- Email: [email protected]
Excretion of juvenile hormone and its metabolites in the locust, Locusta migratoria
J. Insect Physiol., 1975, VoI. 21, pp. 61 to 70. Pergamon Press. Printed in Great Britain
EXCRETION OF JUVENILE HORMONE AND ITS METABOLITES IN THE LOCUST, LOCUSTA
MIGRATORIA DOROTHEE
ERLEY,
SHARON
SOUTHARD,
and HANS EMMERICH*
Zoologisches Institut, 2. Lehrstuhl, Universitiit zu K&n, Germany (Received 3 June 1974)
Abstract-Racemic synthetic 3H-C,s juvenile hormone, dissolved in paraIfin oil, was injected into adult Locusta migratoria and the excreted radioactive material in the faeces was determined. Within 48 hr two-thirds of the injected radioactivity can be recovered in the frass, half of it within 3 hr. The remaining one-third of the injected label is incorporated or is released as water. Adult locusts of either sex or of different ages show no difference in the metabolic pathways of the JH and in its excretion rate. The excreta contain as a degradation product 7-ethyl-3,l l-dimethyl-cis10,ll -epoxy-trans, tram-2,6 trideca-dienoic acid, the corresponding dioldienoic acid and the dioldienoic methyl ester. Unchanged Cecropia JH was also found in the frass. The radioactive hormone, as well as the metabolites, were excreted mainly by the Malpighian tubules; smaller amounts of the radioactive material were also found in the fore-, mid, and hindgut.
INTRODUCTION
THE CHEMICALstructure of the endogenous juvenile hormone (JH) in Locu.rta migratoria is not yet known, but the JH produced in the corpora allata of W&ocerca vagu has been determined as the C,, compound lO,ll,epoxy-3,7,11 trimethyl dodeca-2,6-dienoic methyl ester by JUDYet al. (1973). The synthetic C,, juvenile hormone (Cecropia JH) induces ovarial development in adult allatectomized Locusta (HART-, unpublished). It is bound to a specific carrier lipoprotein in the haemolymph (EMMEFUCH and HARTMANN,1973) similar to that in the silkmoth Cecropia (WHITMOREand GILBERT, 1972). We have found that the radioactive label of the carrier lipoprotein reached a maximum 30 min after injection of the tritiated C&-hormone and then decreased, thus indicating a quick uptake and/or excretion of the hormone. This finding was in clear contrast to that of AJAMI and RIDDIFORD(1973) w h o c1aimed a rather long life of injected juvenile hormone in the pupae of Cecropia. The pupal stage of the moth normally has a very low titre of endogenous JH and the injection of exogenous JH causes a rise of the activity of JH specific JH-esterases in the haemolymph (WHITMOREet al., 1974). * To whom correspondence should be addressed. Present address : Fachbereich Biologie, Technische Hochschule Darmstadt, 61 Darmstadt, Germany. 61
62
DOROTHEE ERLEY,SHARONSOUTHARD, ANDHANSEMMERICH
The titre of JH in adult insects, however, increases from zero during pharate adult development to high titres especially in females, where the ovarian development is under control of the corpora allata. The question is raised whether these changes in the actual titres are due only to an increased rate of hormone synthesis and release or whether the titre is changed in viva by a differential rate of decay. Such a double control has been demonstrated for the titre of the ecdysones during the larval-pupal transition in the blowfly (KAFUSON and BODE,1969). In order to check whether a similar double control of the JH titre occurs in locusts also, we examined the degradation rate of injected C,,-JH in adult locusts of different ages by determination of the ‘half life’ of the circulating C,,- JH, measurement of the excretion rate, and by determining the excreted radioactive substances (ERLEY, 1973). In addition, we tried to elucidate the site of excretion for JH in this insect. MATERIALS AND METHODS The locusts were reared and injected in the neck with the hormone as described previously (EMMERICHand HARTMANN,1973). Each animal received 0.57 &i = 12 x 10e8 g 7-ethyl-l,2-3H-Cecropia hormone (sp. act. 14.1 Ci/m-mole from NEN Chemicals, Dreieichenhain) dissolved in paraffin oil. The radiochemical purity of the hormone was checked by thin-layer chromatography every 2 weeks and the substance purified by preparative TLC when necessary. N-methyl-14C-nitroso-ptoluene sulfoamide (sp. act. 10 mCi/mole from NEN) was used to prepare 14C-diazomethane. Cold, stereochemically pure C,, JH was purchased from Ecocontrol, Cambridge, Mass. Methyl-14C-2-transdodecenoate was prepared from dodecenoic acid (Fluka AG, Buchs) with 14C-diazomethane and purified by preparative TLC. The specific activity obtained was 8.4 mCi/mole. Preparation of reference substances
7-Ethyl-3,11-dimethyI-lO,ll-dihydroxy-2,6-tridecadienoic methyl ester (diolester = DE) and 7-ethyl-3,11-dimethyl-10,ll epoxy-2,6_tridecadionic acid (epoxy acid = EA) were synthetized according to WHITE (1972). After purification by preparative TLC yields of 32 and 29 per cent respectively were obtained. The epoxy acid was esterified back with 14C-diazomethane in order to obtain a This preparation was chemically indouble labelled Cl,-Cecropia hormone. distinguishable from the reference hormone. The corresponding free acid ( = DA) was prepared from the diolester according to WHITE (1972). The purified reference substances were kept in petrol ether under nitrogen at - 20°C to avoid oxidative degradation. Analysis of faeces and tissues
To determine the distribution of labelled material in the haemolymph at the times indicated in Fig. 1 the tarsi of the middle legs were cut off and 10 11 haemolymph was soaked into a Drummond microcap. The haemolymph was dissolved in Packard soluene and counted in a toluene scintillator with an efficiency of 54 per
EXCRJlTION OF JUVENILE
HORMONE
63
hr
(b)
”
c
L)
24
b
hr FIG. 1. ‘Diffusion’ of C,s-JH and dodecenoic methylester out of paraffin oil into haemolymph. (a) Tritium counts of labelled compounds in 5 ~1 haemolymph after injection of 4 ~1 paraffin oil with 057 &i Crs-JH and 0.6 $.2i W-methyldodecenoate. (b) Tritium counts in 5 ~1 haemolymph phosphate buffer 1 : 1 after incubation at 4°C in vitro with 4 $i paraffin oil containing the above-mentioned labelled compounds with 1 ml diluted haemolymph. O-O-0, Haemolymph without haemocytes; O- - - -O- - - -0, haemolymph with haemocytes.
cent. The faeces were collected 2, 4, 6, 8, 12, 24, and 48 hr after injection from separated single individuals to determine the radioactivity in the faeces. They were dried over P,O, at room temperature for 24 hr. Drying at higher temperatures or for prolonged periods had to be avoided, since JH and its metabolites have a rather high vapour pressure. Radioactivity was measured in a Packard liquid scintillation counter (Model 3003) with an efficiency of 28 per cent. No effort was made to estimate the amount of tritiated water from oxidative metabolism of JH in the faeces or in the expired air. For the determination of the excretion sites of the~radioactive material, male locusts were starved for 24 hr and then injected as described above and sacrificed 30 min later. Their digestive tract was dissected and divided as indicated in Fig. 4. The faeces in these pieces were sampled and dried separately. In 5 starved males the gut was ligated by a perlon thread just behind the junction of the Malpighian
64
DOROTHE
ERLEY, SHARON SOUTHARD, AND HANS EMMERICH
tubules under continuous CO, anaesthesia. One hour after recovery the animals were injected with the juvenile hormone and sacrificed 30 min later. Analysis of the metabolites
Fresh faeces were extracted with dichlormethane-methanol 3 : 1. The extract was evaporated to dryness and separated without further purification on kieselgel with benzene-ethyl acetate (4 : 1). A mixture of the reference substances was also run on each plate. The kieselgel was scraped in 5 mm bands directly into counting vials and counted in a toluene scintillator. The quenching effect of the contaminating chlorophylls and xanthophylls was eliminated by standardization of the counting rate by a quenching correction curve with increasing amounts of chlorophylls and xanthophylls. RESULTS
The radioactive hormone diffuses very rapidly from the oil droplet into the haemolymph and reaches a maximum concentration within 30 min either in &JO or
:
5-3 I’
3 *I
2
2 ‘ALull 28
(b)
I1 24
I 48
JLLLU 28
II -
II
IO-
IO
0: -
5 -4-
I
0
(a)
4 -’
1 24
I 48
9
:r
!r 8
7
7
E6
6
P
5
u
4
5 (cl
(d)
4
3
3
2
2 28
24
48
hr
28
24
48
hr
FIG. 2. Excretion of non-volatile labelled material with the faeces of L. migmtoriu after a single injection of 0.57 pCi 8H-C18-JH in 4 pl mineral oil. (a) Females, 2 to 4 days old. (b) Females, 12 to 14 days old. (c) Males, 2 to 4 days old. (d) Males, 12 to 14 days old.
EXCRRTION
OF JUVENILE
65
HORMONE
in haemolymph preparations (see Fig. 1). 1n wivo the amount of labelled material falls exponentially, 50 per cent of it disappears within 90 min. Most of the material is excreted by the Malpighian tubules (see below) but different amounts are incorporated into the tissues of the locust. Especially high concentrations were found in the ovaries (E-RICH and SOUTHARD, unpublished) and the epidermis (see also SCHMIALEK et al., 1973). The hormonally inactive methyl dodecenoate does not diffuse from the oil droplet into the haemolymph either in wivo or in e&-o. In the in vitro experiment we did not check the nature of the radioactive compounds in the watery phase. It is possible that most of them were degraded by haemolymph esterases (see WHITMOREet cd., 1972).
(b)
k
18
\ VI
-.
Cd)
(cl
o_
14
; a u
IO-
6 -”
28
24
48
hr
FIG.
26
24
46
hr
3. Excretion rate of injected CIs-JH per hr of locusts injected with 3H-CIs-JH. For details see legend to Fig. 2.
As shown in Figs. 2 and 3 the excretion of the injected Cecropia hormone starts quickly after the injection-even the first pellets excreted after injection were labelled. Since the amount of excrement obtained in the time intervals varied with each individual, the amounts of radioactivity excreted from a single animal at any given time are quite different. Therefore, the values obtained
DOROTHEE ERLEY,SHARONSOUTHARQAND HANSEMMERICH
66
from the individuals for each time were averaged and their standard deviation indicated. It is seen from Figs. 2 and 3 that there are no significant differences in the excretion rates either for males and females or for animals of different ages. Adults 2 to 4 days and 14 to 16 days old were chosen because it has been shown that shortly after adult emergence the titre of endogenous JH is very low (JOHNSONand HILL, 1973), whereas in 2-week-old females the ovaries develop rapidly, indicating a high titre of endogenous JH. In young adult males and females (2-4 days old) 69.9 per cent of the injected radioactivity was recovered within 48 hr as non-volatile compounds in the faeces. Fifty per cent of the excreted radioactivity was found in the faeces within 3.2 and 3.1 hr respectively. In 14-day-old males and females the recovery was 64.0 and 67.0 per cent respectively; half the excreted radioactive material was recovered within 3.1 hr and 3.0 hr, respectively. The distribution of radioactivity in gut and faeces 30 min after the injection of labelled C,, hormone clearly indicated that most of the labelled material enters the digestive tracts at the Malpighian tubules and/or the hindgut (rectal papillae) (see Figs. 4a and 5a).
-Gut ----Faeces
40-
“!
%J-
; P 0 zo-
L -Gut ----Faeces
!u
FIG. 4. Distribution of tritium label in the digestive tract of male locusts 30 min after injection of 057 /.&i sH-C,s-JH. Mean values from 5 independent experiments. I, Foregut; II, anterior midgut with caeca; III, midgut to the end of the second abdominal segment; IV, midgut from the third and fourth abdominal segments ; V, hindgut till the middle of the sixth segment ; VI, hindgut in sixth and seventh segments; VII, Malpighian tubules. (a) Total label in the different parts. (b) Label per mg dry weight.
67
EXCRETION OF JLWENILE HORMONE
50-
50-
--Gut ----Faeces
;
40CYI a, 3
z. 30k E” P.’ 2 zo; :
(a)
T I I , I , I , , , I ! r_.___:__ # :
2 .? $
40-
-Gut
? ‘0 r
(b)
----Faeces 30-
w’ P ;
20-
:
FIG. 5. Distribution of tritium in the digestive tract of male locusts with a ligature behind the injection of the Malpighian tubules 30 min after the injection of O-57 $i ‘H-CIs-JH. Mean values from 5 independent experiments. For details see Fig. 4.
In order to decide which organ was mainly involved in the excretion of JH we ligated animals just behind the junction at the Malpighian tubules. The results clearly indicate (Figs. 3b and 4b) that the Malpighian tubules secrete most of the radioactivity into the gut. Only small amounts of the label are found in the anterior parts of the digestive tract. The ligation of the midgut, which makes necessary a rather large opening in the third abdominal segment between the tergite and the sternite, does not seem to disturb the distribution of the C,,-JH through the body and its excretion: within 30 min 10.1 per cent of the injected radioactivity was found in the digestive tract as compared with 10.6 per cent in unoperated controls. From the radioactive material excreted with the faeces only 30 per cent can be extracted by dichlormethane-methanol3 : 1. This indicates that two-thirds of the excreted label are metabolized to highly polar compounds, which could not be analysed during these experiments. The extractable lipophilic radioactive compounds were separated by thin-layer chromatography on silica gel. Five major radioactive bands could be detected (Fig. 6), four of them corresponding well with the four reference substances: C,,-JH and its derivatives dioldienoic methyl ester (DE), epoxy dienoic acid (EA), and the diol dienoic acid (DA). When the excreted EA and DA were eluted from the plate, diluted by the cold reference substance, and esterified by 14C-diazo methane, the 3H and the 14C label 3
68
DOROTHEE ERLN, SHARON SOUTHARD,AND HANS EMMERICH
22-
22
Start
Front
20 - DA
” (a)
;i:-
60 mm
(b)
E 122
20
1
IO-
100
-
EA DE JH
20
60
100 mm
140
FIG. 6. TLC of the lipophilic JH metabolites excreted within 8 hr after the injection of *H-Cls-JH into female locusts. DA, Dioldienoic acid derivative of JH; EA, epoxy dienoic acid derivative of JH; DE, dioldienoic methyl ester derivative of JH; JH, CT,,-JH; X, unknown degradation product. (a) Metabolites after injection of 0.57 &i JH. (b) Metabolites after injection of 2.28 &i JH.
always chromatographed together with the R, of C,,-JH and DE respectively. Thus we believe that the demonstrated metabolites were indeed DA and EA. No attempt was made to identify the nature of the apolar compound X. Since a similar product is also found in stored solutions of C,,-JH after a few weeks, it may well be that this compound is not a metabolite but an oxidative degradation product, e.g. a lactone, which was produced during the extraction procedure. When a small amount of the C,,-JH (1.2 x lo-* g/animal) was injected only metabolites of the hormone were recovered in the first 9 hr. By injecting four times the amount of JH (4.8 x 10-s g/ animal) not only did the amount of extractable metabolites in the faeces rise from 30 to 48 per cent but unchanged C,,-JH could also be recovered in the faeces. We did not find any difference in the pattern of excreted metabolites in animals of different sexes. DISCUSSION
In the last 3 years the metabolism of JH and JH analogues has been investigated in several insects (AJAMI and RIDDIFORD,1971, 1973; SLADE and ZIBITT, 1972; WHITE, 1972; WHITMOREet al., 1972, 1974; WEIRICH and WREN, 1973).
In most cases the kinetics of the degradation process were not mentioned. The rate of JH metabolism seems to be influenced by three factors: (a) mode of administration in different solvents, (b) the species and the developmental stage of the test animal, and (c) the site of application. Topical application, by which the lipopholic hormone becomes incorporated within the cuticle, seems to provide the animal with a low but prolonged titre of the hormone, whereas the injected material is
JlXCRETION OF JUVENILE HORMONE
69
much more quickly distributed and metabolized (PATTERSON,1973). Of greatest importance are the properties of the vehicle in which the active compounds are dissolved. Emulgators such as Tween 80 or proteins which are able to carry lipids (like BSA) lead to a very quick distribution and metabolic degradation of the hormone (GR~SMANN et al., 1968; AJAMIand RIDDIFORD,1973), whereas administration in an oily solution gives rise to a slower diffusion of the JH out of the droplet. The speed of this process may vary within different species or different developmental stages. We found after injection of 1.2 x 10-s g/locust in 4 ~1 mineral oil that there was a rather quick equilibration of the haemolymph with the hormone, which reached a maximum after 30 min. AJAMI and RIDDIFORD(1973) showed a very slow equilibration of tritiated JH from mineral oil into the pupal haemolymph of Manduca, but they injected about 100 times the concentration we did, an amount which is beyond the solubility of JH in diluted salt solutions. Therefore, we believe that the old idea (see WIGGLESWORTH,1966), that oil droplets containing JH active compound may act as a depot for the hormone, is true only at unphysiologically high JH concentrations. However, the JH may persist for a prolonged time as an unchanged molecule within the target cells (SCHMIALEKet al., 1973) or within oil droplets of the fat body cells (AJAMIand RIDDIFORD,1973). The time in which half of the totally excreted JH metabolites are recovered is about 3 hr. But even within 1.5 hr after the injection half the injected radioactivity had disappeared from the haemolymph. This is much quicker than in M&a pupae, where half the injected C,,-JH is metabolized in about 24 hr (AJAMI and RIDDIFORD, 1973) ; here the degradation limiting process seems to be the diffusion from the oil phase into the haemolymph. The metabolic pathways for the C,,-JH seem to be very similar to those found for methyl farnesoate-lO,ll-epoxide in 5’chistocercu (WYATT, 1972). The ester bound is split by haemolymph esterases (WHITMOREet al., 1972) and the epoxy group is opened within the fat body cells. We did not follow the consecutive steps of conjugation of these metabolites to glucuronides, glycosides, etc. or a possible oxidation of the hydrocarbon chain. The fact that most radioactivity is excreted via the Malpighian tubules indicates a selective and possibly active process. Only a little labelled material reaches the digestive tract via the different parts of the digestive tract. It is not known whether the metabolites are excreted actively by the gut epithelial cells or whether they penetrate the epithelial membranes passively because of their lipophilic properties. Acknowledgements-This work was supported by the Deutsche Forschungsgemeinschaft and through the Sonderforschungsbereich 74. REFERENCES AJAMI A. M. and RIDDIFORDL. M. (1971) Comparative metabolism of Cecropia juvenile hormone. Am. ZooI. 11,644-645. AJAMI A. M. and RIDDIFORDL. M. (1973) Comparative metabolism of Cecropia juvenile hormone. g. Insect Physiol. 19,635-645. DAVIDSON J. D. and OLWERIOV. T. (1967) Tritium and carbon-14 by oxygen flask combustion. Atomlight 60, 1-12.
70
DOROTHEEERLEY, SHARONSOUTHARD,AND HANS EMMMERICH
ERLEY D. (1973) Experimentelle Untersuchungen tiber die Inaktivierung und Ausscheidung des Juvenilhormons bei der Heuschrecke, Locusta migvatoria. Realschularbeit, Koln. EMMERICHH. and HARTMANNR. (1973) A carrier lipoprotein for juvenile hormone in the haemolymph of Locusta migratoria. J. Insect Physiol. 19, 1663-1675. GRASSMANN A., GEYJXRA., HERDAA., und SCHMIALEKP. (1968) Die Inaktivierung von juvenilhormonwirksamen Substanzen in Insekten. Acta ent. bohe-m. 65, 92-99. JOHNSONR. A. and HILL L. (1973) The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust. J. Insect Physiol. 19, 1921-1932. JUDY K. J., SCHOOLEYD. A., HALL M. S., BERGOTB. J., and SIDDALLJ. B. (1973) Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro. Life Sci. 13, 1511-1516. KARLSONP. und BODE C. (1969) Die Inaktivierung des Ecdysons bei der Schmeissfliege Calliphora erythrocephala Meigen. J. Insect Physiol. 15, 11 l-l 18. PATTERSONJ. W. (1973) The effect of persistence of juvenile hormone mimics on their activity on Rhodniusprolixus. J. Insect Physiol. 19, 1631-1637. SCHMIALEKP., BOROWSKIM., GEYER A., MIOSGAV., NUNDEL M., ROSENBERGE., und ZAPF B. (1973) Die Epidermis von Z’enebrio mo2itor L. Puppen als Zielorgan fur das Juvenilhormon-analoge lO.ll-Epoxy-6.7-tvans-2.3-trans-farnesyl-propeny~ther. Z. Naturforsch. 28 c, 173-177. SLADEM. and ZIBITT C. H. (1972) Metabolism of cecropia juvenile hormone in insects and in mammals. In InsectJuvenile Hormones (Ed. by MENN J. J. and BEROZAM.), pp. 15.5177. Academic Press, New York. WEIRICH G. and WREN J. (1973) The substrate specificity of juvenile hormone esterase from Manduca sexta haemolymph. Life Sci. 13, 213-226. WHITMORED., GILBERT L. J., and ITTYCHERIAHP. J. (1974) The origin of haemolymph carboxylesterases ‘induced’ by the insect juvenile hormone. Mol. Cell. Endocr. 1, 37-54. WHITMORED., WHITMOREE., and GILBERT L. J. (1972) Juvenile hormone induction of esterases: a mechanism for the regulation of juvenile hormone titer. Proc. nut. Acad. Sci., U.S.A., 69, 1592-1595. WHITMOREE. and GILBERT L. J. (1972) Haemolymph lipoprotein transport of juvenile hormone. J. Insect Physiol. 18, 1153-1168. WIGGLESWORTHV. B. (1966) The hormonal regulation of growth and reproduction in insects. Adv. Insect Physiol. 2, 248-336. WHITE A. F. (1972) Metabolism of the juvenile hormone analogue methyl farnesoate 10, ll-epoxide in two insect species. Life Sci. 11, 201-210. WYA-~T G. R. (1972) Biochemical Action of Hormones (Ed. by LITWACK G.), 2, 432-462. Academic Press, New York.
EXCRETION OF JUVENILE HORMONE AND ITS METABOLITES IN THE LOCUST, LOCUSTA
MIGRATORIA DOROTHEE
ERLEY,
SHARON
SOUTHARD,
and HANS EMMERICH*
Zoologisches Institut, 2. Lehrstuhl, Universitiit zu K&n, Germany (Received 3 June 1974)
Abstract-Racemic synthetic 3H-C,s juvenile hormone, dissolved in paraIfin oil, was injected into adult Locusta migratoria and the excreted radioactive material in the faeces was determined. Within 48 hr two-thirds of the injected radioactivity can be recovered in the frass, half of it within 3 hr. The remaining one-third of the injected label is incorporated or is released as water. Adult locusts of either sex or of different ages show no difference in the metabolic pathways of the JH and in its excretion rate. The excreta contain as a degradation product 7-ethyl-3,l l-dimethyl-cis10,ll -epoxy-trans, tram-2,6 trideca-dienoic acid, the corresponding dioldienoic acid and the dioldienoic methyl ester. Unchanged Cecropia JH was also found in the frass. The radioactive hormone, as well as the metabolites, were excreted mainly by the Malpighian tubules; smaller amounts of the radioactive material were also found in the fore-, mid, and hindgut.
INTRODUCTION
THE CHEMICALstructure of the endogenous juvenile hormone (JH) in Locu.rta migratoria is not yet known, but the JH produced in the corpora allata of W&ocerca vagu has been determined as the C,, compound lO,ll,epoxy-3,7,11 trimethyl dodeca-2,6-dienoic methyl ester by JUDYet al. (1973). The synthetic C,, juvenile hormone (Cecropia JH) induces ovarial development in adult allatectomized Locusta (HART-, unpublished). It is bound to a specific carrier lipoprotein in the haemolymph (EMMEFUCH and HARTMANN,1973) similar to that in the silkmoth Cecropia (WHITMOREand GILBERT, 1972). We have found that the radioactive label of the carrier lipoprotein reached a maximum 30 min after injection of the tritiated C&-hormone and then decreased, thus indicating a quick uptake and/or excretion of the hormone. This finding was in clear contrast to that of AJAMI and RIDDIFORD(1973) w h o c1aimed a rather long life of injected juvenile hormone in the pupae of Cecropia. The pupal stage of the moth normally has a very low titre of endogenous JH and the injection of exogenous JH causes a rise of the activity of JH specific JH-esterases in the haemolymph (WHITMOREet al., 1974). * To whom correspondence should be addressed. Present address : Fachbereich Biologie, Technische Hochschule Darmstadt, 61 Darmstadt, Germany. 61
62
DOROTHEE ERLEY,SHARONSOUTHARD, ANDHANSEMMERICH
The titre of JH in adult insects, however, increases from zero during pharate adult development to high titres especially in females, where the ovarian development is under control of the corpora allata. The question is raised whether these changes in the actual titres are due only to an increased rate of hormone synthesis and release or whether the titre is changed in viva by a differential rate of decay. Such a double control has been demonstrated for the titre of the ecdysones during the larval-pupal transition in the blowfly (KAFUSON and BODE,1969). In order to check whether a similar double control of the JH titre occurs in locusts also, we examined the degradation rate of injected C,,-JH in adult locusts of different ages by determination of the ‘half life’ of the circulating C,,- JH, measurement of the excretion rate, and by determining the excreted radioactive substances (ERLEY, 1973). In addition, we tried to elucidate the site of excretion for JH in this insect. MATERIALS AND METHODS The locusts were reared and injected in the neck with the hormone as described previously (EMMERICHand HARTMANN,1973). Each animal received 0.57 &i = 12 x 10e8 g 7-ethyl-l,2-3H-Cecropia hormone (sp. act. 14.1 Ci/m-mole from NEN Chemicals, Dreieichenhain) dissolved in paraffin oil. The radiochemical purity of the hormone was checked by thin-layer chromatography every 2 weeks and the substance purified by preparative TLC when necessary. N-methyl-14C-nitroso-ptoluene sulfoamide (sp. act. 10 mCi/mole from NEN) was used to prepare 14C-diazomethane. Cold, stereochemically pure C,, JH was purchased from Ecocontrol, Cambridge, Mass. Methyl-14C-2-transdodecenoate was prepared from dodecenoic acid (Fluka AG, Buchs) with 14C-diazomethane and purified by preparative TLC. The specific activity obtained was 8.4 mCi/mole. Preparation of reference substances
7-Ethyl-3,11-dimethyI-lO,ll-dihydroxy-2,6-tridecadienoic methyl ester (diolester = DE) and 7-ethyl-3,11-dimethyl-10,ll epoxy-2,6_tridecadionic acid (epoxy acid = EA) were synthetized according to WHITE (1972). After purification by preparative TLC yields of 32 and 29 per cent respectively were obtained. The epoxy acid was esterified back with 14C-diazomethane in order to obtain a This preparation was chemically indouble labelled Cl,-Cecropia hormone. distinguishable from the reference hormone. The corresponding free acid ( = DA) was prepared from the diolester according to WHITE (1972). The purified reference substances were kept in petrol ether under nitrogen at - 20°C to avoid oxidative degradation. Analysis of faeces and tissues
To determine the distribution of labelled material in the haemolymph at the times indicated in Fig. 1 the tarsi of the middle legs were cut off and 10 11 haemolymph was soaked into a Drummond microcap. The haemolymph was dissolved in Packard soluene and counted in a toluene scintillator with an efficiency of 54 per
EXCRJlTION OF JUVENILE
HORMONE
63
hr
(b)
”
c
L)
24
b
hr FIG. 1. ‘Diffusion’ of C,s-JH and dodecenoic methylester out of paraffin oil into haemolymph. (a) Tritium counts of labelled compounds in 5 ~1 haemolymph after injection of 4 ~1 paraffin oil with 057 &i Crs-JH and 0.6 $.2i W-methyldodecenoate. (b) Tritium counts in 5 ~1 haemolymph phosphate buffer 1 : 1 after incubation at 4°C in vitro with 4 $i paraffin oil containing the above-mentioned labelled compounds with 1 ml diluted haemolymph. O-O-0, Haemolymph without haemocytes; O- - - -O- - - -0, haemolymph with haemocytes.
cent. The faeces were collected 2, 4, 6, 8, 12, 24, and 48 hr after injection from separated single individuals to determine the radioactivity in the faeces. They were dried over P,O, at room temperature for 24 hr. Drying at higher temperatures or for prolonged periods had to be avoided, since JH and its metabolites have a rather high vapour pressure. Radioactivity was measured in a Packard liquid scintillation counter (Model 3003) with an efficiency of 28 per cent. No effort was made to estimate the amount of tritiated water from oxidative metabolism of JH in the faeces or in the expired air. For the determination of the excretion sites of the~radioactive material, male locusts were starved for 24 hr and then injected as described above and sacrificed 30 min later. Their digestive tract was dissected and divided as indicated in Fig. 4. The faeces in these pieces were sampled and dried separately. In 5 starved males the gut was ligated by a perlon thread just behind the junction of the Malpighian
64
DOROTHE
ERLEY, SHARON SOUTHARD, AND HANS EMMERICH
tubules under continuous CO, anaesthesia. One hour after recovery the animals were injected with the juvenile hormone and sacrificed 30 min later. Analysis of the metabolites
Fresh faeces were extracted with dichlormethane-methanol 3 : 1. The extract was evaporated to dryness and separated without further purification on kieselgel with benzene-ethyl acetate (4 : 1). A mixture of the reference substances was also run on each plate. The kieselgel was scraped in 5 mm bands directly into counting vials and counted in a toluene scintillator. The quenching effect of the contaminating chlorophylls and xanthophylls was eliminated by standardization of the counting rate by a quenching correction curve with increasing amounts of chlorophylls and xanthophylls. RESULTS
The radioactive hormone diffuses very rapidly from the oil droplet into the haemolymph and reaches a maximum concentration within 30 min either in &JO or
:
5-3 I’
3 *I
2
2 ‘ALull 28
(b)
I1 24
I 48
JLLLU 28
II -
II
IO-
IO
0: -
5 -4-
I
0
(a)
4 -’
1 24
I 48
9
:r
!r 8
7
7
E6
6
P
5
u
4
5 (cl
(d)
4
3
3
2
2 28
24
48
hr
28
24
48
hr
FIG. 2. Excretion of non-volatile labelled material with the faeces of L. migmtoriu after a single injection of 0.57 pCi 8H-C18-JH in 4 pl mineral oil. (a) Females, 2 to 4 days old. (b) Females, 12 to 14 days old. (c) Males, 2 to 4 days old. (d) Males, 12 to 14 days old.
EXCRRTION
OF JUVENILE
65
HORMONE
in haemolymph preparations (see Fig. 1). 1n wivo the amount of labelled material falls exponentially, 50 per cent of it disappears within 90 min. Most of the material is excreted by the Malpighian tubules (see below) but different amounts are incorporated into the tissues of the locust. Especially high concentrations were found in the ovaries (E-RICH and SOUTHARD, unpublished) and the epidermis (see also SCHMIALEK et al., 1973). The hormonally inactive methyl dodecenoate does not diffuse from the oil droplet into the haemolymph either in wivo or in e&-o. In the in vitro experiment we did not check the nature of the radioactive compounds in the watery phase. It is possible that most of them were degraded by haemolymph esterases (see WHITMOREet cd., 1972).
(b)
k
18
\ VI
-.
Cd)
(cl
o_
14
; a u
IO-
6 -”
28
24
48
hr
FIG.
26
24
46
hr
3. Excretion rate of injected CIs-JH per hr of locusts injected with 3H-CIs-JH. For details see legend to Fig. 2.
As shown in Figs. 2 and 3 the excretion of the injected Cecropia hormone starts quickly after the injection-even the first pellets excreted after injection were labelled. Since the amount of excrement obtained in the time intervals varied with each individual, the amounts of radioactivity excreted from a single animal at any given time are quite different. Therefore, the values obtained
DOROTHEE ERLEY,SHARONSOUTHARQAND HANSEMMERICH
66
from the individuals for each time were averaged and their standard deviation indicated. It is seen from Figs. 2 and 3 that there are no significant differences in the excretion rates either for males and females or for animals of different ages. Adults 2 to 4 days and 14 to 16 days old were chosen because it has been shown that shortly after adult emergence the titre of endogenous JH is very low (JOHNSONand HILL, 1973), whereas in 2-week-old females the ovaries develop rapidly, indicating a high titre of endogenous JH. In young adult males and females (2-4 days old) 69.9 per cent of the injected radioactivity was recovered within 48 hr as non-volatile compounds in the faeces. Fifty per cent of the excreted radioactivity was found in the faeces within 3.2 and 3.1 hr respectively. In 14-day-old males and females the recovery was 64.0 and 67.0 per cent respectively; half the excreted radioactive material was recovered within 3.1 hr and 3.0 hr, respectively. The distribution of radioactivity in gut and faeces 30 min after the injection of labelled C,, hormone clearly indicated that most of the labelled material enters the digestive tracts at the Malpighian tubules and/or the hindgut (rectal papillae) (see Figs. 4a and 5a).
-Gut ----Faeces
40-
“!
%J-
; P 0 zo-
L -Gut ----Faeces
!u
FIG. 4. Distribution of tritium label in the digestive tract of male locusts 30 min after injection of 057 /.&i sH-C,s-JH. Mean values from 5 independent experiments. I, Foregut; II, anterior midgut with caeca; III, midgut to the end of the second abdominal segment; IV, midgut from the third and fourth abdominal segments ; V, hindgut till the middle of the sixth segment ; VI, hindgut in sixth and seventh segments; VII, Malpighian tubules. (a) Total label in the different parts. (b) Label per mg dry weight.
67
EXCRETION OF JLWENILE HORMONE
50-
50-
--Gut ----Faeces
;
40CYI a, 3
z. 30k E” P.’ 2 zo; :
(a)
T I I , I , I , , , I ! r_.___:__ # :
2 .? $
40-
-Gut
? ‘0 r
(b)
----Faeces 30-
w’ P ;
20-
:
FIG. 5. Distribution of tritium in the digestive tract of male locusts with a ligature behind the injection of the Malpighian tubules 30 min after the injection of O-57 $i ‘H-CIs-JH. Mean values from 5 independent experiments. For details see Fig. 4.
In order to decide which organ was mainly involved in the excretion of JH we ligated animals just behind the junction at the Malpighian tubules. The results clearly indicate (Figs. 3b and 4b) that the Malpighian tubules secrete most of the radioactivity into the gut. Only small amounts of the label are found in the anterior parts of the digestive tract. The ligation of the midgut, which makes necessary a rather large opening in the third abdominal segment between the tergite and the sternite, does not seem to disturb the distribution of the C,,-JH through the body and its excretion: within 30 min 10.1 per cent of the injected radioactivity was found in the digestive tract as compared with 10.6 per cent in unoperated controls. From the radioactive material excreted with the faeces only 30 per cent can be extracted by dichlormethane-methanol3 : 1. This indicates that two-thirds of the excreted label are metabolized to highly polar compounds, which could not be analysed during these experiments. The extractable lipophilic radioactive compounds were separated by thin-layer chromatography on silica gel. Five major radioactive bands could be detected (Fig. 6), four of them corresponding well with the four reference substances: C,,-JH and its derivatives dioldienoic methyl ester (DE), epoxy dienoic acid (EA), and the diol dienoic acid (DA). When the excreted EA and DA were eluted from the plate, diluted by the cold reference substance, and esterified by 14C-diazo methane, the 3H and the 14C label 3
68
DOROTHEE ERLN, SHARON SOUTHARD,AND HANS EMMERICH
22-
22
Start
Front
20 - DA
” (a)
;i:-
60 mm
(b)
E 122
20
1
IO-
100
-
EA DE JH
20
60
100 mm
140
FIG. 6. TLC of the lipophilic JH metabolites excreted within 8 hr after the injection of *H-Cls-JH into female locusts. DA, Dioldienoic acid derivative of JH; EA, epoxy dienoic acid derivative of JH; DE, dioldienoic methyl ester derivative of JH; JH, CT,,-JH; X, unknown degradation product. (a) Metabolites after injection of 0.57 &i JH. (b) Metabolites after injection of 2.28 &i JH.
always chromatographed together with the R, of C,,-JH and DE respectively. Thus we believe that the demonstrated metabolites were indeed DA and EA. No attempt was made to identify the nature of the apolar compound X. Since a similar product is also found in stored solutions of C,,-JH after a few weeks, it may well be that this compound is not a metabolite but an oxidative degradation product, e.g. a lactone, which was produced during the extraction procedure. When a small amount of the C,,-JH (1.2 x lo-* g/animal) was injected only metabolites of the hormone were recovered in the first 9 hr. By injecting four times the amount of JH (4.8 x 10-s g/ animal) not only did the amount of extractable metabolites in the faeces rise from 30 to 48 per cent but unchanged C,,-JH could also be recovered in the faeces. We did not find any difference in the pattern of excreted metabolites in animals of different sexes. DISCUSSION
In the last 3 years the metabolism of JH and JH analogues has been investigated in several insects (AJAMI and RIDDIFORD,1971, 1973; SLADE and ZIBITT, 1972; WHITE, 1972; WHITMOREet al., 1972, 1974; WEIRICH and WREN, 1973).
In most cases the kinetics of the degradation process were not mentioned. The rate of JH metabolism seems to be influenced by three factors: (a) mode of administration in different solvents, (b) the species and the developmental stage of the test animal, and (c) the site of application. Topical application, by which the lipopholic hormone becomes incorporated within the cuticle, seems to provide the animal with a low but prolonged titre of the hormone, whereas the injected material is
JlXCRETION OF JUVENILE HORMONE
69
much more quickly distributed and metabolized (PATTERSON,1973). Of greatest importance are the properties of the vehicle in which the active compounds are dissolved. Emulgators such as Tween 80 or proteins which are able to carry lipids (like BSA) lead to a very quick distribution and metabolic degradation of the hormone (GR~SMANN et al., 1968; AJAMIand RIDDIFORD,1973), whereas administration in an oily solution gives rise to a slower diffusion of the JH out of the droplet. The speed of this process may vary within different species or different developmental stages. We found after injection of 1.2 x 10-s g/locust in 4 ~1 mineral oil that there was a rather quick equilibration of the haemolymph with the hormone, which reached a maximum after 30 min. AJAMI and RIDDIFORD(1973) showed a very slow equilibration of tritiated JH from mineral oil into the pupal haemolymph of Manduca, but they injected about 100 times the concentration we did, an amount which is beyond the solubility of JH in diluted salt solutions. Therefore, we believe that the old idea (see WIGGLESWORTH,1966), that oil droplets containing JH active compound may act as a depot for the hormone, is true only at unphysiologically high JH concentrations. However, the JH may persist for a prolonged time as an unchanged molecule within the target cells (SCHMIALEKet al., 1973) or within oil droplets of the fat body cells (AJAMIand RIDDIFORD,1973). The time in which half of the totally excreted JH metabolites are recovered is about 3 hr. But even within 1.5 hr after the injection half the injected radioactivity had disappeared from the haemolymph. This is much quicker than in M&a pupae, where half the injected C,,-JH is metabolized in about 24 hr (AJAMI and RIDDIFORD, 1973) ; here the degradation limiting process seems to be the diffusion from the oil phase into the haemolymph. The metabolic pathways for the C,,-JH seem to be very similar to those found for methyl farnesoate-lO,ll-epoxide in 5’chistocercu (WYATT, 1972). The ester bound is split by haemolymph esterases (WHITMOREet al., 1972) and the epoxy group is opened within the fat body cells. We did not follow the consecutive steps of conjugation of these metabolites to glucuronides, glycosides, etc. or a possible oxidation of the hydrocarbon chain. The fact that most radioactivity is excreted via the Malpighian tubules indicates a selective and possibly active process. Only a little labelled material reaches the digestive tract via the different parts of the digestive tract. It is not known whether the metabolites are excreted actively by the gut epithelial cells or whether they penetrate the epithelial membranes passively because of their lipophilic properties. Acknowledgements-This work was supported by the Deutsche Forschungsgemeinschaft and through the Sonderforschungsbereich 74. REFERENCES AJAMI A. M. and RIDDIFORDL. M. (1971) Comparative metabolism of Cecropia juvenile hormone. Am. ZooI. 11,644-645. AJAMI A. M. and RIDDIFORDL. M. (1973) Comparative metabolism of Cecropia juvenile hormone. g. Insect Physiol. 19,635-645. DAVIDSON J. D. and OLWERIOV. T. (1967) Tritium and carbon-14 by oxygen flask combustion. Atomlight 60, 1-12.
70
DOROTHEEERLEY, SHARONSOUTHARD,AND HANS EMMMERICH
ERLEY D. (1973) Experimentelle Untersuchungen tiber die Inaktivierung und Ausscheidung des Juvenilhormons bei der Heuschrecke, Locusta migvatoria. Realschularbeit, Koln. EMMERICHH. and HARTMANNR. (1973) A carrier lipoprotein for juvenile hormone in the haemolymph of Locusta migratoria. J. Insect Physiol. 19, 1663-1675. GRASSMANN A., GEYJXRA., HERDAA., und SCHMIALEKP. (1968) Die Inaktivierung von juvenilhormonwirksamen Substanzen in Insekten. Acta ent. bohe-m. 65, 92-99. JOHNSONR. A. and HILL L. (1973) The activity of the corpora allata in the fourth and fifth larval instars of the migratory locust. J. Insect Physiol. 19, 1921-1932. JUDY K. J., SCHOOLEYD. A., HALL M. S., BERGOTB. J., and SIDDALLJ. B. (1973) Chemical structure and absolute configuration of a juvenile hormone from grasshopper corpora allata in vitro. Life Sci. 13, 1511-1516. KARLSONP. und BODE C. (1969) Die Inaktivierung des Ecdysons bei der Schmeissfliege Calliphora erythrocephala Meigen. J. Insect Physiol. 15, 11 l-l 18. PATTERSONJ. W. (1973) The effect of persistence of juvenile hormone mimics on their activity on Rhodniusprolixus. J. Insect Physiol. 19, 1631-1637. SCHMIALEKP., BOROWSKIM., GEYER A., MIOSGAV., NUNDEL M., ROSENBERGE., und ZAPF B. (1973) Die Epidermis von Z’enebrio mo2itor L. Puppen als Zielorgan fur das Juvenilhormon-analoge lO.ll-Epoxy-6.7-tvans-2.3-trans-farnesyl-propeny~ther. Z. Naturforsch. 28 c, 173-177. SLADEM. and ZIBITT C. H. (1972) Metabolism of cecropia juvenile hormone in insects and in mammals. In InsectJuvenile Hormones (Ed. by MENN J. J. and BEROZAM.), pp. 15.5177. Academic Press, New York. WEIRICH G. and WREN J. (1973) The substrate specificity of juvenile hormone esterase from Manduca sexta haemolymph. Life Sci. 13, 213-226. WHITMORED., GILBERT L. J., and ITTYCHERIAHP. J. (1974) The origin of haemolymph carboxylesterases ‘induced’ by the insect juvenile hormone. Mol. Cell. Endocr. 1, 37-54. WHITMORED., WHITMOREE., and GILBERT L. J. (1972) Juvenile hormone induction of esterases: a mechanism for the regulation of juvenile hormone titer. Proc. nut. Acad. Sci., U.S.A., 69, 1592-1595. WHITMOREE. and GILBERT L. J. (1972) Haemolymph lipoprotein transport of juvenile hormone. J. Insect Physiol. 18, 1153-1168. WIGGLESWORTHV. B. (1966) The hormonal regulation of growth and reproduction in insects. Adv. Insect Physiol. 2, 248-336. WHITE A. F. (1972) Metabolism of the juvenile hormone analogue methyl farnesoate 10, ll-epoxide in two insect species. Life Sci. 11, 201-210. WYA-~T G. R. (1972) Biochemical Action of Hormones (Ed. by LITWACK G.), 2, 432-462. Academic Press, New York.