Archives of Insect Biochemistry and Physiology 28:209-223 (1995) Non-Steroidal Ecdysteroid Agonists: Tools for the Study of Hormonal Action H. Oberlander, D.L. Silhacek, and P. Porcheron liisect Attractants, Behavior and Basic Biology Research Laboratory, Agricultural Research Service, U S D A , Gaiiiesville, Florida (H.O., D.L.S.); Dipartemeizt de Biologic., Uiziversite' Pierre et Marie Curie, Paris, Fruiice (P.P.) The first non-steroidal ecdysteroid agonists are dibenzoyl hydrazines and are typified by the compounds designated RH-5849 and RH-5992. The discovery that these compounds m i m i c 20E in a variety of insect orders, and especially the Lepidoptera, generated great interest from the research and agricultural communities. Such compounds provide important n e w research tools for physiological, biochemical, and molecular studies. In addition, the potential for application to agricultural pests looks very promising, especially for RH-5992 (tebufenozidej. This review evaluates the evidence o n the specificity of the ecdysteroid-like actions of these materials and considers their application for research and pest management. o 1995 WiIey-Liss, Inc * Key words: RH-5849, RH-5992, Dibenzoyl hydrazine, tebufenozide INTRODUCTION The first non-steroidal ecdysteroid agonists, dibenzoyl hydrazines, typified by RH-5849 [1,2-dibenzoyl, I-tert-butyl hydrazone] (Fig. l), were described in 1988 (Wing, 1988; Wing et al., 1988). The discovery that these compounds mimic 20-hydroxyecdysone (20E) in both Manduca sexta and Drosophila rnelanognstev generated great interest from several perspectives. First of all, such compounds provide an important new research tool as the first Acknowledgments: We thank Rohm and Haas Co. (Spring House, PA) for supplying courtesy samples of RH-5849 and RH-5992, and C.R. Carlson (Rohm and Haas C0.j for providing information on these compounds and their agricultural applications. We also thank G.R. Carlson and T. Dhsdialla (Rohm and Haas Co.), A. Retnakaran (Forest Pest Management Institute, Sault Ste. Marie, Ontario, Canada) and K.D. Wing (E.I. du Pont de Nemours & Co., Newark, DE) for reviewing our manuscript. We are grateful lo 1.1. Heller (Roussel Uclaf, Paris, France), J. Koolrnan (Universitat Marburg, Germany), A. Retnakaran, C . Smagghe, and D. Dcgheele (Universiteit Gent, Belgium) tor providing us with copies of their manuscripts that were in press. This article was written while H. Oberlander was a Visiting Professor at Pierre and Marie Curie University (Paris, France), in the laboratory of P. Porcheron. Received August 19, 1994; accepted December 5,1994. Address reprint requests to Herbert Oberlander, USDA/ARS, P.O. Box 1456.5, Gainesville, FL 32604. @ 1995 Wiley-Liss, Inc. *This article is a US Government work and, as such, is in the public domain in the United States of Amerira 21 0 Oberlander et al. non-steroidal materials with ecdysteroid activity. Secondly, with the steroid structure no longer a barrier, there is an opportunity to evaluate the control of insects of economic importance with non-steroidal ecdysteroid agonists. The first compound in this series to be commercialized is RH-5992 [tebufenozide, 3,5-dimethylbenzoic acid 1-1 (1,1-dimethylethyl)-2)4-ethylbenzoyl) hydrazide] (Fig. 2). Thus, the repertoire of insect growth regulators is being expanded from juvenile hormone agonists to include ecdysteroid agonists. Manifestly, both research and agricultural applications of the dibenzoyl hydrazines require confidence in the validity and specificity of their ecdysteroidlike actions. Our objectives in this review are to evaluate the first 6 years of research on RH-5849 and RH-5992 in terms of the specificity of their ecdysteroid-like actions, advantages for research on hormonal action in insects, and opportunities for practical applications to agriculture. SPECIFICITY AS ECDYSTEROID AGONISTS The array of ecdysteroid effects on insects is so vast that we can only look for key signals that demonstrate that the hydrazines are true ecdysteroid mimics. Wing et al. (1988) made the seminal observation that RH-5849 induces molting in Lepidoptera in the absence of a known source of endogenous ecdysteroids when tested in ligated larvae (“isolated abdomens”) of M . sexta. Similarly, they found that RH-5849 caused premature molting in intact M . sexta larvae without a concomitant increase in ecdysteroid titers. These early findings have been extended to other species of Lepidoptera, including Plodia in tevpunctella (Silhacek et al., 19901, Spodoptera littoralis (Smagghe and Degheele, 1992), Spodoptera exempta (Smagghe and Degheele, 1994a), Mamestva brassicase (Davis et al., 19921, Pieris brussicae (Darvas et al., 1992),and Ckoristoneura fumiferana (Retnakaran and Oberlander, 1993). Molting and Feeding The hormonal activity assayed from the prothoracic glands was characterized in the early literature not only as a ”molting hormone” but as a ”growth and differentiation hormone” (Scharrer, 1946) or as a “growth and molting” H RH-5849 Fig. 1 . Structure of RH-5849. Non-Steroidal Ecdysteroid Agonists H CH3- 1 C- I H H 21 1 dH3 \ CH3 RH-5992 Fig. 2 Structure of RH-5992. hormone (Wigglesworth, 1954).Manifestly, the ecdysis of the old cuticle provides a new, larger skeletal framework into which the newly molted larva can grow through feeding during the subsequent intermolt period, while metamorphosis leads to differentiation of the adult structures. Moreover, the cessation of feeding activity by larvae is a normal part of the preparation for molting. In this context, RH-5849 not only induced premature molting, but also reduced feeding activity and weight gain. The decreased body weight of RH-5849-treated larvae has been confirmed in P. interpuncfella (Silhacek et al., 1990) and S. littoralis (Smagghe and Degheele, 1992). Juvenile Hormone Interestingly, the effects of RH-5849 on feeding and molting could be decoupled through the simultaneous application of the juvenile hormone mimic, methoprene, and RH-5849 in the diet of penultimate or final instar larvae. With methoprene treatment only, the larvae continued to feed but did not molt. The addition of both methoprene and RH-5849 resulted in undiminished weight gain by the treated larvae. Moreover, under such a protocol u p to 80% of the larvae treated with both compounds molted into a supernumerary instar (Silhacek et al., 1990). Thus, even under conditions where a juvenile hormone mimic prevented any reduction in feeding, the ecdysteroid-like activity of RH-5849 was pronounced. In the environment of a high titer of juvenoid molting could proceed normally but metamorphosis was prevented. Such supernumerary larvae appeared normal, except that in some cases the wing discs formed a pupal cuticle within the larva (Silhacek et al., 1990). Essentially similar results were obtained by Smagghe and Degheele (199413) after application of RH-5849 or RH-5992 and the juvenile hormone agonist, pyriproxifen, to Spodoptera exigua. The induction of normal larval molting was observed as well in Ostrinia izubilalis larvae, in which topical application of RH-5849 early in the last larval instar induced a supernumerary molt, presumably as a consequence of a high titer of juvenile hormone (Gadenne et al., 1990). Later in the instar the application of RH-5849 induced either larval-pupal intermediates or normal pupae. The induction of perfect supernumerary instars was observed as well when early last instar Galleria rnellonella larvae were 21 2 Oberlander et al. treated topically with RH-5849. However, in this case RH-5849 appeared to increase production or release of juvenile hormone by the corpora allata (Musynska-Pytel et al., 1992). Thus, while the stimulation of premature molting is usually incomplete and therefore fatal, a complete molt can be obtained under conditions of application of RH-5849 and simultaneously elevated juvenile hormone titer. Juvenile hormone counteracted some deleterious effects of dibenzoyl hydrazine, i.e., premature incomplete molting and cessation of feeding. This phenomenon provided increased opportunities for investigating physiological effects of RH-5849. Specificity for Insects and Crustacea The major molting hormone in anthropods is 20E, but other ecdysteroids may serve as the primary hormone in some species (Horn and Begamasco, 1985). Given this diversity of ecdysteroids, it is important to determine to what extent RH-5849 acts as a universal ecdysteroid agonist. Such information is important for evaluation of potential effects on non-target species in pest management applications. Interestingly, RH-5849 displayed ecdysteroidlike activity in some Crustacea, but not in all orders of insects. Clare et al. (1992) reported that RH-5849 accelerated molting in crab zoeae. RH-5849 was not active as an ecdysteroid agonist in Orthoptera (Locusta rnigratoria), or Hemiptera (Podisus sagittal (Smagghe and Degheele, 1994a); also, Darvas et al. (1992) reported low activity against Homoptera (Acyrthosiphon pisum). Whether such differences in activity are due to variations in transport and metabolism, to possible differences in the ecdysteroid receptor, or to differing abilities to mimic specific ecdysteroids in these diverse species, is not clear. For the purposes of determining specificity of ecdysteroid-like actions, we will focus in the remainder of this article on RH-5849/RH-5992 as ecdysteroid agonists with respect to 20E, with emphasis on Lepidoptera. Organ Culture One approach to investigating the direct action of hormonal agents on tissues and cells is to conduct experiments on organ cultures in vitro or on established cell lines (e.g., Oberlander and Miller, 1987). In cultures of wing imaginal discs obtained from last instar larvae of the Indian meal moth, P. interpunctella, RH-5849 stimulated incorporation of I4C-GlcNAc into chitin. In these experiments, 10-100 times as much RH-5849 as 20E was needed for effective stimulation of both chitin synthesis and evagination in vitro (Oberlander et al., 1990; Silhacek et al., 1990). Maximum effectiveness was obtained in these experiments with a limited duration of treatment (24h), just as is required for 20E (Dutkowski and Oberlander, 1974). Thus, while exposure to 20E or RH-5849 was necessary to stimulate chitin synthesis subsequent to the conclusion of the hormone treatment, continued exposure to the hormone or agonist was inhibitory (see also Mikolajczyk et al., 1994; Retnakaran et al., in press). These often observed inhibitory effects of overexposure to 20E or its agonists probably explain the failure to stimulate production of a lamellate endocuticle in the spruce budworm treated with RH-5849, even though precocious molting was induced (Retnakaran and Non-Steroidal Ecdysteroid Agonists 21 3 Oberlander, 1993). Similar results were obtained with integumentary fragments in culture (Oikawa et al., 1993). Thus, not only did RH-5849 mimic 20E by stimulating chitin synthesis in cultured tissues, but like 20E, its period of application must be limited in order to be effective. Tissue culture studies have also pointed to the action of RH-5849 in tissue disintegration. The reorganization of the fat body occurs during metamorphosis and is accompanied by biochemical changes that typify autophagy (Locke, 1980). In this connection Ashok and Dutta-Gupta (1991) showed that RH-5849 was able to stimulate the production of acid phosphatase in culture. It is noteworthy that while 20E stimulated acid phosphatase when injected in vivo, it failed to do so in vitro. The difference in activities in vitro may be due to the greater stability of RH-5849. Thus, it is clear from experiments in vivo and in organ culture that RH5849 induces chitin synthesis in several test systems and precocious molting in a number of species. These effects appear to be direct since RH-5849 was active in ligated larval abdomens as well as in cultured imaginal discs and integumentary fragments. The case for dibenzoyl hydrazines being true ecdysteroid agonists is more compelling when its effectiveness was established at the cellular and receptor levels. In this regard, RH-5849 and RH5992 perform in a manner consistent with the paradigm of 20E action mediated at the cellular level by specific nuclear ecdysteroid receptors. Cellular Effects Wing (1988) was the first to demonstrate the ecdysteroid-like action of RH5849 at the cellular level in experiments with D. melanogaster Kc cells (Echalier and Ohannessian, 1969). The response to RH-5849 consisted of a cessation of proliferation, clumping, and the formation of extended processes, effects that were indistinguishable from the results of exposure to 20E. In these experiments RH-5849 was effective at 100 times the concentration for 20E. Similar results were obtained for the imaginal disc derived cell line from P. znterpunctella, IAL-PID2 (Lynn and Oberlander, 1983), in which RH-5849 inhibited proliferation about 50% at lo-' M, higher than the effective concentration for 20E (Silhacek et al., 1990). In the same cell line RH-5992 inhibited (Oberlander, Hatt proliferation at the same concentration as 20E (2 x and Porcheron, unpublished observations). An adaptation of the typical suppression of proliferation by ecdysteroids in cell lines was reported by Clemente et al. (1993).They showed that in the tumorous blood cell line B-I1 (Gateff et al., 19801, the cell density of the cultures could be measured by assessing the turbidity through absorbance detection on a microtiter plate reader. Under such conditions TH-5849 (lo4 M) reduced absorbance, and thus proliferation, but at substantially higher concentrations than 20E (2 x M). In addition to effects on proliferations, 20E causes more complex alterations in morphogenesis in cell lines that grow as multicellular vesicles. In the case of the Chironoinus tentans cell line (Wyss, 19821, the vesicles shrink in response to either 20E or RH-5849 (Spindler-Barth et al., 1991). The situation for the Trichoplusia ni cabbage looper, imaginal disc-derived cell line, IAL-TNDI (Lynn et al., 19821, is more complex. After ca. the 30th passage the vesicles spontaneously changed to solid clusters, but the vesicle form could be rescued 214 Oberlander et al. through the application of a hemolymph-derived 16 kDa peptide, vesicle promoting factor (VPF) (Ferkovich et al., 1987). The action of VPF was blocked by 20E (Oberlander et al., 19871, and was similarly inhibited by RH-5849 (Oberlander and Leach, unpublished observations). Thus, even quite specific effects of 20E on cell lines can be mimicked by RH-5849. In addition to proliferation and morphogenesis, biochemical changes also typify the action of 20E on cell lines. For example, increased acetylcholinesterase activity is observed after treatment with RH-5849, just as it is following exposure to 20E (Wing, 1988; Spindler-Barth et al., 1991). In another example, 20E inhibits, rather than stimulates, the spontaneous synthesis of chitin by the C. tentans cell line, an effect duplicated by RH-5849 and RH5992 (Spindler-Barth et al., 1991; Spindler et al., 1994). This negative effect on chitin synthesis may be analogous to the inhibitory effects of overexposure to ecdysteroids cited earlier. More typically, the IAL-PID2 cell line responds to 20E with increased uptake of GlcNAc, a precursor of chitin, in much the same manner as the wing imaginal discs from which they are derived (Porcheron et al., 1988).This stimulation of GlcNAc uptake was likewise, induced by RH-5849 (Silhacek et al., 1990). Taken together, it is clear that the entire repertoire of 20E induced effects on proliferation, morphogenesis, enzymatic activity and transport in established cell lines are all duplicated by RH-5849, albeit at higher concentrations than for 20E. Receptor Studies If RH-5849 is indeed an ecdysteroid agonist, it should operate by the same mode of action, i.e., binding to ecdysteroid-specific receptors that are members of the steroid hormone receptor superfamily (Koelle et al., 1991). The approach that has been taken in several laboratories was to compare RH5849 and 20E in competitive binding assays in which the displacement of [3H] ponasterone A is measured. RH-5849 was not recognized by antibody to ecdysteroids, despite the apparent binding to the same receptor domain as ponasterone A. In such an assay with the Drosophila Kc cells, RH-5849 was about 100 times less effective than 20E in displacing radiolabeled ponasterone A from receptors (Wing 1988).Similar results were obtained by Spindler-Barth et al. (1991) for the C. tentans cell line, but RH-5849 was only 4 times less effective than 20E. Recent experiments have shown that RH-5992 was more effective than 20E in the same binding assay (Carlson et al., 1994; Spindler et al., 1994). Compelling evidence for the interaction of RH-5849 with ecdysteroid receptors came from experiments by Wing (1988) in which he showed that Kc cells that are incubated for 4 weeks with either 20E or RH-5849 became resistant to the application of ecdysteroids. Moreover, both of these resistant PO u lations of cells showed a sharp reduction in their capacity to bind [ HI ponasterone A relative to control cells. A direct test of the 20E receptor came from the transient transfection of mammalian cell lines with EcR resulting in HeLa cells that supported expression of a chloramphenicol acetyltransferase (CAT) reporter gene by muristerone A, ponasterone A, and RH5849, though not 20E (Thomas et al., 1993). The relevance of these receptor studies to RH-5849’s action at the organismal level is supported by the find- Y- Non-Steroidal Ecdysteroid Agonists 21 5 ing that the activity of 28 analogues of RH-5849 in inducing premature head capsule apolysis in M . sexta was positively correlated with their ability to bind to Drosophilu Kc cell ecdysteroid receptors (Wing et al., 1988). As an agonist of 20E, RH-5849 should have all of the physiological activities of the natural hormone. In fact, we have seen that RH-5849 mimics 20E actions at the organismal, tissue, cellular, and molecular levels. In this connection, it is noteworthy that the first ecdysteroid receptors were described for Lepidoptera in the course of studies with RH-5849. Nuclear extracts from the IAL-PID2 cell line (Lynn and Oberlander, 1983) yielded saturable, high affinity ponasterone A receptors, to which RH-5849 bound nearly as effectively as 20E, while RH-5992 was more effective than 20E (Wing and Ramsay, 1989; Carlson et al., 1994). Taken together, the results of RH-5849 and RH5992 action are consistent with the hypothesis that these compounds bind to ecdysteroid receptors and consequently lead to the major events associated with molting. UTILITY AS INVESTIGATIONAL TOOLS It is clear that RH-5849 and RH-5992 act as ecdysteroid agonists, and that these molecules provide increased opportunities for studies or hormonal action in insects in general, and particularly among the Lepidoptera. Molecular Studies In this connection, Sobek et al. (1993) described ecdysteroid binding proteins from nuclear extracts of last instar larvae of Galleria rnellonella. They found that RH-5849 was slightly more active than 20E, but 25 times more active than ecdysone in a competition assay with 13H]ponasterone A. This level of binding activity by RH-5849 suggests that such compounds may be particularly useful in future receptor studies. Another application of the interactions of RH-5849 and RH-5992 with 20E receptors involves their ability to mimic molecular actions of ecdysteroids. Extensive research on 20E action at the molecular level in M . sextu has demonstrated that a DNA binding protein, “hormone receptor 3” [MHR3],is a putative transcription factor, found in the epidermis that fluctuates during development as the ecdysteroid titer changes (Palli et al., 1992; kddiford and Truman, 1993). The stimulation of mRNA for MHR3 appears 3 h after treatment with either 20E or RH-5992. Unlike many of the assays with RH-5849, RH-5992 was 10 times more active than 20E in these experiments (Retnakaran et al., in press). Other investigations with M. sexta epidermis show that the larval endocuticle protein gene, LPC14, is expressed during the intermolt period, and this expression is prevented by 20E (Hiruma et al., 1991).In subsequent studies, Retnakaran et al. (in press) showed that RH-5992 also suppressed LCP14. Moreover, when a 17 h treatment period with 20E is followed by a 48 h hormone-free period, LCP14 activity is largely restored. By contrast, a similar protocol with RH-5992 results in persistent suppression of the LCP14 gene. Thus, in these tests RH-5992 was not only more active than 20E but its effects were more persistent, suggesting greater stability of the compound and/or enhanced binding affinity to receptor sites. 21 6 Oberlander et al. Ecdysteroid Biosynthesis The regulation of biosynthesis of 20E is another arena for research with dibenzoyl hydrazines. Ecdysone 20-mono-oxygenase, a cytochrome P450-dependent enzyme, is responsible for hydroxylating ecdysone to form 20E (Smith, 1985). In this process, ecdysone and 20E are not only substrate and product, respectively, but also are implicated in substrate (E) induction or product (20E) inhibition in vivo (Grieneisen, 1994). However, in M. sexta both E and 20E, when applied in vivo, stimulate dramatic increases in midgut 20-mono-oxygenase (Keogh et al., 1989). Certainly, it would be of value to investigate this process with an ecdysteroid agonist that is not itself affected by the enzyme. In this respect, Keogh and Smith (1991) found that RH-5849 was 20 times more active than ecdysone and 50 times more active than 20E in stimulating the dramatic increase in ecdysone 20-mono-oxygenase activity in vivo. The effects of 20E in vitro in M . sexta midgut are quite different from the results obtained in vivo, i.e., 20E acts as an inhibitor in vitro. This system was used to demonstrate the similarity of the action of RH-5849 to 20E in midgut homogenates (Keogh and Smith, 1991; Grieneisen, 1994).Presumably, the inhibition of the cell-free assay of ecdysone 20-mono-oxygenase occurs because the L3H1 ecdysone used as a substrate was dislodged from the enzyme by either 20E or RH-5849. Moreover, Grieneisen et al. (1993) demonstrated that RH-5849 was not simply a general inhibitor of cytochrome P450-dependent enzymes, because it had no effect on assays of prothoracic gland P450 hydroxylases. Structure-Activity It is not clear how 20E and RH-5849 and RH-5992 have similar biological activities based on specific binding to ecdysteroid receptors when their chemistry is so different. However, an analogous situation was pointed out by Riddiford (1994) in that highly active juvenile hormone mimics, fenoxycarb and pyriproxyfen, are structurally unrelated to natural juvenile hormone. Although the structural dissimilarities between the dibenzoyl hydrozine and ecdysteroids are great, they are not perceived as such by insects. Clearly, more research on molecular interactions of the dibenzoyl hydrazines with the ecdysteroid receptors is needed. It is of interest in this connection that Chan et al. (1990) have elucidated the crystal structure of RH-5849 and compared it with its closely related parent compound, 1-2-dibenzoyl hydrazine, which, surprisingly, is biologically inactive. Specific differences observed in the crystal structure of these compounds, in comparison with ecdysteroid structures, may provide insights into understanding the nature of the 20E receptor interaction as a basis for biological activity. Computer modeling studies comparing the molecular structure of RH-5849 with 20E showed promise, but were not definitive (Zander, 1990; Zander and Koolman, 1990). Ecdysteroid receptor binding per se does not automatically confer ecdysteroid agonist activity. The brassinosteroids, derived from plants, bind to ecdysteroid receptors but antagonize rather than mimic 20E (Sobek et al., Non-Steroidal Ecdysteroid Agonists 217 1993; Lehmann et al., 1988; Koolman, 1990). Thus, structural studies of optimal binding of compounds to ecdysteroid receptors may yield antagonists as well as agonists of 20E. Neurotoxicity At least in some species RH-5849 causes neurotoxicity. This effect appears unrelated to its ecdysteroid-like action and occurs at much higher concentrations in Lepidoptera but at the same concentrations in Coleoptera. RH-5849 apparently causes neurotoxicity in certain species by blocking K' channels in nerve and muscle (Salgado, 1992a,b).These findings provide an opportunity for using dibenzoyl hydrazines to investigate K' channels in insects. APPLICATIONS AS INSECT GROWTH REGULATORS There is strong evidence from both laboratory and field studies that RH5992 will be of immense practical value in the control of agriculturally important insect pests, particularly Lepidoptera. Substantial background information for practical applications of the dibenzoyl hydrazines was first obtained with RH-5849. Although RH-5849 is less active than 20E in vitro, it is 30 to 670 times more active than 20E when tested in vivo (Wing et al., 1988). This reversal of activity in vivo has been ascribed to the superior absorption and metabolic stability of RH-5849 relative to 20E. Thus, Wing et al. (1988) found that administration of RH-5849 in the diet (10 yg/gm) of M . sexta larvae resulted in a rapid buildup of the compound in the hemolymph peaking at 16 pM at 6 h, followed by a decline to 3 pM, which was stable over the next 36 h. On the other hand, feeding an excess of 20E (2000 ppm) had no effect on the larvae. Moreover, there seems to be an accumulation of RH-5849 in the epidermis, which is necessarily the primary target tissue for the premature stimulation of a new cuticle (Wing and Aller, 1990). Smagghe and Degheele (1993) demonstrated that after uptake by specific tissues, RH5849 was relatively metabolically stable. The effectiveness of RH-5849 in vivo against agriculturally important insect pests has been demonstrated for a number of species, including the Colorado potato beetle, Leptinotarsa decernlineata (Darvas et al., 1992), the fall armyworm, Spodoptera frugiperda (Monthkan and Potter, 1992), the cabbage moth, Marnestvn bvassicae (Darvas et al., 1992; Smagghe and Degheele, 1993; Smagghe and Degheele, 19944, and the Indian meal moth, P. interpunctella (Silhacek et al., 1990). Importantly, RH-5849 caused premature molting, whether applied topically, by mixing with an artificial diet, or by coating leaves (Darvas et al., 1992; Silhacek et al., 1990; Smagghe and Degheele, 1993; Wing et al., 1988). RH-5849 was even effective systemically when applied as a soil treatment to tomato plants on which L. decernlineata were fed (Wing and Aller, 1990). Wing and Aller (1990) reported that RH-5849 has the desired characteristics of an environmentally benign compound in that it has minimal mammalian toxicity, is negative in the Ames mutagenicity test, and is essentially non-toxic to birds and fish. Although it might be expected that RH-5849 would affect crustacea, whose molting is ecdysteroid-dependent, it was toxic only 21 8 Oberlander et al. at very high concentrations-7 mg/l for Daphnia (Wing and Aller, 19901, and 10 mg/l for crab zoeae (Clare et al., 1992; see also Kreutzweiser et al., 1994). The greater effectiveness of RH-5992 than RH-5849 in tissue culture assays (Retnakaran et al., in press; Oberlander, Hatt and Porcheron, unpublished observations) is consistent with the greater activity of RH-5992 in in vivo assays. For example, Smagghe and Degheele (1994b) showed that RH-5992 was 50 times more active against S. exigua than was RH-5849 in inducing a premature lethal molt, and was 100 times more active than RH-5849 in causing a supernumerary molt when applied simultaneously with pyriproxifen, a juvenile hormone mimic. The effectiveness of RH-5992 against important crop pests also has been demonstrated in laboratory bioassays for the corn earworm, Helicoverpa zea, the rice stem borer, Chilo supressalis (Oikawa et al., 1994),and the fall armyworm, S. frugiperda (Chandler et al., 1992). Most importantly, RH-5992 is more highly active in Lepidoptera than is RH-5849. For example, RH-5992 was 44 times more active against S. exiqun than was RH-5849, based on the concentration required to kill 50% of the treated larvae (Smagghe and Degheele, 199413).On the other hand, RH-5992 had no significant effect on the Colorado potato beetle (Smag he and Degheele, 1994~). Their data on the distribution and metabolism of [5 I C-IRH5992 indicates that these differences in biological activity cannot be explained by metabolic or pharmacokinetic variations (Smagghe and Degheele, 19944. Perhaps, it will be discovered that receptor specificity accounts for the observed differences in species effectiveness. Clearly, RH-5992 is both more active and more selective, with respect to Lepidoptera, than is RH-5849. Moreover, RH-5992 is ”remarkably non-toxic to a wide range of beneficial parasitic and predatory insects” (Carlson et al., 1994).For example, recent tests have shown that RH-5992 was effective against Lepidoptera without effects on predatory mites or braconid parasites (Mattioda et al., 1993; Brown, 1994). These results suggest that RH-5992 has important properties needed for practical use and this has been borne out by field tests. Thus, Heller et al. (1992) demonstrated that RH-5992 provided outstanding control of key pests in orchards (the codling moth, Cydia pornonella, and the oriental fruit moth, Cydia nzolestn), vine (the vine moth, Lobesia botruna, the grape berry moth, Clysia arnbiguella), and vegetables (the beet armyworm, S. exigua). Taken together, it appears that RH-5992 will soon have substantial practical applications in controlling lepidopterous pests, while it does not seem likely that RH-5849 will be developed for commercial use (G. Carlson, personal communication). The remarkable properties of the dibenzoyl hydrazines provide a commonality of interest and benefit for insect endocrinologists and agricultural entomologists alike. The effectiveness of these compounds in the field, their selectivity and environmentally friendly characteristics provide just the sort of application of research on insect hormones predicted by C.M. Williams (1967).Moreover, these compounds represent exciting new tools for investigation of insect physiology and particularly hormone receptor interactions. Such possibilities are underscored by structure activity studies of dibenzoyl hydrazines in which the potency of the compounds depended on Non-Steroidal Ecdysteroid Agonists 21 9 the conformational influence of the substituents and their positions on the benzoyl-benzene rings, thus affecting molecular shape (Oikawa et al., 1994). 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