Degradation and conjugation of Cecropia juvenile hormone by the southern armyworm (Prodenia eridania)

Comp. Biochem. Physiol., 1974, Vol. 49B, pp. 99 to 103. Pergamon Press. Printed in Great Britain

DEGRADATION AND CONJUGATION OF CECROPIA JUVENILE HORMONE BY THE SOUTHERN ARMYWORM

(PRODENIA ERIDANIA) MICHAEL SLADE and C. F. W I L K I N S O N Department of Entomology, Cornell University, Ithaca, N.Y. 14850, U.S.A.

(Received 4 September 1973) Abstract--1. The degradation of Cecropia juvenile hormone by

Prodenia

eridania occurs via two major pathways. 2. Ester hydrolysis can either precede or succeed epoxide cleavage. 3. The responsible esterase and epoxide hydrase are found in a number of tissues. 4. The final degradative product is an acid-diol that is conjugated and excreted as the sulphate. INTRODUCTION ALTHOUGHthe role of juvenile hormone in controlling the development of insects is well established (Wigglesworth, 1970), the regulatory mechanism by which its titer is controlled is less well defined. It is possible that a feedback mechanism is involved, in which the relative levels of juvenile hormone and/or its metabolites play an important part. In order to investigate this possibility, it is necessary to have a clearer understanding of the processes involved in the degradation of juvenile hormone in several insect species. The juvenile hormone of the silkmoth Hyalophora cecropia (JH) has been shown to be rather non-specific in its ability to inhibit metamorphosis in insects (R611er & Dahm, 1968), and because of this the metabolism of JH has been studied quite extensively in a number of insect species representing several orders. Slade & Zibitt (1971, 1972) established that metabolism occurred vfi~ two major pathways. Ester hydrolysis, mediated by an esterase, can either precede or succeed epoxide cleavage, in which an epoxide hydrase converts the epoxide ring to a trans-diol. It was further established that in the case of the tobacco hornworm (Manduca sexta) esterase activity occurs in several tissues, and epoxide hydrase activity is found in the fat body (Slade & Zibitt, 1971, 1972). Some of these results have been confirmed by later observations (Ajami & Riddiford, 1973). In the flesh-fly(Sarcoptmga bullata), it has been shown that the major metabolite, which occurs through epoxide ring cleavage without coincident esterase action, is conjugated as the sulphate (Slade & Zibitt, 1972). However, it has been suggested that JH metabolites and those of structurally related compounds are conjugated 99

100

MICHAEL SLADEAND C. F. WILKINSON

as the glucuronides or glucosides (White, 1972; Ajami & Riddiford, 1973). Clarification is obviously needed about the nature of the conjugates as well as further information concerning the functions of specific tissues in the metabolism of J H . T h i s report covers these areas, with reference to the southern a r m y w o r m (Prodenia

eridania). MATERIALS AND M E T H O D S Materials Racemic JH, labelled with 14C in the 2-position and with a specific activity of 25"3 mCi/ m-mole, was synthesized by the method of Hafferl et al. (1971). ATP (disodium salt), GSH and sulfatase (Type I I I from limpets, low ~-glucuronidase activity) were obtained from Sigma Chemical Company, St. Louis. All other chemicals employed were of analytical reagent grade. Sixth (i.e. last) instar southern armyworm larvae, reared according to previously reported conditions (Krieger & Wilkinson, 1969) from eggs supplied by the Niagara Chemical Division, FMC Corporation, Middleport, N.Y., were used in all the experiments. In vivo experiments Following injection of radiolabelled JH, dissolved in 60% aqueous acetone, into the abdomina of larvae, anesthetized with carbon dioxide, the armyworms were divided into two groups. One group was allowed to incubate at 27°C for 1"0 hr, after which time the contents of the armyworms were extracted with 2 : 1 ether-ethanol, buffered with ammonium sulfate (2 g]ml), and analyzed by thin-layer chromatography (TLC) on silica gel plates using a 35 % ethyl acetate in hexane solvent system. The second group of armyworms was held in a beaker at 27°C and supplied ad lib. with fresh kidney bean leaves. The larval feces was collected during the 24 hr following JH injection. It was then subjected to aqueous extraction, incubated with sulfatase in acetate buffer, pH 5"0, at 37°C for 1"0 hr and analyzed by TLC. In vitro experiments All tissue homogenates were prepared by established procedures (Yang & Wilkinson, 1972). The preparations (0"5 ml of a suspension of two tissues/ml in 0"15 M KC1) were incubated with JH (2"5/zg) in 0"IM Tris-HC1, pH 7"8, for 10 min at 30°C. The total volume of the reaction medium was 5.0 ml. The incubations were extracted and analyzed in a manner similar to that used for the in vivo experiments. RESULTS AND D I S C U S S I O N T h e 35% ethyl acetate in the hexane solvent system separated the radiolabelled components of the organic extract of the whole a r m y w o r m into a n u m b e r of areas associated with the major metabolites of Cecropia juvenile h o r m o n e (Fig. 1). T h e s e areas, well defined by Slade & Zibitt (1971, 1972), are unchanged J H (RI, 0.88), an epoxy-acid (2; Fig. 2; Rf, 0.49), an ester-diol (3; R 1, 0.37) and an acid-diol (4; RI, 0.10). T h e T L C analysis of the in vitro extracts produced a similar pattern, but in degrees that varied according to the tissue under investigation. Prior to incubation with sulfatase at least one other radioactive zone, occurring at the origin, was present in the fecal extracts. Use of the more polar 9 : 1 chlorof o r m - e t h a n o l solvent system failed to move the zone f r o m the origin, but following sulfatase incubation, all the area m o v e d to a zone of R 1 0.45, identifiable as being associated with the acid-diol 4.

CECROPIAJUVENILEHORMONE,DEGRADATIONANDCONJUGATION

101

SOLVENT

FiONT

L__

m

FIo. 1. Major radiolabelled components present in the organic extract from P. eridania 1"0 hr after injection of 2-1~C-Cecropia juvenile hormone, revealed by thin-layer chromatography with a 35% ethyl acetate-hexane solvent system. (1, unchanged JH; 2, epoxy-acid; 3, ester-diol; 4, acid-diol.)

~,/R-COOCH 3 ESTERAS~

1;JH

~/R'--COOH

CONJUGATED?

~

EPOXIDE

j~/R--COOCH 3

H~/R

COOH

CONJUGATED?

I SULPHOTRANSFERASE SULPHATECONJUGATE 5 EXCRETED

FIG. 2. Major metabolic pathways of Cecropia juvenile hormone in the southern

armyworm (P. eridania). The pathways for the metabolism of JH in the southern armyworm are therefore as shown in Fig. 2, and are basically the same as those established for other insects (Slade & Zibitt, 1971, 1972). The acid 2 and the ester-diol 3 may be conjugated and excreted. However, the fact that the acid diol is definitely excreted as the sulphate conjugate 5 was supported by in vitro synthesis of acid-diol sulphate. The acid-diol (72.8 per cent), formed in the absence of cofactors necessary for the generation of "active" sulphate, was converted to its sulphate by the armyworm gut and the sulphotransferase system of Yang & Wilkinson (1972). No evidence for glucuronide or glucoside formation was obtained.

102

MICHAEL SLADEAND C. F. WILKINSON

The data in Table 1 show that esterase activity is found in all tissues of P. eridania, as well as in all tissues of H. cecropia, and in the mid-gut, at least, of G. portentosa (unpublished). This supports the earlier evidence from M . sexta (Slade

& Zibitt, 1971, 1972). Epoxide hydrase activity, which was found to be microsomal, TABLE 1--DISTRIBUTION OF

JH

DEACTIVATING ENZYMES IN VARIOUS INSECTS*

Tissue Enzyme system Esterase Epoxide hydrase Sulphotransferaset

Blood P, H, M ---

Mid-gut

Fat body

P, H, G P, H, G P, H,M, G

P, H, M P, H, M P

Malpighian tubules P, H P, H P

Body wall P, H, M P, H, M --

* P, Prodenia eridania; H, Hyalophora cecropia; M, Manduca sexta; G, Gromphadorhina portentosa. The mid-gut and the malpighian tubules of M. sexta have not been investigated for either esterase or epoxide hydrase activity. The mid-gut is the only tissue that has been studied in detail in G. portentosa. The distribution of sulphotransferase has only been investigated completely in P. eridania. ~"Yang & Wilkinson (1972, 1973). can be detected in a number of tissues, but it is absent from the haemolymph of all the insects studied. High epoxide hydrase activity occurs in the fat body and in the mid-gut, where sulphate conjugation and excretion can occur. Indeed, sulphate conjugation of JH metabolites is almost certainly a purely excretory mechanism, in contrast to the possibility that sulphate formation may play an important role in controlling the availability to insects of the moulting hormone, ecdysone (Yang & Wilkinson, 1973). The precise manner in which the level of endogenous juvenile hormone is controlled to insure normal metamorphosis is unknown. Whitmore et al. (1972) have shown that juvenile hormone induces the esterases capable of breaking down the hormone and have suggested that this may be one mechanism that controls insect development. Although a similar situation may exist with epoxide hydrase, information is lacking in this case. Further knowledge concerning the roles played by the enzymes that degrade J H is definitely needed. Added significance has been given to this by the considerable attention given in recent years to the possibility of controlling insects by interfering with the action of their hormones (Menn & Beroza, 1972). This has resulted in a large number of compounds being synthesized that exhibit activity in juvenile hormone assays and which, in spite of their very limited structural resemblance to natural juvenile hormones, have been considered to be intrinsically hormonal. However, recent findings indicate that the observed morphogenic action of many

CECROPIAJUVENILEHORMONE, DEGRADATIONAND CONJUGATION

103

of these compounds is probably synergistic (Slade & Wilkinson, 1973). T h e compounds inhibit the esterase and the epoxide hydrase responsible for metabolizing J H ; but the relationship between the in vitro activities and the in vivo effects is dependent upon a n u m b e r of complex factors, such as age-related metabolic rate changes. Changes in the rate of J H metabolism with increasing age in P. eridania have been observed and these probably are related to the changes in the activity of the epoxide hydrase with age that were also seen.

Acknowledgements--The authors thank Dr. Raymond S. H. Yang of this laboratory for his critical evaluation of this paper. The investigation was supported in part by Public Health Service Grant No. ES 00098, P.H.S. Grant No. ES 00400 and Rockefeller Foundation Grant No. RF 69073. REFERENCES

AJAMI A. M. ~ RIDDIFORDL. M. (1973) Comparative metabolism of the Cecropia juvenile hormone. J. Insect Physiol. 19, 635-645. HAF~RL W., Zum~Ltra R. & DtrNnAM L. (1971) Radiochemical synthesis--II. The preparation of 14C-labelled juvenile hormone. J. Label. Compounds 7, 331-339. KRIEGERR. I. & WILKINSONC. F. (1969) Microsomal mixed-function oxidases in insects--I. Localization and properties of an enzyme system effecting aldrin epoxidation in larvae of the southern armyworm (Prodenia eridania). Biochem. Pharmac. 18, 1403-1415. MENN J. J. & Bm~ozA M. (1972) (Editors) InsectJuvenile Hormones: Chemistry and Action. Academic Press, New York. ROLLERH. & DAHM K. H. (1968) The chemistry and biology of juvenile hormones. Recent Prog. Horm. Res. 24, 651-680. SLADEM. & WILKINSONC. F. (1973) Juvenile hormone analogs: a possible case of mistaken identity ? Science, Wash. 181, 672--674. SLADEM. & ZIBBITTC. H. (1971) Metabolism of Cecropia juvenile hormone in lepidopterans. In Chemical Releasers in Insects, Proceedings of International 1UPAC Congress 2nd Pesticide Chemistry (Edited by TArlORIA. S.), Vol. 3, pp. 45-58. Gordon & Breach, New York. SLADEM. & ZIBn'T C. H. (1972) Metabolism of Cecropia juvenile hormone in insects and in mammals. In lnsectJuvenile Hormones: Chemistry and Action (Edited by MENN J. J. & BEROZAM.), pp. 155-176. Academic Press, New York. WHITE A. F. (1972) Metabolism of the juvenile hormone analogue methyl farnesoate 10,11epoxide in two insect species. Life Sci. 11, 201-210. WHITMORE D., JR., WHITMOIZEE. & GILBERTL. I. (1972) Juvenile hormone induction of esterases: a mechanism for the regulation of juvenile hormone titer. Proc. natn. Acad. Sci. U.S.A. 69, 1592-1595. WIGGLESWORTHV. B. (1970) lnsect Hormones. Oliver & Boyd, Edinburgh. YANG R. S. H. & WILKINSONC. F. (1972) Enzymic sulphation ofp=nitrophenol and steroids by larval gut tissues of the southern armyworm (Prodenia eridania Cramer). Biochem. ft. 130, 487-493. YANG R. S. H. & WILKINSON C. F. (1973) Sulphotransferases and phosphotransferases in

insects. Comp. Biochem. Physiol. 46B, 717-726.

Key Word Index--Cecropia juvenile hormone; degradation; conjugation; Prodenia eridania.