Journal of Biotechnology and Sericology 71, 35-42 (2002)

Identification of an Imidazole Compound-Binding Protein from Diapausing Pharate First Instar Larvae of the Wild Silkmoth yamamai

Takayuki Shimizu1, Takahiro Shiotsuki2, Atsushi Seino2, Ying An1, Eiichi Kuwano3 and Koichi Suzuki1,*

1 Department of Agro-Bioscience, Faculty of Agriculture, Iwate University, Morioka 020-8550, Japan, 2 National Institute of Agrobiological Sciences, Tsukuba,Ibaraki 305-8634, Japan, and 3Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Fukuoka 812-8581, Japan

(Received September 28, 2001; Accepted December 12, 2001)

A series of 1-substituted and 1,5-disubstituted imidazoles have been shown to terminate diapause in pharate first instar larvae of the wild silkmoth, Antheraea yamamai, and the gypsy , Lymantria dispar. To understand the mode of action for the imidazole compounds, we analyzed imidazole compound (KK-42)-binding proteins by affinity chromatography using a synthetic resin-coupled KK-42 analog. Two binding proteins (KK-42BPs) of 40 and 45 kDa were isolated in soluble fractions prepared from diapausing pharate first instar larvae. The N-terminal amino acid sequence from the first 20 residues was determined for the 45 kDa protein. This 45 kDa protein appeared throughout the periods of pre-diapause and diapause, and it disappeared after the KK-42 application and long period of chilling. It was further demonstrated through specific positive staining with antiserum that the 45 kDa KK-42BP was localized in yolk cells. These results are the first report of a protein associated with artificial hatching in . Key words: Binding protein, imidazole compound, diapause termination, pharate first instar larva, Antheraea yamamai

INTRODUCTION pounds were effective as diapause breaker for this species (Suzuki et al., 1989; Kuwano et al., 1991; Nakamura et al., Insect diapause can sometimes be broken artificially 1998, 1999). KK-42 is also effective in diapausing pharate with chemical compounds. For example, development in first instar larvae of the gypsy moth, Lymantria dispar the silkworm, Bombyx mori, arrests in the late gastrular (Suzuki et al., 1993; Bell, 1996; Lee and Denlinger, 1996). stage of embryonic development, and this diapause can be This imidazole compound has been suggested to prevented or broken with hydrochloric acid. The mechani- function as both antijuvenile hormones (Akai and sm behind this popular artificial method for hatching, Mauchamp, 1989) and anti-ecdysteroids (Kadono-Okuda et however, has not yet been elucidated (Yamashita and al., 1987; Yamashita et al., 1987; Lee and Denlinger, Suzuki, 1991). Diapause of some cricket and grasshopper 1997). We suggested that KK-42 did not function as an anti- eggs can be broken by solutions of urea and organic JH and/or an anti-ecdysteroid and had unknown action(s) solvents (Hogan, 1962; Slifer, 1958). Embryonic diapause in the diapause termination of pharate first instar larvae in in the false melon beetle, Atrachya menetriesi, can be the wild silkmoth (Suzuki et al., 1991). Whatever functions terminated by dipping the eggs into mercuric chloride imidazole compounds exert in diapause termination, it is solution (Kurihara and Ando, 1969). Although many reasonable to speculate that there is KK-42-binding examples of these types are known, the mechanism of protein(s) that works on the target tissues. In this artificial hatchings is not yet understood. investigation, we prepared a resin-coupled imidazole Many anti juvenile hormone agents have been synthesiz- compound with sepharose, and by this affinity chromatogra- ed as new insect growth regulators. Among them, Kuwano phy we then isolated an imidazole compound-binding et al. (1985) have reported a large number of protein from the soluble fraction of diapausing pharate first 1,5-disubstituted imidazoles that induce clear precocious Instar larvae of A. yamamai. We will also discuss what metamorphosis of B. mori. We found that an imidazole clues the isolated protein may provide for understanding compound, 1-Benzyl-5-[(E)-2,6-dimethyl-l,5-heptadienyl] the mechanism of artificial hatching of this insect. imidazole (KK-42) successfully breaks diapause in pharate first Instar larvae of the wild silkmoth, Antheraea MATERIAL AND METHODS yamamai. We demonstrated that mass artificial hatching could be accomplished and that other imidazole com- Insects Larvae of the wild silkmoth, Antheraea yamamai, were *To whom correspondeces should be addressed . bred as described in our previous study (Suzuki et al., Fax: +81-19-621-6177. Tel: +81-19-621-6147. 1990). Eggs 2 to 3 days after oviposition were washed with Email: [email protected] chlorinated lime (0.5%) to eliminate egg glues. Under 36 Shimizu et alL incubation at 25 °C the eggs developed to the stage of larvae were kept at 250C until used, and 74.1% of those diapause initiation of pharate first instar larvae 10 days terminated diapause 4 days after the KK-42 application after oviposition. They were further incubated at 25°C until (Fig. 1C, D). being used for the experiments. To terminate diapause of pharate first instar larvae, intact diapausing eggs were Protein preparation exposed to 5 °C for 90 days and then transferred to 25 °C Naked pharate first instar larvae were stored at -80 °C (This feature of pharate first instar larvae is shown in Fig. until used, and about 200 larvae (1.0 g) were homogenized IA). After 7 days of transfer to 25 °C, 50% of larvae with 5 vol. of a sample buffer (50 mM sodium phosphate dissected from the eggs showed several signs of diapause buffer, pH 7.5, 20% glycerol, 1 mM EDTA, 1 mM PMSF, 1 termination such as the development of yellow color, mM benzamidine, 1 ,u g/ml pepstatin A, I u g/ml leupeptin melanization of integument stripes and legs, coloring and 1 mM DTT). One group of the homogenate was (reddish) of cervical shield, erection of bristles and centrifuged at 10,000g for 10 min at 0 °C, and this locomotion of larval body. After 10 days of transfer to 25 supernatant and the other group of the homogenate were °C , 92% of larvae dissected from the eggs showed these stored at -80'C until used. Only the other group of the indications of diapause termination (Fig. IB). Eight-day homogenate was centrifuged at 105,000g for 60 min at 4 °C old eggs kept at 25-C from oviposition were used for the , and this supernatant was subjected to affinity pre-diapause stage. chromatography. The other group of diapausing pharate first instar larvae Hemolymph of diapausing pharate first instar larvae was was applied using the imidazole compound KK-42 as collected through the ventral neck membrane with a described by Suzuki et al. (1990) and Nakamura et al. disposable micropipette (5 ,u 1) and immediately pooled in a (1998). The chorion of diapausing eggs was removed sample tube containing a few crystals of phenylthiourea. manually with fine forceps, and the naked pharate first Samples were centrifuged at 1000g for 10 min at 0 °C to instar larvae were held on ice at least 2 h and treated with remove hemocytes. The entire central nervous system, 0.1 ,u g/0.5 ,u 1 in acetone to the ventral side. The treated alimentary canal, alimentary content and integument of diapausing individuals were dissected and rinsed well with cold Bombyx saline (Narahashi, 1963) to reduce the contamination of hemolymph and hemocytes. These tissues were homogenized with the sample buffer followed by centrifugation at 10,000g for 10 min at 0 °C. The supernatants were stored at -80°C until used. The protein concentrations were determined with the BCA protein assay reagent kit (Pierce) using bovine serum albumin as a standard.

Syntheses of KK-42 and 5-[(E)-2,6-dimethyl-1,5- heptadienyl]imidazole-affinity resin 1-Benzyl-5-[(E)-2,6-dimethyl-1,5-heptadienyl] imidazole (KK-42) was synthesized by the method of Kuwano et al. (1985). The hydrochloride salt of KK-42 (KK-42-HCI) was obtained by treating a diethyl ether solution of KK-42 with 1 M hydrogen chloride in diethyl ether solution and then collecting the precipitate. 1-(3-Hydroxypropyl)-5-[(E)-2,5-dimethyl-1,7-heptadie- nyl] imidazole (HDHI) was prepared as follows: A mixture of 7.6 g of citral, 4.5 g of 3-amino-1-propanol and 9.0 g of anhydrous MgSO4 in 60 ml of dichloromethane was Fig. 1. Diapausing and diapause-terminated pharate first refluxed for 3 h. MgSO4 was filtered off, and the filtrate instar larvae of A. yamamai. Intact diapausing eggs were was concentrated under reduced pressure. To the residue exposed to 5 °C for 90 days and then transferred to 25 °C. Immediately after the transfer, pharate first instar larvae were dissolved in 60 ml of methanol were added 12 g of K2C03 removed from the eggs and incubated at 25-C. Photographs and 10.8 g of tosylmethylisocyanide, and the mixture was were taken on 0 day (A) and 10 days (B) after incubation. The refluxed for 3 h. After removal of the solvent, 150 ml of other group of naked diapausing pharate first instar larvae was applied with KK-42 and incubated at 25°C. These photographs water was added to the residue. The product was extracted were taken on 0 day (C) and 4 days (D) after incubation. with ethyl acetate, and the ethyl acetate solution was Imidazole Compound-Binding Protein of Antheraea yamamar 37 washed with brine and dried over Na2SO4.After removal of washed with EtOH, 50% EtOH, distilled water and finally the solvent, the residue was chromatographed on silica gel with 0.1 M Tris-HCl buffer, pH 7.6, to block the activated and eluted with ethyl acetate and ethyl acetate-isopropanol site completely. (3:1). Concentration of the ethyl acetate-isopropanol (3:1) eluate yielded 6.9 g (20.4%) of HDHI as an oil. 1H-NMR Affinity chromatography (CDC13)d : 1.62 (3H, s), 1.70 (3H, s), 1.88 (3H, d, J = 1 All chromatographic procedures were performed in a Hz), 1.5-2.4 (6H, overlapped), 3.0 (1H, br), 3.55 (2H, t, J = cold room at 4°C. About 3.0 ml resin poured in a glass 7 Hz), 4.08 (2H, t, J = 7 Hz), 4.9-5.2 (1H, m), 5.95 (1H, s), column (15 mm X 50 mm) (Econo-Column, Bio-Rad) was 6.96 (1H, s), 7.48 (1H, s). equilibrated with 20 vol. of an equilibration buffer (50 mM For preparation of 1- (3-Aminopropyl) - 5 - [(E)-2,5- sodium phosphate buffer, pH 7.5, 20% glycerol and 1 mM dimethyl-l, 7-heptadienyl]imidazole (ADHI), 2.0 g of DTT). The sample (5 ml) was applied onto the gel-bed, and methanesulfonyl chloride was added at 0-5 °C to a solution the column was rotated gently overnight. Then the bed was of 4.0 g of HDHI and 2.4 g of triethylamine in 20 ml of washed with 10 vol. of the equilibration buffer to remove dichloromethane. After stirring for 1 h at room temperatu- non-binding proteins. Additional non-specific binding re, the reaction mixture was poured into 20 ml of water. proteins were washed out with 10 vol, of 1 M NaCI in the The dichloromethane solution was washed with water and equilibration buffer followed by 10 vol. of 25% ethylene brine, dried over Na2SO4, and concentrated. The residue glycol in the same buffer. Elution of KK-42 binding was dissolved in 100 ml of dimethylformamide, and a proteins (KK-42BPs) was accomplished competitively with solution of 1.4 g of NaN3 in 15 ml of water was added to 10 vol. of 0.01% KK-42 • HCl in the equilibration buffer. this solution. The mixture was heated at 80-90 °C for 4 h Eluents containing KK-42BPs were collected and concen- and then cooled to room temperature. One hundred ml of trated by ultrafiltration using the Ultrafree-15 centrifugal water was added to the mixture, and the product was filter unit (MWCO 10,000) (Millipore). Concentrated extracted with ether. The ether fraction was washed with proteins were analyzed on tricine SDS-PAGE. brine, dried over Na2SO4and concentrated. To the residue dissolved in 20 ml of dry tetrahydrofuran was added 1.2 g Tricine SDS-PAGE of LiA1H4at 0-5°C. The mixture was stirred for 2 h at room For electrophoresis, protein samples were mixed with temperature and quenched by adding ether and water. The 4% SDS and 2% ~9-mercaptoethanol, denatured at 100°C product was extracted three times with ether. The for 5 min and run on a tricine SDS-PAGE (Schagger and combined organic extracts were washed with brine and von Jagow, 1987) using 10% acrylamide gel. After dried over Na2SO4. Concentration of the solvent yielded electrophoresis, these gels were stained with Coomassie 0.89 g (22.3%) of ADHI as an oil. 1H-NMR (CDCl3) d: brilliant blue R-250 (CBB) or by silver staining (Poehling 1.62 (3H, s), 1.69 (3H, s), 1.87 (3H, s), 1.7-2.0 (4H, and Neuhoff, 1981) and used in the Western blot analysis. overlapped), 2.1-2.3 (4H, m), 2.69 (2H, t, J = 7 Hz), 4.00 (2H, t, J = 7 Hz), 5.1-5.2 (1H, m), 5.93 (1H, s), 6.97 (1H, Amino acid sequencing s), 7.43 (1H, s). The fraction containing KK-42BPs was examined on ADHI-Sepharose was prepared by coupling ADHI to tricine SDS-PAGE as described above, and the proteins CNBr-activated Sepharose. CNBr-activated Sepharose 4B were electroblotted to PVDF membranes in 10 mM CAPS, was purchased from Pharmacia (Uppsala, Sweden). The pH 11.0 in 10% methanol. The membranes were stained resin was suspended and washed with 1 mM HCI, then with Ponceau S (Aldrich Chemical Company Inc.). The washed with stepwise increasing of EtOH (25%, 50%, stained bands were excised, and partial N-terminal amino 100%) in 1 mM HCI. An EtOH solution of ADHI (0.86 acid sequences of the protein bands were determined by mg/ml) was added to an equal amount of the gel automated Edman degradation with a protein sequencer suspension (100 mg/ml) in EtOH, then the mixture was (PPSQ-21, Shimadzu). shaken gently for 6 h at room temperature. The gel was washed five times with the same amount of EtOH by Antibody production and Western blotting decantation. Aliquots of the prepared gel and original Polyclonal antiserum was raised in rabbits against a CNBr-activated gel were subjected to elementary analyses. synthetic peptide corresponding to the N-terminal 13 The concentration of the activated sites was calculated amino acids of a 45 kDa KK-42BP. This synthetic peptide from the nitrogen ratio in the original CNBr-activated gel was conjugated to bovine serum albumin (BSA) as a (one nitrogen per site) as 1.82 mmol/g gel. The ligand carrier protein (Peptide Institute Inc.). About 500 u g of (ADHI) concentration was estimated from the increase of this conjugate in phosphate buffered saline (PBS) the nitrogen ratio in the obtained gel (four nitogen per site) emulsified with Freund's complete adjuvant (Nacali Tesque as 0.24 mmol/g gel. The resulting affinity gel (Fig. 2A) was Inc.) was injected into Japanese white rabbits. 38 Shimizu et al.

For Western blotting, protein samples were subjected to A tricine SDS-PAGE and electroblotted to PVDF membranes. The blotted membranes were subsequently incubated at 4 °C overnight with TBS-T (20 mM Tris-HC1 buffer, pH 7.5, 0.5 M NaCI and 0.05% Tween-20) containing 5% skim milk. The membranes were incubated for 1 h with the primary antibody in TBS-T. After washing, the membranes were further incubated for I h with goat anti-rabbit IgG B C conjugated with horseradish peroxidase and washed 2 times with TBS-T. The immune complex formation was detected by enhanced chemiluminescence (ECLTM,Amer- sham Pharmacia Biotech).

Immunohistochemistry Whole bodies of diapausing pharate first instar larvae, which were cut open along the dorsal vessel, were fixed in Bouin's fixative. The paraffin sections were processed for immunostaining with the avidin-biotin peroxidase complex (ABC) methods as previously described (An et al., 1998).

Fig. 2. Procedure for isolation of imidazole compound- RESULTS binding proteins from diapausing pharate first instar larvae of A. yamamai. (A) Structure of synthetic resin-coupled KK-42 Identification of KK-42 binding proteins analog used for affinity chromatography. (B) Tricine SDS-PAGE We first designed a purification procedure by affinity analysis of KK-42 binding proteins (KK-42BPs) isolated by chromatography using a KK-42 analog, ADHI as a ligand affinity chromatography. Two protein bands (40 and 45 kDa) stained by silver staining were observed in diapausing pharate (Fig. 2A). Soluble fractions prepared from diapausing first instar larvae (lane 1) and those in 2 days of KK-42 pharate first instar larvae were applied to this affinity resin. application (lane 2). Each sample contained 50 larvae- Binding proteins eluted by the addition of KK-42-HCl equivalent. (C) Western blot analysis of a 45 kDa KK-42BP. Purified binding protein (lane 1) and whole body sample (lane were analyzed on tricine SDS-PAGE followed by silver 2) prepared from diapausing pharate first instar larvae were staining. Two binding proteins of 40 and 45 kDa were subjected to tricine SDS-PAGE followed by Western blotting. obtained as major bands (Fig. 2B). The same chromatogra- The antiserum against a 45 kDa KK-42BP recognized a specific band in Western blotting for both preparations. Purified protein phic methods and SDS-PAGE showed that electrophoretic and whole body sample contained 50 larvae-equivalent and concentration of the two binding proteins isolated from the about 20 tcg of protein, respectively. soluble fraction in diapausing pharate first instar larvae declines 2 days after the application of KK-42 (Fig. 2B). Changes of electrophoretic pattern in 45 kDa KK- Thus we recognized that there were KK-42-binding 42BP proteins (KK-42BPs). Although our preliminary data To investigate the effects of KK-42 on the 45 kDa KK- suggested that the 40 kDa protein resulted in a partially 42BP, we carried out both CBB-stained tricine SDS-PAGE limited degradation of the protein of high molecular mass, and Western blotting on whole body samples prepared we mostly examined the 45 kDa protein in this study. The throughout the periods of pre-diapause, diapause and post- N-terminal amino acid sequence for the first 20 residues of diapause. As shown in Fig. 3A, major protein bands were a KK-42BP (45 kDa) was determined: D-L-E-L-T-N-N-Q- observed with molecular masses greater than 90 kDa and N-T-Q-V-A-T-T-E-D-I-K-K. According to a data base around 35 kDa, irrespective of stages. These protein bands search (BLAST), this N-terminal sequence has no changed after diapause initiation and 3 days after the KK- homology with known proteins. We also prepared an 42 application. On the other hand, Fig. 3B showed that the antiserum against the N-terminal 13 amino acids of a 45 antiserum reacted with the preparations of pre-diapause kDa KK-42BP and used it for Western blotting. As shown and diapause periods, but barely reacts with the preparation in Fig. 2C, the antiserum reacted with the isolated binding of 3 days after the KK-42 application. Only 3.7% of protein and whole body sample prepared from diapausing pharate first instar larvae terminated diapause 3 days after pharate first instar larvae. the KK-42 application. The effects of chilling on 45 kDa KK-42BP were also examined in pharate first instar larvae extracted from the eggs chilled for 30, 60 or 90 days and then incubated at 25 Imidazole Compound-Binding Protein of Antheraea yamamai 39

A B

c D

Fig. 3. Changes of electrophoretic patterns in tricine SDS-PAGE and Western blot analysis throughout the periods of pre-diapause, diapause and post-diapause. (A) and (C), Electrophoretic pattern of whole body samples prepared from various stages and stained by Coomassie Blue. (B) and (D), Western blotting of the 45 kDa KK-42BP from the same gel. In (A) and (B), lane 1, pre-diapause (8 days after oviposition); lane 2, diapausing (14 days after oviposition); lane 3-5, post- diapause (1 to 3 days after KK-42 application to naked pharate first instar larvae dissected from diapausing eggs incubated at 25°C for about one month). In (C) and (D), diapausing eggs were chilled at 5°C for 30, 60 or 90 days from 40 days after oviposition. After a 90-day chilling, the eggs were transferred to 25°C, and then naked pharate first instar larvae were further incubated for 1, 2, 4, 6, 8 or 10 days. Each sample contained about 20tcg of protein. °C . As shown in Fig. 3C, the major protein bands did not results indicated that the disappearance of 45 kDa KK- change during chilling but clearly changed 6 days after the 42BP occurred after the KK-42 application and the transfer transfer to 25°C, and then they disappeared 8 and 10 days to 25°C post-chilling treatment. after the transfer. Fig. 3D showed that the antiserum reacted with all preparations during chilling but the Localization of 45 kDa KK-42BP reaction became weak 4 days after the transfer to 25 °C. To identify the tissue containing the 45 kDa KK-42BP, The positive bands disappeared 6, 8 and 10 days after the we carried out Western blot analysis using hemolymph, the transfer to 25°C. Six days after the transfer to 25°C, 11% of entire central nervous system, alimentary canal, its contents pharate first instar larvae terminated diapause. These and integument prepared from diapausing individuals. As 40 Shimizu et alL

shown in Fig. 4A, the antiserum reacted strongly with the mode of bioactive peptide-like material (Suzuki et al., in 45 kDa protein of gut contents and whole body, and it preparation). These novel sequences and secretory organs reacted weakly with about 90 kDa protein of the central will be reported in the near future. In the present study, in nervous system, alimentary canal and integument. Additio- order to understand the molecular mechanism of how the nally, we immunohistochemically investigated the foregut- imidazole compound causes the diapause termination, we midgut boundary, midgut and its contents (yolk cells) in designed and prepared an imidazole compound coupled to diapausing pharate first instar larvae. The results sepharose and identified a 45 kDa KK-42BP from the demonstrated that only the contents, yolk cells, were soluble fraction in diapausing pharate first instar larvae, strongly stained (Fig. 4B). using affinity chromatography. The antiserum against the 45 kDa KK-42BP also recognized a specific band in diapausing pharate first instar larvae. This is the first DISCUSSION demonstration of a protein responsible for artificial A diapause in the pharate first-instar larvae of A. hatching in insects. yamamai has been proposed to be controlled by the Throughout the entire period of pre-diapause, diapause neurohumoral production of a repressive factor and a and post-diapause, Furusawa et al. (1989, 1993) analyzed maturation factor, and the diapause termination is caused vitellin polypeptides of a major yolk protein in the wild artificially by the imidazole compound (Suzuki et al., silkmoth eggs using SDS-PAGE. The pattern of vitellin 1990). A maturation factor, which may be secreted from degradation and protease activity shows a stage-dependent the 2nd to 5th abdominal segments, is a peptide-like feature during this embryonic development. Changes in the material (Naya et al., 1994). A repressive factor which may electrophoretic pattern of major protein bands (molecular be secreted from the mesothorax, is also really short and masses of greater than 90 kDa and about 35 kDa) in our

Fig. 4. Localization of the 45 kDa KK-42BP in diapausing pharate first instar larvae of A. yamamai. (A) Western blotting for hemolymph (lane 1), entire central nervous system (lane 2), alimentary canal (lane 3), its contents (lane 4), integument (lane 5) and whole body (lane 6). Each sample contained about 20µg of protein. (B) Immunohistochemical observation of diapausing individual gut with the antiserum against 45 kDa KK-42BP. The control was stained with antiserum preabsorbed with antigen (B-1); Specific positive staining (brown color indicated by the arrows) was observed in yolk cells (B-2). F/M indicates the forgut-midgut boundary. fe, forgut epithelium; me, midgut epithelium; Y, yolk cells; Scale bars = 100 um. Imidazole Compound-Binding Protein of Antheraea yamamai 41 results, may represent partially a limited degradation of show that this signal protein may be a novel protein in vitellin. However, although changing patterns of these insects, although the relationship between two proteins is polypeptides are useful for monitoring embryonic develop- not clear. Thus we suggest that further analyses of ment, it is difficult for us to get any information concerning nucleotide and amino acid sequences of the 45 kDa KK- regulatory mechanism from these analyses. As a first step 42BP, and in vitro correlation between imidazole to get a prospective molecule in the regulatory system compound and this protein may provide significant resulting in diapause termination, we found a probe information about the control mechanism of artifitial protein, such as a 45 kDa KK-42BP. hatching and diapause phase. In many insects, diapause duration is terminated by cold acclimation over a long period. More recently Gray et al. ACKNOWLEDGMENTS (2001) proposed a model of diapause where the developmental phase is controlled by two simultaneous This study was supported in part by a grant for Scientific temperature-dependent processes. Yet there is a very little Research (A) (12356002) from Japan Society for the information available about cold-inducible molecules that Promotion of Science and by the Research for the Future load to diapause termination. Some regulatory molecules Program from Japan Society for the Promotion of Science have been suggested to explain the low temperature- (JSPS-RFTF 99L01203). sensitive mechanism involved in diapause termination. In the work on B, mori, cold-inducible genes have been REFERENCES detected that encode sorbitol dehydrogenase and a novel member of the BAG protein family (Niimi et al., 1993; Akai, H., and Mauchamp, B. (1989) Suppressive effects of an Moribe et al., 2001). In fresh fly, Sarcophaga crassipalpis, imidazole derivative, KK-42 on JH levels in hemolymph of Bombyx larvae. J. Seric. Sci. Jpn. 58, 73-74. the isolation of pupal diapause-regulated clones encoding An, Y., Nakajima, T., and Suzuki, K. (1998) Immunohisto- stress proteins may provide valuable tools for under- chemical demonstration of mammalian- and FMRFamide- standing the molecular mechanism of cold acclimation like peptides in the gut innervation and endocrine cells of the (Flannagan et al., 1998; Yocum et al., 1998). A specific wild silkmoth, Antheraea yamamai (: Saturnii- dae) during diapause and post-diapause of pharate first-instar peptide produced during adult diapause of the leaf beetle, larvae. Eur: J. Entomol. 95, 185-196. Gastrophysa atrocyanea, also may enable us to access the Bell, R.A. 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The diapause proteins during the embryonic and diapause development of termination caused by KK-42 occurs faster than the the Japanese silkworm, Antheraea yamamai. J. Seric. Sci. Jpn. 58, 179-185. termination caused by a low temperature event. Disappea- Furusawa, T., Narutaki, A., Mitsuda, K., and Teramoto, N. rance of 45 kDa KK-42BP parallels the diapause (1993) Stage-dependent limited degradation of vitellin and its termination. In addition, the 45 kDa KK-42BP was localization in the Japanese oak silkworm, Antheraea localized inside the gut containing yolk cells. These results yamamai, during embryonic development. Comp. Biochem. support a previous model: chilling and the imidazole Physiol. 104B, 787-794. Gray, DR., Ravlin, F.W., and Braine, J.A. (2001) Diapause in compound may act via the same temperature-sensitive the gypsy moth: a model of inhibition and development. J. receptor localized in the thorax (Suzuki et al., 1994). 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