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Juvenile hormone-mediated termination of larval diapause in the
bamboo borer, Omphisa fuscidentalis
Tippawan Singtripop
a, Somsak Wanichacheewa
a, Sho Sakurai
b,*aDepartment of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand bDepartment 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 is a strategy to survive seasons with environmental conditions that are inadequate for sustain-ing continuous development or maintenance of the organism (Denlinger, 1985). In the tropics, diapause may occur in response to a period of drought which reduces the food supply (Denlinger, 1986; Tauber et al., 1986). The bamboo borer, Omphisa fuscidentalis, is a univol-tine lepidopteran that experiences an annual severe dry season in Northern Thailand, Laos and Myanmar. In Chiang Mai Province, Northern Thailand, adults appear in August, in mid wet season, and lay egg clusters on newly grown bamboo shoots. Newly hatched larvae enter the internode to feed on the inner pulp. After they complete larval growth in September, the larvae enter
* Corresponding author. Tel.:+81-76-264-5713; fax:+ 81-76-264-5977.
E-mail address: [email protected] (S. Sakurai).
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diapause and remain inside the internode of bamboo culm until the following June when they pupate. Bam-boo borer larvae are thus in diapause for nine months, from September until the following June (Singtripop et al., 1999).
The availability of food may be profoundly influenced by seasonal rhythms (Denlinger, 1986). Rains stimulate an increase in plant growth, which provides a wealth of new food resources for many phytophagous insects. The long diapause is, therefore, important in maintaining synchrony between the insect life cycle and the phe-nology of the host plants in the tropics. Bamboo pro-duces new shoots in the wet season, and the shoots become hard by the end of the wet season. Therefore the long period of larval diapause in Omphisa appears to be well adapted to the recurring, annual dry–wet seasons (Singtripop et al., 1999).
dehydroecdysone and/or ecdysone. Such a decrease is caused by depletion of prothoracicotropic hormone (PTTH), a neuropeptide which is produced by two pairs of neurosecretory cells in the brain and which stimulates the prothoracic glands. In larval diapause, a high juvenile hormone (JH) titer in the hemolymph is reported to be involved in suppression of the brain–prothoracic glands axis, preventing the release of ecdysteroids for larval growth and pupation (Denlinger, 1985). In fact, removal of CA from diapausing larvae causes a decrease in JH concentration, which induces an increase in hemolymph ecdysteroid, thus terminating diapause (Yagi and Fukaya, 1974; Yin and Chippendale, 1979).
During the long larval diapause in O. fuscidentalis, the hemolymph ecdysteroid concentration is low (Singtripop et al., 1999). This indicates that JH might be involved in maintaining the larval diapause of the bam-boo borer, as in other lepidopteran larvae (Yin and Chip-pendale, 1973). Application of JH analogue (JHA), how-ever, terminated the larval diapause. In the present study, we report that in O. fuscidentalis, JH is not involved in maintenance of the larval diapause, but rather stimulates the prothoracic glands of the diapausing larvae.
2. Materials and methods
2.1. Animals
Bamboo borer larvae were collected from bamboo,
Dendrocalamus membranaceus, in a forest in Amphur
Maewang, Chiang Mai Province, Thailand and kept in plastic containers (12×14×8 cm) on wet paper towels at 25°C in continuous dark (Singtripop et al., 1999). Larvae used in the present experiments were collected from November through to February.
2.2. Hormones
S–methoprene (.95% stereochemically pure; SDS Biotech, Tokyo) was dissolved in acetone at a concen-tration of 5 mg/ml and kept at235°C as a stock solution. An aliquot of the stock solution was diluted to an appro-priate concentration with acetone, and a 5µl aliquot was topically applied to the dorsal surface of each larva using a 50 µl micro-syringe. Ecdysone and 20–hydroxyecdy-sone (20E) (Sigma, St. Louis, MO) was dissolved in dis-tilled water at 1 mg/ml and stored at 220°C until the used. The 20E stock solution was diluted with distilled water, and a 5 µl aliquot was injected into each larva through the first proleg.
2.3. Preparation of brain extract
A crude extract of brains was used as a PTTH sample. One hundred brains from diapausing larvae were
homo-genized in 500 µl Grace’s insect culture medium (Life Technologies, Gland Island, NY) and heated in boiling water for 3 min. The solution was centrifuged at 10,000
g for 10 min, and the resulting supernatant was kept at
235°C. The brain extract was diluted with Grace’s medium to a concentration of one brain equivalent in 25 µl medium, for use in incubations of prothoracic glands.
2.4. Measurement of hemolymph ecdysteroid concentration
Hemolymph (30µl) was combined with 270µl meth-anol and centrifuged at 10,000 g for 5 min. The super-natant was transferred to a small test tube and dried under reduced pressure at room temperature. The residue was dissolved in water and an aliquot of the aqueous solution was subjected to ecdysteroid radioimmunoassay (RIA) (Sakurai et al., 1998). The cross-reactivity of the antibody to ecdysone and 20E was 1:5 (Yokoyama et al., 1996).
2.5. In vitro incubation of prothoracic glands
Prothoracic glands were individually incubated in 25 µl Grace’s insect culture medium, pH 6.5, adjusted with 1 N NaOH, at 25°C for 6 h. After incubation, the amount of ecdysteroid in the medium was determined by RIA.
3. Results
3.1. Response of diapausing larvae to 20– hydroxyecdysone
Larvae were injected with various doses of 20E and observed for six weeks thereafter for pupal cuticle for-mation (Table 1). Larvae injected with 1–4 µg 20E
Table 1
Response of diapausing larvae to 20-hydroxyecdysone
Dose No. of No. of Mean S.D. Range
a 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
pupal cuticle.
actively moved on the day of injection and also on the following day if touched, but became inactive two days after the injection. They produced frosted frass and a partially ruptured hindgut was occasionally visible. After becoming inactive, the larvae produced a tanned pupal cuticle beneath the larval cuticle but did not shed the old larval cuticle. At higher doses (1–4µg), most larvae deposited a pupal cuticle, but the day of pupal cuticle formation ranged from 7 to 32 days. The first day of pupal cuticle formation was the same with doses of 1, 2 and 4 µg, but the last day was delayed in proportion to the dose injected. The mean day of pupal cuticle for-mation with 4µg was significantly earlier than that with 2 or 1µg (ANOVA, P=0.026), while there was no stat-istical difference between 1 and 2µg (P=0.387). Effects of 20E were less at doses of 0.5 and 0.25 µg. These results showed that the diapausing larvae were com-petent to respond to 20E and that the critical dose to induce a response fell between 0.5 and 1 µg/larva. It should be noted that 20E injection did not induce a stationary molt nor produce larval–pupal intermediates.
3.2. Termination of larval diapause with JHA
When the diapausing larvae were topically treated with 1µg JHA, they turned brown with a hard and pig-mented cuticle, which indicated pupation of these larvae (Fig. 1A). When 0.1 µg JHA was applied, the larvae became inactive prior to formation of the brown cuticle. The body color then changed from creamy to light yel-low, and those larvae were designated as G1. On the following day, the dorsal epidermis became light brown (G2), due to the deposition of pigmented pupal cuticle beneath the old larval cuticle. About one day later, the entire body became brown (G3). The body color turned darker and harder two days after G2, that stage was designated G4. At stage G5, the pupae were approxi-mately three days after deposition of the pupal cuticle (G2). None of the G5 larvae shed the old cuticle. If the old cuticle was removed with forceps (Fig. 1B), the ani-mals possessed evaginated appendages such as antennae, compound eyes, and mandibles; forewings covered with tanned cuticle; hind wings with almost no tanned cuticle; and legs with tanned cuticle. The prolegs with crochets disappeared (data not shown). These morphological characteristics indicate that the animals with tanned cuti-cle were complete pupae.
JHA effects were further examined by applying vari-ous doses of JHA to diapausing larvae, which were then observed for six weeks. When larvae received 0.025 µg JHA or more, they eventually pupated, even though they failed to shed the old cuticle. The JHA effect was dose– dependent, with the lowest effective dose between 0.05 and 0.025 µg/larva (Table 2). The effect of JHA was more pronounced with four applications every other day.
As shown in Fig. 1C, larvae occasionally shed the old cuticle and formed complete pupae.
3.3. Involvement of the brain in the termination of diapause by JHA
In order to determine whether JHA stimulated the brain to release PTTH in the diapausing larvae, brains were removed from larvae 1, 4, 7 or 10 days after treat-ment with 1µg JHA. As shown in Fig. 2A, the day of pupation was not altered by the day of brain removal, suggesting that the brain was not directly involved in the termination of diapause by JHA. This possibility was further examined by application of various doses of JHA on larvae whose brains were removed prior to JHA application. In the brainless larvae, a dose of JHA less than 0.005 µg was still effective (Fig. 2B): nine of 15 larvae pupated, but the time period between JHA appli-cation and pupation was longer than in those treated with 0.05µg or more. Control larvae treated with acetone did not pupate at all.
3.4. Changes in hemolymph ecdysteroid titer after JHA treatment
The hemolymph ecdysteroid titer was determined after treatment with 1 µg JHA (Fig. 3). For the first 12 days after JHA application, the titer remained at the same low level. Although the concentration was low, it was at a measurable level and never declined below the detection limit (0.2 ng/ml). After day 12, the ecdysteroid titer gradually increased to a peak of 10 ng/ml on day 16 after the application. The titer decreased on day 18 and then abruptly increased after day 20. During the 20-day period, the titer in control larvae remained low.
Since pupation occurred in some individuals after day 20, we employed the morphological indicators to deter-mine the physiological age of the larvae, rather than using the actual age in days after JHA application (see Fig. 1). Larvae exhibited G1 morphology 20 days after JHA application. The titer increased to 154 ng/ml on the day of pupation (G2) and to 289 ng/ml in G4 animals. In G5 animals, the titer decreased to a level similar to that of G1–3.
3.5. Secretory activity of prothoracic glands after JHA treatment
Fig. 1. Pupal metamorphosis induced by S–methoprene (JHA) treatment of diapausing larvae of Omphisa fuscidentalis. Larvae were topically treated once with 1µg JHA (A and B) or four times with 0.1µg every other day (C). (A), progression of pupal metamorphosis graded from 1 to 5 (G1–G5 in text). (B) typical pupa produced after a single application of JHA. The old cuticle of a G3 pupa (B1) removed to show evaginated appendages (B2). (C) complete pupa obtained after four applications of 0.1µg JHA every other day. (C1), dorsal view; (C2), ventral view. For B and C: a, antennae; ce, compound eye; fw, forewing; hw, hindwing; hc, larval head capsule; ll, larval thoracic leg; pl, pupal leg: l (in C2), pupal leg; ls, larval spiracle;T1–T3, pro-, meta-, mesothoracic tergites, respectively.
activity was four to five times as much as that of the diapausing larvae.
3.6. Responsiveness of prothoracic glands to PTTH
Four different JHA doses were applied to diapausing larvae, and the prothoracic glands were cultured 1, 4, 7 or 10 days later. As shown in Fig. 5, the secretory activity of the prothoracic glands increased seven days after JHA treatment with any dose applied. In order to determine whether the glands became competent to respond to brain extracts, or PTTH, after the treatment
Table 2
Break of larval diapause with JHA
Single application 4 applicationsc
Dose Na nb Mean SD Na nb Mean SD
(µg) day day
0d 15 0 15 0
0.0005 15 0 15 0
0.001 15 0 15 8 29.8 2.9
0.005 15 0 30 13 32.6 10.7
0.01 15 0 30 14 31.9 3.7
0.025 15 2 28.5 0.5 30 17 31.4 5.4 0.05 15 10 32.1 9.3 30 14 30.3 9.0
0.1 15 11 24.5 3.1 30 16 28.1 5.0
0.3 15 10 21.9 4.4
0.6 15 13 20.5 2.7
1.25 15 8 22.1 1.3
aNumber of larvae used.
b Number of larvae that pupated within 6 weeks after JHA
appli-cation.
c4 applications of each dose indicated. d Acetone (5µl) was applied as a control.
brain extracts on day seven after treatment fell between 0.5 and 1 µg, while a dose between 0.1 and 0.25 µg elicited a response on day 10. Thus, the sensitivity of the glands to brain extracts increased by about 2-fold from seven and 10 days after JHA application.
The responsiveness of the prothoracic glands to brain extracts in vivo was tested by injecting diapausing larvae with five and 10 µl of brain extact containing one and two brain equivalents, respectively. The larvae were observed for six weeks after injection but did not exhibit any change.
4. Discussion
4.1. Developmental stage for entering the larval diapause
Larval diapause in lepidopteran insects occurs at a wide diversity of larval stages from pharate first instars to prepupae but is observed most frequently in the last larval stadium (Denlinger, 1985; Suzuki et al., 1990; Lee and Denlinger, 1997). Even in the last larval stadium, diapause occurs at different stages in different species: during the feeding period, after maturation or after the onset of wandering (prepupa). In Chilo suppresalis (Yagi and Fukaya, 1974; Yagi, 1975), Chilo partellus
(Scheltes, 1978) and Diatraea grandiosella (Yin and Chippendale, 1973; Chippendale and Yin, 1976), larvae enter diapause during the feeding period. In Omphisa, JHA stimulated the prothoracic glands and thereby induced pupal metamorphosis, indicating that the larvae enter diapause after the switch in responsiveness of
pro-Fig. 2. Effects of brain removal on termination of diapause by JHA. (A) Brains removed 1, 4, 7 or 10 days after 1µg JHA application; larvae observed for six weeks. (B) Effects of single application of various doses of JHA on diapausing larvae with brains removed prior to JHA application. Day of pupation=day of formation of pupal cuticle (Grade 2) after JHA treatment. Number in each column=number of pupated larvae. Fifteen larvae used for each day (A) or for each dose (B).
thoracic glands to JH (Sakurai, 1990) and therefore after pupal commitment (Riddiford, 1978; Riddiford et al., 1980). Larvae moved when disturbed, showing that they were not prepupae. In addition, we observed that frosted frass was excreted by larvae two or three days after 20E injection. Frosted frass is the last fecal material excreted by lepidopteran last instars before gut purge (Nijhout and Williams, 1974). This observation indicates that O.
fus-cidentalis larvae enter diapause after they cease feeding
but before they purge their gut contents, probably around the onset of the wandering stage.
4.2. Endocrine mechanisms underlying JH-mediated termination of larval diapause
Fig. 3. Hemolymph ecdysteroid titer after a single application of 1 µg JHA to diapausing larvae of O. fuscidentalis. Day of JHA appli-cation=day 0. Insert, ordinate expanded 10 fold to show changes. Each datum point=mean±SD, n=5. G1 and G5, same as in Fig. 1.
Fig. 4. Increase in ecdysteroid secretory activity of prothoracic glands after single application of 1µg JHA. One, 4, 7 and 10 days after JHA application, one prothoracic gland from each larva was cultured in 25µl Grace’s insect culture medium at 25°C for 6 h, with amount of ecdysteroid produced determined by ecdysone RIA. Day 0 on abscissa-=secretory activity of glands from diapausing larvae before JHA treat-ment. Each datum point=mean±SD, n=5.
induced the production of a pupal cuticle under the larval cuticle. Repeated applications of JHA induced com-plete pupae.
JH is thought to be involved in maintaining larval diapause. A high JH hemolymph titer during the first two-thirds of the diapause period causes the initiation and maintenance of the larval diapause in C.
sup-pressalis (Agui, 1977), Diatraea grandiosella (Yin and
Chippendale, 1979) and Sesamia nonagrioides
(Eizaguirre et al., 1998), while allatectomy elicits pupation of diapausing larvae of these species (Yagi and Fukaya, 1974; Yin and Chippendale, 1979). Accord-ingly, it is thought that a high JH titer inhibits the brain–
Fig. 5. Effects of JHA on responsiveness of the prothoracic glands of diapausing larvae to brain extract. Graded doses of JHA applied to diapausing larvae, with ecdysteroid secretory activity of prothoracic glands determined in vitro. (A) Glands individually cultured 1, 4, 7 or 10 days after JHA application in the absence (open column) or pres-ence (closed column) of brain extracts (1 brain equivalent/25 µl medium) at 25°C for 6 h. (B) Changes in responsiveness of glands, expressed as an activation ratio (Ar: Bollenbacher et al., 1984) after topical application of JHA. Ar=amount of ecdysteroid produced in presence of brain extract/amount of ecdysteroid produced in control medium. Each datum point=mean±SD, n=5.
prothoracic gland axis which maintains larval diapause. Since no study to this time has associated a rise in JH titer with diapause termination, Denlinger (1985) indi-cated that the termination of larval diapause by JH was unlikely. Nevertheless, our present results show that JHA undoubtedly terminates larval diapause in
Omphisa larvae.
Niimi and Sakurai, 1997), and JH application stimulates the secretory activity of brain and/or prothoracic gland, thereby increasing the titer of hemolymph ecdysteroids (Hiruma et al., 1978; Sakurai, 1990). Therefore, JH is possibly involved in termination of Omphisa larval diapause, which must be confirmed by determining the JH titer throughout the diapause period. Larval diapause is thought to be brought about by a shutdown of the brain–prothoracic gland axis and the subsequent sup-pression of the production of ecdysone, which is needed for further differentiation. Larval prothoracic glands are usually competent to respond to brain extracts or PTTH if they are capable of producing even a small amount of ecdysone (Bollenbacher et al., 1984; Okuda et al., 1985). Prothoracic glands of Omphisa diapausing larvae exhib-ited low secretory activity but lost their responsiveness to PTTH, as demonstrated both in vivo and in vitro. This indicates that the larval diapause of O. fuscidentalis is not merely maintained by a deficiency of PTTH.
The endocrine mechanisms for maintaining larval diapause in this species thus appears to be more compli-cated. JHA application to the Omphisa diapausing larvae resulted in an increase in the secretory activity of protho-racic glands in vitro, followed by an increase in the hem-olymph ecdysteroid titer, which resulted in the termin-ation of diapause. The brain may not be involved in the diapause-terminating effects of JHA, since JHA effects were observed in larvae whose brains were removed before JHA treatment. This indicates that the brain of a diapausing larva is not the primary target of the applied JHA. Rather, JHA may directly stimulate prothoracic glands, as suggested in Mamestra and Manduca after wandering (Hiruma et al., 1978; Sakurai, 1990).
A stimulatory effect of JH on prothoracic glands was first demonstrated in diapausing pupae of Hyalophora
cecropia (Gilbert and Schneiderman, 1959; Williams,
1959). In the last larval instar of lepidopteran insects, JHA inhibits the prothoracic glands prior to wandering, but a switch in its effect from inhibitory to stimulatory occurs shortly before or during the wandering stage (Hiruma et al., 1978; Safranek et al., 1980; Sakurai, 1990). Although we did not examine the stimulation of prothoracic glands with JHA in vitro, our results cer-tainly suggest that JHA might directly stimulate the glands of diapausing larvae. However, an increase in prothoracic gland activity and the hemolymph ecdys-teroid titer did not occur directly after JHA application. Increased secretory activity was not observed until seven days after JHA application, and the hemolymph ecdys-teroid titer did not increase until five days after that (12 days after JHA application). This indicates that although JHA may stimulate the prothoracic glands, the stimu-lation is not sufficient initially to provide a significant increase in the hemolymph ecdysteroid titer.
A single 20E injection induced a gut purge-like response in all the larvae, with pupal cuticle formation
occurring 7–32 days after the injection. It is clear that the injected 20E induced the gut purge-like response, but not pupal cuticle formation, since it is unlikely that the injected 20E persisted long enough to provoke a mor-phogenetic response. The delayed effects of the exogen-ous ecdysteroid may be caused by feedback activation of prothoracic glands, as was observed in the diapausing pupae of H. cecropia (Williams, 1952). The long period from JHA application to pupation could be interpreted in the same way. Similar to the effects of JH injection to diapausing pupae of H. cecropia (Gilbert and Schne-iderman, 1959), JHA may stimulate the prothoracic glands to secrete sufficient ecdysone so that positive feedback loops are initiated, operating in a manner simi-lar to that demonstrated in the Manduca prothoracic glands (Sakurai and Williams, 1989). In Manduca, ecdy-steroids stimulate prothoracic glands having very low secretory activity. The secretory activity of the glands of Omphisa diapausing larvae appears similar to that in
Manduca, low enough to be sensitive to positive
feed-back activation. The possibility of feedfeed-back activation of diapausing larval prothoracic glands by ecdysteroids needs to be examined in vitro.
If a similar mechanism exists for prothoracic gland activation by JHA, then why isn’t the low titer of ecdys-teroids found in the hemolymph of diapausing larvae involved in positive feedback of the prothoracic glands throughout the diapause period? If the prothoracic glands of diapausing larvae of O. fuscidentalis do not respond to ecdysteroids, the endocrine mechanism(s) suppressing the positive feedback pathway must be important to the maintenance of diapause and needs to be explored. In conjunction with this, it also needs to be determined whether JHA acts directly on prothoracic gland cells to initiate the positive feedback pathway, resulting in the termination of the larval diapause.
The present study clarifies the following endocrino-logical conditions in diapausing larvae of O.
fusciden-talis: 1) the prothoracic glands exhibit low secretory
activity; 2) the glands are incompetent to respond to PTTH; 3) autoactivation of the glands by ecdysteroids is suppressed and the hemolymph ecdysteroid titer is maintained at low level; and 4) the prothoracic glands in the diapausing larvae can be activated by JHA treat-ment. Thus the hormonal conditions during larval diapause in O. fuscidentalis appear to be similar to those found in pupal diapause of H. cecropia, although as yet there is no information about how PTTH release is inhibited, how positive feedback by ecdysteroids is sup-pressed, and how JHA affects the prothoracic glands.
Acknowledgements
supported by the Thailand Research Fund to T.S. (PDF 4080042) and Grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan to S.S. (09440273, 08276102).
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