Juvenile hormone esterase activity repressive factor in the plasma of parasitized insect larvae.

A proteinaceous factor that represses plasma juvenile hormone esterase activity in parasitized insect larvae has been isolated and partially characterized from last instar larvae of the armyworm Pseudaletia separata parasitized with the wasp Apantales kariyai. Purification procedures consisted of extraction with 25% ethanol, gel filtration and reversed phase high performance liquid chromatography. Plasma juvenile hormone esterase activity in Day 3 last instar larvae was repressed by 50% when larvae were injected on Days 1 and 2 with 6.5 pmol of the purified peptide, which has a molecular weight of about 4,500 Da. The application of the factor also causes more than a 2-day delay in the onset of pupation. The sequence of 23 amino acid residues at the amino terminus of the factor was determined as follows: H-Glu-Asn-Phe-Ser-Gly-Gly-Xaa-Val-Ala-Gly-Tyr-Met- Arg-Thr-Pro-Asp-Gly-Arg-Xaa-Lys-Pro-Thr-Phe-Tyr-Gln-.

Plasma juvenile hormone e&erase activity in Day 3 last instar larvae was repressed by 50% when larvae were injected on Days 1 and 2 with 0.5 pmol of the purified peptide, which has a molecular weight of about 4,500 Da. The application of the factor also causes more than a 2-day delay in the onset of pupation.

Endoparasitic
insects disrupt the metamorphosis of holometabolous host insects with the development often arrested in the larval stages (1). The specific mechanisms whereby parasitism causes suppressed metamorphosis of host insects remain enigmatic and little progress has been made toward resoIving the question until recently.
Last instar larvae of the armyworm Pseudaletia sepparuta parasitized with eggs of the parasitoid wasp Apantuh kmiyui do not initiate metamorphosis and the wasp larvae emerge from the host larvae about 10 days after parasitization (2). Studies on the endocrinological nature of the effect indicate that the parasitoid inhibits prothoracicotropic hormone synthesis and secretion by the host larval brain (2). The role of JH in controlling the release of prothoracicotropic hormone has been investigated (3)(4)(5)(6) and, in early last instar larvae, the absence of JH' from plasma is necessary for release of prothoracicotropic hormone from the brain (3). In last instar larvae of most Lepidoptera, the first burst of JH &erase activity correlates with a decline in plasma JH, after which prothorscicotropic hormone is released and initiates a cascade of events leading to pupation (7,8). In the light of this proposed scheme, it was of interest to examine JH esterase *This work was supported in part by the Akiyama Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduert~~ement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
' The abbreviations used are: JH, juvenile hormone; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography. activity in the plasma of parasitized larvae. In this study I have demonstrated the existence of a repressive factor that blocks the first peak of plasma JH esterase activity in the parasitized last instar larvae. Isolation and characterization of the factor was also carried out. In addition, the effect of the factor on the metamorphosis of the host larva was also tested.

MATERIALS AND METHODS
Animals-l? reparata were reared on an artificial diet at 25 f 1 "C with a photoperiod of 16 h light:8 h dark (2). Parasitization by A. kariyai was carried out by exposing prospective hosts (usuatly Day 0 or Day T last instar larvae) to female wasps. After a single oviposition was observed the parasitized larvae were quickly removed in order to avoid superparasitization and then reared on the artificial diet. Adult wasps were maintained with honey.
C~~GGX&---[~O-~H]JH I (15.5 Ci/mmol) and peptide 4-methylcoumarin amide were purchased from Dupont and Peptide Institute, Inc., Japan, respectively. Preparation of Plasma-The hemolymph of parasitized Day 4-6 last instar larva (3-6 days after parasitization) was collected into a chilled polypropylene test tube containing 500 ~1 of 0.05% phenylthiourea in 0.2 M phosphate buffer, pH 6.0 by the "flushing out" method with an injection of about 0.1 ml of 0.1 M phosphate buffer containing 150 mhi KC1 and 10 mM EDTA, pH 6.0, into each hemocoele (9). Collected hemolymph was immediately centrifuged at 4 "C for 5 min at 500 X g and the supematant was used as the plasma sample for purifying the YH esterase repressive factor. Ten milliliters of plasma was collected from about 80 parasitized last instar hrvae.

Erz2yms
Assay-Ten microliters of the hemolymph was collected from a test larva into a microcentrifuge tube containing 110 pl of 0.02% phenylthiourea in 0.2 M phosphate buffer, pH 6.0, using a micropipette and immediately centrifuged at 4 'C for 5 min at 5M) x g. The supernatant was used as enzyme preparation for measuring plasma eskrase activities.
Plasma degradation of [IO-%]JH I and unlabeled carrier hormone (E,E-cis-JH I) (Sigma) was measured in U&O using the methods of Hammock and Roe (10). Every enzyme preparation (plasma) was first preincubated with 2 x lo-' M &isopropyl fluorophosphate (Sigma) at 25°C for 10 min prior to addition of substrate (final Substrate concentration = 5 X lo-' M). Plasma without preincubating with diisopropyl fluorophospbate was used as enzyme preparation for assay of peptide I-methylcoumarin amide (tert-butoxycarbonyl-Val-Pro-Arg-4-methylcoumarin amide) hydrolyzing activity. The release of 'I-amino-l-methylcoumarin from peptide 4-metbylcoumarin amide during the hydrolaae enzyme reaction was detected using a fluorescence spectropbotometer according to the methods of Morita et al. (11).
Bioassrcy of&press& &&or-The purified factor was diluted with Ringer's aulution to the desired concentration (final volume = 10 ~1) and injected into unparasitized last instar larvae at Day 1 and Day 2. The next day, plasma was prepared from the larvae for assay of JH esterase activity. Controls were injected with 10 pl of Ringer's solution only. One unit of repressive activity decreased 10% of plasma JR esterase activity compared with the control under the conditions described above. Protein Determination and Characterization-Protein was determined by the method of Bradford (12) using the Bio-Rad protein assay kit with bovine serum albumin as standard.
The NH,-terminal amino acid sequence of purified JH eaterase repressive factor was analyzed by automated Edman degradation with a protein sequencer (model 477A, Applied Biosystems) {13). The sequence was verified by analyzing 100-200 pmol of the native purified factor 5 times.
Samples of purified repressive factor were prepared for SDSpolyacrylamide gel electrophoresis by incubating with 80 mM Tris-HCl buffer, pH 8.8, containing 1% SDS and 2.5% @-mercaptoethanol in boiling water for 5 min and developed by the methods of Laemmli JH E&erase Repressive Factor in Parasitized Insect (14). Gel was silver-stained for protein according to the methods of Merril et al. (15). A

AND DISCUSSION
In unparasitized last instar larvae (Day 3 and Day 6) of P. separata, two major bursts of plasma JH esterase activity were observed in agreement with reports for other lepidopterous larvae (7). However, no burst of JH esterase activity occurred in parasitized larval plasma during the last larval instar (Fig.  1). A similar observation has been reported in the larval hemolymph of the tobacco hornworm parasitized by the braconid wasp (16). These results suggest the presence of a factor that inhibits JH esterase activity directly or indirectly in the plasma of parasitized larvae. Although preliminary experiments failed to demonstrate an inhibitor functioning directly against JH esterase, the injection of plasma from parasitized last instar larvae into unparasitized larvae at Days 1 and 2 resulted in repression of JH esterase activity in Day 3 larvae. This finding was interpreted to indicate that a JH esterase repressive factor functions in uiuo in parasitized last instar larval plasma.
The plasma collected from parasitized last instar larvae was mixed with -20 "C ethanol (final concentration = 25%), and, following centrifugation at 4 "C for 15 min at 20,000 x g, the supernatant was concentrated by lyophilization and applied to a 3-ml disposable Cl8 extraction column (J. T. Baker Chemical Co.) equilibrated with 6 ml of distilled water. The column was washed with 5 ml of distilled water and subsequently eluted with 2.5 ml of 30% acetonitrile.
The eluent was concentrated under a stream of nitrogen and applied to gel permeation chromatography on Superose 6 HR lo/30 (Pharmacia LKB Biotechnology Inc.) equilibrated with 12.5 mM phosphate buffer, pH 6.6. The bioactive zone was collected and further resolved by a reversed phase Cs HPLC column (Yamamura Co., Japan; 4.6 mm inner diameter X 250 mm, pore size = 300 A) using gradient elution from 18 to 40% CH,CN in 0.1% CF3COOH/H20 at a flow rate of 0.4 ml/min. The bioactive fraction was again collected and rechromatographed using a reversed phase cyanopropyl-derived silica HPLC column (Yamamura Co.; 4.6 mm inner diameter X 250 A, Cs active fraction chromatographed with CH&N in 0.1% CF&OOH/HzO; B, cyanopropyl active fraction rechromatographed with CH&N in 0.05% CF$F&F&OOH/HzO; C, cyanopropyl active fraction of B rechromatographed with CH&N in 0.075% CF&F&F2COOH/H20. The columns were rnn sequentially. Horizontal bars indicate fractions that show repressive activity against plasma JH esterase. mm, pore size = 300 A) under three elution schedules as follows (Fig. 2). The active fraction of Cs was first eluted using a gradient of CH&N from 15 to 25% in 0.1% CF3COOH/H20 at a flow rate of 0.4 ml/min ( Fig. 2A) and then reapplied and eluted with a gradient of 18 to 25% CH,CN in 0.05% CF&F2CF2COOH/Hz0 at a flow rate of 0.4 ml/ min (Fig. 2B). The fraction with repressive activity eluted in a single peak and was reapplied to the same column and then eluted using a gradient of 20 to 25% CH,CN in 0.075% CF3CFzCF2COOH/HZ0 at a flow rate of 0.4 ml/min (Fig. 2C). Typical data on the purification process of the repressive factor are summarized in Table I. SDS-polyacrylamide gel electrophoresis of the purified repressive factor yielded a single band that indicates a molecular weight of about 4,500 as shown in Fig. 3. The NH*-terminal amino acid sequence for the first 23 residues was determined as follows: H-Glu-Asn-Phe-Ser-Gly-Gly-Xaa-Val-Ala-Gly-Tyr-Met-Arg-Thr-Pro-Asp-Gly-Arg-Xaa-Lys-Pro-Thr-Phe-Tyr-Gln-.
Sequence comparison was made against the National Biomedical Research Foundation Protein Data Bank using Beckman's MicroGenie version 4.0. Limited sequence similarity was observed with internal sequence of several ' One unit of repressive activity decreased 10% of plasma JH esterase activity compared to the control under the conditions described under "Materials and Methods." ' Purification and recovery are based on the specific activity and total activity, respectively, of plasma. ' The volume of plasma collected from about 180 larvae. On Days 1 and 2 at 3 h after lights on the purified repressive factor was injected into unparasitized host larvae. Plasma was prepared from the larvae 24 h after the second injection to assay JH esterase activity by the method described under "Materials and Methods." The amount indicated on the abcissa gives the total amount of purified repressive factor injected into unparasitized larvae for 2 consecutive days (Days 1 and 2). Injection of bovine serum albumin (60 pmol) did not have any significant effect against plasma JH esterase activity 92 -C 11 nmol/ min/ml, n = 3). Each point represents a single determination of plasma JH esterase activity of two larval plasma mixture.
The purified repressive factor shows a dose-dependent capacity to decrease plasma JH esterase activity as shown in Fig. 4. Injection of about 6.5 pmol of the purified factor into " MCA, peptide ii-methylcoumarin amide. *Each enzyme activity is the mean f S.D. of four separate assays. unparasitized last instar larvae at Day 1 and Day 2, respectively, reduced plasma JH esterase activity by 50% compared with control (see "Materials and Methods"). In contrast, the other hydrolase activity, peptide 4-methylcoumarin amide hydrolyzing activity, was not changed at all by the injection (Table II). Furthermore, the application of about 7 pmol of the purified repressive factor at Days 1 and 2, respectively, also resulted in more than a P-day delay in pupation in 80% of the larvae (Table III).
As long as juvenile hormone concentration maintains its high level in insect larval plasma (hemolymph without cells), the insect never undergoes metamorphosis from larva to pupa (8). Since the JH esterase found in larval plasma undoubtedly accounts for the major hydrolysis of JH (7), it can be said that plasma JH esterase is a key enzyme for controlling insect metamorphosis.
The factor that I reported here represses plasma JH esterase activity in the last instar larvae and consequently causes a delay in the onset of pupation. The prolonged application of about 6 pmol of the purified repressive factor into the last instar larvae once every day from Day 0 to Day 5 causes more than a &day delay in pupation (data not shown). This observation together with the results in Table III strongly suggests that a cascade of events leading to pupation will not initiate as long as the repressive factor exists in the host larvae. It is evident that the repressive factor may perturb the endocrinological processes that control normal metamorphosis in the host insect although the site of action of the factor is still unknown. As a result of the action of the repressive factor, the onset of host pupation is delayed and the parasitoid has adequate time in which to complete growth and development, especially when parasitization occurs during the late stages of larval development of the host. In summary, this study clearly shows the existence of a JH