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Haemolymph juvenile hormone esterase activity during the reproductive cycle of the viviparous cockroach Diploptera punctata
Insect Biochem., Vol. 12, No. 3. pp. 263-268, 1982 Printed in Great Britain
0020-1790/82/030263-06503.00/0 © 1982 Pergamon Press Ltd.
H A E M O L Y M P H J U V E N I L E H O R M O N E ESTERASE ACTIVITY D U R I N G THE R E P R O D U C T I V E CYCLE OF THE VIVIPAROUS COCKROACH DIPLOPTERA PUNCTATA D. ROTIN, R. FEYERE1SEN,* J. KOENER* a n d S. S. TOBE+ Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S IA1 (Receiced 19 July 1981; revised 2 November 1981)
Abstract Juvenile hormone esterase (JHE) activity and ~-naphthyl acetate esterase (NAcE) activity were studied in haemolymph of Diploptera punctata. The apparent K,, value for JHE activity was 6.79/2M. Enzymatic activity of JHE and NAcE in females' haemolymph were measured during the first reproductive cycle. NAcE activity increased from emergence to oviposition and remained high during pregnancy. JHE activity was high at adult emergence and declined during the first three days. A sharp peak of JHE activity was observed on day 7 and high levels were maintained during pregnancy, JHE activity was not inhibited by diisopropylfluorophosphate (DFP) whereas NAcE activity was inhibited 90"~, at l mM. JHE activity profiles of virgin and allatectomized females did not show the peak on day 7 typical of normal mated females. Both oocyte growth and high JHE activity were restored in allatectomized females treated with hydroprene, a JH analogue, in a dose-dependent fashion. JH titre may regulate JHE activity at precise times during the reproductive cycle of D. punctata. Key Word Index: Diploptera punctata, juvenile hormone, esterase, corpora allata, hydroprene
INTRODUCTION THE HAEMOLYMPHjuvenile h o r m o n e (JH) titre reflects the difference between JH biosynthesis and JH degradation a n d removal from the haemolymph. Esterolytic catabolism and interaction of J H with specific carrier proteins have been suggested to be major mechanisms for regulation of JH titre in larval Lepidoptera (SANBURG et al., 1975; AKAMATSU et al., 1975). In adult insects, it has been suggested that changes in JH synthesis govern the changes in JH titre (STAY and TOBE, 1977; TOBE and STAY, 1979: DE KOR'r et al., 1978; TOBE, 1980). Use of a short-term radiochemical assay for the synthesis and release of JH by the corpora allata (CA) in vitro has permitted a precise study of CA regulation in the viviparous cockroach, Diploptera punctata, during the reproductive cycle (ToBe, 1980). To assess the respective roles of JH biosynthesis, catabolism, binding to h a e m o l y m p h proteins and clearance through excretion, to the regulation of JH titre in this species, we have studied JH degradation (JH-esterase activityl in the h a e m o l y m p h during the reproductive cycle. CI~JH is the only JH homologue synthesized and released by the CA of D. punctata (TOBE and STAY, 1977) a n d was used in our experiments because JH esterase (JHE) activity may be qualitatively and quantitatively different when unnatural substrates le.g. Cj,,JH as a JH analogue) are used instead of the insect's natural h o r m o n e (PETER et al., 1979; De KORT et al., 1979). In addition to J H E activity, we have also followed the esterolytic activity of D. punctata haemo-
lymph using :~-naphthyl-acetate as substrate. Changes in J H E activity in virgin females, in allatectomized females and following JH analogue treatment have been studied to detect possible regulatory mechanisms for J H E activity in adult female D. punctata. MATERIALS AND METHODS Insects
Cultures of D. punc~ata were maintained as described by STAY and Coop {1973). Newly emerged females were isolated from the stock culture after mating. Completion of mating was confirmed by the presence of a spermatophore. Virgin females were collected separately, after isolating fifth instar female nymphs prior to ecdysis. Haemolymph collection Insects were anaesthetized by chilling for 5-10rain in crushed ice. The haemolymph was collected in capillary pipettes after clipping the antennae, the tarsi, and by incision in the pronotum. The haemolymph was diluted in 0.1 M sodium phosphate buffer, pH 7.2 and held at 0°C until use. To obtain cell-free haemolymph, blood was centrifuged 17 rain, 2500 g) in ice and the supernatant was used.
* Present address: Department of Entomology, Oregon State University. Corvallis, OR 97331, U.S.A. + To whom all correspondence should be addressed. 263
Allatectomy Allatectomy was performed as described by STAY and TOBE (1977). The operation was carried out in a sterile environment on mated females at one or two days of age. Chemicals [10-3H]-C~,JH (JH I11, metfiyl-10-1 l-epoxy-3,7,1 l-trimethyl-2E,6E-dodecadienoate) sp. act. 11.6 Ci/mmol, was obtained from New England Nuclear Corp. It was diluted with unlabelled CI~JH (Calbiochemt to give a final concentration of 250 pmol (25,000 dpm)//d in ethanol. ~-Naphthyl acetate (NAc) and ~-naphthol (NOH) and Fast red TR salt were obtained from Sigma. DFP tdiisopropylfluorophos-
264
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Fig. 1. Reversed-phase HPLC separation of an ethyl acetate extract of an in vitro incubation of [3H]-Ct6JH with D. punctata haemolymph. phate) was obtained from Fluka. Hydroprene (ZR 512) was a gift from Dr. G. B. Staal (Zoecon Corp.). ~-Naphthyl acetate esterase assay "General" carboxylesterase activity was measured using ~-naphthyl acetate as substrate. Five hundred #1 of substrate solution [NAc, 1 mM in 0.1 M phosphate buffer, pH 7.2, containing 1% (v/v) acetone and 5% (v/v) ethanol] were mixed with 500#1 of the enzyme solution (3 #1 of haemolymph in 0.1 M phosphate buffer) to give a final concentration of 0.5 mM NAc. The mixture was vortexed and then incubated for 30 rain at 30°C. The reaction was stopped by adding 50#1 of a 0.5% Fast red TR salt, 5% (w/v) sodium dodecyl sulphate solution and the O.D. measured at 537 nm in a Zeiss PMQ II speetrophotometer. J H esterase assay All glassware was treated with Carbowax 20M as described by HAMNErT and PRATT (1978) to reduce JH binding to the glass (Gz~E et al., 1977). Two gl of [3H-I-Ct6JH in ethanol were added to the enzyme solution, 5 gl of haemolymph in 100#i of phosphate buffer, pH 7.2. Final concentration of C16JH was 5 x i0-6 M. The incubation vials were vortexed and then incubated at 30°C for 30 min. JH and the product JH acid (JHA) were separated using the partition assay of HAMMOCK and SPARKS (1977). We have found that this assay, originally described for C18JH, could be used for C ~ J H as well. Aliquots from the isooctane and the aqueous phase were assayed for radioactivity in a Beckman LS 350 liquid scintillation spectrometer, using Aquasol (NEN) as scintillation fluid.
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Fig. 3. Dependence of NAcE activity and JHE activity on incubation time in vitro. The assay conditions are described in Materials and Methods. Values are means + S.E.M. of three or six assays. The products of the enzymatic reactions were as in Fig. 2.
High-performance liquid chromatography (H PLC) Phosphate buffer-diluted haemolymph was incubated with [3H]-Ct6JH at 30°C. The radioactive JH and its metabolites were extracted with ethyl acetate as described by HAMMOCKand SPARKS(1977). The ethyl acetate extract was dried under N2, taken up in 20 ~1 acetonitrile and a 10 #1 aliquot was analyzed by HPLC under the following conditions: a Varian model 5000 liquid chromatograph equipped with a 300 x 4 m m reversed-phase Micropak MCH-10 column was used with isocratic elution at 65% (v/v) acetonitrile iia water at 1.5 ml/min. Fractions were collected every 12 sec and radioactivity was assayed by liquid scintillation spectrometry as detailed above. RESULTS CtJH
metabolism by D. punctata haemolymph
Reversed-phase H P L C analysis (Fig. 1) of the labelled metabolites obtained after incubation of cellfree h a e m o l y m p h with [10-3H'JC16JH revealed that C16JH was converted to a single metabolite, tentatively identified as C16JH acid. Because n o other metabolites were detected (e.g. J H dial or J H aciddial), it was possible to use the rapid partition assay
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Fig. 2. Dependence of NAcE activity and JHE activity on enzyme concentration in vitro. The assay conditions are described in Materials and Methods. Values are means + S.E.M. of three or five assays. Haemolymph concentration is in pl/100#1 and #1/I ml, 0A M phosphate buffer pH 7.2 for JHE and NAcE activity, respectively. The product of NAcE activity was ~t-naphthol (NOH) and that of JHE activity was JH acid (JHA).
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Fig. 4. Effect of substrate (C~6JH) concentration on JH degradation. Five microlitres of cell free ha,molymph (in 100/,d 0.1 M phosphate buffer, pH 7.2) were incubated in the indicated concentrations of [ ~ H ] - C t j H . Substrate concentration (S) is expressed in #M, and the rate of JHE activity (JH degradation) in pmol/30~h/5#1. Insert: EAD1E and HOFSTEE plot. The K m c a t o t i l l t ~ from the EADIE and HOFSTEE plot is 6,79 #M. Each ~ t represents the mean of three determinations.
265
JH esterase in Diploptera punctata 80
Diisoprop ylfluorophosphate (DF P) inhibition Work on Manduca sexta has shown that DFP can be used to selectively inhibit NAcE activity (SANBURG et al., 1975). Figure 5 shows that DFP did not inhibit JHE activity from D. punctata haemolymph at concentrations up to 10-aM. NAvE activity was considerably inhibited at 10-6M (85}o inhibition) but higher concentrations (up to 10-3 M ) caused only a slight increase in inhibition, indicating that approx. 10°Jo of the haemolymph NAvE activity was not inhibited by 10 -3 M DFP.
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NAvE activity duriny the reproductive cycle NAcE activity was measured daily prior to oviposition and on selected days during the gestation (post-oviposition) period. Figure 6 shows that there was a gradual increase in NAcE activity from adult emergence and mating to oviposition. High values were maintained during pregnancy.
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Fig. 5. Influence of DFP on JHE activity and NAvE activity in vitro. DFP was incubated for 30 min with cell-free diluted haemolymph. Each plot represents the mean _+ S.E.M. of five to seven assays. Absence of vertical bars indicates that the S.E.M. is smaller than the data point. The control samples (arrow) did not contain DFP. The products of the enzymatic reaction were as in Fig. 2.
JHE activity during the reproductive cycle JHE activity was high in newly emerged mated females (70pmol/hr//A) and decreased rapidly to a minimum on day 3 (37 pmol/hr//d) (Fig. 7). JHE activity was higher on days 4-6 and reached a peak on day 7 (85 pmol/hr/gl). Thus JHE activity more than doubled between day 3 and day 7. The peak of JHE activity occurred two days after the peak of C16JH release by CA in vitro. Figure 8 shows the relationship between oocyte length and JHE and CA activity. JHE activity declined after day 7 but remained at high levels throughout the post-oviposition period.
(HAMMOCK and SPARKS, 1977) to measure rates of JH catabolism by the haemolymph.
~t-Naphthyl acetate esterase (NAcE) and JH esterase (JHE) activities in the haemolymph The essential conditions for the assay of NAcE and JHE activities were studied. Figures 2 and 3 show that esterase activities were linear with respect to enzyme (haemolymph) concentration and to incubation time. Haemolymph diluted with phosphate buffer retained its NAcE and JHE activities when kept at 0°C for up to three days. However, loss of NAcE activity was observed upon storage for one day at 4C. Figure 4 shows the effect of increasing substrate (C16JH) concentration upon hydrolysis of JH, From the EADIEand HOFSTEEtransformation of the data, an apparent Km of 6.79 #M for JHE activity was calculated. The concentration of C~oJH used for routine assay (5/~M) was, therefore, close to the value of the apparent Kin, but still almost 10 times lower than the maximal solubility of the hormonc in the enzymatic solution (ROTIN, unpublished observations).
JHE activity in virgin females To determine whether mating [necessary for CA activation and rapid oocyte growth (STAYand TOBE, 1977)] affects JHE activity during the first 12 days following adult emergence, JHE activity was compared in mated females and in virgin females. Figure 9B shows that JHE activity in virgin females was very similar to that of mated females during the first six days after adult emergence. However, the peak of JHE activity observed in mated females on day 7 was absent in virgin females, Instead, very low values were observed on days 7 and 8 (minimum on day 7:
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Fig. 6. NAcE activity during the reproductive cycle. Each point represents the mean + S.E.M. using haemolymph samples from precisely timed insects. Oocyte length of the same insects is also given. Sample size (nt is indicated at points. The arrow indicates the time of oviposition. The product of NAvE activity was NOH.
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Fig. 7. JHE activity during the reproductive cycle. Each point represents the mean + S.E.M. of n different haemolymph samples from precisely time insects. The dotted line indicates the rate of Cz6JH release by CA in vitro for the same developmental period (data from FEYEREISENet a/.. 1981). Basal oocyte length of the same insect is given. The arrow indicates the time of oviposition. The product of JHE activity was JHA. 27 pmol/hr/#l). By day 9, the activity of JHE had, mated females and virgin or allatectomized females again reached values similar to those of pregnant suggested that JH titre influences JHE activity. To females. The oocytes of all virgin females used in this test this hypothesis, females allatectomized on day 2 study were observed to be in the pre-vitellogenic were topically treated with hydroprene (ZR 512) on stage. day 4. On day 7, haemolymph was collected and oocyte length measured. Figure 10 shows that oocyte JHE activity in allatectomized females growth was restored by hydroprene in a dose-depenThe possible influence of the CA on JHE activity dent fashion (see also TOBE and STAY,1979). Similarly, was studied by comparing JHE activity from mated JHE activity increased with increasing hydroprene females and from mated females allatectomized on dose. day 1. As shown in Fig. 9B, JHE activity of allatectomized females was similar to that of virgin females, except on days 4--6 when the JHE activity was found to be even lower than in virgins. To ensure that alla~c~ E O . i I (a) tectomy had been successfully performed, oocytes E o. ~'~"~'~..o..o..~ were measured at the time of haemolymph collection (Fig. 9A); no oocyte growth was observed. T l , , , t , i i , i , i O O
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Fig. 8. JHE activity expressed as a function of basal oocyte length. The results were pooled arbitrarily in classes of oocyte length of 0.1 ram. Each point represents the mean + S.E.M. of n different haemolymph samples. The dotted line indicates the rate of Cz6JH release by CA in vitro (data from FE'n~RE]SI~Net al., 1981). The product of JHE activity was JHA.
Fig. 9. (A) Basal oocyte length in allatectomized females. (B) JHE activity in virgin (e------@) and a!latectomized (O O) insects. Each point represents the mean + S.E.M. of n different haemolymph samples. The stippled line represents the JHE activity of normal mated females (see Fig. 7). The product of JHE activity was JHA BOth oocyte length (see A) and JHE activity were measured on the same allatectomized females.
JH esterase in Diploptera punctata 80
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Fig. 10. Influence of the dose of topically applied hydroprene on oocyte length and JHE activity of the same allatectomized females. The insects were allatectomized on day 2, treated on day 4 and assayed on day 7. Each point represents the mean + S.E.M. of n different insects/haemolymph samples. The arrow indicates sham-operated insects. The product of JHE activity was JHA,
DISCUSSION JH catabolism in D. punctata haemolymph occurs by methyl ester hydrolysis, with the conversion of C16JH to C16JH acid, as shown in numerous other insect species (DE KORT and GRANGER, 1981). Determination of the enantiomeric selectivity of hydrolysis (PETER et al,, 1979) of racemic [3H]-CI6JH used in our study awaits further experimentation. The rate of JHE activity, ranging from 30 to 90pmol/hr//A, is similar to that measured in another cockroach, Periplaneta americana (DE KORT et al., 1979). However, NAcE activity, sometimes referred to as "general esterase" activity, is considerably higher (40-80 nmol/ hr/~l) and is similar to values reported in a variety of insect orders (DE KORT et al., 1979). In M. sexta haemolymph, inhibition by D F P was used by SANBURG et al. (1975) to distinguish between "general esterases'" isensitive to DFP) capable of hydrolyzing free JH and "JH-specific esterases" (relatively DFP-resistant), capable of hydrolyzing free and carrier-bound JH. In other insects, e.g.L, deeemlineata (KRAMER and DE KORT, 1976), Blaberus giganteus (HAMMOCKet al., 1977) or Trichoplusia ni (SPARKSand HAMMOCK, 1979a), DFP-sensitivity yields less clearcut results. In D. punctata haemolymph, DFP does not inhibit JHE activity and inhibits only up to 90~,, of the NAcE activity. Thus, use of D F P cannot help to distinguish between various types of JH esterases in D. punctata. The possibility that DFP-resistant esterases of low substrate specificity can hydrolyze both JH and NAc cannot be ruled out on the basis of our experiments. Partial purification of the esterase populations of D. punctata haemolymph may shed some light on their specificity. The pattern of change of JHE and NAcE activities during the reproductive cycle showed some important differences which provide support for the hypothesis that NAcE activity is unrelated to JHE activity. NAcE activity showed an increase from adult emer-
267
gence to oviposition, with high values during the gestation period. In contrast, JHE activity declines during the first three days but increases thereafter, peaking on day 7. Comparison of JHE activity with the changes in CA activity in vitro indicates that JHE activity is lowest at the time of maximal CA activity. An increase in JHE activity follows the increase in CA activity by two days and JHE activity is highest prior to oviposition, when CA activity has decreased. High JHE activity is observed during pregnancy, when CA activity is low (ToBE and STAY, 1977). Thus, JHE activity appears to be inversely correlated with the presumed JH titre: high levels just after emergence may be related to the low CA activity observed at the end of the last larval stadium (Szmso et al., 1982). Low levels are observed at the time of the JH-dependent oocyte growth and vitellogenesis. High levels occur throughout the gestation period, when presumed low JH titres allow normal embryogenesis to proceed (see STAY and LIN, 19811. Recent studies (TosE, S T A Y , BAKER and SCHOOLEY, unpublished observations) have revealed that peak titres of C~6JH in the haemolymph of D. punctata during the gonotrophic cycle are very high, reaching values in the/~M range. This finding may explain the high apparent Km for JHE activity found in D. punctata. Binding protein(sl may also cause an elevation of the apparent K,,. Since we do not know whether JH-binding protein(s} in the haemolymph modulate the effective JH titre available to target tissues and/or JH esterases, it is premature to discuss the respective roles of CA activity and JHE activity in regulation of JH titre. However, the available evidence (STAY and TOBE, 1977; TOnE and STAY, 1979; TONE, 1980) suggests that changes in CA activity regulate JH titres in adult D. punctata as in adult L. decemlineata (DE KORT et al., 1978). The variations in JHE activity during the reproductive cycle suggest that JH esterase(s) activity is precisely regulated. The difference observed in JHE activity between mated, virgin and allatectomized females suggests that CA activity (JH titre) controls in part JHE activity between day 3 and day 9, Whereas low rates of JH biosynthesis (virgins) may be sufficient to bring about the small surge in JHE activity on days 4 to 6 (compare mated and virgins with allatectomized females), high rates of JH biosynthesis are necessary for the full expression of the JHE peak on day 7 (normal females). The data suggest that this peak of JHE activity may be "induced" by a high titre of JH caused by high CA activity on day 5. The dosedependent induction of JHE activity in allatectomized insects by hydropene provides support for this hypothesis. Induction of JHE activity by JH or functional analogues has been documented in other insects, e.g. pupae of Hyalophora cecropia (WHITMORE et al., 1972) and Galleria mellonella (REDDY et al., 1979, larvae of T.. ni (SPARKS and HAMMOCK, 1979b) and adult females of L. decemlineata (KRAMER, 1978). Induction (i.e. production of new enzyme or activation of preexisting enzyme or release of enzyme from the fat body) by JH itself cannot account for all the changes in JHE activity occurring during the reproductive cycle. The initial three-day decline in JHE
268
l ) Rt?ll~,
activity as well as the high activity after day 8 appear to occur independently of CA activity. Studies on other insects have implicated repression (inhibition) of J H E activity by J H at specific stages of development (KRAMER, 1978; SPARKS a n d HAMMOCK. 1979b; REDDY et al., 1979), a l t h o u g h as with the induction studies, the doses of J H a n d the time of treatment may give unphysiologicat results. N e u r o e n d o c r i n e control of J H E activity has been proposed in larvae of T. ni (SPARKS a n d HAMMOCK, 1979b; JONES et al., 1981). It will be of considerable importance to verify whether the J H - d e p e n d e n t and J H - i n d e p e n d e n t J H esterase activities are mediated by the same or by different enzymes. Acknowledgements--We would like to thank Professor B. STAY for critically reading the manuscript and Dr. C. A. D. DE KORT for his critical comments. This work was supported by operating and strategic grants from the National Sciences and Engineering Research Council of Canada.
REFERENCES AKAMATSU Y., DUNN P. E., KEZDY F, J., KRAMER K. J., LAW J. H., REIBSTEIN D. and SANBURGL. L. (1975) Biochemical aspects of juvenile hormone action in insects. In Control Mechanisms in Development (Ed. by MEINTS R. H. and DAVIES E.), pp. 123-149. Plenum, New York. DE KORT C. A. D., KRAMER S. J. and WIETEN M. (1978) Regulation of juvenile hormone titres in the adult Colorado beetle: interaction with carboxylesterases and carrier proteins. In Comparative Endocrinology (Ed. by GAILLARD P. J. and BOER H. H.), pp. 507-510. Elsevier/ North Holland, Amsterdam. DE KORT C. A. D., WIETEN M. and KRAMER S. J. (1979) The occurrence of juvenile hormone specific esterases in insects. A comparative study. Proc. K. ned. Akad. Wet. 82, 325-331. DE KORT C. A. D. and GRANGER N. A. (1981) Regulation of the juvenile hormone titre. A, Rev. Ent. 26, 1-28. FEYEREISEN R., FRIEDEL T. and TOBE S. S. (1981) Farnesoic acid stimulation of Ct6 juvenile hormone biosynthesis by corpora allata of adult female Diploptera punctata. Insect Biochem. l l , 401-409. GIESE C., SPINDLER K. D. and EMMERICH H. (1977) The solubility of insect juvenile hormone in aqueous solutions and its adsorption by glassware and plastics. Z. Naturf 32C, 158-160. HAMMOCK B, D. and SPARKS T. C. (1977) A rapid assay for insect juvenile hormone esterase activity. Analyt. Biochem. 82, 573-579. HAMMOCK B. D., SPARKS T. C. and MutinY S. M. (1977) Selective inhibition of JH esterases from cockroach hemolymph. Pestic. Biochem. Physiol. 7, 517-530. HAtiNETT A. F. and PRATT G. E. (1978) Use of automated capillary column radio gas chromatography in the identification of insect juvenile hormones. J. Chromat. 158, 387-399.
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al
JONES G., WING K. D., JONES D. and HAMMt)CK 15. D. (1981) The source and action of head factors regulating juvenile hormone esterase in larvae of the cabbage looper, Triehoplusia ni. J. Insect Physiol, 27, 185 191 KRAMER S. J. and DE KORT C. A. D. (1976) Some properties of hemolymph esterases from Leptinotar~, decemlineata Say. Life Sci. i% 211-218. KRAMER S. J. (1978) Regulation of the activity of JH-specific esterases in the Colorado potato beetle, Leminotarsa decemlineata. J. Insect Physiol. 24. 743--747. PETER M. G., GUNAWAN S., GELLISSEN G. and EMMERICH H. (1979) Differences in hydrolysis and binding of homologous juvenile hormones in Locust, miqratori, hemolymph Z. Natur/~ 34, 588 598. Szmao C. M., ROTIN D., FEYER~IS~N R. and °[OBE S. S. (1982) Synthesis and degradation of C~6 juvenile hor65-71. SANBURG L. L., KRAMER K. J., KEZDY F. J. and LAW J. H, (1975) Juvenile hormone-specific esterases in the haemolymph of the tobacco hornworm, Manduca sexta. J. Insect Physiol. 21,873-887. SPARKS T. C. and HAMMOCKB. D. (1979a) A comparison of the induced and naturally occurring juvenile hormone esterases from last instar larvae of Triehoplusia ni. Insect Biochem. 9, 411-421. SPARKS T. C. and HAMMOCK B. D. (1979b) Induction and regulation of juvenile hormone esterases during the last larval instar of the cabbage looper, Trichoplusia hi. J. Insect Physiol. 25, 551-560. STAY B. and Coop A. (1973) Developmental stages and chemical composition in embryos of the cockroach, Diploptera punctata with observations on the effect of diet. J. Insect Physiol. 19, 147-171. STAY B. and TOBE S. S. (1977) Control of juvenile hormone biosynthesis during the reproduction cycle of a viviparous cockroach. I. Activation and inhibition of corpora allata. Gen. Comp. Endocrinol. 33, 531-540. STAY B. and LIN H. L. (1981) The inhibition of milk synthesis by juvenile hormone in the viviparous cockroach, Diploptera punctata. J. Insect Physiol. 27, 551-557. Szmao C. M., ROTIN D., FEYEREISEN R. and TOBE S. S. (1981) Synthesis and degradation of Cl6 juvenile hormone (JH IlI) during the final two stadia of the cockroach, Diploptera punctata. Gen. Comp. Endocrinol. (in press). TOaE S. S. and STAY B. (1977) Corpus allatum activity in vitro during the reproductive cycle of the viviparous cockroach, Diploptera punctata (Eschscholtz). Gen. Comp. Endocrinol. 31, 138-147. TOBE S. S. and STAY B. (1979) Modulation of juvenile hormone synthesis by an analogue in the cockroach. Nature, Lond. 281,481-482, TOBE S. S. (1980) Regulation of the corpora allata in adult female insects. In Insect Biology in the Future (Ed. by LOCKE M. and SMITH D. S.), pp. 345--367. Academic Press, New York. WHITMORE D., WHITMOREE. and GILBERT L. I. (1972) Juvenile hormone induction of esterases: a mechanism for the regulation of juvenile hormone titre. Proc. Bath. Acad. Sci. U.S.A. 69, 1592-1595.
0020-1790/82/030263-06503.00/0 © 1982 Pergamon Press Ltd.
H A E M O L Y M P H J U V E N I L E H O R M O N E ESTERASE ACTIVITY D U R I N G THE R E P R O D U C T I V E CYCLE OF THE VIVIPAROUS COCKROACH DIPLOPTERA PUNCTATA D. ROTIN, R. FEYERE1SEN,* J. KOENER* a n d S. S. TOBE+ Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S IA1 (Receiced 19 July 1981; revised 2 November 1981)
Abstract Juvenile hormone esterase (JHE) activity and ~-naphthyl acetate esterase (NAcE) activity were studied in haemolymph of Diploptera punctata. The apparent K,, value for JHE activity was 6.79/2M. Enzymatic activity of JHE and NAcE in females' haemolymph were measured during the first reproductive cycle. NAcE activity increased from emergence to oviposition and remained high during pregnancy. JHE activity was high at adult emergence and declined during the first three days. A sharp peak of JHE activity was observed on day 7 and high levels were maintained during pregnancy, JHE activity was not inhibited by diisopropylfluorophosphate (DFP) whereas NAcE activity was inhibited 90"~, at l mM. JHE activity profiles of virgin and allatectomized females did not show the peak on day 7 typical of normal mated females. Both oocyte growth and high JHE activity were restored in allatectomized females treated with hydroprene, a JH analogue, in a dose-dependent fashion. JH titre may regulate JHE activity at precise times during the reproductive cycle of D. punctata. Key Word Index: Diploptera punctata, juvenile hormone, esterase, corpora allata, hydroprene
INTRODUCTION THE HAEMOLYMPHjuvenile h o r m o n e (JH) titre reflects the difference between JH biosynthesis and JH degradation a n d removal from the haemolymph. Esterolytic catabolism and interaction of J H with specific carrier proteins have been suggested to be major mechanisms for regulation of JH titre in larval Lepidoptera (SANBURG et al., 1975; AKAMATSU et al., 1975). In adult insects, it has been suggested that changes in JH synthesis govern the changes in JH titre (STAY and TOBE, 1977; TOBE and STAY, 1979: DE KOR'r et al., 1978; TOBE, 1980). Use of a short-term radiochemical assay for the synthesis and release of JH by the corpora allata (CA) in vitro has permitted a precise study of CA regulation in the viviparous cockroach, Diploptera punctata, during the reproductive cycle (ToBe, 1980). To assess the respective roles of JH biosynthesis, catabolism, binding to h a e m o l y m p h proteins and clearance through excretion, to the regulation of JH titre in this species, we have studied JH degradation (JH-esterase activityl in the h a e m o l y m p h during the reproductive cycle. CI~JH is the only JH homologue synthesized and released by the CA of D. punctata (TOBE and STAY, 1977) a n d was used in our experiments because JH esterase (JHE) activity may be qualitatively and quantitatively different when unnatural substrates le.g. Cj,,JH as a JH analogue) are used instead of the insect's natural h o r m o n e (PETER et al., 1979; De KORT et al., 1979). In addition to J H E activity, we have also followed the esterolytic activity of D. punctata haemo-
lymph using :~-naphthyl-acetate as substrate. Changes in J H E activity in virgin females, in allatectomized females and following JH analogue treatment have been studied to detect possible regulatory mechanisms for J H E activity in adult female D. punctata. MATERIALS AND METHODS Insects
Cultures of D. punc~ata were maintained as described by STAY and Coop {1973). Newly emerged females were isolated from the stock culture after mating. Completion of mating was confirmed by the presence of a spermatophore. Virgin females were collected separately, after isolating fifth instar female nymphs prior to ecdysis. Haemolymph collection Insects were anaesthetized by chilling for 5-10rain in crushed ice. The haemolymph was collected in capillary pipettes after clipping the antennae, the tarsi, and by incision in the pronotum. The haemolymph was diluted in 0.1 M sodium phosphate buffer, pH 7.2 and held at 0°C until use. To obtain cell-free haemolymph, blood was centrifuged 17 rain, 2500 g) in ice and the supernatant was used.
* Present address: Department of Entomology, Oregon State University. Corvallis, OR 97331, U.S.A. + To whom all correspondence should be addressed. 263
Allatectomy Allatectomy was performed as described by STAY and TOBE (1977). The operation was carried out in a sterile environment on mated females at one or two days of age. Chemicals [10-3H]-C~,JH (JH I11, metfiyl-10-1 l-epoxy-3,7,1 l-trimethyl-2E,6E-dodecadienoate) sp. act. 11.6 Ci/mmol, was obtained from New England Nuclear Corp. It was diluted with unlabelled CI~JH (Calbiochemt to give a final concentration of 250 pmol (25,000 dpm)//d in ethanol. ~-Naphthyl acetate (NAc) and ~-naphthol (NOH) and Fast red TR salt were obtained from Sigma. DFP tdiisopropylfluorophos-
264
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Fig. 1. Reversed-phase HPLC separation of an ethyl acetate extract of an in vitro incubation of [3H]-Ct6JH with D. punctata haemolymph. phate) was obtained from Fluka. Hydroprene (ZR 512) was a gift from Dr. G. B. Staal (Zoecon Corp.). ~-Naphthyl acetate esterase assay "General" carboxylesterase activity was measured using ~-naphthyl acetate as substrate. Five hundred #1 of substrate solution [NAc, 1 mM in 0.1 M phosphate buffer, pH 7.2, containing 1% (v/v) acetone and 5% (v/v) ethanol] were mixed with 500#1 of the enzyme solution (3 #1 of haemolymph in 0.1 M phosphate buffer) to give a final concentration of 0.5 mM NAc. The mixture was vortexed and then incubated for 30 rain at 30°C. The reaction was stopped by adding 50#1 of a 0.5% Fast red TR salt, 5% (w/v) sodium dodecyl sulphate solution and the O.D. measured at 537 nm in a Zeiss PMQ II speetrophotometer. J H esterase assay All glassware was treated with Carbowax 20M as described by HAMNErT and PRATT (1978) to reduce JH binding to the glass (Gz~E et al., 1977). Two gl of [3H-I-Ct6JH in ethanol were added to the enzyme solution, 5 gl of haemolymph in 100#i of phosphate buffer, pH 7.2. Final concentration of C16JH was 5 x i0-6 M. The incubation vials were vortexed and then incubated at 30°C for 30 min. JH and the product JH acid (JHA) were separated using the partition assay of HAMMOCK and SPARKS (1977). We have found that this assay, originally described for C18JH, could be used for C ~ J H as well. Aliquots from the isooctane and the aqueous phase were assayed for radioactivity in a Beckman LS 350 liquid scintillation spectrometer, using Aquasol (NEN) as scintillation fluid.
l
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time,
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Fig. 3. Dependence of NAcE activity and JHE activity on incubation time in vitro. The assay conditions are described in Materials and Methods. Values are means + S.E.M. of three or six assays. The products of the enzymatic reactions were as in Fig. 2.
High-performance liquid chromatography (H PLC) Phosphate buffer-diluted haemolymph was incubated with [3H]-Ct6JH at 30°C. The radioactive JH and its metabolites were extracted with ethyl acetate as described by HAMMOCKand SPARKS(1977). The ethyl acetate extract was dried under N2, taken up in 20 ~1 acetonitrile and a 10 #1 aliquot was analyzed by HPLC under the following conditions: a Varian model 5000 liquid chromatograph equipped with a 300 x 4 m m reversed-phase Micropak MCH-10 column was used with isocratic elution at 65% (v/v) acetonitrile iia water at 1.5 ml/min. Fractions were collected every 12 sec and radioactivity was assayed by liquid scintillation spectrometry as detailed above. RESULTS CtJH
metabolism by D. punctata haemolymph
Reversed-phase H P L C analysis (Fig. 1) of the labelled metabolites obtained after incubation of cellfree h a e m o l y m p h with [10-3H'JC16JH revealed that C16JH was converted to a single metabolite, tentatively identified as C16JH acid. Because n o other metabolites were detected (e.g. J H dial or J H aciddial), it was possible to use the rapid partition assay
•
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Fig. 2. Dependence of NAcE activity and JHE activity on enzyme concentration in vitro. The assay conditions are described in Materials and Methods. Values are means + S.E.M. of three or five assays. Haemolymph concentration is in pl/100#1 and #1/I ml, 0A M phosphate buffer pH 7.2 for JHE and NAcE activity, respectively. The product of NAcE activity was ~t-naphthol (NOH) and that of JHE activity was JH acid (JHA).
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Fig. 4. Effect of substrate (C~6JH) concentration on JH degradation. Five microlitres of cell free ha,molymph (in 100/,d 0.1 M phosphate buffer, pH 7.2) were incubated in the indicated concentrations of [ ~ H ] - C t j H . Substrate concentration (S) is expressed in #M, and the rate of JHE activity (JH degradation) in pmol/30~h/5#1. Insert: EAD1E and HOFSTEE plot. The K m c a t o t i l l t ~ from the EADIE and HOFSTEE plot is 6,79 #M. Each ~ t represents the mean of three determinations.
265
JH esterase in Diploptera punctata 80
Diisoprop ylfluorophosphate (DF P) inhibition Work on Manduca sexta has shown that DFP can be used to selectively inhibit NAcE activity (SANBURG et al., 1975). Figure 5 shows that DFP did not inhibit JHE activity from D. punctata haemolymph at concentrations up to 10-aM. NAvE activity was considerably inhibited at 10-6M (85}o inhibition) but higher concentrations (up to 10-3 M ) caused only a slight increase in inhibition, indicating that approx. 10°Jo of the haemolymph NAvE activity was not inhibited by 10 -3 M DFP.
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NAvE activity duriny the reproductive cycle NAcE activity was measured daily prior to oviposition and on selected days during the gestation (post-oviposition) period. Figure 6 shows that there was a gradual increase in NAcE activity from adult emergence and mating to oviposition. High values were maintained during pregnancy.
-Logic m
Fig. 5. Influence of DFP on JHE activity and NAvE activity in vitro. DFP was incubated for 30 min with cell-free diluted haemolymph. Each plot represents the mean _+ S.E.M. of five to seven assays. Absence of vertical bars indicates that the S.E.M. is smaller than the data point. The control samples (arrow) did not contain DFP. The products of the enzymatic reaction were as in Fig. 2.
JHE activity during the reproductive cycle JHE activity was high in newly emerged mated females (70pmol/hr//A) and decreased rapidly to a minimum on day 3 (37 pmol/hr//d) (Fig. 7). JHE activity was higher on days 4-6 and reached a peak on day 7 (85 pmol/hr/gl). Thus JHE activity more than doubled between day 3 and day 7. The peak of JHE activity occurred two days after the peak of C16JH release by CA in vitro. Figure 8 shows the relationship between oocyte length and JHE and CA activity. JHE activity declined after day 7 but remained at high levels throughout the post-oviposition period.
(HAMMOCK and SPARKS, 1977) to measure rates of JH catabolism by the haemolymph.
~t-Naphthyl acetate esterase (NAcE) and JH esterase (JHE) activities in the haemolymph The essential conditions for the assay of NAcE and JHE activities were studied. Figures 2 and 3 show that esterase activities were linear with respect to enzyme (haemolymph) concentration and to incubation time. Haemolymph diluted with phosphate buffer retained its NAcE and JHE activities when kept at 0°C for up to three days. However, loss of NAcE activity was observed upon storage for one day at 4C. Figure 4 shows the effect of increasing substrate (C16JH) concentration upon hydrolysis of JH, From the EADIEand HOFSTEEtransformation of the data, an apparent Km of 6.79 #M for JHE activity was calculated. The concentration of C~oJH used for routine assay (5/~M) was, therefore, close to the value of the apparent Kin, but still almost 10 times lower than the maximal solubility of the hormonc in the enzymatic solution (ROTIN, unpublished observations).
JHE activity in virgin females To determine whether mating [necessary for CA activation and rapid oocyte growth (STAYand TOBE, 1977)] affects JHE activity during the first 12 days following adult emergence, JHE activity was compared in mated females and in virgin females. Figure 9B shows that JHE activity in virgin females was very similar to that of mated females during the first six days after adult emergence. However, the peak of JHE activity observed in mated females on day 7 was absent in virgin females, Instead, very low values were observed on days 7 and 8 (minimum on day 7:
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Fig. 6. NAcE activity during the reproductive cycle. Each point represents the mean + S.E.M. using haemolymph samples from precisely timed insects. Oocyte length of the same insects is also given. Sample size (nt is indicated at points. The arrow indicates the time of oviposition. The product of NAvE activity was NOH.
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Fig. 7. JHE activity during the reproductive cycle. Each point represents the mean + S.E.M. of n different haemolymph samples from precisely time insects. The dotted line indicates the rate of Cz6JH release by CA in vitro for the same developmental period (data from FEYEREISENet a/.. 1981). Basal oocyte length of the same insect is given. The arrow indicates the time of oviposition. The product of JHE activity was JHA. 27 pmol/hr/#l). By day 9, the activity of JHE had, mated females and virgin or allatectomized females again reached values similar to those of pregnant suggested that JH titre influences JHE activity. To females. The oocytes of all virgin females used in this test this hypothesis, females allatectomized on day 2 study were observed to be in the pre-vitellogenic were topically treated with hydroprene (ZR 512) on stage. day 4. On day 7, haemolymph was collected and oocyte length measured. Figure 10 shows that oocyte JHE activity in allatectomized females growth was restored by hydroprene in a dose-depenThe possible influence of the CA on JHE activity dent fashion (see also TOBE and STAY,1979). Similarly, was studied by comparing JHE activity from mated JHE activity increased with increasing hydroprene females and from mated females allatectomized on dose. day 1. As shown in Fig. 9B, JHE activity of allatectomized females was similar to that of virgin females, except on days 4--6 when the JHE activity was found to be even lower than in virgins. To ensure that alla~c~ E O . i I (a) tectomy had been successfully performed, oocytes E o. ~'~"~'~..o..o..~ were measured at the time of haemolymph collection (Fig. 9A); no oocyte growth was observed. T l , , , t , i i , i , i O O
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The difference in JHE activity observed between ~. so
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Fig. 8. JHE activity expressed as a function of basal oocyte length. The results were pooled arbitrarily in classes of oocyte length of 0.1 ram. Each point represents the mean + S.E.M. of n different haemolymph samples. The dotted line indicates the rate of Cz6JH release by CA in vitro (data from FE'n~RE]SI~Net al., 1981). The product of JHE activity was JHA.
Fig. 9. (A) Basal oocyte length in allatectomized females. (B) JHE activity in virgin (e------@) and a!latectomized (O O) insects. Each point represents the mean + S.E.M. of n different haemolymph samples. The stippled line represents the JHE activity of normal mated females (see Fig. 7). The product of JHE activity was JHA BOth oocyte length (see A) and JHE activity were measured on the same allatectomized females.
JH esterase in Diploptera punctata 80
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DISCUSSION JH catabolism in D. punctata haemolymph occurs by methyl ester hydrolysis, with the conversion of C16JH to C16JH acid, as shown in numerous other insect species (DE KORT and GRANGER, 1981). Determination of the enantiomeric selectivity of hydrolysis (PETER et al,, 1979) of racemic [3H]-CI6JH used in our study awaits further experimentation. The rate of JHE activity, ranging from 30 to 90pmol/hr//A, is similar to that measured in another cockroach, Periplaneta americana (DE KORT et al., 1979). However, NAcE activity, sometimes referred to as "general esterase" activity, is considerably higher (40-80 nmol/ hr/~l) and is similar to values reported in a variety of insect orders (DE KORT et al., 1979). In M. sexta haemolymph, inhibition by D F P was used by SANBURG et al. (1975) to distinguish between "general esterases'" isensitive to DFP) capable of hydrolyzing free JH and "JH-specific esterases" (relatively DFP-resistant), capable of hydrolyzing free and carrier-bound JH. In other insects, e.g.L, deeemlineata (KRAMER and DE KORT, 1976), Blaberus giganteus (HAMMOCKet al., 1977) or Trichoplusia ni (SPARKSand HAMMOCK, 1979a), DFP-sensitivity yields less clearcut results. In D. punctata haemolymph, DFP does not inhibit JHE activity and inhibits only up to 90~,, of the NAcE activity. Thus, use of D F P cannot help to distinguish between various types of JH esterases in D. punctata. The possibility that DFP-resistant esterases of low substrate specificity can hydrolyze both JH and NAc cannot be ruled out on the basis of our experiments. Partial purification of the esterase populations of D. punctata haemolymph may shed some light on their specificity. The pattern of change of JHE and NAcE activities during the reproductive cycle showed some important differences which provide support for the hypothesis that NAcE activity is unrelated to JHE activity. NAcE activity showed an increase from adult emer-
267
gence to oviposition, with high values during the gestation period. In contrast, JHE activity declines during the first three days but increases thereafter, peaking on day 7. Comparison of JHE activity with the changes in CA activity in vitro indicates that JHE activity is lowest at the time of maximal CA activity. An increase in JHE activity follows the increase in CA activity by two days and JHE activity is highest prior to oviposition, when CA activity has decreased. High JHE activity is observed during pregnancy, when CA activity is low (ToBE and STAY, 1977). Thus, JHE activity appears to be inversely correlated with the presumed JH titre: high levels just after emergence may be related to the low CA activity observed at the end of the last larval stadium (Szmso et al., 1982). Low levels are observed at the time of the JH-dependent oocyte growth and vitellogenesis. High levels occur throughout the gestation period, when presumed low JH titres allow normal embryogenesis to proceed (see STAY and LIN, 19811. Recent studies (TosE, S T A Y , BAKER and SCHOOLEY, unpublished observations) have revealed that peak titres of C~6JH in the haemolymph of D. punctata during the gonotrophic cycle are very high, reaching values in the/~M range. This finding may explain the high apparent Km for JHE activity found in D. punctata. Binding protein(sl may also cause an elevation of the apparent K,,. Since we do not know whether JH-binding protein(s} in the haemolymph modulate the effective JH titre available to target tissues and/or JH esterases, it is premature to discuss the respective roles of CA activity and JHE activity in regulation of JH titre. However, the available evidence (STAY and TOBE, 1977; TOnE and STAY, 1979; TONE, 1980) suggests that changes in CA activity regulate JH titres in adult D. punctata as in adult L. decemlineata (DE KORT et al., 1978). The variations in JHE activity during the reproductive cycle suggest that JH esterase(s) activity is precisely regulated. The difference observed in JHE activity between mated, virgin and allatectomized females suggests that CA activity (JH titre) controls in part JHE activity between day 3 and day 9, Whereas low rates of JH biosynthesis (virgins) may be sufficient to bring about the small surge in JHE activity on days 4 to 6 (compare mated and virgins with allatectomized females), high rates of JH biosynthesis are necessary for the full expression of the JHE peak on day 7 (normal females). The data suggest that this peak of JHE activity may be "induced" by a high titre of JH caused by high CA activity on day 5. The dosedependent induction of JHE activity in allatectomized insects by hydropene provides support for this hypothesis. Induction of JHE activity by JH or functional analogues has been documented in other insects, e.g. pupae of Hyalophora cecropia (WHITMORE et al., 1972) and Galleria mellonella (REDDY et al., 1979, larvae of T.. ni (SPARKS and HAMMOCK, 1979b) and adult females of L. decemlineata (KRAMER, 1978). Induction (i.e. production of new enzyme or activation of preexisting enzyme or release of enzyme from the fat body) by JH itself cannot account for all the changes in JHE activity occurring during the reproductive cycle. The initial three-day decline in JHE
268
l ) Rt?ll~,
activity as well as the high activity after day 8 appear to occur independently of CA activity. Studies on other insects have implicated repression (inhibition) of J H E activity by J H at specific stages of development (KRAMER, 1978; SPARKS a n d HAMMOCK. 1979b; REDDY et al., 1979), a l t h o u g h as with the induction studies, the doses of J H a n d the time of treatment may give unphysiologicat results. N e u r o e n d o c r i n e control of J H E activity has been proposed in larvae of T. ni (SPARKS a n d HAMMOCK, 1979b; JONES et al., 1981). It will be of considerable importance to verify whether the J H - d e p e n d e n t and J H - i n d e p e n d e n t J H esterase activities are mediated by the same or by different enzymes. Acknowledgements--We would like to thank Professor B. STAY for critically reading the manuscript and Dr. C. A. D. DE KORT for his critical comments. This work was supported by operating and strategic grants from the National Sciences and Engineering Research Council of Canada.
REFERENCES AKAMATSU Y., DUNN P. E., KEZDY F, J., KRAMER K. J., LAW J. H., REIBSTEIN D. and SANBURGL. L. (1975) Biochemical aspects of juvenile hormone action in insects. In Control Mechanisms in Development (Ed. by MEINTS R. H. and DAVIES E.), pp. 123-149. Plenum, New York. DE KORT C. A. D., KRAMER S. J. and WIETEN M. (1978) Regulation of juvenile hormone titres in the adult Colorado beetle: interaction with carboxylesterases and carrier proteins. In Comparative Endocrinology (Ed. by GAILLARD P. J. and BOER H. H.), pp. 507-510. Elsevier/ North Holland, Amsterdam. DE KORT C. A. D., WIETEN M. and KRAMER S. J. (1979) The occurrence of juvenile hormone specific esterases in insects. A comparative study. Proc. K. ned. Akad. Wet. 82, 325-331. DE KORT C. A. D. and GRANGER N. A. (1981) Regulation of the juvenile hormone titre. A, Rev. Ent. 26, 1-28. FEYEREISEN R., FRIEDEL T. and TOBE S. S. (1981) Farnesoic acid stimulation of Ct6 juvenile hormone biosynthesis by corpora allata of adult female Diploptera punctata. Insect Biochem. l l , 401-409. GIESE C., SPINDLER K. D. and EMMERICH H. (1977) The solubility of insect juvenile hormone in aqueous solutions and its adsorption by glassware and plastics. Z. Naturf 32C, 158-160. HAMMOCK B, D. and SPARKS T. C. (1977) A rapid assay for insect juvenile hormone esterase activity. Analyt. Biochem. 82, 573-579. HAMMOCK B. D., SPARKS T. C. and MutinY S. M. (1977) Selective inhibition of JH esterases from cockroach hemolymph. Pestic. Biochem. Physiol. 7, 517-530. HAtiNETT A. F. and PRATT G. E. (1978) Use of automated capillary column radio gas chromatography in the identification of insect juvenile hormones. J. Chromat. 158, 387-399.
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