Effects of RH 5849 the first nonsteroidal ecdysteroid agonist on larvae of Spodoptera littoralis Boisd. LepidopteraNoctuidaeкод для вставкиСкачать
Archives of insect Biochemistry and Physiology 21 :119-128 (1992) Effects of RH 5849, the First Nonsteroidal Ecdysteroid Agonist, on larvae of Spodoptera littoralis (Boisd.) (Lepidoptera: Noctuidae) Guy Smagghe and Danny Degheele Laboratory of Agrozwlogy, Faculty of Agricultural Sciences, University of Ghent, Ghent, Belgium The effects of RH 5849 on the larval molt and larval-pupal metamorphosis in Spodoptera littoralis (Boisd.) were examined. Application of RH 5849 to newly ecdysed 3rd and 6th (last) instar larvae induced a larval molt within the first day after application. Symptoms included cessation of feeding and larval weight, extrusion of gut, loss of hernolymph, and a developmentally abnormal and subsequent lethal larval ecdysis. Treated larvae died shortly afterwards. Treated 6th instar larvae, which did not appear to be affected before pupation, showed an abnormal and lethal pupation process. Differences in the protein pattern of the cuticle between treated vs. untreated 6th instar larvae, demonstrated by using PAGE, indicated that in the newly induced larval cuticle some proteins were missing or expressed with less intensity. The lack of several bands in the pattern of cuticular and hernolymph proteins of treated vs. untreated 6th instar larvae, probably from proteins specific for the pupal instar, is suggested as a cause of unsuccessful pupation in the treatment. D 1992 Wiley-Liss, Inc. Key words: toxicity, physiology, larval molting, larval-pupal metamorphosis, development, cuticular proteins INTRODUCTION During the last decade many investigations have been carried out concerning the possible use of ER* as original target sites for new insecticides [ 1 4 ] . Acknowledgments: The authors express their grateful thanks to H.E. Aller (Rohm and Haas Company, Spring House, Pennsylvania)for providing a sample of RH 5849 of technical grade for this study. Received April 6, 1992; accepted June 29, 1992. Address reprint requests to D. Degheele, Laboratory of Agrozoology, Faculty of Agricultural Sciences, University of Ghent, Coupure Links, 653, 8-9000 Ghent, Belgium. *Abbreviationsused: ER = ecdysteroid receptors; ICR’s = insect growth regulators; IPM = integrated pest management; LC-p line = log(concentration)-probitline; LC25, LC50 and L& = concentrations required to kill 25, 50 and 95% of larvae treated, respectively; RH 5849 = 2’-benroyLl’-tertbutylbenzohydrazide. 0 1992 Wiley-Liss, Inc. 120 Smagghe and Degheele The binding of ecdysteroids (e.g., 20-hydroxyecdysone)to the ER induces the molting process in invertebrate larvae (e.g., caterpillars). Disturbance of this process by using ecdysteroid agonists may be a tool in controlling resistant pest insects and may be used in IPM-programs. RH 5849 was the first nonsteroidal ecdysteroid agonist to be described [5-71. It was demonstrated that RH 5849 binds to the ER in a manner competitive with the natural ecdysteroids [6,8]. The ecdysteroid mimic acted specifically against caterpillars and induced a premature and subsequently lethal larval molt [6,8-lo]. In the study described here, RH 5849 was applied to larvae of the Egyptian cotton leafworm, Spodopteru littoralis (Boisd.). The aim of the study was to evaluate the toxicity of RH 5849 and the changes in development of 3rd and 6th (last) instar larvae caused by this compound. Changes in the pattern of cuticular and hemolymph proteins of treated vs. untreated 6th instar larvae were also examined. The data obtained may be an indication of the usefulness of RH 5849 and help in better understanding its activity. MATERIALS AND METHODS Insects The strain of Spodopteru littoralis (Boisd.) was originally provided by R. Neumann (Ciba-Geigy,Basel, Switzerland). The insects were reared on castor bean leaves (Ricinus communis L.) under standard laboratory conditions of 23"C, 75%RHand a photoperiod of 16:8 (L:D) [ll]. Chemicals and Treatment RH 5849 (99.9%technical) was purchased from Rohm and Haas Company (Spring House, Pennsylvania). Suitable solutions of RH 5849 were made in water with 0.02% Triton X-100. Castor bean leaves were dipped in the solution for 10 s, allowed to dry for 45 min at room temperature in a fume hood and fed to the larvae. Freshly treated leaves were supplied daily. All assays were kept under standard conditions as specified above. Toxicity Assay At each assessment, larvae were classed as unaffected, i.e., giving a normal response when gently stimulated by touch, or either as dead or affected. The latter gave an abnormal response to stimulation or showing abnormal growth or behavior as compared to controls. Mortality percentages included both dead and affected . The treated leaves were placed in glass jars (ca. 300 rnl) containing ten 3rd or five 6th instar newly ecdysed (0-2 h) larvae. At the end of the 6th instar, some wood shavings were placed in the jars to allow pupation. For each concentration, 30 insects were used. Mortalities were scored after 4 days (3rd instar) and 8 days (6th instar). These data were corrected for untreated mortalities using Abbott's formula  and analyzed with the probit option of POLO afterwards. Probit analysis is the standard method for analysis of toxicity data. LC50 is the concentration which corresponds to probit 5 or 50% mortality on the LC-p line. The larvae were Effects of RH 5849 on Spodoptera littoralis larvae 121 examined at 12 h intervals for weight gain, molting, pupation and specific symptoms due to the treatment. Protein Characterization Assays were conducted with 6th instar larvae treated with a concentration of RH 5849 used to obtain the LC5o-value. Treated larvae were dead at day 5 of the 6th instar. After soaking two cuticles in cold distilled water for 10 min, they were homogenized in a glass homogenizer with distilled water (100 mg wet weight of cuticle per 1 ml water). The suspension was centrifuged at 10,OOOg for 10 min. The supernatants, which contained the water-soluble fraction of cuticular proteins, was used for PAGE. Following Auda’s example , who used Bradford’s method  to determine protein content, a electrophoreticseparation of 10 pl supernatant was carried out on an ultrathin pore-gradient polyacrylamide gel (250 x 120 x 0.5 mm and T = 5.5-11.1%) and later stained with Coomassie blue R-250. Hemolymph was collected from a wound made by cutting off a left thoracic leg of 6th instar larvae, which were anesthetized with C02. An electrophoreticseparation of 5 p1 supernatant was performed as described for the larval cuticle proteins to study the pattern of hemolymph protein. RESULTS Toxicity of RH 5849 on 3rd and 6th Instar Larvae Toxicity values of RH 5849 on 3rd and 6th instar larvae of S. tifforalis are presented in Table 1. Generally, last instar larvae were more susceptible than 3rd instar larvae. The LC5o-values for 6th and 3rd instar were 21.94 and 84.15 ppm, respectively. Effects of RH 5849 on Development, Molting, and Larval Growth of 6th Instar Larvae When high concentrations were applied (333 ppm), external symptoms of an induced molt were visible within the first 24 h of continuously feeding RH 5849 to newly ecdysed larvae. Clear symptoms of head capsule apolysis were seen and the treated larvae underwent head capsule slippage shortly afterwards. However, such larvae did not ecdyse successfully thereby double head capsules were visible. Figure 1A shows a well-formed double head capsule of a 2nd-day-old 6th instar larvae treated with 100 ppm. Underneath the new head capsule, the mandibles of the old cuticle were hidden thereby preventing TABLE 1. The Lc25-, LCw-, and LCg5-Values (ppm), the 95%Confidence-Limits(CL95) (ppm) and the Slope of the LCp Line of RH 5849 for 3rd and 6th Instar Larvae of S. littoralis Larval instar LC25 Cb5 LCSO c L 9 5 LC95 cL95 Slope 3rd 6th 46.22 12.96 39.72-53.78 11.0G15.19 84.15 21.94 73.07-96.91 19.40-24.82 364.83 79.60 244.67-544.00 62.67-101.11 2.57 2.93 122 Smagghe and Degheele Effects of R H 5849 on Spodoptera httoralis larvae 123 the larva any further feeding. Food intake and weight gain ceased in comparison to the control and dose dependently, from the moment clear signs of the induced molt were visible. In larvae fed RH 5849 at concentrations 233 pprn larval growth was markedly inhibited (Table 2). In such larvae other symptoms of an unsuccessful molt were visible. Treated larvae showed an extrusion of hind gut and also lost a lot of hemolymph. The old cuticle was incompletely shed and an adhesion of remnants of the old cuticle onto the new cuticle was seen (Fig. lB,C). Before dying, treated larvae turned black, probably due to oxidation. Treated 6th instar larvae died in their old larval cuticle at approximately 36 h into the 6th instar. Larvae treated with lower concentrations, 3 and 10 ppm, died later in the 6th instar. Head capsule apolysis was observed at about 60 h in the 6th instar. At the moment such larvae showed double head capsules, weight gain ceased (Table 2). Treated larvae also did not shed their old cuticle completely and died shortly afterwards. Treated larvae, which did not appear to be affected before pupation, showed an abnormal and lethal pupation. Such larvae were unable to synthetize or to deposit a normal pupal cuticle, nor to ecdyse successfully from the last larval cuticle. When 1 ppm of RH 5849 was applied, no visible symptoms were observed during the last larval instar. Prepupation and successful pupal ecdysis took place at the same time as in the control. Effects of RH 5849 on Development, Molting, and Larval Growth of 3rd Instar Larvae Larvae fed RH 5849 at 100 ppm, showed symptoms of an induced molt within the first 24 h of the 3rd instar. Head capsule slippage took place shortly afterwards, however double head capsules were formed. Food-intake and weight gain ceased from that moment in comparison to the control (Table 3). Other symptoms of toxicity included extrusion of gut, loss of hemolymph, and an incomplete shedding of the old cuticle. Treated larvae died shortly afterwards. No visible effects were observed at doses of 1, 3, 10, and 33 ppm R H 5849. Successful larval ecdysis appeared in about 90% in each treatment at the same time as in the control. Effect of R H 5849 on the Pattern of Cuticular Proteins of 6th Instar Larvae The pattern of cuticular proteins comprised 24 bands. Between 30 and 36 h the intensity of all the bands in the treatment had greatly diminished. Between 42 and 48 h, bands 4 and 5 had disappeared and bands 12,13,18,19, and 20 were nearly absent in the RH 5849 treated larvae vs. untreated larvae (Fig. 2). In the controls, the pattern of cuticular proteins revealed a remarkable change between 42 and 72 h into the 6th instar. Such change is probably related to apolysis. From day 3 onwards, bands 14, 6, 7, 11-24 were observed on day Fig. 1 . Symptoms of 6th instar 5. littoralis larvae treated with 100 ppm RH 5849. (A) first symptoms of the prematurely induced larval molt: head capsule apolysis, double head capsule; and the old cuticle shed incompletely. Remnants of the old cuticle remained on the new cuticle: lateral view of ( 6 )front and (C)hind segments. 12 110.36a 112.49a 108.09a 110.63a 110.57a 109.09a 0 83.55a 81.52a 82.79a 83.41a 81.71a 83.73a 190.75a 190.39a 190.29a 2O5.93a 187.89a 113.77b 24 227.47a 232.05a 231.83a 235.61a 200.81b 110.39~ 36 302.23a 318.30a 324.75a 344.15a 197.5513 100.63c 60 378.37ab 424.56a 402.93a 349.30b 182.46~ 82.09d Hours into 6th instar 48 459.59a 473.58a 483.77a 344.38b 197.08~ 73.85d 72 210.31~ 71.65d 344.8213 461.53a 492.45a 484.86a 84 -e 373.69a 389.01a 390.27a 369.45a 218.03b 96 “Average larval weight was obtained by weighing 6th instar larvae in 6 groups of 5 larvae for each treatment; multiple range test by Duncan I191 was camed out; values in the same column with the same letter (a,b,c,d) are not significantly different (P < 0.05). ‘AU larvae treated were dead. 0 1 3 10 33 100 (p??m) RH 5849 TABLE 2. Effect of RH 5849 on the Fresh Weight (mg) of 6th Instar Larvae of S. littoralis (For living l a m e only, larval weight is shown)* Effects of R H 5849 on Spodoptera liftoralis Larvae 125 TABLE 3. Effect of RH 5849 on the Fresh Weight (mg) of 3rd Instar Larvae of S. littoralis (For living larvae only, larval weight is shown)* RH5&19(ppm) 0 1 3 10 33 100 0 1.41a 1.43a 1.37a 1.39a 1.39a 1.43a 12 2.07a 2.13a 2.18a 2.16a 2.29a 2.21a Hours into 3rd instar 24 36 48 2.95a 3.04a 3.15a 3.17a 3.14a 2.7% 4.65a 4.70a 5.08a 4.84a 4.99a 3.77b 5.52a 5.51a 6.16a 5.86a 5.55a 3.82b 60 5.45a 5.29a 5.53a 5.79a 5.47a 3.66b 72 5.39a 5.31a 5.65a 5.65a 5.41a 3.60b *Average larval weight was obtained by weighing 3rd instar larvae in 3 groups of 10 larvae for each treatment; multiple range test by Duncan was carried out; values in the same column with the same letter (a,b) are not significantly different (P < 0.05). 3 in untreated larvae. This pattern remained until day 5. On day 6 bands 1-3, 18, and 23 were no longer observed in the pattern of the control. In the treated larvae bands 1 3 , 1 4 1 7 , and 24 could not be observed on day 3. Bands 9 and 24 were expressed with less intensity in comparison with the control at the same time. On the fourth day, the protein pattern of the treated larvae was hazy: only bands 4, 19, 20, and 22 appeared, but were very pale (Fig. 2). Effect of R H 5849 on the Pattern of Hemolymph Proteins of 6th Instar Larvae The pattern of hemolymph proteins comprised 14 bands. In treated larvae bands 7 and 8 never appeared although they were continuously seen in the control from day 3 to day 6 (Fig. 3). DISCUSSION Differences in sensitivity to RH 5849 between 6th and 3rd instar larvae of S. Zitfordis were observed. Although there were differences in toxicity, the Fig. 2. Pattern of cuticular proteins of untreated (A) and treated (B) sixth instar 5. littoralis larvae revealed by use of PAGE. 126 Smagghe and Degheele Fig. 3. Pattern of hemolymph proteins of untreated (A) and treated (B) sixth instar 5. littoralis larvae revealed by use of PAGE. same prime activity was observed on 6th and 3rd instar larvae. It was very surprising that an induction of a larval molt appeared in both instars when equal concentrations of RH 5849 were continuously fed to the larvae. The LC5o-values obtained with RH 5849 are relatively high for insecticidal use. However, all toxicity experiments were performed with technical product. Our results suggested that RH 5849 can stimulate the epidermal cells to undergo apolysis and to synthesize proteins. Probably, at the moment RH 5849 binds to the ER of the epidermal cells, a new molt was induced. However, to assure a successful molting process, the ecdysteroid titer must decline after reaching peak levels [17,18]. In contrast to 20-hydroxyecdysone, RH 5849 is easily absorbed into the hemolymph and epidermal cells and had a great longevity in the larval body [6,7]. In the initial stage of RH 5849 binding to ER the epidermal cells supposedly respond by shutting off the synthesis of intermolt products and prepare for the deposition of new larval cuticle. A correlation between the prematurely induced molt in treated larvae and the changes in the pattern of cuticular proteins could be evaluated. Malformation of the new larval cuticle was confirmed by differences observed between the pattern of cuticular proteins of untreated larvae during the first hours after the 5th ecdysis and the pattern of treated larvae about two days after the 5th ecdysis. Absence of such proteins in the new cuticle may be the cause of loss of hemolymph, as seen in treated larvae. Cuticle proteins play an important role in cuticle formation. Continuous binding of RH 5849 to ER is probably the reason for disruption of development of the treated larvae. From apolysis to prepupation, the cuticle of treated last instar larvae differed greatly from the cuticle in the control larvae. With regard to the pattern of hemolymph proteins several protein bands never appeared in treated last instar larvae, although these bands appeared in the controls from apolysis until prepupation. The absence of such Effects of RH 5849 on Spodoptera littoralis larvae 127 bands, which are probably specific for pupal cuticle proteins, can therefore be related to an unsuccessful pupation process that had been observed in treated larvae. The results presented here agree with the proposed activity of RH 5849, namely that of imitating the natural ecdysteroids. RH 5849 and its analogues are therefore important insecticides worth further investigation. Moreover, they can provide a better understanding of larval molting and larval-pupal metamorphosis. LITERATURE CITED 1. 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