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Epoxide hydrase activity and its relationship to development in the southern armyworm, Prodenia eridania
J. Insect Physiol.,
1976, Vol. 22, pp. 619 to 622. Pergamon Press. Printed in Great Britain.
EPOXIDE HYDRASE ACTIVITY AND ITS RELATIONSHIP TO DEVELOPMENT IN THE SOUTHERN ARMYWORM, PRODENIA ERIDANIA MICHAEL SLADE,* H. KRYSTYNAHETNARSKI,and C. F. WILKINSON Department
of Entomology, Cornell University, Ithaca, N.Y. 14850, U.S.A. (Received 26 April 1975; revised 30 October 1975)
Abstract-Enzymatic epoxide hydration of the cyclodiene insecticide HEOM by the southern armyworm was investigated throughout the late larval, pupal and adult stages of development. Epoxide hydrase activity reaches a maximum in the period between ecdysis of the last instar and the larval-pupal ecdysis, decreases during pupal life and becomes essentially zero following adult emergence. Comparison was made with variations occurring in juvenile hormone hydrase activity, and the significance of these age-dependent changes in relation to regulation of insect development are discussed.
INTRODUCTION ENZYMATIC epoxide hydration is a mechanism of established importance in mammals in the metabolism of biologically active epoxides and arene oxides arising from the microsomal oxidation of a variety of olefinic and aromatic substrates (OESCH, 1972). It
has been shown also to exist in a number of insect species (BROOKS et al., 1970; BROOKS 1973, 1974; HAMMOCK et al., 1974; SLADE et al., 1975), and epox-
ide hydrase is one of two major enzymes involved in the metabolic degradation of juvenile hormone (SLADE and ZBI~, 1971, 1972; SLADE and WILKINSON,1974). Since the presence or absence of juvenile hormone in insects critically determines the precise stage of their development (GILBERTand KING, 1973), and because this control could result from age-related differences in metabolism rates that reflect changes in the titres of the deactivating enzymes, epoxide hydration potentially constitutes an important insect regulatory mechanism. In the case of the second major deactivating mechanism of juvenile hormone, ester hydrolysis, agedependant variations have been observed in the haemolymph of the tobacco hornworm (Manduca sex@ (WEIRICH et al., 1973) and it has been suggested that
tivity on insect age is lacking. The present report investigates this area and is particularly concerned with changes that occur in the ability to hydrate HEOM in relation to insect development and comparison of this with variations in juvenile hormone hydrase activity. Emphasis is placed on activity modifications occurring during the last larval instar, pupal and early adult stages of P. eridania. Any attempt to evaluate changes in juvenile hormone hydrase activity is complicated by the susceptibility of the hormone to simultaneous metabolic attack by the hydrase and esterase (SLADEand ZIBITT, 1971, 1972; SLADE and WILKINSON, 1974). It is assumed that the hydrase responsible for conversion of juvenile hormone to a dihydroxy ester is the same as that which converts the epoxy acid (juvenile hormone acid), formed through esterase attack on the hormone, to a dihydroxy acid. MATERIALS AND METHODS Chemicals Radiolabelled Cecropia juvenile hormone (JH) was prepared by the method of HAFFERL et al. (1971) and
was used to synthesize juvenile hormone acid (JH acid) labelled with 14C in the 2-position and with a specific activity of 2.5.3mCi/m-mole. Hydrolysis of JH was effected with 0.5 N sodium hydroxide in 50% aqueous ethanol for 24hr at room temperature (SLADEand ZIBITT, 1972) but in low yield. However, since JH can be hydrolyzed without further degration by esterases present in the haemolymph of various insects (SLADE and ZIBITT, 1972), JH acid was produced quantitatively by incubating JH aerobically concerning the dependence of epoxide hydrase ac- with haemolymph of P. eridania in 0.1 M Tris-HCl (pH 7.8) for 1 hr at 30°C. Extraction and thin-layer chromatography (TLC) * Present address: Central Toxicology Laboratory, I.C.I. procedures used for clean-up have been described by Ltd., Alderley Park, Near Macclesfield, Cheshire, England. 619 2?/5 the esterase responsible for these changes is involved in the regulation of hormone titre (WHITMORE et al., 1972; WEIRICH et al., 1973). However, although the in vitro requirements of the hydrase responsible for the cleavage of the cyclodiene insecticide HEOM in pupal homogenates of the blowfly (Calliphoru erythrocephakz) and in larval mid-gut preparations of the southern armyworm (Prodenia eridukz) have been defined (SLADE et al., 1975), comparable information
I.I.P.
A
620
MICHAELSLADE,H. KRYSTYNAHETNARSKI, AND C. F. WILKINSON
SLADE and ZIBITT (1972). The preparation of HEOM (1,2,3,4,9,9-hexachloro-6,7-epoxy-1,4,4a,5,6,7, 8,8a-octahydro-l,4-methanonaphthalene) was by the method of BROOKSand HARRISON(1964). All other chemicals employed were of analytical reagent grade. Insects The P. eridania culture was reared from eggs supplied by the Niagara Chemical Division, FMC Corporation, Middleport, N.Y., and maintained under previously described greenhouse conditions (YANG and WILKINSON, 1972). In all experiments, 300 larvae were isolated over a period of 12 hr just prior to and during ecdysis of the 5th (last) instar and, depending on size, up to 20 insects were used at that time and at the same hr of each succeeding day for enzyme preparation and assay. Enzyme preparation and assay Mid-gut homogenates, prepared by established procedures (YANG and WILKINSON, 1972) constituted the enzyme source in the larval experiments. For the pupal, pharate adult and adult stages, the total body contents were removed, following decapitation, and suspended in ice-cold 0.15 M KC1 in a hand-operated all-glass Potter Elvehjem homogenizer. A 10% homogenate was prepared for use in the experiments. Protein concentrations were determined by the modified Lowry method of CHAYKIN (1966). Due to differences in the pH optima for the hydration of HEOM and of JH and JH acid (SLADE et al., 1975), glycine-sodium hydroxide buffer (pH 9.0) was used for studies conducted with HEOM and Tris-HCl buffer (pH 7.8) was employed for those with JH and JH acid. The standard reaction mixture consisted of 0.5 ml of the enzyme preparation, 4.5 ml of appropriate buffer and either 100 pg HEOM in 25 ~1 ethanol or 2.5 pg 2-14C-JH or 2-14C-JH acid in 10~1 ethanol. The incubations were carried out aerobically for 10 min at 30°C. The HEOM reaction was terminated by the addition of 7.0ml acetone and, following the addition of 10 ml of 4% sodium sulfate, the whole mixture was extracted with two 5.0 ml aliquots of ether-hexarie (1:l). The combined organic extracts were dried over anhydrous sodium sulphate and estimation of HEOM diol in the extracts was achieved, after conversion to the corresponding trimethyl silyl ether (TMS) derivative, by gas chromatographic analysis as previously reported (SLADE et al., 1975). Termination and extraction of the JH and JH acid reactions with ether-ethanol (2: 1) in the presence of &nmonium sulphate and subsequent TLC analysis of the extracts was by described methods (SLADE and ZIBITT, 1972). Radioactive zones were detected by scanning the TLC plates on a Packard Radiochromatogram Scanner Model 7201 and for quantitation the radioactive gel regions were eluted with a scintillation mixture (0.55% w/v PPO and 0.015’~ w/v dimethyl POPOP in toluene-Triton X-100, 2: 1) for direct radiocarbon
assay with a Packard Tri-Carb Spectrometer, Model 2425.
Liquid
Scintillation
RESULTS The activity of the epoxide hydrase responsible for the hydration of HEOM, measured by HEOM diol formation, was determined daily with tissue preparations from P. eridania, beginning with larvae just prior to ecdysis of the 5th instar, continuing with pupae and ending with adults after emergence. The results (Fig. l), which represent the average of 3 replicate experiments, clearly indicate that the enzyme activity is age-dependent, increasing rapidly at larval ecdysis, attaining a maximum between that ecdysis and larval-pupal ecdysis, and decreasing throughout pupal and pharate adult life to become essentially zero following adult emergence. Although the hydration of JH acid to the corresponding diol was investigated with homogenates from P. eridania only between ecdysis of the 5th instar and larval-pupal ecdysis, a dependence of hydrase activity on insect age comparable to that for HEOMhydrase is evident (Fig. 2). The juvenile hormone hydrase titre is highest during the period between the larval ecdysis and the larval-pupal ecdysis, with maximum activity occurring at the same time as that for HEOM-hydrase: The data in Fig. 2 resulted from experiments conducted during very humid and very hot (31 to 34°C) summer weather whereas those shown in Fig. 1 were obtained during the bright, warm (18 to 21°C) springtime, and so there is a difference between HEOM diol formation in Fig. 2 compared with that in Fig. 1. However, in spite of the seasonal variations in the developmental period of the insects, the patterns of epoxide hydrase activity are essentially the same. As is also shown in Fig. 2, the recovery of JH from the in vitro incubations, which is the inversely proportional measure of the rate of JH metabolism, follows a course during the last larval instar t&at is, up to the time preparatory to puparium formation (burrowing), the inverse of that of epoxide hydrase activity. Thus the rate of JH metabolism is highest in mid-ins&, the time of highest epoxide hydrase activity. In contrast to this, the rate of formation of JH acid from JH, a measure of juvenile hormone esterase activity, remains low throughout the instar but increases very rapidly at larval-pupal ecdysis.
DISCUSSION Age-dependent changes in the activity of the enzyme that catalyzes the hydration of HEOM to the corresponding diol have been demonstrated in P. eridania for the period from just prior to the onset of ecdysis of the 5th instar to early adult formation and encompassing the last larval instar and pupal stages of development. Dependence of activity on age between the larval ecdysis and larval-pupal ecdysis
Epoxide
hydrase
activity
and insect development
621
0.3 Dial
produced uglmg
protein
i 10
min 0.2
Fig. 1. Age-dependent
changes in the enzymatic hydration of the epoxide HEOM pupal and early adult development of the southern armyworm.
during
late larval,
1.5
has also been observed rase.
The
similarity
with juvenile hormone hydof these changes to those of
HEOM hydrase during that period suggests that, in spite of certain differences in properties and substrate requirements for the 2 enzymes (SLADE et al., 1975) a pattern of activity for the hydration of juvenile hormone comparable to that of HEOM could exist throughout all development stages. In spite of the well-established role of juvenile hormone in controlling insect development (GILBERT and KING, 1973) the mechanism by which its titre is regulated is less clearly defined. Evidence has been presented for the participation of neural, neuroendocrine and humoral factors in the control of JH production (ENGELMANN,1965; HIGHNAM, 1967) and it has been suggested that result from the
stimulation stimulation
of
JH
synthesis
1.0
Activity
0.5
could
of RNA synthesis in the corpora allata that has been reported as occurring with ecdysone (SIEW and GILBERT, 1971) although this conclusion has been challenged (WILLIS, 1974). Binding of JH to a lipoprotein, necessary for JH transport in the haemolymph, has been demonstrated (WHITMOREand GILBERT, 1972; EMMERICHand HARTMANN, 1973) and it is possible that regulation of the availability of the carrier protein could provide a method of modulating JH titre (SLADE and ZIBIIT, 1972; TRAUTMANN, 1972); or an esterase known to specifically degrade the hormone-carrier complex could be involved in the control of metamorphosis (SANBURG et al., 1975). However, the metabolism of JH itself by esterases present in the haemolymph could control the hormone level as well (SLADE and ZIBITT, 1971; WHITMORE et al., 1972; WEIRICH et al., 1973). Indeed, inverse correlations between haemo-
I
#
I
1 , I
2 Burrowing Time
I
1
0
V. instar (days
after
Pupa ecdysis
)
Fig. 2. Age-dependent changes in the metabolism of Cecropia juvenile hormone (JH) and comparison with JH acid formation (juvenile hormone e&erase activity) and with HEOM hydrase and juvenile hormone hydrase activities during the late larval development of the southern armyworm. The activity is expressed as relative recovery of compound/mg protein/l0 min [JH, l ; x 10-rPJH acid (esterase), W ; x 10-rPJH acid diol (JH hydrase), q] or as diol produced/mg protein/l0 min [x 10-Z-HEOM diol (HEOM hydrase), 01.
622
MICHAEL SLADE, H. KRYSTYNA HETNARSKI,AND C. F. WILKINSON
lymph esterase and JH levels have been observed in the tobacco hornworm; esterase activity increases from a relatively low level soon after the 5th (last) larval ecdysis to a maximum in the period of the 5th instar, then drops to a low level just prior to larvalpupal ecdysis and is followed by somewhat higher activity at the time of larval-pupal ecdysis and, to a much lesser extent, during the adult stage (WEXRICH et al., 1973). The results of the current study suggest that juvenile hormone hydrase could be equally involved in the JH regulatory mechanism. In fact, on the basis of the data in Fig. 2 it is tempting to suggest that epoxide hydrase activity is of primary importance in the control of JH titre throughout the last larval instar, but during the approach to larval-pupal ecdysis, when hydrase activity decreases, the rapid increase in esterase activity is clearly more significant in maintaining the relatively low JH level necessary to ensure pupation. These notions concerning the greater importance of hydrase activity in determining JH levels during the critical period of larval insect development are supported by selective inhibition of the epoxide hydrase and esterase. During early instar, hydrase inhibition is much more effective in stabilizing JH in the presence of a degradative enzyme system than is esterase inhibition (SLADE, in preparation). Although the precise mechanism regulating insect hormone titre is still not established, age-dependent changes in the activities of the enzymes that metabolize juvenile hormone, especially those changes associated with epoxide hydrase, clearly constitute an important element in the complex combination of factors involved. The exact manner in which these activity changes are controlled, however, requires further investigation. Acknowledgements-This work was supported in part by grants from the U.S. Public Health Service (ES 000098 and ES 00400) and the Rockefeller Foundation (RF 69073).
REFERENCES BROOKS G. T. (1973) Insect epoxide hydrase inhibition by juvenile hormone analogues and metabolic inhibitors. Nature, Lond. 245, 382-384. BROOKS G. T. (1974) Inhibitors of cyclodiene epoxide ring hydrating enzymes of the blowfly, Calliphora erythrocephala. Pestic. Sci. 5, 177-183. BRCICIKSG. T. and HARRI~QN A. (1964) The effect of pyrethrin synergists, especially sesamex, on the insecticidal potency of hexachlorocyclopentadiene derivatives (‘cyclodiene’ insecticides) in the adult housefly, Musca domestica L. Biochem. Pharmacol. 13, 827-840. BRINKS G. T., HARRISON A., and LEWIS ‘S. E. (1970) Cyclodiene epoxide ring hydration by; microsomes from mammalian liver and houseflies. Biochem. PharmacoI. 19, 255-273. CHAYKIN S. (1966) In Biochemistry Laboratory Techniques, p. 20. Wiley, New York. EMMERICH H. and HARTMANN R. (1973) A carrier lipopro-
tein for juvenile hormone in the haemolymph of Locusta migratoria. J. Insect Physiol. 19, 1663-1675. ENGELMANN F. (1965) The mode of regulation of the corpus allatum in adult insects. Arch. Anat. microsc. Morph. exp. 54, 387-404. GILBERT L. I. and KING D. S. (1973) Physiology of growth and development: endocrine aspects. In The Physiology of Insecta (Ed. by ROCKSTEIN M.), 2nd. ed., 1, 249-370. Academic Press, New York. HAFFERL W., ZURFLVJH R., and DUNHAM L. (1971) Radiochemical synthesis II. The preparation of 14C-labelled juvenile hormone. J. Label. Compounds 7, 331-339. HAMMOCK B. D., GILL S. S., and CASIDA J. E. (1974) Insect metabolism of a phenyl epoxygeranyl ether juvenoid and related compounds. Pestic. Biochem. Physio2. 4, 393aO6. HIGHNAM K. C. (1967) Insect hormones. J. Endocr. 39, 123-150. OESCH F. (1972) Mammalian epoxide hydrases: inducible enzymes catalysing the inactivation of carcinogenic and cytotoxic metabolites derived from aromatic and oletiic compounds. Xenobiotica 3, 305-340. SANBURG L. L., KRAMER K. J., KEZDY F. J., LAW J. H., and OBERLANDER H. (1975) Role of juvenile hormone esterases and carrier proteins in insect development. Nature, Land. 253, 266-267. SIEW Y. C. and GILBERT L. I. (1971) Effects of moulting hormone and juvenile hormone on insect endocrine gland activity. J. Insect Physiol. 17, 2095-2104. SLADE M. and WILKINSON C. F. (1974) Degradation and conjugation of Cecropia juvenile hormone by the southern armyworm (Prodenia eridunia). Comp. Biochem. Physiol. 49B, 99-103. SLADE M. and ZIBITT C. H. (1971) Metabolism of Cecropia juvenile hormone in lepidopterans. In Chemical -Releasers in Insects, Proce&n& of 2nd International IUPAC Congress of Pesticide Chemistrv (Ed. bv TAHORI A. S.) 3, 45-k Gordon & Breach, New‘York: SLADE M., and ZIBITT C. H. (1972) Metabolism of Cecropia juvenile hormone in insects and in mammals. In Insect Juvenile Hormones: Chemistry and Action (Ed. by MENN J. J. and BEROZA M.), pp. 155-176. Academic Press, New York. SLADE M., BROOKS G. T., HETNARSKI H. K., and WILKINSON C. F. (1975) Inhibition of the enzymatic hydration of the epoxide HEOY in insects. Pestic. Biochem. Physiol. 5, 3546. TRAUTMANN K. H. (1972) In vitro studium der tragerproteine von 3H-markierten juvenilhormonwirksamen verbindingen in der hzmolymphe von Tenebrio molitor L. larven. Z. Naturjorsch. 27, 263-273. WEIRICH G., WREN J., and SIDDALL J. B. (1973) Developmental changes of the juvenile hormone esterase activity in haemolymph of the tobacco hornworm, Manduca sexta. Insect Biochem. 3, 397-407. WHITMORE D., Jr., WHITMORE E., and GILBERT L. I. (1972) Juvenile hormone induction of esterases: a mechanism for the regulation of juvenile hormone titer. Proc. naf. Acad. Sci. U.S.A. 69, 1592-1595. WHITMORE E. and GILBERT L. I. (1972) Haemolymph lipoprotein transport of juvenile hormone. J. Insect Physiol. 18, 1153-1168. WILLIS J. H. (1974) Morphogenetic action of insect hormones. A. Rev. Ent. 19, 97-115. YANG R. S. H. and WILKINSON C. F. (1972) Enzymic sulphation of p-nitrophenol and steroids by larval gut tissues of the southern armyworm (Prodenia eridania Cramer). Biochem. J. 130, 487493.
1976, Vol. 22, pp. 619 to 622. Pergamon Press. Printed in Great Britain.
EPOXIDE HYDRASE ACTIVITY AND ITS RELATIONSHIP TO DEVELOPMENT IN THE SOUTHERN ARMYWORM, PRODENIA ERIDANIA MICHAEL SLADE,* H. KRYSTYNAHETNARSKI,and C. F. WILKINSON Department
of Entomology, Cornell University, Ithaca, N.Y. 14850, U.S.A. (Received 26 April 1975; revised 30 October 1975)
Abstract-Enzymatic epoxide hydration of the cyclodiene insecticide HEOM by the southern armyworm was investigated throughout the late larval, pupal and adult stages of development. Epoxide hydrase activity reaches a maximum in the period between ecdysis of the last instar and the larval-pupal ecdysis, decreases during pupal life and becomes essentially zero following adult emergence. Comparison was made with variations occurring in juvenile hormone hydrase activity, and the significance of these age-dependent changes in relation to regulation of insect development are discussed.
INTRODUCTION ENZYMATIC epoxide hydration is a mechanism of established importance in mammals in the metabolism of biologically active epoxides and arene oxides arising from the microsomal oxidation of a variety of olefinic and aromatic substrates (OESCH, 1972). It
has been shown also to exist in a number of insect species (BROOKS et al., 1970; BROOKS 1973, 1974; HAMMOCK et al., 1974; SLADE et al., 1975), and epox-
ide hydrase is one of two major enzymes involved in the metabolic degradation of juvenile hormone (SLADE and ZBI~, 1971, 1972; SLADE and WILKINSON,1974). Since the presence or absence of juvenile hormone in insects critically determines the precise stage of their development (GILBERTand KING, 1973), and because this control could result from age-related differences in metabolism rates that reflect changes in the titres of the deactivating enzymes, epoxide hydration potentially constitutes an important insect regulatory mechanism. In the case of the second major deactivating mechanism of juvenile hormone, ester hydrolysis, agedependant variations have been observed in the haemolymph of the tobacco hornworm (Manduca sex@ (WEIRICH et al., 1973) and it has been suggested that
tivity on insect age is lacking. The present report investigates this area and is particularly concerned with changes that occur in the ability to hydrate HEOM in relation to insect development and comparison of this with variations in juvenile hormone hydrase activity. Emphasis is placed on activity modifications occurring during the last larval instar, pupal and early adult stages of P. eridania. Any attempt to evaluate changes in juvenile hormone hydrase activity is complicated by the susceptibility of the hormone to simultaneous metabolic attack by the hydrase and esterase (SLADEand ZIBITT, 1971, 1972; SLADE and WILKINSON, 1974). It is assumed that the hydrase responsible for conversion of juvenile hormone to a dihydroxy ester is the same as that which converts the epoxy acid (juvenile hormone acid), formed through esterase attack on the hormone, to a dihydroxy acid. MATERIALS AND METHODS Chemicals Radiolabelled Cecropia juvenile hormone (JH) was prepared by the method of HAFFERL et al. (1971) and
was used to synthesize juvenile hormone acid (JH acid) labelled with 14C in the 2-position and with a specific activity of 2.5.3mCi/m-mole. Hydrolysis of JH was effected with 0.5 N sodium hydroxide in 50% aqueous ethanol for 24hr at room temperature (SLADEand ZIBITT, 1972) but in low yield. However, since JH can be hydrolyzed without further degration by esterases present in the haemolymph of various insects (SLADE and ZIBITT, 1972), JH acid was produced quantitatively by incubating JH aerobically concerning the dependence of epoxide hydrase ac- with haemolymph of P. eridania in 0.1 M Tris-HCl (pH 7.8) for 1 hr at 30°C. Extraction and thin-layer chromatography (TLC) * Present address: Central Toxicology Laboratory, I.C.I. procedures used for clean-up have been described by Ltd., Alderley Park, Near Macclesfield, Cheshire, England. 619 2?/5 the esterase responsible for these changes is involved in the regulation of hormone titre (WHITMORE et al., 1972; WEIRICH et al., 1973). However, although the in vitro requirements of the hydrase responsible for the cleavage of the cyclodiene insecticide HEOM in pupal homogenates of the blowfly (Calliphoru erythrocephakz) and in larval mid-gut preparations of the southern armyworm (Prodenia eridukz) have been defined (SLADE et al., 1975), comparable information
I.I.P.
A
620
MICHAELSLADE,H. KRYSTYNAHETNARSKI, AND C. F. WILKINSON
SLADE and ZIBITT (1972). The preparation of HEOM (1,2,3,4,9,9-hexachloro-6,7-epoxy-1,4,4a,5,6,7, 8,8a-octahydro-l,4-methanonaphthalene) was by the method of BROOKSand HARRISON(1964). All other chemicals employed were of analytical reagent grade. Insects The P. eridania culture was reared from eggs supplied by the Niagara Chemical Division, FMC Corporation, Middleport, N.Y., and maintained under previously described greenhouse conditions (YANG and WILKINSON, 1972). In all experiments, 300 larvae were isolated over a period of 12 hr just prior to and during ecdysis of the 5th (last) instar and, depending on size, up to 20 insects were used at that time and at the same hr of each succeeding day for enzyme preparation and assay. Enzyme preparation and assay Mid-gut homogenates, prepared by established procedures (YANG and WILKINSON, 1972) constituted the enzyme source in the larval experiments. For the pupal, pharate adult and adult stages, the total body contents were removed, following decapitation, and suspended in ice-cold 0.15 M KC1 in a hand-operated all-glass Potter Elvehjem homogenizer. A 10% homogenate was prepared for use in the experiments. Protein concentrations were determined by the modified Lowry method of CHAYKIN (1966). Due to differences in the pH optima for the hydration of HEOM and of JH and JH acid (SLADE et al., 1975), glycine-sodium hydroxide buffer (pH 9.0) was used for studies conducted with HEOM and Tris-HCl buffer (pH 7.8) was employed for those with JH and JH acid. The standard reaction mixture consisted of 0.5 ml of the enzyme preparation, 4.5 ml of appropriate buffer and either 100 pg HEOM in 25 ~1 ethanol or 2.5 pg 2-14C-JH or 2-14C-JH acid in 10~1 ethanol. The incubations were carried out aerobically for 10 min at 30°C. The HEOM reaction was terminated by the addition of 7.0ml acetone and, following the addition of 10 ml of 4% sodium sulfate, the whole mixture was extracted with two 5.0 ml aliquots of ether-hexarie (1:l). The combined organic extracts were dried over anhydrous sodium sulphate and estimation of HEOM diol in the extracts was achieved, after conversion to the corresponding trimethyl silyl ether (TMS) derivative, by gas chromatographic analysis as previously reported (SLADE et al., 1975). Termination and extraction of the JH and JH acid reactions with ether-ethanol (2: 1) in the presence of &nmonium sulphate and subsequent TLC analysis of the extracts was by described methods (SLADE and ZIBITT, 1972). Radioactive zones were detected by scanning the TLC plates on a Packard Radiochromatogram Scanner Model 7201 and for quantitation the radioactive gel regions were eluted with a scintillation mixture (0.55% w/v PPO and 0.015’~ w/v dimethyl POPOP in toluene-Triton X-100, 2: 1) for direct radiocarbon
assay with a Packard Tri-Carb Spectrometer, Model 2425.
Liquid
Scintillation
RESULTS The activity of the epoxide hydrase responsible for the hydration of HEOM, measured by HEOM diol formation, was determined daily with tissue preparations from P. eridania, beginning with larvae just prior to ecdysis of the 5th instar, continuing with pupae and ending with adults after emergence. The results (Fig. l), which represent the average of 3 replicate experiments, clearly indicate that the enzyme activity is age-dependent, increasing rapidly at larval ecdysis, attaining a maximum between that ecdysis and larval-pupal ecdysis, and decreasing throughout pupal and pharate adult life to become essentially zero following adult emergence. Although the hydration of JH acid to the corresponding diol was investigated with homogenates from P. eridania only between ecdysis of the 5th instar and larval-pupal ecdysis, a dependence of hydrase activity on insect age comparable to that for HEOMhydrase is evident (Fig. 2). The juvenile hormone hydrase titre is highest during the period between the larval ecdysis and the larval-pupal ecdysis, with maximum activity occurring at the same time as that for HEOM-hydrase: The data in Fig. 2 resulted from experiments conducted during very humid and very hot (31 to 34°C) summer weather whereas those shown in Fig. 1 were obtained during the bright, warm (18 to 21°C) springtime, and so there is a difference between HEOM diol formation in Fig. 2 compared with that in Fig. 1. However, in spite of the seasonal variations in the developmental period of the insects, the patterns of epoxide hydrase activity are essentially the same. As is also shown in Fig. 2, the recovery of JH from the in vitro incubations, which is the inversely proportional measure of the rate of JH metabolism, follows a course during the last larval instar t&at is, up to the time preparatory to puparium formation (burrowing), the inverse of that of epoxide hydrase activity. Thus the rate of JH metabolism is highest in mid-ins&, the time of highest epoxide hydrase activity. In contrast to this, the rate of formation of JH acid from JH, a measure of juvenile hormone esterase activity, remains low throughout the instar but increases very rapidly at larval-pupal ecdysis.
DISCUSSION Age-dependent changes in the activity of the enzyme that catalyzes the hydration of HEOM to the corresponding diol have been demonstrated in P. eridania for the period from just prior to the onset of ecdysis of the 5th instar to early adult formation and encompassing the last larval instar and pupal stages of development. Dependence of activity on age between the larval ecdysis and larval-pupal ecdysis
Epoxide
hydrase
activity
and insect development
621
0.3 Dial
produced uglmg
protein
i 10
min 0.2
Fig. 1. Age-dependent
changes in the enzymatic hydration of the epoxide HEOM pupal and early adult development of the southern armyworm.
during
late larval,
1.5
has also been observed rase.
The
similarity
with juvenile hormone hydof these changes to those of
HEOM hydrase during that period suggests that, in spite of certain differences in properties and substrate requirements for the 2 enzymes (SLADE et al., 1975) a pattern of activity for the hydration of juvenile hormone comparable to that of HEOM could exist throughout all development stages. In spite of the well-established role of juvenile hormone in controlling insect development (GILBERT and KING, 1973) the mechanism by which its titre is regulated is less clearly defined. Evidence has been presented for the participation of neural, neuroendocrine and humoral factors in the control of JH production (ENGELMANN,1965; HIGHNAM, 1967) and it has been suggested that result from the
stimulation stimulation
of
JH
synthesis
1.0
Activity
0.5
could
of RNA synthesis in the corpora allata that has been reported as occurring with ecdysone (SIEW and GILBERT, 1971) although this conclusion has been challenged (WILLIS, 1974). Binding of JH to a lipoprotein, necessary for JH transport in the haemolymph, has been demonstrated (WHITMOREand GILBERT, 1972; EMMERICHand HARTMANN, 1973) and it is possible that regulation of the availability of the carrier protein could provide a method of modulating JH titre (SLADE and ZIBIIT, 1972; TRAUTMANN, 1972); or an esterase known to specifically degrade the hormone-carrier complex could be involved in the control of metamorphosis (SANBURG et al., 1975). However, the metabolism of JH itself by esterases present in the haemolymph could control the hormone level as well (SLADE and ZIBITT, 1971; WHITMORE et al., 1972; WEIRICH et al., 1973). Indeed, inverse correlations between haemo-
I
#
I
1 , I
2 Burrowing Time
I
1
0
V. instar (days
after
Pupa ecdysis
)
Fig. 2. Age-dependent changes in the metabolism of Cecropia juvenile hormone (JH) and comparison with JH acid formation (juvenile hormone e&erase activity) and with HEOM hydrase and juvenile hormone hydrase activities during the late larval development of the southern armyworm. The activity is expressed as relative recovery of compound/mg protein/l0 min [JH, l ; x 10-rPJH acid (esterase), W ; x 10-rPJH acid diol (JH hydrase), q] or as diol produced/mg protein/l0 min [x 10-Z-HEOM diol (HEOM hydrase), 01.
622
MICHAEL SLADE, H. KRYSTYNA HETNARSKI,AND C. F. WILKINSON
lymph esterase and JH levels have been observed in the tobacco hornworm; esterase activity increases from a relatively low level soon after the 5th (last) larval ecdysis to a maximum in the period of the 5th instar, then drops to a low level just prior to larvalpupal ecdysis and is followed by somewhat higher activity at the time of larval-pupal ecdysis and, to a much lesser extent, during the adult stage (WEXRICH et al., 1973). The results of the current study suggest that juvenile hormone hydrase could be equally involved in the JH regulatory mechanism. In fact, on the basis of the data in Fig. 2 it is tempting to suggest that epoxide hydrase activity is of primary importance in the control of JH titre throughout the last larval instar, but during the approach to larval-pupal ecdysis, when hydrase activity decreases, the rapid increase in esterase activity is clearly more significant in maintaining the relatively low JH level necessary to ensure pupation. These notions concerning the greater importance of hydrase activity in determining JH levels during the critical period of larval insect development are supported by selective inhibition of the epoxide hydrase and esterase. During early instar, hydrase inhibition is much more effective in stabilizing JH in the presence of a degradative enzyme system than is esterase inhibition (SLADE, in preparation). Although the precise mechanism regulating insect hormone titre is still not established, age-dependent changes in the activities of the enzymes that metabolize juvenile hormone, especially those changes associated with epoxide hydrase, clearly constitute an important element in the complex combination of factors involved. The exact manner in which these activity changes are controlled, however, requires further investigation. Acknowledgements-This work was supported in part by grants from the U.S. Public Health Service (ES 000098 and ES 00400) and the Rockefeller Foundation (RF 69073).
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