Journal of Insect Biotechnology and Sericology 71, 35-42 (2002) Identification of an Imidazole Compound-Binding Protein from Diapausing Pharate First Instar Larvae of the Wild Silkmoth Antheraea 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 moth, 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 insects. 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.