Relationship Between Morphogenetic Activity and Metabolic Stability of Insect Juvenile Hormone Analogues*

7. Insect Physiol., 1972, Vol. 18, pp. 2019 to 2028. Pergamon Press. Printed in Great Britain

RELATIONSHIP BETWEEN MORPHOGENETIC ACTIVITY AND METABOLIC STABILITY OF INSECT JUVENILE HORMONE ANALOGUES*

GUNDA REDDY and A. KRISHNAKUMARAN

Department of Biology, Marquette University, Milwaukee, Wisconsin, 53233

(Received 3 March 1972)

Abstract-The relationship between morphogenetic activity and metabolic stability of natural Cecropia juvenile hormone and some of its synthetic analogues (methylenedioxyphenoxy, chlorophenoxy analogues, and ethyl-3,7,11- trimethyldodeca-2,4-dienoate) was studied in Tenebn’o pupae by parabiosis and in Galleria larvae by determination of the time course of decay of injected hormones. Parabiosis of dihomojuvenate or methylenedioxyphenoxy analogue treated Tenebrio pupae to fresh pupae 6 days after application of the hormone showed that the natural hormone but not the analogue was metabolized during the S-day period. Determination of the persistence of juvenile hormonal agents injected into O-day-old Gulleriu last instar larvae revealed that the natural hormone was inactivated much more rapidly when compared to methylenedioxyphenoxy, chlorophenoxy, and ethyl-3,7,11-trimethyldodeca-2,4-dienoate analogues. The significance of the positive correlation between greater stability and higher activity of juvenile hormone analogues is discussed.

INTRODUCTION THE DETERMINATIONof the structures of the two Cecropia juvenile hormones, methyl-12,14-dihomojuvenate (methyl-lo-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate) (R~~LLERet al., 1967), and methyl-1Zhomojuvenate (methyl-lo- epoxy-3,7,11-trimethyl-2,6-tridecadienoate) (MEYER et al., 1968) was followed by the synthesis of several other analogues with juvenile hormone activity (BOWERS, 1969; WIGGLESWORTH,1969; SLLMA et al., 1970; SCHWARTZet al,, 1970; PALLOS et al., 1971; CRUICK~HANKand PALMERE,1971; STAAL et uZ., 1971). In several bioassays including the Tenebrio bioassay, some of the synthetic JH analogues showed higher morphogenetic activity than the natural Cecropia hormones. Two such potent analogues were the methylenedioxyphenoxy derivative of 12,14- dihomojuvenate (BOWERS, 1969) and ethyl-3,7,1 l-trimethyldodeca-2,4-dienoate, a compound lacking epoxide moiety (STAALet al., 1971). These compounds were about 100-fold as active as methyl-12,14-dihomojuvenate in Tmebrio and/or GuZZeriubioassays (BOWERS,1969; STAALet al., 1971). The greater potency of hormonal analogues is a phenomenon well known to mammalian endocrinologists (ERCOLI and FALCONI, 1967) and was shown to be + This research is supported by N.S.F. research grant No. G.B. 19629.

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66 2020 GUNDAREDDY ANDA. KRISHNAKUMARAN true of insect moulting hormones (OHTAKI and WILLIAMS, 1970). Greater potency of a hormonal analogue can be accounted for either by its stereo chemical structure that is more suited for its function in a cell or by its greater metabolic stability. The higher activity of the analogues of mammalian gonadal hormones and of ecdysones was shown to stem from greater resistance of the analogues to enzymatic degradation (cf. STEELMANand HIRSCHMANN,1967). One consequence of this resistance to degradation is that the half life of the analogue was higher than that of the true hormone. Since a knowledge of the physiological basis of the higher activity of JH analogues is important for the proper utilization of these chemicals as insecticides, we sought to determine whether higher activity of the more potent JH analogues is a consequence of their greater stability in the test animal. Our study indicates that the more potent JH analogues are stable and persist longer in Tenebrio pupae and GuZZe-rialarvae than the natural hormone.

MATERIALS AND METHODS Experimental animals Larvae of the wax moth, Galleria mellonella, were reared in the laboratory according to standard procedures (BECK, 1960; KRISHNAKUMARAN,1972). Newly ecdysed last instar (seventh instar) larvae, recognized by the size of the head capsule, weight (BECK, 1960), and dark pigmentation of the integument, were collected and maintained in plastic Petri dishes with ample food. Under these culture conditions, the seventh instar plus pharate pupa lasts about 9 days, of which the last 2 days represent the pharate pupal stage. In all the experiments reported here only seventh instar larvae were used and the term larva is used in the text to denote this stage. Mature larvae of Tmebrio molitor were purchased from bait dealers and maintained in the laboratory at 25°C and 70% r.h. Fresh pupae less than 6 hr old, were used in these studies.

Chemicals Cecropia juvenile hormone, methyl-la, 14-dihomojuvenate (Roller compound), methylenedioxyphenoxy (Bowers’ compound), and chlorophenoxy derivatives of juvenile hormone were generously supplied by Messrs. Hoffmann La Roche, Inc., Nutley, N. J. Ethyl-3,7,1 l-trimethyldodeca-2,4-dienoate was kindly supplied by Dr. J. B. Siddall of Zoecon Corp., Palo Alto, California. These compounds were mixed cis, tram isomers and were 99 per cent pure. As the dihomojuvenate was a mixture of isomers it was only about one-fifth as active as a sample of pure hormone kindly supplied by Professor Andre S. Meyer of Case Western Reserve University. Experimental methods Two different methods were used to determine the persistence or decay of JH and its analogues. ACTIVITY AND METABOLIC STABILITY OF JUVRNILE HORMONES 2021

In the first method, JH treated Tenebrio pupae were parabiosed with untreated pupae, 5 days after topical application of the hormone. Application of about 0.5 pg of Bowers’ compound or 6 pg of dihomojuvenate produced a more or less perfect second pupa. Five days after application of the hormonal agent a second pupal cuticle was formed but the second pupa never extricated itself from the old cuticle. These second pupae were tail-to-tail parabiosed to fresh Tenebrio pupae following the surgical methods outlined for parabiosis of Saturniid pupae (SCHNEIDERMAN, 1966). The parabiosed pupae (usually both the partners) deposited a new cuticle 6 to 8 days after parabiosis (Fig. 1). The nature of the cuticle deposited by the parabiosed pupae reflected the level of the persistent JH activity at the time of the parabiosis. If the parabiosed partners deposited a cuticle with pupal features, it was indicative of the persistence of the JH applied to one of the partners 6 days prior to the parabiosis. The absence of pupal characters in the parabionts at this apolysis was suggestive of the decay of the applied hormonal agent. Furthermore, quanti- tative estimation of the persistent JH activity was based on the morphological features of the cuticle of the parabionts and scoring them according to the procedures described by BOWERSand THOMPSON(1963). The second method consisted of injection of a known quantity of the test chemical into a freshly ecdysed O-day-old (10 to 12 hr old) seventh stadium Galleria larva and the determination of the persistent hormonal activity, at successive time intervals. For this purpose we injected a known quantity (because of differences in the morphogenetic potency of the various juvenile hormone analogues the amount of the test chemical injected into each larva was varied; Cecropia hormone was injected at a dose of 25 pg/larva, while methylenedioxyphenoxy and chlorophenoxy analogues were injected at a dose of 5 pg/larva, ethyl-trimethyl-dodeca-dienoate was applied at a dose of 2 ,ug/larva) of the test chemical in 1~1 of peanut oil into each larva and at different time intervals after injection (0, 4, 8, 24, 48, and 72 hr) the larvae were frozen, homogenized in diethyl ether, and extracted in soxhlet for 3 hr. The lipid extract was washed according to the procedures described by GILBERT and SCHNEIDERMAN(1961) and assayed for morphogenetic activity in Tenebrio pupae (ROSE et al., 1968). Since the amount of lipid extracted at each stage was different, the amount of hormone applied to each Tenebrio pupa was adjusted to give equivalent concentration based on per mg of the lipid extracted. The scoring system was slightly modified from that of Bowers, in that the range of the score was 0 to 5 with increments of 0.5. The percentage of pupae that retained pupal features together with the individual scores of the pupae reflected the recovered JH activity. The JH activity recovered at 0 hr was regarded as 100 per cent of the injected hormone; and based on this the percentage recovered hormonal activity at different time intervals was calculated. To determine the efficiency of the extraction procedures we added a known quantity of the Cecropia hormone (12,14-dihomojuvenate) to a homogenate of the Galleria larvae prior to extraction. These studies revealed that the recovery of the hormone was slightly over the amount added, which was attributed to the fact that there is some endogenous JH in these larvae at this stage. Lipid extracts from 2022 GUNDAREDDY ANDA. KRISHNAKUMARAN peanut oil-injected controls showed no juvenilizing effect in the Tenebrio bioassay, although some morphogenetic effect was noticed in the more sensitive Gulleria wax test (SCHNEIDERMANet al., 1965). Thus we are measuring in these bioassays not only the persistent injected hormonal analogue but also the endogenous JH of the larvae. As our studies revealed that GaZZeria larval lipids do not have syner- gistic or inhibitory effects on the activity of the hormonal agents tested and because the endogenous hormone was undetectable in Tenebrio bioassay we believe that our data are valid reflections of the half life of the synthetic hormones in the larval milieu.

RESULTS Inactivation of juvenile hormones in Tenebrio pupae The results reported in Table 1 show that all the Tenebrio pupae grafted to second pupae obtained by application of 6 pg of methyl-12,14-dihomojuvenate developed into adults with no pupal characters. This clearly shows that natural JH (6 pg) applied to the pupae was inactivated in the 5 days that elapsed from the time of application of hormone to the time of parabiosis. There was not even a pupal patch on the sternites, a feature often noticed when as little as 0.1 pg of 12,14-dihomojuvenate was applied. In addition, the third cuticle deposited by the pupa, that was originally exposed to the hormone, was of the adult type.

TABLE ~-PERSISTENT JWFWLEHORMONEACTIVITYIN Tenebriowpm 5 DAYSAPTERTOPICAL APPLICATION

No. that showed Amount of No. of living morphogenetic Morphogenetic hormone parabiosed effects effect applied pairs (percentage in (average Chemical OLg) examined parentheses) score)

12,14-Dihomojuvenate 6 16 0 (0) 0 Methylenedioxyphenoxy 0.5 15 9 (66) 1.5 compound 1 15 13 (87) 3

The hormonal agent was applied to fresh (> 6 hr) Tenebrio pupa and 5 days later was parabiosed to another fresh pupa. The morphogenetic effect on the pupa was determined at the ecdysis that occurs 6 to 8 days after parabiosis.

In contrast to this, over 60 per cent of the pupae parabiosed to second pupae produced by application of 0.5 pg of methylenedioxyphenoxy compound, showed pupal features at the succeeding ecdysis. At the apolysis that occurred after parabiosis, most of the pupae retained gin traps, and had untanned pupal patches on the sternites (Fig. 1B). This morphogenetic effect was quantified by deter- mining the average score, which was about 1.5. Moreover the third cuticle deposited by the pupae, that were originally exposed to the hormonal agent was pupal 2023

FIG. 1. Effect of JH persistent 5 days after application to a pupa on adult development of a parbiosed > 6 hr old Tenebrio pupa. (A) Parabiosed to a pupa treated with 6 pg of 12,14-dihomojuvenate. Note the absence of pupal features. (B) Parabiosed to a pupa treated with 1 pg of methylenedioxyphenoxy analogue. Note the retention of pupal features. ACTIVITYAND METABOLIC STABILITY OF JLWBNILEHORMONES 2025

in nature. This clearly demonstrates that unlike 12,14-dihomojuvenate, methylenedioxyphenoxy compound persisted in the Tenebrio for at least 5 days. In limited experiments, application of lower (2 pg) and higher (10 pg) doses of 12,14-dihomojuvenate gave similar results as application of 6 pg per pupae. However, on application of higher (1 pg) dose of methylenedioxyphenoxy compound a higher percentage (about 85 per cent) of the parabiosed pupae showed morphogenetic effects. Even the average individual score of morphogenetic effect on the parabiosed pupae increased from about 1.5 to about 3-O.

Relative rates of inactivation of JH analogues in O-day-old last instar Galleria larvae The data presented in Table 2 demonstrate that the natural JH, 12,14-dihomojuvenate was inactivated more or less completely in about 8 hr after injection into a GaZZerialarva. In contrast to this, methylenedioxyphenoxy analogue and ethyl- trimethyl-dodeca-dienoate were persistent at least for 24 hr, and some activity was recovered even 48 hr after injection. The chlorophenoxy analogue was inter- mediate in its stability in these larvae. Thus it was concluded that the three analogues tested in this study persisted longer than the natural hormone in Gal&a larvae.

TABLE ~-DECAY OF JWENILE HORMONE ACTIVITY IN EARLY LAST INSTARGalleriu LARVAE INJECTRDWITH VARIOUSANALOGUES

Percentage activity recovered at different Amount injected intervals (in hr) after injection per larva Compound OLg) 0 4 8 24 48 72

12,14-Dihomojuvenate 2.5 100 33 4 o* 0 - Methylenedioxyphenoxy 5 100 98 83 8 5 Ox analogue Ethyl-3,7,11- 2 100 100 100 6 2 0” trimethyldodeca- dienoate Chlorophenoxy 5 100 50 39 8 O* 0” analogue

* Although no juvenile hormone activity was detected in Tenebrio bioassay, it is possible that a low level of JH not adequate to be detected in this assay may be persistent. Thirty larvae were used for each estimation.

DISCUSSION Metabolic stability of JH and its analogues The data presented in this report clearly show that the half life of the more potent JH analogues, in Tenebrih pupae and Galleria larvae, was longer than that of the natural JH. The longer half life of the JH analogues is apparently due to their 2026 GUNDA REDDY ANDA. KRI~~NAIUJMARAN greater metabolic stability. An alternative explanation for the apparent longer half life of the analogues is that these compounds may stimulate the native allata to synthesize and release JH. But we consider this unlikely because endocrine glands are usually subject to negative feed back controls; although application of JH to Saturniid pupae increased the allatal activity ( SIEW and GILBERT, 1971) ; and unless the level of production of the hormone is unusually high the endogenously produced hormone is unlikely to be detected in the Tenebrio bioassay. Hence we suggest that the longer half life of the analogues is associated with the greater metabolic stability of these chemicals. In the in vivo metabolism of juvenile hormones in insects, mixed function oxidases were implicated. Insects which were resistant to insecticides and had enhanced mixed function oxidase activity were also shown to be resistant to JH analogues (BENSKINand PLAPP, 1971). Moreover, JH is enzymatically degraded in vivvoby hydroxylation of the epoxide or by oxidation of the methyl moiety of the molecule, both steps possibly catalysed by mixed function oxidases (Casida, personal communication). Since the JH analogues used in this study either contain known antagonists of mixed function oxidases, viz. methylenedioxyphenoxy or chlorophenoxy moiety; or lack the epoxide group, an easy target of the mixed function oxidases, it is not surprising that these analogues are more resistant to enzymatic breakdown in insects. It will be interesting to know whether alkylamine derivates are also more potent analogues of the JH (CRUICKSHANKand PALMERE,1971) for similar reasons.

Relationship between metabolic stability and morphogenetic potency The positive correlation between metabolic stability and higher morphogenetic activity of JH analogues suggests that JH need be present in the insect for a long period of time to affect the target tissues. Although the reasons for the prolonged presence of JH are not clear, there are three possibilities. The hormone is required to trigger not one, but a series of successive cellular events to bring about its morphogenetic effect. Alternatively the target cells in different regions of the insect body become responsive to JH at different times. A third and less likely possibility is that the transport of the hormone to the site of its action is a slow process requiring the presence of hormones over a long period. In view of the fact that morphogenetic effects of JH can be expressed only in the simultaneous presence of ecdysone (cf. SCHNEIDERMANand GILBERT, 1964) and because the insect pupal cells require a source of ecdysones for prolonged periods (WILLIAMS, 1952) it is suggested that not all insect cells become sensitive to JH at one time. This may be the underlying cause for the higher potency of the more stable JH analogues. Lastly, these results have a bearing on the application of JH as insecticides. Recent findings of JH analogues that seem to show morphogenetic effect only in one or two related species (SL~A et al., 1970) led to the belief that there may be species specific juvenile hormones. It was further suggested that this species specificity may provide a valuable method for the selective elimination of specific ACTIVITYAND METABOLICSTABILITY OF JWENILEHORMONES 2027 insect pests. In view of the present finding, that hormonal activity may be corre- lated with metabolic stability, the apparent species specificity of JH analogues may be based on differences in the ability of insects to metabolize the various analogues, and not on the diversity of juvenile hormones in insects.

Acknowledgements-Our sincere thanks are due to Dr. KFSTUTISTAUTVYDAS and Dr. BRIAN R. UNSWORTHfor critical reading of the manuscript and help with the English. Thanks are due to Dr. J. C. BAUERNFEINDof Hoffmann La Roche, Nutley, N.J. and Dr. JOHN B. SIDDALLof Zoecon Corporation for generously providing us with the juvenile hormone and its analogues. GUNDA REDDY was supported by the National Science Foundation through their grant No. G.B. 19629.

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