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Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris
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Journal of Insect Physiology 50 (2004) 1075–1081 www.elsevier.com/locate/jinsphys
Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris Florence N. Munyiri, Yukio Ishikawa Laboratory of Applied Entomology, Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Received 9 June 2004; received in revised form 18 September 2004; accepted 20 September 2004
Abstract At 251C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of 1.1 ng/ ml was maintained even 30–60 days into the instar, when they were in diapause. Ecdysteroid titers remained 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers. r 2004 Elsevier Ltd. All rights reserved. Keywords: Psacothea hilaris; Juvenile hormone; Ecdysteroid; Larval diapause; Metamorphosis
1. Introduction Juvenile hormone (JH) and ecdysteroids have long been known to regulate molting, metamorphosis, and diapause induction in insects (see Denlinger, 1985; Truman and Riddiford, 2002, for reviews). JH esterase is also involved in this regulation through the control of the hemolymph JH titer (see de Kort and Granger, 1996, for review). There is considerable evidence that the onset of metamorphosis is preceded by a decrease in the biosynthesis of JH and an increase in JH esterase Corresponding author. Tel.: +81 3 5841 5061; fax: +81 3 5841 5060. E-mail address: [email protected] (F.N. Munyiri).
0022-1910/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.09.009
activity (de Kort and Granger, 1996). This then sets the stage for the elevation of ecdysteroid titers (Mizoguchi et al., 2002). Under non–diapause conditions, ecdysteroid titers peak during the wandering stage, shortly before the pupal ecdysis. Several authors have also reported the occurrence of an additional small ecdysteroid peak in the final larval instar, well before the pupal ecdysis, and this has been associated with pupal commitment (Mizoguchi et al., 2002). Ecdysteroids play a key role in the regulation of larval diapause. The low ecdysteroid titer due to the inactivation of the prothoracic glands is thought to be the cause of the failure of cell differentiation and thus the consequent developmental arrest in diapause larvae (see Chippendale, 1977, for review; Makka et al., 2002). JH is also suggested to be involved in the
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initiation and maintenance of larval diapause since relatively high JH titers are maintained all through the pre-diapause and diapause period in many species (Chippendale, 1977; Singtripop et al., 2000). In a cerambycid beetle, Psacothea hilaris, temperature and photoperiod play a key role in the regulation of non-diapause growth and diapause (Shintani et al., 1996a, b). Under 25 1C and a long-day photoperiod (15 h light : 9 h dark), about 50 percent of normally fed P. hilaris larvae pupate after the 4th instar; the rest molt to the 5th instar and pupate thereafter (Shintani et al., 1996a, b). Under a short-day photoperiod (12 h light : 12 h dark), on the other hand, most (492%) of the 5th instar larvae repeat one or two larval molts, and 69% and 23% of larvae enter diapause in the 6th and 7th instars, respectively (Watari et al., 2002). Our recent studies have shown the importance of nutritional conditions and larval weight, in addition to environmental factors, in defining larval destiny (Shintani et al., 2003; Munyiri et al., 2003, 2004). Under a long-day photoperiod, starvation of 4th instar larvae induces premature pupation in larvae exceeding a threshold weight, resulting in the formation of small but morphologically normal adults, while those animals weighing less cannot pupate and eventually die (Munyiri et al., 2003). We have also obtained evidence indicating the presence of a threshold weight for entering diapause under the short-day photoperiod (Munyiri et al., 2004). Despite our knowledge of the environmental and nutritional effects on the postembryonic development of P. hilaris, little is known of how growth and metamorphosis in this species are regulated at the endocrine level. The purpose of the present study was to gain basic information on the endocrine changes associated with metamorphosis and diapause. Specifically, we investigated the changes in the hemolymph JH titer, JH esterase activity, and ecdysteroid titer in the pupationdestined 5th instar, pre-diapause 5th instar, and diapause-destined 7th instar P. hilaris larvae.
2. Materials and methods 2.1. Insect rearing The colony of P. hilaris was established from 50 adults collected from a mulberry field at Ino town, Kochi Prefecture (33.51N, 133.41E) in 1996. Insects were reared on commercial artificial diets for the silkworm (Silkmate 2S and Insecta LF, Nosan Corp., Yokohama) as described in Shintani et al. (1996a). Neonate larvae placed individually in Petri dishes (5.5 cm in diameter, containing 3 g fresh diet) were reared continually under either a long-day (LD, 15 h light and 9 h dark) or shortday (SD, 12 h light and 12 h dark) photoperiod at 60% relative humidity and 25 1C. Larvae were checked daily
for molting and/or pupation. The date of each molt and the weight of the individual were recorded. 2.2. Staging of the larvae To obtain synchronously growing animals, newly molted 5th instar larvae were removed from the colony every day during the 6th–8th h of the photophase and weighed. Only those animals weighing 0.4–0.6 g were selected for experiments and staged as day 0. They were then provided with food and reared individually in plastic Petri dishes. These 5th instars all pupated under long-day conditions, and almost all of them repeated at least one larval molt under the short-day photoperiod. We did not use 6th instar larvae because the fate of individual larvae, i.e., whether they will enter diapause in this instar or repeat one more larval molt, is unpredictable. Staging by weight was not conducted for the 7th instar larvae, almost all of which entered diapause without a further molt. 2.3. Reagents and glassware JH I, II, and III were purchased from SciTech (Praha, Czech), and purified by thin layer chromatography before use. CD3OD and 20-hydroxyecdysone were obtained from Sigma–Aldrich (St. Louis, MO). [10-3H(N)]-JH III and a-[23, 24-3H(N)]-ecdysone were purchased from NEN Life Science Products Inc. (Boston, MA). Trifluoroacetic acid (TFA) and Clear sol ITM were purchased from Nacalai Tesque Inc. (Kyoto, Japan). All solvents were of residual pesticide analysis grade (Wako Pure Chemicals, Osaka, Japan). All glassware was sequentially rinsed with distilled water, acetone and hexane, and baked overnight at 230 1C before use. 2.4. Hemolymph collection Hemolymph was collected in the 6th–8th h of the photophase. Each of the larvae was tightly tied between the 8th and 9th abdominal segments with a piece of thread. A small incision was made in the neck near the head capsule. The larva was suspended inside a 10-ml glass tube containing 10 ml of 100 nM EDTA. The tubes were centrifuged at 160g for 6 min, allowing the larvae to bleed. Hemolymph was considered free of fat body cells when no white deposits were seen on the surface. Hemolymph was transferred to pre-weighed cryogenic vials (placed in an ice bath), frozen in liquid nitrogen immediately after recording its weight, and then stored at 80 1C prior to use. The amount of hemolymph collected from each larva under these conditions ranged from 140 to 180 ml. We measured JH and ecdysteroid titers in the same hemolymph samples for which the JH esterase assay had been performed.
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2.5. JH esterase assay JH esterase activity was measured as described by Hammock and Sparks (1977). Frozen hemolymph was thawed slowly, and a 4-ml aliquot was transferred into a siliconized microcentrifuge tube containing 240 ml of assay buffer (0.1 M phosphate buffer, 0.01% phenylthiourea, pH 7.4). The buffered hemolymph was centrifuged at 5 1C and 8000g for 5 min. Then 100 ml of the supernatant was transferred into a siliconized glass tube (4 cm long 0.5 cm in diameter) and kept at 5 1C. Two microliters of labeled JH III (diluted to 5 mM and 3.8 1013 Bq/mol with cold JH III) was added to each tube, and following incubation at 30 1C for 30 min, the non-degraded JH was extracted with hexane. The aqueous layer (50 ml) containing JH acid was mixed with 2 ml of Clear sol I, and the radioactivity was measured using a liquid scintillation counter (LSC-6100, Aloka, Tokyo, Japan). The measurement was made in duplicate. Two blanks (substrate without enzyme) were included in each series of assays. JH esterase enzyme activity was expressed in nmoles of JH III degraded by 1 ml of hemolymph/min. 2.6. Hemolymph processing for JH measurement The method of Okuda et al. (1996) was modified for the quantification of JH. Hemolymph diluted with 1.5 ml of methanol was transferred into a clean glass tube. Authentic JH I (9 ng) was added as an internal standard for the quantification of JH III. The extraction of JH from the hemolymph was achieved by repeating the partition of 0.5 ml hexane and 1.5 ml 2% NaCl solution twice. Purification of the JH III extract was conducted using a Pasture pipette packed with 1.0 g of aluminum oxide (activity grade III, ICN Ecochrom, Germany) previously conditioned with hexane. After loading the extract and washing with 2 ml of 10% ether in hexane, JH was eluted with 2 ml of 30% ether in hexane and was then dried under a stream of nitrogen. The JH was derivatized to its methoxyhydrine in 50 ml of CD3OD and 1 ml of TFA at 60 1C for 15 min. Derivatized JH was loaded on the aluminum oxide column, washed with 30% ether in hexane, and eluted with 2 ml of 50% ethyl acetate in hexane. The solution was concentrated to 10 ml under a stream of nitrogen, and a 2 ml aliquot was subjected to GC/MS analysis. 2.7. GC/MS analysis of JH III The analysis of the derivatized JH III was carried out using a GC/MS system (QP5050, Shimadzu) equipped with a DB-35 ms column (J&W Inc., 0.25 mm 30 m, He flow rate of 1.2 ml/min). The column oven temperature was maintained at 120 1C for 2 min, raised to 240 1C at 7 1C /min, and then maintained at 240 1C for 10 min.
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Ions m/z=76 and m/z=90 were monitored for JH III and for JHs I and II, respectively. JH III appeared at a retention time of 20.20 min while JH I (internal standard) appeared at 22.78 min. The hemolymph JH III titer was expressed as ng/ml. Under these conditions, the detection limit of JH was 10 pg/sample and the average recovery rate of JH I was ca. 80%. 2.8. Ecdysteroid analysis The hemolymph ecdysteroid titer was determined with a scintillation proximity assay (Mizoguchi, 2001). Hemolymph (100 ml) was mixed with methanol (1500 ml) and the mixture was centrifuged at 1600g for 5 min. A known volume of the supernatant was evaporated to dryness using a vacuum centrifuge (VEC-100, Iwaki, Japan) and the residue was dissolved in assay buffer (0.5% bovine serum albumin and 0.05% sodium azide in 50 mM borate buffer, pH 8.4). Fifty microliters of the sample was then pipetted into a mini scintillation vial and mixed with 50 ml of a-[23, 24-3H(N)]- ecdysone in assay buffer (ca. 12,000 cpm), 50 ml of 1 : 1000 diluted anti-ecdysteroid rabbit antiserum (Research Plus Inc., Bayonne, NJ), and 50 ml of scintillation proximity assay (SPA) reagent conjugated with anti-rabbit Ig G (RPN140, Amersham, UK). The mixture was incubated overnight at room temperature with continuous shaking, and the amount of tracer bound to the SPA reagent was determined using a liquid scintillation counter (LSC-6100). 20-Hydroxyecdysone (20HE, 1–100 ng/ml) was used as the standard, and the titer was expressed as ng of 20HE equivalent per ml. Each sample was assayed in duplicate. 2.9. Data analysis Statistical analyses were conducted using a software package, Statviews (SAS Institute Inc., NC, USA). Data were analyzed with the t-test. In the figures, values are presented as means7standard errors.
3. Results 3.1. Time course of development in the 5th instar larvae We monitored the changes in larval body weight and metamorphosis-related behavior in the 5th instar in order to relate the JH titer, JH esterase activity, and ecdysteroid titer to the developmental events that occur in the pupation-destined larvae. Newly ecdysed 5th instar larvae weighing 0.4–0.6 g started feeding nearly 8 h after ecdysis and gained weight progressively as they continued feeding. Under the long-day photoperiod, the larvae ceased feeding and purged their guts on day 9, and their weight began to steadily decline. On day 13,
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the larvae entered the pre-pupal stage and acquired the characteristic soft and curved body shape. Pupation occurred 5 days thereafter. Under the short-day photoperiod, most of the 5th instar larvae were destined to enter diapause after one or two additional larval molts. The duration of the 5th instar in these pre-diapause larvae was prolonged, and the majority (90%) molted to the 6th instar on day 20. 3.2. JH titer and JH esterase activity in 5th instar larvae under long day JH III, but not JH I or JH II, was detected in the hemolymph of P. hilaris (data not shown). The hemolymph JH titer on day 1 in the 5th instar was 1.3 ng/ml and declined thereafter, although it remained detectable until day 11 (Fig. 1a). The hormone became virtually undetectable after day 11, when the larvae started preparing for the pre-pupa stage. 5
Long day
There was a steady increase in the hemolymph JH esterase activity as the instar progressed (Fig. 1b). The level of activity was substantially elevated on day 11 in the instar, when JH became nearly undetectable in the hemolymph. JH esterase activity reached its highest level, 1.3 nmol JH hydrolyzed/min/ml of hemolymph, in the pre-pupa stage on day 13 (Fig. 1b). 3.3. JH titer and JH esterase activity in 5th instar larvae under short day We found large individual variations in the hemolymph JH titers in the pre-diapause 5th instar larvae (Fig. 1a). The beginning of the instar was marked by JH titers of over 1.0 ng/ml. There was a peak of 3.3 ng/ml on day 11 followed by a drop on day 13, but the titers remained high throughout the period measured. The JH esterase activity remained below 0.7 nmol/min/ml throughout the period measured in spite of the changes in hormone titers (Fig. 1b), suggesting a lack of regulation by the esterase.
Short day
JH (ng/ml)
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3.4. Ecdysteroid titer in 5th instar larvae under long day Hemolymph ecdysteroid titers in the 5th instar pupation-destined larvae were below 8 ng 20HE eq/ml for the first 7 days after ecdysis (Fig. 2). A small but significant increase in the ecdysteroid titers to 10.5 ng 20HE eq/ml was observed on day 8, which was followed by a significant drop on day 9. On day 10 of the 5th instar, the ecdysteroid titers began to increase and attained a large peak value of 72 ng 20HE eq/ml on day 11.
3
2
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PP
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0 0 (b)
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Fig. 1. Hemolymph JH titer (a) and JH esterase activity (b) during the 5th instar of P. hilaris larvae reared under 25 1C, 15L : 9D (long day) and under 25 1C, 12L : 12D (short day). Values are plotted as the mean7SE. Each data point represents the average of 4–6 separate measurements. GP=gut purge; PP=pre-pupa.
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Days in the 5th instar Fig. 2. Hemolymph ecdysteroid titers during the 5th instar of P. hilaris larvae reared under 25 1C, 15L : 9D (long day) and under 25 1C, 12L : 12D (short day). Values are plotted as the mean7SE. Each data point represents the average of 4–6 separate assays. GP=gut purge; PP=pre-pupa; E6=ecdysis to the 6th instar; 20HE=20hyroxyecdysone.
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3.5. Ecdysteroid titer in 5th instar larvae under short day
4. Discussion
Ecdysteroid titers in the 5th instar pre-diapause larvae remained low (o5 ng 20HE eq/ml) for the first 16 days of the instar (Fig. 2). The ecdysteroid titer rose slightly on day 17 and reached a significant peak of 39 ng 20HE eq/ml on day 18. The hormone titers then sharply fell during the subsequent 2 days to 10 ng 20HE eq/ml on day 20, the day of ecdysis to the 6th instar (Fig. 2).
4.1. Changes in JH titer and JH esterase activity in pupation-destined larvae
3.6. JH titer in diapause-destined 7th instar larvae The 7th instar diapause-destined larvae did not display a large individual variation in JH titers (Fig. 3) as compared with the 5th instar pre-diapause larvae (Fig. 1a). A relatively small peak of 1.9 ng/ml appeared on day 3, after which the titers decreased on day 5 to 0.9 ng/ml and remained around this level until day 60, the time when these larvae were expected to be in diapause (Fig. 3).
3.7. Ecdysteroid titer in diapause-destined 7th instar larvae The developmental profile of ecdysteroid titers in the 7th instar larvae (Fig. 3) shows that the titers remain at levels lower than those in the 5th instar prediapause larvae (Fig. 2). The ecdysteroid titer was 0.0270.01 ng 20HE eq/ml 1 day after ecdysis, and was maintained at a low level with no significant peaks thereafter (Fig. 3).
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In many species of Lepidoptera, two peaks of JH occur in the final instar. For example, in Ostrinia nubilalis, the first peak of JH is observed early in the last instar, and a second one during the wandering stage (Bean et al., 1982). This reappearance of JH in the prepupal stage is thought to suppress precocious development in the pupa (see de Kort and Granger, 1996, for review). In contrast to the lepidopteran species, no second peak is evident in the prepupal stage of P. hilaris. Likewise, in the Colorado potato beetle, Leptinotarsa decemlineata, no second JH peak is observed in the 4th (last) instar (de Kort et al., 1982). The lack of the second JH peak during the wandering stage in coleopteran species may indicate important physiological and developmental differences from Lepidoptera, and warrants further research. Studies on the changes in JH esterase activity in coleopteran species, L. decemlineata (Vermunt et al., 1997) and Tenebrio molitor (Connat, 1983), have revealed only one peak. The occurrence of only one JH esterase peak (on day 13) in P. hilaris is consistent with the finding in those two species; however, the timing of the peak differed greatly. In L. decemlineata, the peak was observed on day 3 of the last instar (Vermunt et al., 1997), while in T. molitor, the enzyme activity peaked at the beginning of the pupal stage (Connat, 1983). Although the synthesis and release of JH by the corpora allata are known to be the major regulatory factors for the hemolymph JH titer, the JH binding proteins and the rate of JH degradation in the hemolymph and tissues also affect the titer (see de Kort and Granger, 1996 for review). In P. hilaris, hemolymph JH levels decline prior to the activation of esterase, suggesting that this decline is not regulated by the esterase. The very high level of JH esterase activity at the pre-pupal stage appears to be significant only in clearing the remaining traces of JH from the larval hemolymph at this stage and thereby, setting the stage for metamorphosis. 4.2. Hemolymph ecdysteroid titers and metamorphosis
0
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30 60
Fig. 3. Hemolymph JH and ecdysteroid titers during the 7th instar of P. hilaris larvae reared under 25 1C, 12L : 12D (short day). Values are plotted as the mean7SE. Each data point represents the average of 4–6 separate measurements. D=diapause; 20HE=20-hyroxyecdysone.
P. hilaris larvae exhibited two ecdysteroid peaks, the first one on day 8 and the second one on day 11 of the 5th instar. Several insect species exhibit two ecdysteroid peaks in the last instar (see Smith, 1985, for review). In Maduca sexta, the first peak is associated with commitment to pupal development and the second one with apolysis and pupation (Truman and Riddiford, 1974; Nijhout, 1976; Riddiford, 1978). Aribi et al. (1997) also
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reported a small ecdysteroid peak in the last instar larvae of a tenebrionid beetle, Zophobas atratus, before the major one, and argued that the peak is associated with pupal commitment. The first ecdysteroid peak in P. hilaris occurred at the time of a very low hemolymph JH titer. Presumably, a small ecdysteroid peak in the presence of very low (nearly undetectable) JH titers may be sufficient to commit the larvae to pupal development. This assumption is supported by our recent findings in starved P. hilaris larvae: starvation induces a rapid decline in the JH titer and a small ecdysteroid peak in the following 48 h. Such larvae do not re-feed even if food is reintroduced. Instead, they progressively advance into metamorphosis (Munyiri et al., 2003; Munyiri, unpublished data). Presumably, these larvae have already become irreversibly committed to metamorphosis, and the small ecdysteroid peak is responsible for the change in commitment. 4.3. JH titer in pre-diapause larvae The occurrence of a larval–larval molt, but not a larval–pupal molt, in the 5th instar larvae reared under the short-day photoperiod is consistent with the elevated hemolymph JH titers in these larvae. The presence of high JH titers suggests that the physiological state of these pre-diapause larvae has already deviated from that of the non-diapause larvae and is closer to the diapause state. Individual variations in JH titers were very large in the 5th instar pre-diapause larvae. In P. hilaris, not all larvae under the same environmental conditions enter diapause in the same instar. At 25 1C under a short-day photoperiod, most of the normally fed P. hilaris larvae repeat an additional larval molt, but a small fraction (8%) enters diapause in the 5th instar (Shintani and Ishikawa, 1997; Watari et al., 2002). Therefore, the JH titer variations in the 5th instar larvae under short-day conditions probably reflect these individual variations in fate. 4.4. Ecdysteroid titer in diapause larvae We noted that the ecdysteroid titers in the early 5th instar pre-diapause larvae (reared under short day) were comparable to those in their non-diapause counterparts (reared under long day). In contrast, ecdysteroids are virtually undetectable in the early 7th instar diapausedestined larvae, indicating that the prothoracic glands are inactivated from the beginning of the instar in which diapause will occur. Low ecdysteroid titers have been reported during larval diapause, and the titers have been shown to increase with the completion of diapause (Peypelut et al., 1990). The endocrine situation at the completion of diapause in P. hilaris remains to be investigated.
In conclusion, the present study clarified the chronological order of the endocrine events in P. hilaris larvae that are destined to undergo metamorphosis or enter larval diapause, although the control of JH synthetic activity of the corpora allata remains to be elucidated.
Acknowledgments We thank Mr. W. Asano for help in insect rearing, and Ms. R. Shinjo and Dr. T. Okuda (National Institute of Agrobiological Resources) for help in establishing the JH analysis method. We are also grateful to Professor S. Tatsuki (University of Tokyo) for fruitful discussions and encouragement throughout the course of this work. References Aribi, N., Quennedey, A., Pitoizet, N., Delbecque, J.P., 1997. Ecdysteroid titers in a tenebrionid beetle, Zophobas atratus: effects of grouping and isolation. Journal of Insect Physiology 43, 815–821. Bean, D.W., Beck, S.D., Goodman, B.W., 1982. Juvenile hormone esterase in diapause and nondiapause larvae of the European corn borer, Ostrinia nubilalis. Journal of Insect Physiology 28, 485–492. Chippendale, G.M., 1977. Hormonal-regulation of larval diapause. Annual Review of Entomology 22, 121–138. Connat, J.L., 1983. Juvenile hormone esterase activity during the last larval and pupal stages of Tenebrio molitor. Journal of Insect Physiology 29, 515–521. Denlinger, D.L., 1985. Hormonal control of diapause. In: Kerkut, G., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 8. Pergamon Press, Oxford, pp. 353–412. Hammock, B.D., Sparks, T.C., 1977. A rapid assay for insect juvenile hormone esterase activity. Analytical Biochemistry 82, 573–579. de Kort, C.A.D., Granger, N.A., 1996. Regulation of JH titers: the relevance of degradative enzymes and binding proteins. Archives of Insect Biochemistry and Physiology 33, 1–26. de Kort, C.A.D., Bergot, B.J., Schooley, D.A., 1982. The nature and titer of juvenile hormone in the Colorado potato beetle, Leptinotarsa decemlineata. Journal of Insect Physiology 28, 471–474. Makka, T., Seino, A., Tomita, S., Fujiwara, H., Sonobe, H., 2002. A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori. Archives of Insect Biochemistry and Physiology 51, 111–120. Mizoguchi, A., 2001. Effects of juvenile hormone on the secretion of prothoracicotropic hormone in the last- and penultimate-instar larvae of the silkworm Bombyx mori. Journal of Insect Physiology 47, 767–775. Mizoguchi, A., Dedos, S.G., Fugo, H., Kataoka, H., 2002. Basic pattern of fluctuation in hemolymph PTTH titers during larval– pupal and pupal–adult development of the silkworm, Bombyx mori. General and Comparative Endocrinology 127, 181–189. Munyiri, F.N., Asano, W., Shintani, Y., Ishikawa, Y., 2003. Threshold weight for starvation-triggered metamorphosis in the yellowspotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae). Applied Entomology and Zoology 38, 509–515. Munyiri, F.N., Shintani, Y., Ishikawa, Y., 2004. Evidence for the presence of a threshold weight for entering diapause in the yellowspotted longicorn beetle, Psacothea hilaris. Journal of Insect Physiology 50, 295–301.
ARTICLE IN PRESS F.N. Munyiri, Y. Ishikawa / Journal of Insect Physiology 50 (2004) 1075–1081 Nijhout, H.F., 1976. Role of ecdysone in pupation in Manduca sexta. Journal of Insect Physiology 22, 453–463. Okuda, T., Tanaka, S., Kotaki, T., Ferenz, H.-J., 1996. Role of the corpora allata and juvenile hormone in the control of imaginal diapause and reproduction in three species of locusts. Journal of Insect Physiology 42, 943–951. Peypelut, L., Beydon, P., Lavenseau, L., 1990. 20–Hydroxyecdysone triggers the resumption of imaginal wing disc development after diapause in the European corn borer Ostrinia nubilalis. Archives of Insect Biochemistry and Physiology 15, 1–19. Riddiford, L.M., 1978. Ecdysone-induced change in cellular commitment of epidermis of tobacco hornworm Manduca sexta, at initiation of metamorphosis. General and Comparative Endocrinology 34, 438–446. Shintani, Y., Ishikawa, Y., 1997. Diapause avoidance induced by low temperature in the yellow-spotted longicorn beetle Psacothea hilaris. Entomologia Experimentalis et Applicata 85, 11–15. Shintani, Y., Ishikawa, Y., Tatsuki, S., 1996a. Larval diapause in the yellow -spotted longicorn beetle, Psacothea hilaris Pascoe (Coleoptera: Cerambycidae). Applied Entomology and Zoology 31, 489–494. Shintani, Y., Tatsuki, S., Ishikawa, Y., 1996b. Geographic variation of photoperiodic response in larval development of the yellow-spotted longicorn beetle, Psacothea hilaris Pascoe (Coleoptera: Cerambycidae). Applied Entomology and Zoology 31, 495–504. Shintani, Y., Munyiri, F.N., Ishikawa, Y., 2003. Change in significance of feeding during larval development in the yellow-spotted long-
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icorn beetle, Psacothea hilaris. Journal of Insect Physiology 49, 975–981. Singtripop, T., Wanichacheewa, S., Sakurai, S., 2000. Juvenile hormone-mediated termination of larval diapause in the bamboo borer, Omphisa fuscidentalis. Insect Biochemistry and Molecular Biology 30, 847–854. Smith, S.L., 1985. Regulation of ecdysteroid titer: synthesis. In: Kerkut, G.A.G., Gilbert, L.I. (Eds.), Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 7. Pergamon Press, New York, pp. 295–341. Truman, J.W., Riddiford, L.M., 1974. Physiology of insect rhythms. III. The temporal organization of the endocrine events underlying pupation of the tobacco hornworm. Journal of Experimental Biology 60, 371–382. Truman, J.W., Riddiford, L.M., 2002. Endocrine insights into the evolution of metamorphosis in insects. Annual Review of Entomology 47, 467–500. Vermunt, A.M.W., Vermeesch, A.M.G., de Kort, C.A.D., 1997. Purification and characterization of juvenile hormone esterase from hemolymph of the Colorado potato beetle. Archives of Insect Biochemistry and Physiology 35, 261–277. Watari, Y., Yamanaka, T., Asano, W., Ishikawa, Y., 2002. Prediction of the life cycle of the west Japan type yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae) by numerical simulations. Applied Entomology and Zoology 37, 559–569.
Journal of Insect Physiology 50 (2004) 1075–1081 www.elsevier.com/locate/jinsphys
Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris Florence N. Munyiri, Yukio Ishikawa Laboratory of Applied Entomology, Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan Received 9 June 2004; received in revised form 18 September 2004; accepted 20 September 2004
Abstract At 251C and under a long-day photoperiod, all 5th instar Psacothea hilaris larvae pupate at the next molt. Under a short-day photoperiod, in contrast, they undergo one or two additional larval molts and enter diapause; the 7th instar larvae enter diapause without further molt. The changes in hemolymph juvenile hormone (JH III) titers, JH esterase activity, and ecdysteroid titers in pupation-destined, pre-diapause, and diapause-destined larvae were examined. JH titers of the 5th instar pupation-destined larvae decreased continuously from 1.3 ng/ml and became virtually undetectable on day 13, when JH esterase activity peaked. Ecdysteroids exhibited a small peak on day 8, 1 day before gut purge, and a large peak on day 11, 2 days before the larvae became pre-pupae. The two ecdysteroid peaks are suggested to be associated with pupal commitment and pupation, respectively. JH titers of the 5th instar pre-diapause larvae were maintained at 1.5 ng/ml for 5 days and then increased to form a peak (3.3 ng/ml) on day 11. JH esterase activity remained at a low level throughout. Ecdysteroid levels exhibited a large peak of 40 ng/ml on day 18, coincident with the larval molt to the 6th instar. JH titers of the 7th instar diapause-destined larvae peaked at 1.9 ng/ml on day 3, and a level of 1.1 ng/ ml was maintained even 30–60 days into the instar, when they were in diapause. Ecdysteroid titers remained 0.02 ng/ml. Diapause induction in this species was suggested to be a consequence of high JH and low ecdysteroid titers. r 2004 Elsevier Ltd. All rights reserved. Keywords: Psacothea hilaris; Juvenile hormone; Ecdysteroid; Larval diapause; Metamorphosis
1. Introduction Juvenile hormone (JH) and ecdysteroids have long been known to regulate molting, metamorphosis, and diapause induction in insects (see Denlinger, 1985; Truman and Riddiford, 2002, for reviews). JH esterase is also involved in this regulation through the control of the hemolymph JH titer (see de Kort and Granger, 1996, for review). There is considerable evidence that the onset of metamorphosis is preceded by a decrease in the biosynthesis of JH and an increase in JH esterase Corresponding author. Tel.: +81 3 5841 5061; fax: +81 3 5841 5060. E-mail address: [email protected] (F.N. Munyiri).
0022-1910/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2004.09.009
activity (de Kort and Granger, 1996). This then sets the stage for the elevation of ecdysteroid titers (Mizoguchi et al., 2002). Under non–diapause conditions, ecdysteroid titers peak during the wandering stage, shortly before the pupal ecdysis. Several authors have also reported the occurrence of an additional small ecdysteroid peak in the final larval instar, well before the pupal ecdysis, and this has been associated with pupal commitment (Mizoguchi et al., 2002). Ecdysteroids play a key role in the regulation of larval diapause. The low ecdysteroid titer due to the inactivation of the prothoracic glands is thought to be the cause of the failure of cell differentiation and thus the consequent developmental arrest in diapause larvae (see Chippendale, 1977, for review; Makka et al., 2002). JH is also suggested to be involved in the
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initiation and maintenance of larval diapause since relatively high JH titers are maintained all through the pre-diapause and diapause period in many species (Chippendale, 1977; Singtripop et al., 2000). In a cerambycid beetle, Psacothea hilaris, temperature and photoperiod play a key role in the regulation of non-diapause growth and diapause (Shintani et al., 1996a, b). Under 25 1C and a long-day photoperiod (15 h light : 9 h dark), about 50 percent of normally fed P. hilaris larvae pupate after the 4th instar; the rest molt to the 5th instar and pupate thereafter (Shintani et al., 1996a, b). Under a short-day photoperiod (12 h light : 12 h dark), on the other hand, most (492%) of the 5th instar larvae repeat one or two larval molts, and 69% and 23% of larvae enter diapause in the 6th and 7th instars, respectively (Watari et al., 2002). Our recent studies have shown the importance of nutritional conditions and larval weight, in addition to environmental factors, in defining larval destiny (Shintani et al., 2003; Munyiri et al., 2003, 2004). Under a long-day photoperiod, starvation of 4th instar larvae induces premature pupation in larvae exceeding a threshold weight, resulting in the formation of small but morphologically normal adults, while those animals weighing less cannot pupate and eventually die (Munyiri et al., 2003). We have also obtained evidence indicating the presence of a threshold weight for entering diapause under the short-day photoperiod (Munyiri et al., 2004). Despite our knowledge of the environmental and nutritional effects on the postembryonic development of P. hilaris, little is known of how growth and metamorphosis in this species are regulated at the endocrine level. The purpose of the present study was to gain basic information on the endocrine changes associated with metamorphosis and diapause. Specifically, we investigated the changes in the hemolymph JH titer, JH esterase activity, and ecdysteroid titer in the pupationdestined 5th instar, pre-diapause 5th instar, and diapause-destined 7th instar P. hilaris larvae.
2. Materials and methods 2.1. Insect rearing The colony of P. hilaris was established from 50 adults collected from a mulberry field at Ino town, Kochi Prefecture (33.51N, 133.41E) in 1996. Insects were reared on commercial artificial diets for the silkworm (Silkmate 2S and Insecta LF, Nosan Corp., Yokohama) as described in Shintani et al. (1996a). Neonate larvae placed individually in Petri dishes (5.5 cm in diameter, containing 3 g fresh diet) were reared continually under either a long-day (LD, 15 h light and 9 h dark) or shortday (SD, 12 h light and 12 h dark) photoperiod at 60% relative humidity and 25 1C. Larvae were checked daily
for molting and/or pupation. The date of each molt and the weight of the individual were recorded. 2.2. Staging of the larvae To obtain synchronously growing animals, newly molted 5th instar larvae were removed from the colony every day during the 6th–8th h of the photophase and weighed. Only those animals weighing 0.4–0.6 g were selected for experiments and staged as day 0. They were then provided with food and reared individually in plastic Petri dishes. These 5th instars all pupated under long-day conditions, and almost all of them repeated at least one larval molt under the short-day photoperiod. We did not use 6th instar larvae because the fate of individual larvae, i.e., whether they will enter diapause in this instar or repeat one more larval molt, is unpredictable. Staging by weight was not conducted for the 7th instar larvae, almost all of which entered diapause without a further molt. 2.3. Reagents and glassware JH I, II, and III were purchased from SciTech (Praha, Czech), and purified by thin layer chromatography before use. CD3OD and 20-hydroxyecdysone were obtained from Sigma–Aldrich (St. Louis, MO). [10-3H(N)]-JH III and a-[23, 24-3H(N)]-ecdysone were purchased from NEN Life Science Products Inc. (Boston, MA). Trifluoroacetic acid (TFA) and Clear sol ITM were purchased from Nacalai Tesque Inc. (Kyoto, Japan). All solvents were of residual pesticide analysis grade (Wako Pure Chemicals, Osaka, Japan). All glassware was sequentially rinsed with distilled water, acetone and hexane, and baked overnight at 230 1C before use. 2.4. Hemolymph collection Hemolymph was collected in the 6th–8th h of the photophase. Each of the larvae was tightly tied between the 8th and 9th abdominal segments with a piece of thread. A small incision was made in the neck near the head capsule. The larva was suspended inside a 10-ml glass tube containing 10 ml of 100 nM EDTA. The tubes were centrifuged at 160g for 6 min, allowing the larvae to bleed. Hemolymph was considered free of fat body cells when no white deposits were seen on the surface. Hemolymph was transferred to pre-weighed cryogenic vials (placed in an ice bath), frozen in liquid nitrogen immediately after recording its weight, and then stored at 80 1C prior to use. The amount of hemolymph collected from each larva under these conditions ranged from 140 to 180 ml. We measured JH and ecdysteroid titers in the same hemolymph samples for which the JH esterase assay had been performed.
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2.5. JH esterase assay JH esterase activity was measured as described by Hammock and Sparks (1977). Frozen hemolymph was thawed slowly, and a 4-ml aliquot was transferred into a siliconized microcentrifuge tube containing 240 ml of assay buffer (0.1 M phosphate buffer, 0.01% phenylthiourea, pH 7.4). The buffered hemolymph was centrifuged at 5 1C and 8000g for 5 min. Then 100 ml of the supernatant was transferred into a siliconized glass tube (4 cm long 0.5 cm in diameter) and kept at 5 1C. Two microliters of labeled JH III (diluted to 5 mM and 3.8 1013 Bq/mol with cold JH III) was added to each tube, and following incubation at 30 1C for 30 min, the non-degraded JH was extracted with hexane. The aqueous layer (50 ml) containing JH acid was mixed with 2 ml of Clear sol I, and the radioactivity was measured using a liquid scintillation counter (LSC-6100, Aloka, Tokyo, Japan). The measurement was made in duplicate. Two blanks (substrate without enzyme) were included in each series of assays. JH esterase enzyme activity was expressed in nmoles of JH III degraded by 1 ml of hemolymph/min. 2.6. Hemolymph processing for JH measurement The method of Okuda et al. (1996) was modified for the quantification of JH. Hemolymph diluted with 1.5 ml of methanol was transferred into a clean glass tube. Authentic JH I (9 ng) was added as an internal standard for the quantification of JH III. The extraction of JH from the hemolymph was achieved by repeating the partition of 0.5 ml hexane and 1.5 ml 2% NaCl solution twice. Purification of the JH III extract was conducted using a Pasture pipette packed with 1.0 g of aluminum oxide (activity grade III, ICN Ecochrom, Germany) previously conditioned with hexane. After loading the extract and washing with 2 ml of 10% ether in hexane, JH was eluted with 2 ml of 30% ether in hexane and was then dried under a stream of nitrogen. The JH was derivatized to its methoxyhydrine in 50 ml of CD3OD and 1 ml of TFA at 60 1C for 15 min. Derivatized JH was loaded on the aluminum oxide column, washed with 30% ether in hexane, and eluted with 2 ml of 50% ethyl acetate in hexane. The solution was concentrated to 10 ml under a stream of nitrogen, and a 2 ml aliquot was subjected to GC/MS analysis. 2.7. GC/MS analysis of JH III The analysis of the derivatized JH III was carried out using a GC/MS system (QP5050, Shimadzu) equipped with a DB-35 ms column (J&W Inc., 0.25 mm 30 m, He flow rate of 1.2 ml/min). The column oven temperature was maintained at 120 1C for 2 min, raised to 240 1C at 7 1C /min, and then maintained at 240 1C for 10 min.
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Ions m/z=76 and m/z=90 were monitored for JH III and for JHs I and II, respectively. JH III appeared at a retention time of 20.20 min while JH I (internal standard) appeared at 22.78 min. The hemolymph JH III titer was expressed as ng/ml. Under these conditions, the detection limit of JH was 10 pg/sample and the average recovery rate of JH I was ca. 80%. 2.8. Ecdysteroid analysis The hemolymph ecdysteroid titer was determined with a scintillation proximity assay (Mizoguchi, 2001). Hemolymph (100 ml) was mixed with methanol (1500 ml) and the mixture was centrifuged at 1600g for 5 min. A known volume of the supernatant was evaporated to dryness using a vacuum centrifuge (VEC-100, Iwaki, Japan) and the residue was dissolved in assay buffer (0.5% bovine serum albumin and 0.05% sodium azide in 50 mM borate buffer, pH 8.4). Fifty microliters of the sample was then pipetted into a mini scintillation vial and mixed with 50 ml of a-[23, 24-3H(N)]- ecdysone in assay buffer (ca. 12,000 cpm), 50 ml of 1 : 1000 diluted anti-ecdysteroid rabbit antiserum (Research Plus Inc., Bayonne, NJ), and 50 ml of scintillation proximity assay (SPA) reagent conjugated with anti-rabbit Ig G (RPN140, Amersham, UK). The mixture was incubated overnight at room temperature with continuous shaking, and the amount of tracer bound to the SPA reagent was determined using a liquid scintillation counter (LSC-6100). 20-Hydroxyecdysone (20HE, 1–100 ng/ml) was used as the standard, and the titer was expressed as ng of 20HE equivalent per ml. Each sample was assayed in duplicate. 2.9. Data analysis Statistical analyses were conducted using a software package, Statviews (SAS Institute Inc., NC, USA). Data were analyzed with the t-test. In the figures, values are presented as means7standard errors.
3. Results 3.1. Time course of development in the 5th instar larvae We monitored the changes in larval body weight and metamorphosis-related behavior in the 5th instar in order to relate the JH titer, JH esterase activity, and ecdysteroid titer to the developmental events that occur in the pupation-destined larvae. Newly ecdysed 5th instar larvae weighing 0.4–0.6 g started feeding nearly 8 h after ecdysis and gained weight progressively as they continued feeding. Under the long-day photoperiod, the larvae ceased feeding and purged their guts on day 9, and their weight began to steadily decline. On day 13,
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the larvae entered the pre-pupal stage and acquired the characteristic soft and curved body shape. Pupation occurred 5 days thereafter. Under the short-day photoperiod, most of the 5th instar larvae were destined to enter diapause after one or two additional larval molts. The duration of the 5th instar in these pre-diapause larvae was prolonged, and the majority (90%) molted to the 6th instar on day 20. 3.2. JH titer and JH esterase activity in 5th instar larvae under long day JH III, but not JH I or JH II, was detected in the hemolymph of P. hilaris (data not shown). The hemolymph JH titer on day 1 in the 5th instar was 1.3 ng/ml and declined thereafter, although it remained detectable until day 11 (Fig. 1a). The hormone became virtually undetectable after day 11, when the larvae started preparing for the pre-pupa stage. 5
Long day
There was a steady increase in the hemolymph JH esterase activity as the instar progressed (Fig. 1b). The level of activity was substantially elevated on day 11 in the instar, when JH became nearly undetectable in the hemolymph. JH esterase activity reached its highest level, 1.3 nmol JH hydrolyzed/min/ml of hemolymph, in the pre-pupa stage on day 13 (Fig. 1b). 3.3. JH titer and JH esterase activity in 5th instar larvae under short day We found large individual variations in the hemolymph JH titers in the pre-diapause 5th instar larvae (Fig. 1a). The beginning of the instar was marked by JH titers of over 1.0 ng/ml. There was a peak of 3.3 ng/ml on day 11 followed by a drop on day 13, but the titers remained high throughout the period measured. The JH esterase activity remained below 0.7 nmol/min/ml throughout the period measured in spite of the changes in hormone titers (Fig. 1b), suggesting a lack of regulation by the esterase.
Short day
JH (ng/ml)
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3.4. Ecdysteroid titer in 5th instar larvae under long day Hemolymph ecdysteroid titers in the 5th instar pupation-destined larvae were below 8 ng 20HE eq/ml for the first 7 days after ecdysis (Fig. 2). A small but significant increase in the ecdysteroid titers to 10.5 ng 20HE eq/ml was observed on day 8, which was followed by a significant drop on day 9. On day 10 of the 5th instar, the ecdysteroid titers began to increase and attained a large peak value of 72 ng 20HE eq/ml on day 11.
3
2
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PP
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0 0 (b)
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Fig. 1. Hemolymph JH titer (a) and JH esterase activity (b) during the 5th instar of P. hilaris larvae reared under 25 1C, 15L : 9D (long day) and under 25 1C, 12L : 12D (short day). Values are plotted as the mean7SE. Each data point represents the average of 4–6 separate measurements. GP=gut purge; PP=pre-pupa.
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8
10
12 14 16
18 20
22
Days in the 5th instar Fig. 2. Hemolymph ecdysteroid titers during the 5th instar of P. hilaris larvae reared under 25 1C, 15L : 9D (long day) and under 25 1C, 12L : 12D (short day). Values are plotted as the mean7SE. Each data point represents the average of 4–6 separate assays. GP=gut purge; PP=pre-pupa; E6=ecdysis to the 6th instar; 20HE=20hyroxyecdysone.
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3.5. Ecdysteroid titer in 5th instar larvae under short day
4. Discussion
Ecdysteroid titers in the 5th instar pre-diapause larvae remained low (o5 ng 20HE eq/ml) for the first 16 days of the instar (Fig. 2). The ecdysteroid titer rose slightly on day 17 and reached a significant peak of 39 ng 20HE eq/ml on day 18. The hormone titers then sharply fell during the subsequent 2 days to 10 ng 20HE eq/ml on day 20, the day of ecdysis to the 6th instar (Fig. 2).
4.1. Changes in JH titer and JH esterase activity in pupation-destined larvae
3.6. JH titer in diapause-destined 7th instar larvae The 7th instar diapause-destined larvae did not display a large individual variation in JH titers (Fig. 3) as compared with the 5th instar pre-diapause larvae (Fig. 1a). A relatively small peak of 1.9 ng/ml appeared on day 3, after which the titers decreased on day 5 to 0.9 ng/ml and remained around this level until day 60, the time when these larvae were expected to be in diapause (Fig. 3).
3.7. Ecdysteroid titer in diapause-destined 7th instar larvae The developmental profile of ecdysteroid titers in the 7th instar larvae (Fig. 3) shows that the titers remain at levels lower than those in the 5th instar prediapause larvae (Fig. 2). The ecdysteroid titer was 0.0270.01 ng 20HE eq/ml 1 day after ecdysis, and was maintained at a low level with no significant peaks thereafter (Fig. 3).
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In many species of Lepidoptera, two peaks of JH occur in the final instar. For example, in Ostrinia nubilalis, the first peak of JH is observed early in the last instar, and a second one during the wandering stage (Bean et al., 1982). This reappearance of JH in the prepupal stage is thought to suppress precocious development in the pupa (see de Kort and Granger, 1996, for review). In contrast to the lepidopteran species, no second peak is evident in the prepupal stage of P. hilaris. Likewise, in the Colorado potato beetle, Leptinotarsa decemlineata, no second JH peak is observed in the 4th (last) instar (de Kort et al., 1982). The lack of the second JH peak during the wandering stage in coleopteran species may indicate important physiological and developmental differences from Lepidoptera, and warrants further research. Studies on the changes in JH esterase activity in coleopteran species, L. decemlineata (Vermunt et al., 1997) and Tenebrio molitor (Connat, 1983), have revealed only one peak. The occurrence of only one JH esterase peak (on day 13) in P. hilaris is consistent with the finding in those two species; however, the timing of the peak differed greatly. In L. decemlineata, the peak was observed on day 3 of the last instar (Vermunt et al., 1997), while in T. molitor, the enzyme activity peaked at the beginning of the pupal stage (Connat, 1983). Although the synthesis and release of JH by the corpora allata are known to be the major regulatory factors for the hemolymph JH titer, the JH binding proteins and the rate of JH degradation in the hemolymph and tissues also affect the titer (see de Kort and Granger, 1996 for review). In P. hilaris, hemolymph JH levels decline prior to the activation of esterase, suggesting that this decline is not regulated by the esterase. The very high level of JH esterase activity at the pre-pupal stage appears to be significant only in clearing the remaining traces of JH from the larval hemolymph at this stage and thereby, setting the stage for metamorphosis. 4.2. Hemolymph ecdysteroid titers and metamorphosis
0
0 0
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30 60
Fig. 3. Hemolymph JH and ecdysteroid titers during the 7th instar of P. hilaris larvae reared under 25 1C, 12L : 12D (short day). Values are plotted as the mean7SE. Each data point represents the average of 4–6 separate measurements. D=diapause; 20HE=20-hyroxyecdysone.
P. hilaris larvae exhibited two ecdysteroid peaks, the first one on day 8 and the second one on day 11 of the 5th instar. Several insect species exhibit two ecdysteroid peaks in the last instar (see Smith, 1985, for review). In Maduca sexta, the first peak is associated with commitment to pupal development and the second one with apolysis and pupation (Truman and Riddiford, 1974; Nijhout, 1976; Riddiford, 1978). Aribi et al. (1997) also
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reported a small ecdysteroid peak in the last instar larvae of a tenebrionid beetle, Zophobas atratus, before the major one, and argued that the peak is associated with pupal commitment. The first ecdysteroid peak in P. hilaris occurred at the time of a very low hemolymph JH titer. Presumably, a small ecdysteroid peak in the presence of very low (nearly undetectable) JH titers may be sufficient to commit the larvae to pupal development. This assumption is supported by our recent findings in starved P. hilaris larvae: starvation induces a rapid decline in the JH titer and a small ecdysteroid peak in the following 48 h. Such larvae do not re-feed even if food is reintroduced. Instead, they progressively advance into metamorphosis (Munyiri et al., 2003; Munyiri, unpublished data). Presumably, these larvae have already become irreversibly committed to metamorphosis, and the small ecdysteroid peak is responsible for the change in commitment. 4.3. JH titer in pre-diapause larvae The occurrence of a larval–larval molt, but not a larval–pupal molt, in the 5th instar larvae reared under the short-day photoperiod is consistent with the elevated hemolymph JH titers in these larvae. The presence of high JH titers suggests that the physiological state of these pre-diapause larvae has already deviated from that of the non-diapause larvae and is closer to the diapause state. Individual variations in JH titers were very large in the 5th instar pre-diapause larvae. In P. hilaris, not all larvae under the same environmental conditions enter diapause in the same instar. At 25 1C under a short-day photoperiod, most of the normally fed P. hilaris larvae repeat an additional larval molt, but a small fraction (8%) enters diapause in the 5th instar (Shintani and Ishikawa, 1997; Watari et al., 2002). Therefore, the JH titer variations in the 5th instar larvae under short-day conditions probably reflect these individual variations in fate. 4.4. Ecdysteroid titer in diapause larvae We noted that the ecdysteroid titers in the early 5th instar pre-diapause larvae (reared under short day) were comparable to those in their non-diapause counterparts (reared under long day). In contrast, ecdysteroids are virtually undetectable in the early 7th instar diapausedestined larvae, indicating that the prothoracic glands are inactivated from the beginning of the instar in which diapause will occur. Low ecdysteroid titers have been reported during larval diapause, and the titers have been shown to increase with the completion of diapause (Peypelut et al., 1990). The endocrine situation at the completion of diapause in P. hilaris remains to be investigated.
In conclusion, the present study clarified the chronological order of the endocrine events in P. hilaris larvae that are destined to undergo metamorphosis or enter larval diapause, although the control of JH synthetic activity of the corpora allata remains to be elucidated.
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