Isolation and identification of a pheromonotropic neuropeptide from the brain-suboesophageal ganglion complex of Lymantria dispar: A new member of the PBAN family

Insect Biochem. Molec. Biol. Vol. 24, No. 8, pp. 829-836, 1994

Pergamon

0965-1748(94)E0005-2

Copyright :~ 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0965-1748/94 $7.00 + 0.00

Isolation and Identification of a Pheromonotropic Neuropeptide from the Brain-Suboesophageal Ganglion Complex of Lymantria dispar: A New Member of the PBAN Family? E. P. MASLER,:~§ A. K. RAINA,:I: R. M. WAGNER,¶ J. P. KOCHANSKY~ Received 7 September 1993; revised and accepted I1 January 1994

A pheromonotropic peptide was isolated from brain-suboesophageal ganglion complexes of the adult female gypsy moth, Lymantria dispar, using a 5-step HPLC purification protocol and an in vivo bioassay in Helicoverpa zea. The intact peptide was sequenced by automated Edman degradation. The L. dispar pheromone biosynthesis activating neuropeptide (Lyd-PBAN) is a C-terminally amidated 33-amino acid peptide with a molecular weight of 3881. The peptide was synthesized using Fmoc procedures. Lyd-PBAN has sequence homology with Hez-PBAN (81.8%) and Bom-PBAN-I (66.7%). All three PBANs share the C-terminal hexapeptide sequence, Tyr-Phe-Ser-Pro-Arg-Leu-NH2. In addition, the C-terminal pentapeptide sequences of Pseudaletia pheromonotropin (Pss-PT), Bombyx diapause hormone (Bom-DH), the Iocustamyotropins (Lom-MT) and leucopyrokinin (Lem-PK) are identical or have a high degree of homology to the C-terminus of PBANs. Amino acid sequence Gypsy moth Lymantria dispar homology Pheromonotropic peptides

INTRODUCTION Female moths use species-specific sex pheromones to attract their mates. Production of these pheromones is under neuroendocrine control, mediated by at least one neuropeptide, the pheromone biosynthesis activating neuropeptide (PBAN, see review by Raina, 1993). Two 33-residue pheromonotropic molecules have been isolated, Hez-PBAN (Raina et al., 1989) from Helicoverpa zea and Bom-PBAN-I (Kitamura et al., 1989) from Bombyx mori. Each has the identical C-terminal hexapeptide sequence, Tyr-Phe-Ser-Pro-Arg-Leu-NH2. The C-terminal pentapeptide sequence has been shown to be the minimal sequence essential for pheromonotropic activity (Raina and Kempe, 1990). Another pheromonotropic §Author for correspondence. t M e n t i o n of a proprietary product does not constitute endorsement by the USDA. ++Insect Neurobiology and H o r m o n e Laboratory, Plant Sciences Institute, U S D A - A R S , Beltsville M D 20705-2350, U.S.A. ¶Livestock Insects Laboratory, Livestock and Poultry Sciences Institute, U S D A - A R S , Beltsville, M D 20705-2350, U.S.A. 829

Neuropeptideisolation

PBAN sequence

peptide was isolated from B. mori and identified as Bom-PBAN-II, a 34-residue peptide, identical to BomPBAN-I except for the presence of an additional residue (Arg) at the amino-terminus (Kitamura et al., 1990). An additional pheromonotropic peptide was isolated from Pseudaletia separata (Matsumoto et al., 1992a). The PssPT has 18 residues with the C-terminal sequence Phe-ThrPro-Arg-Leu-NH2. The female gypsy moth, Lymantria dispar, produces a single-component sex pheromone, disparlure (Bierl and Beroza, 1970), which begins to accumulate in the pheromone glands within the first 24 h following adult emergence (Giebultowicz et al., 1992). A daily titer rhythm was observed with pheromone levels low at night and high during the day. Overall titers increased as the female aged (Giebultowicz et al., 1992). Hollander and Yin (1985) reported the involvement of the brain in the release of pheromone in L. dispar. Extracts of the brain suboesophageal ganglion (SOG) complex stimulated significant pheromone production when injected into H. zea females (Raina and Klun, 1984; Raina et al., 1987), although Hez-PBAN showed only slight

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Elution Time (minutes) F I G U R E I. Fractionation of Sep-Pak extract on HPLC System 1. Approximately 100 brain SOG equivalents were loaded in 10% CH3CN/0.1% TFA. Fractions (1 min) were collected and aliquots (1/5 of each fraction) were removed, dried in silicone coated polypropylene tubes and dissolved in saline for bioassay. The remainder of each fraction was stored, without drying, at -20"C. Solid bar indicates pheromonotropic activity. Following location of activity, fractions from subsequent runs were not aliquoted but pooled for further processing.

stimulation of pheromone production in L. dispar (Raina et al., 1989). The pheromonotropic factor in L. dispar brain-SOG extracts is a peptide (Masler and Raina, 1993) with characteristics similar to Hez-PBAN and Bom-PBAN. Based upon preliminary investigations, it appears that a PBAN-like molecule is involved in the control of pheromone production in L. dispar, but the exact mechanism through which it operates may be different from that in H. zea and B. mori (Raina, 1993). In order to facilitate the investigation of the control of sex pheromone production in L. dispar, we sought to further characterize the L. dispar pheromonotropic neuropeptide. We report here its isolation and identification.

MATERIALS AND M E T H O D S

Animals, bioassay, tissue collection L. dispar were reared and maintained as previously described (Bell et al., 1981; Masler et al., 1991). Pupae were segregated by size, held in paper cups until emergence, and adults were collected daily. Day 1 2 females were used as the source of brain-SOG complexes. L. dispar brain-SOG complexes were dissected in saline (188mM NaCI, 20.1mM KC1, 9 m M CaCI2, l m M MgSO4), and collected on dry ice as previously described (Masler and Raina, 1993). Tissues were held in 1.5 ml polypropylene tubes at - 8 0 ° C until extraction. Typically, 10-30 tissue complexes were collected per tube. H.

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Elution Time (minutes) F I G U R E 2. HP-SEC fractionation of active fractions from System 1. Fractions eluting from 23 to 25 min (Fig. l) were pooled from 20 runs (total o f c. 2000 brain-SOG equivalents), and fractionated on System 2. Pheromonotropic activity (solid bar) eluted at 17 min. Active fractions from four identical runs were pooled. External standards used for molecular weight markers were: A, carbonic anhydrase; B, parvalbumin; C, aprotinin; D, insulin chain B; E, little gastrin; F, adipokinetic hormone-l. Input for the chromatogram shown represents 500 brain SOG equivalents.

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FIGURE 3. Reverse-phase fractionation of active fractions from System 2. Pooled, active fractions from the HP-SEC step (Fig. 2) were fractionated in two batches on System 3. Active fractions (solid bar) were pooled from two runs. Input for chromatogram shown represents 1000 brain SOG equivalents. zea were reared as described (Raina et al.,

1986). Samples were dissolved in saline for bioassay. The bioassays were conducted in H. zea females (Raina et al., 1986) and the pheromone quantified by gas chromatography (Raina and Kempe, 1992). Tissue e x t r a c t i o n

B r a i n - S O G complexes were homogenized on ice in 60% acetonitrile (CH3CN; Fisher Scientific, Springfield Va) in 0.1% aqueous trifluoroacetic acid (TFA, Aldrich Chemical Co., Milwaukee, Wis.) using a Polytron PT-7 generator (Brinkman Instruments, Westbury, N.Y.) set at 50% m a x i m u m power for 15 s. Extractions were made with batches of 10-30 tissue complexes in 20/~1 of 60% CH3CN/0.1% T F A per complex. Ten extractions were pooled and centrifuged (16,000g, 20min, 4°C). The supernatant was decanted, saved on ice, and the pellet was re-extracted in 10 ml of 60% CH3CN/0.1% T F A as

above. The two supernatants were pooled and concentrated under vacuum using a Speed-Vac (Savant Instruments, Farmingdale, N.Y.) to remove organic solvent and retain an aqueous concentrate, now frozen, representing about 30% of original volume. The concentrate ( 3 4 ml) was thawed, diluted with an equal volume of 0.1% TFA, and then processed using a Sep-Pak C~8 cartridge (Waters Assoc., Milford, Mass.). S e p - P a k processing

Approximately 2000 b r a i n - S O G equivalents were processed on a single Sep-Pak cartridge. Details of cartridge activation and equilibration, and sample elution, have been described elsewhere (Masler et al., 1983; Masler and Raina, 1993). Following activation and equilibration, a sample representing c. 100 brain SOG equivalents is applied to the cartridge, eluted in steps of 0.1% TFA, 10% CH3CN/0.1% T F A and 60%

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FIGURE 4. Reverse-phasefractionation of active fractions from System 3. Fractions eluting between 15 and 16 min (Fig. 3) were pooled from two runs and processed on System4. Pheromonotropic activity (solid bar) was localized and a peak eluting at 35% CH3CN was collected for further processing. Input represents 2000 brain-SOG equivalents.

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FIGURE 5. Purification of Lyd-PBAN. The active peak at 24 min (Fig. 4) was desalted on System 5. Input was c. 2000 brain-SOG equivalents. The major peak (arrow) was collected for sequencing. CH3CN/0.1% TFA. The cartridge is re-equilibrated and used for the next sample. Material eluting in the 60% CH3CN/0.1% T F A step is dried in the Speed-Vac and held at - 2 0 ° C until use. Chromatographic

fractionation

(1) Batches of 100 brain SOG equivalents from the Sep-Pak processing were fractionated on System 1, consisting of a Supelco LC18-DB C~8 column (4.6 x 300 mm) with a Supelguard LC18-DB C~8 Supelcosil guard column (4.6 x 20 mm) (Supelco, Bellefont, Pa). Flow rate was 1 ml/min with a linear gradient from 10% CH3CN to 60% CH3CN in 0.1% T F A over 50 rain. Fractions were collected in 4.5 ml polypropylene tubes. All fractions and aliquots were dried in the Speed-Vac and stored at - 2 0 ° C for future use.

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Fractions from Systems 2 to 4 were collected in 1.5 ml polypropylene tubes coated with silicone (Sigmacote; Sigma Chemical Co., St Louis, Mo). Unless otherwise noted, all fractions also contained 5 ~tl of insect Ringer's (Okuda e t al., 1985) to facilitate peptide recovery. (2) Active fractions (fraction numbers 24 and 25; 23-25 rain) from System 1 (Fig. 1) were processed by HP-SEC. System 2 comprised two Waters 1-125 Protein Pak columns (Waters Assoc., Milford, Mass.) in series (7.8 x 600mm) with an 1-125 guard column (3.9 x 50 mm). Mobile phase was 40% CH3CN in 0.1% T F A with a flow rate of 1 ml/min. Samples were processed in four batches, each batch consisting of c. 500 b r a i n - S O G equivalents. The column system was calibrated with the following molecular weight markers; carbonic anhydrase (29,000), parvalbumin (12,000),

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FIGURE 6. Activity of synthetic Lyd-PBAN in vivo. Synthetic Lyd-PBANwas dissolved in saline for bioassay in H. zea. The bioassay was performed as described. Doses ranged from 0.15 to 100 pmol Lyd-PBANinjected per female. Each bar represents the mean of five replicates + SE.

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aprotinin (6500), insulin chain B (3496), all from Sigma, and little gastrin (2126) and locust adipokinetic hormone-I (1159) from Peninsula Laboratories (Belmont, Calif.). Fractions (0.5 min) were collected, aliquoted as needed, and dried in the Speed-Vac. (3) Active fractions from System 2 (Fig. 2; fraction numbers 9 and 10; 17-18 min) were fractionated, in two batches, on System 3, consisting of a 2.1 x 220mm Aquapore RP-300 C 8 column (Applied Biosystems, San Jose, Calif.), and a linear gradient of 45 mM sodium phosphate, pH 6.0 in 10% CH3CN to 20 mM sodium phosphate, pH 6.0, in 60% CH3CN over 50 min with a flow rate of 0.2 ml/min. (4) Systems 4 and 5: active fractions (numbers 17-19; 15-16 min) from the C8 column (Fig. 3) were processed on a Delta Pak C,8 (3.9 x 150mm) column (Waters Assoc. Milford, Mass.). Flow rate was 0.78 ml/min with a linear gradient of 10% CH3CN in 0.1% TFA to 60% CH3CN in 0.1% TFA over 50 min (System 4). Individual peaks were collected and activity localized. Final purification and desalting were done on System 5 using a Vydac 4.6 x 150 mm C4 column (Vydac, Cotati, Calif.), 10% CH3CN in 0.1% TFA t o 60% CH3CN in 0.1% TFA, 20min, 0.5ml/min. Purified material was then sequenced.

Amino acid sequence determination Purified peptide was sequenced by the automated Edman method using an Applied Biosystems (Foster City, Calif.) Model 477A pulsed liquid-phase sequencer. The system had on-line HPLC analysis using an Applied Biosystems Model 120A phenylthiohydantoin (PTH) analyzer.

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Peptide synthesis Lyd-PBAN was synthesized on a Milligen/Biosearch 9600 solid phase peptide synthesizer (Millipore, Burlington, Mass.). Amino group protection was with 9fluorenylmethyloxycarbonyl (Fmoc), and side chain protection was as follows: Arg, pentamethylchroman-6sulphonyl (PMC); Asn and Glu, trimethoxybenzyl (Tmob); Asp, Glu, Ser, Thr, and Tyr, tert-butyl (tBu); Lys, tert-butyloxycarbonyl (Boc). Couplings were performed with diisopropylcarbodimide (DIC) in dimethylformamide (DMF) and methylene chloride (CH2C12) for 1 or 2 h, depending upon both the residues being coupled and the preceding residues. The manufacturer's protocols were used. After cleavage of the completed peptide from the resin with TFA/5%H20/5% triethylsilane (1 h, room temperature) the peptide was purified by HPLC (System 4). Identity of the synthetic peptide was confirmed by sequencing. RESULTS

Peptide purification We had previously shown that pheromonotropic activity could be recovered from L. dispar brain-SOG

834

E.P. MASLERet al.

complexes through reverse-phase, step-wise fractionation (Masler and Raina, 1993) and applied this property to the first isolation step. Activity eluted in fractions 24 and 25 on the Supelco C~8 system (Fig. 1). This elution profile was highly reproducible, enabling us to collect and pool the active fractions from all subsequent fractionations with System 1 (c. 100 tissue equivalents per fractionation) without the routine need to locate activity via the bioassay. Successive runs were made until 2000 brain-SOG equivalents had been collected. We found it most practical to collect the fractions from System 1 in two sets of tubes, concentrating each set on the SpeedVac prior to the next collection. This resulted in two sets of fractions of c. 1000 equivalents each. Corresponding fractions from each set were combined in preparation for System 2. Despite the good resolution of the Supelco Ct8 column, and the fact that pheromonotropic activity eluted prior to the bulk of the sample components (Fig. 1), fractions 24 and 25 contained large amounts of other peptides. Initially, our next step was to process these fractions on a second reverse phase system using a shallow acetonitrile gradient in TFA. However, pilot tests (not shown), revealed that resolution was not usefully increased over System 1, and a significant portion of the material in fractions 24 and 25 was of a larger molecular weight than the pheromonotropic material. This led to the selection of size exclusion chromatography as the next step. The relatively crude preparation of pooled fractions from System 1 was tested with the bioassay to verify the location of pheromonotropic activity in fractions 24 and 25, and pooled fractions were processed on the size-exclusion system, effecting a considerable clean-up of the sample (Fig. 2). Activity consistently eluted in two fractions between 17 and 18 min (bar; elution volume = 17 ml) corresponding to an estimated molecular weight of 3500. A pool of aliquots from each of the two active fractions was examined on a shallow gradient version of System 1 (i.e. C~8 column with CH3CN/TFA mobile phase), but little useful enhancement of separation was observed. The mobile phase was adjusted by using sodium phosphate buffered to pH 6.0 in place of the TFA, changing the protonation of the proteins. Acetonitrile was the eluting solvent. This mobile phase, in combination with a C8 column, effected a significant improvement in component separation over the acetonitrile-TFA system. Pheromonotropic activity (Fig. 3, bar) eluted at a more hydrophilic position (25% CH3CN ) than in System 1 (35% CH3CN). The use of sodium phosphate buffer salts was feasible because mobile phase volatility was not a concern at this point. The resolution obtained with System 3 allowed collection of fractions for final purification. Active fractions from System 3 were concentrated on the Speed-Vac and fractionated on the Delta-Pak C18 system (System 4). An isolated peak with pheromonotropic activity was collected at 24 rain, eluting at 35% CH3CN (Fig. 4). The processing of aliquots of fractions from systems 2, 3 and 4 for bioassay included drying followed by dissolution in assay saline.

Even with the use of polypropylene tubes coated with silicone, losses of activity became a serious problem. Inclusion of 5 #1 of Ringer's in each fraction tube greatly enhanced recovery, and was routinely employed in fraction collection from these three systems. Following detection of activity in fraction aliquots from System 4, the remaining pheromonotropic material was desalted on a Vydac C4, for a final purification (Fig. 5). The PBAN peak (Fig. 5, arrow) was then submitted for amino acid sequencing. Amino acid sequencing

An initial sample yielded the sequence: Leu-Ala-AspAsp-Met-Pro-Ala-Thr-Xxx-Ala-Asp-Gln-Glu-Val-TyrArg-Pro-Glu-Pro-Glu-Gln-Ile-Asp-Ser-Arg-Asn-LysTyr-Phe-Ser-Pro-Arg-Leu, with ambiguity at position 9. A second sample confirmed the initial sequence and identified methionine at position 9. We did not determine the nature of the C-terminal residue. However, based upon the data for Hez-PBAN, Bom-PBAN and Pss-PT (Raina et al., 1989; Kitamura et al., 1989; Matsumoto et al., 1992a), we synthesized Lyd-PBAN with an amidated C-terminus, calculated a molecular weight of 3881, and propose the sequence for LydPBAN indicated in Table 1. The synthetic, amidated peptide was purified using System 4, and its identity was confirmed by amino acid sequencing. Biological activity

The synthetic sequence was active in the H. zea PBAN bioassay, yielding a linear dose-response from 0.15 to 0.6pmol per female (Fig. 6). Doses between 0.6 and 5 pmol gave no further increase in response. Above 5pmol, the response appeared to increase but was variable. Activity was also detected in L. dispar using a bioassay now under development in our laboratory (see Discussion). DISCUSSION We have isolated and characterized a 33-amino acid peptide obtained from brain-SOG complexes of adult L. dispar. The synthetic peptide, termed Lyd-PBAN, is active in the H. zea pheromone production bioassay. The amino acid sequence of Lyd-PBAN is homologous with those of both Hez-PBAN (81.8%) and Bom-PBAN-I (66.7%) (Table 1). Five residue positions were unique to Lyd-PBAN (Ala2, Metg, Vall4, Gluls, Asn26) when compared with the sequences of the other two PBANs. Bom-PBAN also has five unique residue positions (Glu3, Gln16, Met=, Glu23, Arg27). In contrast, Hez-PBAN has one unique residue position (Gln~7) (Table 1). Lyd-PBAN and Hez-PBAN each have two methionine residues, while Bom-PBAN has three. Lyd-PBAN and Hez-PBAN have identical C-terminal heptapeptides and all three PBANs have the identical C-terminal hexapeptide. The significance of the C-terminal pentapeptide, Phe-Ser-Pro-Arg-Leu-NH2, has been established as the minimal sequence capable of eliciting a

GYPSY MOTH PBAN p h e r o m o n o t r o p i c response (Raina and Kempe, 1990; Kuniyoshi et al., 1991). It is also well documented that the C-terminal P h e - X x x - P r o - A r g - L e u - N H 2 sequence is associated with m y o t r o p i c activity ( N a c h m a n and Holman, 1991; F o n a g y et al., 1992; Kuniyoshi et al., 1992; Schools et al., 1992), embryonic diapause (Imai et al., 1991) and cuticular tanning ( M a t s u m o t o et al., 1990, 1992b). P h e r o m o n o t r o p i c activity is present in the b r a i n - S O G complexes o f larval and pupal L. dispar (Masler and Raina, 1993). It is possible that the ' p h e r o m o n o t r o p i n ' present in those pre-adult animals is associated with melanization. It is interesting to note that although neuropeptides with divergent physiological activities share the P h e - X x x - P r o - A r g - L e u - N H 2 C-terminal sequence and are considered to represent a peptide family (Kuniyoshi et al., 1992), those neuropeptides considered primarily as P B A N (Hez-, Bom-, and L y d - P B A N s ) maintain significant sequence h o m o l o g y through residues 1-28 whereas those peptides associated primarily with diapause or melanization have little or no sequence h o m o l o g y with the P B A N s outside o f the C-terminal pentapeptide (Table 1). In addition, within the span o f residues 1-27, there are 15 positions where residues are the same for the P B A N s o f all three species (consensus positions). O f the 12 positions where there is not a consensus (2, 3, 9, 14, 16-18, 21-23, 26, 27; Table 1), eight are between positions 15 and 28. Thus, the internal sequences appear to be the most variable. It is o f interest that the internal pentapeptide 15-19 from H e z - P B A N is an extremely potent p h e r o m o n o t r o p i n in the H. zea bioassay (Raina and Kempe, 1992). In contrast, the pentapeptide o f residues 15-19 from BomP B A N - I , which differs from H e z - P B A N at positions 16 and 17, is inactive in H . zea (Raina and Kempe, 1992). The variability o f the internal sequences o f P B A N and the differing biological activities suggest some species specificity (Raina and Kempe, 1992; Raina, 1993). L y d - P B A N clearly stimulates p h e r o m o n o t r o p i c activity in H . zea (Masler and Raina, 1993; Fig. 6, this paper). The distinct dose-response followed by a leveling-off and a second increase in response (Fig. 6) is similar to the H . zea response to synthetic H e z - P B A N (Raina et al., 1989). Synthetic L y d - P B A N was tested in an in vivo bioassay being developed in our laboratory (Thyagaraja et al., unpubl, observ.) utilizing adult female L. dispar. Decapitated females, which produce essentially no p h e r o m o n e (disparlure) respond to synthetic L y d - P B A N in a dose-dependent manner, producing 5.7 and 2.1 ng disparlure in response to 5 and 1 pmol, respectively, o f peptide. Decapitated females treated with saline only (no peptide) yield 0.2 ng disparlure. C o n t r o l levels for untreated females are typically a b o u t 7 ng disparlure. Thus, as little as 1 pmol o f L y d - P B A N stimulated a 30% response c o m p a r e d with controls. A detailed report on the development and specifics o f the bioassay will be published elsewhere. L y d - P B A N appears to belong to a superfamily o f neuropeptides ( K a w a n o et al., 1992), the members o f

835

which share a high degree o f h o m o l o g y at the Cterminus and can exhibit multiple biological activities. Investigations into potential multiple activities o f LydP B A N , and structure-function studies, are underway.

REFERENCES

Beli R. A., Owens C. D., Shapiro M. and Tardif R. (1981) Development of mass-rearing technology. In The Gypsy Moth: Research Toward Integrated Pest Management (Edited by Doane C. C. and McManus M. L.), pp. 597-633. USDA Technical Bulletin 1584. Bierl B. A. and Beroza M. (1970) Potent sex attractant of the gypsy moth: its isolation, identification and synthesis. Science 170, 87-89. Fonagy A., Schoofs L., Matsumoto S, DeLoof A. and Mitsui T. (1992) Functional cross-reactivities of some locustamyotropins and Bombyx pheromone biosynthesis activating neuropeptide. J. Insect Physiol. 38, 6514557. Giebultowicz J. M., Webb R. E., Raina A. K. and Ridgway R. L. (1992) Effects of temperature and age on daily changes in pheromone titer in laboratory-reared and wild gypsy moth (Lepidoptera: Lymantriidae). Environ. Ent. 21, 822-826. Hollander A. L. and Yin C.-M. (1985) Lack of humoral control in calling and pheromone release by brain, corpora cardiaca, corpora allata and ovaries of the female gypsy moth, Lymantria dispar (L.) J. Insect Physiol. 31, 159 163. Imai K., Konno T., Nakazawa Y., Komiya T., Isobe M., Koga K., Goto T., Yaginuma T., Sakakibara K., Hasegawa K. and Yamashita O. (1991) Isolation and structure of diapause hormone of the silkworm, Bombyx mori. Proc. Japan Acad. 67B, 98 101. Kawano T., Kataoka H., Nagasawa H., Isogai A. and Suzuki A. (1992) cDNA cloning and sequence determination of the pheromone biosynthesis activating neuropeptide of the silkworm, Bombyx mori. Biochem. biophys, res. Commun. 189, 221-226. Kitamura A., Nagasawa H., Kataoka H., Ando T. and Suzuki A. (1990) Amino acid sequence of pheromone biosynthesis activating neuropeptide-II (PBAN-II) of the silkmoth, Bombyx mori. Agrie. biol. Chem. 54, 2495-2497. Kitamura A., Nagasawa H., Kataoka H., Inoue T., Matsumoto S., Ando T. and Suzuki A. (1989) Amino acid sequence of pheromone biosynthesis activating neuropeptide (PBAN) of the silkworm, Bombyx mori. Biochem. biophys, res. Commun. 163, 520-526. Kuniyoshi H., Nagasawa H., Ando T., Suzuki A., Nachman R. J. and Holman G. M. (1992) Cross-reactivity between pheromone biosynthesis activating neuropeptide (PBAN) and myotropic pyrokinin insect peptides. Biosci. biotech. Biochem. 56, 167-168. Kuniyoshi H., Kitamura A., Nagasawa H., Chumar T., Shimazaki K., Ando T. and Suzuki A. (1991) Structure-activity relationship of pheromone biosynthesis activating neuropeptide (PBAN) from the silkmoth, Bombyx mori. In Peptide Chemistry 1990 (Edited by Shimarishi Y.), pp. 251-254. Protein Research Foundation, Osaka, Japan. Masler E. P. and Raina A. K. (1993) Pheromonotropic activity in the gypsy moth Lymantria dispar: evidence for a neuropeptide. J. comp. Physiol. B 163, 259-264. Masler E. P., Hagedorn H. H., Petzel D. H. and Borkovec A. B. (1983) Partial purification of egg development neurosecretory hormone with reverse-phase liquid chromatographic techniques. Life Sci. 34, 1925-1932. Masler E. P., Bell R. A., Thyagaraja B. S., Kelly T. J. and Borkovec A. B. (1991) Prothoracicotropic hormone-like activity in the embryonated eggs of gypsy moth, Lymantria dispar (L.). J. comp. Physiol. B 161, 37~,1. Matsumoto S., Fonagy A., Kurihara M., Uchiumi K., Nagamine T., Chijimatsu M. and Mitsui T. (1992a) Isolation and primary structure of a novel pheromonotropic neuropeptide structurally related to leucopyrokinin from the armyworm larvae, Pseudaletia separata. Biochem. biophys, res. Commun. 182, 534~539.

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Acknowledgements The authors wish to thank Drs R. A. Jurenka and R. J. Nachman for their critical readings of the manuscript.