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Effects of RH 5849 the first nonsteroidal ecdysteroid agonist on larvae of Spodoptera littoralis Boisd. LepidopteraNoctuidae

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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 [12]. 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 [13] and analyzed
with the probit option of POLO [14]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
[15], who used Bradford’s method [16] 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 [19]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. Robbins WE, Kaplanis JN, Thompson JN, Shortino TJ, Cohen CF, Joyner SC: Ecdysones
and analogs: Effects on development and reproduction of insects. Science 161, 1158(1968).
2. Robbins WE, Kaplanis JN, Thompson JN, Shortino TJ, Joyner SC: Ecdysones and synthetic
analogs: Molting hormone activity and inhibitive effects on insect growth, metamorphosis
and reproduction. Steroids 16, 105 (1970).
3. Bergamasco R, Horn DHS: The biological activities of ecdysteroids and ecdysteroid analogues. In: Progress in Ecdysone Research. Hoffmann JA, ed. Elsevier, Amsterdam, pp
299-324 (1980).
4. Galbraith MN, Horn DHS, Kelly BA, Kinnear JF, Martin M-D, Middelton EJ, Virgonia CTF:
Molting hormones. LIII. The synthesis and biological activity of some ecdysone analogues.
Austr J Chem 34, 2607 (1981).
5. Wing KD: RH 5849, a nonsteroidal ecdysone agonist: Effects on a Drusophila cell line. Science
241, 467 (1988).
6. Wing KD, Slawecki RA, Carlson GR: RH-5849, a nonsteroidal ecdysone agonist: Effects on
larval lepidoptera. Science 241, 470 (1988).
7. Aller HE, Ramsay JR: RH 5849-A novel insect growth regulator with a new mode of action,
BCPC-Pests and Diseases 5, 511 (1988).
8. Wing KD, Ramsay JR: Other hormonal agents: Ecdysone agonists. Progress and Prospects
in Insect Control. BCPC Monograph Nu 43, 107 (1989).
9. Kiuchi M: Effects of an insect growth regulator, RH 5849, on the larval molt and larval-pupal
metamorphosis of the silkworm, Bombyx mori. Abstract Seventh International Congress of
Pesticide Chemistry, Hamburg, August 1990.
10. Silhacek DL, Oberlander H, Porcheron P: Action of RH 5849, a non-steroidal ecdysteroid
mimic, on Plodiu interpunctella (Hiibner) in vivo and in vitro. Arch Insect Biochem Physiol
15, 201 (1990).
11. Auda M, Degheele D: Joint action of pyrethroids with chitin synthesis inhibitors, organo-
phosphorus and carbamate insecticides on a susceptible and resistant strain of Spodopteru
littoralis. Med Fac tandbouwwet Rijksuniv Gent 50, 751 (1985).
12. GIFAP: Insect Resistance Action Committee, Newsletter No 5: Insecticide/acaricidesusceptibility tests, Irac-Method No 7, pp 15-16 (1990).
13. Abbott WS: A method of computing the effectiveness of an insecticide. J Econ Entomol 18,
265 (1925).
128
Srnagghe and Degheele
14. LeOra Software: POLO-PC. A user’s guide to probit or logit analysis. LeOra Software Inc.,
Berkeley, California (1987).
15. Auda M: Critical approach of the insecticide resistance in Spodopteru littotalis Boisd. and
Muscu dornesticu L. PhD thesis, Fac Landbouwwet, Univ Gent, 199 pp (1986).
16. Bradford MM A rapid and sensitive method for quantification of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248 (1976).
17. Riddiford LN: Hormone action at cellular level. In: Comprehensive Insect Physiology
Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon, Oxford, Vol8, pp
37-84 (1985).
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Insect Physiology Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon,
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