Biocontrol Science and Technology

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Applied Biological Sciences, University of Gent, Coupure Links 653,B-9000 Gent, ... emulsifiable concentrate, 3.2% azadirachtin, Sipcam Inagra, Spain) via.
Biocontrol Sci Tech 10 (2) 175-187.

Laboratory Effects of Ingestion of Azadirachtin by Two pests (Ceratitis capitata and Spodoptera exigua) and Three Natural Enemies (Chrysoperla carnea, Opius concolor and Podisus maculiventris)

E. VIÑUELA1, A. ADÁN1, G. SMAGGHE2, M. GONZÁLEZ1, Mª.P. MEDINA1, F. BUDIA1, H. VOGT3 AND P. DEL ESTAL1

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Protección de Cultivos, Escuela Técnica Superior de Ingenieros Agrónomos, E-28040 Madrid, Spain.

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Laboratory of Agrozoology, Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, University of Gent, Coupure Links 653,B-9000 Gent, Belgium 3

Federal Biological Research Centre for Agriculture and Forestry, Institute for Plant Protection in Fruit Crops, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany Running title: Azadirachtin effects on natural enemies and pests Key words: azadirachtin, toxicity, side-effects, Ceratitis capitata, Spodoptera exigua, Chrysoperla carnea, Opius concolor, Podisus maculiventris. Corresponding author: Professor Elisa Viñuela, Protección de Cultivos, E.T.S.I. Agrónomos, E-28040-Madrid, Spain. phone: 34 91 336 57 74 fax: 34 91 336 58 66 E-mail: [email protected]

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SUMMARY The effects of azadirachtin on two pests: neonate larvae and newly emerged adults of Ceratitis capitata (Wiedemann) and last-instar larvae of Spodoptera exigua (Hübner); and three natural enemies: newly emerged adults of Opius concolor Szèpligeti, second-instar larvae of Chrysoperla carnea (Stephens), and fifth-instar nymphs of Podisus maculiventris (Say) were studied in laboratory. Adult insects were exposed to a non-oil formulation of azadirachtin (Align®, emulsifiable concentrate, 3.2% azadirachtin, Sipcam Inagra, Spain) via their drinking water and immature instars were reared in the presence of the insecticidetreated diet. The natural enemies were exposed to at least, the maximum field recommended concentration of the insecticide (0.15% v/v). Azadirachtin was highly toxic to neonate larvae of C. capitata and prevented adult emergence at a concentration of 1 mg a.i./l totally. When adults were fed the insecticide at the maximum recommended concentration, their survival was not affected but egg laying was totally inhibited. Last-instar S. exigua larvae were also very susceptible (LC50 = 7.7 mg a.i./l) and at a concentration of 10 mg a.i./l fecundity of surviving adults and egg fertility were reduced by 72% and 85% respectively. Effects on O. concolor were large and significant reductions in longevity, percentage of attacked hosts and progeny size per female were recorded. The predator P. maculiventris was much less sensitive to azadirachtin, but slight reductions in survival of emerged adults and of reproductive parameters occurred. The insecticide had no significant effect on C. carnea larvae fed with treated Sitotroga cerealella (Oliver) eggs, probably because of its inability to penetrate inside the egg. INTRODUCTION Biological control is nowadays an essential component of integrated pest management (IPM) and integrated production of crops. However because a sole method of pest control is seldom insufficient to keep pests under economic threshold levels, requirements of this control tactic must be integrated with the needs of different techniques such as chemical pesticides (Hokkanen, 1997). An essential premise for the use of pesticides in IPM is to ascertain their compatibility with beneficial organisms, because it is known that pesticides can modify the interrelationships of species in ecosystems and that frequently parasitoids and predators suffer greater mortality than do their phytophagous hosts (Pimentel, 1992). But not all pesticides produce the same harmful effects on beneficials and the simultaneous use of enemies and pesticides can be achieved by applying selective control agents or by making an appropriate 2

timing of those products that only affect certain life stages of the beneficial (Jacas & Viñuela, 1994a). Among the more environmentally friendly candidates to the use of broad-spectrum chemicals for the control of pests of economic importance are the naturally derived control products. Azadirachtin, a highly oxidized limonoid, is a botanical pesticide formed by a group of closely related isomers mainly obtained from the seed kernels of the neem tree Azadirachta indica A. Juss (Schmutterer, 1990). Its main mode of action seems to be inhibition of release of prothoracicotropic hormones and allatotropins (Banken & Stark, 1997). The compound has gained more and more attention in recent years as a component of integrated pest management programmes because it has been reported to be less toxic to beneficials than to pests (Stark et al., 1992; Sipcam Inagra, 1996; Banken & Stark, 1997). The Mediterranean fruit fly Ceratitis capitata (Wiedemann) (Diptera, Tephritidae) and the beet armyworm Spodoptera exigua (Hübner) (Lepidoptera, Noctuidae) are two worldwide important pests. The former attacks more than 250 subtropical and deciduous fruits in the Mediterranean region (Fimiani, 1989) and in Spain since 1955, the severity of damage caused to citrus crops led to the establishment of mandatory control measures (Adán et al.,1996). The beet armyworm S. exigua is a polyphagous noctuid of worldwide importance in agriculture, horticulture and ornamentals. This pest is very much feared in western Europe and the Mediterranean region due to the severe losses it causes in greenhouses (Van de Vrie, 1977; Marco & Viñuela, 1994). Moreover, during the last decades, the extensive use of classical insecticides has resulted in failure of control due to development of resistance (Viñuela, 1998; Smagghe et al., 1999). Opius concolor Szèpligeti (Hymenoptera, Braconidae) is an endoparasitoid of the olive fly Bactrocera oleae (Gmelin), one of the key pest of this crop in the Mediterranean region, which is easily mass-reared in laboratory in the substitution host C. capitata (Jacas & Viñuela, 1994b). Chrysoperla carnea (Stephens) (Neuroptera, Chrysopidae) is a general entomophagous predator, which is commercially produced in many countries for use as a biocontrol agent against aphids. This species has been selected as one of the relevant beneficials to be tested within pesticide registration in the European Union (Barret et al.,1994). Podisus maculiventris (Say) (Hemiptera, Pentatomidae) is a generalist predatory pentatomid that feeds actively on larvae of many lepidopterans of economic importance (De 3

Clercq et al.,1995). Its commercial use has been recently started in European greenhouses aiming at controlling S. exigua larvae. In this study we have examined the effects of azadirachtin on two pests and three natural enemies, representatives of different orders, families and genera. The compound was applied via ingestion and we studied toxicity and changes in development in the life stage treated and in some of the insects, on reproduction by evaluating fecundity and egg viability. MATERIALS AND METHODS Insecticides The non-oil commercial formulation of azadirachtin, Align® (3.2% azadirachtin, emulsifiable concentrate, Sipcam Inagra, Spain), was used in all experiments. In all the assays, fresh solutions were prepared in distilled water. Insects The C. capitata and O. concolor specimens used in the tests were obtained from laboratory cultures maintained in Madrid following the standard procedures of Albajes and Santiago-Álvarez (1980) and Jacas and Viñuela (1994b). Colonies of C. carnea and P. maculiventris originated from eggs obtained from the Institute for Plant Protection in Fruit Crops of Dossenheim (Germany) and University of Gent (Belgium) respectively, and were mass-reared in Madrid for at least three generations prior to the assays. C. carnea larvae were reared on Sitotroga cerealella (Oliver) eggs, and adults were fed a nutritive mixture of 1egg, 1yolk, 30g honey, 30g brewer’s yeast flakes, 50g wheat germ, 20g fructose, 15 ml condensed milk and 50ml distilled water as described by Vogt et al. (1998a,b). Nymphs and adults of P. maculiventris were fed Spodoptera littoralis (Boisduval) larvae according to De Clercq et al. (1988) and Viñuela et al. (1998). Rearing conditions were 25±2ºC, 75±5% R.H. and 16 h light photoperiod. All developmental stages of a continuous colony of S.exigua were maintained at 23±2ºC, 70±5% R.H. and 16 h light photoperiod in Gent. Larvae were fed a Poitout based artificial diet and adults a 15% solution of honey in water according to Smagghe and Degheele (1994). Test methods Every experiment consisted of at least three replicates of 10 to 15 insects per concentration level (expressed in active ingredient), and control specimens were treated with 4

distilled water. For pests, concentrations and treatment methods were chosen in accord with our previous experience and biological characteristics of the species. Following IOBC recommendations (Hassan, 1994), beneficials were exposed to a concentration of at least 48 mg a.i./l which is equivalent to the maximum field recommended concentration of Align® (150 cc/hl). Experiments were always done with the most exposed life stage of the enemies: adults of the parasitoid and larvae or nymphs of the predators. In reproductive studies, fecundity was based on scores of 4 replicates of 3-5 pairs/cage for C. capitata, at least two replicates of 2-3 pairs/cage for S. exigua and of 8 replicates of individual pairs for the other species. Fertility was based on the hatching of about 100 eggs per concentration level on two different days. Adult survival was also monitored during life span in O. concolor and during a 7 to 10-day period, for the other species. Ceratitis capitata: Our study focused on two of the vulnerable stages of this fly: neonate larvae (0-12 h), that can be found in recently stored fruits, and adults, which are the current target of field treatments (Adán et al., 1996). Effects on neonate larvae and newly emerged adults (0-24 h) of this species were studied following the methods of Viñuela et al. (1993) for larvae and of Budia and Viñuela (1996) for adults. Larvae were reared in the presence of azadirachtin-treated diet at concentrations of 0.1 and 1 mg/l. Azadirachtin was also fed ad libitum to groups of 5 newly emerged pairs of flies for a 7-day period, at concentrations of 48 and 100 mg/l. Insecticide solutions were offered continuously from adult emergence in glass troughs covered by Parafilm® with a piece of Spontex® wiper. A mixture of sucrose and autolysed brewer’s yeast was supplied as food in plastic containers. Spodoptera exigua: Newly moulted (0-12 h) last (L5)-instar larvae were orally treated in accordance with Smagghe and Degheele (1994). Ten different concentrations of azadirachtin ranging from 0.1 up to 100 mg/l were prepared in water, and the surface of the artificial diet was uniformly treated with 50 µl. Controls were treated with water alone. Mortality was scored 7 days after treatment. Control specimens had entered the pupal stage at this time. In addition, the fresh weight gain of larvae and pupae was measured using an analytical Sartorius balance, and means and SE were expressed as percentages of the control groups. Adult eclosion of surviving pupae was recorded, and expressed as a percentage of the total number of larvae treated. Groups of 2-3 pairs were kept in plastic containers with the inside walls covered with paper providing oviposition places (Smagghe & Degheele, 1994) and adult longevity, fecundity (calculated as the number of eggs deposited during 10 days of oviposition) and fertility were evaluated. 5

Opius concolor: Groups of 15 newly emerged (0-12 h) females per concentration were exposed continuously to azadirachtin in their drinking water at a dose of 48 mg/l. Insects were also provided with a mixture of brewer’s yeast and sugar (1:4). Longevity and beneficial capacity of the wasps (measured as the percentage of attacked hosts and progeny size per female), were studied following the method of Jacas and Viñuela (1994b). Chrysoperla carnea: Newly moulted (0-12 h) L2-larvae were individually placed in an uncovered 9 cm Petri dish with Fluon coated walls to avoid insect escape with a filter paper on the bottom. Insects were fed continuously with S. cerealella eggs treated at concentrations ranging from 10 to 10,000 mg/l azadirachtin following a modification of the method described by Vogt et al. (1998a,b). Eggs were treated under the Potter Precision Spray Tower, with a standard deposit of 1.89±0.05 mg/cm2 (1 ml; 50 kPa) and were allowed to dry at room temperature in a fume hood for 24h before being offered to larvae. Applications rates were calculated using the PIEC formula (the predicted initial environmental concentration of a pesticide) developed by Barret et al. (1994), considering a 0.4 factor and 1,000 l/ha. Podisus maculiventris: Newly moulted (0-12 h) N5-nymphs were placed individually in a 9 cm Petri dish lined with a filter paper and were fed the insecticide continuously in their drinking water at a concentration of 48 mg/l. After the first 24 h (to ensure that they had taken the insecticide initially), the predatory nymphs were provided with S. littoralis L5 larvae ad libitum (De Clercq et al., 1995; Viñuela et al., 1998). Statistical analysis Depending on the species, the following parameters were recorded: mortality during larval, nymphal, pupal and/or adult stage; duration of the instar; weight; malformations, adult longevity, fecundity and fertility. In C. capitata, larval mobility was also scored by counting the number of popped pupae (pupae which jump off the diet to pupate in a drier substratum) as a percentage of the total number of pupae recorded. Data, presented in tables as means with standard errors, were analysed by 1-way analysis of variance using Statgraphics (STSC, 1994). Where appropriate, percentages were transformed and means were separated by the least significant difference (LSD) option (P 5,000 mg/kg) and environmentally friendly, being its EPA toxicity class IV (Copping, 1998). The product was rather harmful in laboratory tests for adults of O. concolor, so its joint use together with the susceptible life stage of the enemy only seems to be possible with an appropriate timing (Jacas & Viñuela, 1994a have reported that pesticides are totally harmless for the protected life stage of this enemy). However before reaching final conclusions, it seems to be necessary to complete the whole IOBC sequential testing scheme for adults (Hassan, 1994). A first series of preliminary semi-field tests recently indicated that Align® was compatible with adults of the beneficial (González, unpublished results); however, further testing is required before publication. The insecticide was harmless for fully grown nymphs of P. maculiventris, but several delayed effects on emerged adults were observed. Hence, it is necessary more completely determine its effects on the reproduction of the bug before reaching a conclusion on its suitability for use with this predator. No conclusive results could be drawn from the results scored in C. carnea, but Align® could be compatible with the predator as NeemAzal-T/S, an oil formulation of azadirachtin, was found to be totally harmless in the field for C. carnea L2-larvae (Viñuela et al., 1996; Vogt et al., 1998a). In summary it can be concluded that Align® could be compatible with the natural enemies studied but further studies should investigate the total risk of using this compound together with them, because the insecticide can exhibit differential toxicity to different life stages and ages of the targeted species. ACKNOWLEDGEMENTS The authors gratefully acknowledge the research support provided by the Spanish Ministry of Education and Culture (Concerted action Spain-Germany HA97-0005 and project AGF98-0715) and the Autonomous Community of Madrid (project 06M/022/96) to E. Viñuela, and by the DAAD (Deutscher Akademischer Austauschdienst) to H. Vogt. M. González and Mª.P. Medina are recipient of grants from the Autonomous Community of Madrid. G. Smagghe acknowledges the Belgian National Fund of Scientific Research 12

(Brussels) for a post-doctoral fellowship. We also thank Dr. J. Jacas for suggesting improvements to the manuscript. REFERENCES ADÁN, A., DEL ESTAL, P., BUDIA, F., GONZÁLEZ, M. & VIÑUELA, E. (1996) Laboratory evaluation of the novel naturally derived compound spinosad against Ceratitis capitata. Pesticide Science. 48, 261-168. ADÁN, A., SORIA, J., DEL ESTAL, P., SÁNCHEZ-BRUNETE, C. & VIÑUELA, E. (1998) Differential action of two azadirachtin formulations on the developmental stages of Ceratitis capitata. Boletín Sanidad Vegetal. Plagas 24, 1009-1018. (In Spanish). ALBAJES, R. & SANTIAGO-ÁLVAREZ, C. (1980) Effects of larval density and food in the sex ratio of Ceratitis capitata. Anales INIA/Serie Agrícola 13, 175-182. (In Spanish). BANKEN, J.A.O. & STARK, J. (1997) Stage and age influence on the susceptibility of Coccinella septempunctata after direct exposure to Neemex, a neem insecticide. Journal of Economic Entomology 90,1102-1105. BARRET, K.L., GRANDY, N., HARRISON, E.G., HASSAN S. & OOMEN, P. (Eds) (1994) Guidance document on regulatory testing procedures for pesticides with non-target arthropods. Society of Environmental Toxicology and Chemistry-Europe, U.K. BUDIA, F. & VIÑUELA, E. (1996) Effects of cyromazine on adult C. capitata on mortality and reproduction. Journal of Economic Entomology 89, 826-831. CARTON, B., SMAGGHE, G., MOURAD, A.K. & TIRRY, L. (1998) Effects of RH-2485 on larvae and pupae of Spodoptera exigua (Hübner). Medelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen,Universiteit Gent 63, 537-545. COPPING, L.G. (Ed) (1998) The biopesticide manual. British Crop Protection Council. UK. CROFT, B.A. (Ed) (1990) Arthropod biological control agents and pesticides. John Wiley & Sons, New York. DE CLERCQ, P., DE COCK, A., TIRRY, L., VIÑUELA, E. & DEGHEELE, D. (1995) Toxicity of diflubenzuron and pyriproxyfen to the predatory bug Podisus maculiventris. Entomologia Experimentalis et Applicata 74, 17-22. DE CLERCQ, P., KEPPENS, G., ANTHONIS, G. & DEGHEELE, D. (1988) Laboratory rearing of the predatory stinkbug Podisus sagitta (F.). Medelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen,Universiteit Gent 53, 12131217. ERMEL, K. & KLEEBERG, H. (1995) Commercial products, in The neem tree (SCHMUTTERER, H., Ed) VCH, Weinheim, pp. 375-384. FIMIANI, P. (1989) Mediterranean region, in World crop pests. Fruit flies, vol. 3A (ROBINSON, A.S. & HOOPER, A.G., Eds) Elsevier, Amsterdam, pp. 37-47 HASSAN, S.A. (1994) Activities of the IOBC/WPRS working group “Pesticides and Beneficial Organisms”. IOBC/WPRS Bulletin 17 , 1-5. HOKKANEN, H.M.T. (1997) Role of biological control and transgenic crops in reducing use of chemical pesticides for crop protection, in Techniques for reducing pesticide use. Economic and environmental benefits (PIMENTEL, D. Ed) Wiley, Chichester, pp. 103127. ISMAN, M.B. (1995) Lepidoptera: butterflies and moths, in The neem tree (SCHMUTTERER, H. Ed) VCH, Weinheim, pp. 299-318 JACAS, J. & VIÑUELA, E. (1994a) Side-effects of pesticides on Opius concolor, a parasitoid of the olive fruit fly. IOBC/WPRS Bulletin 17, 143-146. 13

JACAS, J. & VIÑUELA, E. (1994b). Analysis of a laboratory method to test the effects of pesticides on adult females of Opius concolor, a parasitoid of the olive fruit fly, Bactrocera oleae. Biocontrol Science and Technology 4,147-154. LEORA SOFTWARE, POLO-PC (1994) User’s guide to probit or logit analysis. LeOra Software Inc., Berkeley, CA. MARCO, V. & VIÑUELA, E. (1994) Effects of hexaflumuron on fecundity, fertility and longevity of Ephestia kuehniella Zeller and Spodoptera exigua (Hübner). Medelingen Faculteit Landbouwkundige en Toegepaste Biologische Wetenschappen,Universiteit Gent 59, 457-463. PIMENTEL, D. (1992) Ecological effects of pesticides on non-target species in terrestrial ecosystems, in Methods to assess adverse effects of pesticides on non-target organisms (TARDIFF, R.G. Ed) John Wiley & Sons, England, pp. 171-190. RIDDIFORD, L. (1985) Hormonal action at the cellular level, in Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 8 (KERKUT, G.A & GILBERT, L.I. Eds) Pergamon Press, Oxford, pp. 37-84. RUIZ, A., PRADES, J., CANO, F.J. & ABRIL, E. (1998) Align, un bioinsecticida de origen vegetal, biodegradable, compatible con el medio ambiente y los enemigos naturales de las plagas, in Agricultura ecológica y desarrollo rural, (SEAE Ed) Actas del II Congreso de la SEAE, Pamplona, pp. 205-211. SAXENA, R.C. (1987) Neem seed oil - a potential antifeedant against insect pests of rice, in Pesticide Science and Biotechnology (GREENHALGH, R. & ROBERTS, T.R. Eds) Blackwell Scientific, England, pp. 139-144 SCHMUTTERER, H. (1990) Properties and potential of natural pesticides from the neem tree, Azadirachta indica. Annual Review of Entomology 35, 271-297. SCHMUTTERER, H. (1995): Side-effects on beneficials and other ecologically important non-target organisms. Introduction, in The neem tree (SCHMUTTERER, H. Ed) VCH, Weinheim, pp. 495-517 SCHMUTTERER, H., SAXENA, R.C. & HEYDE, V.D.J. (1983) Morphogenetic effects of some partially-purified fractions and methalic extracts of neem seeds on Mythimna separata (Walker) and Cnaphalocrosis medinalis (Guenée) Zeitschrift für Angewandte Entomologie 46, 230-237. SHIMIZU, T. (1988) Suppressive effects of azadirachtin on spermatogenesis of the diapausing cabbage armyworm, Mamestra brassicae, in vivo. Entomologia Experimentalis et Applicata 46, 197-199. SIPCAM INAGRA (1996) Align®. Technical information. (In Spanish). SMAGGHE, G. & DEGHEELE, D. (1994) Action of a novel nonsteroidal ecdysteroid mimic, tebufenozide (RH-5992), on insects of different orders. Pesticide Science 42, 85-92. SMAGGHE, G., MEDINA, Mª.P., SCHUYESMANS, S., TIRRY, L. & VIÑUELA, E. (1999). Insecticide resistance monitoring and potential of novel insect growth regulators for managing the beet armyworm (Spodoptera exigua Hübner), in Combating insecticide resistance (DENHOLM, I. & IOANNIDIS, P.M. Ed) Enmaria, Hellenic Entomological Society & Irac, Thessaloniki, pp. 70-78. STARK, J., VARGAS, R.I. & THALMAN, R.K. (1990) Azadirachtin: effects on metamorphosis, longevity and reproduction of three tephritid fruit fly species. Journal of Economic Entomology 83, 2168-2174. STARK, J., WONG, T.T.Y., VARGAS, R.I. & THALMAN, R.K. (1992) Survival, longevity and reproduction of tephritid fruit fly parasitoids (Hym. Braconidae) reared from fruit flies exposed to azadirachtin. Journal of Economic Entomology 85, 1125-1129. 14

STSC (1987) Statgraphics user’s guide, version 5.0. Graphic software system, STSC, Rockville, MD. TRUMAN, J.W. (1992) The eclosion hormone system of insects. Progress Brain Research 134, 344-355. VAN DE VRIE, M. (1977) Spodoptera exigua (Lepidoptera: Noctuidae) in siergewassen. Gewasbeschermingsgids 8, 67-70. VIÑUELA, E. (1998) Insecticide resistance in horticultural pests in Spain, in Pesticide resistance in horticultural crops (CUADRADO, I.Mª. & VIÑUELA, E. Eds) FIAPA, Almería, pp. 19-30. VIÑUELA, E., ADÁN, A., GONZÁLEZ, M., BUDIA, F., SMAGGHE, G. & DEL ESTAL, P. (1998) Spinosad and azadirachtin: effects of two naturally derived pesticides against Podisus maculiventris (Say). Boletín Sanidad Vegetal. Plagas 24, 57-66 . (In Spanish). VIÑUELA, E., BUDIA, F., JACAS, J., ADÁN, A., MARCO, V. & DEL ESTAL, P. (1993) Differential larval age susceptibility of the medfly Ceratitis capitata to cyromazine. Journal of Applied Entomology 115, 355-362. VIÑUELA, E., HÄNDEL, U. & VOGT, H. (1996) Evaluation under field conditions of the sideeffects of two naturally derived pesticides: a natural pyrethrine and a neem extract in Chysoperla carnea. Boletín Sanidad Vegetal. Plagas 22, 97-106. (In Spanish). VOGT, H., DEGRANDE, P., JUST, J., KLEPA, S., KÜHNER, C., NICKLESS, A., UFER, A., WALDBURGER, M., WALTERSDORFER, A. & BIGLER, F. (1998a) Side-effects of pesticides on larvae of Chrysoperla carnea: actual state of the laboratory method, in Ecotoxicology: pesticides and beneficial organisms (HASKELL, P.T. Ed) Chapman & Hall, England, pp. 123-138. VOGT, H., GONZÁLEZ, M., ADÁN, A., SMAGGHE G. & VIÑUELA, E. (1998b) Side-effects of azadirachtin via residual contact, in young larvae of the predator Chrysoperla carnea. Boletín Sanidad Vegetal. Plagas 24, 67-78. (In Spanish).

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Ceratitis

capitata

Neonate larvae fed on treated diet % Pupal % Popped Concentrations % Larval mortality1 pupae1* mg a.i./l mortality1 Control 20.7± 3.6a 1.3± 1.4a 93.1± 2.0a 0.1 24.7± 6.8a 4.0± 2.0ab 91.9± 0.8a 1 86.7± 4.1b 13.3± 4.1b 11.4± 6.0b Adults exposed via ingestion in their drinking water Concentrations % Mortality at 7 days2 Eggs/female, mg a.i./l 8 days1** Control 10.0± 4.1a 235.4± 28.8a 48 16.0± 7.1a 0±0b 100 15.0± 2.9a 1.7± 1.7b

% Adult emergence1* 78.0± 4.7a 71.3± 4.5a 0±0b % Eclosion 87.5± 1.3 0±0

Within the same column, data followed by the same letter do not differ significantly. (P=0.05; 1 LSD 2 Bonferroni mean separation). * Compared with total number of larvae. ** Data represent the mean of 4 replicates of 3-5 pairs of flies/ cage.

Table 1: Influence of the developmental stage on the susceptibility of Ceratitis capitata to azadirachtin.

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Opius concolor Adults exposed via ingestion in drinking water Concentrations Longevity Beneficial capacity* mg a.i./l (days) % Attacked hosts % Progeny Control 31.4± 20.7a 87.5± 4.8a 65.0± 7.9a 48 4.9± 0.1b 64.1± 8.2b 43.9± 4.8b Within the same column, data followed by the same letter do not differ significantly (P=0.05; LSD mean separation). * Data represent the mean of 8 replicates of 3-day pesticide-exposed and isolated females. Twenty L3 C. capitata larvae were daily offered to them for 2 h during a period of 4 days.

Table 2: Effects of feeding azadirachtin in water to adult Opius concolor during life span on the longevity and the beneficial capacity of the parasitoid.

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Chrysoperla carnea Concentrations mg a.i./l Control 10 100 1,000 10,000

L2 larvae fed on Sitotroga cerealella treated eggs Eggs/female, % Eclosion % Larval % Pupal % Adult 10 days** mortality mortality emergence* 13.3± 6.6a 10.0± 6.8 a 82.1± 11.8a 501.4± 56.7a 92.2± 3.5a 6.6± 4.2a 30.0± 4.4a 65.0± 3.1a 523.0± 56.2a 92.0± 6.1a 10.0± 4.4a 23.3± 12.0a 60.0± 9.2a 555.5± 108.5a 83.5± 7.5a 13.3± 4.2a 16.6± 6.1a 65.8± 8.7a 618.6± 38.5a 87.2± 7.8a 30.0± 8.4a 26.6± 4.2a 48.1± 12.0a

Within the same column, data followed by the same letter do not differ significantly (P=0.05; Bonferroni mean separation). * Compared with pupae. ** Data represent the mean of 8 replicates of pairs kept individually.

Table 3: Toxicity of azadirachtin to the predator Chrysoperla carnea when L2 larvae were fed continuously Sitotroga cerealella treated eggs.

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Podisus maculiventris N5 nymphs exposed via ingestion in the drinking water Nymphal weight at Duration of the Concentrations % Nymphal 5 days (mg)2 instar (days)2 mg a.i./l mortality1 Control 0a 76.0± 3.3a 7.0± 0.2a 48 12.5± 6.3b 88.6± 4.2a 7.4± 0.1a Within the same column, data followed by the same letter do not differ significantly (P=0.05; 1 LSD 2 Bonferroni mean separation)

Table 4: Effects of azadirachtin on N5 nymphs of the predatory bug Podisus maculiventris exposed via ingestion in the drinking water

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Podisus maculiventris N5 nymphs exposed via ingestion in the drinking water Concentrations % Adult mg a.i./l emergence

Control 48

100± 0.0a 87.5± 6.3b

% Malformed adults1*

0a 36.6± 3.5b

% Adult mortality Eggs/female, at 8 days1 8 days2**

10.0± 4.1a 46.7± 8.7b

119.7± 14.8a 58.0± 27.3a

% Fertile females

% Eclosion2

100 44

60.4± 10.4a 33.8± 11.3a

Within the same column, data followed by the same letter do not differ significantly. * Compared with total number of emerged adults. ** Data represent the mean of 8 replicates of pairs kept individually. (P=0.05; 1 LSD mean separation; 2 Bonferroni mean separation).

Table 5: Effects of azadirachtin on Podisus maculiventris adults when N5 nymphs were fed the insecticide in the drinking water.

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Figure 2: Daily cumulative mortality in Podisus maculiventris adults emerged from N5 nymphs fed Align® ad libium at 45 mg/l via the drinking water. Figure 1: Effects of Align® on larval mortality (A), larval weight gain (B), pupal weight (C), adult survival (D), fecundity of surviving adults (E), and fertility of eggs deposited (F) of Spodoptera exigua when treated in the last-larval instar. Data are expressed as means±SE.

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