Behavourial Responses of the Endoparasitoid Apoanagyrus Lopezi to Odours of the Host and Host's Cassava Plants Entomologica Experimentalis Et Applicata, 90, 215-220

Short communication Behavioural responses of the endoparasitoid Apoanagyrus lopezi to odours of the host and host's cassava plants

R. Souissi' & B. Le Rii2 1Laboratoire d'Ecobiologie des Insectes Parasitoides, Universite de Rennes I, Campus de Beaulieu, 35042 Rennes Cedex, France; 2Laboratoire d'Entomologie, ORSTOM, 1286 Pointe Noire, Congo

Accepted: October 27, 1998

Key words: Apoanagyrus lopezi, Phenacoccus manihoti, cassava, resistance, tritrophic interaction, behaviour, olfactometer

Introduction We tested the response of females of A. lopezi to volatiles, emanating from four different mealy Apoanagyrus lopezi (De Santis) (Hymenoptera: En bug host plants and from mealybugs reared on the cyrtidae) is an endoparasitoid indigenous to South four plants. Three of the host plants studied be America, which was introduced to sub-Saharan Africa long to the Euphorbiaceae: two cassava (M. escu in 1981 as a biological control agent against the cas lenta) varieties (Incoza and MM79) and a hybrid of sava mealybug, Phenacoccus manihoti Matile-Ferrero M. esculenta and M. glaziovii Muel. Arg. (the Faux (Homoptera: Pseudococcidae), a pest of cassava caoutchouc (FC». The fourth host plant was Talinum Manihot esculenta Crantz (Euphorbiaceae) (Herren (Talinum triangulare Jack., Portulacaceae). These four & Neuenschwander, 1991). Apoanagyrus lopezi is plants are characterised by different levels of antibi monophagous on P. manihoti, which feeds almost otic and antixenotic resistance towards P. manihoti exclusively on cassava. The mealybug is mainly a (Tertuliano et al., 1993). Laboratory experiments have phloem-feeding insect and reproduces by thelytok demonstrated that these mealybug host plants influ ous parthenogenesis with three nymphal instars (Ca ence differently the development and the reproduction latayud et al., 1994). These cassava-mealybug and of A. lopezi (Souissi & Le Ru, 1997). However, their mealybug-parasitoid systems have been studied exten effect on the locomotor behaviour of the parasitoid is sively, both in the laboratory and in the field (Fabres, still unknown. In this study, the locomotor behaviour 1981; Lohr et al., 1988; Nenon, 1992). Four studies ofA. lopezi was investigated in a tubular olfactometer. have analysed the influence of cassava on the search For each plant, three odour sources were tested: unin ing behaviour of A. lopezi. Olfactometer studies have fested plants, infested plants, and mealybugs reared on demonstrated that female parasitoids are attracted by the four host plants. the odour of infested cassava leaves (Nadel & van Alphen, 1987; van Baaren & Nenon, 1996; Souissi et al., 1998) and spend significantly more time search Materials and methods ing for their host on cassava leaves contaminated with waxy secretions from mealybugs than on uninfested Plants. Plants were obtained from cuttings 20 cm leaves (Langenbach & van Alphen, 1986). There is high, planted in pots with 2/3 oftheir length in soil and not much information on the tritrophic interactions placed in a controlled room at 25 ± 1 "C, 70 ± 3% r.h., between cassava, host and parasitoid and there are and LI2:DI2. They were watered twice weekly. Light no data on the behaviour of A. lopezi in response to intensity was 1700-1800 lux (artificial light source). odours produced by different uninfested or infested Plants 20-30 cm in height (8-10 leaves, about 9-10 cassava cultivars characterised by different levels of weeks old) were used for the experiments. antixenotic resistance towards the mealybug. 216

Table 1. Response of Apoanagyrus /opezi females to c1ean air and J odours from four uninfested plants: Incoza, MM79, Faux-caoutchouc (Fe) and Talinum (X ± SE)a

Plants Odors tested Mean time spent in Mean number of the 6th section (s) section changes

Incoza PA 46.3 ± 5.9 3.4 ± 0.14 GA 49.3 ± 7.2 4.3 ± 0.13 P NS < 0.05

MM79 PA 47.4 ± 6.6 3.0 ± 0.14 GA 50.9 ± 6.6 4.1 ± 0.19 P NS <0.01

Fe PA 41.6 ± 6.5 3.4 ± 0.16 GA 45.6 ± 5.8 4.4±0.19 P NS <0.001

Talinum PA 45.5 ± 5.7 3.7±0.16 GA 48.1 ± 6.6 4.6±0.18 P NS <0.01

aData obtained in pure air (PA) and in odorous air (GA) were cornpared with a Wilcoxon test.

/nsects. Strains of P manihoti and A. lopezi were the air fiow. The olfactometer was connected ta a Y from field populations in Congo maintained since shaped glass tube 1.5 cm in diameter. The base and 1992 and reared in separate controlled insect rearing the two arms of the Y-tube are 5 cm long. Each arm rooms at 25 ± 1°C, 70 ± 3% r.h., and LI2:D12. was connected to a flowmeter and an odour source Four distinct clones of mealybugs reared on the four container which consisted of a glass cylinder, 25 cm different hast plants were used in these experiments. in diameter and 40 cm in height, large enough ta hold Mealybugs at the L3 stage which is the preferred stage a whole potted cassava plant. Parafilm was used ta for parasitism (Lohr et al., 1988), were used. caver the soil ofthe pot, thus isolating it from the plant Apoanagyrus lopezi was reared on mealybugs foliage through which the airstream was blown. With feeding on several varieties of cassava different from air pressure, an air flow was generated through an hu those mentioned above. For the experiments, mum midifier bottle. The humidified airflow was led firstly mies were harvested from the parasitoid culture and through an empty container and secondly through an kept individually in clear gelatine capsules. Vpon odour source container. After that, the clean or odour emergence, female parasitoids were placed singly with fiow was led through one arm of the Y-shaped tube 2 or 3 males for 24 h in clear polystyrene tubes before entering the tubular olfactometer. In aIl experi (15 x 1.5 cm) where honey was available. AIl experi ments the air flow was 20 l/h which was checked with a ments were carried out with naive (no oviposition and fiowmeter. A preliminary experiment showed that the no contact with cassava plants), mated females that locomotor behaviour of females was the same in the were 1 to 3 days old. presence ofan air fiow at the above rate and in still air. For each test, the behaviour of the same female was Olfactometer set-up and bioassay procedure. A observed; firstly for 4 min in the presence of clean tubular olfactometer with a continuous air fiow, similar air and subsequently for 4 min in the presence of air ta the one described by Cortesero et al. (1993) and scented with the odour ta be tested. At the beginning van Baaren & Nénon (1996) was used ta analyse the of the test, the female was placed in the first section of sensitivity of A. lopezi to the volatile chemicals ema the olfactometer (Cl) and a fiow of clean air was in nating l'rom the four plants and from hasts reared on troduced through the opposite end (C6). After making the different plants. The tubular olfactometer, 1.5 cm observations of the female's behaviour in the clean air in diameter, was divided into 6 sections ofequallength fiow, the female was put back into section (Cl) and an (5 cm) labeIled Cl to C6, Cl being the exit section of odorous air flow was subsequently circulated. In order • 217

Table 2. Response ofApoallagyrus lopezi females to clean air and odours from four infestcd plants: Incoza, MM79, Faux-caoutchouc (Fe) and Taliuum (X ± SEla

Plants Odors tested Mean time spent in Mean number of the 6th section (s) section changes

Inoza PA 40.1 ± 5.3 3.4 ± 0.17 OA 48.5 ± 5.4 5.0 ± 0.21 P <0.01 <0.001

MM79 PA 27.7 ± 4.8 3.0 ± 0.15 OA 36.2 ±4.2 4.6 ± 0.17 P <0.01 <0.01

Fe PA 39.4 ± 4.1 3.6 ± 0.17 OA 50.1 ± 6.6 5.1 ±0.18 P <0.01 <0.001

1àlinium PA 36.2 ± 3.1 3.3 ±0.16 OA 47.0 ± 7.2 4.1 ±0.18 P <0.01 <0.001

3Data obtained in pure air (PA) and in odorous air (OA) were compared with a Wilcoxon test.

to describe its locomotor behaviour, two parameters sisted of an empty odour source container, (2) unin were recorded for each female; attraction, expressed fested plant. Plants were approximately 9-10 weeks by the length of time spent in the sixth section of the old (8-10 leaves; 20-30 cm high). They were used as tube, and locomotor activity, expressed by the number a standard clean cassava odour source, (3) the com of section changes. plete mealybug-infested plant. Plants were infested Thirty females were bioassayed for each odour with 20 unparasitized third instar mealybugs placed test. Each female was tested only once, i.e., in both in groups of 5 on four different leaves. Leaves were control and test odours. The attractiveness ofthe tested chosen randomly and test was run 3 days after infes odours was determined for each series of 30 females tation to enhance the damage done by the mealybugs, by comparing the total time spent in section C6 in the (4) a group of20 third instar mealybugs reared on each presence or absence of odour. The locomotor activ of the four different types of plants, isolated from the ity was analysed by comparing the total number of plants 20 min prior to the experiment and placed in visits in aU sections of the olfactometer in the pres cylindrical glass tubes (4 x 1.5 cm). ence or absence of odour. Values obtained in clean For each odour source, 5 to 6 plants of the same air and odorous air were compared using a Wilcoxon type or groups of mealybugs reared on the same plant matched-pairs signed-ranks test. In aU cases Cl = 0.05 were used in experiments. Pive to 6 insects per day was used to determine significant differences. were individuaUy tested in the tubular olfactometer in The olfactometer tube and the Y-shaped tube were the presence ofone plant or one group of hosts. washed with alcohol after testing 5 females. Tests were carried out 2 to 5 h after the beginning of the pho tophase, which corresponds to the most active period Results and discussion of A. lopezi females (van Baaren & Nénon, 1996). The experiments were carried out at 25 ± 1 oC and In our study, the odours of the four uninfested plants 70 ± 5% r.h. in a box painted white, with an artificial did not attract the females of A. lopezi but they stim light source consisting ofa single 35w fluorescent tube ulated their locomotor activity (Table 1). The females placed above the olfactometer. showed an oriented movement towards the source of the odour. However, the time spent in the sixth sec Odour sources. For each of the four plants, four tion of the olfactometer was not significantly different odour sources were tested: (1) clean air, which con- from controls (Table 1). By contrast, van Baaren & 218

Table 3. Response ofApoollagyrus lopezi females to ciean air and odours ..' from P. mallihoti reared on four plants: Incoza, MM79, Faux-caoutchouc (FC) and Talinuffi (X ± SE)"

Plants Odors tested Mean time spent in Mean number of the 6th section (s) section changes

Incoza PA 35.4 ± 6.3 3.8 ± 0.21 OA 48.1 ± 5.3 5.0 ± 0.15 P <0,001 <0.001

MM79 PA 31.8 ± 2.7 3.2 ± 0.16 OA 45.1±5.0 4.6 ± 0.18 P <0.001 <0.001

FC PA 45.1±5.3 2.9 ± 0.14 OA 61.3±9,0 3.8 ± 0.16 P <0.001 <0.01

Talinium PA 51.5 ± 7.8 3.0 ± 0.15 OA 52.2 ± 7.1 3.9±0.19 P NS

"Data obtained in pure air (PA) and in odorous air (OA) were compared with a WiJcoxon test.

Nénon (1996) reported in a tubular olfactometer sim reported by Nadel & van Alphen (1987) and van ilar to ours that A. lopezi females were attracted by Baaren & Nénon (1996), where females of A. lopezi the odour of host-plant (M'Pembé cassava variety) were attracted by infested cassava plants. Damage by alone. This discrepancy might be due to the variety herbivores can increase the emission of plant volatiles of cassava used or to a different experimental pro enormously (Dicke et al., 1990). In our study, the cedure. The composition of volatile blends emitted increased response of A. lopezi to mealybug-infested by plants is mostly plant-determined, and may de plants could be due to an increased production of pend on the species or the variety, and their quality volatiles or to a changed, more attractive volatile is determined by the age and the growing conditions blend. Cassava undergoes physiological changes in re (Takabayashi et aL, 1991), Moreover, it is likely that sponse to mealybug infestation, as shown by a signifi the whole plants, used in our tests, produced larger cant increase in the levels ofthree flavonoid glycosides quantities of volatiles than did isolated plant parts, (Calatayud et aL, 1994) and by a change in the cyanide such as the apex used in the experiments ofvan Baaren content ofleaves, stems and tubers (Ayanru & Sharma, & Nénon (1996). Therefore, with our experimental 1985). Such changes couId result in the emission of set-up, a greater response from the parasitoids should volatiles and explain why A. lopezi (this study) and the have been obtained. Furthermore, the A. lopezi fe related parasitoid Apoanagyrus diversicornis Howard males used in our study had no previous experience (van Alphen et aL, 1990) are attracted by P. manihoti with either mealybugs or mealybug host plants. In the infested cassava plants. These results indicate that the experiments of van Baaren & Nénon (1996) females mealybug infested-cassava plant is a great source of of A. lopezi were given experience by exposure to attractive chemicals for cassava rnealybug parasitoids. hosts 24 h before the test Previous experience with Only the odours of mealybugs reared on the plants either host or plant have been reported to enhance of the Manihot genus attracted the female parasitoids, the response of parasitoids to test components of the resulted in a significant increase in the time spent in plant-host complex (Turlings et al., 1990). Therefore, the sixth section (Table 3). However, the analysis of it is possible that results reported previously were the number of section changes showed that the loco amplified by the previous experience ofA. lopezi. motor activity of the females was stimulated by the Odours from the four infested plants were attrac odours of mealybugs reared on aIl four plants (Ta tive to A. lopezi females and stimulated their locomo ble 3). This first result concurs with the one reported tor activity (Table 2). Our results concur with those by Langenbach and van Alphen (1986) where females • 219

of A. lapezi reacted to the odours of P. manilIafi and References spent significantly more time searching for their host on cassava leaves contaminated with waxy secretions Alphcn, J. J. M. van. A. R. Kraaijcveld & Xu. C. Ren, 1990. A from mealybugs than on uninfested leaves. In our ex comparaison of Epidinocarsis lopezi and E. diversicornis: A possible explanation for the failcd introduction of E. diversicor periment, mealybugs are removed from cassava plants nis against cassava mcalybug Phenacoccl/s maniho/i inlo Africa. 20 min prior to testing. Souissi et al. (1998) demon Mededelingen Fakulteit Landbouwwetenschappen Rijksuniver strated that mealybugs removed from cassava plants siteit Gent 55: 276-287. Ayanru, D. K. G., & V. C. Sharma, 1985. Changes in total 24 h before the beginning of the test were not attrac cyanide content of tissues from cassava plants infested by mites tive to female parasitoids. Therefore, in the present (Mononychellus tanajoa) and mealybugs (Phenacoccus mani experiment the positive response of A. lapezi females hoti). Agricultural Ecosystems and Environment 12: 35-46. to mealybugs removed from cassava plants might be Baaren, J. van & J. P. Nénon, 1996. Host location and discrimination mediated through olfactory stimuli in two species of Encyrtidae. due to volatiles originating from the cassava plant, Entomologia Experimentalis et Applicata 86: 61--69. adsorbed by the mealybugs and remitted during the Calatayud, P. A., Y. Rhabé, B. Delobel, F. Khuong-Huu, M. Ter test. A time interval of 20 min might not be suf tuliano & B. Le Rü, 1994. Influence of secondary compounds in ficient to dissipate plant derived volatile substances the phloem sap ofcassava on expression ofantibiosis towards the mealybug Phenacoccus manihoti. Entomologia Experimentalis contaminating the mealybugs. Read et al. (1970) re et Applicata 72: 47-57. ported a similar result with females of the braconid Cortesero, A. M., J. P. Monge & J. Huignard, 1993. Response Diaeretiella rapae (M'Intosh) which were attracted to of the parasitoid Eupelmus vui/leti to the odours of the phy the cabbage aphid Brevicaryne brassicae (L.) freshly tophagous host and its host plant in an olfactometer. Entomologia Experimentalis et Applicata 69: 109-116. removed from coUards (15 min before the test) but not Dicke, M., M. W. Sabelis, J. Takabayashi, J. Bruin & M. A. to aphids removed from the host plants 24 h before the Posthumus, 1990. Plant strategies of manipulating predator-prey test. interactions through allelochemicals: prospects for application in The quality and quantity of volatile chemicals re pest control. Journal of Chemical Ecology 16: 3091-3118. Fabres, G., 1981. Première quantification du phénomène de grada leased from the host's secretions and from honeydew tion des populations de Phenacoccus manihoti Matile Ferrero may vary according to the nutritional quality of host (Homo., Pseudococcidae) en République populaire du Congo. plant on which the insect fed. This hypothesis may Agronomie 1: 483-486. Herren, H. R. & P. Neuenschwander, 1991. Biological control of explain the lower attractiveness of mealybugs reared cassava pests in Africa. Annual Review of Entomology 36: 257 on Talinum, on which the level of mealybug infesta 283. tion is much lower than on cassava (Neuenschwander Langenbach, G. E. J. & J. J. M. van Alphen, 1986. Searching be & Madojemu, 1986). The four plants studied here haviour of Epidinocarsis lopezi (Hymenoptera; Encyrtidae) on cassava: Effect of leaf topography and a kairomone produced by have different leaf contents in carbohydrates, amino its host, the cassava mealybug (Phenacoccus manihoti). Med acids and secondary compounds such as cyanated and edelingen Fakulteit Landbouwwetenschappen Rijksuniversiteit phenolic glycosides (Tertuliano et al., 1993). There Gent 51: 1057-1065. fore, it is possible that the range of odours emitted Lohr, B., P. Neuenschwander, A. M. Vare1a & B. Santos, 1988. Interaction between the female parasitoid Epidinocarsis lopezi by mealybugs and by their secretions depends on the De Santis (Hymenoptera: Encyrtidae) and its host the cassava nutritional quality ofthe host plant and elicits different mealybug Phenacoccus manihoti Matile-Ferrero (Homoptera: behavioural responses in A. lapezi. Pseudococcidae). Journal ofApplied Entomology 105: 403-412. Results of our study indicate that females of Nadel, H. & J. J. M. van Alphen, 1987. The role of host- and host-plant odours in the attraction of a parasitoid, Epidinocarsis A. lapezi are attracted by odours emanating from in lopezi, to the habitat ofits host, the cassava mealybug, Phenacoc fested cassava plants and from mealybugs reared on cus manihoti. Entomologia Experimentalis et Applicata 45: this host. This innate response is probably advanta 181-186. geous for A. lapezi females for locating suitable host Nénon, J. P., 1992. Lutte biologique en Afrique contre la coche nille du manioc: conséquences écologiques et agronomiques habitats because cassava is the most predominant host de l'introduction de l'Hyménoptère sud-américain Epidinocarsis plant of P. manihati in Africa. lopezi (Hyménoptère Encyrtidae). Mémoire de la Société Royale Belge d'Entomologie 35: 447-456. Neuenschwander, P. & E. Madojemu, 1986. Mortality of the cassava mealybug, Phenacoccus manihoti Mat. Ferr. (Hom., Pseudococ Acknowledgements cidae), associated with an attack by Epidinocarsis lopezi (Hym. Encyrtidae). Bulletin de la Société Entomologique Suisse 59: The authors are grateful to the three reviewers and 57--62. Read, D. K., P. P. Feeney & R. B. Boot, 1970. Habitat selection by to Dr A. M. Cortesero for valuable comments on the the aphid parasite Diaretiella rapae (Hymenoptera; Braconidae) manuscript. • 220

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