Insect Biochemistry and Molecular Biology 30 (2000) 847–854 www.elsevier.com/locate/ibmb Juvenile hormone-mediated termination of larval diapause in the bamboo borer, Omphisa fuscidentalis Tippawan Singtripop a, Somsak Wanichacheewa a, Sho Sakurai b,* a Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand b Department of Biology, Faculty of Science, Kanazawa University, Kanazawa 920-1192, Japan Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000 Abstract Larvae of the bamboo borer, Omphisa fuscidentalis are in diapause for more than nine months (Singtripop, T., Wanichaneewa, S., Tsuzuki, S., Sakurai, S. 1999. Larval growth and diapause in a tropical moth, Omphisa fuscidentalis Hampson. Zool. Sci. 16, 725–733). To examine the endocrine mechanisms underlying this larval diapause, we assayed the responsiveness of the diapausing larvae to 20–hydroxyecdysone (20E) and a juvenile hormone analogue (JHA: S–methoprene). 20E injection caused the larvae to halt movement, followed by deposition of a pupal cuticle. Topical application of JHA induced pupation in a dose-dependent manner. JHA also induced pupation of the larvae whose brains were removed before JHA application. In those larvae, the prothoracic glands became active and competent to respond to brain extracts within seven days after JHA treatment, and the hemolymph ecdysteroid concentration began to increase 12 days after JHA application. These results indicate that JHA stimulates the prothoracic glands of diapausing Omphisa larvae, terminating larval diapause, in contrast with previous findings that JH inhibits the brain–prothoracic gland axis and thus maintains the larval diapause. Current results therefore suggest a novel regulatory mechanism for larval diapause in this species. 2000 Elsevier Science Ltd. All rights reserved. Keywords: Methoprene; Prothoracic gland; 20–hydroxyecdysone; Ecdysteroid titer; Prothoracicotropic hormone 1. Introduction diapause and remain inside the internode of bamboo culm until the following June when they pupate. Bam- Diapause is a strategy to survive seasons with boo borer larvae are thus in diapause for nine months, environmental conditions that are inadequate for sustain- from September until the following June (Singtripop et ing continuous development or maintenance of the al., 1999). organism (Denlinger, 1985). In the tropics, diapause may The availability of food may be profoundly influenced occur in response to a period of drought which reduces by seasonal rhythms (Denlinger, 1986). Rains stimulate the food supply (Denlinger, 1986; Tauber et al., 1986). an increase in plant growth, which provides a wealth of The bamboo borer, Omphisa fuscidentalis, is a univol- new food resources for many phytophagous insects. The tine lepidopteran that experiences an annual severe dry long diapause is, therefore, important in maintaining season in Northern Thailand, Laos and Myanmar. In synchrony between the insect life cycle and the phe- Chiang Mai Province, Northern Thailand, adults appear nology of the host plants in the tropics. Bamboo pro- in August, in mid wet season, and lay egg clusters on duces new shoots in the wet season, and the shoots newly grown bamboo shoots. Newly hatched larvae become hard by the end of the wet season. Therefore enter the internode to feed on the inner pulp. After they the long period of larval diapause in Omphisa appears to complete larval growth in September, the larvae enter be well adapted to the recurring, annual dry–wet seasons (Singtripop et al., 1999). Diapause in the larval or pupal stage is usually main- * Corresponding author. Tel.: +81-76-264-5713; fax: +81-76-264- 5977. tained by a decrease in the hemolymph ecdysteroid titer E-mail address: [email protected] (S. (Denlinger, 1985), due to a decrease in the biosynthetic Sakurai). activity of the prothoracic gland, which produces 3– 0965-1748/00/$ - see front matter 2000 Elsevier Science Ltd. All rights reserved. PII: S0965-1748(00)00057-6 848 T. Singtripop et al. / Insect Biochemistry and Molecular Biology 30 (2000) 847–854 dehydroecdysone and/or ecdysone. Such a decrease is genized in 500 µl Grace’s insect culture medium (Life caused by depletion of prothoracicotropic hormone Technologies, Gland Island, NY) and heated in boiling (PTTH), a neuropeptide which is produced by two pairs water for 3 min. The solution was centrifuged at 10,000 of neurosecretory cells in the brain and which stimulates g for 10 min, and the resulting supernatant was kept at the prothoracic glands. In larval diapause, a high juvenile Ϫ35°C. The brain extract was diluted with Grace’s hormone (JH) titer in the hemolymph is reported to be medium to a concentration of one brain equivalent in 25 involved in suppression of the brain–prothoracic glands µl medium, for use in incubations of prothoracic glands. axis, preventing the release of ecdysteroids for larval growth and pupation (Denlinger, 1985). In fact, removal 2.4. Measurement of hemolymph ecdysteroid of CA from diapausing larvae causes a decrease in JH concentration concentration, which induces an increase in hemolymph ecdysteroid, thus terminating diapause (Yagi and Hemolymph (30 µl) was combined with 270 µl meth- Fukaya, 1974; Yin and Chippendale, 1979). anol and centrifuged at 10,000 g for 5 min. The super- During the long larval diapause in O. fuscidentalis, natant was transferred to a small test tube and dried the hemolymph ecdysteroid concentration is low under reduced pressure at room temperature. The residue (Singtripop et al., 1999). This indicates that JH might be was dissolved in water and an aliquot of the aqueous involved in maintaining the larval diapause of the bam- solution was subjected to ecdysteroid radioimmunoassay boo borer, as in other lepidopteran larvae (Yin and Chip- (RIA) (Sakurai et al., 1998). The cross-reactivity of the pendale, 1973). Application of JH analogue (JHA), how- antibody to ecdysone and 20E was 1:5 (Yokoyama et ever, terminated the larval diapause. In the present study, al., 1996). we report that in O. fuscidentalis, JH is not involved in maintenance of the larval diapause, but rather stimulates 2.5. In vitro incubation of prothoracic glands the prothoracic glands of the diapausing larvae. Prothoracic glands were individually incubated in 25 µl Grace’s insect culture medium, pH 6.5, adjusted with 2. Materials and methods 1 N NaOH, at 25°C for 6 h. After incubation, the amount of ecdysteroid in the medium was determined by RIA. 2.1. Animals Bamboo borer larvae were collected from bamboo, 3. Results Dendrocalamus membranaceus, in a forest in Amphur Maewang, Chiang Mai Province, Thailand and kept in 3.1. Response of diapausing larvae to 20– plastic containers (12×14×8 cm) on wet paper towels at hydroxyecdysone 25°C in continuous dark (Singtripop et al., 1999). Larvae used in the present experiments were collected from Larvae were injected with various doses of 20E and November through to February. observed for six weeks thereafter for pupal cuticle for- mation (Table 1). Larvae injected with 1–4 µg 20E 2.2. Hormones Table 1 S–methoprene (Ͼ95% stereochemically pure; SDS Response of diapausing larvae to 20-hydroxyecdysone Biotech, Tokyo) was dissolved in acetone at a concen- tration of 5 mg/ml and kept at Ϫ35°C as a stock solution. Dose No. of No. of Mean S.D. Range b An aliquot of the stock solution was diluted to an appro- (µg) larvae used larvae that day (days) responded a priate concentration with acetone, and a 5 µl aliquot was topically applied to the dorsal surface of each larva using 4 15 14 8.1 1.4 7–11 a50µl micro-syringe. Ecdysone and 20–hydroxyecdy- 2 15 15 11.6 4.4 7–21 sone (20E) (Sigma, St. Louis, MO) was dissolved in dis- 1 15 13 11.6 6.5 7–32 tilled water at 1 mg/ml and stored at Ϫ20°C until the 0.5 15 3 10 0 10 0.25 15 6 21 8.4 10–33 used. The 20E stock solution was diluted with distilled 0.1 15 0 water, and a 5 µl aliquot was injected into each larva 0.05 15 0 through the first proleg. 0c 15 0 a 2.3. Preparation of brain extract Larvae injected with 20E did not shed the old cuticle but produced a tanned pupal cuticle. b Mean day was calculated only for the larvae that produced A crude extract of brains was used as a PTTH sample. pupal cuticle. One hundred brains from diapausing larvae were homo- c Water (5 µl) was injected as a control. T. Singtripop et al. / Insect Biochemistry and Molecular Biology 30 (2000) 847–854 849 actively moved on the day of injection and also on the As shown in Fig. 1C, larvae occasionally shed the old following day if touched, but became inactive two days cuticle and formed complete pupae. after the injection. They produced frosted frass and a partially ruptured hindgut was occasionally visible. After 3.3. Involvement of the brain in the termination of becoming inactive, the larvae produced a tanned pupal diapause by JHA cuticle beneath the larval cuticle but did not shed the µ old larval cuticle. At higher doses (1–4 g), most larvae In order to determine whether JHA stimulated the deposited a pupal cuticle, but the day of pupal cuticle brain to release PTTH in the diapausing larvae, brains formation ranged from 7 to 32 days. The first day of were removed from larvae 1, 4, 7 or 10 days after treat- pupal cuticle formation was the same with doses of 1, ment with 1 µg JHA. As shown in Fig. 2A, the day of µ 2 and 4 g, but the last day was delayed in proportion pupation was not altered by the day of brain removal, to the dose injected.