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Ecdysteroid titers and molting aberrations in last-stage Oncopeltus nymphs treated with insect growth regulators
PEUICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
18,
351-356 (1982)
Ecdysteroid Titers and Molting Aberrations in Last-Stage Oncopeltus Nymphs Treated with Insect Growth Regulators1,2 R. E. REDFERN,~ Agricultural
Environmental
Quality
T. J.
KELLY,~
A. B.
BORKOVEC,~
Institute, Agricultural Research Beltsville. Maryland 20705
D. K.
AND
Service,
U.S.
HAYES~
Department
of Agriculture.
Received February 11, 1982; accepted July 21, 1982 The ecdysteroid titers of insect growth regulator (IGR)-treated and untreated large milkweed bug nymphs, Oncopeltus fasciatus (Dallas) (Heteroptera: Lygaeidae), were determined by radioimmunoassay. All of the IGRs tested affected the ecdysteroid levels and the ecdysis of both sexes in some way. AI3-63604, a juvenile hormone mimic, accelerated ecdysteroid production and its subsequent decline, shortened the stadium, and induced a supernumerary nymphal molt. Diflubenzuron had no effect on the onset of ecdysteroid production but slightly retarded the decline in ecdysteroid titers before adult ecdysis, and the ecdysis was incomplete. Ecdysteroid production was delayed and erratic in nymphs treated with azadirachtin and AI3-63967, but the effect on ecdysis was different for each compound. Azadirachtin caused incomplete adult ecdysis, whereas AI3-63967 completely prevented any attempts at ecdysis. The large peak of exdysteroid activity associated with apolysis was absent in starved insects, and they made no attempt to ecdyse.
caused either an incomplete ecdysis (5) or no attempt at ecdysis (6). In view of the previous correlation between ecdysial inhibition and ecdysteroid titers, we investigated the possibility that the inhibitory activity of all IGRs might be correlated with an effect on the ecdysteroid titers.
INTRODUCTION
Insect growth regulators (IGRs) vary widely in their effects on different insect species. This is especially true for juvenile hormone (JH) active IGRs (1) with effects ranging from inhibition of ecdysis and metamorphosis to defective metamorphosis or defective adult emergence. In at least one instance (2), JH treatment of the lastinstar German cockroach, Blattella germanica (L.), caused the formation of permanent larvae apparently through a reduction in the ecdysteroid titers. These titers were suppressed below the levels necessary for ecdysis and metamorphosis. In evaluating some IGRs on last-stage Oncopeftus nymphs, we observed that JHactive IGRs decreased the duration of the last stadium and produced supernumerary (sixth instar) nymphs (3, 4). Other compounds prolonged the fifth stadium and
MATERIALS
AND
METHODS
Test compounds. Pure AI3-63604 (2,6difluoro-l\r-[[4-[(3-fluorophenyl)methoxy]phenyl]methyl]benzenamine) and A13-63967 (1-pyrrolidinecarbothioic acid, 2-[ I-(2-pyridinyl)ethylidene]hydrazide), were supplied by A. B. DeMilo, Insect Reproduction Laboratory. Pure azadirachtin was supplied by J. D. Warthen, Jr., Biologically Active Natural Products Laboratory, Beltsville, Maryland, and diflubenzuron (technical grade, 96% pure) was kindly supplied by Thompson-Hayward Chemical Company, Kansas City, Kansas. Test insects. Newly ecdysed (pale orange) fifth-instar Oncopeltus fasciatus nymphs were collected from our laboratory stock culture that was maintained in 9.5 x 17 x 30-cm polystyrene boxes at 27 -+ 1°C
I Heteroptera:Lygaeidae. ’ Mention of a proprietary product does not constitute an endorsement by the U.S. Department of Agriculture. 3 Livestock Insects Laboratory. 4 Insect Reproduction Laboratory. 351
0048-3.575182106035l-06$02.00/0
352
REDFERN
and 50 2 5% RH under a 14 hr light:lO-hr dark photophase. Stock insects were provided with field-collected milkweed seeds and water in cotton-stoppered vials ad fibitam. The test insects were collected over a 4- to 6-hr period beginning at 7 AM on the day they were to be used. Collected nymphs were held in Erlenmeyer flasks without food or water until treatment with the test compounds a few hours later. Bioassay. AI3-63967 was dissolved in a mixture (1:l vol) of acetone and dimethyl sulfoxide (DMSO) because the solubility in acetone was marginal. All other compounds were dissolved in acetone. The dose of all compounds was 10 pg/nymph; only diflubenzuron was applied at a dose of 9 &nymph because of its limited solubility in acetone. These dose levels were selected to give clear and characteristic effects in all treated insects. The test solutions (1 Flinymph) were applied topically with a calibrated glass micropipet to the ventral side of the last three abdominal segments of the nymph. Treated and control nymphs were confined in 240-ml paper cartons capped with a clear plastic petri dish lid (9 cm) and supplied with milkweed seed and with water in cotton-stoppered vials. Insects were inspected daily until they ecdysed or died or the test was terminated. The treated nymphs from which hemolymph was collected were confined in a similar manner, except that one set of untreated controls was starved for the duration of the test. Hemolymph was collected from three nymphs of each sex every 24 hr.
Ejjkts
of Topically
Applied
Insect
Grmvth
TABLE Regrtlators
ET AL.
Extraction and radioimmanoassay of hemolymph ecdysteroids. Three males and three females were bled by leg or antenna1 amputation every 24 hr. The resulting l-p1 hemolymph samples were collected in l- to ~-PI borosilicate capillary glass pipets and dispensed into borosilicate test tubes (10 x 75 mm) that were closed with parafilm and frozen immediately at -20°C. For extraction of ecdysteroids, hemolymph samples were thawed, diluted to 500 ~1 with 75% methanol, vortexed vigorously, and centrifuged at 3000g for 30 min. The supernatant was carefully removed and either stored at -20°C or a 100~~1 sample was analyzed immediately by radioimmunoassay (RIA) (7). The antibody, prepared from the hemisuccinate of ecdysone derivatized at the C-22 hydroxyl only, was a gift from W. E. Bollenbacher, University of North Carolina at Chapel Hill. RESULTS
AND
DISCUSSION
All four IGRs used in this study prevented normal adult ecdysis (Table 1). The juvenile hormone mimic (JHM), AI363604, produced perfectly ecdysed sixthinstar (supernumerary) nymphs (Fig. 1A) beginning on the fifth day post-treatment. These nymphs either lived 7-10 days before they attempted to ecdyse to the adult (20%) or died without any attempt at ecdysis. The ecdysis inhibitor, AI3-63967, prevented any attempt at ecdysis on the seventh day or at anytime before death several days later. Removal of the old partially digested cuticle from these insects revealed 1 OII Ecdysis
of Fifth-Stage
Oncopeltus Ecdysis
IGR compounds
Dose b.&vmph)
AI3-63604 A13-63967 Diflubenzuron Azadirachtin Acetone Acetone:DMSO ’ Represents
average
IO 10 9 10 1 4 1 PI of four
replicates;
five
nymphs/test.
type (5% ecdysis) Sixth instar (100) None (0) Incomplete (100) Incomplete (100) Normal adults (100) Normal adults (100)
fusciatus”
ECDYSTEROID
TITERS,
FIG. 1. The effects of topically \lirh four IGRs at IO pglpllnymph (B) AI3-63967. (C) d$?ubenzaron,
MOLTING
ABERRATIONS,
AND
Oncupehs
NYMPHS
treating rzewly ecdysed fifth-instar Oncopeltus fasciatus nymphs. except diflubenzrrron ,r%ich +vas at 9 p~lplln~mph. (A) AI3-63604, and (II) a:adirachtirr.
a newly formed cuticle with adult pigmentation on the fourth abdominal sternite (Fig. 1B). The chitin synthesis inhibitor, diflubenzuron, and the antifeedant, azadirachtin, caused incomplete ecdysis (Figs. 1C and D). The diflubenzuron-treated insects attempted ecdysis beginning on the seventh day; while the azadirachtin-treated insects attempted ecdysis beginning on the ninth day. In either case, death usually occurred within 24 hr after the attempt began,
and dissection revealed adult cuticle. In control insects, untreated or solvent treats :d, ecdysis began on the seventh day and Mias completed in all animals by the eighth d:iY. No ecdysis was observed in the untreatf :d, starved nymphs which died from 6 to 12 days after the beginning of the stadium. The ecdysteroid titers were affected to some extent by all the test compounds, tcIa slight extent in acetone-treated animals (co Impare Fig. 2A with C), and slowly drop1 Ied
354
REDFERN
to undetectable levels in starved controls (Fig. 2G). The JHM, AI3-63604, was the only compound tested that accelerated ecdysteroid production and its subsequent decline (compare Figs. 2A and C with E). Smith and Nijhout (8) observed similar ef-
ET
AL.
fects in fifth-instar 0. fa.sciat44s treated with the JHM, methoprene. In our tests, this pattern of ecdysteroid titers correlates with the shortening of the duration of the fifth instar in JHM-treated nymphs. Continuous exposure of the nymphs to AI3-63604
ECDYSTEROID
TITERS.
MOLTING
ABERRATIONS,
(R. E. Redfern, unpublished results), even up to levels of 4000 pgIcm*, did not produce permanent larvae such as those found with JH-active IGR treatment of last-instar B. germanica (2). Contrary to the suggestion by Vogel er al. (1) that in last-stage hemipteran nymphs treated with JH-active IGRs the amount of ecdysone inducing the ecdysis is too low for metamorphosis, we agree with Smith and Nijhout (8) that the JH-active IGRs stimulate premature ecdysteroid production; however, the mechanism of these processes remains unclear. In holometabolous insects, JHMs appear to suppress the events induced by a transient ecdysteroid reprogramming peak (9, 10); however, such a peak was not apparent in our study (Fig. 2A). A more critical analysis of ecdysteroid titers in a synchronous population of last-instar 0. fascialus may resolve this question. Diflubenzuron had no effect on ecdysteroid production, but it slightly retarded the decline in ecdysteroid titers before the attempted adult ecdysis (compare Figs. 2A and C with F). This decline in the ecdysteroid titers correlates with the attempted ecdysis. Diflubenzuron acts by blocking the terminal polymerization step in chitin formation (11, 12). However, it has also been suggested that diflubenzuron affects chitin production by interfering with the metabolism of 20-hydroxyecdysone, thereby reducing its rate of inactivation and, thus, stimulating chitinase production (13, 14). Our results agree in part with this latter suggestion, that diflubenzuron may slightly retard ecdysteroid degradation. Furthermore, the demonstration by Hajjar and Casida (12) that metabolism of radiolabeled ecdysone and 20-hydroxyecdysone in fifth-instar 0. fusciatus nymphs is unaffected by diflubenzuron must be reexamined in light of the recent discovery that makisterone A is the principal molting hormone in fifth-instar 0. fusciatlls (15). Azadirachtin affected both ecdysteroid production and the decline in ecdysteroid titers before the attempted adult ecdysis (compare Figs. 2A and C with D). There
AND
Oncoprltus
NYMPHS
355
was no correlation between the variation in ecdysteroid titers and attempted ecdysis. In fact, the ecdysteroid titers varied strikingly between sexes and even between individuals within the same sex as evidenced by the large standard errors. The ecdysial inhibitor, AI3-63967, delayed the increase in ecdysteroid titers until Day 5 or 6 whereupon the titers increased to about half the normal peak level (compare Figs. 2A and C with B). This response was similar to the effect caused by azadirachtin, and again there was a large variation between sexes and between individuals within the same sex. The lack of any attempt at ecdysis in AI363967-treated nymphs suggests, however, that the mode of action of these two compounds may be different with respect to ecdysis. Slama (16) has demonstrated that the reduction in ecdysteroid titers that normally occurs at the end of an instar is necessary to allow the ecdysial contractions, which result in splitting of the old cuticle. Truman (17) has further shown that this same reduction in ecdysteroid titers is necessary for eclosion hormone release that triggers the subsequent behavioral and physiological events culminating in the shedding of the old cuticle. Since AI3-63967 prevents this reduction in ecdysteroid titer and no attempt at ecdysis occurs, it is possible that AI3-63967 acts by a similar mechanism in preventing eclosion hormone release. Azadirachtin, on the other hand, must be acting through a different mechanism since the ecdysteroid titers remain elevated and ecdysis still occurs. The similarity between the morphological effects of diflubenzuron and azadirachtin suggests that chitin synthesis may be blocked by azadirachtin. Furthermore, the structural similarity of azadirachtin to ecdysone suggests that it may act as an ecdysone analog (18) or possibly as a competitive inhibitor. The suppression of the feedback inactivation of ecdysteroids following their peak production (19) and blocking of the suppression of eclosion hormone release by high titers of ecdysteroids may be some of the effects of
356
REDFERN
azadirachtin. Unfortunately, varying hormone titers in the of new cuticle synthesis and ecdysis are still undetermined
the roles of programming the resultant (10).
ACKNOWLEDGMENTS
We thank G. D. Mills, Jr. (Livestock ratory) and Suzanne M. Spalding (Insect Laboratory) for technical assistance.
Insets LaboReproduction
REFERENCES
1. W.
Vogel, P. Masner. 0. Graf, and S. Dorn, Types of response of insects on treatment with juvenile hormone active insect growth regulators, Experientia 35, 1254 (1979). 2. P. Masner, W. Hangartner, and M. Suchy, Reduced titers of ecdysone following juvenile hormone treatment in the German cockroach, Blattella germanica. J. Insect. Physiol. 21, 1755 (1975).
R. E. Redfern, T. P. McGovern, R. Sarmiento, and M. Beroza. Juvenile hormone activity of mixed ethers containing a phenyl and a terpenoid moiety applied topically to the large milkweek bug and the yellow mealworm. J. Econ. Entomol. 64, 374 (1971). 4. A. B. DeMilo and R. E. Redfern, New juvenile hormone mimics: Aromatic Schiff bases and related compounds against the large milkweed bug and yellow mealworm, J. Agr. Food Chem.
ET
AL.
8. W. A. Smith and H. F. Nijhout. Effects of a juvenile hormone analog on the duration of the fifth instar in the milkweed bug, Oncopeltus fusciatus. J. Insect Physiol. 27, 169 (1981). 9. L. M. Riddiford, Ecdysone-induced change in cellular commitment of the epidermis of the tobacco hornworm, Manduca sexta, at the initiation of metamorphosis, Gen. Comp. Endocrinol. 34, 438 (1978). IO. R. L. Dean, W. E. Bollenbacher, M. Locke, S. L. Smith. and L. I. Gilbert, Haemolymph ecdysteroid levels and cellular events in the intermoultimount sequence of Culpodes ethlius. J. Insect Physiol. 26, 267 (1980). 11. N. P. Hajiar and J. E. Casida, Insecticidal benzoylphenyl ureas: Structure-activity relationships as chitin synthesis inhibitors. Science 200,
3.
27,
760
(1979).
R. E. Redfern. J. D. Warthen Jr., G. D. Mills Jr., and E. C. Uebel, “Molting Inhibitory Effects of Azadirachtin on Large Milkweed Bug,” U.S. Department of Agriculture, Science and Education Administration, Agricultural Research Results, Northeastern Series. No. 5, 1979. 6. T. J. Kelly, R. E. Redfern, A. B. DeMilo, and A. B. Boikovec, Inhibition of ecdysis on Oncopeltrts fLIsciatus by 2-acetylpyridine thiosemicarbazones. Pest. Biochem. Physiol.. in press. 7. D. W. Borst and J. D. O’Connor. Arthropod molting hormone: Radioimmunoassay, Science 5.
178,
418
(1972).
1499
(1978).
12. N. P. Hajjar and J. E. Casida, Structure-activity relationships of benzoylphenyl ureas as toxicants and chitin synthesis inhibitors in Oncopeltus,fasciutus. Pest. Biochem. Physiol. 11, 33 (1979).
13. S. J. Yu and L. C. Terriere, Activities of hormone metabolizing enzymes in house flies treated with some substantial urea growth regulators, L(fe Sci. 17, 619 (1975). 14. S. J. Yu and L. C. Terriere, Ecdysone metabolism by soluble enzymes from three species of Diptera and its inhibition by the insect growth regulator TH-6040, Pest. Biochem. Physiol. 7, 48 (1977).
15. T. J. Kelly, C. W. Woods, R. E. Redfern, and A. B. Boikovec, Makisterone A: The molting hormone of larval Oncopeltus? J. Exp. Zool. 218,
127 (1981).
K. Slama, Homeostatic function of ecdysteroids in ecdysis and oviposition, Acta entomologicu hohemoslorwcn 77. 145 (1980). 17. J. W. Truman, Interaction between ecdysteroid, eclosion hormone, and bursicon titers in Mundrrca scxtu. Amer. Zool. 21, 655 (1981). 18. K. Leuschner. Effects of an unknown plant substance on a shield bug, Natur~Gsensc~huften 16.
59, 19.
T.
217
(1972).
Ohtaki. Ecdysteroid metabolism Amer. Zoo/. 21, 727 (1981).
in insects,
BIOCHEMISTRY
AND
PHYSIOLOGY
18,
351-356 (1982)
Ecdysteroid Titers and Molting Aberrations in Last-Stage Oncopeltus Nymphs Treated with Insect Growth Regulators1,2 R. E. REDFERN,~ Agricultural
Environmental
Quality
T. J.
KELLY,~
A. B.
BORKOVEC,~
Institute, Agricultural Research Beltsville. Maryland 20705
D. K.
AND
Service,
U.S.
HAYES~
Department
of Agriculture.
Received February 11, 1982; accepted July 21, 1982 The ecdysteroid titers of insect growth regulator (IGR)-treated and untreated large milkweed bug nymphs, Oncopeltus fasciatus (Dallas) (Heteroptera: Lygaeidae), were determined by radioimmunoassay. All of the IGRs tested affected the ecdysteroid levels and the ecdysis of both sexes in some way. AI3-63604, a juvenile hormone mimic, accelerated ecdysteroid production and its subsequent decline, shortened the stadium, and induced a supernumerary nymphal molt. Diflubenzuron had no effect on the onset of ecdysteroid production but slightly retarded the decline in ecdysteroid titers before adult ecdysis, and the ecdysis was incomplete. Ecdysteroid production was delayed and erratic in nymphs treated with azadirachtin and AI3-63967, but the effect on ecdysis was different for each compound. Azadirachtin caused incomplete adult ecdysis, whereas AI3-63967 completely prevented any attempts at ecdysis. The large peak of exdysteroid activity associated with apolysis was absent in starved insects, and they made no attempt to ecdyse.
caused either an incomplete ecdysis (5) or no attempt at ecdysis (6). In view of the previous correlation between ecdysial inhibition and ecdysteroid titers, we investigated the possibility that the inhibitory activity of all IGRs might be correlated with an effect on the ecdysteroid titers.
INTRODUCTION
Insect growth regulators (IGRs) vary widely in their effects on different insect species. This is especially true for juvenile hormone (JH) active IGRs (1) with effects ranging from inhibition of ecdysis and metamorphosis to defective metamorphosis or defective adult emergence. In at least one instance (2), JH treatment of the lastinstar German cockroach, Blattella germanica (L.), caused the formation of permanent larvae apparently through a reduction in the ecdysteroid titers. These titers were suppressed below the levels necessary for ecdysis and metamorphosis. In evaluating some IGRs on last-stage Oncopeftus nymphs, we observed that JHactive IGRs decreased the duration of the last stadium and produced supernumerary (sixth instar) nymphs (3, 4). Other compounds prolonged the fifth stadium and
MATERIALS
AND
METHODS
Test compounds. Pure AI3-63604 (2,6difluoro-l\r-[[4-[(3-fluorophenyl)methoxy]phenyl]methyl]benzenamine) and A13-63967 (1-pyrrolidinecarbothioic acid, 2-[ I-(2-pyridinyl)ethylidene]hydrazide), were supplied by A. B. DeMilo, Insect Reproduction Laboratory. Pure azadirachtin was supplied by J. D. Warthen, Jr., Biologically Active Natural Products Laboratory, Beltsville, Maryland, and diflubenzuron (technical grade, 96% pure) was kindly supplied by Thompson-Hayward Chemical Company, Kansas City, Kansas. Test insects. Newly ecdysed (pale orange) fifth-instar Oncopeltus fasciatus nymphs were collected from our laboratory stock culture that was maintained in 9.5 x 17 x 30-cm polystyrene boxes at 27 -+ 1°C
I Heteroptera:Lygaeidae. ’ Mention of a proprietary product does not constitute an endorsement by the U.S. Department of Agriculture. 3 Livestock Insects Laboratory. 4 Insect Reproduction Laboratory. 351
0048-3.575182106035l-06$02.00/0
352
REDFERN
and 50 2 5% RH under a 14 hr light:lO-hr dark photophase. Stock insects were provided with field-collected milkweed seeds and water in cotton-stoppered vials ad fibitam. The test insects were collected over a 4- to 6-hr period beginning at 7 AM on the day they were to be used. Collected nymphs were held in Erlenmeyer flasks without food or water until treatment with the test compounds a few hours later. Bioassay. AI3-63967 was dissolved in a mixture (1:l vol) of acetone and dimethyl sulfoxide (DMSO) because the solubility in acetone was marginal. All other compounds were dissolved in acetone. The dose of all compounds was 10 pg/nymph; only diflubenzuron was applied at a dose of 9 &nymph because of its limited solubility in acetone. These dose levels were selected to give clear and characteristic effects in all treated insects. The test solutions (1 Flinymph) were applied topically with a calibrated glass micropipet to the ventral side of the last three abdominal segments of the nymph. Treated and control nymphs were confined in 240-ml paper cartons capped with a clear plastic petri dish lid (9 cm) and supplied with milkweed seed and with water in cotton-stoppered vials. Insects were inspected daily until they ecdysed or died or the test was terminated. The treated nymphs from which hemolymph was collected were confined in a similar manner, except that one set of untreated controls was starved for the duration of the test. Hemolymph was collected from three nymphs of each sex every 24 hr.
Ejjkts
of Topically
Applied
Insect
Grmvth
TABLE Regrtlators
ET AL.
Extraction and radioimmanoassay of hemolymph ecdysteroids. Three males and three females were bled by leg or antenna1 amputation every 24 hr. The resulting l-p1 hemolymph samples were collected in l- to ~-PI borosilicate capillary glass pipets and dispensed into borosilicate test tubes (10 x 75 mm) that were closed with parafilm and frozen immediately at -20°C. For extraction of ecdysteroids, hemolymph samples were thawed, diluted to 500 ~1 with 75% methanol, vortexed vigorously, and centrifuged at 3000g for 30 min. The supernatant was carefully removed and either stored at -20°C or a 100~~1 sample was analyzed immediately by radioimmunoassay (RIA) (7). The antibody, prepared from the hemisuccinate of ecdysone derivatized at the C-22 hydroxyl only, was a gift from W. E. Bollenbacher, University of North Carolina at Chapel Hill. RESULTS
AND
DISCUSSION
All four IGRs used in this study prevented normal adult ecdysis (Table 1). The juvenile hormone mimic (JHM), AI363604, produced perfectly ecdysed sixthinstar (supernumerary) nymphs (Fig. 1A) beginning on the fifth day post-treatment. These nymphs either lived 7-10 days before they attempted to ecdyse to the adult (20%) or died without any attempt at ecdysis. The ecdysis inhibitor, AI3-63967, prevented any attempt at ecdysis on the seventh day or at anytime before death several days later. Removal of the old partially digested cuticle from these insects revealed 1 OII Ecdysis
of Fifth-Stage
Oncopeltus Ecdysis
IGR compounds
Dose b.&vmph)
AI3-63604 A13-63967 Diflubenzuron Azadirachtin Acetone Acetone:DMSO ’ Represents
average
IO 10 9 10 1 4 1 PI of four
replicates;
five
nymphs/test.
type (5% ecdysis) Sixth instar (100) None (0) Incomplete (100) Incomplete (100) Normal adults (100) Normal adults (100)
fusciatus”
ECDYSTEROID
TITERS,
FIG. 1. The effects of topically \lirh four IGRs at IO pglpllnymph (B) AI3-63967. (C) d$?ubenzaron,
MOLTING
ABERRATIONS,
AND
Oncupehs
NYMPHS
treating rzewly ecdysed fifth-instar Oncopeltus fasciatus nymphs. except diflubenzrrron ,r%ich +vas at 9 p~lplln~mph. (A) AI3-63604, and (II) a:adirachtirr.
a newly formed cuticle with adult pigmentation on the fourth abdominal sternite (Fig. 1B). The chitin synthesis inhibitor, diflubenzuron, and the antifeedant, azadirachtin, caused incomplete ecdysis (Figs. 1C and D). The diflubenzuron-treated insects attempted ecdysis beginning on the seventh day; while the azadirachtin-treated insects attempted ecdysis beginning on the ninth day. In either case, death usually occurred within 24 hr after the attempt began,
and dissection revealed adult cuticle. In control insects, untreated or solvent treats :d, ecdysis began on the seventh day and Mias completed in all animals by the eighth d:iY. No ecdysis was observed in the untreatf :d, starved nymphs which died from 6 to 12 days after the beginning of the stadium. The ecdysteroid titers were affected to some extent by all the test compounds, tcIa slight extent in acetone-treated animals (co Impare Fig. 2A with C), and slowly drop1 Ied
354
REDFERN
to undetectable levels in starved controls (Fig. 2G). The JHM, AI3-63604, was the only compound tested that accelerated ecdysteroid production and its subsequent decline (compare Figs. 2A and C with E). Smith and Nijhout (8) observed similar ef-
ET
AL.
fects in fifth-instar 0. fa.sciat44s treated with the JHM, methoprene. In our tests, this pattern of ecdysteroid titers correlates with the shortening of the duration of the fifth instar in JHM-treated nymphs. Continuous exposure of the nymphs to AI3-63604
ECDYSTEROID
TITERS.
MOLTING
ABERRATIONS,
(R. E. Redfern, unpublished results), even up to levels of 4000 pgIcm*, did not produce permanent larvae such as those found with JH-active IGR treatment of last-instar B. germanica (2). Contrary to the suggestion by Vogel er al. (1) that in last-stage hemipteran nymphs treated with JH-active IGRs the amount of ecdysone inducing the ecdysis is too low for metamorphosis, we agree with Smith and Nijhout (8) that the JH-active IGRs stimulate premature ecdysteroid production; however, the mechanism of these processes remains unclear. In holometabolous insects, JHMs appear to suppress the events induced by a transient ecdysteroid reprogramming peak (9, 10); however, such a peak was not apparent in our study (Fig. 2A). A more critical analysis of ecdysteroid titers in a synchronous population of last-instar 0. fascialus may resolve this question. Diflubenzuron had no effect on ecdysteroid production, but it slightly retarded the decline in ecdysteroid titers before the attempted adult ecdysis (compare Figs. 2A and C with F). This decline in the ecdysteroid titers correlates with the attempted ecdysis. Diflubenzuron acts by blocking the terminal polymerization step in chitin formation (11, 12). However, it has also been suggested that diflubenzuron affects chitin production by interfering with the metabolism of 20-hydroxyecdysone, thereby reducing its rate of inactivation and, thus, stimulating chitinase production (13, 14). Our results agree in part with this latter suggestion, that diflubenzuron may slightly retard ecdysteroid degradation. Furthermore, the demonstration by Hajjar and Casida (12) that metabolism of radiolabeled ecdysone and 20-hydroxyecdysone in fifth-instar 0. fusciatus nymphs is unaffected by diflubenzuron must be reexamined in light of the recent discovery that makisterone A is the principal molting hormone in fifth-instar 0. fusciatlls (15). Azadirachtin affected both ecdysteroid production and the decline in ecdysteroid titers before the attempted adult ecdysis (compare Figs. 2A and C with D). There
AND
Oncoprltus
NYMPHS
355
was no correlation between the variation in ecdysteroid titers and attempted ecdysis. In fact, the ecdysteroid titers varied strikingly between sexes and even between individuals within the same sex as evidenced by the large standard errors. The ecdysial inhibitor, AI3-63967, delayed the increase in ecdysteroid titers until Day 5 or 6 whereupon the titers increased to about half the normal peak level (compare Figs. 2A and C with B). This response was similar to the effect caused by azadirachtin, and again there was a large variation between sexes and between individuals within the same sex. The lack of any attempt at ecdysis in AI363967-treated nymphs suggests, however, that the mode of action of these two compounds may be different with respect to ecdysis. Slama (16) has demonstrated that the reduction in ecdysteroid titers that normally occurs at the end of an instar is necessary to allow the ecdysial contractions, which result in splitting of the old cuticle. Truman (17) has further shown that this same reduction in ecdysteroid titers is necessary for eclosion hormone release that triggers the subsequent behavioral and physiological events culminating in the shedding of the old cuticle. Since AI3-63967 prevents this reduction in ecdysteroid titer and no attempt at ecdysis occurs, it is possible that AI3-63967 acts by a similar mechanism in preventing eclosion hormone release. Azadirachtin, on the other hand, must be acting through a different mechanism since the ecdysteroid titers remain elevated and ecdysis still occurs. The similarity between the morphological effects of diflubenzuron and azadirachtin suggests that chitin synthesis may be blocked by azadirachtin. Furthermore, the structural similarity of azadirachtin to ecdysone suggests that it may act as an ecdysone analog (18) or possibly as a competitive inhibitor. The suppression of the feedback inactivation of ecdysteroids following their peak production (19) and blocking of the suppression of eclosion hormone release by high titers of ecdysteroids may be some of the effects of
356
REDFERN
azadirachtin. Unfortunately, varying hormone titers in the of new cuticle synthesis and ecdysis are still undetermined
the roles of programming the resultant (10).
ACKNOWLEDGMENTS
We thank G. D. Mills, Jr. (Livestock ratory) and Suzanne M. Spalding (Insect Laboratory) for technical assistance.
Insets LaboReproduction
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