Juvenile hormone degradation in the female locust (Locusta migratoria): Evidence for new catabolites

J. Insect Physd.

Pergamon

0022-1910(94)00054-9

Vol. 40, No. I I. pp. 975-982, 1994 Copyright (0 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0022-1910/94 $7.00+0.00

Juvenile Hormone Degradation in the Female Locust (Locusta migratoria): Evidence for New Catabolites STfiPHANE

DEBERNARD,*

FRANCK

COUILLAUD*t

Received 7 March 1994; revised 18 April 1994

Catabolism of (10R)-[3Hjjuvenile hormone III (JH III), the labelled natural enantiomer of JH III was studied in uiuo in the adult female Locusta migruforiu. Reverse phase liquid chromatography (RPLC) at pH 7.4 coupled with on line radiodetection, revealed JH III acid diol and an unknown compound (X) as major end products of JH III catabolism. RPLC analysis at pH 3 revealed an additional end-product (Z) that comigrates with JH III acid diol at pH 7. Both X and Z lose their carboxyl methyl group and Z was proposed as the diol form of X. A lower rate of in situ degradation for the (10R) enantiomer when compared with racemic JH III was demonstrated when JH degradation was followed quantitatively by using a partition assay. In short-term experiments, inhibition of JH biosynthesis using fluvastatin (an HMG-CoA reductase inhibitor) or surgical allatectomy, increased the rate of disappearance of labelled JH. However, 24 h after allatectomy, the rate of disappearance of the label was similar to that in sham-operated animals, suggesting substantial changes in JH III catabolism in response to the operation. Juvenile hormone

Enantiospecificity

Biosynthesis

INTRODUCTION

Locust

Insect

(Baker et al., 1987; Zimowska et al., 1989; Halarnkar and Schooley, 1990; Halarnkar et al., 1993) and that JH degradation still remains an open question. Data on JH catabolism in the locust are sparse. The pioneering work of Slade and Zibitt (1972) and Erley et a2. (1975) using racemic JH I, provided a general scheme for JH degradation in the locust. However, the physiological relevance of this data has to be re-evaluated in the light of more recent findings. It has been shown that JH III is the sole JH in locusts (Bergot et al., 198 l), probably in the 10R configuration as demonstrated in Munduca (Judy et al., 1973). In the hemolymph, JH III binds to a JH-binding protein (JHBP). Locust JHBP has been fully characterized and found to preferentially bind the natural enantiomer (10R) of JH III (Peter et al., 1979; De Bruijn et al., 1986; De Kort and Koopmanschap, 1986a, b; Koopmanschap and De Kort, 1988). From the dissociation constant and the total concentration of JHBP binding sites, it can be calculated that the ratio between bound and free hormone is approx. 2OOO:l(De Kort, 1990). Thus, almost all JH III in the hemolymph is associated with JHBP. Binding is probably important for JH III protection against degradation by hemolymph esterases. Hemolymph carboxyesterases in the locust are not JH-specific esterases. They are general esterases which also degrade JH III. Their contribution to the regulation of JH titre is uncertain (De Kort et al.,

Juvenile hormone (JH) plays a crucial role in insect reproduction and development. The de nova synthesis of this hormone is limited to the corpora allata (see Tobe and Feyereisen, 1983; Feyereisen, 1985; Schooley and Baker, 1985). Changes in the rate of JH biosynthesis are believed to govern fluctuations in JH titre and thus the physiological response. However, changes in the rate of JH degradation also contribute significantly to the modulation of JH titres at particular times in the life cycle. During the last larval stadium for example, JH biosynthesis is reduced but does not cease until pupation (see De Kort and Granger, 198 1; Hammock, 1985). Degradation reduces the levels of JH in specific tissues, and may ensure that JH titres are maintained at a low level even when JH biosynthesis is not totally halted. During the last few years, studies on JH degradation have focused on hemolymph JH esterase (JHE) in Lepidoptera (Hammock, 1985) leading to the identification of a cDNA fragment encoding JHE in Hefiothis virescens (Hanzlik et al., 1989). Surprisingly, few studies have examined JH degradation in situ. Recent work suggests that hemolymph JHE may not play a major role *Laboratoire de Neuroendocrinologie, UniversitC de Bordeaux CNRS 1138, Avenue des Facultts , 33405 Talence Cedex tTo whom correspondence should be addressed.

Degradation

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, France

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1979; Koopmanschap and De Kort, 1989; De Kort, 1990). These data prompted us to re-examine JH-metabolism in the locust. In the present paper, we report results from in situ injections of minute doses of (10R)-[3H-10]JH III into adult female Locusta migratoria migratorioides. The labelled tracer is expected to label the endogenous pool of JH III and to provide information on JH metabolism in situ when assayed at different times after injection. MATERIALS

AND METHODS

COUILLAUD

In vivo study

Fluvastatin diluted in water, was injected (4 ~1, 200 pg per animal) through the intersegmental membrane between the fourth and fifth abdominal sternites. Control insects were treated similarly but with distilled water only. Labelled JH dissolved in acetone (5 ~(1, approx. 70,000 dpm; 2 ng) was injected in a similar manner. After incubation at 30°C for various time periods, insects were homogenized using an Ultra-turrax T25 homogenizer at 13,500 rpm (IKA, Staufen, Germany), for 30 s at 4°C. Reverse phase liquid chromatography (RPLC)

Animals and surgical procedures Locusta migratoria migratorioides were reared at 30°C

under crowded conditions. They were fed every morning on fresh wheat and bran. All experimental females were isolated on the first day of adult life. Allatectomy was performed using Pascheff Wolff scissors on COz anaesthetized females. Sham-operated animals underwent the same surgical procedure except for gland ablation. Operated and sham-operated females were individually labelled and reared together. Chemicals

JH III was purchased from Sigma (TauIkirchen, Germany). (10RS)-[10-3H]JH III (444 Bq/pmol) was purchased from NEN (Wilmington, DE). Labelled (10R)-[12-3H]JH III (555 Bq/pmol) was a gift from Professor Glenn Prestwich (Stony Brook, New York). Fluvastatin was a gift from Dr F. Kathawala (Sandoz Research Institute, Hanover, NJ). Farnesol was a gift of Professor R. Feyereisen (Tucson, AZ). JH diol was prepared by hydrolysing JH with 0.01 N H2S04 in 5:3 tetrahydrofuran-water (Halarnkar and Schooley, 1990). JH was converted to JH acid using I ~1 hemolymph from Leptinotarsa decemlineata in 0.05 M phosphate buffer (30 min; 28°C; kindly provided by Dr Stan De Kort, Wageningen, The Netherlands). Unconverted JH was first extracted with isooctane, then JH acid was extracted with ethyl acetate (recovery is around 80%). JH acid diol was prepared from JH acid using the same procedure as for JH diol preparation. In vitro biosynthesis of (lOR)-[14C methyl]JH

and FRANCK

III

( lOR)-[14C methyl]JH III was produced by incubating individual pairs of corpora allata from adult female locusts for 4 h in TC- 199 containing [‘4C]methyl-methionine (Amersham, UK; final conditions 0.3 mM; 80 dpm/pmol) and farnesol(300 PM). Farnesol enhances JH production by locust corpora allata up to 200 pmol per pair of corpora allata per hour (Couillaud et al., 1988). At the end of the incubation, the products were extracted with isooctane and subjected to normal phase liquid chromatography. Radioactivity was monitored using solid scintillation on a Berthold LB 506 detector (Berthold, Postfach, Germany) and peaks corresponding to ( 10R)-[‘4C methyl]JH III were collected.

Experimental and analytical conditions were essentially those described by Halarnkar and Schooley (1990). Insects were homogenized in 5 ml acetonitrile containing triethylamine (40 ~1 per 100 ml acetonitrile), centrifuged for 5 min, at 6000g at 4°C and 1 ml of the supernatant was evaporated under a stream of nitrogen, the sample was redissolved in 50 ~1 of buffer A-buffer B (see below for composition) (7:3) and injected into the RPLC system. The remaining 4 ml acetonitrile of each sample was pooled and stored at - 18°C. We used a Beckman System Gold HPLC system (Palo Alto, CA) with a Beckman 166 UV detector. A 50 x 4.6 mm polymer column (PLRP-5, 5 pm, 100 A) was used, together with a 5 x 3 mm PLRP-5 guard cartridge (Polymer Labs, Amherst, MA). We used 2 buffer systems. The first one was Hepes buffer at pH 7.4 as described by Halarnkar and Schooley (1990): buffer A was 5 mM Hepes adjusted to pH 7.4. Buffer B was 5 mM Hepes buffer (final concentration) in 80% acetonitrile. In the second buffer system Hepes was replaced by citrate buffer 5 mM at pH 3. Samples were separated at a flow rate of 1 ml/min using a linear gradient from 5 to 100% buffer B in 20 min. UV absorption was monitored at 245 nm. Radioactivity was monitored on line at 25% efficiency after adding scintillation fluid (Flow Zinker, 3 ml/min; Zinsser analytic, Frankfurt, Germany) using a Berthold LB 506 detector (Berthold). Partition assay

For the partition assays (Share and Roe, 1988), insects were homogenized in 10 ml methanol-water (50:50). Isooctane (5 ml) was added and the sample was shaken vigorously. After centrifugation (5 min; 3OOOg),radioactivity from both aqueous and isooctane phases was determined by liquid scintillation counting of a 2 ml aliquot using Beckman Ready Safe scintillation fluid and Beckman LS 2800 spectrometer. RESULTS

Route for (lOR)-JH

III degradation in vivo

In a first series of experiments, we injected labelled ( 10R)-[3H]JH III into lo-day-old adult females of Locusta migratoria. After 1 h, each insect was homogenized and an aliquot of the sample was injected into the RPLC using

JH DEGRADATION

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FIGURE 1. Radio-RPLC chromatograph using Hepes pH 7.4 solvent system of (IOR#H]JH III and its metabolites, 1h after injection into lo-day-old adult female of L. migratoria. Sample was prepared as described under Materials and Methods. Peaks were identified according to the retention time of the corresponding standard. dpm for JH III acid dial= 7360: X = 4330; JH III acid = 1440: JH III = 14170.

FIGURE 3. Radio-RPLC chromatograph using citrate pH 3 solvent system of (10R)-[3H]JH III and its metabolites, 2 h after injection into lo-day-old adult female of L. migratoria. Sample was prepared as described under Materials and Methods. Peaks were identified according to the retention time of the corresponding standard. dpm for Z= 3894; JH III acid dial= 1484; Y= 1504; JH III= 611.

the Hepes buffer method developed by Halarnkar and Schooley (1990). Figure 1 illustrates the typical pattern of the radio-RPLC trace. Within 1 h, radioactivity was obtained in four peaks which eluted at retention times similar to JH III (l&30) JH acid diol (5:36) and JH acid (8:12). A significant amount of radioactivity was also found in a peak (named X) which eluted between acid diol and acid (7: 12). No peak was detected at a retention time similar to the JH diol standard (12:24). To further characterize the unknown peak X, we injected both (lOR)-[ 12-3H]JH III and ( lOR)-[14C methyl]JH III into adult females to provide information about the persistence of the carboxymethyl group. The time between injection and extraction was increased up to 2 h to promote the formation of end-products. Then, the sample was analysed for both 3H and 14C isotopes. As

shown in Fig. 2, the 3H trace revealed the occurrence of unchanged JH III (l&30), JH acid diol(5:36) and peak X (7:12). The 14Ctrace revealed only unchanged JH III. This suggests that the unknown compound identified as peak X has lost the labelled carboxyl methyl group and that it is not JH III diol phosphate recently identified in Manduca sexta (Halarnkar et al., 1993). Carboxylic acids are known to change their chromatographic mobility according to changes in pH of the mobile phase (Van de Venne et al., 1978). Using citrate buffer (pH 3), the retention time for JH acid shifted from 8:12 to 13:36 and for JH acid diol from 536 to 9:24. The elution times of JH III and JH diol did not change significantly with respect to pH. As shown in Fig. 3 the radio-RPLC trace for organic extract from females injected with (10R)-[3H]JH III analysed using citrate buffer at pH 3 revealed 4 peaks. Two of them had retention times similar to JH III (l&30) and JH acid diol (9:24) standards, respectively. However, two peaks (Y and Z) had retention times different from known standards. One of them probably corresponded to peak X. This was investigated further. We have addressed this problem using a large pool of organic extract from more than 30 females individually injected with (10R)-[3H]JH III. The pool was passed through a Sep Pak Cl8 cartridge (Waters) and vacuum evaporated. When an aliquot of this pool was analysed using Hepes buffer at pH 7.4, we observed 4 peaks identified as JH III (18:30), JH acid (8: 12) JH acid diol (5:36) and peak X (7: 12) [Fig. 4(A)]. When an aliquot of the same pool was injected on the RPLC system using citrate buffer pH 3, we found 5 peaks corresponding to JH III (18:30), JH acid (13.36), JH acid diol(9:24) and peaks Y and Z (lo:06 and 7:36, respectively) [Fig. 4(B)]. Using another aliquot from the same pool, the sample was separated in Hepes buffer and the peaks were collected separately according to their retention time. Peaks were dried under vacuum using a speed-vat concentrator and

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TIME (mn) FIGURE 2. Radio-RPLC chromatograph using Hepes pH 7.4 solvent system of (lOR#H]JH III and (10R)-[14C-methyl]JH III and its metabolites, 2 h after injection into lo-day-old adult females of L. migraforia. Sample was prepared as described under Materials and Methods. Peaks were identified according to the retention time of the corresponding standard. 3H-dpm for JH III acid diol = 7832; X = 1523; JH III= 384; W-dpm for JH III= 353.

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FIGURE 4. Radio-RPLC chromatograph of (10R)-[3H]JH III and its metabolites from the same pool resulting from injection into lo-day-old adult females of L. migratoria. Sample was prepared as described under Materials and Methods. Peaks were identified according to the retention time of the corresponding standard. Trace A was obtained from an aliquot of the pool separated using Hepes buffer at pH 7.4: dpm for JH III acid dial= 10,758; X = 4262; JH Iii acid = 3513; JH III = 5587. Traces B, C and D were obtained using citrate buffer at pH 3: trace B was obtained from an aliquot of the pool; dpm for Z = 9683; JH III acid dial= 3574; Y = 3689; JH III acid = 2592; JH III = 3907. Trace C was obtained when peak named JHAD in Hepes buffer was collected, dried and injected in citrate buffer, dpm for Z= 20,099; JH III acid dial= 8739. Trace D was obtained when the peak named X in Hepes buffer system was collected, dried and injected in citrate buffer, dpm for X = Y = 7222.

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We compared JH metabolism in lo-day-old adult females injected with either (10R)-[3H]JH III or (10RS)-[3H]JH III. As shown in Fig. 5 the natural tracer [(lOR)-JH III] decreased more slowly than the racemic mixture. During the first 20 min, the racemic mixture disappeared quickly, leading to an apparent half-life of about 25 min. However, for (lOR)-JH III, the half-life is around 60 min, in agreement with our observations using HPLC analysis. The main difference between the two curves was observed during the first 20 min when the titre of the racemic tracer was decreasing very quickly. 0

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TIME (mb) FIGURE 5. In viuo time-dependent degradation of( 10R)-[3H]JH Ill (*) and racemic [‘H]JH Ill(m) when injected into lo-day-old adult females. Undegraded JH was determined using the partition assay as described under Materials and Methods. Each time point is the mean ( f SE) of 3 insects.

one third of each peak was re-injected into the RPLC system (Hepes 7.4) to confirm the accuracy of the collection procedure (not shown). The remaining two thirds of each sample were injected into the RPLC system using citrate buffer. As expected, JH acid shifted from R,= 8:12 in Hepes to 13:36 in citrate buffer. The peak indicated as JH acid diol in the Hepes system (5:36) provided 2 peaks after citrate buffer separation [Fig.4(C)]. The first one is JH acid diol(9:24) and a second one is peak Z (7:36). Peak X with a retention time of 7:12 in Hepes appeared as peak Y with R, lo:06 in citrate buffer [Fig. 4(D)]. Finally, we incubated JH acid for 1 h in 50 ~1 acidic buffer (pH 1) to hydrolyse the 10-l 1 epoxy group. When analysed in the citrate RPLC system, JH acid (13.36) had been converted into the corresponding JH acid diol(9:24). When peak X = Y (R, = 10:06) was subjected to the same procedure, peak X was completely converted into peak Z (7:36) suggesting that peak Z could be the diol form of X (not shown). No-changes were observed when either the JH acid diol or peak Z was submitted to the same acidic treatment. Together these experiments suggest a new pathway for JH III catabolism in locust females. This pathway leads to the formation of 2 compounds identified as peaks (X and Z) whose identity are currently unknown. We propose that Z could be the diol form derived from X. Both X and Z have lost the carboxyl methyl group since they are never formed from (10R)-[‘4C methyl]JH III.

Experimental changes of JH-balance When minute amounts of ( 10R)-[3H]JH III are injected into the female, labelled JH is believed to label the endogenous pool and to undergo the same fate as endogenous JH. Labelled degraded molecules will be replaced by unlabelled JH produced by the corpora allata and the specific activity of the JH will gradually decrease. The labelled tracer will disappear more and more slowly. Suppression of JH biosynthesis will result in a constant specific activity of JH, a progressive decrease in the endogenous JH pool and theoretically in an increase in the rate of disappearance of the labelled tracer. To check that the labelled tracer really reflects JH metabolism in situ, we suppressed JH biosynthesis by the corpora allata and examined the repercussions on the rate of disappearance of labelled-tracer. In one experiment, lo-day-old females were injected with the HMG-CoA reductase inhibitor: fluvastatin. When injected into locusts, fluvastatin inhibited JH biosynthesis by the corpora allata (Debernard et al., 1994). Twenty minutes later, (10R)-[3H]JH III was injected and undegraded [3H]JH III was measured 120 and 180 min after JH injection. As shown in Fig. 6, the amount of undegraded C3H]JHIII is significantly lower in fluvastatin treated insects than in controls. When the

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Racemic and (IOR)-JH III degradation in vivo According to our radio-RPLC profiles using either Hepes or citrate buffers, JH III is the only nonpolar labelled peak. Thus, for quantitative studies of JH III degradation, we switched from RPLC analysis to a partition assay developed by Share and Roe (1988). JH partitions into the isooctane phase and the JH metabolites into the aqueous methanol phase (Share and Roe, 1988).

u

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POST-INJECTION DELAY (mn) FIGURE 6. In viuo time-dependent degradation of (10R)-[3H]JH Ill in 1O-day-old adult females injected 20 min earlier ($ solid line) or 180 min earlier (* dashed line) with either 200 pg fluvastatin to inhibit JH production or sham-injected(m). Undegraded JH was determined using the partition assay as described under Materials and Methods. Each time point is the mean (f SE) of 6 insects.

STBPHANE

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FIGURE 7.

Sham

DEBERNARD

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of (10R)-[3H]JH III after a 2 h incubation in adult females allatectomized or sham-operated on day 10. Part A compares degradation of JH in 2 h in both allatectomized (hatched bars) and sham-operated (open bars) females 2 h after surgical procedure. Part B compares the degradation of JH after 2 h, 24 h after the surgical procedures. Undegraded JH was determined using the partition assay as described under Materials and Methods. Each point is the mean (+ SE) of 6 insects. In

aiuo degradation

period of fluvastatin inhibition was increased from 20 to 180 min before JH injection, the rate of disappearance of labelled JH also increased. However, locust corpora allata escape from the inhibiting effect of fluvastatin after 5-6 h (Debernard et al., 1994). To study the effect of a prolonged lack of JH biosynthesis on JH metabolism, we allatectomized females on day 10 after fledging. After 2 h, females were believed to have recovered from the surgical trauma and were injected with labelled JH. As revealed in Fig. 7(A), 120 min after JH injection, allatectomized females exhibited an apparent lower amount of undegraded [3H]JH III than sham-operated females as reported above for fluvastatin-treated animals. However, when labelled JH was injected 24 h after allatectomy and then assayed 120 min later, allatectomized females (expected to possess a JH-pool around zero) exhibited a similar apparent amount of undegraded JH to sham-operated females [Fig. 7(B)]. The level of undegraded [3H]JH III, 120 min after JH-injection, was higher in females allatectomized for 24 h than in females allatectomized for 2 h suggesting an adjustment in JH metabolism in situ following allatectomy. JH degradation in allatectomized or fluvastatin-treated females was investigated using RPLC with Hepes and citrate buffer systems. After suppression of JH biosynthesis, both allatectomized and fluvastatin-treated females revealed the same pattern of JH catabolism as controls, including formation of peaks X and Z (not shown). DISCUSSION

Our in uivo study provided important information on JH catabolism in the female locust Locusta migratoria. Of particular importance was our finding that JH III degradation leads to the formation of 2 unknown compounds. The first unknown compound was peak Z, the major end product of JH catabolism in the locust. Peak Z

and FRANCKCOUILLAUD comigrated with JH acid diol at pH 7.4 but could be separated from it when the pH of the mobile phase was brought to a more acidic pH. We chose citrate buffer at pH 3 to achieve this separation. A pH lower than 3 resulted in spontaneous hydrolysis of the 10-l 1 epoxy ring of the JH skeleton. The second unknown compound called X, was isolated using both Hepes pH 7.4 and citrate pH 3 buffer systems. When subjected to a pH lower than 1, peak X was converted into peak Z. In the same way, JH acid was converted to JH acid diol and JH to JH diol. Accordingly, we suggest that Z is the diol form of X. A diol-phosphate conjugate has recently been identified as the major end product of JH catabolism in Manduca sexta (Halarnkar et al., 1993) and its mobility in the RPLC Hepes system is consistent with peak X. However, from our experiments using JH labelled on the carboxyl methyl group, it is clear that X and Z are not methylated. Thus, X and Z are not the same as the JH diol-phosphate from Manduca sexta. Studies are currently in progress in our laboratory to identify compounds X and Z. It is interesting that following injection of small amounts of radiolabelled JH III radioactivity in JH acid is very low and radiolabelled JH diol was not detected. This suggests that both JH esterase and JH epoxy-hydrolase are very active in forming the JH acid diol. Hemolymph esterases in the locust are not JH specific (Koopmanschap and De Kort, 1989) and epoxide hydrolases, which are membrane-associated enzymes (Casas et al., 1991) have been reported to be absent from the hemolymph of a variety of insects (see Hammock, 1985) including Locusta (S. Debernard, unpublished). Our in vivo finding that JH acid production was very low, suggests that tissues other than hemolymph were involved in JH degradation. Our in uivo experiments, however, did not allow us to assign a functional role to specific tissues in JH catabolism. In the locust, both the JHBP and hemolymph esterase showed enantiospecificity (Peter et al., 1979,1983). When JHBP and esterase are present in the same sample, the rate of hydrolysis of the natural enantiomer is markedly reduced because the enzyme preferentially hydrolyses (lOS)-JH III (Peter et al., 1983). We have injected minute amounts of JH in vivo and due to the large number of expected JHBP binding sites (De Kort, 1990), we suggest that all exogenous hormone will probably be bound. However, JHBP affinity for the (1OR) enantiomer is higher than for the 10s form (De Kort and Koopmanschap, 1986a). This was probably responsible for the difference in the rate of degradation between the 10R and racemic mixture. Degradation of the racemic mixture occurred more rapidly because the unnatural enantiomer is less well protected by JHBP. This is not always the case in other insect species, in Nauphoeta cinerea for example, the natural (lOR)-JH III is degraded more rapidly than the racemic mixture (Lanzrein el al., 1993). To support the hypothesis that in vivo the JH titre is controlled by degradation, we have experimentally manipulated JH titre and found changes in the rate of

JH DEGRADATION

degradation. Corpora allata are the unique source of JH in the locust and suppression of JH biosynthesis can be easily performed. We used chemical inhibition of JH biosynthesis by fluvastatin (Debernard et al., 1994) and surgical allatectomy. When injection of labelled JH tracer occurred 3 h or less after suppression of JH production, disappearance of labelled JH increased compared with controls. In short-term experiments, the difference between the experimental and control insects increased as the delay between the time of suppression of JH biosynthesis and the determination of unchanged JH increased. This difference is believed to result from both the gradual reduction of the endogenous pool of JH subsequent to JH biosynthesis suppression and from the constant specific activity of the labelled JH which is no longer diluted by the JH produced by the corpora allata. It is interesting that in the short-term experiment, fluvastatin and allatectomy resulted in the same effect on JH metabolism. Neither allatectomy nor fluvastatin resulted in modification of the RPLC pattern. Thus, our present data provides an additional argument that fluvastatin does inhibit JH biosynthesis in vivo in the locust. Because injection of fluvastatin had no effect on the JH-regulated maintenance of the larval status and JH-regulated ovarian maturation (Debernard et al., 1994), we suggest that the locust can undergo a severe and transitory reduction in JH biosynthesis without detectable modifications of JH-regulated physiological processes such as oocyte maturation and maintenance of the larval status. When females were assayed for JH catabolism 1 day after allatectomy, the disappearance of the labelled JH tracer was similar to that in sham-operated animals. This suggests that the rate of JH catabolism has been considerably decreased after allatectomy. Removal of the copora allata on day 1 resulted in the absence of JH on day 8 (Couillaud et ul., 1985). After allatectomy on day 8 Joly (1960) demonstrated that females were unable to initiate a new oocyte maturation cycle but no data are available for JH titre following allatectomy on day 8. Thus, it seems reasonable to expect a very low or zero JH titre 24 h after allatectomy in our insects. In 24h-allatectomized females the rate of JH degradation is markedly reduced compared to that of controls suggesting some changes in JH catabolism. Changes may result from several causes: (i) changes in enzyme activities or enzyme turnover induced by allatectomy or by the low JH titre; (ii) very low JH concentrations below the K, of the enzymes; or (iii) the injected JH may be protected in some wav. Our data suggest that the tracing of minute quantities of ( 10R)-[3H]JH III could provide a valuable method for the analysis of JH degradation in vivo. The data argue that metabolism of JH can be modified within a few hours in response to drastic changes in JH synthesis. However, additional experiments are required to determined the nature of the changes. At the same time, our RPLC methods

using

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interesting

variation of the protocol developed by Halarnkar

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Schooley (1990). In the locust, this procedure allowed us to demonstrate the existence of a novel pathway for JH catabolism and opened the door for additional studies in this insect.

REFERENCES Baker F. C., Tsai L. W., Reuter C. C., and Schooley D. A. (1987) In rive fluctuation of JH acid and ecdysteroid titer and JH esterase activity during development of fifth stadium Manduca sexta. Insect Biochem. 17, 989-996. Bergot J., Schooley D. A., and De Kort C. A. D. (1981) Identification of JH III as principle juvenile hormone in Locusta migratoria. Experientia 37, 909-9 10 Casas J., Harshman L. G., and Hammock B. D. (1991) Epoxide Hydrolase Activity on Juvenile Hormone in Manduca sexta. Insect Biochem. 21, 17-26. Couillaud F., Mauchamp B. and Girardie A. (1985) Regulation of juvenile hormone titer in African locust. Experientia 41, 1165-l 167. Couillaud F., Mauchamp B., Girardie A. and De Kort C. A. D. (1988) Enhancement by farnesol and farnesoic acid of JH biosynthesis in induced low-activity locust corpora allata. Arch. Insect Biochem. Phwiol. 7. 133~-143. Debernard S., Couillaud F., and Rossignol F. (1994) The HMG-Coa reductase inhibitor. fluvastatin, inhibits insect juvenile hormone biosynthesis in vivo and in vitro. Gen. camp. Endocrin. 95, 92-98. 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. Ser. C. 82, 325-331. De Kort C. A. D. and Granger N. A. (1981) Regulation of juvenile hormone titer. Ann. Rer. Entomol. 26. l-28. De Kort C. A. D and Koopmanschap A. B. (1986a) Specificity of binding of juvenile hormone III to hemolymph proteins of Leptinotarsa decemlineata and Lorasta migratoria. E.xperientia 43, 904-905. De Kort C. A. D. and Koopmanschap A. B. (198613) High molecular transport proteins for JH-III in insect hemolymph. E.uperientia 42, 834-836. De Kort C. A. D. (1990) Juvenile Hormone and insect reproduction. In ‘Advances iti Invertebrate Reproduction’, Hoshi M. and Yamashita 0. eds. pp 187-192. De Bruijn S. M., Koopmanschap A. B., and De Kort C. A. D. (1986) High-molecular-weight serum proteins from Locusta migratoria: identification of a protein specifically binding juvenile hormone III. Physiol. Entomology 11, 7-16. Erley D., Southard S. and Emmerich H. (1975) Excretion of juvenile hormone and its metabolisms in the Locust, Locusta migratoria. J. Insect Physiol. 21, 61-70. Feyereisen R. (1985) Regulation ofjuvenile hormone titer: Synthesis. In ‘Comprehensive Insect physiology, biochemistry and pharmacology’ (Kerkut G. A. and Gilbert L. I. eds) (Pergamon press, Oxford) pp. 391429. Halarnkar P. P. and Schooley D. A. (1990) Reversed-Phase Liquid Chromatographic Separation of Juvenile Hormone and Its Metabolites, and Its Application for an in vivo Juvenile Hormone Catabolism Study in Manduca sexta. Anal. Biochem. 188, 394397. Halarnkar P. P.. Jackson G. P.. Straub K. M., and Schooley D. A. (1993) Juvenile Hormone Catabolism in Manduca-sesta-Homologue Selectivity of Catabolism and Identification of a Diol-Phosphate Conjugate as a Major End Product. E.xperientia 49, 988-994. Hammock B. D. (1985) Regulation of juvenile hormone titer: degradation. In ‘Comprehensive Insect physiology. biochemistry and pharmacology‘(Kerkut G.A. and Gilbert L. I. eds) (Pergamon press, Oxford) pp. 431472. Hanzlik T. N., Abdelaal Y. A. I., Harshman L. G., and Hammock B. D. (1989) Isolation and sequencing of cDNA clones coding for juvenile hormone esterase from Heliothis virescens: Evidence for a catalytic mechanism for the serine carboxylesterases different from that of the serine proteases. J. hiol. Chem. 264, 12419-12425.

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Acknowledgements-We

thank Professor Glenn Prestwich (Stony Brook, New York) for the gift of labelled juvenile hormone, Dr F. Kathawala (Sandoz research Institute, Hanover, NJ) for providing fluvastatin, Professor Barry Lougthon (York Univ., Toronto, Canada) and Dr Lionel Peypelut (Univ. Bordeaux, France) for reading the manuscript. We gratefully acknowledge F. Rossignol for her technical assistance and Dr Stan De Kort (Univ. Wageningen, The Netherlands) for constant and stimulating discussions over the last 10 years. Supported by E.C. contract TS3.CT93.0208.