Toxicity and Absorption of Azadirachtin, Diflubenzuron, Pyriproxyfen ...

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Toxicity and Absorption of Azadirachtin, Diflubenzuron, Pyriproxyfen, and Tebufenozide after Topical Application in Predatory Larvae of Chrysoperla carnea (Neuroptera: Chrysopidae) P. MEDINA,1 G. SMAGGHE,2 F. BUDIA,1 L. TIRRY,2

AND

˜ UELA1, 3 E. VIN

Environ. Entomol. 32(1): 196Ð203 (2003)

ABSTRACT Susceptibility of the generalist predator, the lacewing Chrysoperla carnea (Stephens), to the insect growth regulators azadirachtin, dißubenzuron, pyriproxyfen, and tebufenozide was tested in the laboratory. Third instars were topically treated with different doses of formulated materials of each compound by direct topical exposure. At maximum Þeld-recommended dose, pyriproxyfen and tebufenozide were harmless to C. carnea, whereas azadirachtin and dißubenzuron were harmful (respective LD90s were 24.5 and 6.9 ng active ingredient [AI] per insect). At sublethal doses of azadirachtin and dißubenzuron, females laid fertile eggs, but azadirachtin caused a slight negative effect on oviposition. Pyriproxyfen and tebufenozide had no effect on oviposition and egg fertility. As a second approach of this study, toxicity data are discussed in relation to the rate of penetration and excretion after topical application. One hour after administration, ⬇80% of pyriproxyfen had penetrated; whereas for dißubenzuron and tebufenozide, only percentages of 10 Ð20% were recorded in the same time interval. However, although pyriproxyfen penetration was fast and high, most of the compound was also quickly eliminated via excretion. Our data suggest that the use of azadirachtin and dißubenzuron in combination with C. carnea in integrated pest management (IPM) programs should be carefully evaluated. Pyriproxyfen and tebufenozide are considered to be safe for C. carnea. KEY WORDS Chrysoperla carnea, IGRs, side-effects, cuticular penetration, excretion

ONE OF THE commonly used strategies of integrated pest management (IPM) is the joint use of biological and selective chemical control, because at present, the complex of the pests attacking a crop cannot be controlled only with biological control. In this strategy, it is obvious that the biological control of one pest and the chemical control of another should not interfere. Therefore, the knowledge on the activity of insecticides toward the pests, the nontargets insects, and the environment, including humans is a necessity. Larvae of the lacewing Chrysoperla carnea (Stephens) have long been considered important naturally occurring predators in many horticultural and agricultural cropping systems (Canard et al. 1984), and currently it is one of the most commonly used and commercially available natural enemies (Tauber et al. 2000). Azadirachtin, dißubenzuron, pyriproxyfen ,and tebufenozide are insect growth regulators (IGRs) with different modes of action. Azadirachtin, one of the most common triterpenoids found in Meliaceae 1 Proteccio ´ n de Cultivos, Escuela Te´ cnica Superior de Ingenieros Agro´ nomos, E-28040, Madrid, Spain. 2 Laboratory of Agrozoology, Department of Crop Protection, Faculty of Agricultural and Applied Biological Sciences, Gent University, Coupure Links 653, B-9000 Gent, Belgium. 3 (E-mail: [email protected])

(e.g., the neem tree Azadirachta indica A. Juss) produces two kind of effects in insects: It alters insect behavior because of an antifeedant and repellent action, and it modiÞes insect development by inhibiting the release of prothoracicotropic hormones and allatotropins (Mordue and Blackwell 1993, Williams and Mansingh 1996). Dißubenzuron is known to inhibit chitin synthesis in insects that results in a disruption of the molting process (Retnakaran and Wright 1987, Ishaaya and Horowitz 1998). The potent juvenile hormone mimic pyriproxyfen interferes with the hormonal balance and provokes a strong suppression of embryogenesis, metamorphosis, and adult formation (Ishaaya et al. 1994). The dibenzoylhydrazine tebufenozide belongs to the group of nonsteroidal ecdysone agonists and interacts with the insect molting hormone receptor, especially in Lepidoptera, to induce premature and lethal molting (Chandler et al. 1992, Smagghe and Degheele 1994, Dhadialla et al. 1998, Carlson 2000). These IGRs are used against different pests in fruit-trees where C. carnea is an important and representative natural enemy. Our goals in this paper were (1) to evaluate the compatibility of these four IGRs with the predatory larvae of C. carnea at doses related with those used in the Þeld to be able to recommend or not the joint use of these compounds with the enemy in IPM; and (2)

0046-225X/03/0196Ð0203$04.00/0 䉷 2003 Entomological Society of America

February 2003

MEDINA ET AL.: TOXICITY OF IGRS IN Chrysoperla LARVAE

to expand our basic knowledge about the impact of penetration through the larval cuticle on the toxicity observed. The latter may contribute to our understanding of the resistance or tolerance of natural enemies and beneÞcial insects against pesticides, especially IGRs. Materials and Methods Insects. A continuous laboratory colony of C. carnea (25 ⫾ 2⬚C, 75 ⫾ 5% RH, and a photoperiod of 16 L: 8 D) was established in 1997 with eggs obtained from the Institute for Plant Protection in Orchards (Dossenheim, Germany); and introductions of new insects have been done yearly since then. Under our conditions, the predator has ⬇1 generation per month. Larvae were fed on Sitotroga cerealella (Oliver) eggs, and adults were provided with an artiÞcial diet (Vogt et al. 1998a). Chemicals. The IGRs tested were Align (3.2% azadirachtin, nonoil EC, Sipcam Inagra, Valencia, Spain), Dimilin25 (25% dißubenzuron, WP, AgrEvo, Valencia, Spain), Juvinal (10% pyriproxyfen, EC, Kenogard, Barcelona, Spain), and Mimic (24% tebufenozide, SC, Rohm and Haas, Barcelona, Spain). Treatment to Assess Mortality and Effects on Oviposition and Egg Fertility. Third-instars (9 d old) were topically treated on the thoracic dorsum with 0.5 ␮l of an acetonic concentration of each IGR with the use of an Arnold hand microapplicator equipped with a 1-ml glass syringe Þtted with a 30-gauge hypodermic needle (Burkard, UK). Controls were treated with acetone alone. A preliminary assay with the maximum Þeldrecommended doses showed very high toxicity with azadirachtin and dißubenzuron, whereas no toxicity was observed for tebufenozide and pyriproxyfen. Maximum Þeld-recommended doses are based on maximum Þeld-recommended concentration when the applied drop size is 0.5 ␮l. (azadirachtin: 150 cm3/ hectoliter [cm3/hl]; dißubenzuron: 60 g/hl; pyriproxyfen: 75 cm3/hl; tebufenozide: 75 cm3/hl, expressed as commercial product. water amount: 1,000 liter/ha). In a Þrst series of assays, two doses were used to evaluate the side-effects of the four IGRs (on the basis of maximum Þeld-recommended concentration and the double). Mortality was negligible for pyriproxyfen and tebufenozide and very high for azadirachtin and dißubenzuron, so for these two last compounds, in a second series of assays, we tested a range of six (2Ð 48 ng AI/insect) and nine (0.5Ð75 ng AI/ insect) doses, respectively, to calculate LD50s. After treatment, insects were kept individually in 2 ⫻ 1.8 cm3 cylindrical plastic vials to pupate, and after cocoon-spinning, specimens belonging to the same replicate were removed from the single wells and kept in bigger plastic cages (11 cm diam ⫻ 5 cm high) over folded paper to make it easier for newly emerged adult to stretch wings (slightly modiÞed from Shour and Crowder 1980). Four replicates of 10 or 15 larvae were treated for each dose and chemicals. The mean ⫾ SD fresh weight of 9-d-old untreated larvae was 7.53 ⫾ 2.26 mg based on 75 individual measurements. Larval

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mortality (they did not move when slightly touched with a Þne camel brush), failure to pupate successfully (larvae were unable to totally spin the cocoon), pupal mortality inside or outside the cocoon (black cocoons and dead pharate adults, respectively), and adult emergence were recorded. In a second experiment, the effects of the four IGRs were evaluated on the oviposition rate and the percentage of egg hatch in accord with Medina et al. (2001). Emerged adults were kept in oviposition plastic cages (3 pairs per cage) (11 cm diam ⫻ 5 cm high) covered by a gauze kept in place with the lid, which had a hole 7.5 cm in diam, and provided with food and water. For tebufenozide and pyriproxyfen, we used the maximum Þeld dose and the double, but for azadirachtin and dißubenzuron, we only tested sublethal doses because mortality was very high at the Þeld doses. To study fecundity, eggs were collected and counted every 1Ð2 d from Þrst oviposition. Mean number of eggs per female per day, during a 7-d period, was recorded to compare fecundity among treatments. Five replicates of at least three pairs of adults per dose and compound were used. To assess effects on fertility, eggs deposited at day 5 after the Þrst oviposition were collected and placed in plastic cages (9 cm diam ⫻ 3 cm high) containing S. cerealella eggs to prevent cannibalism among neonates before being counted. The number of emerged larvae was evaluated 5Ð 6 d later. Cuticular Penetration of 14C-isotopes after Topical Exposure. The radiolabeled isotopes used were 14Cdißubenzuron (spec. act. 15 mCi/g; Duphar B.V., Weesp, The Netherlands), 14C-pyriproxyfen (spec. act. 181 mCi/g; Sumitomo, Osaka, Japan) and 14Ctebufenozide (spec. act. 23.06 mCi/g; Rohm and Haas, Spring House, PA), and were diluted in acetone. Third-instars (9 d old) were selected from the continuous colony and treated topically with 0.5 ␮l of every solution using a Hamilton microapplicator (Hamilton, Bonaduz, Switzerland). The average amount of radioactivity on larvae was 113,996 ⫾ 18,122 cpm, 629 ⫾ 168 cpm and 4,854 ⫾ 358 cpm, for dißubenzuron, pyriproxyfen and tebufenozide respectively. At least nine larvae per time interval and compound were used. After treatment, larvae were randomly divided in groups of three and kept in plastic petri dishes (␾ 9 cm) and provided with S. cerealella eggs ad libitum. At several intervals (varied with the compound), the amount of radioactivity absorbed and the rate of penetration were determined by washing groups of three larvae twice in 1 ml of acetone for 15 min in a 20-ml plastic scintillation vial, as was optimized by Farinos et al. (1999). Acetone was concentrated to dryness, and the amount of radioactivity was determined after adding 10 ml LumaSafe⫹ (Lumac, Mechelen, Belgium) in a Kontron liquid scintillation counter (LKB, Brussels, Belgium) (Smagghe and Degheele 1993). The lack of correlation between the low toxicity of pyriproxyfen and the high penetration rate observed led us to design a new experiment to ascertain pyriproxyfen excretion and accumulation in different parts of the body. Sixteen larvae were topically treated

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Table 1. Effects of pyriproxyfen and tebufenozide on pupal and adult development (mean ⴞ SD) when topically applied to third-instars of C. carnea at single and double field-recommended dose Insecticide

Dosea (ng AI/insect)

Pupation (%)

Adult emergenceb (%)

Eggs/female/d

Egg hatch (%)

0 37.5 75 0 90 180

100 96.4 ⫾ 2.0 100 96.5 ⫾ 1.9 98.3 ⫾ 1.6 96.5 ⫾ 1.9

89.8 ⫾ 2.7 90.0 ⫾ 5.7 93.3 ⫾ 2.7 94.0 ⫾ 3.9 93.0 ⫾ 3.9 98.1 ⫾ 1.8

36.4 ⫾ 2.2 34.5 ⫾ 2.6 41.2 ⫾ 1.9 39.8 ⫾ 1.6 39.3 ⫾ 1.9 31.5 ⫾ 3.6

87.0 ⫾ 4.4 78.2 ⫾ 4.1 82.4 ⫾ 4.4 81.3 ⫾ 2.4 82.7 ⫾ 2.0 87.2 ⫾ 4.2

Pyriproxyfen Tebufenozide

Means within same column for each insecticide were not signiÞcantly different (P ⫽ 0.05, Bonferroni mean separation). n ⫽ 60 larvae per dose. Data of adult emergence are expressed as a percentage of the total number of spun cocoons.

a

b

with 1 ␮l of the radiolabeled solution, kept individually in plastic petri dishes with Þlter paper at the bottom, and provided with S. cerealella eggs. The average amount of radioactivity on larvae was 4,368 ⫾ 437 cpm. To measure the excretion, 24 h after treatment, larvae were dissected to separate the gut, the Malpighian tubules, and the rest of body including fat body, hemolymph, and integument. In addition, the excreted ßuids were collected separately in the Þlter paper. Then, samples were combusted in a Packard Biological Material Sample Oxidizier model 306 (BMO 306, Perkin Elmer Life Sciences, Zaventem, Belgium), the combusted gases (⫹14C-dioxide) were collected with 8 ml of Carbosorb⫹ and 10 ml of Permaßuon⫹ (Canberra Packard, Belgium), and the amounts of radioactivity (in cpm) were measured in an LKBscintillation counter (Smagghe and Degheele 1993). Statistical Analysis. Toxicity data, presented in tables as means ⫾ SD, were analyzed by one-way analysis of variance (ANOVA) using Statgraphics (STSC 1987). Means were separated by the least signiÞcant difference (LSD) multiple range test (P ⬍ 0.05); and in those cases where the F value from ANOVA was not signiÞcant, a Bonferroni test was applied (Milliken and Johnson 1984). Mortality for dißubenzuron and azadirachtin, evaluated after survivors became adults, were subjected to POLO-PC analysis to estimate probit regressions (LeOra Software 1994). Lethal doses in ng AI/insect (LD10, LD50, and LD90) and 95% Þducial limits were calculated. In the penetration bioassay, data were expressed as means ⫾ SD in terms of percentage of the mean value ⫾ SD of the applied amount of 14C-isotope in cpm. Penetration curves were estimated using a polynomial regression curve and the quality of Þtting was evaluated by the curve correlation coefÞcient R (Excel, Microsoft). In the excretion bioassay, data are Table 2.

expressed as mean ⫾ SD percentages of radioactivity recovered (in cpm) on the basis of four replicates of four tissues per sample. Data of excretion violated the premises for performing an ANOVA; therefore, a Kruskal-Wallis test was applied for medians separation, using Statgraphics (STSC 1987). Results Mortality, and Effects on Oviposition and Egg Fertility. For pyriproxyfen and tebufenozide, the percentages of normal pupal formation and adult emergence, and the oviposition and egg fertility data did not differ signiÞcantly from those of the controls, even at double the maximum Þeld-recommended dose (Table 1). For dißubenzuron, a dose as low as 2.26 ng AI/insect caused 50% mortality (Table 2). Treated larvae were able to spin the cocoon at any dose (values ranging between 92.5 and 100%; F ⫽ 1.60; df ⫽ 9,30; P ⫽ 0.1594). However, the pupa died inside the cocoon. Mortality was ⬇100% for doses ⬎10 ng AI/insect. The lowest concentration tested (0.5 ng AI/insect) was used to evaluate the effects on reproduction. The mean number of eggs laid per female and per day and the percentage of egg hatch did not differ signiÞcantly from those of the controls (Table 3). Azadirachtin was also harmful, but at higher doses (Table 2). Treated larvae spun the cocoon at any dose as well (values ranging from 96.6 to 100%; F ⫽ 1.47;df ⫽ 6,21;P ⫽ 0.2365). Mortality occurred during pupal development. Most treated specimens died inside the cocoon. In some cases, the pupae fully developed but failed to ecdyse to the adult stage and died as a pharate adult. At the maximum tested dose (48 ng AI/insect), all the insects died inside the cocoon (Table 4). Surviving adults, assessed when the emergence was ⬎50% (2 and 8 ng AI/insect), laid fertile eggs. But, at 8 ng

Toxicity of azadirachtin and diflubenzuron applied topically to third instars of C. carnea

Insecticide

No.

Slope ⫾ SE

LD10 (95% FL)

LD50 (95% FL)

LD90 (95% FL)

Chi-square

Azadirachtin Dißubenzuron

40 40

1.98 ⫾ 0.42 1.32 ⫾ 0.16

5.55 (2.85Ð7.49) 0.74 (0.43Ð1.05)

11.66 (9.19Ð14.04) 2.26 (1.74Ð2.78)

24.51 (19.26Ð40.43) 6.87 (5.48Ð9.34)

2.13 0.77

Data are based on assesed mortality after survivors became adults. LD-values and slopes (in ng AI/insect) were calculated by probit analysis (Leora Software, Polo-PC, 1994).

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Table 3. Effects on reproduction at sublethal doses of azadirachtin and diflubenzuron when topically applied to third instar of C. carnea Insecticide Azadirachtin Dißubenzuron

proxyfen (1Ð1.5%) was recovered from the Malpighian tubules.

Dose Eggs/female/day Egg hatch (%) (ng AI/insect) 0 2 8 0 0.5

38.7 ⫾ 2.3a 33.1 ⫾ 2.2ab 28.9 ⫾ 2.3b 36.7 ⫾ 3.4a 34.5 ⫾ 1.1a

81.4 ⫾ 1.6a 85.3 ⫾ 4.5a 72.8 ⫾ 3.6a 81.5 ⫾ 3.9a 81.2 ⫾ 4.7a

Within the same column and insecticide, data (means ⫾ SD) followed by the same letter are not signiÞcantly different (P ⫽ 0.05, LSD, Bonferroni mean separation).

AI/insect, oviposition and egg fertility were slightly lower compared with controls, although only the decrease in egg laying was signiÞcant (P ⫽ 0.05) (Table 3). Cuticular Penetration Profiles. After topical application on the dorsal thorax, penetration of dißubenzuron, pyriproxyfen, and tebufenozide was followed over a period of 96 h (Fig. 1). Among the three components, pyriproxyfen showed a conspicuously high rate of penetration. One h after administration, ⬇80% had penetrated, whereas for dißubenzuron and tebufenozide only percentages of 10 Ð20% were recorded in the same time interval. The half-life values (T one-half: the moment that 50% of absorption in the insect body is reached) were calculated using the regression curves (Fig. 1). T [one/half] for pyriproxyfen, dißubenzuron, and tebufenozide was 0.004 h, 57.5 h, and 140.8 h, respectively. Furthermore, the amount penetrated at the end of the experiment (96 h) was ⬇55% for dißubenzuron and 45% for tebufenozide, showing a difference of 10% for these two IGRs. In contrast, 98% of the applied amount of pyriproxyfen had penetrated 24 h after treatment. Excretion of Pyriproxyfen via the Alimentary Canal and Malpighian Tubules. At 24 h after topical application, 98% of 14C-pyriproxyfen had penetrated in the body of C. carnea third instars. However, because 66% of the radioactivity was recovered from the ßuids excreted after 24 h (Fig. 2), it seems that the bigger part of the penetrated material is quickly eliminated via the excretion. About 18% was retained in the gut after 24 h. In contrast, a very low level of 14C-pyri-

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Discussion Predatory insects may be affected by pesticide applications in several ways. Predators may be exposed to a pesticide indirectly by feeding on contaminated prey or directly by insecticide spray and by residual contact with treated plant/substrate material. In this study, we have evaluated four currently used IGRs on their compatibility with the generalist predator C. carnea. In addition, we have evaluated the impact of absorption through the cuticle of three of these IGRs to explain differences in toxicities. After topical application, azadirachtin was highly toxic during the formation of pupae leading to inhibition of adult formation. The LD50-value of this nonoily EC-formulation of azadirachtin (Align) was calculated to be 11.7 ng AI/insect. LD90Õs mortality was estimated at 24.5 ng AI/insect, about the maximum Þeld-recommended dose. Total loss of adult emergence was scored with a dose as low as 48 ng AI/larva. The current results agree with those of Vogt et al. (1998b) showing that residual contact in laboratory conditions with Þrst-instar larvae at maximum Þeldrecommended rate prevented pupation and adult emergence. However, it should be noted that these high mortalities have not been validated in the Þeld for the nonoily EC-formulation Align (Vin˜ uela et al. 1996). In contrast, toxicity assays with developmental stages of C. carnea other than third-instars demonstrated that azadirachtin was harmless for adults (Ruiz et al. 1998), eggs, and pupae (Medina et al. 2001). On the contrary, the current assays conÞrmed the high toxicity against immature stages using this nonoily formulation of Align. When applied topically, dißubenzuron was more toxic to C. carnea larvae than was azadirachtin. The LD50- and LD90-values were estimated to be 2.26 and 6.87 ng AI/insect, respectively. Similarly, when dißubenzuron was applied via ingestion to the last instar of Podisus maculiventris (Say), the predator was strongly affected with a LD50 and LD90 of 7.2 and 37.7 ␮g/ml (De Clercq et al. 1995a). However, in the literature, strong variations in susceptibility to dißuben-

Table 4. Abnormalities in pupation, and inhibition of adult formation of C. carnea, topically treated as third instars with different doses of azadirachtin Concentration (mg AI/liter)

Dose (ng AI/insect)

0 4 16 24 32 48 96

0 2 8 12 16 24 48

a b

Mortalitya (%) No cocoon formation

Death inside cocoon

Death pharate adult

5.0 ⫾ 5.0 0.0 ⫾ 0.0 2.5 ⫾ 2.5 2.7 ⫾ 2.7 2.5 ⫾ 2.5 3.5 ⫾ 2.0 1.5 ⫾ 1.5

7.7 ⫾ 2.5 32.7 ⫾ 10.9 30.0 ⫾ 12.2 43.0 ⫾ 12.8 58.7 ⫾ 14.1 50.2 ⫾ 6.9 98.2 ⫾ 1.7

2.7 ⫾ 2.7 2.5 ⫾ 2.5 12.5 ⫾ 6.2 18.2 ⫾ 6.8 12.7 ⫾ 6.2 24.5 ⫾ 6.9 0.0 ⫾ 0.0

Adult emergenceb (%) 84.2 ⫾ 6.6a 64.5 ⫾ 10.0ab 55.0 ⫾ 6.4bc 33.0 ⫾ 10.9cd 28.5 ⫾ 8.4d 20.5 ⫾ 8.9de 0.0 ⫾ 0.0e

Distribution of total mortality ⫽ No cocoon formation ⫹ death inside cocoon ⫹ death pharate adult. n ⫽ 40 larvae per concentration. Within the same column, means ⫾ sd followed by the same letter are not signiÞcantly different (P ⫽ 0.05; LSD mean separation).

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Fig. 2. Distribution of 14C-pyriproxyfen in the alimentary canal, the Malpighian tubules and the ßuids excreted of third instars of C. carnea at 24 h after topical application (P ⫽ 0.05, Kruskal-Wallis test). At this time interval, nearly all (98%) radioactivity applied had penetrated in the insect body. Data are expressed as percentage (means ⫾ SD) of the applied 14C-radioactivity recovered in the tissues.

Fig. 1. Penetration of (A)14C-dißubenzuron, (B) 14Cpyriproxyfen and (C) 14C-tebufenozide in third instar of C. carnea at different time intervals after treatment. Data are expressed as percentage (means ⫾ SD) of the applied 14Cradioactivity absorbed through the cuticle.

zuron have been noted between predatory insects and the path of exposure. Wilkinson et al. (1978) reported that adults and nymphs of Geocoris punctipes (Say) and larvae and adults of Hippodamia convergens Guerin-Meneville were unaffected by topical application at doses of 10,000 ppm. Ravensberg (1981) reported that repeated treatments of dißubenzuron in apple orchards did not affect numbers of predatory bugs, lady beetles, and lacewings, but they did considerably reduce the abundance of the earwing Forficula auricularia L. Nymphs of the predatory bug Orius niger Wolff were not appreciably affected when exposed to sweet pepper plants treated with dißubenzuron at 300 ppm (Van de Veire 1992). Other chitin synthesis inhibitors, teßubenzuron and trißumuron, were also reported to be very toxic by residual contact treatments toward different instars of Neuroptera, such as Chrysopa oculata (Say), Micromus tasmaniae

Walker and also C. carnea (Broadbent and Pree 1984, Bigler and Waldburger 1994, Vogt 1994, Rumpf et al. 1997, Shuja et al. 1997). As was the case in our assays with C. carnea, treated larvae in the last instar were able to spin normal cocoons, but then died during the molting/metamorphosis process and adult formation was inhibited. The juvenile hormone mimic pyriproxyfen was not toxic to third-instars of C. carnea after topical treatment with doses representing 0.5, 1, and 2 times the Þeld-recommended dose. The low toxicity of pyriproxyfen concurred with the Þndings of Nagai (1990) who studied the side effects on a predatory anthocorid bug of genus Orius. No detrimental effects of pyriproxyfen applied to larvae at recommended Þeld rates of 100 ppm were reported. In addition, the fertility of Orius eggs laid on plants (Solanum melongena L.), that were previously dipped in a 100-ppm concentration, was not different from that of the controls. Likewise, Delbeke et al. (1997) noted that pyriproxyfen was harmless to Orius laevigatus (Fieber) when Þfth-instars were exposed to concentrations up to 1000 ppm via ingestion and residual contact. In contrast, pyriproxyfen caused high mortality to the predatory insects Coccinella septempunctata L., and P. maculiventris (Yakti and Poehling 1988, De Clercq et al. 1995b). For the ecdysone agonist tebufenozide, no abnormalities were observed for third-instar, pupation and adult development, and oviposition and egg fertility. These results agree with Dhadialla et al. (1998) and Carlson (2000) who reported that tebufenozide has no adverse effects against various beneÞcial organisms after screening ⬎150 insect species. The latter data conÞrmed that tebufenozide was very target-selective toward Lepidoptera, which makes this compound a perfect tool for IPM programs using natural enemies. Van De Veire et al. (1996) reported no negative effects in Orius spp. when tebufenozide was used at the Þeldrecommended rate and the insect was exposed by residual contact. Similarly, Smagghe and Degheele

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(1994) found no effects on the development of Orius insidiosus (Say) when the Þfth-larval and adult stage were fed on fresh eggs of Spodoptera spp. treated with 1,000 ppm. tebufenozide. In a second part of this study, we tried to explain differences in toxicity by evaluating the impact of IGR absorption through the cuticle. For the three compounds tested, clear differences in penetration patterns were shown. For chitin synthesis inhibitors in general, it has been shown that these compounds were more active by ingestion than by contact or topical application because their cuticular absorption is relatively low. This phenomenon has often been claimed as the reason for their selectivity against beneÞcial insects (Retnakaran and Wright 1987). However, in our test, dißubenzuron was absorbed for ⬇40% after 24 h which may explain its high toxicity. In Spodoptera spp. larvae, 15Ð30% of the topically applied dißubenzuron, had penetrated the cuticle after 24 h (Smagghe et al. 1997). Thus, these high percentages of penetration may explain the high toxicity of dißubenzuron. In addition, we cannot exclude the possible role of metabolism. However, as was seen for Spodoptera spp. larvae (Smagghe et al. 1997), this was of minor importance for dißubenzuron. In contrast with dißubenzuron, ⬇80 Ð90% pyriproxyfen was absorbed through the cuticle within the Þrst 6 Ð12 h after topical application. In spite of this very rapid absorption, neither toxicity nor effects on fecundity and egg fertility were observed. Dißubenzuron and pyriproxyfen caused severe deformities at ecdysis in nymphs of the predator P. maculiventris (De Clerq et al. 1995a), even though relatively small amounts of radiactivity were ingested by the enemy (8 and 15%, respectively of the amount orally applied to the prey) when sucking on Spodoptera exigua (Hu¨ bner) caterpillars (De Clerq et al. 1995b). Trybou (1993) found that most (75%) of pyriproxyfen was present as nonmetabolized compound, clearly explaining the high toxicity of pyriproxyfen toward P. maculiventris. To explain the lack of toxicity in our study, we could demonstrate that the high amounts of absorbed pyriproxyfen ⬎90%) were rapidly cleared via the ßuids excreted. Surprisingly, very low radioactivity was recovered from the Malpighian tubules, although these ßuids are produced by this excretory organ as an adhesive during locomotion and, at the same time, an excretory end product (Chapman 1998). Obviously, this yellow-brownish ßuid is not stored in the Malpighian tubules and is quickly and repeatedly discharged through the anus. Additionally, it can be hypothesized for larvae of C. carnea that amounts of retained pyriproxyfen can be present as low metabolites. For the ecdysone agonist tebufenozide, penetration was relatively slow and low (only ⬍45%). In contrast, absorption in caterpillars was much more rapid with a T1⁄2 for tebufenozide of about 3 h after application to last-instar S. exigua and Spodoptera littoralis (Boisduval) (Smagghe et al. 1999, 2001). Consequently, we suggest that the low penetration of tebufenozide helps to explain its nontoxicity to C. carnea larvae, but in addition, target site differences in

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the molting hormone receptor might play a major role. The latter hypothesis is consistent with the concept that structure and biochemical properties of ecdysteroid receptors may differ among insect species (Bidmon and Sliter 1990, Carlson 2000). Taken together, the current data with 14C-labeled isotopes of dißubenzuron, pyriproxyfen, and tebufenozide indicated that no simple relationship on C. carnea larvae could be drawn between toxicity and cuticle penetration. It is safe to suggest that the tolerance or resistance of natural enemies toward pesticides involves a complex of processes besides penetration. Once the insecticide has penetrated the cuticle, it is subjected to metabolism and excretion on its path to its target site in the insect body. In addition, differences in the biochemical mode of action of the studied compounds and target site variations may also have caused the different potencies. It should also be noted that extrapolation from oral feeding toxicity results to topical toxicity data should be evaluated with caution, especially for IGRs used in IPM programs. In conclusion, although it has been reported that the studied IGRs exhibit good selectivity toward several beneÞcial insects, our results suggest that the generalist predator C. carnea may be harmed by direct contact with azadirachtin and dißubenzuron. So, the application of these insecticides must be considered carefully for use in IPM programs involving populations of C. carnea. However, it is clear that more research is needed to test the side-effects on both compounds against different developmental stages. Also, this laboratory experiment should be veriÞed in Þeld conditions. For the juvenile hormone mimic pyriproxyfen and the ecdysone agonist tebufenozide, no adverse effects were observed, and these compounds can be considered as safe for use together with C. carnea. Acknowledgments This work was supported by the Spanish Ministry of Education and Culture (proyect AGF 99 Ð1135) to EV. PM is recipient of a grant from the Autonomous Community of Madrid (Spain). GS acknowledges the Fund for ScientiÞc Research (Brussels, Belgium) for a postdoctoral grant.

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