variance (ANOVA). The abundance of P. rainfall and infestation levels of P. xylostella
xylostella larvae and pupae, parasitoid populations, (r = –0.198, p = 0.247). There was a signiicant
temperature and rainfall among the seasons were difference between rainfall during the rainy and
dry seasons (F = 13.75, p = 0.001, Tab. 1). Rainfall
Table 1. Overall relationship between the abundance of was higher during the rainy season than the dry
Plutella xylostella larvae and pupae, parasitism, adults
season (Fig. 2). There was a signiicant correlation
of parasitoid species, temperature and rainfall from June
2009 to April 2011 between temperature and P. xylostella populations
(r = –0.405, p = 0.014). There was also a signiicant
Season difference between temperatures during the rainy
Parameter
dry rainy and dry seasons (F = 65.37, p = 0.0001, Tab. 1).
P. xylostella larvae/pupae 474.0 ± 71.4 a* 29.1 ± 9.2 b Temperatures were higher (30 to 42°C) during the
Parasitism (%) 5.5 ± 1.6 a 0.4 ± 0.1 b rainy season than the dry season (Fig. 2).
Oomyzus sokolowskii 7.7 ± 1.5 a 0.9 ± 0.3 b Relationship between P. xylostella populations
Apanteles litae 10.3 ± 2.3 a 0.6 ± 0.2 b and the age of the cabbage
Cotesia plutellae 3.9 ± 2.1 a 1.1 ± 0.7 a There was a negative correlation between the young
Brachymeria citrae 0.0 a 0.1 ± 0.1 a larval stages of P. xylostella with the cabbage age
Mean temperature (°C) 23.8 ± 1.5 b 29.7 ± 2.6 a (r = –0.340, p = 0.038). These young stages (L2 and
Total rainfall (mm) 0.0 b 498.0 ± 0.0 a L3) decreased when the age of the cabbage was 50
*Values marked with the same letters do not differ signiicantly to 55 days in the dry season. On the other hand,
at p = 0.05 the number of old stage (L4 and pupae) increased

57
6 Relationship between Plutella xylostella and its environment

800
700 Pupae
Fourth instar larvae
No. of P. xylostella

600
Third instar larvae
500 Second instar larvae
400
300
200
100
0
25.06.09
04.07.09
14.07.09
24.07.09
04.08.09
14.08.09
24.08.09
15.09.09

25.10.09
04.11.09
14.11.09
24.11.09
15.12.09
25.12.09
04.01.10
14.01.10

15.07.10
25.07.10
04.08.10
14.08.10
24.08.10
03.09.10
13.09.10
23.09.10

06.02.11
16.02.11
26.02.11
08.03.11
18.03.11
28.03.11
07.04.11
17.04.11
Rainy season Dry season Rainy season Dry season

Figure 1. Abundance of P. xylostella larvae and pupae on unsprayed cabbage ields during the rainy and dry seasons
from June 2009 to April 2011

120 Rainfall 45
40
100 Temperature
35

Temperature( °C)
Rainfall (mm)

80 30
25
60
20
40 15
10
20
5
0 0
04.07.09

24.11.09
25.06.09

14.07.09
24.07.09
04.08.09
14.08.09
24.08.09
15.09.09

25.10.09
04.11.09
14.11.09

15.12.09
25.12.09
04.01.10
14.01.10

15.07.10
25.07.10
04.08.10
14.08.10
24.08.10
03.09.10
13.09.10
23.09.10

06.02.11
16.02.11
26.02.11
08.03.11
18.03.11
28.03.11
07.04.11
17.04.11

Rainy season Dry season Rainy season Dry season

Figure 2. Total rainfall and mean temperature recorded at Malika (Senegal) during the rainy and dry seasons from June
2009 to April 2011

900
Abundance of immature stages

800

700

600
500

400
300
200
100
0
10 20 30 40 50 60 70 80
Days after transplanting
Second instar larvae Third instar larvae Fourth instar larvae Pupae

Figure 3. Relationship between the abundance of immature stages of P. xylostella and the age of the cabbage in the dry
season. Arrow indicates the beginning of hearting (cabbage maturation)

58
Gallo Sow, Karamoko Diarra, Laurence Arvanitakis, Dominique Bordat 7

100%
90%
Pecentage of total

80%
70%
60%
50%
40%
30%
20%
10%
0%
10 20 30 40 50 60 70 80
Days after transplanting
Second instar larvae Third instar larvae Fourth instar larvae Pupae

Figure 4. Relationship between the relative abundance of immature stages of P. xylostella and the age of the cabbage
in the dry season. Arrow indicates the beginning of hearting (cabbage maturation)

with the age of the cabbage (r = 0.44, p = 0.007) the parasitoid complex (Fig. 5). It was the only
(Figs 3 and 4). There was a signiicant correlation gregarious parasitoid recorded during this study.
between the diameter of the cabbage plants and pest The population density of O. sokolowskii varied
populations (r = 0.39, p = 0.018). The diameter of signiicantly between the dry and rainy seasons
cabbage plants increased with plant age. (F = 14.49, p = 0.001, Tab. 1). Apanteles litae
Relationships between natural enemies and Dixon (Braconidae), a larval parasitoid, was most
climatic factors predominant in the dry season (F = 7.55, p = 0.009,
Effects of season Tab. 1). Cotesia plutellae Kurdjumov (Braconidae),
a larval parasitoid, was also recorded throughout
Parasitoid populations were signiicantly different
the year, but its activity was sporadic (Fig. 5). There
depending on the seasons (F = 29.81, p = 0.0001).
The average parasitism varied signiicantly between was no signiicant difference between the seasons
seasons. It was high in the dry season, and very low (F = 1.71, p = 0.19, Tab. 1). Only two specimens
in the rainy season (Tab. 1). of Brachymeria citrae Westwood (Chalcididae),
Four indigenous parasitic Hymenoptera were a pupal parasitoid, were recorded and there were
found in the pest populations (Fig. 5). Oomyzus no signiicant differences between the seasons (F =
sokolowskii Kurdjumov (Eulophidae), a larval- 0.23, p = 0.06, Tab. 1). Hyperparasitoids were not
pupal, was active throughout the year and dominated recorded.

30
Oomyzus sokolowskii
25 Apanteles litae
Cotesia plutellae
No. of parasitoids

20 Brachymeria citrea

15

10

0
04.08.10

16.02.11
25.06.09
04.07.09
14.07.09
24.07.09
04.08.09
14.08.09
24.08.09
15.09.09

25.10.09
04.11.09
14.11.09
24.11.09
15.12.09
25.12.09
04.01.10
14.01.10

15.07.10
25.07.10

14.08.10
24.08.10
03.09.10
13.09.10
23.09.10

06.02.11

26.02.11
08.03.11
18.03.11
28.03.11
07.04.11
17.04.11

Rainy season Dry season Rainy season Dry season

Figure 5. Abundance of parasitoids associated with P. xylostella on unsprayed cabbage ields during the rainy and dry
seasons from June 2009 to April 2011

59
8 Relationship between Plutella xylostella and its environment

Effects of rainfall and temperature between cabbage age and O. sokolowskii (r = –0.44,
No signiicant correlation was found between p = 0.007, Fig. 7). No signiicant correlation was
rainfall and parasitoid populations (r = –0.27, p found between the age of the cabbage and A. litae
= 0.1) (Fig. 2). There was a negative correlation (r = –0.23, p = 0.16), C. plutellae (r = –0.32, p =
between temperature and parasitoid populations 0.056) or B. citrae (r = –0.041, p = 0.8).
(r = –0.34, p = 0.04). There was also a negative Relationships between P. xylostella populations
correlation between temperature and A. litae (r = and natural enemies
–0.35, p = 0.032). There was a positive correlation The correlation between pest populations and total
between temperature and B. citrae (r = 0.36, p = parasitism was positive (r = 0.36, p = 0.003)(Figs
0.03). However, no signiicant correlation was 3 and 7). There was a positive correlation between
found between temperature and O. sokolowskii P. xylostella populations and O. sokolowskii (r =
(r = –0.29, p = 0.08). There was also no correlation 0.38, p = 0.02), A. litae (r = 0.98, p = 0.0001) and
between temperature and C. plutellae (r = –0.13, p C. plutellae (r =0.77, p = 0.0001). No signiicant
= 0.4). correlation was found between P. xylostella
Relationships between natural enemies populations and B. citrea (r = 0.08, p = 0.6).
and the age of the cabbage
The correlation between the age of the cabbage DISCUSSION
and total parasitism was signiicant (r = –0.65, p = The relationships between P. xylostella populations,
0.0001, Fig. 6). There was a negative correlation climatic factors, cabbage phenology and the natural

7
Percentage parasitism

0
10 20 30 40 50 60 70 80
Days after transplanting

Figure 6. Relationship between P. xylostella parasitism and the age of the cabbage in the dry season. Arrow indicates
the beginning of hearting (cabbage maturation)

30
Abundance of parasitoids

25

20

15 Cotesia plutellae
Apanteles litae
10
Oomyzus sokolowskii
5

0
10 20 30 40 50 60 70 80
Days after transplanting

Figure 7. Relationship between the abundance of natural enemies and the age of the cabbage in the dry season. Arrow
indicates the beginning of hearting (cabbage maturation)

60
Gallo Sow, Karamoko Diarra, Laurence Arvanitakis, Dominique Bordat 9

enemy fauna were examined. The importance populations (Elliott et al. 2002), and the parasitoid
of climatic factors in the population dynamics of complexes develop more in relation to plant
this pest has been emphasised by several authors succession (Price 1973). Temperature can have
(Cohen 1982, Vickers et al. 2004). The P. xylostella considerable effects on host susceptibility and/
population was low in the rainy season. It is or parasite virulence with parasitoids (Matthew
possible that rainfall may cause the mortality of and Blanford 2003). Our study showed that the
immature stages of P. xylostella; but it is unlikely temperatures in the dry season (20 to 25°C) were
to be a major factor in the reduction of the pest a favourable range for the development, survival,
populations during this period. The detrimental and reproduction of parasitoids particularly for O.
effect of rainfall and high temperature on pest sokolowskii (Wang et al. 1999).
populations has been reported by several authors However, the impact of parasitoids on P. xylo-
(Wakisaka et al. 1992, Lui et al. 2000, Shirai stella populations was low. Parasitoid populations
2000, Waladde et al. 2001). In the present study, were not able to control this pest. Shepard et al.
we observed that the temperature inluenced the (1999) noted that in Southeast Asia, indigenous
dynamics of the P. xylostella population. The pest parasitoids of cabbage moth were not able to regulate
population increased when the temperature fell. populations of this pest. Many agro-ecosystems are
In the rainy season, the mean temperature was unfavourable environments for natural enemies due
29.7°C, in the transient season it was 26.8°C and to high levels of disturbance (Landis et al. 2000).
in the dry season it was 23.8°C. These data conirm These results may be due to the particular
Atwal (1955), where the optimal temperature for P. location of the cabbage plots, but the importance
xylostella development was 17 to 25°C. However, of the selection pressures present in each agro-
several authors have reported that P. xylostella ecosystem and the effects of natural selection
is a more important pest in tropical areas than in on the totality of viable species and the change
a temperate climate. This pest has a high number of in their behaviour during successive generations
generations per year in tropical areas; 20 in Taiwan should be recognised. For example, C. plutellae
and 28 in Malaysia (Miyata et al. 1986, Talekar and populations control P. xylostella larval populations
Shelton 1993). In Senegal (semi-urban Dakar area), in South Africa (Smith and Villet 2002) and in some
P. xylostella larvae damage is most important in localities in Benin (Goudegnon et al. 2000), have
the dry season, probably due to many consecutive decreased a few larval populations in Martinique
cabbage crops growing in this area for a long period Island (Smeralda 2000) and have had no incidence
of time. In the rainy season, vegetable farmers do in Dakar Niayes.
not grow cabbages continuously. Generally, the immediate environment of
A signiicant relationship was found between a cultivated plot is more inluential (beneicial or
the immature stages of P. xylostella and the age not) on the population of pests than on natural
of the cabbage. The number of young larval stages enemy populations (Burel et al. 2000). Further
decreased on aged plants, whereas pupal stages studies in other environmental conditions in the
increase considerably. According to Nofemela ield will be conducted to conirm the inluence of
and Kir (2005), the preponderance of younger the selection pressures on P. xylostella populations
individuals is an indication of a growing population, and their natural enemies in a cabbage crop agro-
whereas the high incidence of older individuals system. Despite four species of natural enemies,
is an indication of a declining population. This
the low parasitism rates found on P. xylostella
phenomenon is probably also due to the low
immature stages cannot control pest populations
attraction of old cabbages to ovipositing females
and may necessitate additional control measures.
because the glucosinolates produced by the cabbage
decrease in concentration during tissue maturation
CONCLUSIONS
(Hopkins et al. 1998, Spencer et al. 1999), and
the effect of declining resource quality (Campos 1. The present study showed that climatic factors
et al. 2006). According to Campos et al. (2003), inluenced the dynamics of P. xylostella
plant ageing increased pre-imaginal mortality and populations and their natural enemies. The
reduced the larval development rate and fecundity. density of pests increased when the temperature
Relationships were found between P. xylostella was low. Females of P. xylostella oviposit on
and parasitoid populations. Generally, the abundance relatively young cabbages where the presence
of natural enemy populations increases with host of young larvae was important, whereas older

61
10 Relationship between Plutella xylostella and its environment

immature stages were mainly found in older scale brassicas and tomatoes. Natural Resources
cabbages. Institute (NRI), Kent, UK: 179.
elliOTT n.C., KieCKheFer r.w., MiChels G.J., Giles
2. Farmers can avoid chemical treatments 40 days
K.l., 2002. Predator abundance in alfalfa ields
after planting; the cabbages were not attractive to in relation to Aphids, within-ield vegetation, and
female pests. The limitation of these treatments landscape matrix. Environ. Entomol. 31(2): 253-260.
promotes the survival of parasitoid fauna, GOudeGnOn A.e., KirK A.A., ArvAniTAKis l., BOrdAT d.,
despite its low observed incidence. 2004. Status of the diamondback moth and Cotesia
3. For treatments against larval populations, plutellae, its main parasitoid, in the Cotonou and
farmers should use bacterial insecticides, such Porto Novo periurban areas of Benin. In: Improving
as Bacillus thuringiensis (Bt) to control them. biocontrol of Plutella xylostella. A.A. Kirk and D.
Bordat (eds), Proc. Intl. Symp., 21-24 October 2002,
These products have no effect on natural
Montpellier, France: 172-178.
enemies.
GOudeGnOn A.e., KirK A.A., sChiFFers B., BOrdAT d.,
2000. Comparative effects of deltamethrin and neem
ACKNOWLEDGEMENTS kernel solution treatments on diamondback moth
and Cotesia plutellae (Hym., Braconidae) parasitoid
The authors are grateful for the support of the AUF
populations in the Cotonou peri-urban area in Benin.
through the program “Horizons Francophones”. We
J. Appl. Entomol. 124: 141-144.
would also like thank two anonymous reviewers for GrzywACz d., rOssBACh A., rAuF A., russel d.A.,
comments on the manuscript. srinivAsAn r., shelTOn A.M., 2010. Current
control methods for diamondback moth and other
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12 Relationship between Plutella xylostella and its environment

wAlAdde s.M., leuTle M.F., villeT M.h., 2001. zhOu l., huAnG J., Xu h., 2011. Monitoring resistance
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Plutella xylostella. BioControl 44: 391-402. Received August 15, 2012; accepted November 21, 2012

64
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

ARTICLE 2

Life history traits of Oomyzus sokolowskii Kurdjumov (Hymenoptera:

Eulophidae), a parasitoid of the diamondback moth

Gallo SOW, Laurence ARVANITAKIS, Saliou NIASSY, Karamoko DIARRA,

and Dominique BORDAT

African Entomology, 2013, 21: 231-238

Life history traits of Oomyzus sokolowskii Kurdjumov (Hymenoptera: Eulophidae), a
parasitoid of the diamondback moth

G. Sow *, L. Arvanitakis , S. Niassy , K. Diarra & D. Bordat

1 2 1,3 1 2

1
Equipe Production et Protection Intégrées en Agroécosystèmes horticoles, Département de Biologie Animale,
Faculté des Sciences et Techniques, Université Cheikh Anta Diop de Dakar (UCAD), BP 5005, Dakar, Senegal
2
Laboratoire de Biodiversité des Agrosystèmes Horticoles TAB/L, Campus International de Baillarguet, CIRAD,
34398 Montpellier Cedex 5, France
3
International Centre for Insect Physiology and Ecology, P.O. Box 30772-00100, Nairobi, Kenya

In this study the life-history of Oomyzus sokolowskii (Kurdjimov), a parasitoid of the

diamondback moth (DBM) Plutella xylostella (L. ) as characterized. The life cycle, adult size,
fecundity, ovigeny, parthenogenesis, host age preference and host-searching behaviour by
parasitoid females were studied under laboratory conditions. Oomyzus sokolowskii’s life cycle
lasted 15. 6 days. Sexual dimorphism was recorded, with females being bigger than males.
The species is synovigenic. The parasitism rate was significantly different between mated
and unmated females, which implied that mating stimulated the behaviour of parasitism.
Thelythokous parthenogenesis was not recorded. Females could parasitize all larval stages
and pre-pupae, but the parasitism rate was higher in the fourth larval stages of DBM. The
host-seeking behaviour was influenced by host spatial patchiness; O. sokolowskii females
performed better when they were placed in a 7 cm3 oviposition box. This study gives a better
understanding of the life history traits of O. sokolowskii, which has been neglected in the
biological control of DBM in tropical regions. The study suggests the use of O. sokolowski as a
promising candidate for the management of DBM in cabbage in combination with other
IPM strategies.
Key words: Plutella xylostella, sexual dimorphism, ovigeny, parthenogenesis, koinobiont,
behaviour, biological control.

INTRODUCTION
Parasitoids are insects that feed on the body of parasitoid development is correlated with
another insect or arthropod during the larval stage parasitoid life history traits (Mayhew & Blackburn
of their life cycle. The host organism will die as a 1999; Jervis et al. 2003).
result (Jervis et al. 2001). The study of parasitoid Most parasitic wasps reproduce sexually as well
biology and behaviour was first motivated by as parthenogenetically. Apart from a number of
their interest as auxiliaries in biological control species or strains that reproduce by thelythokous
programmes (van Alphen & Jervis 1996). The life- parthenogenesis, most parasitic wasps reproduce
history traits are directly related to the organism’s by arrhenotokous parthenogenesis (Wenseleers
fitness, hence to its reproductive success and sur- & Billen 2000). In arrhenotokous species, mated
vival (Roitberg et al. 2001; Le Lann et al. 2011). For females store sperm in their spermatheca and may
example, the hind tibia length is the best indicator have the ability to control the sex ratio of their
of body size among parasitoids and is usually offspring by modifying the proportion of fertilized
correlated with fitness (Riddick 2005; Da Rocha eggs laid (Ratnieks & Keller 1998).
et al. 2007). In parasitoids, egg production has been identi-
Parasitoids can be koinobiont, i.e. parasitoids fied as an important life history trait (Rosenheim
whose larvae are associated with the development et al. 2000; Jervis et al. 2001). Flanders (1950) classi-
of their hosts and emerge at the end of their fied parasitic wasps into two groups: pro-ovigenic
development, and idiobiont, which kill or paralyse species whose eggs mature prior to laying, and
their host at the time of parasitism and use the re- synovigenic species that continue to mature eggs
sources available at the time of oviposition throughout their reproductive lives. Ovigeny is a
(Quicke1997). Comparative studies on a wide concept that helps in the understanding of the
number of species have shown that the mode of evolution of life history strategies in insects, and it
*Author for correspondence. E-mail: [email protected] is measured by the ovigeny index which ranges
African Entomology 21(2): 231–238 (2013)
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232 African Entomology Vol. 21, No. 2, 2013

from 0 to 1 and is defined as the ratio of the mature the adult females to oviposit on brown mustard
egg load at emergence (initial egg load) on maxi- plants (Brassica juncea L. Czern. ) in oviposition
mum potential lifetime fecundity (Jervis et al. 2001). boxes (50 cm × 50 cm × 50 cm). Egg clutches were
Parasitoid foraging behaviour affects the number collected every 24 hours. At hatching, larvae were
of successfully developing offspring (Mackauer & placed on fresh leaves of cauliflower (Brassica
Völkl 1993; Godfray1994). For this reason, knowl- oleracea L. var. botrytis). Mature larvae were trans-
edge of parasitoid foraging behaviour can enhance ferred to new leaves in a large plastic box (28 cm ×
the implementation of a successful biological con- 27 cm × 8 cm) where they pupated. The pupae
trol programme (Godfray 1994). To find their host, were collected daily. At emergence, adults were
parasitoids may use chemical signals such as host placed in oviposition boxes and fed with water
sex pheromones (Leal et al. 1995) or aggregation and honey.
pheromones (Yasuda & Tsurumachi 1995), or the Cultures of O. sokolowskii were maintained by
volatile compounds produced by the infested exposing fourth-instar DBM larvae to parasitoid
plant (Choh et al. 2008; Kawazu et al. 2010). females in a clear plastic container (5 cm high, 8 cm
Oomyzus sokolowskii (Kurdjumov) (Hymenoptera: diameter). After 24 hours exposure, all larvae were
Eulophidae) is an important parasitoid and a removed and placed in an identical container.
potential biocontrol agent of the diamondback Adult parasitoids emerging from parasitized pupae
moth (DBM) Plutella xylostella (L.) (Lepidoptera: were recovered and fed with honey.
Plutellidae), a major pest of Brassicaceae (Fitton & All rearing and experiments were conducted at a
Walker 1992). This gregarious parasitoid is adapted constant 25 °C, 60 % relative humidity and 12L/
to high temperature conditions and has been 12D photoperiod.
introduced in tropical and subtropical zones to
control DBM (Talekar & Hu 1996), where it has Development of O. sokolowskii
been recorded as an effective parasitoid (Ooi 1988; Fifteen one-day-old females and 30 fourth-
Liu et al. 1997). In Senegal, it is the most common instar DBM larvae were placed in a clear plastic
parasitoid of DBM (Sall-Sy et al. 2004). Therefore, container (5 cm high, 8 cm diameter) daily. After
studies are needed to improve the knowledge of 24 hours of exposure, all pupae were recovered and
the biology and ecology of this beneficial for its placed individually in clear plastic pill bottles
integration in a DBM management programme (1 cm high, 3 cm diameter). To determine the
in cabbage. immature stages of O. sokolowskii, 30 parasitized
In this study the life history traits of O. sokolowskii pupae were dissected and observed under a micro-
were determined under laboratory conditions. scope. The development time in days of the
These included the parasitoid development cycle, parasitoid was measured from oviposition to adult
adult size, fecundity, ovigeny, parthenogenesis, eclosion.
host age preference and foraging behaviour of
females. The establishment of these traits in
Adult size and ovigeny
O. sokolowski will be useful for the improvement
The size of adult O. sokolowskii was assessed by
of biocontrol strategies against DBM in tropical
measuring the length of the hind tibiae of 30
areas.
one-day-old males and 30 one-day-old females
MATERIAL AND METHODS using a microscope equipped with an ocular
micrometer.
Insect rearing The ovigeny index was calculated using the
The study was conducted at the Entomology formula of Jervis et al. (2001). To determine the
Laboratory of the Centre for International Coop- initial egg load, we used virgin females obtained
eration in Agronomic Research in Development by dissecting them as pupae out of parasitized
(CIRAD) in Montpellier, France. DBM pupae and placing them in separate pill
The parasitoid population was obtained from boxes (1cm × 3 cm). Upon emergence, 30 virgin
parasitized pupae of DBM collected in 2011 from females were dissected and the number of eggs in
cabbage crops (Brassica oleracea L. var. capitata) in each was counted. Maximum potential lifetime
the Niayes area, situated in northwest Senegal fecundity was determined by presenting 30 one-
(12°54’44”N 12°8’84”W, and at 189 m altitude). day-old mated female parasitoids, each with two
Cultures of DBM were maintained by allowing new fourth-instar DBM larvae every day until the

68
 Sow et al.: Life history traits of Oomyzus sokolowskii, a parasitoid of the diamondback moth 233

female died. After 24 hours of contact, formed (ANOVA) using Statview version 4. 55 (Statview
pupae were placed individually in pill boxes. The 1996). The sizes of parasitoid adults, female pro-
number of eggs laid in host larvae and the duration ductivity, parasitism rates, and progeny sex ratios
of female oviposition were recorded. Each dead from mated and unmated females were compared
female was dissected and the number of eggs with t-tests. Parasitism rates of the different stages
remaining was counted. of DBM were analysed using one-way ANOVA.
Female productivity, parasitism rate, sex ratio,
Parthenogenesis and offspring development time from the three
Thirty parasitized DBM pupae were dissected to oviposition boxes were analysed using one-way
recover parasitoid pupae, and each was isolated in ANOVA. Means were separated using the Stu-
a pill box (1 cm high, 3 cm diameter). Thirty of the dent-Newman-Keuls test (XLSTAT software ver-
resulting unmated females were each presented sion 2012.1.01). The sex ratio (% female) was
with two fourth-instar DBM larvae. The same calculated using the formula by Silva-Torres et al.
experiment was performed using 30 mated females. (2009). In all statistical analyses P-values < 0. 05
After 24 hours, each DBM larva that successfully were considered significant.
pupated was placed individually in a pill box and
monitored until the emergence of parasitoids or RESULTS
adult moths. Parasitoids were sexed, and the para-
sitism rate, the number of parasitoids produced Development of O. sokolowskii
and the sex ratio (% females) was calculated and The incubation period was 3.2 ± 20 days. The
compared with offspring of unmated and mated eggs were aggregated to each other forming one
females. or more clusters of 5 to 20 units . Most eggs were
located in the back of the host larvae but isolated
Host age preference eggs were sometimes observed. Vermiform larvae
Five stages of immature DBM (L2, L3, L4, hatched three days after host parasitism and con-
prepupae, and pupae) were exposed to 24h-old tinued growing until filling the entire body of the
unmated parasitoid females. Thirty individuals of host pupae. The duration of the larval stage
one stage were exposed to 15 female parasitoids in ranged from four to seven days (mean 4.6 ± 0.24).
a clear plastic container (5 cm high, 8 cm diameter). The parasitoid prepupae were whitish with red
After 24 hours of exposure, the immature DBM eyes, and the pupae were dark. The nymphal
were recovered and placed individually in pill stage lasted 7.8 days on average. Adults emerged
boxes, where they were monitored until emergence. from the pupae parasitized by drilling several
Five replicates were performed for each DBM holes through the host cuticle. Males and females
stage. The parasitism rate was calculated from the of the parasitoid emerged simultaneously from
number of parasitized larvae and pupae recovered. DBM pupae. The overall development time of
O. sokolowskii from egg to adult was 15.6 ± 0. 40
Foraging behaviour days (Fig. 1).
To study foraging behaviour, three different
oviposition boxes; 3 cm3 (A), 7 cm3 (B) and 40 cm3 Adult size and ovigeny
(C) were used. In each box, one 24h-old female Females were significantly larger than males
O. sokolowskii was exposed to two fourth-instar (Table 1), and the size difference was significant
larvae of DBM for 24 hours. DBM pupae were (t = –8.71, d.f. = 58, P < 0. 0001). After the emer-
placed individually in pill boxes, and were moni- gence, O. sokolowskii females had an average initial
tored until emergence. Ten replicates were per- egg load of 14 eggs. The average potential fecundity
formed for each oviposition box size. Parasitism was four times higher. The ovigeny index of this
rate, female productivity, number eggs laid per species was 0.3 (Table 1).
female, sex ratio (% female) and offspring devel-
opment time were compared among the three Parthenogenesis
oviposition boxes. The parasitism rate was significantly different
between unmated and mated females (t = 6.39,
Statistical analysis d.f. = 6, P = 0. 0007). The mated females produced
Data were normalized by logarithmic transfor- normal sexual offspring (males and females) while
mation before performing an analysis of variance unmated females produced only males (Table 2).

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234 African Entomology Vol. 21, No. 2, 2013

Fig. 1. Development time in days of Oomyzus sokolowskii from egg to adult at 25 °C. Different letters indicate signifi-
cant differences (Student-Newman-Keuls, P < 0.05).

Table 1. Sexual dimorphism, fecundity and ovigeny index (mean ± S.E.) of Oomyzus sokolowskii.

Tibia length (mm) Initial egg-load Potential fecundity Ovigeny index

Male 0 3 ± 0 01 a – – –
Female 0 4 ± 0.01 b 14. 1 ± 1 68 53. 6 ± 1. 60 0.3

Means in columns followed by the same letters are not significantly different (t-test, P > 0.05).

Host age preference P <0. 05) than in the boxes A and C. Ten females
The parasitism rate varied significantly with the laid in B, whereas in A and C, 3 and 1, respectively.
host age (F4.16 = 26.23, P <0.0001). It was signifi- The number of male and female offspring in box B
cantly higher at the L4 larval stages (P < 0. 05). was significantly different from those in boxes A
However, there was no significant difference and C (F2.18 = 5.87, P = 0.008, and F2.18 = 10.00,
between L2 and L3 stages (Fisher, P > 0. 05). The P = 0. 001, respectively). Similarly, the total number
parasitism rate was significantly lower in prepupae of offspring was significantly higher in B (F2.18 =
and no parasitism was recorded on pupae (Table 3). 10.29, P = 0. 001). The sex ratio of offspring was
not significantly different between the three ovi-
Foraging behaviour position boxes (F2.18 = 1.42, P = 0.28). The offspring
The parasitism rate was significantly different development time was significantly higher in
in the three oviposition boxes (F2.18 = 15.87, P < box C, not statistically different in A and B (F2.18
0. 0001). It was significantly higher in box B (Fisher, = 9.01, P = 0.004) (Table 4).

Table 2. Offspring productivity, parasitism rate and sex ratio (mean ± S.E.) between mated and unmated Oomyzus
sokolowskii females.

Males Females Total progeny Parasitism (%) Sex ratio

Mated female 1. 8 ± 0.41 a 8. 4 ± 0.73 a 10.2 ± 1.04 a 45. 6 ± 3. 92 a 83. 0 ± 2. 03 a

Unmated female 10.3 ± 0.87 b 0.0 ± 0.00 b 10.3 ± 0.86 a 12. 2 ± 0.14 b 0.0 ± 0.00 b

Means in columns followed by the same letters are not significantly different (t-test, P > 0.05).
Sex ratio corresponding to percentage females.

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 Sow et al.: Life history traits of Oomyzus sokolowskii, a parasitoid of the diamondback moth 235

Table 3. Host age preference (immature DBM stages) of are some exceptions (Harvey & Strand 2003;
female Oomyzus sokolowskii. Da Rocha et al. 2007). The size of a parasitoid is
considered one of the most important life-history
Host age (instars) Parasitism (%) Range (%) traits contributing to the success of biological control
of a pest (Aruna & Manjunath 2010). Moreover, it
Second 39.9 ± 7.57 b 23.3–63.3 is related to other relevant factors such as fecundity,
Third 54.7 ± 8.71 b 23.3–73.3 longevity and their fitness (Jervis & Copland 1996;
Fourth 75.9 ± 2.43 c 70.0–83.3 Eijs & van Alphen 1999; Mayhew & Glaizot 2001).
Prepupa 15.3 ± 5.86 a 0.0–36.7 Our results showed that mated females and virgin
Pupa 0.0 ± 0.00 a 0.0–0.0 females produced the same number of offspring.
They are similar to those reported by Metzger et al.
Means (± S.E.) in columns followed by the same letters are not (2008) on Venturia canescens Gravenhorst (Hyme-
significantly different according to the Student-Newman-Keuls test.
noptera: Ichneumonidae) which produced the
DISCUSSION same number of offspring.
After emergence, O. sokolowskii females have few
Oomyzus sokolowskii is a larval-pupal endopara- eggs to lay. However, they do not seem to be
sitoid and its life cycle takes place entirely inside limited in the number of eggs for the first attack.
its host larvae, P. xylostella. Males and females Our results also show that female parasitoids con-
emerge simultaneously, unlike in Tetrastichus tinue maturing eggs throughout their reproduc-
howardi (Olliff) another Eulophidae parasitoid tive life. According to Jervis et al. (2001), the
where females are the first to emerge (Birot et al. number of eggs that a female lays is determined by
1999). Under our experimental conditions, the the interaction of three factors: the number of suit-
cycle duration was 15.6 days, which is similar to able hosts encountered, the number of mature
the development time reported by Wang et al. eggs during the female’s life and the behavioural
(1999). manipulation of the rate of egg laying. The work of
According to La Barbera (1989), sexual dimor- Ellers & Jervis (2003) on parasitic wasps revealed
phism in respect to size is commonly observed in an initial egg load between 15 and 435 eggs and
the animal kingdom, including wasps. Our study potential lifetime fecundity between 40 and
confirms that females of O. sokolowskii are gener- 835 eggs. In Oomyzus sokolowskii, the ovigeny
ally bigger than males. These results have been index was less than unity; thus, it is a synovigenic
reported in many species of hymenoptera. For species according to the classification of Jervis et al.
example, Bokonon-Ganta et al. (1995) and Aruna & (2001). However, the average number of eggs
Manjunath (2010) showed that females of Anagyrus corresponding to the O. sokolowskii lifetime seems
mangicola (Noyes) (Hymenoptera: Encyrtidae) relatively low as compared to those described by
and Nesolynx thumus (Girault) (Hymenoptera: Eller & Jervis (2003). Mayhew & Blackburn (1999)
Eulophidae) are bigger than males. Mackauer & have shown that koinobiont species have a shorter
Sequeira (1993) and Thompson (1993) attributed adult lifetime, higher fertility levels and a greater
the sexual dimorphism in the hymenopteran oviposition rate than idiobiont species. According
parasitoids to their development time and to the to Jervis et al. (2001), the ovigeny index seems to
efficiency of their metabolism. The males emerge be related to the mode of development of the
first and are smaller than females, though there parasitoid. Koinobiont parasitoids tend to have an

Table 4. Oviposition box volume effect on the parasitism percentage and Oomyzus sokolowskii female production
(mean ± S.E.).

Laying box % Parasitism Females Males Females Total adults Cycles (days) Sex ratio
laid

3(A) 30.0 ± 15.31 b 3 0.7 ± 0.34 a 5.9 ± 3.01 a 6.6 ± 3.43 a 14.3 ± 0.35 a 89.5 ± 0.84 a
7(B) 85.5 ± 7.60 c 10 2.1 ± 0.63 b 13.6 ± 1.70 b 15.7 ± 2.04 b 15.7 ± 0.30 a 86.8 ± 2.36 a
40(C) 5.0 ± 3.93 a 1 0.2 ± 0.12 a 0.8 ± 0.54 a 1.0 ± 0.61 a 18.0 ± 0.04 b 80.0 ± 0.02 a

Means in columns followed by the same letters are not significantly different according to the Student-Newman-Keuls test.
Sex ratio corresponding to percentage females.

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236 African Entomology Vol. 21, No. 2, 2013

ovigeny index on average higher than idiobiont parts, ovipositor and tarsi for host detection
species. However, O. sokolowskii is a koinobiont (Frings & Frings 1949; Weseloh 1972; Barlin &
gregarious larval-pupal parasitoid. According to Vinson 1981). Therefore, host distribution on a
Talekar & Hu (1996) and Wang et al. (1999), O. soko- given substrate may play an important role in the
lowskii parasitizes all larval stages and even the success of foraging and parasitism. Female O. soko-
prepupae of DBM, which is confirmed by our lowskii seem more sensitive to the presence of the
study. Koinobiont parasitoids have developed host in the 7 cm3 box. In the 3 cm3 box a saturation
various adaptive mechanisms to operate a wide of chemoreceptors of the female due to a high con-
range of host stages, such as altering the host’s centration of chemical signals from the host may
behaviour (Slansky 1986), manipulating the host’s explain the decrease in the female ovipositions, re-
development (Vinson & Iwantsch 1980) and con- sulting in a lower parasitism rate. Otherwise, a low
trolling the host’s immune responses (Strand & concentration of chemical signals in the 40 cm3 box
Pech 1995). This mode allows parasitoid develop- could be the cause of the low parasitism rate and
ment to increase diverted food resources. Koino- oviposition, resulting in lower female productiv-
biont species can manipulate their physiology and ity. The behavioural process of host-seeking by fe-
feeding behaviour of their host to their advantage males of O. sokolowskii is an important factor that
(Harvey et al. 1999). Strand (2000) found that determines the number of offspring of a female;
koinobiont parasitoids have developed strategies therefore, it is directly linked to the success of
to make the host resources more predictable. reproduction and to fitness (Godfray 1994).
The parasitism rate is higher in the fourth larval The results presented in this study provide valu-
stages of DBM. Nakamura & Noda (2001) found able information on some life history traits of
that the larval stages were more appropriate for O. sokolowskii, which could be exploited effi-
O. sokolowskii because they produce more interfer- ciently for the management of DBM in Africa. For
ence than other stages. This could be explained by instance, in laboratory mass-rearing programmes,
the increase in resources in the larger larvae. it is advisable to maintain O. sokolowski with late
According to Harvey et al. (2004) large hosts are larval stages of DBM in adequate containers. In
more profitable than small ones because they have the case of mass-release programmes, the study
more resources available for development of encourages the release of mated females, which
the parasitoid offspring. Older larval stages are are shown to be more efficient in host foraging.
preferable because they are likely to escape Therefore, O. sokolowskii stands as a promising
superparasitism close to pupation (Silva Torres candidate for the control of DBM in Senegal and
et al. 2009). other tropical areas.
Several authors have shown that volatile chemi-
cals such as kairomones, pheromones or allo- ACKNOWLEDGEMENTS
mones from the host can influence the parasitoid’s
behaviour (Mattiacci et al. 2000; van Alphen et al. This work was funded by Agence Universitaire
2003). The stimuli diffuse through the air and the de la Francophonie (AUF) through the programme
receivers are sensitive to very small amounts of ‘Horizons Francophones’. We thank the Centre for
products (Roux et al. 2007). In most parasitoids, International Cooperation in Agronomic Research
patch quality is closely related to the proportion of in Development (CIRAD) in Montpellier, France,
good (unparasitized hosts) and bad (parasitized for providing all the research facilities. Many
hosts). Parasitoid insects are able to evaluate the thanks to A.A. Kirk, researcher at the European
quality of the patch in which they are currently Biological Control Laboratory, United States
searching for hosts and the travel time between Department of Agriculture, Agricultural Research
patches (Pierre et al. 2002). Insects may also rely on Service (EBCL USDA-ARS), Montpellier, for cor-
chemoreceptors found on the antennae, mouth- recting our English.

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sequential decisional sampling? Behavioral Ecology sitism of Plutella xylostella (Lep., Plutellidae) by
and Sociobiology 54: 147–155. Oomyzus sokolowskii (Hym., Eulophidae). Entomo-
QUICKE, D.L.J. 1997. Parasitic Wasps. Chapman and Hall, phaga 41: 45–52.
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ness of transient egg limitation in insects. Proceedings WANG, X., LIU, S., GAO, S. & LIN, W. 1999. Effect of host
of the Royal Society, London, Series B 267: 1565–1573. stages and temperature on population parameters of
ROU X , O. , G E RS, C ., T EN E- G H O M S I , J .N ., Oomyzus sokolowskii, a larval-pupal parasitoid of
ARVANITAKIS, L., BORDAT, D. & LEGAL, L. 2007. Plutella xylostella. BioControl 44: 391–402.
Chemical characterization of contact semiochemicals WENSELEERS, T. & BILLEN, J. 2000. No evidence for
for host-recognition and host-acceptance by the Wolbachia-induced parthenogenesis in the social
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SALL-SY, D., DIARRA, K. & TOGUEBAYE, B.S. 2004. WESELOH, R.M. 1972. Spatial distribution of the gypsy
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2002. Montpellier, France. pp. 163–166. host-searching of the parasitoid, Gryon pennsylvaricum
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BARROS, R. 2009. Superparasitism and host size Entomology and Zoology 30: 139–144.
Accepted 14 April 2013

74
 Chapitre II : Interaction entre Plutella xylostella et le parasitoïde Oomyzus sokolowskii

ARTICLE 3

Performance of the parasitoid Oomyzus sokolowskii (Hymenoptera:

Eulophidae) on its host Plutella xylostella (Lepidoptera: Plutellidae)
under laboratory conditions

Gallo SOW, Laurence ARVANITAKIS, Saliou NIASSY, Karamoko DIARRA

and Dominique BORDAT

International Journal of Tropical Insect Science, 2013, 33: 38-45

International Journal of Tropical Insect Science Vol. 33, No. 1, pp. 38–45, 2013 doi:10.1017/S1742758412000422
q icipe 2013

Performance of the parasitoid Oomyzus

sokolowskii (Hymenoptera: Eulophidae)
on its host Plutella xylostella
(Lepidoptera: Plutellidae) under
laboratory conditions
Gallo Sow1*, Laurence Arvanitakis2, Saliou Niassy1,
Karamoko Diarra1 and Dominique Bordat2
1
Laboratoire de Parasitologie, Département de Biologie Animale,
Faculté des Sciences et Techniques, Equipe Production et Protection
Intégrées en Agroécosystèmes horticoles, Université Cheikh Anta Diop
de Dakar (UCAD), BP 5005, Dakar, Senegal; 2Laboratoire de Biodiversité
des agrosystèmes horticoles TAB/L, Campus international de
Baillarguet, CIRAD, 34398 Montpellier Cedex 5, France

(Accepted 14 November 2012)

Abstract. Oomyzus sokolowskii (Kurdjumov) is a gregarious larval –pupal parasitoid of the

diamondback moth Plutella xylostella (L.). The objective of this study was to investigate the
interactions between host and parasitoid by examining the effects of biotic factors such as
gregariousness, host origin and stages, and female parasitoid age on the parasitism rate,
developmental time, the number of offspring and the offspring sex ratio of O. sokolowskii
under laboratory conditions. The percentage of parasitism and the number of parasitoids
increased with the number of O. sokolowskii females. Oomyzus sokolowskii preferred fourth
larval instars over other larval stages. The parasitism rate and the progeny production of
O. sokolowskii decreased with parasitoid age; however, the developmental time and the sex
ratio of the offspring were not significantly different. Our results confirm previous
findings on larval preferences of O. sokolowskii. The study also confirmed the importance
of geographical origin of the host on the performance of O. sokolowskii.

Key words: parasitoids, Plutella xylostella, cabbage, rearing, parasitism rate, host stage,
biological control

Introduction 2006). Annual costs for pest management expenses

are estimated at US$ 1 billion (Grzywacz et al., 2010)
Cabbage is an important agricultural crop that exclusively for synthetic insecticide applications.
occupies a central role in the economy of many Complete dependence on a chemical-based strategy
countries, especially in Asia and Africa (Grzywacz for crop protection is, however, not sustainable.
et al., 2010). The diamondback moth (DBM) Plutella Also, the DBM has developed resistance to many
xylostella (L.) (Lepidoptera: Plutellidae) is an synthetic insecticides and biopesticides, including
oligophagous pest, considered as the most import- Bacillus thuringiensis (Berliner) formulations (Liu
ant source of crop losses in the Brassicaceae family et al., 1997; Zhou et al., 2011). As a result, attention
(van Loon et al., 2002; Shelton, 2004; Sarfraz et al., has been directed towards alternative methods that
could be used as components of integrated pest
*E-mail: [email protected] management systems.

77
 Effects of host on parasitism by Oomyzus sokolowskii 39
Biological control using parasitoids (Lim, 1992) The O. sokolowskii colony was obtained from
is an alternative to chemical control. Among other parasitized DBM pupae collected from cabbage
natural enemies of DBM, the larval parasitoid plantations in Pikine (near Dakar), Senegal. The
Oomyzus sokolowskii Kurdjumov (Hymenoptera: rearing consisted of exposing fourth-instar DBM
Eulophidae) is one of the most important endo- larvae to parasitoid females in transparent plastic
parasitoids, used as a biological control agent for boxes (8 £ 5 cm) for oviposition for 24 h. The DBM
DBM management in various parts of the world larvae were removed after parasitism and placed in
(Wang et al., 1999; Ferreira et al., 2003). identical boxes for their development until pupa-
Efficient biological control is only possible tion. Fresh leaves of cauliflower were provided as
through knowledge of the biology and ecology source of food. Adult parasitoids emerging from
of both the pest and its natural enemies (Andow parasitized pupae were collected and maintained in
et al., 1997; Martinez-Castillo et al., 2002). DBM a plastic box and fed with honey drops deposited
populations from different localities present differ- on the mesh cover.
ences in biological behaviour (Arvanitakis et al., Climatic conditions of all insect rearing were
2002; Kirk et al., 2004). Previous studies have maintained at 25 ^ 18C temperature, 60 ^ 5%
demonstrated the existence of genetic variations relative humidity and 12 h light-12 h dark
between various DBM populations (Pichon et al., photoperiod.
2004; Roux et al., 2007). A parasitoid host can affect
its performance, especially in gregarious species
Gregariousness of O. sokolowskii
(Silva-Torres et al., 2009b). To date, little is known
about the performance of O. sokolowskii, taking Females of O. sokolowskii (24 h old) were used in
into account those biotic factors. The knowledge of this experiment. In brief, 1, 5, 10, 15 and 20 females
such differences could be used in biological control were placed, respectively, with 2, 10, 20, 30 and 40
or in conservation biological control programmes fourth-instar DBM larvae (from Senegal) for 24 h.
against the DBM. The number of host larvae was increased to avoid
Hence, the aim of this study was to describe the risk of superparasitism. The larvae were then
the interactions between P. xylostella larvae and O. transferred individually into clear plastic pill boxes
sokolowskii females under laboratory conditions. We (3.5 £ 1 cm) with a leaf disc of cabbage as food
focused on the effect of gregarious behaviour, host supply, and monitored daily until the emergence of
origin and stages, and female parasitoid age on the adult parasitoids. The parasitism rate was calcu-
parasitism rate, developmental time, the number lated from the number of parasitized larvae and
of offspring and the sex ratio of O. sokolowskii. pupae. The developmental time (from egg to adult),
and the number of offspring and the sex ratio were
Materials and methods also compared in each cohort. The experiment was
replicated seven times.
Study site
The experiments were conducted at the Labora- Host origin effects
tory of Entomology for International Cooperation
in Agronomic Research for Development Centre Two populations of P. xylostella were used
(CIRAD) in Montpellier, France. in this experiment: a population from Senegal
(from the Niayes area) and another one from
Martinique Island (Saint Pierre, 148440 3000 N and
Insect rearing
618100 3300 W; 1200 m elevation). For each population,
DBM larvae were previously collected from the size of the pupae was measured using a
cabbage plantations (Brassica oleracea var. capitata L.) dissecting microscope equipped with an ocular
in the Niayes area in Senegal (128540 4400 N and micrometer. Thirty pupae were used for each DBM
12880 8400 NW; 189 m elevation). The colony was population.
maintained on Chinese mustard (Brassica juncea Fifteen 24-h-old O. sokolowskii females were
(L.) Czern.), where gravid females were allowed to exposed to 30 fourth-instar DBM larvae from each
oviposit. Emerging larvae were placed on the fresh of the regions. After 24 h of exposure, all larvae
leaves of cauliflower (B. oleracea var. botrytis L.) were collected and placed individually in clear
for their development. The larvae were later plastic pill boxes and followed up until the
transferred onto the fresh leaves placed on the emergence of adult parasitoids. The parasitism
bottom of a large transparent plastic box rate was calculated from the number of parasitized
(28 £ 26 £ 15 cm), for pupation, and pupae were larvae and pupae of each population. The develop-
collected daily. Newly emerged adults were placed mental time (from egg to adult), and the number
in cages (50 £ 50 £ 50 cm) and fed with water and sex ratio (% females) of the offspring were
and honey. calculated. Bioassays were replicated seven times.

78
40 G. Sow et al.
DBM stage effects proportion of females. In all statistical analyses, the
level of significance was kept at 5%.
Thirty DBM larvae (second, third and fourth),
pre-pupae and pupae were exposed to 15 O.
sokolowskii females in a clear plastic pill box for Results
24 h. The DBM population in this experiment was Gregariousness of O. sokolowskii
native to Senegal. Honey drips were deposited on
the cover mesh as food supply. Parasitized larvae The parasitism rate varied significantly accord-
were transferred separately into pill boxes, with a ing to the number of females (F(4,24) ¼ 11.35;
leaf disc of cabbage as food supply. The newly P , 0.0001; Table 1). The parasitism rate was
formed pupae were monitored until the emergence significantly lower at one female parasitoid and
of parasitoid adults. The parasitism rate for each increased as the number of females increased.
DBM stage was calculated from the number of However, there were no significant differences
parasitized pupae. The developmental time (from between the 5, 10, 15 and 20 females (P , 0.05).
egg to adult), the number of offspring and the sex There were significant differences between the
ratio (% female) of parasitoids were compared number of female parasitoids on the number of
among the different host stages. The experiment adults (F(4,24) ¼ 17.80; P , 0.0001), and the number
was replicated 10 times for each DBM stage. of females (F(4,24) ¼ 16.50; P , 0.0001). A single
female produced fewer offspring than grouped
females. The developmental time and the sex ratio of
Effect of female parasitoid age the progeny were not significantly affected by the
Fifteen 1-, 5-, 15- and 28-day-old O. sokolowskii number of female parasitoids (F(4,24) ¼ 1.32; P ¼ 0.29
females were, respectively, exposed to 30 fourth- and F(4,24) ¼ 3.94; P ¼ 0.05, respectively; Table 1).
instar DBM larvae for 24 h. The larvae were
collected and transferred individually into pill
boxes. They were monitored until the emergence Host origin effects
of parasitoids. The parasitism rate, developmental The difference in size between DBM pupae from
time, the number of offspring and the sex ratio Senegal and DBM pupae from Martinique was
(% females) were compared. For each cohort, the significant (t ¼ 6.091; degree of freedom (df) ¼ 58;
experiment was replicated five times. P , 0.0001). DBM populations from Senegal were
morphologically bigger than those from Martinique
Statistical analysis (Table 2).
There was a significant difference in parasitism
Data were normalized by logarithmic trans- rate between DBM populations from Senegal and
formation before being subjected to an analysis of Martinique (t ¼ 22.62; df ¼ 12; P ¼ 0.022; Table 3).
variance (ANOVA). Parasitism rate, the total However, there was no significant difference in the
number of adult offspring, developmental time total production of adults and the production of
and offspring sex ratios were compared for the females (t ¼ 2 1.31; df ¼ 12; P ¼ 0.215 and
different treatments using one-way ANOVA. Means t ¼ 2 0.52; df ¼ 12; P ¼ 0.61, respectively). The
were separated using the Student–Newman–Keuls number of males in the offspring was significantly
test (XLSTAT software, version 2012.1.01). Parasit- different between the two populations (t ¼ 2 2.68;
ism rate, developmental time, parasitoid offspring df ¼ 12; P ¼ 0.02). There was no significant differ-
number and sex ratio between host populations ence in developmental time between the two
of different origins were compared with t-tests populations (t ¼ 0.60; df ¼ 12; P ¼ 0.56). The sex
(STATVIEW, 1996). The sex ratio was calculated ratio was significantly different between the two
using the Silva-Torres et al. (2009b) formula as the populations (t ¼ 2.52; df ¼ 12; P ¼ 0.026; Table 3).

Table 1. Mean (^ SE) parasitism rate, productivity, developmental time and sex ratio (% female) of Oomyzus sokolowskii on
DBM larvae
Number of females Parasitism (%) Females Total number Cycle (days) Sex ratio (%)
1 14.30 ^ 4.27b 1.30 ^ 1.28b 1.45 ^ 1.42b 15.00 ^ 0.30a 90.00 ^ 0.04a
5 52.86 ^ 6.80a 8.57 ^ 0.72a 9.86 ^ 0.80a 15.46 ^ 0.26a 86.91 ^ 1.47a
10 71.43 ^ 5.64a 9.72 ^ 0.47a 12.29 ^ 0.68a 15.19 ^ 0.12a 79.30 ^ 1.51a
15 74.29 ^ 3.47a 10.43 ^ 0.65a 12.57 ^ 0.87a 15.21 ^ 0.10a 83.30 ^ 1.18a
20 77.90 ^ 3.43a 10.71 ^ 1.36a 13.14 ^ 1.67a 15.10 ^ 0.14a 81.64 ^ 1.35a
Means in columns followed by the same letters are not significantly different (P . 0.05; Student – Newman – Keuls test).

79
 Effects of host on parasitism by Oomyzus sokolowskii 41
Table 2. Mean size of DBM pupae (mean ^ SE) from related to superparasitism, which, in turn, favours a
different geographic origins higher number of offspring (Gu et al., 2003; Silva-
Host origin Size (mm) Range (mm)
Torres and Matthews, 2003; Keasar et al., 2006).
According to Silva-Torres et al. (2009b), gregarious-
Senegal 6.14 ^ 0.05a 5.53– 6.67 ness and superparasitism can adversely affect
Martinique 5.65 ^ 0.06b 4.76– 6.33 parasitoid fitness. These behaviours appear to be
Means in columns followed by the same letter are not of advantage to the parasitoid (Yamada and
significantly different (P . 0.05; t-test). Miyamoto, 1998). In our study, the parasitism rate
and the number of offspring increased with the
number of parasitoid females. These results
corroborate those of Hirashima et al. (1990) who
DBM stage effects found that host availability was a favourable factor
to high parasitism rates. The proportion of males in
The parasitism rate varied significantly accord- the offspring also increased with the number of
ing to host stage (F(4,36) ¼ 26.23; P , 0.0001). It was female parasitoids. These results are similar to those
higher in L4 larvae and lower in pre-pupae by 75.9 reported by Chen et al. (2008).
and 15.3%, respectively. However, there were no The DBM population from Senegal seemed to be
significant differences between the L2 and L3 larval of higher fitness than the population from Martini-
stages (Table 4). Similarly, there were no significant que. According to Chown et al. (2009), large-sized
differences in the total production of adults insects have a higher chance of survival and
(F(3,27) ¼ 0.50; P ¼ 0.68) and females (F(3,27) ¼ 0.69; reproduction. In our study, the parasitism rate
P ¼ 0.57) between the host stages. Developmental was significantly higher in the population from
time and sex ratio were not different between the Martinique; however, female productivity was
host stages (F(3,27) ¼ 1.39; P ¼ 0.28 and F(3,27) ¼ 0.56; similar in both populations. Our results do not
P ¼ 0.65, respectively; Table 4). seem to be in agreement with previous findings.
Edwards (1954) has reported the effect of host size.
The author discovered that most female parasitoids,
Effect of female parasitoid age
especially in gregarious species, first estimate the
The parasitism rate was significantly affected by size of the host before ovipositing. This trait is one
the age of O. sokolowskii females (F(3,12) ¼ 21.32; of the most important indicators used by female
P , 0.0001; Table 5). It was higher in 1- and 5-day- parasitoids for reproduction (Aruna and Manju-
old females. The number of females was signifi- nath, 2010). Zaviezo and Mills (2000) made the same
cantly affected by the age of female parasitoids assertion; the offspring production in gregarious
(F(3,12) ¼ 4.70; P ¼ 0.02); it was higher in 5-day-old parasitoids is often correlated with host size. In our
female parasitoids. The number of adults was also results, DBM pupae from Senegal are larger than
significantly affected (F(3,12) ¼ 4.24; P ¼ 0.03). How- the DBM from Martinique, which would imply that
ever, the developmental time and the sex ratio were the female parasitoid would oviposit more in
not affected by the age of female parasitoids Senegalese larvae. Yet, DBM individuals from
(F(3,12) ¼ 1.01; P ¼ 0.42 and F(3,12) ¼ 1.55; P ¼ 0.25, Senegal, which were bigger in size, were less
respectively). parasitized (2 15%). Although the number of
females was similar, the number of males in the
offspring was higher in DBM individuals from
Discussion
Martinique. As a result, the sex ratio was higher in
Knowledge on the biology and ecology of Senegalese DBM, which implies that O. sokolowskii
biological control agents is of paramount import- could be a more efficient biological control agent in
ance in the management of pests such as DBM. Our Senegal than in Martinique. In addition to the
results suggest that gregariousness promotes difference in size between the DBM populations,
increased parasitism rates and increased number the difference in altitude between the regions could
of offspring per female. In fact, gregariousness is explain the difference in the performance of

Table 3. Effect of host origin on the parasitism rate, progeny, developmental time and sex ratio (mean ^ SE) of Oomyzus
sokolowskii
Host origin Parasitism (%) Females Total progeny Cycle (days) Sex ratio (%)
Senegal 65.94 ^ 4.85b 9.71 ^ 0.61a 11.57 ^ 0.78a 15.56 ^ 0.21a 84.23 ^ 1.56a
Martinique 81.43 ^ 3.36a 10.14 ^ 0.55a 12.86 ^ 0.60a 15.37 ^ 0.22a 78.76 ^ 1.51b
Means in columns followed by the same letters are not significantly different (P . 0.05; t-test).

80
42 G. Sow et al.
Table 4. Effect of host stages on the parasitism rate, progeny, developmental time and sex ratio of the DBM parasitoid
Oomyzus sokolowskii
Instars Parasitism (%) Females Total progeny Cycle (days) Sex ratio (%)
Second 39.98 ^ 7.60b 10.40 ^ 0.60a 13.40 ^ 1.03a 17.58 ^ 0.44a 78.10 ^ 1.74a
Third 54.66 ^ 8.74b 10.20 ^ 0.97a 12.40 ^ 1.03a 16.14 ^ 0.33a 82.65 ^ 5.04a
Fourth 75.98 ^ 2.45a 14.40 ^ 2.02a 17.40 ^ 3.30a 15.08 ^ 0.32a 85.18 ^ 3.51a
Pre-pupae 15.32 ^ 5.93c 13.40 ^ 4.53a 17.70 ^ 6.12a 12.96 ^ 3.24a 81.80 ^ 5.27a
Pupae 0d 0b 0b 0b 0b
Means in columns followed by the same letters are not significantly different (P . 0.05; Student – Newman – Keuls test).

O. sokolowskii (Shelly et al., 2003). It has been shown fourth-stadium hosts. Yet, our results did not
that distributions of both hosts and their parasitoids corroborate their findings.
are influenced by altitude (Sivinski et al., 2000, The success of parasitism depends on the age of
2004), which presumably is, in turn, due to the female parasitoid (Medeiros et al., 2000); generally,
temperature and moisture gradients. parasitism rate decreased with age as shown in our
The study revealed the importance of host stage study that 5-day-old females lay more eggs in their
as an important ecological parameter to be host than older females. This decrease in productivity
considered in the biology of parasitoids (Bai et al., can be explained by a decrease in physiological
1992; Godfray, 1994; Islam and Copland, 1997; activity (Giron and Casas, 2003). Silva-Torres et al.
Fidgen et al., 2000). It has also been shown that the (2009a) showed that the parasitism rate was higher in
quality of the host affects the fitness of offspring in 2- to 4-day-old females of O. sokolowskii. According to
parasitoids (Godfray, 1994). Our results indicate Rajapakse (1992), the ideal age for Cotesia margin-
that the parasitism rate was higher in the fourth iventris (Cresson) (Hymenoptera: Braconidae) to
larval stages of DBM, which seems to be the most properly parasitize larvae of Spodoptera frugiperda
suitable stage for the development of O. sokolowskii. (Smith) (Lepidoptera: Noctuidae) ranged from 48 to
Host stages of bigger size contain more resources 96 h, whereas the ideal age of parasitism for
for the parasitoid larval stages (Harvey et al., 2004). Ceratogramma etiennei (Delvare) (Hymenoptera: Tri-
These observations are similar to previous work chogrammatidae) varied from 1 to 2 days post-
reported by Talekar and Hu (1996). emergence (Amalin et al., 2005). Our results are
There were no significant differences in the similar to the work of Silva-Torres et al. (2009a) and
number of offspring, developmental time and Chen et al. (2008), who showed that the progeny
progeny sex ratio of O. sokolowskii in relation to produced per female and the number of parasitoids
the age of host stages, corroborating findings of emerging per host significantly decreased according
Wang et al. (1999), possibly due to the gregarious- to age. Similarly, Li et al. (1993) argued that offspring
ness and superparasitism behaviour of O. sokolows- production from females of Trichogramma minutum
kii. Although there were differences in parasitism Riley (Hymenoptera: Trichogrammatidae) decreases
rate between the stages of DBM, potential with age. However, the developmental time and sex
competition for resources between larval para- ratio of offspring were not different between the
sitoids could affect the number of offspring. different age groups of the parasitoid. Our results are
Gregariousness and superparasitism can adversely similar to the findings of Riddick (2003), who
affect the number of offspring (Silva-Torres et al., demonstrated that the age of Anaphes iole Girault
2009b). Consequences of such behaviour could (Hymenoptera: Mymaridae) does not affect the sex
greatly affect the fitness of progeny. On the other ratio of the parasitoid.
hand, Nakamura and Noda (2002) reported that The parasitism rate tended to decrease drasti-
the number of O. sokolowskii tends to increase with cally in the 5-days-old females, suggesting that
host age and is significantly higher in the late beyond a certain period of maturity, O. sokolowskii

Table 5. Effect of female Oomyzus sokolowskii age on parasitism rate, the number of offspring, developmental time and sex
ratio (mean ^ SE)
Female age (days) Parasitism (%) Females Number Cycle (days) Sex ratio (%)
1 82.10 ^ 3.20a 9.25 ^ 0.25b 11.25 ^ 0.25ab 15.34 ^ 0.12a 82.38 ^ 3.26a
5 71.38 ^ 4.96a 12.75 ^ 1.60a 15.50 ^ 2.40a 15.08 ^ 0.42a 83.43 ^ 3.00a
15 41.10 ^ 4.23b 8.00 ^ 1.23b 9.50 ^ 1.50b 15.75 ^ 0.28a 84.35 ^ 1.05a
28 31.35 ^ 7.52b 8.00 ^ 0.41b 9.00 ^ 0.41b 15.25 ^ 0.25a 88.85 ^ 0.51a
Means in columns followed by the same letters are not significantly different (P . 0.05; Student – Newman – Keuls test).

81
 Effects of host on parasitism by Oomyzus sokolowskii 43

females are less efficient in controlling DBM Chen R. X., Zhang F., Huangfu W. G., Yao H. Y., Zhou J. B.
populations. Therefore, biological control of DBM and Kuhlmann U. (2008) Reproductive attributes of
cannot rely entirely on the use of parasitoids when the eulophid Oomyzus sokolowskii, a biological control
they are too old (Persad and Hoy, 2003; Amalin et al., agent of diamondback moth, Plutella xylostella
2005), and other methods need to be envisaged. (Lepidoptera: Plutellidae). Biocontrol Science and
Technology 18, 753 – 765.
Chown S. L., Jumbam K. R., Sorensen J. G. and Terblanche
Conclusion J. S. (2009) Phenotypic variance, plasticity and
The present study confirms the importance of O. heritability estimates of critical thermal limits depend
sokolowskii as a promising biological control agent on methodological context. Functional Ecology 23,
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Edwards R. L. (1954) The effect of diet on egg maturation
the management of DBM populations in cabbage
and resorption in Mormoniella vitripennis (Hymenop-
production. However, performance could be
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affected by host geographical origin. Moreover,
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age of female parasitoids could be a limiting factor Ferreira S. W. J., Barros R. and Torres J. B. (2003)
for parasitoid production; therefore, other par- Exigências térmicas e estimativa do nùmero de
ameters should be combined during DBM manage- geraçoes de Oomyzus sokolowskii (Kurdjumov) (Hyme-
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necessary to know in real time the best period for cruciferas em Pernambuco. Neotropical Entomology 32,
parasitoid mass release to ensure successful para- 407 – 411.
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Acknowledgements fitness of Elachertus cacoeciae attacking a low-density
population of spruce budworm Choristoneura fumifer-
This work was funded by the French Agency ana larvae. Ecological Entomology 25, 156 – 164.
‘Agence Universitaire de la Francophonie’ through Giron D. and Casas J. (2003) Mothers reduce egg
the programme ‘Horizons Francophones’. We thank provisioning with age. Ecology Letters 6, 273 – 277.
the French Agricultural Research for Development Godfray H. C. J. (1994) Parasitoids: Behavioral and
Centre (CIRAD) in Montpellier for providing all Evolutionary Ecology. Princeton University Press,
the research facilities. We thank Dr A. A. Kirk, Princeton, NJ. 473 pp.
researcher at the EBCL USDA-ARS in Montpellier, Grzywacz D., Rossbach A., Rauf A., Russell D. A.,
for English translation. Srinivasan R. and Shelton A. M. (2010) Current control
methods for diamondback moth and other brassica
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 CHAPITRE III

Interaction entre Plutella xylostella et le parasitoïde

Cotesia vestalis
 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

1. Introduction

L’espèce Cotesia vestalis (Haliday) a subi des modifications taxonomiques et dans les
articles elle peut avoir été dénommée Cotesia plutellae (Kurdjumov). Les travaux présentés
dans ce chapitre sont présentés ici sous la forme de trois articles, dont un soumis et deux
publiés.

Une étude a été réalisée dans un premier temps en plein champ au Bénin et présentée
dans l’article 4. Nous avons voulu déterminer quels sont les ennemis naturels de P. xylostella
qui peuvent jouer un rôle dans un environnement tropical, et comprendre quand et
comment l’interaction hôte-parasitoïde pouvait influencer la dynamique des populations de P.
xylostella en l’absence d’insecticide. En second lieu, nous avons voulu comparer l'impact
direct des parasitoïdes avec celui indirect des conditions abiotiques tropicales, puisque les
précipitations sont, d’après plusieurs auteurs, un facteur important de contrôle de la teigne.
Pour atteindre ces objectifs, nous avons étudié pendant 39 mois consécutifs dans la zone
périurbaine de Cotonou (Bénin), les variations d’abondance de P. xylostella, dans un champ
de choux pommés non traités, ainsi que celles des autres ravageurs également inféodés aux
Brassicacées et de leurs ennemis naturels.

Ensuite, nous nous sommes intéressés au système de reconnaissance qui lie C. vestalis
à P. xylostella, en conditions de laboratoire. Cette étude porte d’abord sur le mode de
détection et d’identification de l’hôte par le parasitoïde en recherchant chez C. vestalis quels
sont les organes impliqués (article 5), puis nous avons tenté de déterminer la nature exacte du
stimulus permettant cette identification et l’initiation de l’oviposition (article 6).
En effet, pour qu’un parasitoïde réussisse son infestation et son développement, il est
communément admis que plusieurs étapes chronologiques doivent être franchies avec succès
(Doutt 1959 ; Vinson 1976). Nous nous sommes intéressés plus particulièrement à la phase
qui correspond à la perception par une femelle, de la série de stimuli qui vont lui permettre de
réduire progressivement son aire de recherche pour aboutir à la découverte d’un hôte et à son
acceptation en tant que site de ponte (on parle alors d’oviposition). Les étapes de cette
première phase, qualifiées de pré-ovipositionnelles, sont très importantes car elles dépendent
du comportement des femelles adultes (Vinson 1981) et conditionnent l’impact du parasitisme
sur la dynamique des populations hôtes.

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

Bien qu’une préférence ait été démontrée chez des femelles de C. vestalis d’une part pour
l’odeur émise par le chou et par un régurgitât de chenille de P. xylostella (Shiojiri et al. 2000
a, b, 2001) et d’autre part par l’odeur des sécrétions laissée sur la feuille (Reddy et al. 2002),
les mécanismes de reconnaissance au contact de l’hôte restent à ce jour encore mal connus.
Décrite comme spécialiste, cette espèce devrait pouvoir avec exactitude localiser et
surtout reconnaitre son hôte. Pour comprendre le mécanisme par lequel les femelles effectuent
cette tâche, nous avons procédé par étapes:

- (1) Observation du comportement de recherche et de reconnaissance à l’aide

d’enregistrements vidéo afin de déterminer quels stimuli pouvaient être à l’origine du
réflexe d’oviposition,
- (2) Essai d’identification des différents organes sensoriels des femelles de C. vestalis
impliqués dans la perception de ces stimuli,
- (3) Etude de l’équipement sensillaire de C. vestalis en observant en microscopie
électronique à balayage les antennes des mâles et des femelles.

Dans un deuxième temps, nous avons tenté de déterminer la nature chimique exacte du
stimulus impliqué lors de la rencontre physique entre C. vestalis et P. xylostella.
Selon Nelson & Charlet (2003), les substances impliquées dans la reconnaissance de
l’hôte par les parasitoïdes sont des lipides cuticulaires non volatils. Vinson (1976) annonçait
déjà que des hydrocarbures cuticulaires étaient les substances principalement impliquées dans
ces phénomènes. Pour vérifier cela, nous avons analysé par chromatographie à phase gazeuse
couplée à une spectrométrie de masse (GC-SM) un extrait cuticulaire et réalisé une série de
tests comportementaux visant à tester les réactions du parasitoïde vis-à-vis d’extraits
cuticulaires de la chenille hôte appliqués sur des leurres (chenille non-hôte).

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

2. Etude de terrain : le Bénin

2.1. Présentation générale

Le Bénin (Fig. 14) se situe dans la zone intertropicale de l’Afrique de l’Ouest qualifiée
de « Diagonale de sécheresse », caractérisée par la faiblesse relative des précipitations
annuelles. De forme étirée entre le fleuve Niger au nord et la plaine côtière dans le sud,
le relief de l'ensemble du pays est peu accidenté. Le nord du pays est principalement constitué
de savane et de montagnes semi-arides. Le sud du pays est constitué d'une plaine côtière basse
parsemée de marécages, lacs et lagunes. C’est un petit pays d’une longueur de 700 km et
d’une largeur de 120 km au sud et de 300 km au nord. Le climat varie fortement du sud au
nord. Le sud a un climat subéquatorial (type Guinéen) qui se caractérise par une forte
humidité (1 200 mm de pluie par an), par deux saisons sèches et deux saisons pluvieuses
(avril à juillet et septembre à octobre) et par une température comprise entre 25°C et 30° C.
Au nord, le climat est tropical (type Soudanien), marqué par des températures plus élevées
pouvant atteindre 46° C, des précipitations annuelles plus faibles (900 mm de pluie) et par
l’alternance de deux saisons, dont une pluvieuse (mai à octobre). La partie nord-ouest,
occupée par la chaîne de l’Atacora, bénéficie d’un climat particulier où les températures sont
plus fraîches et les précipitations plus élevées que dans le reste du pays.

Depuis une quinzaine d'année, le chou pommé est rentré dans la cuisine traditionnelle
du Bénin et constitue une composante importante des régimes alimentaires quotidiens. Ces
feuilles coriaces sont bien adaptées aux cuissons longues de cette cuisine. Ce légume est
devenu incontournable et est particulièrement consommé en temps que met traditionnel pour
la période de Noël. Les pommes de ces choux sont de petite taille comparées à celles des
variétés cultivées en Europe. C'est une culture à haute valeur ajoutée actuellement en
extension qui génère des sources importantes de revenus, particulièrement dans les zones
urbaines et périurbaines. Cependant les opportunités économiques offertes par ces légumes
sont souvent affaiblies par des dommages, provoqués par des nuisibles affectant leur
production et leur commercialisation.

2.2. Statut de Plutella xylostella au Bénin

Au Bénin, P. xylostella est le principal ravageur des cultures de chou. L’ampleur des
dégâts qu’il provoque peut aller jusqu’à la destruction totale de la culture (observations

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

personnelles). Les agriculteurs appliquent de manière inappropriée des pesticides, utilisés

souvent pour les cultures de coton, qui sont de toute évidence inefficaces contre la teigne du
chou. Au Bénin comme dans la plupart des autres pays africain, des stratégies alternatives à
l’utilisation de pesticides sont nécessaires mais peu ou pas développées à ce jour. A propos de
la lutte biologique, les agriculteurs béninois sont peu sensibilisés à ce mode de lutte car la
plupart ne connaissent pas les différences qu’il y a entre un auxiliaire et un ravageur.

Figure 14 : Carte du Bénin avec localisation (en rouge) du site d’étude dans la zone
périurbaine de Cotonou

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

2.3. Site d’expérimentation

Notre étude s’est déroulée sur le site de Kouhounou (Fig. 15), dans la zone périurbaine
de Cotonou. Nous avons effectué des prélèvements sur des parcelles de choux pommés non
traités durant 39 mois consécutifs. Dans cette zone, les agriculteurs cultivent le chou toute
l’année sans arrêt de culture.

Figure 15 : Parcelle de choux sur le site de Kouhounou dans la zone périurbaine de Cotonou.

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

3. Synthèse des résultats

Nous avons pu établir la composition des communautés de ravageurs et d’ennemis

naturels associées à la culture du chou au Bénin, dans la zone périurbaine de Cotonou. Le
système hôte-parasitoïde comprend presque exclusivement P. xylostella et son parasitoïde
larvaire Cotesia vestalis (Haliday) (Hymenoptera: Braconidae). Ces deux espèces ont des
niveaux similaires d'abondance (en moyenne 7,5 ± 0,3 et 7,2 ± 0,3 individus par plante,
respectivement). Plutella xylostella et C. vestalis ont montré des oscillations d’abondance
couplées, avec un décalage d'environ deux semaines entre les pics de l’hôte et ceux du
parasitoïde. Une forte abondance de parasitoïdes a entraîné une diminution significative de
l'abondance de la teigne pendant plusieurs semaines. Toutefois, la population de parasitoïde
diminuant à son tour n'a pas pu empêcher par la suite la remontée des effectifs de la teigne du
chou. L’abondance de la teigne au cours des saisons n’est pas corrélée avec les variables
météorologiques (précipitations et températures), même si de fortes précipitations durant la
principale saison des pluies ont pu temporairement affecter le ravageur.

Des expériences d’ablation ont montré que les antennes jouaient un rôle prédominant
dans l’induction de l’oviposition. L’analyse des séquences vidéo a révélé que l’oviposition ne
pouvait avoir lieu sans un contact antennaire préalable. La femelle dispose ses antennes en
crosse lors du comportement de recherche. Les observations en microscopie électronique à
balayage ont révélé sept types de sensilles sur les antennes, parmi lesquels un type particulier
est 4,5 fois plus présent chez les femelles que chez les mâles.

Le stimulus à l’origine de la réponse de la femelle parasitoïde (oviposition) est bien

d’origine gustative. Les lipides cuticulaires des chenilles semblent impliqués puisque la
femelle réagit à un extrait lipidique complet appliqué sur une chenille non-hôte. Le
fractionnement de cet extrait inhibe l’effet des lipides cuticulaires car aucune des deux
fractions engendrées (hydrocarbure et non-hydrocarbure) ne permet l’oviposition. L’analyse
en chromatographie à phase gazeuse des deux fractions a révélé la présence de 44 composés.
L’identification des composés cuticulaires en spectrométrie de masse n’a révélé que des
produits très communs dans la fraction contenant les hydrocarbures, excepté un triterpenoïde
apparenté à l’amyrine.

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

4. Conclusion

Aucun contrôle stable du ravageur n’a été observé durant la longue période de mesures
effectuées. Dans les conditions tropicales du Bénin, les populations de P. xylostella croissent
rapidement, avec une forte probabilité de recolonisation à partir des zones environnantes. La
lutte biologique en utilisant un parasitoïde spécialiste bien établi, comme C. vestalis, montre
ici ses limites et des recherches de mesures de contrôle supplémentaires paraissent
nécessaires.
Seules les sensilles trichoïdes de type II révèlent un dimorphisme sexuel qui laisse
sous-entendre qu’elles jouent un rôle tout particulier dans les processus menant à l’oviposition
(localisation et identification de l’hôte). Leur absence chez d’autres espèces proches, comme
C. glomerata et C. rubecula, suggère qu’elles sont spécifiques à l’espèce C. vestalis. Ces
sensilles ont très certainement une fonction gustative (Vinson 1985 ; Schmidt & Smith 1989).
Cependant, l’influence du contact chimique est difficile à séparer de l’influence du toucher et
de la texture (Vinson 1976).
Nous avons pu identifier avec exactitude que le stimulus impliqué dans la
reconnaissance de l’hôte et dans le déclenchement du réflexe d’ovipostion était bien d’origine
gustative et qu’il était matérialisé par les lipides cuticulaires de la chenille hôte.

Cotesia vestalis est la seule espèce de parasitoïde rencontrée dans notre site d’étude
dans le sud du Bénin. Malgré des abondances importantes et un fort taux de parasitisme, C.
vestalis n’arrive pas à contrôler la teigne. Les précipitations ne sont pas un facteur de
régulation des populations du ravageur. Nous avons pu mettre en évidence trois points
importants du système de reconnaissance chez C. vestalis envers son hôte : (1) les femelles
détectent et reconnaissent leur hôte à partir de leur signature chimique, composée par les
lipides cuticulaires ; (2) le stimulus chimique constituant le signal de reconnaissance est
composé de plusieurs molécules appartenant à deux classes de lipides et agissant en synergie ;
(3) ce stimulus chimique est perçu par des sensilles gustatives implantées sur les antennes des
femelles du parasitoïde.

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

ARTICLE 4

Incomplete control of the diamondback moth, Plutella xylostella, by the

parasitoid Cotesia vestalis in a cabbage field under tropical conditions

Laurence ARVANITAKIS, Jean-François DAVID and Dominique BORDAT

Soumis à BioControl
 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

Incomplete control of the diamondback moth, Plutella xylostella,

by the parasitoid Cotesia vestalis in a cabbage field under tropical
conditions.

L. Arvanitakis1, J-F. David2 and D. Bordat1

1
CIRAD-Persyst, UPR HortSys, Campus International de Baillarguet, TA B 103/L, 34398
Montpellier cedex 5, France.
2
Centre d'Ecologie Fonctionnelle & Evolutive, CNRS, 1919 route de Mende, 34293
Montpellier cedex 5, France

Corresponding author:
Laurence Arvanitakis
CIRAD-Persyst, UPR HortSys, Campus International de Baillarguet, TA B 103/L, F-34398
Montpellier cedex 5, France.
Phone : 00 33 (0)4 67 59 31 12
E-mail : [email protected]

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

Abstract
Immature Plutella xylostella (Lepidoptera: Plutellidae) and parasitoids were sampled for 39
months in an unsprayed cabbage field near Cotonou, Benin, to determine how and when host-
parasitoid interactions influence the population dynamics of the moth in a tropical
environment. Eighty-three samples were taken at approximately two-week intervals. There
were no seasonal patterns in moth abundance, which was not correlated with weather
variables, although heavy rainfall during the principal rainy season may have temporarily
affected the population. The host-parasitoid system consisted almost exclusively of P.
xylostella and its larval parasitoid Cotesia vestalis (Hymenoptera: Braconidae), both species
occurring at similar levels of abundance (on average 7.5 ± 0.3 and 7.2 ± 0.3 individuals per
plant, respectively). The tendency for host-parasitoid dynamics to cycle was apparent in the
field. Plutella xylostella and C. vestalis showed coupled oscillations in abundance, with a time
lag of about two weeks between host and parasitoid peaks. High parasitoid abundance
resulted in significant decreases in moth abundance over several weeks. However, the
parasitoid population in turn decreased, could not prevent the moth from rebounding, and
there was no stable control of the pest. We conclude that under tropical conditions in which P.
xylostella populations grow rapidly, combined with a high probability of recolonization from
surrounding areas, biological control by a well-established specialist parasitoid reaches its
limits and additional control measures are necessary.

Keywords: Plutella xylostella, Plutellidae, Cotesia vestalis, Braconidae, Host-parasitoid

dynamics, Tropical agro-ecosystems.

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

INTRODUCTION

The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is the most
destructive pest of Brassicaceae, especially Brassica vegetable crops, in many parts of the
world (Furlong et al. 2013). Insecticide resistance is high in this species, which was the first
crop pest to develop resistance to DDT and has now developed field resistance to a variety of
products, including Bacillus thuringiensis-based products (Talekar and Shelton 1993; Furlong
et al. 2013). This problematic situation has stimulated research activities on alternative
methods of control, particularly on classical biological control programmes utilizing
parasitoids.
Many species of parasitoid wasps (Hymenoptera) are known to attack P. xylostella
(Delvare 2004; Sarfraz et al. 2005). Larval parasitoids in the genera Cotesia (Braconidae),
Diadegma (Ichneumonidae) and Microplitis (Braconidae) have the greatest control potential,
although a few prepupal and pupal parasitoids of the genus Diadromus (Ichneumonidae) also
contribute to reduce populations of this pest (Sarfraz et al. 2005). Their impact on moths was
clearly demonstrated in some studies. In South Africa, for example, Kfir (2004) showed that
the suppression of parasitoids following the use of a selective insecticide in the field resulted
in significantly higher levels of infestation by P. xylostella. In other studies, however,
parasitism was insufficient to prevent severe outbreaks in unsprayed cabbage fields (Guilloux
et al. 2003). Introductions of parasitoids against the diamondback moth in a number of
countries also resulted in both successes and failures. Following releases of large numbers of
the larval parasitoid Cotesia vestalis (Haliday) [= C. plutellae (Kurdjumov)] and the pupal
parasitoid Diadromus collaris (Gravenhorst) on the island of St Helena, P. xylostella
infestations remained so low that chemical control became unnecessary (Kfir 2011).
However, unsuccessful introductions have also been reported and their causes are generally
poorly understood (e.g. Mitchell et al. 1997; Upanisakorn et al. 2011). Failures have been
mainly attributed to (1) the use of broad-spectrum insecticides that affect both the pest and its
parasitoids; and (2) the inability of parasitoids to maintain abundant populations in the target
population's environment, presumably due to the use of inappropriate species or ecotypes
(Sarfraz et al. 2005; Kfir 2011; Furlong et al. 2013). However, host-parasitoid population
dynamics is not straightforward, depends on many abiotic and biotic factors, and it is
uncertain whether well-established populations of parasitoids necessarily translate into
effective pest control (Vickers 2004). For example, host plant quality can indirectly influence

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

the development time and performance of specialist parasitoids (Sarfraz et al. 2009; Fathi et
al. 2012), and obligate hyperparasitoids can limit the impact of primary parasitoids (Nofemela
2013). In Australia, Furlong et al. (2004) found largely unpredictable variations in the rates of
parasitism among P. xylostella populations and concluded that more research was needed to
understand the processes underlying the activity of natural enemies.
In the present study, we monitored changes in the abundance of the diamondback moth,
other insect herbivores, and their natural enemies, in an unsprayed cabbage field in Cotonou,
Benin, with the following objectives: (1) to determine which natural enemies of P. xylostella
play a key role in this tropical area; (2) to clarify how and when host-parasitoid interactions
can influence the population dynamics of P. xylostella in the absence of any insecticide; and
(3) to compare the impact of parasitoids with the effects of hot and humid conditions in
Benin, since rainfall has been reported to be an important factor of control of the moth in a
number of studies (Guilloux et al. 2003; Kobori and Amano 2003; Tonnang et al. 2010).
Some results of this field study were summarized elsewhere (Goudegnon et al. 2004) but the
present paper gives the first detailed analysis of the data.

MATERIAL AND METHODS

Study site and sampling

This study was conducted near Cotonou, Benin, in western tropical Africa, where the
mean annual temperature is 27.2°C, with a low temperature range between the hottest month
(28.9°C in March) and the coolest (25.6°C in August). Annual rainfall averages 1360 mm in
the region, with two rainy seasons. The principal rainy season is from April to July, the peak
rainfall occurring in June, and a less intense rainy period occurs in October. The driest months
are December and January.
Cabbage (Brassica oleracea L.) is grown throughout the year in the periurban areas of
Cotonou. Our study was conducted in an unsprayed cabbage field located at Kouhounou
(6°24' N; 2°31' E), in which the KK Cross cultivar was grown in monoculture. Cultivation
was done by local farmers who transplanted, watered and fertilized cabbages as usual, and
were compensated for crop losses due to the presence of pests.
All lepidopteran larvae (from the second instar onwards) and pupae, as well as pupal
cocoons of hymenopteran parasitoid wasps, were sampled over a 39-month period (January

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

1995-March 1998). Aphids (Hemiptera: Aphididae) and hoverfly larvae (Diptera: Syrphidae)
were also sampled for 36 months over the same period (January 1995-December 1997).
Eighty-three samples were taken, mostly at two-week intervals, with some temporal
irregularity and two gaps in June and December 1996. The elapsed time between two samples
was on average 14.0 ± 0.3 days but ranged from 7 to 20 days. On each sampling date, about
20 randomly selected cabbages were collected from even-aged plots 1.5 month after
transplanting. In total, 1737 cabbages were examined, i.e. 21 ± 1 per sampling date. Leaves
were thoroughly inspected and the numbers of insect larvae and pupae found in each plant
were recorded. Aphid abundance was estimated semi-quantitatively on a scale of 1 to 5.
Sub-samples of lepidopteran larvae were taken to the laboratory, where they were
maintained under ambient temperature, humidity and photoperiod conditions, and fed on fresh
cabbage leaves until pupation. Pupae and parasitoid cocoons were then kept in plastic boxes
(2.5 cm in diameter) until adult emergence to identify the primary parasitoids and
hyperparasitoids associated with each species. Specimens were identified at the CIRAD
laboratory, Montpellier, France.

Statistical analyses
Abundance data were expressed as mean numbers of individuals per plant (plus or minus the
standard error of the mean). Relationships between moth abundance and weather variables
were tested using the Pearson correlation coefficient (r). Shapiro-Wilk tests for normality
were performed beforehand and variables were power-transformed when necessary. Pearson's
r was also used to test the correlations between host and parasitoid abundances after square-
root transformation. These correlations were assessed for host and parasitoid abundances in
the same samples (lag 0) and after shifting one variable several lags forwards or backwards
(lagged correlation). Differences in moth abundance before and after parasitoid population
peaks were tested using unpaired t-tests on square-root transformed data. All calculations
were done using Stata statistical software (StataCorp 2005).

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

RESULTS

Community composition on cabbage leaves

Larvae and pupae of four moth species were collected: Plutella xylostella, Hellula undalis
(Fabricius) (Crambidae), Spodoptera littoralis Boisduval and Chrysodeixis chalcites (Esper)
(both Noctuidae). Mean abundance during the study period was 8.0 ± 0.3 immature moths per
plant, 93.4% of which belonged to P. xylostella. The other three species accounted for only
3.6%, 2.9% and 0.1% of the moths, respectively. Cocoons from three primary parasitoid
wasps (Hymenoptera) were also collected: Cotesia vestalis, Meteorus laphygmae Viereck
(Braconidae) and Euplectrus laphygmae Ferrière (Eulophidae). Mean abundance was 7.2 ±
0.3 cocoons per plant, 99.9% of which belonged to C. vestalis. Laboratory cultures showed
that C. vestalis was strictly associated with P. xylostella, M. laphygmae and E. laphygmae
with S. littoralis. No parasitois were found in C. chalcites. Therefore, the host-parasitoid
system at this site was strongly dominated by the P. xylostella-C. vestalis association, both
species occurring at similar levels of abundance.
The aphid Lipaphis pseudobrassicae Davis (Hemiptera: Aphididae) was often abundant
on cabbage leaves (mean score 1.8 ± 0.1 on the scale of 1 to 5; range 0 to 5), as well as larvae
of Ischiodon aegyptius (Wiedemann) (Diptera: Syrphidae) (on average 2.2 ± 0.1 larvae per
plant), which is an important predator of aphids.
Only small numbers of hymenopteran hyperparasitoids were obtained from C. vestalis
cocoons in the laboratory, the most common being Aphanogmus reticulatus (Fouts)
(Ceraphronidae) and Trichomalopsis orizae (Risbec) (Pteromalidae). Five other
hyperparasitoids were recorded sporadically: Aphanogmus fijiensis (Ferriere)
(Ceraphronidae), Elasmus sp. (Elasmidae), Hockeria sp. (Chalcidae), Notanisomorphella
borborica (Giard) and Pediobius aff. afronigripes Kerrich (both Eulophidae).

Seasonal aspects
The monthly mean abundance of P. xylostella did not show consistent seasonal fluctuations
(Fig. 1). Temporal variation followed a different pattern in each year and the highest monthly
mean abundance values were observed under a variety of weather conditions, namely in
August 1996 (cool with moderate rainfall), January 1998 (quite hot and very dry), March
1997 (hot and dry) and September 1996 (cool and dry). After two periods of heavy rainfall (>
500 mm) in June 1996 and 1997, abundance was very low in samples taken in late June-early

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

35 a
30
Number per plant

25
20
15
10
5
0
J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J F M

600 b 35
500

Temperature (°C)
Rainfall (mm)

400 30
300
200 25
100
0 20
J F M A M J J A S O N D J F M A M J J A S O N D J F M A M J J A S O N D J F M

Fig. 1 (a) Variation in the monthly mean abundance of Plutella xylostella (larvae 2 to 4 and
pupae) on Brassica crops in Cotonou, compared to (b) monthly rainfall (bars) and mean
temperatures (line) during the period of study (January 1995-March 1998)

July (0.3 ± 0.3 larvae and 1.3 ± 0.3 pupae per plant). However, there were no significant
correlations between diamondback moth abundance and monthly precipitation (r = -0.07),
temperature (r = 0.12) or relative humidity (r = -0.13) during the study period.

Host-parasitoid dynamics
Changes in the mean abundance of P. xylostella and C. vestalis in the 83 cabbage samples are
shown in Fig. 2. Populations of both species fluctuated markedly over short time intervals.
Lagged correlation analysis showed that the abundance of C. vestalis was strongly correlated
with that of P. xylostella in the preceding sample (r = 0.74; P < 0.001) (Fig. 2). This indicates
that high and low population densities of the host tended to be followed two weeks later by
high and low population densities of the parasitoid, respectively. There was a highly
significant linear relationship between the abundance of the parasitoid and that of the host at
lag 1 (Fig. 3).

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

60
1
50
0
Number per plant

40 -2 -1 0 1 2

30

20

10

0
0 120 240 360 480 600 720 840 960 1080 1200
Time (days)

Fig. 2 Variation in the mean abundance of Plutella xylostella (larvae 2 to 4 and pupae) (solid
line) and Cotesia vestalis cocoons (dotted line) in 83 cabbage samples collected at ca. two-
week intervals in Cotonou. The inset shows correlation coefficients between the two variables
at lag 0 and at neighbouring lags

The impact of C. vestalis on P. xylostella can be assessed by comparing the mean abundance
of immature moths before, during and after peaks in parasitoid abundance. For example, the
results given in Table 1 were calculated for all peaks ≥ 10 C. vestalis cocoons per plant (n =
20). They show that, on average, the abundance of P. xylostella, which was about 16
individuals per plant in samples immediately preceding parasitoid population peaks,
decreased significantly in subsequent samples (t ≥ 2.54; P < 0.05). The abundance of larvae
decreased first, during parasitoid peaks and two weeks later, while the abundance of pupae
was still significantly reduced four weeks after parasitoid peaks (Table 1).

Relationships between P. xylostella and other insect herbivores

There was no significant correlation between the mean abundance of immature P. xylostella
and that of the other three moth species. There was however a significant negative correlation
between the mean abundance of young larvae of P. xylostella (second instar) and that of the
aphid L. pseudobrassicae (r = -0.35; P < 0.01). Second-instar larvae were found in abundance
on cabbage leaves only when aphids were absent or not very numerous.

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

Parasitoids in sample n+1

6

0
0 1 2 3 4 5 6 7
Hosts in sample n

Fig. 3 Linear relationship between the mean abundance of Cotesia vestalis in a sample and
that of Plutella xylostella in the preceding sample (square-root transformed data). The
equation of the regression line is y = 0.02 + 0.86 x (P < 0.001; r2 = 0.53)

Table 1 Variation in the mean abundance of Plutella xylostella (number of individuals per
plant ± SE) before, during and after abundance peaks in Cotesia vestalis (≥ 10 cocoons per
plant). Moth abundance was high in samples n, just before the occurrence of parasitoid peaks
in samples n+1, and subsequently declined. Asterisks indicate significant decreases in moth
abundance in samples n+1, n+2 and n+3 (P < 0.05)

samples n samples n+1 samples n+2 samples n+3

(parasitoid peaks)

larvae and pupae 15.9 ± 2.2 11.5 ± 2.3 9.4 ± 1.9 * 9.4 ± 1.7 *

larvae 9.6 ± 1.8 5.1 ± 1.7 * 5.1 ± 1.8 * 5.8 ± 1.8

pupae 6.3 ± 1.0 6.4 ± 0.9 4.3 ± 0.7 3.6 ± 0.6 *

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

DISCUSSION

Climate conditions in Cotonou are favourable for the year-round persistence of the
diamondback moth. In the present study, samples that contained at least eight immature moths
per cabbage plant were collected in any month of the year, even during the principal rainy
season from April to July. Although several studies showed that large numbers of eggs and
larvae could be washed off by rainfall or watering with sprinklers (Wakisaka et al. 1992;
Kobori and Amano 2003), we found no correlation between diamondback moth abundance
and monthly precipitation in Cotonou. The only obstacle to P. xylostella due to weather
conditions in southern Benin may be heavy rains that occasionally pour down on the crops
during the principal rainy season, which seems to affect moth populations for a few weeks, as
in Ghana (Cobblah et al. 2012). Climate conditions are also favourable for the year-round
persistence of the larval endoparasitoid C. vestalis, which is obviously an important natural
enemy of P. xylostella in the system studied. This braconid wasp is common in western and
southern Africa and has already been reported as the most abundant parasitoid of the
diamondback moth in South Africa (Nofemela and Kfir 2008) and Ghana (Cobblah et al.
2012). Although C. vestalis was relatively less abundant in the first year of our study, possibly
because of relatively dry conditions and former spraying of insecticides, the species
subsequently reached higher population densities and its rate of parasitism in P. xylostella
larvae was frequently well above 60% (Goudegnon et al. 2004).
At first sight, our results show aseasonal, apparently erratic fluctuations in the
population of P. xylostella. However, the results also show that P. xylostella and C. vestalis
populations are linked together by coupled oscillations in abundance, with a time lag of about
two weeks between abundance peaks in the two species. This may reflect the inherent
tendency of an insect host to show boom-and-bust cycles in the presence of a specialist
parasitoid (Snyder and Ives 2009). Such oscillations are predicted by a number of models
involving one prey species and one predator or parasitoid species (Ricklefs and Miller 2000;
Begon et al. 2006). Similar oscillations were observed in laboratory microcosms involving an
insect species and its parasitoid (Utida 1957; Begon et al. 1996), notably in laboratory
cultures of P. xylostella and C. vestalis under controlled conditions (Karimzadeh et al. 2004).
To our knowledge, this phenomenon has not been reported so far for a P. xylostella
population attacked by a parasitoid under field conditions. In the cabbage field studied near
Cotonou, the community of moths and parasitoids is strongly dominated by P. xylostella and

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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

C. vestalis and close to a one-host-one-parasitoid system. Although C. vestalis can develop in

other lepidopteran hosts in the laboratory (Cameron et al. 1997), it is generally regarded as
highly specific to P. xylostella. In theory, this situation is conducive to oscillations. Indeed,
abundance peaks in immature P. xylostella were often followed by abundance peaks in C.
vestalis cocoons two weeks later, and high parasitoid population densities significantly
reduced diamondback moth population densities over several weeks. When moths became
scarce, the parasitoid population also declined strongly, which could make the crop
susceptible to subsequent pest infestations.
In our field study, oscillations were much less regularly spaced out than some models
predict, which can be explained in several ways: (1) not all samples were collected at regular
time intervals; (2) due to the destructive sampling design, insects were not observed on the
same individual cabbage plants on each date, which could influence the results when species
distributions were patchy; (3) in the field, many biotic and abiotic factors can potentially
interfere with host-parasitoid dynamics and cause erratic patterns of population density
change; for example, our results indicate that the presence of aphids may affect the abundance
of second-instar larvae of P. xylostella on cabbage leaves.
Coupled oscillations occurred repeatedly at our study site, in contrast with what occurs
in laboratory studies, in which populations of hosts and parasitoids often crash rapidly (e.g.
Karimzadeh et al. 2004 for P. xylostella and C. vestalis). The persistence of interactions in the
field may be favoured by dispersal from and to surrounding areas. Adult diamondback moths
move readily among fields (Shirai and Nakamura 1994; Schellhorn et al. 2008a). As cabbage
fields are quite common around Cotonou, there are probably many places that may serve as
sources of recolonization when population density is very low at a given site. Cotesia vestalis
also moves readily among fields to find the host required for its reproduction. Feeding
damage by P. xylostella leads to increased amounts of volatiles released by plants, which
attracts the parasitoid (Potting et al. 1999). The host-parasitoid system may thus function not
at the field scale, but as a metapopulation system at the landscape or regional scales, and
further studies are needed to fully understand its dynamics in a spatial context (Schellhorn et
al. 2008b).
As regards the efficiency of C. vestalis as a biological control agent, significant decreases in
diamondback moth abundance after parasitoid population peaks suggest that C. vestalis is
able to stop pest outbreaks. However, the parasitoid had only short-term effects at our study
site and there was no stable control of the moth population at a sufficiently low density. The
key problem is that the abundance of C. vestalis strongly decreases when there are few P.

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xylostella, so that the parasitoid cannot prevent a resurgence of the pest. Under temperature
conditions prevailing in Cotonou, the intrinsic rate of increase of P. xylostella is nearly at a
maximum (Golidazeh et al. 2009) and, in the absence of parasitoids, small numbers of adult
moths can produce considerable numbers of larvae in one or two generations. As C. vestalis
numerically responds with a time lag roughly equal to two weeks, the pest can reach high
densities before the parasitoid population becomes large enough to be effective. Therefore, C.
vestalis is unable to control moth populations before damage exceeds acceptable levels.
Although it is likely that cabbage infestation by P. xylostella would have been more severe in
the absence of parasitism (Kfir 2011), the population density recorded in the presence of C.
vestalis was on average 7.5 immature moths per plant in our study, which is hardly tolerable
for Brassica vegetable growers. Clearly, additional control measures are necessary to reduce
the damage caused by the diamondback moth in this system.

CONCLUSION

The inherent tendency for host-parasitoid dynamics to cycle can become apparent in
field populations of P. xylostella attacked by a specialist parasitoid. Although C. vestalis
populations are well established in the Cotonou region and seem able to contain diamondback
moth outbreaks by drastically reducing large populations, this parasitoid cannot prevent small
populations of the pest from rebounding rapidly. This may be linked to the environmental
conditions in southern Benin, i.e. a tropical climate in which moth populations can grow very
rapidly when parasitoid density is low, combined with a high probability of recolonization
from surrounding areas. Under such conditions, biological control by a specialist parasitoid
reaches its limits and additional control measures − preferably compatible with C. vestalis in
an integrated pest management programme should be taken against the diamondback moth.

Acknowledgements
We are greatly indebted to E. Goudegnon who was instrumental in collecting the data in
Cotonou, and to G. Delvare for identifying the specimens of parasitoids and hyperparasitoids
collected. We are very grateful to A.A. Kirk for checking the English in the manuscript.

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ARTICLE 5

Antennal structure and oviposition behavior of the Plutella xylostella

specialist parasitoid : Cotesia plutellae

Olivier ROUX, Joan Van BAAREN, Charles GERS, Laurence ARVANITAKIS

and Luc LEGAL

Microscopy Research and Technique, 2005, 68:36-44

 MICROSCOPY RESEARCH AND TECHNIQUE 68:36–44 (2005)

Antennal Structure and Oviposition Behavior of the

Plutella xylostella Specialist Parasitoid: Cotesia plutellae
OLIVIER ROUX,1 JOAN VAN BAAREN,2 CHARLES GERS,1 LAURENCE ARVANITAKIS,3 AND LUC LEGAL1*
1
Laboratoire Dynamique de la Biodiversité, UMR 5172, Université Paul Sabatier, 118 route de Narbonne,
31062 Toulouse cedex 9, France
2
Ethologie Evolution Ecologie, UMR 655 Université de Rennes I, Campus de Beaulieu, Avenue de Général Leclerc,
35042 Rennes cedex, France
3
CIRAD, UPR 17, Sécurisation de l’approvisionnement horticoles des villes, TA 40/L, CSIRO
Campus international de Baillarguet, 34398 Montpellier Cedex 5, France

KEY WORDS SEM; sensilla; flow charts on factorial maps; diamondback moth; Braconidae
ABSTRACT Although several species of the genus Cotesia are used in biological control pro-
grams against insect caterpillars throughout the world, little is known of their oviposition behav-
ior. We describe here the types and distribution of antennal sensilla in Cotesia plutellae, a larval
parasitoid of Plutella xylostella, and we analyze its oviposition behavior. Seven types of sensilla
were found on both males and females. Only sensilla trichodea type II, with a putative contact che-
moreceptive function, was significantly more abundant in females than in males, and its morphol-
ogy and position on antennomeres were linked to the antennation behavior used by females during
host search. We conclude that gustatory stimulus following antennal contact is probably the key
stimulus inducing oviposition behavior. The sensilla type assumed to be implied in oviposition
behavior was present in C. plutellae but not in two closely related species (C. glomerata and
C. rubecula), which is discussed. Microsc. Res. Tech. 68:36–44, 2005. V 2005 Wiley-Liss, Inc.
C

INTRODUCTION 1999; Reddy et al., 2002; Vuorinen et al., 2004a,b).

The genus Cotesia comprises 400 described species After landing on a cabbage leaf, C. plutellae females
among an estimated total number of nearly 1,000 spe- appear to search randomly until they find a host (Guil-
cies worldwide (Shaw and Huddleston, 1991). Several loux, 2000). There is no fine description of the oviposi-
Cotesia species are used for biological control of pest tion behavior in this species.
caterpillars throughout the world, while some are used Cotesia glomerata, a gregarious and generalist para-
as key model organisms in studies of the physiology sitoid, is phylogenetically closer to C. plutellae (solitary
and molecular biology of host-parasitoid interactions, and specialist) than to Cotesia. rubecula, a solitary par-
behavioral ecology, and the ecology and genetics of asitoid specialist of Pieris rapae larvae (Michel-Salzat
metapopulations in fragmented habitats (Michel-Sal- and Whitfield, 2004). Significant differences have been
zat and Whitfield, 2004). shown between C. glomerata and C. rubecula in the
Cotesia plutellae (Kurdjumov) (Hymenoptera: Braco- number of some sensilla types, but not among types
nidae) is a primary solitary larval endoparasitoid, spe- and topographical arrangement (Bleeker et al., 2004).
cialist of the Diamondback Moth (DBM), Plutella xylo- In the present study, we describe the antennal equip-
stella (L.) (Lepidoptera: Yponomeutidae) (Velasco, 1982; ment of male and female C. plutellae, using scanning
Verkerk and Wright, 1996). C. plutellae originates from electron microscopy. The complete oviposition sequence
Europe (Oatman, 1978) but has been reported world- of C. plutellae is also described and series of experi-
wide. It is largely used as biological control agent for ments were performed to determine which organs were
DBM management (Talekar and Shelton, 1993), and really involved in host recognition. We hypothesized
numerous attempts have been made to introduce it into that a specific sensilla type was linked to oviposition
different areas of the world, with mixed results (Talekar behavior sensus lato and to the detection of the host
and Shelton, 1993; Velasco, 1982; Verkerk and Wright, sensus stricto. This possible adaptive link is discussed,
1996). In Asia, it is the only larval parasitoid of DBM, as well as the possible contribution of comparative and
able to survive in tropical and subtropical plains (Tale- phylogenetic analysis to the test of this hypothesis.
kar and Shelton, 1993; Verkerk and Wright, 1996). MATERIALS AND METHODS
Hymenopteran parasitoids commonly find their Plants and Insects
hosts, using chemical stimuli produced by the host or
by the plant (Vet and Dicke, 1992; Vinson, 1976). When C. plutellae and its host DBM originated from Coto-
searching for hosts, wasps use different stimuli in a nou in Benin and were collected on cabbage, Brassica
hierarchical process that lead them gradually to the
host habitat, the host plant, and finally the host itself *Correspondence to: Dr. Luc Legal, Laboratoire Dynamique de la Biodiversité,
(see for review, Quicke, 1997; Vinson, 1976). During UMR 5172 Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse
cedex, France. E-mail: [email protected]
long range searching, C. plutellae females use monoter- Received 18 April 2005; accepted in revised form 6 July 2005
penes and glucosilonate produced by wounds left by DOI 10.1002/jemt.20220
herbivores or mechanical damages (Potting et al., Published online in Wiley InterScience (www.interscience.wiley.com).

V
C 2005 WILEY-LISS, INC.

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 ANTENNAL STRUCTURE AND OVIPOSITION BEHAVIOR 37
TABLE 1. Patterns of oviposition behavior of Cotesia plutellae Behavioral Oviposition Sequences
and abbreviations used in text and figures
Ten second instar caterpillars feeding on a piece of
Pattern Definition cabbage leaf (33 mm in diameter) were individually
walk The female walks continuously with antennal exposed to a parasitoid female in a glass Petri dish
movements (right/left, up/down, U-shape position (33 mm in diameter and 8 mm depth). Each host was
with dorsal side or tip in contact with the leaf)
ant-c Antennal contact with external, internal, or dorsal
attacked once (a host was considered as being attacked
side or with the tip when the female had stung and departed from it). All
line The female takes place in line parallel to the caterpillars were dissected in a saline solution a few
caterpillar body hours after oviposition so as to confirm that an egg has
reab The female moves toward the caterpillar and
begins to bend the abdomen
been deposited by the female wasp. Ten females were
ex-ca Extension of abdomen, outspread wings, and tested (10 ovipositions per female for a total of 100
antennal contact observations). Each oviposition sequence (from search-
dset-abd The abdomen is deep-set in the host, wings are ing for host to departure of the female) was video-
perpendicular to the body and antennal contact
ovip Oviposition (the abdomen is quickly extended)
recorded with a camera (Canon Powershot A80),
sting Sting without oviposition (the abdomen is quickly mounted on a binocular microscope (Wild Heerbrugg),
extended, no egg was found) and analyzed image by image (Pierre and Kasper,
b-ant Antennae back to normal position (above the head) 1990; Van Baaren et al., 1993, 2002, 2003, 2004). On
b-abd Abdomen back to normal position (in the same
line as thorax)
the basis of these observations, we obtained a descrip-
b-wing1 Wings back to outspread position tion of the sequential structure of behavioral patterns
b-body Body back to normal position (from the interpretation of factorial axes), and the pat-
groo Grooming (wasp rubs its antennae and terns were placed in factorial space, their distance
abdomen with legs)
fly The female flies away being inversely related to the frequency of their tempo-
ov-att Oviposition attempt on the leaf ral succession. This analysis yielded a flow chart on
chase The caterpillar escapes and the female chases it factorial maps in which two patterns occurring fre-
wigg The caterpillar wiggles to escape from the quently in succession will appear closely and linked
wasp after contact or sting
eject The female is ejected by the caterpillar with thick arrows. Conversely, two patterns occurring
rarely in succession will be represented far apart and
linked with thin arrows. The behavioral patterns, used
in the analysis, are described in Table 1.
oleracea var. capitata. DBM (adults and larvae) was Sequences with oviposition (one egg was found) and
reared on Indian mustard, Brassica juncea, in Plexi- with sting without oviposition (no egg was found) were
glas cages of 50 3 50 3 50 cm, with water and honey analyzed separately and relative frequencies of pat-
provided separately ad libitum for adults. During ovi- terns were compared with a v2 test, using R statistical
position of DBM, a new cabbage was placed in the software (Ihaka and Gentleman, 1996). After a first
cages every day to avoid larval stages overlapping. Lar- analysis with all patterns, some rare patterns (i.e.,
vae were transferred on a new cabbage every day. recorded less than 10 times) were pooled to reduce the
Cocoons were collected regularly and kept in a plastic number of low values. Four classes of behavioral pat-
box until emergence. terns were defined as follows: the first comprised rare
Parasitoids were reared in Plexiglas cages of 50 3 patterns linked to preoviposition behaviors, the second,
50 3 50 cm for mating and oviposition, with water and patterns linked to oviposition, the third, patterns
honey provided ad libitum. For oviposition, a cabbage linked to avoidance of defensive behaviors by the cater-
with L2 larvae was introduced in the cage. Nymphs of pillar, and the last, behaviors following oviposition,
parasitoid were collected and kept in a plastic box until before the female returned to host location behavior.
emergence. DBM and parasitoids were maintained in a
climatic room at 25 6 18C, 40–50% RH, and a 12 h
light–dark photoperiod. All insects used were spring Determination of Organs Involved
from second rearing generation. in Host Detection
Six tests were performed to evaluate the importance
of chemical and contact detection of different organs,
vision, hearing, and perception of movement in suc-
Scanning Electron Microscopy cessful ovipositions: in each test, six second instar cat-
Newly emerged parasitoids were killed by freezing erpillars were exposed to a parasitoid female in a glass
and serially dehydrated in alcohol (70, 75, 80, 90, 95, Petri dish (50 mm in diameter) for 2 h. For the first
and 100%), then placed in 100% acetone for 15 min, test, females were untreated (control), for tests 2–6,
and in 100% chloroform at 608C for 4 h. Preparations antennae were cut off in test 2, tarsus on first pair of
were kept in 100% acetone until the observation. legs were cut off in test 3, palps were cut off in test 4,
Before observations, samples were critical point dried glass Petri dish were exposed in dark in test 5, and cat-
using CO2 with a Balzers CPD-010 (Balzers, Liechten- erpillars were killed by freezing in test 6. Fifteen repli-
stein) and gold/palladium coated using a JEOL JFC- cates were done for each test. To perform ablations,
1100 sputter unit (JEOL Ltd., Tokyo, Japan). Observa- females were anesthetized on ice and manipulated
tions were performed at 2.5 kV with a JEOL SEM- under a binocular microscope (Wild Heerbrugg). All
6400. caterpillars were dissected in a saline solution a few
We will use the sensilla denomination and classifica- hours after oviposition so as to determine the total
tion of Bleeker et al. (2004), Zachuruck (1980, 1985), number of eggs the female oviposited in each test. To
and Keil (1999). compare tests, data were normalised by decimal loga-

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38 O. ROUX ET AL.

in a socket, and had a grooved surface (Figs. 2c, 2e, 2f,

4e, and 4f). Type I had a classical conical shape with
two pores on the tip (Fig. 4e). It had a length of 22 lm
on first antennomere vs. 18 lm on the last one in
males, and 15 lm in females, with a diameter of 1 lm
at the base. These type I sensilla trichodea were
present on all antennomeres in two rings, one on the
middle of the antennomere and one (with more sen-
silla) at the distal part of the antennomere. At the apex
of the last antennomere stood three TP type I sensilla,
longer than those on last distal antennomeres (Figs. 4a
and 4b).
Type II sensilla trichodea was characterized by a
cuticular projection over apical pores. This cuticular
projection had an opercula shape and the opening was
turned towards the proximal end of the antennomere
(Fig. 2c). It had a length of 19 lm on proximal antenno-
meres vs. 16 lm on distal antennomeres in females,
and a constant length of 16 lm in males. They were
thicker than Type I sensilla (Fig. 2c) with a diameter of
Fig. 1. Length (lm) of male and female antennomeres, and length
(lm) of their sensilla placodea, from the proximal (1) to the distal (16) 2 lm at the base. Pores were numerous and distributed
part of the antenna. in an oval ring under the cuticular projection (Figs. 4e
and 4f). These sensilla were 4.5 times more abundant
in females than in males. They appeared on all anten-
rithm (log (x þ 1)) or square transformations (Hx) nomeres in females and only on antennomeres 6–16 in
tested with the Kolmogorov–Smirnov nonparametric males (Table 2). In males, the cuticular projection
test (Lilliefors method) (SYSTAT 8.0, 1998). An seemed to be less developed than in females (Fig. 2f).
ANOVA was performed and the post hoc Bonferroni These type II sensilla stood always in a ring on the
test were used (SYSTAT 8.0, 1998). middle part of antennomeres, but never on the ventral
side. In females, they appeared in two rings on the last
RESULTS antennomere (Figs. 4a and 4b). They were distributed
Microscopy equally on dorsal and lateral sides. These sensilla
Antennae. Antennae of males and females had a appeared sometimes under a slightly different mor-
long uniform slender shape and had both 16 antenno- phology (Fig. 4f): the cuticular projection seemed to be
meres whose length decreased from that of the proxi- malformed or broken. This different morphological
mal to the distal one (Fig 1). Antennae of males were type appears everywhere on the antennomeres.
thicker (diameter of antennomeres 52.6–82.0 lm) than Sensilla Placodea (or Multiporous Plate Sen-
those of females (49.2–65.5 lm), and their antenno- silla). These sensilla were found on all antennomeres
meres were longer than those of females (Fig. 1). The and were distributed into two regular rings on each
space between antennomeres was larger in females antennomere, one proximal, and one distal (Fig. 3a).
than in males (Figs. 2a and 2b). They were more numerous in males (Table 2). They
Sensilla Types. Seven different types of sensilla had a carina shape and had a length of 61.6–81.5 lm in
were found on both male and female antennae, and the females and 69.5–87.0 lm in males, a width of 3 lm
numbers on each antennomere are given in Table 2. and 2 lm high. In comparison to antennomeres, they
had a relative constant length (Fig. 1). Therefore, the
Sensilla Trichodea Nonporous (NP). They were more the antennomeres shortened, the more sensilla
the most abundant on all antennomeres and were dis- placodea become imbricate (Fig. 3b). This phenomenon
tributed in several rows between each sensilla placo- was more evident in females because of their smaller
dea. They were inserted in a socket, had a grooved sur- antennomeres. On the last female antennomeres, the
face, and a droplet shape at the tip (Fig. 2c). Their repartition became random (Fig. 4a). They were cov-
length was about 35 lm and their diameter 2 lm at the ered by pores of 20 nm in diameter. These pores were
base on the first antennomere, and they became thin- found in V-shaped rows (Fig. 4c).
ner and shorter toward the distal end of antennae (L 
24 lm, Ø  1 lm). Sensilla Coeloconica Type I. They were described
as a peg protruding from a pit in a donut-shaped ring
Sensilla Trichodea With Wall Pores (WP). These (Bleeker et al., 2004). They had at maximal a width of
sensilla had a smooth cuticle, covered by small pores, 1 lm and 4 lm in length, and the pit had an external
and had a socket (Fig. 2d and Table 3). They were diameter of 6 lm. The peg was deeply striated (Fig.
slightly bulbous at the base with a diameter of 2 lm 4g). They were present in males and females on the
and had a conical tip. They were only distributed ventral side of antennomeres 6–15 (only one on each)
around the distal side of antennomeres and were and were situated at the end of the proximal half part
absent in the last one (Figs. 3a, 3b, and Table 2). of them (Fig. 4d).
Sensilla Trichodea With Tip Pores (TP). Two dif- Sensilla Coeloconica Type II. These sensilla had
ferent types were identified. They were both inserted an oval donut shape of 7.5 lm long and 5 lm wide with

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 ANTENNAL STRUCTURE AND OVIPOSITION BEHAVIOR 39

Fig. 2. Sensilla trichodea NP (thin black arrows), s. placodea showing s. trichodea WP. The box shows pores on the cuticle. (e) S.
(stars), s. trichodea TP type I (white arrows), sensilla trichodea TP trichodea TP type I showing the double pores on the tip. (f) S. tricho-
type II (white double shafted arrow). (a) Apical part of male antenna. dea TP type II on the 9th male antennomere showing a thin cuticular
(b) Apical part of female antenna. (c) Detail of 16th female antenno- projection.
mere. (d) Detail of the apical part of the 2nd female antennomere

TABLE 2. Number of different types of sensilla on each antennomere in males (M) and females (F) of C. plutellae,
from the proximal (1) to the distal (16) part of antenna
Antennomere 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Total
s. trichodea NP F Numerous on each antennomere
M
s. placodea F 19 22 22 23 23 25 25 26 26 27 25 24 22 20 20 13 362
M 37 39 40 41 42 40 38 38 40 37 35 35 30 28 26 24 570
s. trichodea WP F 20 14 14 11 12 11 13 16 17 22 18 21 22 17 21 — 249
M 15 15 11 9 8 11 10 13 17 13 14 15 16 14 10 — 191
s. trichodea TP type I F 7 6 7 7 8 8 8 10 10 9 8 8 11 10 10 24 151
M 5 6 6 6 5 7 6 7 8 8 9 7 8 9 9 17 123
s. trichodea TP type II F 1 1 2 2 2 2 3 4 6 6 6 6 6 6 8 13 74
M — — — — — 1 — 1 1 1 1 2 2 3 2 2 16
s. coeloconica I F — — — — — 1 1 1 1 1 1 1 1 1 1 — 10
M — — — — — 1 1 1 1 1 1 1 1 1 1 — 10
s. coeloconica II F — — 1 — 1 — 1 — 1 — 1 — 1 — — — 6
M — — 1 — 1 — 1 — 1 — 1 — 1 — — — 6

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40 O. ROUX ET AL.

TABLE 3. Differences between sensilla of C. glomerata, C. rubecula (Bleeker et al., 2004), and C. plutellae (this article)
C. glomerataa C. rubeculab C. plutellaeb
Body size 6–7 mm 6–7 mm 4–5 mm
Length of antennae in 2.8–3.7 mm 3.8–3.9 mm 2.1–2.9 mm
female and male
s. trichodea NP No differences
s. trichodea WP Length: 30 lm; pore Length: 30 lm; pore Length: 23 lm; Pore
diameter: 20 nm diameter: 20 nm diameter: 30 nm
s. trichodea TP type I Length: 30 lm; Length: 30 lm; Length: 15–22 lm;
diameter 2 lm diameter 2 lm Diameter: 1 lm
s. trichodea TP type II Absent Absent Present
s. placodea Length: 70–132 lm Length: 81–132 lm Length: 61–87 lm
Arrangement of pores: in rows Arrangement of pores: in rows Arrangement of pores: in V-shape
s. coeloconica type I Peg: 2–3 lm width; pit diameter: Peg: 2–3 lm width; pit diameter: Peg: 1 lm width; pit diameter: 6 lm
8 lm on antennomeres 2–15 8 lm on antennomeres 2–15 on antennomeres 6–15
s. coeloconica type II Diameter: 9 lm Diameter: 9 lm Maximal diameter: 7.5 lm
a
Generalist and gregarious.
b
Specialist and solitary.

a small bulb of 0.8 lm of diameter in the middle (Fig. tion. This effect is representative of a low variability in
4h). Only one was present on distal ventral side of pattern succession between different repetitions. How-
antennomeres 3, 5, 7, 9, 11, and 13 (Fig. 4d) in males ever, this sequence was sometimes modified by the
and females. defensive behavior of the host. DBM larvae can react
violently to the presence of the parasitoid (wigg) or can
Behavioral Sequences let themselves fall along a silk line. Although these
The typical oviposition sequence was divided into defensive behaviors could temporarily interrupt the
three parts that appear clearly on the factorial map: (1) sequence, the female parasitoid rapidly reacted by the
host detection and self positioning before oviposition, insertion of the ovipositor (dset-abd) before the cater-
(2) oviposition, and (3) return to normal position. The pillar escaped, or chased it (chase).
first axis separated the patterns linked to host detec- Dissections of host larvae showed that 84% of ovipo-
tion and positioning (negative side) from the patterns sitions were successful. No behavioral differences were
linked to the behaviors following oviposition (positive observed between ovipositions and stings without ovi-
side). The second axis separated the searching pattern position, except for the following patterns: at the end of
(walk) (negative side) from oviposition (positive side). the sequences, the grooming frequency (groo) was sig-
Once the female landed on a leaf, it started to walk nificantly superior in case of oviposition and the walk
rapidly in a random pattern with antennal contact with frequency (walk) was superior in case of sting without
leaf, feces, and silk. Most of the time, the antennae oviposition (v2 (5) ¼ 11.53; P ¼ 0.042).
were U-shaped with the dorsal side of apical an-
Organs Involved in Host Detection
tennomeres (or sometimes only the tips) in contact
with the leaf. This typical searching behavior (walk) The ANOVA performed on rate of parasitism and on
(Table 1, Fig. 5), ended when the female had an anten- the number of eggs by the female revealed that treat-
nal contact (ant-c) with the host. This antennation ments were significantly different (F5,84 ¼ 14.116 (P <
always occurred with the dorsal or lateral side or the 0.001); R2 ¼ 45.7% and F5,84 ¼ 12.974 (P < 0.001); R2 ¼
tip of antennae, but never with the ventral side. Imme- 43.6%, respectively) and the Bonferroni post hoc
diately after this contact, the female moved towards adjustment showed that treatments 2 and 3 were dif-
the caterpillar and began to bend its abdomen under ferent from the control (Table 4).
the thorax (reab). The abdomen was then pushed far-
ther on and the wings were slightly outspread above DISCUSSION
the abdomen (ex-ca). Oviposition was divided into two We described the antennal sensilla of the larval para-
patterns. During the first one, the abdomen was firmly sitoid C. plutellae, and its behavioral oviposition on its
deep-set in the host, the wings were perpendicularly host DBM. Seven different types of sensilla were found
erected under the thorax, and the antennae were always to be common in both sexes. Six of these sensilla types
in contact with the host (dset-abd). The second pattern had already been described in other hymenopteran
was the oviposition sensus stricto. The female quickly families (Ochieng et al., 2000; Van Baaren et al., 1996,
and violently extended the abdomen (ovip), and during 1999). The putative function of sensilla can be deduced
this movement antennal contact was lost. After oviposi- from the number of pores (Bleeker et al., 2004; Keil,
tion, the body returned to a normal position in three 1999). Sensilla trichodea NP are considered to be
steps, starting with antennae moved back above the head mechanoreceptors, TP type to be contact chemosensi-
(b-ant), abdomen back in line with the thorax (b-abd), tive and involved in gustatory function, and WP type,
and finally wings starting to regain their resting position such as sensilla coeloconica type I, to be olfactory sen-
(b-wing 1). Then, the female regained normal position (b- silla. Sensilla coeloconica type II could be thermo- or
body), began grooming (groo), and walked again (walk). hygroreceptors (Altner and Prillinger, 1980), while sen-
This sequence of patterns shows a clear Guttman silla placodea have probably an olfactory function. The
effect (Guttman, 1941) on the factorial map (Fig. 5). remaining type described in our study, the sensilla tri-
This effect results in a hyperbola-shape of majority pat- chodea TP type II, had a particular tip that we can
terns corresponding to the ideal sequence of oviposi- describe as a series of wide pores protected by a cuti-

119
 ANTENNAL STRUCTURE AND OVIPOSITION BEHAVIOR 41

Fig. 3. Dorsal side of the 14th male (a) and female (b) antennomeres showing classical disposition of
s. trichodea TP II (white double shafted arrows), s. trichodea TP (white arrows), s. trichodea WP (thick
black arrows), and imbrications of s. placodea (stars) in females.

cular excrescence. Similar sensilla tips have been Ablation experiments showed that antennae were
described in Braconidae and Encyrtidae and were con- essential in host detection. The effect of ablation of tar-
sidered to be contact chemosensitive (Ochieng et al., sus on first pair of legs was also significantly different
2000; Van Baaren et al., 1996). from controls, but observations revealed that these dif-

120
42 O. ROUX ET AL.

Fig. 4. Sensilla placodea (stars), s. trichodea TP type I (white ar- the 9th female antennomere. (e) Detail of the tip of a s. trichodea TP
rows), sensilla trichodea TP type II (white double shafted arrows), s. coe- type II showing the pore series in oval shape disposition. (f) S. trichodea
loconica type I (white open arrow), s. coeloconica type II (black open TP type II showing a broken cuticular projection. (g) Detail of s. coelo-
arrow). (a) Dorsal side (on top) of the 16th female antennomere. (b) conica type I on 7th male antennomere. (h) Detail of s. coeloconica type
Dorsal side (on top) of the 16th male antennomere. (c) Detail of s. placo- II on 7th female antennomere.
dea showing the V-shape stripe pores organization. (d) Ventral side of

ferences are probably due to a physical handicap when We also showed that an antennal contact was obliga-
females tried to oviposit on their host. tory to initiate the oviposition sequence. This antennal
contact was the last step of host research and the first
step of positioning for oviposition, and it was always
Antennal Sensilla and Behavior done using the external side, the tip, or the dorsal side
Our results showed that antennal structure, sensilla of the antennae. It should be noted that the sensilla tri-
number, and topography were different between males chodea TP type II are distributed on the dorsal and lat-
and females and were probably linked to the use of eral sides of antennomeres. These sensilla, that are
antennae during the searching behavior of C. plutellae. probably contact chemosensory sensilla, are perpendic-
First, when the female walks searching for its host, its ularly erected from the cuticle and emerge from the
antennae form a U-shape. This position was never layer of other sensilla, and are more numerous in
observed in male antennae (pers. obs.) that had most females than in males. This suggests that the sensilla
often a straight erected or slightly curved position. The trichodea TP type II are closely related to the gustatory
antennomeres in the distal half part of the male anten- recognition of the host. Moreover, this type of sensilla
nae are more narrowly spaced than in females, prob- have normally their opening oriented backwards, but
ably preventing the male’s antennae from adopting the in the U-shape antennal position, these opening are
U-shape position. oriented toward the front and are the more exposed to

121
 ANTENNAL STRUCTURE AND OVIPOSITION BEHAVIOR 43
observed in C. plutellae, in which they were slightly
smaller. The distribution of sensilla on antennomeres
was very similar in the three species except for sensilla
coeloconica type I. In C. glomerata and C. rubecula, a
single sensilla coeloconica type I was found on each
antennomere from the 2nd to the 15th one. In C. plutel-
lae, they were less numerous and were found distrib-
uted from the 6th to the 15th antennomere (Table 4).
In C. plutellae, an additional sensilla was described,
the sensilla trichodea TP type II, which represent the
major difference with C. glomerata and C. rubecula.
This sensilla type seems to be important in oviposition
behavior (detection and identification of host).
In C. glomerata, as in C. plutellae, female antennae
are shorter than in males, but not in C. rubecula where
the length of antennae is equal in both sexes. This sex-
ual dimorphism seems to be common in other hyme-
nopteran (Ochieng et al., 2000; Van Baaren et al.,
1999). The length of antennae is directly linked to the
length of the antennomeres in the three species and
with the length of sensilla placodea in both C. glomer-
ata and in C. plutellae. Nevertheless, the decrease in
antennomere length toward apical side of female
antennae was not proportionally reflected in the length
of s. placodea. Sensilla were slightly shorter, but it was
the imbrications of proximal and distal s. placodea that
Fig. 5. Flow chart on factorial map obtained with 100 oviposition allowed the narrowing of antennomeres. The length
sequences of Cotesia plutellae. Behavioral patterns correspond to the
abbreviations given in Table 1. Axis I and II are the two first axes of
difference of s. placodea between sexes was greater in
the Factorial Correspondence Analysis. The circles represent, from C. glomerata and C. plutellae than in C. rubecula.
the smallest to the largest, respectively less than 10, 11–30, 31–60, A particular type of sensilla has been observed in C.
61–80, 81–90 and more than 90 behavioral patterns. The arrows rep- plutellae, and neither in C. rubecula nor in C. glomer-
resent the number of successions between two patterns. Small dashed
lines represent less than 10 successions; solid lines are directly pro- ata. It is tempting to hypothesize a correspondence
portional to the number of successions (10–90). between sensillar equipment and types of parasitism
(e.g., generalist vs. specialist). In the present case, C.
plutellae and C. rubecula are specialists, while C. glom-
chemical contacts. The dorsal location of contact che- erata is a generalist, hence it does not seem that these
mosensory sensilla is uncommon. Usually, in parasi- sensilla are a general adaptation to specialization in
toids (Encyrtidae, Mymaridae, Trichogrammatidae), parasitism. However, it could be an adaptation to a spe-
sensory equipment linked to oviposition are present on cial character of the specific host DBM, like kairo-
the tip or on the ventral side of apical antennomeres mones or some defensive behavior. To test these points
(Cônsoli et al., 1999; Isidoro et al., 2001; Van Baaren
further on, other close relative species should be
et al., 1995, 1996) and an increase in the variety of
studied concerning their type of parasitism, host char-
types is observed from the proximal antennomeres to
the distal ones (Sen and Mitchell, 2001; Van Baaren acteristics, and sensillar equipment. Also, a phyloge-
et al., 1996, 1999). In the present case, we observed five netic perspective could help determining whether the
sensilla types present from the first antennomere on, presence of some particular sensilla types is a histori-
including sensilla trichodea type II involved in oviposi- cal novelty (apomorphy), hence, a possible adaptation
tion behavior. Moreover, the ablation of antennae sup- relatively to other species equipment. Phylogenetic
pressed totally the oviposition behavior. hypotheses are available for the genus Cotesia (Michel-
The egg passage through the ovipositor may be Salzat and Whitfield, 2004; Papp, 1986, 1987). The
observed in several parasitoid species, enabling the presently available data concern only three species,
observer to distinguish oviposition from insertion of the which does not allow any phylogenetic conclusion con-
ovipositor without oviposition (Van Baaren et al., 1995). cerning evolutionary scenarios of biological characters
Although dissection of DBM larvae showed that some (Brooks and MacLennan, 1991; Harvey and Pagel,
females inserted their ovipositor without depositing an 1991; Martins, 1996). A promising research program
egg, in behavioral sequences, no differences were found would consist in analyzing the relevant biological traits
between sequences with or without oviposition. Only in species of the C. rubecula and C. glomerata group
the pattern ‘‘grooming’’ was frequently linked to true (Michel-Salzat and Whitfield, 2004) so as to both check
oviposition, but this connection was not exclusive. for further possible correspondence between morphol-
ogy and specialization in this parasitoid group, as
Comparison With C. glomerata and C. rubecula already shown in Aphidiinae, Encyrtidae, Aphelinidae,
In C. glomerata and C. rubecula, six types of sensilla Trichogrammatidae, Pteromalidae, Eucoilidae, Chari-
were found: sensilla trichodea NP, WP, TP type I, sen- pidae, and Megaspilidae by Le Ralec et al. (1996), and
silla placodea, and sensilla coeloconica type I and II polarize the evolutionary scenarios for traits of interest
(Bleeker et al., 2004). They were similar to those into possible adaptations.

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44 O. ROUX ET AL.

TABLE 4. Effects of the six treatments performed on female parasitoids and described in Material and Methods section, on the proportion of
parasitized hosts and the mean number of eggs laid by each tested individuals
Treatments (sense or organ tested)
Movement and
Control Antennae Tarsus Palps Vision hearing
Na 89 90 90 90 88 90
% of parasitized caterpillars 62.92 0.00** 20.00* 56.67 52.27 34.44
Mean number of eggs laid by female 6 SEM 5.2 6 3.6 0.0 6 0** 1.5 6 2.1** 8.9 6 12.1 4.263.2 3.6 6 1.7
a
The number of caterpillars.
Values significantly different from control (ANOVA, followed by post hoc Bonferroni tests for 2 3 2 comparisons): *P  0.01; **P  0.001.

ACKNOWLEDGMENTS Potting RPJ, Poppy GM, Schuler TH. 1999. The role of volatiles from
cruciferous plants and pre flight experience in the foraging behav-
The authors are grateful to Jo Le Lannic for SEM iour of the specialist parasitoid Cotesia plutellae. Entomol Exper
technical assistance, to Guy Boivin and Pierre Dele- Appl 93:87–95.
porte for helpful comments and for revising the English Quicke DLJ. 1997. Parasitic wasps. London: Chapman & Hall. 470 p.
Reddy GVP, Holopainen JK, Guerrero A. 2002. Olfactory responses of
version of the manuscript. Plutella xylostella natural enemies to host pheromone, larval frass,
and green leaf cabbage volatiles. J Chem Ecol 28:131–143.
Sen A, Mitchell BK. 2001. Olfaction in the Colorado potato beetle:
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 Chapitre III : Interaction entre Plutella xylostella et le parasitoïde Cotesia vestalis

ARTICLE 6

Chemical characterization of contact semiochemicals for host-recognition

and host-acceptance by the specialist parasitoid Cotesia plutellae
(Kurdjumov)

Olivier ROUX, Charles GERS, Josephe Nathan TENE-GHOMSI, Laurence

ARVANITAKIS, Dominique BORDAT and Luc LEGAL

Chemoecology, 2007, 17 : 13-18

Chemoecology 17: 13–18 (2007)
0937-7409/07/010013-6 CHEMOECOLOGY
© Birkhäuser Verlag, Basel, 2006
DOI 10.1007/s00049-006-0351-y

Chemical characterization of contact semiochemicals for host-recognition

and host-acceptance by the specialist parasitoid Cotesia plutellae
(Kurdjumov)
Olivier Roux1, Charles Gers1, Josèphe Nathan Tene-Ghomsi2, Laurence Arvanitakis3, Dominique Bordat3
and Luc Legal1
1
Université Paul Sabatier, Laboratoire Dynamique de la Biodiversité, UMR-CNRS 5172, Bat 4R3. 31062 Toulouse cedex 9, France
2
École Nationale de Formation Agronomique, 2 route de Narbonne, 31326 Castanet-Tolosan cedex, France
3
CIRAD, UPR Horticulture, TA 40/L, CSIRO. Campus international de Baillarguet, 34398 Montpellier Cedex 5, France

Summary. Cotesia plutellae is a specialist parasitoid of moth (DBM) (Lepidoptera: Plutellidae), the greatest pest of
Plutella xylostella. This specificity is potentially under the crucifers in many parts of the world (Velasco 1982; Verkerk
control of several factors before and after oviposition. & Wright 1996). It is largely used as a biological control
Thereby, the stimuli that lead female parasitoids to host agent for DBM management and numerous attempts have
locations and to oviposition, might be at the basis of the been made to introduce it into different areas of the world,
specificity. We explore here the response of C. plutellae with mixed results (Velasco 1982; Talekar & Shelton 1993;
females exposed to host cuticular lipids. A total cuticular Verkerk & Wright 1996). In Asia, it is the only larval para-
lipid extract of host caterpillars was fractionated into a sitoid of DBM able to survive in tropical and subtropical
hydrocarbon fraction and a non-hydrocarbon fraction. plains (Talekar & Shelton 1993; Verkerk & Wright 1996).
Neither fraction alone had any effect on oviposition behav- The females of C. plutellae are primarily attracted by
iour in C. plutellae but the hydrocarbon fraction alone did green leaf volatiles produced by wounds left by herbivores or
seem to have a positive effect on the rate of antennal contact mechanical injury (Potting et al. 1999; Liu & Jiang 2003).
by the females. To induce oviposition behaviour, both frac- They also tend to show a specific response toward the
tions were necessary and reflect cooperation between at host/plant versus non-host/plant complex odour (Shiojiri et al.
least one compound in each fraction. Identification of cutic- 2000a; 2001). After landing on a damaged cabbage leaf,
ular lipids shows that hydrocarbons were dominant (77%). C. plutellae females have the ability to recognize a plant
Non-hydrocarbon compounds were mainly represented by infested by its host through antennal contacts with
15-nonacosanone (18% of the total lipid extract). This kairomones. Host regurgitant, frass and plant contain the info-
ketone is rare in insect cuticle lipids and is thought to origi- chemicals and are important in host recognition (Shiojiri et al.
nate from the cabbage epicuticle where it is dominant with 2000b; Reddy et al. 2002). In contrast, encounters with non-
n-C29 and 14- and 15-nonacosanol also found among the host kairomones put an end to the searching behaviour of
cuticular lipids of the host caterpillar. females (Shiojiri et al. 2001). Despite all these mechanisms of
host location, females are likely to encounter non-host insects
Key words. Cotesia plutellae - Plutella xylostella - cuticu- on cabbage. To ensure that eggs are laid in a suitable host,
lar lipids - host-recognition - host-acceptance - parasitoid. cues perceptible at very short range are necessary.
Cuticular lipids are often involved in relationships
between insects (Howard & Blomquist 1982; Blomquist
et al. 1998). In social Hymenoptera, the cuticular lipids used
Introduction in insect/insect relationships are mainly hydrocarbons and
are involved in a variety of functions such as nest-mate
Hymenopteran parasitoids commonly find their hosts using recognition, reproduction, or parasitism (Dettner & Liepert
various chemical stimuli produced by the host or by the 1994; Turillazzi et al. 2000; Ruther et al. 2002; Dani et al.
plant (Vinson 1976; Vet & Dicke 1992). These different 2005). In parasitoid wasps, non-volatile host cuticular lipids
stimuli lead the wasps to the host habitat, then, in a hierar- are used in very short range and in specialist parasitoids
chical process, to the host plant and finally to the host itself serve as chemical recognition signals to identify host
(Vinson 1976; Quicke 1997). species (Vinson 1976; Rutledge 1996; Howard et al. 1998;
Cotesia plutellae (Kurdjumov) (Hymenoptera: Padmavathi & Paul 1998; Kumazaki et al. 2000) or to dis-
Braconidae) is a primary solitary larval endoparasitoid, and criminate suitable individuals for oviposition (Vinson &
a specialist of Plutella xylostella (L.), the diamondback Guillot 1972; Buckner & Jones 2005).
We have recently shown that the females of C. plutellae
Correspondence to: Luc Legal, e-mail: [email protected] detect their hosts through a short antennal contact, and we

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14 O. Roux et al. CHEMOECOLOGY

hypothesize that gustatory stimuli are elicitors of oviposition were observed and counted: short antennal contact followed by an
behaviour (Roux et al. 2005). In the present paper, we oviposition movement, as described in Roux et al. (2005), and
short antennal contact without oviposition movement. Any female
investigate the role of cuticular lipids as gustatory stimuli which showed no searching behavior during the first minute (i.e.
for host acceptance. We characterized the cuticular lipid inactive or running around the box) was discarded and replaced by
composition of DBM caterpillars and assessed their biologi- another one.
cal activity on the oviposition behaviour of C. plutellae Statistical Analysis. Data were analysed in two manners: (1)
females through behavioural tests. We used bioassays to Counting the number of each response and (2) the proportions of
each response. The number and proportions of each response to the
isolate active fractions and to detect additive or cooperative treatments were analyzed using one-way ANOVA. Before analysis
processes among them in eliciting behavioural responses in the number of events was normalised by square root transformation
C. plutellae. (√x) and proportions by the arcsine of the square root (arcsine √p).
Data normalisation was tested with the Kolmogorov-Smirnov non-
parametric test (Lilliefors method) (SYSTAT 8.0, 1998). ANOVA were
performed and the post-hoc Bonferroni test used with the two data
Methods and Materials sets (SYSTAT 8.0, 1998).
Chemical Analysis. The total extract and the two fractions was
Plants and Insects. Cotesia plutellae and its host DBM originated analysed on an HP 5890 series II gas chromatograph (GC) with a
from Cotonou (Benin) and were initially collected in the field on split/splitless injector at 280 °C, an apolar HT5 capillary column
Brassica oleracea var. capitata. DBM (adults and larvae) were (25 m × 0.22 mm ID, 0.1 µm film thickness, 5 % diphenyl and 95%
reared in the laboratory on Indian mustard, Brassica juncea, in dimethylpolysiloxane), and a flame ionization detector (FID) at
Plexiglas cages of 50 × 50 × 50 cm, with water and honey provided 320 °C. The GC was coupled to a computer and data were
separately ad libitum for adults. During oviposition of the DBM, a processed with HP millennium software. One µl was injected into
new potted plant was placed in the cages every day to avoid over- the column and elution was carried out with helium at 1 ml/min.
lapping of larval stages. Larvae were transferred to a new potted The oven temperature was programmed from 50 °C (for 1 min)
plant every day to provide them sufficient fresh leaves for optimal to 140 °C at 20 °C/min, from 140 °C to 280 °C at 4 °C/min and
development. Cocoons were collected regularly and kept in a plas- 280 °C for 5 min.
tic box until emergence. The determinations and checks were performed on a Finnigan
Parasitoids were also reared in 50 × 50 × 50 cm Plexiglas cages Trace GC-MS 2000 Series chromatograph directly coupled to a
for mating and oviposition, with water and honey provided sepa- mass spectrometer quadrupole detector. The whole system was
rately ad libitum. For oviposition, a cabbage with second stage lar- controlled by the Xcalibur data system, version 1.2. MS spectra
vae (L2) was introduced into the cage. Larvae in their final stage were recorded in EI mode (70 eV), over a mass range of 50-550
leave the host to form cocoons; these were collected and kept in a mass units with 2 scans per second. One µl was injected in splitless
plastic box until emergence of the imago. DBM and parasitoids mode with an injector temperature of 280 °C and a detector
were maintained in a climatic room at 25±1 °C, 40-50 % RH and temperature of 300 °C. An apolar Rtx®-5MS capillary column (30
a 12L:12D photoperiod. m × 0.25 mm ID × 0.25 µm film thickness, 5 % diphenyl and 95 %
Cuticular Lipid Extraction and Filtration. Cuticular lipids of dimethylpolysiloxane) was used. Carrier gas conditions and oven
200 P. xylostella larvae (stage L2) were extracted in 600 µl of temperature were the same as for GC analyses. Identifications were
hexane. Of this 200µl were put aside and used as total lipid extract. processed with a spectral database and with a series of standard
The 400 µl remaining were fractionated on a silica gel column (1 cm, linear alkanes. The standard linear alkanes were also used for
70-230 mesh, 60Å). Elution was done successively with 2 ml of alignment of GC profiles.
hexane, 1 ml of chloroform and 2 ml of methanol. The hexane frac-
tion contained only hydrocarbons. The chloroform and methanol
fractions contained non-hydrocarbon lipids and were pooled. The
two resulting fractions (hydrocarbon and non-hydrocarbon) were Results
dried under nitrogen and re-dissolved in 200 µl of hexane.
Bioassay. All bioassays were performed with dead caterpillars Bioassay. The behaviour of 27 females of C. plutellae was
(killed by freezing) or dummies. Dummies were made of second observed for each treatment. Results of the oviposition pro-
instar DBM larvae killed by freezing, washed twice in hexane for
1 min and dried under a stream of nitrogen. In order to exclude any portions (Fig. 1) are presented in two separate figure panels
ambiguities that could be linked to the possibility of incomplete for better readability of two-by-two comparisons, but only
washing of the host caterpillars, a second kind of dummy was pre- one ANOVA was performed. The first part (Fig. 1a) presents
pared similarly but using Ostrinia nubilalis (Hübner) (Lepidoptera: positive (total hexane extract) and negative (washed) con-
Pyralidae), a non-host caterpillar from a mass-reared laboratory trols on host and non-host dummies and the second part
strain maintained by the Institut National de Recherche
Agronomique (INRA) “Le Magneraud” (Aquitaine, France). (Fig. 1b) presents tests of the different host extract fractions
Nine different treatments were performed: (1) Untreated DBM applied to non-host dummies. ANOVA showed that oviposi-
caterpillar (Host); (2) Untreated O. nubilalis caterpillar (Non- tion proportions differed between treatments (F8, 234 =
Host); (3) DBM dummies + 3 µl pure hexane added per dummy 18.011; P = 0.001). As expected, non-host caterpillars were
(Washed Host); (4) O. nubilalis dummies + 3 µl pure hexane
(Washed Non-Host); (5) DBM dummies + 3 µl total lipid extract significantly less attractive than hosts. Washed host caterpil-
(Host + total extract); (6) O. nubilalis dummies + 3 µl total lipid lars were also less attractive than untreated hosts (Fig. 1a.).
extract (Non-Host + total extract); (7) O. nubilalis dummies + 3 µl When the total extract was applied to host dummies, no sig-
hydrocarbon fraction (Non-Host + Hc); (8) O. nubilalis dummies + nificant difference was observed with untreated host cater-
3 µl non-hydrocarbon fraction (Non-Host + n-Hc) and (9) O. nubi- pillars. Nevertheless, activity tended to be not fully
lalis dummies + 3 µl recombined fractions (Non-Host + Hc + n-
Hc). The amount of 3 µl was the equivalent to the extract obtained recovered. The application of total extract on non-host dum-
from one caterpillar giving the dummies physiological concentra- mies provided the same activity as an application on host
tions of lipids. dummies. In fact, washed host caterpillars were not signifi-
For each treatment, 3 identical dummies or untreated caterpil- cantly different from host dummies with total extract. To
lars were exposed to a mated naïve parasitoid female in a glass
Petri dish (33 mm in diameter and 8 mm depth) for 3 min. test fractionated extract, we used only non-host dummies to
Caterpillars or dummies were placed on corners of a triangular be sure that no active compounds remained on them. The
piece of cabbage leaf with 1.5 cm edges. Two kinds of response two fractions of total extract tested separately on non-host

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Vol. 17, 2007 Host-recognition in Cotesia plutellae 15

a. Fig. 1 Relative proportions of

oviposition (± SE) produced by
Cotesia plutellae females after
Host a antennal contacts with different
treatments applied to caterpil-
Non -Host b
lars. Treatments with different
letters were significantly differ-
ent (P<0.01). The results are
Non -Host + total extract a, c separated into two parts for a
better readability of compari-
sons.
Host + total extract a, c a. Comparison of effects of
total lipid extract when applied
Non -Host ( Washed ) b, d to host and non-host dummies.
b. Comparison of effects of
hydrocarbon fraction (Hc) and
Host ( Washed ) c, d non-hydrocrabon fraction (n-Hc)
when applied to non-host dum-
100 90 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 90 100 mies. Treatments marked by an
% of contact without % of contact followed asterisk were the same as in (a.)
oviposition by oviposition and were repeated to facilitate
comparison

b. *Host b

*Non -Host + total extract b

Non - Host + Hc + n-Hc b

Non -Host + n-Hc a

Non -Host + Hc a

*Non -Host ( Washed ) a

100 90 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 90 100
% of contact without % of contact followed
oviposition by oviposition

Fig. 2 Mean number of anten-

35 a nal contacts (± SE) produced
by Cotesia plutellae females
Mean n u m b er of a n ten n a l con tacts / femelle

30 with the different treatments

applied to caterpillars. Treat-
25
ments with the same letter
were not significantly different
b
b
(P< 0.01). Hc: hydrocarbon frac-
20 b tion; n-Hc: non-hydrocrabon
b
fraction
15
c c
10 c

5 c

0
Host Host Host Non-Host Non-Host Non-Host Non-Host Non-Host Non-Host
+ (Washed) + + Hc (Washed) + n-Hc + Hc
Total extract Total extract + n-Hc

dummies were inactive. The hydrocarbon and non- the females. The ANOVA revealed that the mean number of
hydrocarbon fractions had the same activity as solvent alone contacts produced differed among treatments (F8, 234 =
applied to washed non-host caterpillars (Fig. 1b). However, 29.521; P = 0.001). Greater the numbers of antennal con-
when the two fractions were recombined, the oviposition tacts associated with greater oviposition responses, suggest-
proportion was the same as with the total lipid extract. ing the female’s interest in the caterpillars. The results were
Mean number of antennal contacts (Fig. 2), also slightly different from those obtained with the observation
indicated that the treatments differed in acceptability to of oviposition proportions. As previously, it was for

129
16 O. Roux et al. CHEMOECOLOGY

Table 1. Cuticular lipid compounds of Plutella xylostella caterpillars detected by GC analysis and identified by GC-MS analysis

Compound Retention time Relative abundance Diagnostic ions m/z

HC fraction
n-tricosane (nC23) 19.63 0.6 −
9-methyltricosane (9MeC23) 20.52 0.2 140/141, 224/225
Unknown 21.45 0.4 −
n-tetracosane (nC24) 22.15 tr −
n-pentacosane (nC25) 24.72 1.3 −
9-methylpentacosane (9MeC25) 25.61 2.5 140/141, 252/253
7-methylpentacosane (7MeC25) 25.74 2.2 112/113, 280/281
5-methylpentacosane (5MeC25) 25.94 1.4 84/85, 308/309
3-methylpentacosane (3MeC25) 26.58 1.6 56/57, 336/337
n-hexacosane (nC26) 27.33 0.4 −
9-methylhexacosane (9MeC26) 28.04 0.9 140/141, 266/267
Unknown 28.21 0.3 −
n-heptacosane (nC27) 29.75 0.8 −
11-methylheptacosane (11MeC27) 30.64 13.1 168/169, 252/253
9-methylheptacosane (9MeC27) 30.71 3.6 140/141, 280/281
7-methylheptacosane (7MeC27) 30.82 3.7 112/113, 308/309
9,14-dimethylheptacosane (9,14diMeC27) 31.22 3.2 140, 211, 224, 296
7,16-dimethylheptacosane (7,16diMeC27) 31.42 6.8 112, 182, 252, 323
10,16-dimethylheptacosane (10,16diMeC27) 31.62 1.9 182, 253, 267, 281
n-octacosane (nC28) 32.25 1.1 −
11-methyloctacosane (11MeC28) 32.92 0.7 168/169, 266/267
n-nonacosane (nC29) 34.80 21.4 −
13-methylnonacosane (13MeC29) 35.29 1.8 196/197, 252/253
7,16-dimethylnonacosane (7,16diMeC29) 36.12 3.1 113, 211, 253, 351
Unknown 36.92 0.6 −
Unknown triterpenoidb 39.24 2.3 −
Unknownsa − 0.93 −

Non-HC fraction
15-nonacosanone 40.36 18.2 224/225
14+15-nonacosanol 40.50 0.9 213, 226/227, 241
Unknown 43.13 1.4
Unknownsa − 2.79 −
a
Unknowns which were present in traces in GC analyses and were not detected by GC-MS analyses were pooled, the superscript digit indi-
cating the number of pooled compounds.
b
non-hydrocarbon compound but not retained in a silica gel column on elution with hexane.

untreated host caterpillars that the interest of females was fraction, which contributed to 22.5 % of the cuticular lipids,
highest and significantly different from other treatments was composed of 11 compounds, but despite pooling the
with a mean of 30 antennal contacts per females (P = 0.001). cuticular lipids extracted from 200 caterpillars, only 2 com-
Host dummies, non-host dummies with total extract, washed pounds were present in sufficient quantities to be identified
host and non-host with hydrocarbon fraction showed a high by GC-MS. Two compounds were unusual in insect cuticu-
number of antennal contacts. Female C. plutellae produced lar lipids, a ketone (15-nonacosanone) and an unspecified
fewer antennal contacts for non-host caterpillars, washed triterpenoid belonging to the amyrin family.
non-host dummies and non-host dummies with the non-
hydrocarbon fraction. With approximately 4 antennal con-
tacts recorded per females, non-host dummies with Discussion
recombined fractions were the less attractive.
Chemical Analyses. Forty compounds were detected Most of the compounds identified by GC-MS analyses are
by the GC analyses, but only 28 were sufficiently concen- common in cuticular lipids of insects. Only two compounds
trated for analysis by GC-MS (Table 1). The chain length of (15-nonacosanone and a triterpenoid belonging to the
these compounds ranged from 23 to 29 carbon atoms. The amyrin family) present in high quantities are unusual in
cuticular lipid pool was mainly composed of linear, insects. 15-Nonacosanone is one of the most abundant lipids
monomethyl-branched, or dimethyl-branched alkanes with in the epicuticular waxes of cabbage together with n-C29 and
an odd-number of carbons in the chain. Four compounds 14- and 15-nonacosanol (Terroine 1936; Eigenbrode et al.
were dominant and represented more than 59 % of the total 1991). The triterpenoid was eluted with the hydrocarbon
cuticular lipid extract: nC29 (21.4 %), 15-nonacosanone fraction on the silica gel column. To our knowledge, only α-
(18.2 %), 11MeC27 (13.1 %) and 7,16diMeC27 (6.8 %). The and β-amyrin (triterpenol) have been described as con-
hydrocarbon fraction represented more than 77 % of the stituents of the epicuticular waxes in cabbage (Eigenbrode
amount of total cuticular lipids. The non-hydrocarbon et al. 1991). These two compounds were not identified in

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Vol. 17, 2007 Host-recognition in Cotesia plutellae 17

our extract but two unknown non-hydrocarbon peaks References

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Received 6 June 2006; accepted 12 September 2006.

Published Online First 25 November 2006.

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 CHAPITRE IV

Structuration génétique de
Plutella xylostella
 Chapitre IV : Structuration génétique de Plutella xylostella

1. Introduction

Dans les deux précédents chapitres, nous nous sommes plus particulièrement penchés
sur la biologie et l’écologie de deux parasitoïdes de la teigne. Nous avons également étudié
l’influence des facteurs biotiques et abiotiques de l’environnement tropical, au Sénégal et au
Bénin, sur la dynamique des populations du ravageur et de ses ennemis naturels. Nous allons
dans ce dernier chapitre nous intéresser tout particulièrement au ravageur.
Plutella xylostella est une espèce cosmopolite dont l’aire de répartition est mondiale.
L’étendue de sa propagation est due essentiellement à son importante capacité de déplacement
par migration passive, à l’extension des cultures de Brassicacées due à l’activité humaine et à
son importante capacité d’adaptation à des conditions de vie les plus extrêmes et les plus
variées. Cela a pour conséquence de former un véritable « patchwork » de populations aux
contours souvent indéterminés, ce qui rend son contrôle encore plus délicat. Pour autant, ce
ravageur est toujours considéré comme une seule et même espèce. Quelques études ont
montré une certaine variabilité génétique au sein de populations de P. xylostella originaires
d’une même région (Corée, Sud-Est de l’Australie et de la Chine) (Kim et al. 2000 ; Endersby
et al. 2005 ; Wei et al. 2013). Cependant, aucune étude sur la variabilité et la structuration des
populations de P. xylostella à l’échelle mondiale n’avait été menée à ce jour.
On peut supposer que les populations présentes dans des régions géographiques
éloignées peuvent être différenciées génétiquement. Des études sur la résistance aux
pesticides chez P. xylostella ont mis en évidence des niveaux de sensibilité différents entre
des populations séparées par moins de dix kilomètres dans des régions des îles Hawaii et de
Taïwan (Cheng 1981 ; Liu et al. 1982 ; Tabashnik et al. 1987). Ces variations sont
probablement induites par des variations de la pression de sélection dues aux différents
composés insecticides utilisés. A Hawaii, les flux géniques entre les populations ne sont pas
suffisants pour réduire les différences de sensibilité aux insecticides. Toutefois, les adultes des
populations situées en zone tempérée sont capables de migrer sur de longues distances, par
exemple entre le sud de la Finlande et l’Angleterre (McKenzie 1958) ou entre le sud des
Etats-Unis et le Canada (Harcourt 1986). Dans certaines régions, les flux de gènes entre les
populations pourraient donc réduire les effets d’une dérive génétique au sein de ces
populations.

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 Chapitre IV : Structuration génétique de Plutella xylostella

Notre étude a pour objectif de déterminer si des populations d’origines très différentes
au niveau mondial sont génétiquement différenciées, d’évaluer lesquelles semblent les plus
isolées génétiquement et de déterminer s’il existe une relation entre les distances génétiques et
les distances géographiques entre les populations.
Nous avons pour cela comparé des populations de P. xylostella d’origines
géographiques différentes à l’échelle intercontinentale (Ile de la Réunion, Afrique du Sud,
Bénin, Egypte, Brésil, Etats-Unis, Canada, Martinique, France, Roumanie, Autriche,
Ouzbékistan, Japon, Philippines, Hong Kong, Laos, et quatre localités australiennes). Pour
réaliser cette étude, nous avons utilisé deux marqueurs moléculaires : les isozymes et les ISSR
(Inter Simple Sequence Repeat). Ces résultats font l’objet des articles 7 et 8.

2. Synthèse des résultats

Marqueur enzymatique : sur 21 enzymes étudiées, seulement sept (IDH, MDHP,

G6PDH, MPI, PGM, HK, AAT) ont révélé des loci polymorphes avec des bandes clairement
lisibles permettant une interprétation. Les tests d’Hardy-Weinberg ont montré que les
populations de P. xylostella sont en déséquilibre pour de nombreux loci. Les déviations de
l’équilibre sont dues à un déficit d’hétérozygotes pour quatre loci (IDH, MDHP, G6PGH et
MPI) et à un excès pour un locus (AAT).
Marqueur ISSR : sept amorces ont été testées mais trois d’entre elles n’ont produit que
des « smears » en raison d’une trop faible spécificité. A partir des quatre amorces restantes,
188 bandes ont été sélectionnées et utilisées dans les différentes analyses. La variabilité
observée entre les populations est maximale (100% de polymorphisme) et les populations sont
hautement différenciées sur le plan génétique (Gst = 0,238). Toutefois, la plus grande part de
la variabilité est exprimée entre les individus au sein des populations mais les 19 populations
étudiées restent cependant parfaitement identifiables.
Avec les deux marqueurs, on ne constate pas de corrélation entre les distances
géographiques et les distances génétiques. Par contre, on observe clairement une structuration
génétique des populations à l’échelle mondiale. Cependant avec les isozymes, cette
structuration génétique semble plus cohérente. En effet, on distingue un premier groupe
réunissant toutes les populations australiennes, un second groupe représenté uniquement par
la population du Japon et un troisième groupe réunissant le reste des autres populations

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 Chapitre IV : Structuration génétique de Plutella xylostella

étudiées. Si on fait un lien avec le type de climat, les populations sont réparties en deux
groupes : les populations originaires des régions tropicales (Brésil, Philippines, Bénin et
Réunion) et les populations originaires des régions tempérées (Australie, Ouzbékistan,
France, USA, Afrique du Sud).

3. Conclusion

Les résultats obtenus avec les deux marqueurs moléculaires ont décrit une très forte
variabilité au sein de chacune des populations. Malgré tout, la variabilité entre les populations
reste très importante puisqu’elles sont parfaitement bien séparées. Il existe une structuration
génétique des populations de P. xylostella à l’échelle mondiale. On peut penser que cette
structuration est influencée par le type de climat : tropical ou tempéré.
Ces résultats étaient attendus en raison de la grande échelle géographique couverte par
notre étude. Cependant, même des populations relativement proches géographiquement
(moins de 600 km) présentent une forte différenciation là où Endersby et al. (2006)
concluaient à des populations identiques en utilisant des microsatellites comme marqueurs.
Cette très haute variabilité intra et inter-populations trouve son origine dans la nature
du marqueur utilisé (ISSR) ainsi que dans la biologie du ravageur. En effet, les ISSR étant des
fragments d’ADN situés sur des portions non-codantes (marqueurs neutres), ils tendent à
accumuler les mutations qui apparaissent au cours des générations. Ces mutations peuvent
apparaitre en très grand nombre en raison, d’une part, de l’usage d’insecticides, agents
mutagènes par excellence, d’autre part, d’un grand nombre de générations (plus de 20)
susceptibles de se succéder au cours d’une même année sous des conditions climatiques de
type tropical.

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 Chapitre IV : Structuration génétique de Plutella xylostella

ARTICLE 7

Genetic differentiation among various populations of the diamondback

moth, Plutella xylostella (Lepidoptera : Yponomeutidae)

Apolline PICHON, Laurence ARVANITAKIS, Olivier ROUX, Alan KIRK, Claude

ALAUZET, Dominique BORDAT and Luc LEGAL

Bulletin of Entomological Research, 2006, 96: 137-144

Bulletin of Entomological Research (2006) 96, 137–144 DOI: 10.1079/BER2005409

Genetic differentiation among various

populations of the diamondback moth,
Plutella xylostella (Lepidoptera:
Yponomeutidae)
A. Pichon1, L. Arvanitakis2, O. Roux1, A.A. Kirk3,
C. Alauzet1, D. Bordat2 and L. Legal1 *
1
Laboratoire dynamique de la biodiversité, UMR UPS/CNRS 5172,
Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 4,
France: 2CIRAD/AMIS, Laboratoire Entotrop, CSIRO Campus
International de Baillarguet, TA 40/L, 34398 Montpellier Cedex 5, France:
3
USDA/ARS, European Biological Control Laboratory, Montferrier sur Lez,
34398 St Gély du Fesc, France

Abstract

Genetic variation among 14 populations of Plutella xylostella (Linnaeus) from

USA (Geneva, New York), Brazil (Brasilia), Japan (Okayama), The Philippines
(Caragan de Oyo), Uzbekistan (Tashkent), France (Montpellier), Benin (Cotonou),
South Africa (Johannesburg), Réunion Island (Montvert), and five localities in
Australia (Adelaide, Brisbane, Mareeba, Melbourne, Sydney) were assessed by
analysis of allozyme frequencies at seven polymorphic loci. Most of the populations
were not in Hardy–Weinberg equilibrium and had a deficit in heterozygotes. The
global differentiation among populations was estimated by the fixation index (Fst)
at 0.103 for the 14 populations and at 0.047 when populations from Australia and
Japan, which differed most and had a strong genetic structure, were excluded from
the analysis. By contrast, the populations from Benin (West Africa) and Brazil
(South America) were very similar to each other. Genetic differentiation among the
populations was not correlated with geographical distance.

Keywords: Plutella xylostella, allozyme, population differentiation

Introduction Populations of diamondback moth in tropical areas cause

severe damage (as in Southeast Asia), but in temperate areas
Plutella xylostella (Linnaeus) (Lepidoptera: Yponomeuti-
(e.g. Canada, USA, UK) damage to crops is usually less
dae), the diamondback moth, is a major pest of Brassica crops
dramatic (Lim, 1986). Measures to control this pest were
and has a worldwide distribution (Talekar & Shelton, 1993).
estimated globally at US$ 1 billion in 1992 (Talekar &
Its geographical origin is considered to be in the eastern
Shelton, 1993). The main strategy of management has
Mediterranean region or South Africa because of the number
traditionally been insecticides (Talekar & Shelton, 1993). As
of wild and endemic brassicas in these regions and the
a major consequence, resistance to synthetic insecticides has
presence of a high number of known parasitoid species
appeared in a relatively short period of time in the field (Sun
(Tsunoda, 1980; Kfir, 1998).
et al., 1986). In addition P. xylostella has become the first
species to develop field resistance to some of the toxins of
Bacillus thuringiensis Berliner (Eubacteriales) (Tabashnik
*Author for correspondence et al., 1987; Talekar & Shelton, 1993).
Fax: 00 33 (0)5 61 55 61 96 Genetic differences and gene flow between populations
E-mail: [email protected] of P. xylostella have been investigated in the context of

141
138 A. Pichon et al.

Table 1. Location and date of collection of Plutella xylostella.

Country Locality Latitude Longitude Abbreviation Date of collection

0
0
South Africa Johannesburg 26 08 S 27 54 E SA 07/1999
Benin Cotonou 6
290 N 2
370 E BEN 09/1998
Réunion Island Montvert 20
520 S 55
280 E REU 08/1998
Brazil Brasilia 15
520 S 47
550 W BRA 08/1998
United States Geneva, NY 42
510 N 76
590 W USA 08/1999
France Montpellier 43
380 N 3
530 E FRA 08/1998
Uzbekistan Tashkent 41
190 N 69
150 E UZB 06/1999
Japan Okayama 34
390 N 133
550 E JAP 07/1999
The Philippines Caragan de Oyo 7
040 N 125
360 E PHI 01/1999
Australia Adelaide 34
460 S 138
320 E AUAd 06/1999
Australia Brisbane 27
300 S 153
100 E AUBr 05/1999
Australia Mareeba 17
000 S 145
260 E AUMa 08/1999
Australia Melbourne 37
520 S 145
080 E AUMe 03/1999
Australia Sydney 33
550 S 151
170 E AUSy 06/1999

insecticide resistance. Differences have been shown in the investigate possible reasons for these differences. Allelic
level of susceptibility to insecticide between populations less variation was measured in populations from 14 different
than 10 km apart, both in Taiwan and in Hawaii (Cheng, areas: USA, Brazil, South Africa, Benin, Réunion Island,
1981; Liu et al., 1982; Tabashnik et al., 1987). These differences France, Uzbekistan, Japan, the Philippines and five areas in
were probably induced by local selection pressure due to Australia (Sydney, Brisbane, Melbourne, Mareeba, and
local variations in the insecticide treatments and the Adelaide).
compounds used. Gene flow might not have been sufficient
to overcome the differences in insecticide susceptibility
between the populations in Hawaii (Tabashnik et al., 1987). Materials and methods
Therefore it could be assumed that in some regions, isolated Sampling
populations of P. xylostella may differentiate from each other,
because of a strong selection pressure and because of Strains of P. xylostella were obtained from 14 localities
reduced migration and therefore reduced gene flow. worldwide (table 1). Each sample comprised a minimum of
However, adults can migrate over very long distances; 100 larvae and pupae. One of us (A. Kirk) and foreign
mass migrations have been reported in temperate climates, collaborators collected pupae and larvae (second and third
between the south of Finland and England (Mackenzie, instars) by hand from plants from one field at each locality.
1958), The Netherlands and England (Chapman et al., 2002) They were immediately placed in plastic dishes with leaves
and between the southern USA and Canada (Harcourt, of their host plant (cabbage) and dispatched by air to the
1986). In these areas, the gene flow may be sufficient to CIRAD laboratory (Centre de Coopération Internationale en
overcome differences between the existing populations; Recherche Agronomique pour le Développement, France)
however, differences in levels of insecticide resistance within 48 h. Samples received were reared at 25
C, +2
C,
between populations may also result from different selection 75% relative humidity, and a photoperiod of 12L/12D on a
pressures due to local variation in insecticide use. sequence of cultivated Brassica species. Different Brassica
The genetic variation among world populations of species were used in order to maintain moths in good
P. xylostella using neutral alleles was not estimated. It was condition at each stage of their development. We observed
assumed that environmental factors, such as variations of that second instar larvae developed better when placed on
temperature and rainfall could affect the selection pressure. fresh leaves of cabbage (B. oleracea L. var. Chateaurenard),
Therefore it could be considered that genetic differences whereas third instar larvae did best on cauliflower leaves
between populations of P. xylostella could also occur on (B. oleracea L. var botrytis) until pupation. All pupae were
genes which were not linked to insecticide resistance. In this placed in a plastic box until emergence of adults (males and
study, these genetic differences between widely separated females) which were placed in a Plexiglas cage with Chinese
populations of P. xylostella were investigated with the mustard B. juncea (L.) plant. Chinese mustard was used for
objective of determining whether they were related to the oviposition and first instar larval development. When the
geographical distance between populations. An earlier study number of eggs was sufficient to establish a laboratory
of the estimate of gene flow from neutral or quasi-neutral colony, the adults were deep frozen in liquid nitrogen
allelic variation among populations from Hawaii, Wisconsin and conserved at x80
C. If the number of individuals
and Florida in the USA showed low levels of differentiation was insufficient for a complete analysis, offspring of the
and suggested a substantial gene flow occurring between moths were reared under the same conditions for another
them (Caprio & Tabashnik, 1992). The enzyme electrophor- generation. Adults were deep frozen in liquid nitrogen and
esis technique, used in the study has been used to analyse conserved at x80
C.
geographical variations among populations of lepidopterous
species (Daly & Gregg, 1985; Buès et al., 1994; Wainhouse &
Enzyme electrophoresis
Juke, 1997).
The aims of the present study were to measure Horizontal starch gels (13%) were prepared following the
differences among P. xylostella populations worldwide and protocol of Pasteur et al. (1987) and stored at 4
C for up to

142
 Differentiation among populations of Plutella xylostella 139

Table 2. Enzymes screened in populations of Plutella xylostella and running conditions for electrophoresis.

Buffer Enzyme stained EC Abbreviation

Tris-citrate pH 8 Isocitrate dehydrogenase 1.1.1.42 IDH
NADPH+ malate dehydrogenase 1.1.1.40 MDHP
Glucose-6-phosphate dehydrogenase 1.1.1.49 G6PDH
Histidine pH 6 Mannose-6-phosphate isomerase 5.3.1.8 MPI
Phosphoglucomutase 5.4.2.2 PGM
Tris-maleate EDTA pH 7.4 Hexokinase 2.7.1.1 HK
Adenylate kinase 2.7.4.3 AK
Acid phosphatase 3.1.3.2 ACP
Glucose-6-phosphate isomerase 5.3.1.9 GPI
Glutamate dehydrogenase 1.4.1.2 GTDH
Hydroxybutyrate dehydrogenase 1.1.1.30 HBDH
Phosphogluconate dehydrogenase 1.1.1.44 PGDH
Glycerol-3-phosphate dehydrogenase 1.1.1.8 G3PDH
(S)-2-hydroxy-acid-oxidase 1.1.3.15 HAOX
Tris-ctrate pH 8.7 Aspartate amino transferase 2.6.1.1 AAT
Superoxide dismutase 1.15.1.1 SOD
Glucose dehydrogenase 1.1.1.118 GCDH
Xanthine dehydrogenase 1.1.1.204 XDH
Alcohol dehydrogenase 1.1.1.1 ADH
Gluthatione reductase 1.6.4.2 GR
Pyruvate kinase 2.7.1.40 PK

one day. Gel buffers used were Tris-citrate pH 8, histidine the proportion of heterozygotes observed (Ho). Differences
pH 6, Tris-citrate pH 8.7, Tris-maleate-EDTA pH 7.4. among allelic frequencies for all pairs of populations were
Each entire frozen adult was homogenized in 20 ml of a tested using Fisher’s method, i.e. when genotypic frequen-
NADP 2.3% solution using a pestle and mortar. This solution cies were not independent, due to a linkage disequilibrium
was then centrifuged for 10 min at 13000 rpm and 4
C. between loci, these were not maintained for the analysis of
Fifteen ml of the resulting supernatant was maintained at population structures. Deviations from equilibrium were
x20
C or used immediately. estimated using Wright’s F statistics (Weir & Cockerham,
Each sample (15 ml of supernatant) was loaded on a wick 1984). Values of Fis (deviation from random mating within a
of Whatman no. 3 filter paper and placed into a well of the population), Fit (deviation from random mating in all
gel. Each gel consisted of 15 wells equivalent to 15 adults populations) and Fst or ‘fixation index’ (deviation from
from five populations, therefore three adults per population. random mating among populations) were given. The mean
Migration (150 V, 70 mA) lasted 6 h at 4
C. Migration buffers number of migrants (Nm) from each generation was
used were: Tris-citrate pH 8, Tris-maleate EDTA pH 7.4, estimated using the inverse relationship between Nm and
borate/NaOH pH 8.25, Tris-citrate pH 8.7 (Pasteur et al., Fst (Wright, 1951): Nm = [(1/Fst)x1]/4, where N is the size
1987; Hillis et al., 1996). of the population and m the mean migration ratio.
Soon after migration, each gel was horizontally sliced to An unrooted neighbour-joining tree was constructed with
four layers, 2 mm thick. Stains were prepared as required the values of Fst among each population pair, using DARwin
following the procedure of Pasteur et al. (1987) and Hillis version 3.6.40 (Perrier & Jacquemoud-Collet, 2000).
et al. (1996). Different enzymes were tested (table 2) and each
enzyme was stained in one slice. The following seven enzymes
exhibited discernable bands and polymorphic loci: isocitrate Results
dehydrogenase, NADPH+ malate dehydrogenase, glucose-6-
phosphate dehydrogenase, mannose-6-phosphate isomerase, Of 21 enzymes screened in P. xylostella, four loci did not
phosphoglucomutase, hexokinase and aspartate aminotrans- show a band: superoxide dismutase (EC 1.15.1.1), alcohol
ferase. The banding patterns characteristic of the seven dehydrogenase (EC 1.1.1.1), glutamate dehydrogenase (EC
enzymes were observed in moths. Soon after their appearance, 1.4.1.2) and phosphogluconate dehydrogenase (EC 1.1.1.44).
bands were noted and a scan of the gel was done to conserve a Some loci had diffuse bands: xanthine dehydrogenase (EC
permanent recording. For each population and each enzyme, a 1.1.1.204), acid phosphatase (EC 3.1.3.2), adenylate kinase
minimum of 30 adults was used. (EC 2.7.4.3), hydroxybutyrate dehydrogenase (EC 1.1.1.30),
(S)-2-hydroxy-acid oxidase (EC 1.1.3.15), glucose dehydro-
genase (EC 1.1.1.118), glutathione reductase (EC 1.6.4.2) and
glucose-6-phosphate isomerase (EC 5.3.1.9). Sometimes,
Data analysis
bands had a good resolution but loci appeared to be
Bands were interpreted in terms of loci and alleles monomorphic in each population: glycerol-3-phosphate
and the allelic frequencies were determined. GENEPOP dehydrogenase (EC 1.2.1.12) and pyruvate kinase (EC
version 1.2 (Raymond & Rousset, 1995) was used to test for: 2.7.1.40). Enzymes with polymorphic loci that could be
(i) departures from the Hardy–Weinberg equilibrium (using clearly interpreted were: isocitrate dehydrogenase (IDH,
Fisher’s exact test); and (ii) the proportion of heterozygotes EC 1.1.1.42), NADPH+ malate dehydrogenase (MDHP, EC
estimated under a Hardy–Weinberg equilibrium (He) and 1.1.1.40), glucose-6-phosphate dehydrogenase (G6PDH, EC

143
140 A. Pichon et al.

Table 3. Allelic frequencies of the seven polymorphic loci in the 14 Plutella xylostella populations.

Locus SA BEN BRA FRA JAP USA UZB PHI REU AUAd AUBr AUMA AUMe AUSy
N 32 31 30 30 33 35 32 34 32 33 34 35 35 35
Allele
IDHf 1 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000
2 0.984 1.000 0.983 1.000 0.924 1.000 0.984 0.812 1.000 0.985 0.941 0.943 0.871 0.900
3 0.016 0.000 0.017 0.000 0.076 0.000 0.016 0.188 0.000 0.015 0.059 0.057 0.129 0.100
IDHs 1 0.016 0.000 0.017 0.000 0.045 0.043 0.016 0.191 0.000 0.000 0.059 0.057 0.000 0.086
2 0.984 1.000 0.983 1.000 0.955 0.928 0.937 0.794 1.000 0.970 0.941 0.929 1.000 0.914
3 0.000 0.000 0.000 0.000 0.000 0.029 0.047 0.015 0.000 0.030 0.000 0.014 0.000 0.000
MDHPf 1 0.433 0.383 0.517 0.383 0.576 0.469 0.464 0.375 0.219 0.803 0.206 0.000 0.500 0.500
2 0.567 0.617 0.483 0.617 0.424 0.531 0.536 0.625 0.781 0.197 0.794 1.000 0.500 0.500
MDHPs 1 0.203 0.339 0.167 0.100 0.182 0.057 0.000 0.221 0.047 0.470 0.044 0.057 0.029 0.071
2 0.484 0.548 0.550 0.467 0.455 0.586 0.516 0.632 0.406 0.485 0.897 0.943 0.971 0.929
3 0.313 0.113 0.283 0.433 0.363 0.357 0.484 0.147 0.547 0.045 0.059 0.000 0.000 0.000
G6PDH 1 0.078 0.210 0.217 0.100 0.000 0.029 0.125 0.103 0.094 0.030 0.191 0.043 0.029 0.143
2 0.359 0.371 0.367 0.350 0.227 0.143 0.234 0.279 0.297 0.318 0.279 0.329 0.200 0.343
3 0.422 0.419 0.416 0.550 0.697 0.529 0.547 0.618 0.563 0.652 0.471 0.500 0.557 0.514
4 0.141 0.000 0.000 0.000 0.076 0.300 0.094 0.000 0.047 0.000 0.059 0.129 0.214 0.000
MPI 1 0.141 0.113 0.000 0.033 0.000 0.100 0.000 0.059 0.031 0.000 0.015 0.257 0.000 0.043
2 0.141 0.113 0.200 0.300 0.455 0.129 0.188 0.221 0.141 0.318 0.088 0.357 0.014 0.114
3 0.359 0.323 0.333 0.300 0.545 0.214 0.531 0.265 0.344 0.500 0.882 0.329 0.714 0.443
4 0.203 0.290 0.317 0.200 0.000 0.457 0.188 0.412 0.250 0.152 0.015 0.043 0.229 0.300
5 0.078 0.129 0.100 0.117 0.000 0.086 0.031 0.044 0.109 0.030 0.000 0.000 0.043 0.100
6 0.078 0.032 0.050 0.050 0.000 0.014 0.063 0.000 0.125 0.000 0.000 0.014 0.000 0.000
PGM 1 0.016 0.016 0.017 0.000 0.000 0.014 0.016 0.000 0.000 0.000 0.000 0.000 0.000 0.000
2 0.062 0.032 0.117 0.017 0.000 0.114 0.047 0.000 0.016 0.031 0.000 0.000 0.014 0.043
3 0.656 0.597 0.583 0.450 1.000 0.715 0.656 0.647 0.734 0.939 0.985 0.986 0.986 0.671
4 0.250 0.307 0.200 0.533 0.000 0.143 0.187 0.235 0.219 0.030 0.015 0.014 0.000 0.200
5 0.016 0.048 0.083 0.000 0.000 0.014 0.094 0.118 0.031 0.000 0.000 0.000 0.000 0.086
HK 1 0.000 0.097 0.000 0.050 0.000 0.071 0.031 0.015 0.031 0.000 0.103 0.000 0.014 0.014
2 0.828 0.790 0.900 0.900 0.424 0.843 0.844 0.926 0.844 0.697 0.735 0.800 0.700 0.757
3 0.172 0.113 0.100 0.050 0.576 0.086 0.125 0.059 0.125 0.303 0.162 0.200 0.286 0.229
AAT 1 0.234 0.452 0.417 0.100 0.000 0.057 0.016 0.471 0.422 0.091 0.000 0.186 0.086 0.214
2 0.766 0.532 0.583 0.850 0.803 0.943 0.984 0.529 0.578 0.909 0.897 0.814 0.914 0.686
3 0.000 0.016 0.000 0.050 0.197 0.000 0.000 0.000 0.000 0.000 0.103 0.000 0.000 0.100

See tables 1 and 2 for abbreviations.

N, number of adults analysed for each population.

1.1.1.49), mannose-6-phosphate isomerase (MPI, EC 5.3.1.8), MDHPf showed variation between relatively close popula-
phosphoglucomutase (PGM, EC 5.4.2.2), hexokinase (HK, EC tions such as those from Adelaide and Mareeba. The mean
2.7.1.1) and aspartate aminotransferase (AAT, EC 2.6.1.1). heterozygosity observed (Ho) ranged from 0.183 (Brisbane)
Some enzymes had several loci: IDH with loci IDHf and to 0.424 (Philippines). These values were lower than those
IDHs; MDHP with loci MDHPf and MDHPs. Allelic estimated from the allelic frequencies, following the Hardy–
frequencies obtained for each locus in each population are Weinberg equilibrium (He), except for the populations from
given in table 3. Tests of the Hardy–Weinberg equilibrium Melbourne, Sydney and Adelaide (table 5).
showed that populations of P. xylostella were unbalanced at Allelic frequencies were significantly different for all
numerous loci. Frequencies observed differed from those pairs of populations using the Fisher test, except for the pair
estimated in 70 of the 126 comparisons (P < 0.05). They were Benin/Brazil (x2 = 28.029, P = 0.06162).
caused by heterozygote deficits in the loci IDHs, MDHPs, Some loci showed unbalanced linkages: IDHs and IDHf,
G6PDH and MPI, and by an excess of heterozygotes in locus MEf and G6PDH, MEf and MPI. Loci IDHf and MEf were
AAT (table 4). Fis values were significantly different from 0 not used in later analyses.
for the loci MDHPs (x2 = 194.4, df = 28, P = 0.000), G6PDH For all populations, Fst = 0.103+0.025, and P
(x2 = 268.2, df = 28, P = 0.000), MPI (x2 = 1, df = 28, P = 0.000), (Fst = 0) < 0.001. Values of Fst obtained for pairs of populations
and AAT (x2 = 96.1, df = 26, P = 0.000). ranged from 0 (Benin–Brazil) to 0.230 (France–Melbourne)
The loci IDHf and IDHs were monomorphic in popula- (table 6). In the Australian population group, Fst ranged from
tions of P. xylostella from Benin, France, Reunion Island and 0.039 (Brisbane–Melbourne) to 0.126 (Adelaide–Brisbane). The
Melbourne. A low number of heterozygotes was observed values of Fst between the populations from Australia and the
for the locus PGM: it was monomorphic in the population other populations were over 0.100, except for the population
from Japan and some of its alleles had low frequencies from Sydney (table 6). Fst calculated for the population of Japan
in populations from Adelaide, Brisbane, Mareeba and was also relatively high, it ranged from 0.072 (Japan–Adelaide)
Melbourne in Australia. Allelic frequencies of the locus to 0.195 (Japan–Philippines). The estimated Fst for other pairs

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 Differentiation among populations of Plutella xylostella 141

Table 4. F statistics for all Plutella xylostella populations. considered as under reproductive isolation; whereas popula-
tions from Australia and Japan had a higher value of Fst,
Locus Fis Fst Fit suggesting non-random mating and a low degree of gene flow
IDHf x0.001 0.056 0.047 with other populations. When populations from Australia and
IDHs 0.103 0.049 0.147 Japan were excluded from the analysis, Fst was 0.047+0.020,
MDHPf x0.024 0.140 0.120 with P (Fst = 0) < 0.001.
MDHPs 0.191 * 0.173 0.331 Populations differed the most about the loci MDHPs
G6PDH 0.484 * 0.020 0.494 (Fst = 0.173), PGM (Fst = 0.136) and AAT (Fst = 0.155). Of
MPI 0.227 * 0.092 0.298
the loci analysed, AAT was the only one that exhibited an
PGM 0.008 0.136 0.144
HK 0.026 0.087 0.111 excess of the heterozygotes observed, compared to the
AAT x0.417 * 0.155 x0.197 estimate. To assess its impact on the values of Fst, analyses
All 0.151 * 0.103 * 0.238 were conducted without the allelic frequencies of AAT. The
values of the heterozygosity observed were inferior to
See table 2 for abbreviations. the values expected from the allelic frequencies, except in the
population from Adelaide in Australia (table 7). The estimate
of Fst for all the populations was 0.095+0.027, with P
Table 5. Mean observed and expected heterozygosity at seven (Fst = 0) < 0.033. Concerning population pairs, Fst ranged
loci in Plutella xylostella populations. from 0 (Benin–Brazil) to 0.241 (France–Melbourne). The
Population Ho He x2 df P values of Fst were within the same estimates as previous
analyses, but lower in the majority of the pairs of populations.
South Africa 0.315 0.425 1 14 0.000 When the populations from Australia and Japan were
Benin 0.406 0.436 73 14 0.000 excluded from the analysis, the mean Fst = 0.02. However,
Réunion Island 0.323 0.384 95.1 14 0.000
Brazil 0.374 0.429 77.2 14 0.000
the populations from Australia and Japan remained very
USA 0.311 0.373 70 16 0.000 different from the other populations.
France 0.289 0.378 70.8 14 0.000 The estimated comparative numbers of migrants per
Uzbekistan 0.271 0.363 63.6 14 0.000 generation each year (Nm) has been calculated for some
Japan 0.307 0.350 57.2 16 0.000 populations. When a population is in a suitable environment
The Philippines 0.424 0.451 93.1 18 0.000 (25
C), a new generation is produced every four weeks. Nm
Australia was from 2 to 6 between populations in Australia and was
Adelaide 0.316 0.310 45.7 16 0.000 estimated at 15 between populations from Benin and South
Brisbane 0.183 0.253 50 16 0.000 Africa. The mean number of migrants was around seven
Mareeba 0.222 0.261 34 14 0.002 individuals per generation for the populations from France
Melbourne 0.314 0.274 62.6 14 0.000 and Uzbekistan. The mean numbers of migrants estimated
Sydney 0.409 0.406 89.4 18 0.000 between widely separated populations were not significantly
Ho, observed heterozygosity; He, estimated heterozygosity
different. The South African population exhibited a rela-
under Hardy–Weinberg equilibrium. tively high migration potential.

of populations ranged from 0.007 (Brazil–South Africa) to 0.104

Discussion
(Uzbekistan–Philippines). The genetic differences between the The enzymes corresponded to 11 loci of which nine were
populations were shown in an unrooted tree calculated with polymorphic. Plutella xylostella can therefore be considered a
the values of Fst, using the neighbour-joining method (fig. 1). highly polymorphic species. No alleles were found that
No relationship was observed between the geographic discriminated one population from another. The analyses
distances and the values of Fst. Populations from Benin and did not reveal a locus linked to the sex, or the presence of
Brazil exhibited no differences (Fst = 0.0002), or were not null alleles.

Table 6. Fst estimated for all pairs of Plutella xylostella populations.

Population SA BEN BRA FRA JAP USA UZB PHI REU AUAd AUBr AUMa AUMe
BEN 0.017
BRA 0.007 0.000
FRA 0.024 0.064 0.043
JAP 0.124 0.186 0.175 0.185
USA 0.032 0.093 0.067 0.067 0.166
UZB 0.026 0.102 0.063 0.036 0.122 0.035
PHI 0.042 0.019 0.013 0.079 0.195 0.081 0.104
REU 0.016 0.040 0.013 0.049 0.150 0.068 0.052 0.046
AUAd 0.067 0.101 0.107 0.139 0.072 0.117 0.098 0.116 0.127
AUBr 0.136 0.180 0.173 0.217 0.166 0.176 0.119 0.196 0.197 0.126
AUMa 0.086 0.121 0.117 0.169 0.151 0.115 0.128 0.113 0.146 0.097 0.103
AUMe 0.131 0.176 0.172 0.230 0.175 0.135 0.132 0.180 0.193 0.124 0.039 0.081
AUSy 0.047 0.044 0.043 0.097 0.159 0.082 0.085 0.044 0.092 0.096 0.088 0.056 0.069

See table 1 for abbreviations.

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142 A. Pichon et al.

JAP Table 7. Mean observed and expected heterozygosities at six loci

(AAT excluded) in Plutella xylostella populations.

Population Ho He x2 df P
South Africa 0.365 0.489 1 10 0.000
Benin 0.445 0.586 57.8 10 0.000
Réunion Island 0.400 0.523 53.2 10 0.000
Brazil 0.311 0.472 56 10 0.000
AUAd USA 0.457 0.523 59.5 10 0.000
France 0.320 0.531 63 10 0.000
Uzbekistan 0.370 0.438 37 10 0.000
FRA Japan 0.394 0.449 43.9 10 0.000
UZB
The Philippines 0.350 0.501 56.6 12 0.000
AUMa
AUSy USA Australia
Adelaide 0.379 0.374 35 10 0.000
SA Brisbane 0.191 0.275 41.6 10 0.000
BRA Mareeba 0.248 0.318 32.8 10 0.000
AUBr PHI Melbourne 0.276 0.300 18.8 10 0.043
AUMe Sydney 0.324 0.418 40.1 12 0.000
REU BEN

Fig. 1. Unrooted tree for fixation index (Fst) among pairs of Ho, observed heterozygosity; He, estimated heterozygosity
populations of Plutella xylostella, calculated using the under Hardy–Weinberg equilibrium.
unweighted neighbour-joining method. See table 1 for
abbreviations.
and Kim et al. (1999) estimated Fst = 0.0215. The differentia-
Deviations from the Hardy–Weinberg equilibrium tion index for populations excluding those from Australia
were caused by heterozygote deficits, except for one locus and Japan (Fst = 0.047) was similar to these values. Values
(aspartate aminotransferase) where an excess of hetero- of Fst for populations of other lepidopterous species vary
zygotes was found. However, the results seemed to reveal a from 0.109 for pine beauty moth Panolis flammea (Denis &
Walhund effect (resulting from a mix of several panmictic Schiffermüller) (Lepidoptera: Noctuidae) (Wainhouse &
sub-populations for which initial allelic frequencies are fairly Juke, 1997), Fst = 0.080 for bog fritillary Proclossiana eunomia
different) and may be explained by the various following (Esper) (Lepidoptera: Nymphalidae) (Nève et al., 2000),
causes which are not mutually exclusive. Departures from Fst = 0.007 in the populations of the migrating black
the Hardy–Weinberg equilibrium could be attributed to the antworm Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae)
sampling method, which included several populations of (Buès et al., 1994) and among African and European
related species with different allelic frequencies. Another populations of the cotton bollworm Helicoverpa armigera
explanation was that the low heterozygosity could be (Hübner) (Lepidoptera: Noctuidae) (Nibouche et al., 1998).
derived from a small founding population. An example In the present study, estimates of Fst between the
would be that a low number of migrants could have populations were not correlated with the geographic
established a new population in an area. This hypothesis distances between them. Populations from Australia and
was an explanation for the reduction of the heterozygosity Japan were the most different, whereas populations from
observed in the corn earworm Helicoverpa zea (Boddie) Brazil and Benin exhibited very similar allelic frequencies.
(Lepidoptera: Noctuidae) (Mallet et al., 1993). This phenom- This result has to be confirmed using DNA data to elucidate
enon might have happened in the population from the USA the phylogeny of these populations. Results of the enzyme
since Geneva, New York is in northeastern USA, on the electrophoresis were not informative enough to assume that
migration pathway of adults of P. xylostella from the these populations had the same geographical origin, or if the
southern states to Canada (Dosdall et al., 2002). Another allelic frequencies were related to a high gene flow.
example would be that climatic conditions could consider- Gene flow between populations was limited, as confirmed
ably reduce the size of a population in an area. In the by the number of migrants, particularly between populations
northern region of Japan during winter, densities of in Australia. Nevertheless, migrations in tropical and subtro-
P. xylostella are around five individuals to ten cabbage plants pical areas (e.g. South Africa/Benin) appeared to be more
(Honda et al., 1992). In Benin, where cabbage production is important than in temperate areas (e.g. Uzbekistan/France).
maintained throughout the year, the population size Populations from Australia and Japan were different
decreases from June to September, because of the impact of from other populations and between themselves. The mean
heavy rains (Bordat & Goudegnon, 1997). A third explanation number of migrants estimated among the Australian
is that heterozygote deficits could be a consequence of populations was reduced despite the short distances
reproductive isolation. Kim et al. (1999) considered this separating these populations. The differences could be the
hypothesis, as they observed a high Fis in populations from consequence of a small size of the population and a reduced
South Korea. In the present study, there were not sufficient gene flow. When populations are small, effects of genetic
data to evaluate population dynamics throughout the year, or drift are more important. As a consequence, a higher number
to ascertain the level of migration. Therefore, none of the three of migrants are necessary so gene flow can counterbalance
hypotheses could be confirmed. genetic drift (Allendorf & Phelps, 1981). These populations
The global Fst was relatively high (Fst = 0.103), compared could be in a condition of high reproductive isolation.
to the values obtained in previous studies on P. xylostella. In addition, variations of the genetic structure of
Caprio & Tabashnik (1992) obtained Fst values of 0.028–0.034 populations observed through their allelic frequencies could

146
 Differentiation among populations of Plutella xylostella 143

be linked to insecticide resistance. The role of esterases in References

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appearance of this isozyme was caused by genetic selection Stabilité du polymorphisme enzymatique dans les popula-
and not by a physiological induction. As in the present tions d’un Lépidoptère migrant, Agrotis ipsilon. Entomologia
study, no data on resistance in the populations sampled Experimentalis et Applicata 73, 187–191.
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between the resistance and the allelic frequencies observed estimate gene flow among populations of diamondback
has not been confirmed. But, considering the results, it is moth (Lepidoptera: Plutellidae) in Hawaii. Annals of the
possible that the isozyme linked to the locus of Bt resistance Entomological Society of America 21, 808–816.
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P. xylostella (migrations, reduction of size due to environ- species H. armigera (Hübner) and H. punctigera Wallengren
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environment and migration in temperate areas where they (2002) The origins of infestation of diamondback moth,
probably do not overwinter. Gene flow could maintain the Plutella xylostella (L.), in canola in western Canada., pp. 95–
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distance apart or the gene flow over long distances, probably Natural Resources and Environment.
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time, according to population size, which itself varies due to moth in southern Ontario. p. 3. in Talekar, N.S. & Griggs,
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Recent work on the genetic basis of insecticide resistance 1985, Tainan, Taiwan, Asian Vegetable Research and
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abundance and the possibility of spring immigration of the
diamondback moth, Plutella xylostella (Linnaeus) (Lepidop-
tera: Yponomeutidae) in Morioka City, Northern Japan.
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Allozyme polymorphism in the cotton bollworm Helicoverpa Esterase electrophoretic variation in Bemisia tabaci (Genn.)
armigera (Lepidoptera: Noctuidae): comparison of African (Hom., Aleyrodidae) among host plants and localities in
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 Chapitre IV : Structuration génétique de Plutella xylostella

ARTICLE 8

ISSR-PCR: Tool for discrimination and genetic structure analysis of

Plutella xylostella populations native to different geographical areas

Olivier ROUX, M. GEVREY, Laurence ARVANITAKIS, Charles GERS, Dominique

BORDAT and Luc LEGAL

Molecular phylogenetics and Evolution, 2007, 43:240-250

 Molecular Phylogenetics and Evolution 43 (2007) 240–250
www.elsevier.com/locate/ympev

ISSR-PCR: Tool for discrimination and genetic structure analysis of

Plutella xylostella populations native to different geographical areas q
O. Roux a, M. Gevrey a, L. Arvanitakis b, C. Gers a, D. Bordat b, L. Legal a,*

a
Laboratoire Dynamique de la Biodiversité, Université Paul Sabatier Toulouse III, UMR-CNRS 5172, Bat 4R3, 31062 Toulouse cedex 9, France
b
CIRAD, UPR Horticulture, TA 40/L, CSIRO Campus international de Baillarguet, 34398 Montpellier Cedex 5, France

Received 25 March 2006; revised 31 August 2006; accepted 27 September 2006

Available online 7 October 2006

Abstract

The diamondback moth (DBM), Plutella xylostella (L.) is considered as the most destructive pest of Brassicaceae crops world-wide.
Its migratory capacities and development of insecticide resistance in many populations leads to more difficulties for population manage-
ment. To control movement of populations and apparitions of resistance carried by resistant migrant individuals, populations must be
identified using genetic markers. Here, seven different ISSR markers have been tested as a tool for population discrimination and genetic
variations among 19 DBM populations from Canada, USA, Brazil, Martinique Island, France, Romania, Austria, Uzbekistan, Egypt,
Benin, South Africa, Réunion Island, Hong Kong, Laos, Japan and four localities in Australia were assessed. Two classification methods
were tested and compared: a common method of genetic distance analyses and a novel method based on an advanced statistical method
of the Artificial Neural Networks’ family, the Self-Organizing Map (SOM). The 188 loci selected revealed a very high variability between
populations with a total polymorphism of 100% and a global coefficient of gene differentiation estimated by the Nei’s index (Gst) of
0.238. Nevertheless, the largest part of variability was expressed among individuals within populations (AMOVA: 73.71% and mean
polymorphism of 94% within populations). Genetic differentiation among the DBM populations did not reflect geographical distances
between them. The two classification methods have given excellent results with less than 1.3% of misclassified individuals. The origin of
the high genetic differentiation and efficiency of the two classification methods are discussed.
 2006 Elsevier Inc. All rights reserved.

Keywords: Decision-making tool; Inter Simple Sequence Repeat; Genetic differentiation; Plutella xylostella; Self-Organising Map; Pest management;
Population analysis

1. Introduction of brassica and other crucifer crops in many areas of the

world. DBM can live under wide climatic conditions and
The diamondback moth (DBM), Plutella xylostella (L.) is known to migrate across the world (Chu, 1986; Honda,
(Lepidoptera; Plutellidae), is the major cosmopolitan pest 1990; Honda et al., 1992; Chapman et al., 2002; Coulson
et al., 2002). DBM is a prolific species in tropical climates,
q
This study forms a part of O. Roux’s PhD thesis on the relationship where it can have more than 20 generations a year. DBM
between DBM and a larval parasitoid Cotesia plutellae. The authors are can cause more than 90% crop loss (Verkerk and Wright,
part of a university laboratory which has for interest diversity and
evolution in agro-ecosystem and part of an Agricultural Research Centre
1996) and only few fourth stage larvae on a cabbage can
for International Development where some laboratories are implicated in make it unsaleable (Shelton et al., 1983; Maltais et al., 1998).
IPM in tropical areas. M. Gevrey was developing and adapting neural Extensive insecticide applications are used for its man-
network to molecular work. L. Arvanitakis and D. Bordat are IPM agement which has led to a rapid increase in DBM resis-
entomologists were providing us the background of knowledge on DBM, tance. Resistance to DDT appeared in 1953 (Ankersmit,
reared in laboratory all populations from fields. This work was directed by
L. Legal and C. Gers who are both evolutionary ecologists.
1953) and to Bt in 1980 (Tabashnik et al., 1990; Shelton
*
Corresponding author. Fax: +33 5 61 55 61 96. et al., 1993a,b; Tabashnik, 1994). The cost of DBM
E-mail address: [email protected] (L. Legal). populations control worldwide has been estimated as

1055-7903/$ - see front matter  2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.ympev.2006.09.017

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 O. Roux et al. / Molecular Phylogenetics and Evolution 43 (2007) 240–250 241

approximately one billion US $ annually (Talekar and non-linear relationships are present in the analysed system
Shelton, 1993). to classify complex data (Lek et al., 1996; Lek and Guégan,
Many authors have noticed differences in susceptibility 2000; Park et al., 2003a). SOM provides an alternative to
to many insecticides between DBM strains (Dı́az-Gomez traditional statistical methods as Principal Component
et al., 2000; Gonzalez-Cabrera et al., 2001; Mohan and Analysis, Polar Ordination, Correspondence Analysis and
Gujar, 2002, 2003; Liu et al., 2003). However, migration Multidimensional Scaling (Foody, 1999; Giraudel and
capabilities of the pest cause difficulties for strains determi- Lek, 2001; Brosse et al., 2001). SOM’s are used widely
nation and delay the use of IPM programs. The develop- for knowledge discovery, pattern recognition, clustering
ment of some markers is necessary to identify and and visualisation of large multi-dimensional datasets
characterize DBM strains. Such markers have to be low (Ferran and Ferrara, 1992; Chon et al., 1996; Park et al.,
cost and to give fast results. 2003b; Gevrey et al., 2004). To our knowledge only a few
This topic study explores the usefulness of Inter Simple recent studies have used SOM with genetic data (Giraudel
Sequence Repeat (ISSR) markers to identify and discrimi- et al., 2000; Zhao et al., 2006), but it has been successfully
nate several populations of DBM worldwide through used over the last few decades in biology (Lek and Guégan,
genetic variations. The ISSR is known to evolve rapidly 2000; Recknagel, 2003; Lek et al., 2005). This study
and consequently generate a large number of polymorphic attempts to associate a molecular marker and a classifica-
bands at the intraspecific level. Bands are generated by a tion statistical method to provide a complete decision-mak-
single-primer PCR reaction where the primer is a repetition ing tool in DBM invasion management.
of a di-, tri- or tetranucleotide and the amplified region is a
portion of genome between two identical microsatellite 2. Materials and methods
primers with an opposite orientation on the DNA strand.
These primer sequences are broadly distributed on the gen- 2.1. Plants and insects
ome. Therefore, the ISSR-PCR technique permits to screen
quickly a wide part of the genome without prior DNA DBM populations native to 16 countries and 19 different
sequence knowledge. As for RAPD (Random Amplifica- localities given in Table 1 were collected on cabbage, Bras-
tion of Polymorphic DNA), ISSR bands are considered sica oleracea var. capitata. DBM females laid on Indian
as dominant markers but have higher reproducibility (Fang mustard, Brassica juncea, in Plexiglas 50 · 50 · 50 cm cag-
and Roose, 1997; Nagaoka and Ogihara, 1997). The dialle- es. Water and honey were provided as food ad libitum. All
lique interpretation (presence/absence) may cause matters. larval stages were reared on B. oleracea var. capitata. DBM
Indeed the absence of a band can correspond to one or sev- populations were maintained in climatic rooms at 25 ±
eral divergences in primer site or to a chromosomal rear- 1 C, 40–50% RH and a 12L:12D photoperiod. All adults
rangement (Wolfe and Liston, 1998) and presence of two used to this study were spring from the first rearing
bands with the same weight does not necessarily affirm sim- generation.
ilarity, as the variability is probably underestimated. Nev- For molecular analysis, DBM adults were killed in
ertheless, ISSR has already been used in numerous liquid nitrogen and conserved to 80 C until DNA
organisms for genetic characterization (Reddy et al., extraction.
1999; Sobhian et al., 2003; Cano et al., 2005), to assess
genetic diversity (Qiu et al., 2004; Wang et al., 2005; Lu 2.2. DNA extraction
et al., 2006; Zhang et al., 2006), to identify genetic trait loci
(Zietkiewicz et al., 1994; Ratnaparkhe et al., 1998; Blair Abdomens were cut from dead and frozen DBM males
et al., 1999; Arcade et al., 2000), and for understanding and incubated 12 h at 50 C in 350 lL of lyses buffer B
phylogenetic and/or interspecific relationships (Wolfe and (10 mM Tris, pH 7.5, 25mM EDTA, and 75 mM NaCl)
Liston, 1998; Josh et al., 2000; Wolfe and Randle, 2001; with 500 lg of Proteinase K and 20 lL of 20% SDS. Pro-
Datwyler and Wolfe, 2004; Wu et al., 2005). teins and residues were precipitated with 200 lL of saturat-
Several families of Lepidoptera have been investigated ed NaCl solution and centrifuged at 1400 rpm for 30 min.
using ISSR markers; Noctuidae, Pyralidae, Pieridae and DNA from supernatant was saved and precipitated with
Sphingidae (Luque et al., 2002; Hundsdoerfer et al., 2005; 400 lL of cold isopropanol and centrifuged at 1400 rpm
Hundsdoerfer and Wink, 2005). An interesting element is for 40 min at 2 C. The isopropanol was eliminated and
that depending the type of population studied (open or the precipitate was washed with 500 lL of 70% ethanol,
close), variability and number of informative bands varies centrifugated at 1400 rpm for 10 min at 2 C, dried and
from 50% for the localized species: Diarsia brunnea (Noc- redissolved in 100 lL of TE buffer and conserved at
tuidae) (Luque et al., 2002) to 100% for a quasi cosmopol- 28 C until utilization.
itan species such as Pieris rapae (Pieridae) (Hundsdoerfer
and Wink, 2005). 2.3. ISSR-PCR and electrophoresis
The Self-Organizing Map (SOM) (Kohonen, 1982),
which is an advanced statistical method of Artificial Neural Inter Simple Sequence Repeat (ISSR) analysis was per-
Networks’ family, is an efficient method when complex formed using seven different primers listed in Table 2.

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242 O. Roux et al. / Molecular Phylogenetics and Evolution 43 (2007) 240–250

Table 1
Location and date of collection of the 19 DBM populations
Country Site Abbreviation code Site coordinate Date of collection
Latitude Longitude
Canada Beaverlodge Bea 55130 N 119250 O 01-2002
United States Geneva, NY Gen 42520 N 76590 O 09-1998
Brazil Brasilia Bra 15470 S 47530 O 09-2000
Martinique Island Le Carbet Car 14410 N 61110 O 02-2001
France Montpellier Mon 43370 N 03520 E 02-1999
Romania Iasi Ias 47100 N 27350 E 10-2003
Austria Seibersdorf Sei 47580 N 16310 E 12-2002
Uzbekistan Tashkent Tas 41170 N 69160 E 07-1998
Egypt El Fayoun ElF 29190 N 30500 E 04-2000
Benin Cotonou Cot 06220 N 02260 E 02-2001
South Africa Pretoria Pre 25440 S 28120 E 09-1999
Réunion island Piton Hyacinthe P.Hy 21130 S 55310 E 11-1999
Hong Kong Hong Kong HK 22230 N 114130 E 12-1998
Laos Vientiane Vie 17570 N 102370 E 02-1999
Japan Okayama Oka 34400 N 133550 E 09-1999
Australia Adelaide Ade 34570 S 138340 E 06-1998
Melbourne Mel 37510 S 144570 E 09-1999
Brisbane Bri 27270 S 153300 E 06-1998
Sydney Syd 33520 S 151120 E 06-1999

Table 2
SSR primer screened for ISSR-PCR and their annealing temperature
Primer code Primer sequence (50 fi 30 ) Primer abbreviation Annealing TC
CA CACACACACACACA (CA)7 47
CA+ CACACACACACACARY (CA)7RY 50
+CA RYCACACACACACACA RY(CA)7 50
+ACA BDBACAACAACAACAACA BDB(ACA)5 50
ACA+ ACAACAACAACAACABDB (ACA)5BDB 50
+GACA WBGACAGACAGACAGACA WB(GACA)4 58
GACA+ GACAGACAGACAGACAWB (GACA)4WB 58
With B = T, C or G; D = A,T or G; R = A or G; W = A or T; Y = C or T.

The reaction mixture (25 lL) contained 50 ng of DNA tem- The binary matrix was used under the Hardy–Weinberg
plate, 50 lM of primer, 5 mM of PCR nucleotide Mix equilibrium to calculate the percentage of polymorphism
(C144H, Promega), 2.5 lL of 10· PCR buffer (10 mM (P), Nei’s gene diversity (H), Shannon’s information index
Tris–HCl, pH 9, 50 mM KCl, and 0.1% Triton X-100), (i), total gene diversity (Ht), within population gene diver-
3.5 lL of MgCl2 (25 mM) and 2 units Taq polymerase sity (Hs), between population gene diversity (Dst), coeffi-
(M166A, Promega). PCRs were carried out on a T3 Ther- cient of gene differentiation (Gst), the level of gene flow
mocycler Biometra. The cycling conditions were: initial (Nm), Nei (1972) genetic identity (I) and genetic distance
denaturation of 4 min at 94 C, 39 cycles of 45 s at 94 C, (D) using POPGEN 1.32 (Yeh et al., 1997). In order to
45 s at 50 or 58 C and 2 min at 72 C, one last step of describe genetic structure and variability among and
10 min at 72 C, followed by 4 C storage. between populations, the non-parametric Analysis of
Primers were first tested in order to identify those produc- Molecular Variance (AMOVA) was performed using Arle-
ing a clear amplified product. Seven micro-litres of amplified quin software (version 2.000, Schneider et al., 2000) with
products were mixed with 3 lL of bromophenol blue and 10,000 permutations and Euclidian distances.
electrophoresis was performed on a 2% agarose gel using
1· Tris acetate EDTA buffer at 130 V and on 10 cm, detect- 2.4.1. Classical method
ed with ethidium bromide under UV light and digitized. Most of time the UPGMA distance method was used to
treat the band data. However, this method does not fully
2.4. Data analysis take into account evolutionary patterns and is not search-
ing for optimal tree. Therefore a heuristic search for an
Smeared and weak bands obtained with certain primers optimal tree was carried out by TBR (Tree–bisection–re-
were excluded. Reproducible bands were scored as 1 (pres- connection) branch swapping. Distance (minimum evolu-
ence) or 0 (absence) for individuals, and matrices generated tion) measure uses the mean character difference.
by each primer were assembled. Distance analysis was performed using PAUP version

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 O. Roux et al. / Molecular Phylogenetics and Evolution 43 (2007) 240–250 243

4.0b10 (Swofford, 2001). Negative branch lengths were where t is the number of iterations, and RVk(t) is a real
allowed, but set to zero for tree-score calculation. Steepest vector. In other words, RVk(t) is a vector of the values of
descent options were not in effect. Starting tree(s) were the input neurons at iteration t, VVj(t) is a virtual vector
obtained via neighbour joining and no out-group was used. that represents the weights between a neuron j of the out-
put layer and all the neurons of the input layer at iteration
2.4.2. SOM method t, g (t) is the learning rate that is a decreasing function of
An unusual statistical method for ISSR data analysis iteration t and N(t, r) is the neighbourhood function with
was also used to evaluate differentiation of populations, r representing the distance in the map between the winning
the Self-Organizing Map (SOM) (Kohonen, 1982). Its clas- neuron and its neighbouring neurons. This function defines
sification potential using such data was assessed. This arti- the size of the neighbourhood of the winning neuron
ficial neural network method is powerful and adaptive. It (BMU) to be updated during the learning process. The
used an unsupervised learning algorithm that is efficient learning algorithm is broken down into two parts: (i)
in modelling complex non-linear relationships. SOM per- the VV are widely modified in a large neighbourhood of
forms a non-linear projection of the multi-dimensional the BMU (large values of g), the ordering phase; (ii)
data space onto 2D space. Two layers (input and output) only the VV adjacent of the BMU are modified (t much
of elements called neurons constitute this artificial network. larger than the former phase and g decreasing very slowly
The input layer is associated to the Real Vectors (RV) rep- toward 0), the tuning phase.
resented by the samples of the data, previously randomly More details of the SOM algorithm could be found in
mixed. There are as many neurons in this layer as element Kohonen (2001) and Chon et al. (1996). Giraudel and
in the samples. The output layer is often represented by a Lek (2001) or Park et al. (2003b) are example studies that
map or a rectangular grid with l by m neurons, laid out also detailed this method.
in a hexagonal lattice in order to not favour horizontal The aim of this method is an organisation and visualisa-
and vertical directions (Kohonen, 2001). Each neuron of tion of the real vectors according to their similarities by
this layer is associated with a Virtual Vector (VV) com- arranging the distribution to the samples onto a 2D space
posed of as many elements as neurons in the input layer. represented by the map. The samples placed in the same
The neurons of both layers are connected by links that output neuron are considered similar. Moreover, the sam-
are called weights. ples that are neighbours on the map are also expected to be
The SOM algorithm can be summarised as follows: more similar to each other and then belong to the same
cluster. A cluster analysis is then applied to the virtual vec-
• The virtual vectors (VVj, 1 6 j 6 c) are initialised with a tors to define the cluster boundaries on the map, which are
random sample. the groups of similar output neurons. Commonly a hierar-
• The virtual vectors are updated in an iterative way. chical cluster analysis with a Ward linkage method is
 A real vector (RVk) is chosen as an input vector. applied. We used the functions implemented in the SOM
 The Euclidean distance between this RVk and each toolbox for Matlab (MathWorks, 2001).
VV (each output neuron) is computed.
 The VV closest to the RVk is selected and called ‘‘best 2.5. Validation test
matching unit’’ (BMU).
 The BMU and its neighbours are moved slightly To check the validity of the results, cluster and map, a
towards the RVk using this rule: special validation procedure was established for this study.
For each of the 19 DBM populations, new individuals were
created (Fig. 1). Fifteen individuals from each population
VV j ðt þ 1Þ ¼ VV j ðtÞ þ gðtÞ  N ðt; rÞðRV k ðtÞ  VV j ðtÞÞ were extracted randomly to build five new individuals per

a 0011000100010001110001… …010000111101001000010… … 00001111011010010100010

b 0100100100001110100010… …111001101011001000010… … 01110010010010100010010

c 010000111101001000010… …100100111010010010011… … 01110011100100100100010

z 0011000100010001110001… …111001101011001000010… … 01110011100100100100010

Fig. 1. Creation of the ‘‘new’’ individual for the validation of methods. One part of the sequence of 188 bands was selected in three individuals (a–c) of the
same population. Each part was complementary such that when they were joined, they formed a ‘‘new’’ individual (z). Limits between each part were
randomly selected for all ‘‘new’’ individuals.

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244 O. Roux et al. / Molecular Phylogenetics and Evolution 43 (2007) 240–250

population. The 95 new individuals were then introduced in less, the largest variability was expressed among individuals
the matrix data set of PAUP and into the calibrated SOM within populations (73.71%, df = 520, P < 105).
map. If the obtained results are powerful, these individuals The mean percentage of polymorphic loci (P) within
have to be classified in the clusters and neurons where indi- populations was 94%, ranging from 84% in Seibersdorf
viduals of the same population have been classified. and Okayama populations to 99% in the Beaverlodge pop-
ulation. The Shannon’s index (i), Nei’s gene diversity (H)
3. Results and the percentage of polymorphism (P) within each pop-
ulation are summarized in Table 3. The coefficient of gene
3.1. ISSR profile differentiation (Gst) and the gene flow (Nm) between pop-
ulations by pair is given in Table 4. The Okayama popula-
Seven ISSR primers were initially tested on the 19 DBM tion seems to be the most differentiated with a mean Gst of
populations. All gave amplified products but the three less- 0.235. Genetic identity (I) among populations ranged from
er specific primers (CA, +CA and CA+) produced smears. 0.795 to 0.950, with an average of 0.888 ± 0.032, and the
Only anchored tri-nucleotide SSR primers produced clear genetic distance (D) between populations varied from
and reproducible fragments. From these, a total of 188 0.051 between Tashkent and Iasi to 0.230 between Coto-
scorable ISSR loci (bands) were selected in the 539 DBM nou and Okayama with an average of 0.119 ± 0.036.
individuals screened from the 19 populations. Within pop-
ulations, the number of loci amplified per primer ranged 3.3. Population’s classification and cluster analysis
from 28 to 49 (Table 3), with an average of 42.37 loci. Each
of the 539 individuals presented a unique ISSR genotype, 3.3.1. Classical distance analysis
indicating extensive genetic variation within populations. Unrooted tree obtained by distance analysis provided a
clear discrimination of the 19 populations. For each popu-
3.2. Genetic structure and diversity lation, individuals were regrouped in a spindle-shape except
for one individual of the Iasi population (Iasi 09) which was
At the species level (all populations), the total polymor- inserted between Australian populations of Brisbane and
phism was maximal (100%). The total gene diversity (Ht) Sydney (Fig. 2a). Branches that support OTU (Operational
was 0.347 ± 0.016, gene diversity within populations (Hs) Terminal Unit) i.e. individuals, were longer than branches
was 0.265 ± 0.009 and between populations was that separate populations. The Okayama population had
0.082 ± 0.007. The global coefficient of gene differentiation the longest branch (Fig. 2b) but also the shortest individual
(Gst) was 0.238. branches (Fig. 2a) that show a great differentiation with
AMOVA indicated a highly significant genetic difference other populations on the one hand but a relative similarity
among populations (26.29%, df = 18, P < 105). Neverthe- of individuals within the population on the other. At the

Table 3
Genetic variability parameters of the 19 DBM populations for each usable SSR primer
+ACA ACA+ +GACA GACA+ P mean He ± SD i ± SD
N P N P N P N P
Beaverlodge (26)a 47 100 49 98 42 100 43 100 99 0.319 ± 0.147 0.482 ± 0.189
Geneva (30) 47 91 49 96 40 98 35 97 96 0.273 ± 0.170 0.417 ± 0.232
Brasilia (30) 47 100 48 96 42 100 48 94 97 0.354 ± 0.145 0.524 ± 0.186
Le Carbet (30) 45 98 48 96 41 98 42 88 95 0.303 ± 0.179 0.452 ± 0.239
Montpellier (30) 45 96 45 96 40 100 40 95 97 0.252 ± 0.178 0.387 ± 0.240
Iasi (26) 39 95 45 93 41 100 43 91 95 0.244 ± 0.168 0.379 ± 0.232
Seibersdorf (27) 47 89 44 98 40 98 28 50 84 0.226 ± 0.184 0.346 ± 0.260
Tashkent (28) 44 86 46 89 41 100 40 95 93 0.267 ± 0.174 0.407 ± 0.239
El Fayoun (30) 39 79 44 95 42 100 45 98 93 0.252 ± 0.167 0.390 ± 0.231
Cotonou (27) 44 93 47 96 41 98 43 91 94 0.297 ± 0.178 0.444 ± 0.240
Pretoria (30) 44 89 47 96 39 97 44 93 94 0.256 ± 0.169 0.396 ± 0.230
Piton Hyacinthe (25) 45 91 48 98 42 100 45 89 94 0.284 ± 0.156 0.436 ± 0.210
Hong Kong (30) 47 100 45 98 40 100 46 87 96 0.306 ± 0.162 0.461 ± 0.215
Vientiane (26) 46 98 47 96 40 98 41 93 96 0.268 ± 0.168 0.413 ± 0.226
Okayama (29) 32 81 42 86 34 88 33 82 84 0.192 ± 0.196 0.292 ± 0.276
Adelaide (29) 40 93 45 96 42 100 44 98 96 0.249 ± 0.170 0.387 ± 0.231
Brisbane (26) 30 80 44 89 37 95 39 92 89 0.216 ± 0.185 0.332 ± 0.262
Melbourne (30) 41 98 48 94 39 92 46 96 95 0.273 ± 0.181 0.414 ± 0.242
Sydney (30) 29 86 41 88 38 100 44 91 91 0.197 ± 0.180 0.308 ± 0.253
Mean 42 92 46 94 40 98 42 90 – – –
a
Number of individual; N: Number of loci (bands); P: percentage of polymorphism; He: Nei’s gene diversity; i: Shannon’s information index; SD:
standard deviation.

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 O. Roux et al. / Molecular Phylogenetics and Evolution 43 (2007) 240–250 245

other extreme, Brasilia and Beaverlodge populations have

3.117
2.580
2.974
2.368
2.172
3.125
2.080
2.881
1.840
2.819
2.757
3.102
2.584
2.803
1.379
3.333
2.816
2.303
****
Syd

very long OTU branches that revealed a great differentia-

tion of individuals in these populations. Genetic distances
observed on the tree do not reflect geographic distance
3.094
2.464
3.510
3.388
2.886
3.005
1.991
2.936
2.334
3.004
2.160
2.881
4.342
3.065
1.925
2.841
2.709

0.178
****
Mel

between populations. Only three Australian populations

(Brisbane, Sydney and Adelaide) were grouped in the same
2.990
2.646
2.491
2.569
1.996
3.466
1.833
3.002
2.101
2.708
2.850
3.162
2.553
2.912
1.446
3.071

0.156
0.151
cluster and have a geographic proximity.

****
Bris

3.3.2. SOM
5.758
4.611
4.223
2.882
3.082
4.663
2.174
5.287
2.873
3.527
3.343
3.924
3.269
3.302
1.597

0.140
0.150
0.131
****
The SOM neural network was constituted of 188 neu-
Ade

rons (one for each band) in the input layer linked to the
539 DBM individuals such that the representation of the
1.964
1.549
2.274
1.931
1.722
1.469
1.278
1.526
1.411
1.523
1.406
1.732
2.102
1.690

0.238
0.257
0.206
0.266
****
Oka

presence/absence of the 188 bands for each individual

Coefficient of gene differentiation (Gst) below the diagonal and estimated gene flow per generation (Nm) above the diagonal between pairs of populations

formed 539 real vectors. The output layer comprised 456

neurons organised in an array with 24 rows and 19 col-
5.633
3.926
3.931
3.865
3.542
4.359
3.160
4.251
3.654
3.158
4.014
5.514
5.799

0.228
0.132
0.147
0.140
0.151
****
Vie

umns. Each neuron of the output layer is linked to the

input neurons (i.e. 188) by weights forming a virtual vector.
During the learning process, a virtual individual is then
4.907
3.712
4.972
5.663
4.434
4.208
2.763
3.814
3.772
3.709
3.450
6.214

0.079
0.192
0.133
0.164
0.103
0.162
****
HK

computed in each neuron. Several maps were created using

different sizes. We used the topographic and the quantiza-
5.408
4.276
3.722
4.129
3.743
5.180
2.994
4.218
3.694
3.947
3.865

0.075
0.083
0.224
0.113
0.137
0.148
0.139
P.Hy

tion errors (Kohonen, 2001) to determine final map size.

****

For the final map size (456 neurons), these errors were suf-
ficiently low, 0.0019 and 4.943 respectively. The conver-
4.717
3.502
3.962
3.093
2.853
3.498
2.313
3.943
2.910
4.103

0.125
0.127
0.111
0.262
0.130
0.149
0.188
0.154
****

gence was mostly reached in 725 iterations with a

Pre

learning rate beginning from 0.5 for the ordering phase

and for the tuning phase, from a learning rate of 0.05,
3.982
4.254
4.157
3.268
3.069
3.657
2.599
4.951
3.054

0.109
0.112
0.119
0.137
0.247
0.124
0.156
0.143
0.151
****
Cot

the maximum number of iterations was 2765. Each individ-

ual was classified in a neuron of the output layer (the map).
In general, the individuals coming from the same
3.736
3.670
3.203
3.087
2.655
3.806
2.442
3.481

0.141
0.150
0.119
0.117
0.120
0.262
0.148
0.192
0.177
0.214
****
ElF

population are in the same neuron or in neighbour neu-

rons. The Pretoria population is the most concentrated
with all the individuals classified into 5 neighbouring neu-
5.296
5.195
3.825
3.444
3.453
6.131
2.631

0.126
0.092
0.113
0.106
0.116
0.105
0.247
0.086
0.143
0.146
0.148
****
Tas

rons. The Beaverlodge population is the most widespread

with its individuals classified into 16 neurons (Fig. 3a).
2.580
2.713
2.524
2.522
2.136
2.997

0.160
0.170
0.161
0.178
0.143
0.153
0.137
0.281
0.187
0.214
0.201
0.194
****

A hierarchical cluster analysis was applied to the 456

Sei

virtual vectors of this map. As many clusters as popula-

tions were chosen, i.e. 19, to test the quality of the classifi-
4.564
4.564
3.192
3.250
2.997

0.143
0.075
0.116
0.120
0.125
0.088
0.106
0.103
0.254
0.098
0.126
0.143
0.138
****

cation method (Fig. 3b). Individuals are relatively well

Iasi

classified into the 19 clusters according to their origin pop-

ulation. However, few individuals were considered as badly
5.235
3.064
3.615
3.597

0.143
0.190
0.127
0.159
0.140
0.149
0.118
0.101
0.124
0.225
0.140
0.200
0.148
0.187
Mon

****

classified, i.e. when these individuals were found in neuron/

cluster mainly occupied by individuals of a different popu-
lation. One Adelaide individual was classified in the cluster
4.221
2.929
4.056

0.122
0.133
0.165
0.127
0.139
0.133
0.139
0.108
0.081
0.115
0.206
0.148
0.163
0.129
0.174
****
Car

of Iasi, two individuals of Piton Hyacinthe were classified

in the Le Carbet and in Vientiane clusters, two individuals
5.619
3.771

0.110
0.122
0.135
0.165
0.112
0.135
0.107
0.112
0.118
0.091
0.113
0.180
0.106
0.167
0.125
0.144

from Iasi were classified in the cluster of Brisbane and

****
Bra

Vientiane, while one Vientiane individual was classified in

the Tashkent cluster and one Beaverlodge individual in
5.593

0.117
0.146
0.140
0.099
0.156
0.088
0.120
0.105
0.125
0.105
0.119
0.113
0.244
0.098
0.159
0.169
0.162
****

See Table 1 for abbreviations.

the Cotonou cluster.

Gen

3.4. Validation
0.082
0.082
0.106
0.087
0.099
0.162
0.086
0.118
0.112
0.096
0.085
0.093
0.082
0.203
0.080
0.143
0.139
0.138
****
Bea

A special procedure has been used to validate the quality

of the cluster and the map using new individuals created
randomly (see Fig. 1 and ‘‘validation test’’ Section 2).
Table 4

Ninety five new individuals were introduced into the matrix

P.Hy
Mon

Oka
Gen

Ade
Bris
Mel
HK
Bea

Car

Syd
ElF
Cot
Bra

Tas
Iasi

Pre

Vie
Sei

data set of PAUP and into the SOM calibrated model, i.e.

156
246 O. Roux et al. / Molecular Phylogenetics and Evolution 43 (2007) 240–250

a Geneva TashkentIasi
Cotonou Pretoria

Brisbane
Brasilia Iasi 09
Sydney

Melbourne Adelaide

Okayama Seibersdorf Brisbane

Tashkent
Cotonou Pretoria
Geneva Iasi 09
Le Carbet Iasi
Vientiane Sydney

Brasilia Adelaide
Hong Kong El Fayoun
Montpellier Piton
0.1 Melbourne
Beaverlodge hyacinthe Seibersdorf

Okayama Vientiane

Pi
to
n
Le Carbet Hong Kong

hy
Beaverlodge

ac
El Fayoun

in
th
e
0.1 Montpellier b

Fig. 2. Unrooted tree obtained by distance analyses of the 19 DBM populations. (a) Complete unrooted tree; (b) unrooted tree simplified and enlarged on
population separation part.

the map. All 95 were correctly classified into the cluster and scale where Endersby et al. (2006) found no differentiation
neurons of their origin population. For example, the five with microsatellites. Beside geographic distances, this level
new individuals of Brisbane have been correctly classified of divergence between and within populations can be
into the Brisbane cluster using either PAUP or SOM. attributed to other things. First, biological characteristics
The new tree, obtained by a completely new computation of DBM may be involved. In tropical areas, there may be
of distances with PAUP, was not different from global more than 20 generations a year. This results in an increase
arrangement of population clusters from the previous tree. in appearance of mutations and therefore increases diver-
gence between individuals within populations. Secondly,
massive uses of insecticides create bottlenecks in popula-
4. Discussion tions increasing divergence between populations by select-
ing different haplotypes. All chemical contaminants and
4.1. Origin of genetic diversity insecticides are known for their high mutagen power, this
also increases the number of mutations in resistant individ-
Most studies of population genetic characterisation on uals. ISSR markers are mainly constituted of non-codant
DBM have used allozymes (Caprio and Tabashnik, 1992; DNA that preserves all mutations. In the long term, these
Kim et al., 1999), RAPDs (Heckel et al., 1995), mitochon- successive increases and massive disappearances of haplo-
drial gene encoding cytochrome oxidase I (COI) (Chang types, occurs independently in each population, and have
et al., 1997; Pichon, 2004), enzyme electrophoresis (Arvani- a negative effect on the conservation of phylogenetic infor-
takis et al., 2002; Pichon et al., 2002, 2006), oviposition mation increasing the population genetic differentiation.
behaviour (Arvanitakis et al., 2002), morphology (Chacko Such effects are increased if populations are separated in
and Narayanasamy, 2002) and microsatellites (Endersby time without sufficient genetic flow.
et al., 2002, 2005, 2006) with mixed results. The best results DBM shows an overall observed polymorphism of
have been found with highly variable markers, such as 100%. Similar to that of Pieris rapae (Hundsdoerfer and
RAPDs, microsatellites and enzymes despite the fact that Wink, 2005). It seems that crop pests, where man has an
morphological characters on Indian populations (Chacko important influence in their distributions are loosing their
and Narayanasamy, 2002) and behavioural characters on geographical population genetic structure. Nonetheless,
populations of Benin (Arvanitakis et al., 2002) have also another type of population genetic structure is generated
given good results in terms of population discrimination. and accessible for analysis using ISSR markers. ISSRs
Despite a very high variability at the intra-population can be used as tools to evaluate human influence on natural
level, ISSR seems to permit differentiation of each of the pest populations and their level of dispersal.
studied populations very easily. All populations had high
coefficients of genetic differentiation (Gst). That was not
surprising given the large scale covered. Nevertheless, the 4.2. Gene flow and long range migrations
resolution of ISSR patterns allowed genetic differentiation
(Gst = 0.150) of Australian populations separated by only Although massive annual DBM migration has been
620 km (distance between Melbourne and Sydney), at a described in several parts of the world (Smith and Sears,

157
 O. Roux et al. / Molecular Phylogenetics and Evolution 43 (2007) 240–250 247

MELBOURNE HONG KONG

OKAYAMA LE CARBET

1
TASHKENT BRASILIA

IASI
SYDNEY

BEAVERLODGE

4 MONTPELLIER
SEIBERSDORF

PITON
HYACINTHE GENEVA
BRISBANE

COTONOU

VIENTIANE

EL FAYOUN PRETORIA
ADELAIDE
5-6 7

b
Tashkent
a
Iasii Seibersdorf
rd r
E
El Fayoun
a

Vientiane
Sydney
n
Piton Hyacinthe
Brisbane

Le Carbet

Kong Kong Geneva

Adelaide

Brasilia
Cotonou
Pretoria

Beaverlodge
Montpellier
Melbourne

Okayama

Fig. 3. Classification of 539 DBM individuals from 19 populations using Self-Organising Map (SOM) method. (a) The patterned SOM map. Based on
similarities between ISSR bands, the individuals are ordinated into the map. Similar individuals are in the same or neighbour neurons. The 19 clusters
issued from b are differentiated by bold lines and gradient of grey colour. Individuals misclassified are numbered 1–7: with individual 1 from Adelaide, 2
from Vientiane, 3 and 6 from Piton Hyacinthe, 4 and 5 from Iasi, and 7 from Beaverlodge. (b) Hierarchical classification of the SOM map. The unrooted
tree has been simplified in the figure and only the 19 clusters are represented.

158
248 O. Roux et al. / Molecular Phylogenetics and Evolution 43 (2007) 240–250

1982; Chu, 1986; Honda et al., 1992; Chapman et al., been introduced in the model, it would be classified in a
2002), very long range migrations (at the scale of our sam- neuron of the map to the neuron closest to it, based on
pling) are probably irregular or anecdotal elsewhere. In our band similarity. If the individual came from a new popula-
study, despite values greater than one successful migrant tion however, the model nonetheless is obliged to classify it.
per generation, the gene flow (Nm) seems to be insufficient This is the drawback of this method. It can however be
to effectively homogenise populations as Slatkin (1987) pre- avoid using a recent algorithm, the Evolving Self-Organiz-
dicted, probably because of interactions between character- ing Map, which creates a new neuron and evolves the map
istics of DBM and ISSR fragments described above. Very accordingly (Deng and Kasabov, 2003).
limited migrations have been described in Hawaiians pop- Foody (1999) compared SOM with three other classifica-
ulations of DBM that were separated by less than ten kilo- tion methods (K-means, Hierarchical clustering and fuzzy
metres (Tabashnik et al., 1987). Mo et al. (2003) have also c-means) and with ordination method (Principal compo-
shown that dispersal of DBM rarely occurs beyond 200 nent analysis (PCA)) in community data analysis. Giraudel
meters in healthy cabbage patch exploitation. When grow- and Lek (2001) compared SOM with Polar ordination,
ing conditions are favourable, moths do not migrate. PCA, correspondence analysis and multidimensional scal-
Moreover, in areas free of massive annual migration com- ing for ecological community ordination. Finally Brosse
mercial and cultural cabbage movement probably has more et al. (2001) used SOM and PCA to study fish assemblages.
impact on DBM genetic structure than natural gene flows. They all showed that SOM can be successfully applied to
The absence of correlation between genetic and geographic complex data and constitute a useful alternative to common
distances found here is not unusual in crop pest popula- multivariate statistical analysis. In this paper, SOM is for
tions (Chang et al., 1997) and has already been observed the first time compared to a genetic classification method.
in DBM (Arvanitakis et al., 2002; Pichon et al., 2006). The worst misclassification individual rate was only of
Our study suggests no phylogenetic inference occurred. 1.3%. Classical distances and SOM analyses gave similar
results for population discrimination but produced different
4.3. Classification methods clusters between populations. This difference comes proba-
bly from their respective algorithms. With the heuristic
The observed branch length on the dendrogram from method, individuals are compared between them, while in
the classical distance analysis shows that distances between the SOM, they are compared to virtual individuals which
individuals are longer than between populations (Fig. 2a). are modified progressively during the training.
This reflects an acceleration of diversification of non-cod-
ing DNA in recent times, without exchanges between pop- 5. Conclusion
ulations. Greater distances between individuals over
populations probably reflect the massive and recurrent With this study, we present a complete decision-making
use of insecticides. Such evolutionary change is of concern tool. The ISSR-PCR technique combines the two qualities
because it favours insecticide resistance. It would not occur required for routine analyses i.e. low cost and fast process-
with the use of a biological agent to control the pest. ing with minimum equipment requirements. This study has
The discrimination power of this analysis was excellent shown that the population genetic of DBM (described by
with only one individual misclassified. Nevertheless, the ISSR bands) can be mapped by the SOM artificial neural
computation time was very long with more than 20 h on networks method; faster than other methods. The high var-
a modern desktop computer. The validation test of the iability obtained with ISSR markers can also allows assess-
method required are computation of results and gave a ment at low geographic scales.
new tree for distance analysis with PAUP. We think that
the insertion of a completely new population will probably Acknowledgment
produce a new cluster.
After the iterative learning phase in the SOM analysis, The authors thank Dr. Andy Sheppard CSIRO for Eng-
each of the 539 DBM individuals was associated with an lish correction of the manuscript.
output neuron. Moreover, mapping individuals according
to their ISSR band similarities into the SOM map revealed References
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DISCUSSION GENERALE & PERSPECTIVES
 Discussion générale et perspectives

Les questions de recherche développées dans le présent travail peuvent être réunies
sous une même thématique, celle des relations tritrophiques en milieu tropical, avec comme
modèle le chou - la teigne des Brassicacées (Plutella xylostella) - et les auxiliaires (en
particulier Oomyzus sokolowskii et Cotesia vestalis) dans deux pays d’Afrique de l’Ouest, le
Sénégal et le Bénin.
L’intensification de la culture du chou en zone tropicale, plus particulièrement en
Afrique, n'est pas très ancienne. Dans les années 1970, cette espèce était cultivée mais
souffrait durant sa culture des conditions climatiques tropicales (fortes chaleurs et forte
humidité), non adaptées à sa croissance, les variétés utilisées à l'époque étant des cultivars
européens. Depuis une trentaine d'années, l'arrivée sur le marché de cultivars "tropicalisés"
d'origine japonaise a fait prendre un essor important à cette espèce légumière, à tel point
qu'elle entre maintenant de façon courante dans l'alimentation locale des familles de ces deux
pays. Il faut savoir également que les africains, particulièrement les béninois, sont de grands
consommateurs de "légumes feuilles", dont le chou fait partie et dont les pommes récoltées se
conservent très bien lors des transports routiers effectués sur les pistes, contrairement à
d'autres légumes plus fragiles. Les surfaces plantées en choux ont donc augmenté et il n'est
pas rare actuellement de voir chez les producteurs des parcelles cultivées dépassant 5 000 m2
et cela tout au long de l'année.
Malheureusement, cette multiplication des surfaces cultivées de façon continue tout au
long de l'année (présence au même moment de choux à tous les stades de la culture) offre à
son principal ravageur, le lépidoptère défoliateur Plutella xylostella, l'occasion de se
multiplier de façon exponentielle, dérégulant ainsi certains agrosystèmes précédemment en
équilibre.
Au niveau mondial, pour tenter de contrôler les populations de chenilles de ce
ravageur, les cultivars de chou pommé ont fait l'objet d'une sélection variétale basée sur des
changements de la structure des cires épicuticulaires dont l'effet recherché était de diminuer
l’appétence du chou pour les chenilles de P. xylostella. Cependant, ces variétés n’ont pas été
exploitées car les modifications induites favorisent d’autres ravageurs et donnent un aspect
brillant aux feuilles (glossy leaves), ce qui est peu apprécié des consommateurs.
La teigne du chou est recensée en plus ou moins grand nombre sur tous les continents
et à quasiment toutes les latitudes, partout où il y a des cultures de Brassicacées (Shelton
2004). Dans les conditions tropicales, les populations de P. xylostella sont très importantes en
nombre d'individus et de ce fait difficilement contrôlables. En effet, les températures élevées
et l’humidité de ces zones favorisent la multiplication des populations qui effectuent entre 20

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et 24 générations par an suivant les endroits, alors qu'elles n'en ont que trois en zones
tempérés.
Du fait de sa répartition mondiale, les populations de ce ravageur subissent des
pressions locales de sélection dépendant du climat, des ennemis naturels présents, des
systèmes de culture des agriculteurs, etc.
La question fondamentale qui se pose est la suivante : ces populations sont-elles
identiques au niveau de leurs "performances" en tant que ravageur ? Il est impossible de
mettre au point une lutte "générique" dans tous les pays où sévit la teigne et les différentes
formes de luttes mises en place pour contrôler les populations de chenilles ne sont pas
efficaces partout : (1) la lutte chimique intensive entraîne dans de nombreux pays l'apparition
de populations résistantes. Elle est cependant très employée et encore efficace dans certains
pays, du fait de l'apparition des nouvelles molécules mises sur le marché ; (2) la lutte variétale
est inefficace ; (3) la lutte biologique de conservation semble être actuellement la plus
employée au niveau mondial, mais sa réussite dépend de l'efficacité des espèces locales
d’auxiliaires présentes.
Des études en laboratoire (marqueurs isozymes) ont montré qu'au niveau mondial les
populations de P. xylostella semblent être structurées génétiquement en plusieurs groupes
homogènes, comme c’est le cas pour l'Australie et le Japon, au contraire des autres
populations originaires d'Europe, d'Afrique et du continent américain qui sont nettement
différenciées. En analysant ces groupes, nous nous sommes aperçus que toutes les populations
provenant d'une localité tropicale (zone de basses latitudes comprises entre 0 et 23°) étaient
regroupées dans un même ensemble, alors que celles natives de zones non tropicales (zone de
moyennes et hautes latitudes entre 24 et 63°) constituaient un autre groupe bien séparé. Des
groupes structurés ont été mis en évidence de la même manière lors de notre étude avec les
ISSR. Chacun de ces deux marqueurs a montré de fortes divergences entre les populations
étudiées, permettant ainsi de les différencier. D’un marqueur à l’autre, les distances
génétiques calculées entre les populations ne sont pas équivalentes, probablement en raison de
l’évolution indépendante de ces deux marqueurs mais surtout parce que les isozymes,
considérés comme marqueurs neutres, peuvent quand même être soumis à une pression de
sélection alors que les ISSR ne le sont pas. Dans les deux cas, l’information apportée par ces
marqueurs n’est pas corrélée avec les distances géographiques, principalement en raison des
caractéristiques de chacun d’eux.
Récemment, suite à des identifications d'individus de P. xylostella récoltés sur des
choux, des chercheurs australiens utilisant le "Barcoding" ont identifié des individus d'une

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autre espèce, différente de P. xylostella. Il s’est avéré que les génitalias des mâles et des
femelles n'étaient pas identiques à celles de P. xylostella, alors que la couleur et la
morphométrie étaient rigoureusement semblables. Ils ont décrit cette nouvelle espèce comme
P. australiana (Landry & Hebert 2013). Cette information est capitale car P. xylostella est
peut-être constitué d’un complexe d’espèces ou de biotypes équivalent au complexe de la
"Mouche blanche" Bemisia tabaci (Gennadius) (Hemiptera : Aleyrodidae), non encore mises
en évidence, ce qui pourrait expliquer les différences observées dans le comportement et le
contrôle du ravageur.
Au delà de ces différences observées au niveau génétique entre les populations, avec la
possibilité d’espèces cryptiques en mélange, les essais au laboratoire ont montré de grandes
différences dans le nombre d'œufs pondus par les femelles de P. xylostella et dans la stratégie
de ponte entre les populations originaires de cinq localités du Bénin (Arvanitakis et al. 2004).
Cependant, il n’y a pas de corrélation entre les différences génétiques et les différences
biologiques. Cela nous conforte dans l’idée que les populations de la teigne ne sont pas
identiques entre elles et que leur contrôle ne peut se faire de façon générique, mais de manière
spécifique au niveau de chaque population présente dans son environnement naturel.
Nos résultats ont montré l'importance des auxiliaires entomophages qui, sans être
d'une efficacité totale dans le contrôle des populations de la teigne, excepté en Afrique du Sud
(Kfir 2011), permettent cependant de réduire les populations du ravageur. La lutte biologique
par conservation montre là toute sa nécessité et devient donc d'une grande utilité.
L’efficacité des parasitoïdes dépend également des pressions de sélection par leur
environnement. Par exemple, O. sokolowskii, endoparasite grégaire larvo-nymphal, peut
conduire à plus de 80% de parasitisme en conditions de laboratoire (conditions optimum),
alors que ce taux ne dépasse pas 10% dans les conditions naturelles au Sénégal (Sow et al.
2013). Le pourcentage de parasitisme par cette espèce est de l'ordre de 40 à 50 % en Asie du
Sud-Est (Srinivasan & Moorthy 1992).
Cette différence aperçue dans le pourcentage de parasitisme peut être due à plusieurs
causes : (1) les conditions de laboratoire sont très favorables à une bonne croissance des
populations des parasitoïdes (pas de recherche de l’hôte, pas de concurrence pour la ressource,
absence d'antagonistes, conditions climatiques optimales), ce qui n'est pas le cas en milieu
naturel ; (2) comme pour l'hôte, les capacités biologiques peuvent être différentes suivant leur
origine ; (3) ils peuvent être porteurs de bactéries endosymbiotiques qui pourraient perturber
leur efficacité.

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En effet, de nombreuses espèces d’Hyménoptères sont naturellement infectées par des

bactéries endosymbiotiques du genre Wolbachia (Werren et al. 1995). Ces endosymbiotes
manipulent la reproduction de leurs hôtes, soit en biaisant la sex-ratio, soit en empêchant
certains types de croisements, pouvant ainsi induire jusqu'à une différenciation des
populations (Bordenstein et al. 2001). Ceci pourrait entrainer une baisse d’efficacité
parasitaire chez les parasitoïdes infectés. C'est effectivement le cas de notre population d'O.
sokolowskii en provenance du Sénégal qui était infectée par Wolbachia.
L'espèce C. vestalis est un endoparasite larvaire solitaire qui possède pratiquement la
même répartition géographique que P. xylostella, ce qui en fait le principal ennemi naturel de
ce ravageur. Dans ce cas encore, des différences importantes apparaissent dans son potentiel
d'efficacité envers les populations de chenilles de la teigne. Au cours d'essais effectués au
laboratoire sur sa réponse fonctionnelle, des femelles de ce parasitoïde provenant du Bénin
pouvaient parasiter en 24 heures 80 chenilles sur 120 présentées, alors que des femelles
originaires de Martinique n'en parasitaient que 40. Nous noterons ici que les femelles de
l'hyménoptère issues de la population des Antilles étaient infectées par Wolbackia (Rincon et
al. 2006) et de ce fait elles étaient moins performantes par rapport à celles du Bénin qui
étaient saines.
Au Sénégal, en condition de plein champ, C. vestalis est peu présente, mis à part en
période d’hivernage où les températures sont assez élevées (30°C en moyenne). Son taux de
parasitisme n'excède cependant pas 5% (Sow et al. 2013). Par contre cette espèce peut aboutir
à près de 90% de parasitisme dans la zone maritime du Bénin où, malgré ce taux extrêmement
élevé, elle ne contrôle pas les populations de chenilles de P. xylostella (Arvanitakis et al.
2013, soumis à BioControl). Comme pour son hôte P. xylostella et le parasitoïde O.
sokolowskii, les populations de C. vestalis sont hétérogènes au niveau génétique. Des études
effectuées avec des marqueurs isozymes sur des individus de populations d'origines
géographiques différentes (Ile de la Réunion, Taïwan, Bénin, Afrique du Sud et Martinique)
ont montré un éloignement génétique entre les populations, ce qui pourrait influencer
l'efficacité des différentes femelles sur leurs hôtes respectifs. Nous noterons également que le
climat "doux" de la région de Dakar n'est peut être pas très favorable à C. vestalis, celui-ci
préférant les environnements chauds et humides présents dans la zone maritime de Cotonou,
où il est fortement présent sur les chenilles de P. xylostella.
Nous noterons également que les choux cultivés dans la zone maritime de Cotonou
sont fortement infestés par P. xylostella. Il n'est pas rare de dénombrer plus de 100 chenilles
par pied de chou. Le climat ne subissant que très peu de différences au cours de l'année tant

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 Discussion générale et perspectives

au niveau de la température que de l'humidité, celui-ci est très favorable à la croissance des
chenilles de P. xylostella. Au Sénégal les différences de températures et d'humidité dues aux
deux saisons (sèche et humide) ne sont pas très favorables au ravageur, ce qui entraîne des
populations de chenilles beaucoup moins importantes qu'au Bénin.
Une co-évolution pourrait être survenue au Bénin où la sélection naturelle aurait
favorisé les femelles de C. vestalis les plus performantes et éliminé les moins efficaces. La
présence de Wolbachia dans les adultes, qui peut influencer sur son aptitude au parasitisme,
ne peut être mise en cause puisque les populations du Sénégal et du Bénin ne sont pas
infectées.
En zones tropicales, le comportement imprévisible des infestations de ce ravageur au
niveau des parcelles cultivées (Zalucki & Furlong 2011 ; Wei et al. 2013) et surtout le
système de culture des producteurs, qui favorisent la croissance des populations de la teigne
dans les parcelles, sont des freins au maintien à un niveau faible des populations de P.
xylostella. En effet, les agriculteurs laissent les pieds de choux en place lors de la récolte des
pommes, ce qui favorise le redémarrage des repousses favorables au développement des
chenilles. Ils se désintéressent également des foyers de chenilles présents sur le feuillage des
planches cultivées en navet (ils ne vendent que les racines), qui sont souvent proches des
planches de choux.
On connaît les risques pour la santé humaine et l'environnement découlant des
applications anarchiques d'insecticides utilisés par des acteurs souvent mal informés, voire
ignorants des effets néfastes de ces pesticides (Furlong et al. 2012). La mise en place de
programmes de libération d'auxiliaires biologiques (entomophages, champignons, virus etc.)
est très coûteuse et nécessite une compétente technique importante qui est malheureusement
absente chez ces producteurs. La solution à ces problèmes serait : (1) de favoriser la lutte
biologique par conservation de la faune utile, en remplaçant par exemple les applications
d'insecticides de synthèse par des formulations à base de produits naturels (huile de "neem")
ou biologiques (B.thuringiensis) ; (2) d'effectuer des recherches sur les effets que pourraient
apporter l'utilisation des plantes compagnes en association avec les espèces maraîchères
cultivées qui possèdent un effet piège (moutarde chinoise, Pak choï…), un effet répulsif
(oignon, ail, basilic…), ou neutre (salade, carotte…), mais qui pourraient perturber les
femelles de P. xylostella à la recherche de sites de ponte (Sheehan 1986). Ces espèces
cultivées pourraient également apporter un supplément de revenus et la mise en place de cette
stratégie serait également en phase avec les compétences horticoles que possèdent les
producteurs.

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 Discussion générale et perspectives

En effet, la majorité des agriculteurs sénégalais sont illettrés et ils connaissent la

technique de la culture des légumes par leurs ascendants qui eux-mêmes la connaissent de
leurs parents, voire grands-parents. Ils n'ont aucune connaissance phytopharmaceutique et
appliquent sur leurs cultures tous les produits disponibles sur le marché, en particulier ceux
utilisés en cultures cotonnières et qui sont dangereux pour les utilisateurs et les
consommateurs. La pollution de la nappe phréatique est également concernée par ces
méthodes, d’autant plus, que l'eau de cette nappe est régulièrement réintroduite lors des
arrosages journaliers des cultures. Ils ne connaissent pas l'existence de la "faune auxiliaire" et
pratiquent des cultures associées non pas par souci « agroécologique » mais par manque
d'espace au niveau de leurs planches. La mise en place de programmes basés sur l'association
d'espèces végétales cultivées et non sauvages, car entrant en concurrence avec elles au niveau
de l'eau, et qui perturberaient les femelles du ravageur à la recherche de sites de ponte,
seraient facilement acceptée par les agriculteurs, la technique de culture étant en rapport avec
leurs compétences.
Comme nous avons pu le constater au Sénégal et au Bénin, la teigne du chou n’est pas
contrôlée par ses ennemis naturels. Bien que ces deux pays se situent en zone tropicale, le
nombre d’espèces de parasitoïdes et les taux de parasitisme sont variables. En conditions de
laboratoire, l’efficacité parasitaire des deux espèces étudiées, O. sokolowskii et C. vestalis,
vis-à-vis de leur hôte n’est pas remise en cause puisqu’elles sont performantes. On peut donc
penser que le problème peut venir, d’une part, des populations du ravageur puisque nous
avons montré qu’elles étaient différentes et structurées génétiquement à l’échelle mondiale et,
d’autre part, des conditions climatiques qui varient d’un pays à l’autre.
J’ai eu l’occasion de faire une expertise en Bretagne (France) auprès de producteurs
qui produisaient des choux porte-graines sous serre. La teigne du chou est présente en
Bretagne mais n’est pas considérée comme un ravageur à cause du climat. Les adultes
provenant des champs avoisinants rentrent dans les serres et y trouvent des conditions plus
clémentes (températures élevées) et donc favorables à leur développement. Les producteurs
étaient infestés par la teigne malgré un vide sanitaire de deux mois entre deux périodes de
cultures et l’application de 23 traitements chimiques sur une saison. Cet exemple permet de
voir que si on « tropicalise » artificiellement un agrosystème, il devient favorable au
développement de cette espèce qui devient alors un ravageur.
Il peut être intéressant d’essayer de modifier les agrosystèmes par des aménagements à
l’échelle de la parcelle, voire du paysage. On s’est rendu compte par exemple que les choux
sont moins attaqués quand on les cultive en association avec de la carotte ou de l’oignon, ou

170
 Discussion générale et perspectives

bien quand ils sont dans des parcelles plus ombragées. Dans cet objectif d’action, une
approche agroécologique est peut-être l’une des solutions pour contrôler la teigne du chou en
régions tropicales.

171
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ANNEXES
 Interactions entre Plutella xylostella (L.), Lepidoptera :
Plutellidae et la température, la plante hôte et les parasitoïdes
Sow Gallo1 Diarra Karamoko1
Arvanitakis Laurence2
Bordat Dominique2
1 Equipe 2PIAH, Département
de Biologie animale, FST / UCAD,
U fait de sa capacité de multiplication (> 20 générations/an), la lutte biologique Dakar, Sénégal.
2 UR-HortSys, TA B-103, Campus
International de Baillarguet, CIRAD,
contre P. xylostella n'est pas efficace en zones tropicales. Nous avons donc 34398 Montpellier Cedex, France

priorisé une approche basée sur la recherche d'interactions existantes entre [email protected]

le ravageur et son environnement (température, plante hôte et parasitoïdes).

M a teriels et méthodes
Cette étude est réalisée à Malika (Dakar, Sénégal). La parcelle d'échantillonnage
comporte 200 choux non traités. Tous les dix jours, 10 plantes prélevées au hasard sont
décortiquées. Les chenilles de P. xylostella récupérées, sont maintenues en élevage
jusqu'à l'apparition de l'adulte ou d'un parasitoïde. Avant chaque prélèvement, le
diamètre du chou est mesuré. La température est relevée tous les jours.

R ésultats Figure 1. Influence de la température sur

La température n'influe pas sur la dynamique des populations du ravageur. Les les populations de P. xylostella (r = - 0,294 ns).
populations semblent même augmenter quand la température chute (figure 1).
Il y a une forte corrélation entre les chenilles et le diamètre de la plante hôte (figure 2).
Il y a également une forte corrélation entre les stades jeunes des chenilles (L2 + L3) et l'age de la plante hôte.
Le nombre de jeunes chenilles diminue lorsque la plante vieillie et les stades plus âgés
(L4 + Nymphes) sont plus nombreux quand elle approche de la maturation (figure 3).
Trois espèces de parasitoïdes sont présentes sur les chenilles de P. xylostella. Cotesia plutellae (Kurdjumov) et
Apanteles litae (Dixon), deux Braconidae endo-parasites larvaires et Oomyzus sokolowskii (Kurdjumov), Eulophidae,
parasitoïde larvo-nymphal.
Le pourcentage de parasitisme naturel est faible au cours des trois cultures consécutives, respectivement 5,2 %
pour la première, 34,9 % pour la seconde et 1,4 % pour la dernière (tableau 1). Ce pourcentage de parasitisme
est principalement dû à O. sokolowskii, puis à C. plutellae. A. litae n'est que faiblement représenté (figure 4).

Figure 2. Interactions entre le nombre de chenilles de

P. xylostella et le diamètre de la plante hôte (r = 0,661 ; P < 0,01).

Tableau 1. Nombre d'adultes de parasitoïdes et pourcentage de parasitisme

obtenus sur les populations de chenilles de P. xylostella.
Cultures Nombre de Nombre de Nombre d' * Nombre d' Nombre de %
consécutives P. xylostella C. plutellae A. litae O. sokolowskii parasitoïdes Parasitisme
25/06-24/08/08 591 16 7 8 31 5,2

15/09-24/11/08 166 10 1 47 58 34,9

15/12-13/02/09 623 1 1 7 9 1,4

* Nombre de nymphes de P. xylostella parasitées (en moyenne 8 adultes de O. sokolowskii / nymphe)

C. plutellae = Cotesia plutellae (Kurdjumov) ; A. litae = Apanteles litae (Dixon) ;
O. sokolowskii = Oomyzus sokolowskii (Kurdjumov)

C o nclusion
Conception : CIRAD, Martine Duportal, Avril 2010 — Photos : © Dominique Bordat
Figure 3. Interactions entre le nombre de chenilles de P. xylostella
et l'age de la plante hôte (r = - 0,561 ; P < 0,01 pour
les L2 + L3, r = - 0,561 ; P < 0,01 pour les L4 + Nymphes).

Contrairement à de nombreuses zones tropicales, les populations de P. xylostella n'augmentent pas dès que la température s'élève. Ce fait est
probablement dus au micro climat de Dakar (20-25°C), à l'exception des 4 mois d'hivernage où la température est de 30°C. L'espèce se serait adaptée
à ces températures. Il faut noter que les surfaces cultivées en chou augmentent considérablement dès que la saison sèche arrive,
ce qui favorise l'augmentation des populations du ravageur.
Pour les femelles de P. xylostella, la taille du plant semble favoriser la ponte, plus le diamètre du plant augmente plus le nombre
de chenilles est important. L'attractivité des choux pour la femelle est due au ratio entre la taille du plant et les
glucosinates produits par celui-ci, qui sont également favorables au développement des chenilles. Dès que le
chou pomme, la production de glucosinate diminue et les femelles vont à la recherche d'autres plantes plus
jeunes pour y pondre.
Le parasitisme naturel est faible (1,4 % à 34,9 %). La température ambiante de la zone de Dakar (20-25°C) favorise la
multiplication des populations d'O. sokolowskii, mais elle pénalise celles de C. plutellae qui préfèrent des températures
plus élevées. On trouve cette espèce en plus grand nombre en hivernage (30°C), où les populations d'O. sokolowskii sont moins nombreuses. Le climat
de la zone de Dakar n'ait pas favorable à la multiplication d'A. litae, qui est plutôt présent dans les zones sahéliennes.

Conférence Internationale Francophone d'Entomologie (CIFE), du 5 au 10/07 à Louvain la neuve (Belgique)

www.cirad.fr
Several life traits history of Oomyzus sokolowskii
parasitoid of Diamondback moth.
HE species Oomyzus sokolowskii (Kurdjumov), a major
G. SOW1, L. ARVANITAKIS2, S. NIASSY1,
parasitoid of Diamondback moth (DBM) Plutella xylostella (L.)
K. D IARRA1, D. BORDAT2
pest of Brassicaceae is a potential biological control agent against
this species. These species are gregarious and cosmopolitan like its 1
Equipe production et protection intégrées
en Agro-écosystèmes horticoles, Département
host (Fitton and Walker, 1992). Female in laying position de Biologie animale, Faculté des Sciences et Techniques,
Université Cheikh Anta Diop de Dakar (UCAD), BP 5005,
The aim of this work is to study under laboratory conditions some life on Plutella xylostella
larva Dakar, SENEGAL
history traits of this Hymenoptera species. The life-traits knowledge is very 2
Laboratoire de Biodiversité des agrosystèmes horticoles,
TAB/L, Campus international de Baillarguet, CIRAD,
important to build entomophagous programs to control populations DBM in 34398 Montpellier Cedex 5, FRANCE.

cabbage crops field farmers in tropical areas. contact: [email protected]

Material and Methods

■■ The parasitoid population was obtained from parasitized pupae of DBM collected in cabbage crop
(Brassica oleracea var. capitata) in Fass Boye, in the "Niayes" area, situated in North West of Senegal. The population
of DBM is native to the same locality.
O. sokolowskii
■■ The rearing of the host and its parasitoid were conducted in climate rooms with the following conditions: 25 °C adults emergence.
temperature, 60% relative humidity and 12L/12D photoperiod.
■■ Traits such as development stage cycle, reproductive mode, host age preference, foraging behaviour of the female
were assessed. All tests performed in this study were realized in the laboratory of Entomology for International
Cooperation in Agronomic Research for Development Center (CIRAD) in Montpellier (France).
■■ All data were analyzed with the software StatView 4.55.

Table 1. Developmental stage of parasitism

and O. sokolowskii biological cycle.

Results J0
J+1
J+2
Laying inside L4 larvae
Eggs inside L4 or pupae
Eggs inside pupae
■■ The duration of the larval stage is between 4 and 7 days. The pupal stage during 7 days. In our J+3
J+4
Hatching
Young larvae, visible holes inside the pupae
study conditions, the development time from egg to adult is 15 days. (Table 1) J+5 Idem
J+6 Ponctued larvae visible trough the pupae
■■ Parasitism rate was significantly different between unmated and mated females (t = 6.391, J+7 Prepupae
df = 6, P = 0.0007).The mated females produced normal sexual offspring (male and female) while J+8 White pupae
unmated females have produced only males. (Table 2) J+9 Idem
J+10 Pupae eyes begin red
■■ The parasitism rate varies significantly with age of the host (F = 26.23, df = 4.16, P <0.0001). J+11 All pupae eyes are red
J+12 Pupae body become black
This rate is significantly higher at the L4 larval stages. (Table 3) J+13 All pupae body are black
J+14 Idem
■■ The parasitism rate was significantly different in the three laying-boxes (F = 15.87, df = 2.18, J+15 Adults emergence
P <0.0001). Male offspring number was significantly different among the three laying-boxes
(F = 5.87, df = 2.18, P = 0.008). Female offspring number was significantly different among the three Table 3. Host age preference in O. sokolowskii
laying-boxes (F = 10, df = 2.18, P = 0.001). The offspring development time was significantly females on immature DBM stage.
different between the laying-boxes (F = 9.01, df = 2.18, P = 0.004). (Table 4) Host age Parasitism % Min / Max
2nd 39,9 ± 7,6 b 23,3 / 63,3
3rd 54,7 ± 8,7 b 23,3 / 73,3
Table 2. Offspring productivity, parasitism percentage and sex ratio (% female) between
4th 75,9 ± 2,4 c 70,0 / 83,3
mated and unmated O. sokolowskii female.
Prepupa 15,3 ± 5,9 a 0,0 / 36,7
Males Females Total progeny Parasitism (%) Sex ratio Pupa 0,0 ± 0,0 a 0/0
Mated female 1.80 ± 0.40 a 8.40 ± 0.70 a 10.20 ± 1.00 a 45.60 ± 3.90 a 83.00 ± 2.00 a Values are mean ± SE. Numbers in columns
Unmated female 10.30 ± 0.90 b 0.00 ± 0.00 b 10.30 ± 0.90 a 12.20 ± 0.10 b 0.00 ± 0.00 b followed by the same letter are not
significantly different at P = 0.05 (ANOVA;
Values are mean ± SE. Numbers in columns followed by the same letter are not significantly different (P > 0.05). Fisher). Min = Minimum; Max= Maximum.

Table 4. Laying box volume effect on the parasitism percentage and the O. sokolowskii female production.
Laying box Parasitism % Females laid Males Females Total adults Cycles (days) Sex-ratio
3 (A) 30,0 ± 15,3 b 3 0,7 ± 0,3 a 5,9 ± 3,0 a 6,6 ± 3,4 a 14,3 ± 0,3 a 89,5 ± 0,8 a
Conception : CIRAD, Martine Duportal, May 2012 — © photos Cirad

7 (B) 85,5 ± 7,6 c 10 2,1 ± 0,6 b 13,6 ± 1,7 b 15,7 ± 2,0 b 15,7 ± 0,3 a 86,8 ± 2,3 a
40 (C) 5,0 ± 5,0 a 1 0,2 ± 0,1 a 0,8 ± 0,5 a 1,0 ± 0,6 a 18,0 ± 0,0 b 80,0 ± 0,0 a

Conclusion Values are mean ± SE. Numbers in columns followed by the same letter are not significantly different at P = 0.05 (ANOVA; Fisher).

■■ The O. sokolowskii life cycle lasted 15 days. The parasitism rate is significantly different between
mated and unmated females which imply that mating stimulates the behaviour of parasitism. Females can
parasitize all larval stages including prepupae of DBM. However, the parasitism rate was higher in the
fourth larval stages. The host-seeking behaviour is influenced by volume.
■■ The results presented in this study provide valuable information on some O. sokolowskii life traits
history, a major natural enemies of DBM, pest of Brassicaceae. This information can help a better
understanding on the biology of this species and allow more efficient use of this parasitoid in the
programs of population management of DBM in the release of entomophagous.

Colloque national des Entomophagistes, du 23 au 25 mai 2012 à Montpellier (France)