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Endocrine changes associated with the starvation-induced premature metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris
General and Comparative Endocrinology 144 (2005) 150–155 www.elsevier.com/locate/ygcen
Endocrine changes associated with the starvation-induced premature metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris Florence N. Munyiri, Yukio Ishikawa ¤ Laboratory of Applied Entomology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan Received 25 December 2004; revised 6 May 2005; accepted 23 May 2005 Available online 15 July 2005
Abstract Under 25 °C and a long day photoperiod, about half of the fourth instar Psacothea hilaris larvae molt to the Wfth instar on day 13 and pupate about 18 days later; the rest pupate without a further larval molt with a mean fourth instar period of 24 days. However, starvation of fourth instar larvae exceeding a threshold weight induces premature pupation, resulting in the formation of small but morphologically normal adults. To clarify the endocrine basis for this premature pupation, hemolymph juvenile hormone (JH) and ecdysteroid titers were quantiWed during the fed and the starved periods. Normally fed fourth instar larvae exhibited two populations with regard to JH and ecdysteroid titers, one having JH titers ranging from 1.2 to 2.1 ng/ml through to day 13, the other, similarly high titers in the early part of the instar but low titers reaching 0.1 ng/ml on day 13. One population had ecdysteroid titers with a peak of 43 ng/ml on day 10, coinciding well with the period when some larvae normally molt to the Wfth instar (day 13), the other, a small peak of 14 ng/ml on day 14 and a large peak of 70 ng/ml on day 17 coinciding well with the period just before the prepupa stage. When fourth instar larvae were starved after 4 days of feeding, JH titers decreased sharply in the next 24 h and never recovered, and a small but signiWcant increase (to 21 ng/ml) in ecdysteroid titers was observed on day 6, followed by a large peak of 63 ng/ml on day 11. Altogether, these results suggest that starvation induces a rapid decline in the JH titer, and this cues the early occurrence of a small ecdysteroid peak that commits larvae to early metamorphosis. 2005 Elsevier Inc. All rights reserved. Keywords: Psacothea hilaris; Starvation; Premature pupation; Juvenile hormone; Ecdysteroid; Threshold weight
1. Introduction In many insect species, the availability of food during larval development is critical for metamorphosis (Cymborowski et al., 1982; Jones et al., 1990; Morita and Tojo, 1985; Nijhout, 1975). Under normal rearing conditions, Manduca sexta larvae are committed to pupation in the early Wfth instar (Nijhout, 1975) but when newly ecdysed Wfth instar larvae are starved for 3 days and thereafter refed, 90% molt to the sixth instar
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Corresponding author. Fax: +81 3 5841 5060. E-mail address: [email protected] (Y. Ishikawa).
0016-6480/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2005.05.006
(Cymborowski et al., 1982; Jones et al., 1980; Nijhout, 1975). Allatectomy prior to starvation abolishes the supernumerary molt (Bhaskaran and Jones, 1980). Hemolymph juvenile hormone (JH) titers in the starved lepidopteran larvae appear to be maintained at a high level (see Cymborowski et al., 1982 for M. sexta; see Bogus and Szolajska, 1995 for Galleria mellonella; see Jones et al., 1990 for Trichoplusia ni), ensuring that a larval molt instead of a pupal one is induced (Cymborowski et al., 1982; Jones et al., 1980). Recently, Kamimura and Kiuchi (2002) showed that combining starvation and an application of fenoxycarb (a JH analog) consistently induced supernumerary molts in Bombyx mori larvae.
F.N. Munyiri, Y. Ishikawa / General and Comparative Endocrinology 144 (2005) 150–155
These observations suggest that the program of metamorphosis in the last instar can be altered by starvation. Bhaskaran and Jones (1980) showed that the high JH titer in starved M. sexta larvae is due to activation of the corpora allata (CA) by allatotropin secreted from the brain medial neurosecretory cells. More recently, Lee and Horodyski (2002) showed that starvation of M. sexta results in an increase of allatotropin mRNA in the larval nerve cord. Under 25 °C and a long day photoperiod (15 h light:9 h dark, 15L:9D), about 50% of fourth instar Psacothea hilaris larvae (Coleoptera; Cerambycidae) molt to the Wfth instar on day 13 and pupate about 18 days thereafter; the rest pupate without a larval molt with a mean fourth instar period of 24 days (Shintani et al., 1996). However, Munyiri et al. (2003) found that starvation of fourth instar larvae not only strongly suppresses molting to the Wfth instar but also induces premature (»5 days earlier than normal) pupation in fourth instar larvae exceeding a threshold weight (180 mg), resulting in the formation of unusually small but morphologically normal adults, while larvae weighing less cannot pupate and eventually die. Induction of premature pupation upon starvation was also reported in the scarabaeid, Onthophagus taurus (ShaWei et al., 2001). Hence it is now emerging that insects respond to starvation in diverse ways. Although starvation induces a supernumerary molt in the above-mentioned lepidopteran species and premature metamorphosis in the coleopterans, P. hilaris and O. taurus, the physiological and the endocrine mechanisms initiating such divergent responses have not been reported in these coleopteran species. To understand the basis for this premature metamorphosis in P. hilaris, we investigated the changes in the hemolymph JH and ecdysteroid titers of fourth instar larvae that were subjected to a starvation regimen that induces 100% premature metamorphosis (feeding 4 or 6 days prior to starvation). For comparison, we also quantiWed hemolymph JH and ecdysteroid titers of fourth instar larvae starved from the day of ecdysis to the fourth instar, which neither molt to the Wfth instar nor pupate but eventually die (Munyiri et al., 2003; Shintani et al., 2003).
2. Materials and methods 2.1. Insect rearing and staging of larvae The colony of P. hilaris was established from adults collected from a mulberry Weld in Ino (33.5°N, 133.4°E), Kochi Prefecture, Japan in 1996. Insects were reared on artiWcial diets for silkworms (Silkmate 2S and Insecta LF, Nosan, Yokohama), following the method of Shintani et al. (1996). Newly hatched larvae were reared
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individually in Petri dishes (6 cm in diameter) at 25 °C, at 50–60% relative humidity, and under a 15L:9D photoperiod. Upon each molt, the date and the weight of the individual were recorded. The larvae that had ecdysed to the fourth instar were removed daily between the sixth and the eighth hour in the photophase, and those weighing 0.15–0.20 g were selected for the experiments and staged as day 0. These larvae were assigned at random to either the experimental or control group. Control larvae had unlimited access to food for the entire larval development. Experimental larvae were transferred into clean Petri dishes with no food or moisture source on day 0 or day 4 of the fourth instar. To assess the inXuence of water intake on the hemolymph JH and ecdysteroid titers, another group of larvae was isolated on the day of ecdysis and given access to water (in the form of 2% agar) but deprived of food throughout the experimental period. To conWrm our results on the induction of premature metamorphosis by removal of food, we set up another experimental group by isolation and starvation of larvae on day 6 of the fourth instar and then quantiWed their hemolymph JH and ecdysteroid titers. 2.2. Reagents and glassware JH I and JH III were purchased from SciTech (Praha, Czech Republic), and puriWed by thin-layer chromatography before use. CD3OD and 20-hydroxyecdysone were obtained from Sigma-Aldrich (St. Louis, MO). -[23,24-3H(N)]ecdysone was purchased from NEN Life Science Products (Boston, MA). TriXuoroacetic acid (TFA) was purchased from Nacalai Tesque (Kyoto, Japan). Aluminum oxide (activity grade III) was obtained from ICN Ecochrom (Germany). All solvents were of residual pesticide analysis grade purchased from Wako Pure Chemicals (Osaka, Japan). All glassware was sequentially rinsed with distilled water, acetone and hexane, and baked overnight at 230 °C before use. 2.3. Hemolymph collection Each of the larvae was tightly tied between the eighth and ninth 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 l of 100 nM EDTA. The tubes were centrifuged at 1000 rpm for 6 min, allowing the larvae to bleed. The amount of hemolymph collected from each larva under these conditions ranged from 60 to 80 l. We measured ecdysteroid in the same hemolymph samples for which JH measurements had been performed. 2.4. JH measurement The method for quantifying JH was described in detail by Munyiri and Ishikawa (2004). In brief, after
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addition of JH I (internal standard, 9 ng) to a hemolymph sample, JH was extracted using hexane and puriWed on a small aluminum oxide column. The JH sample was then derivatized to its methoxyhydrine using CD3OD and TFA. The JH derivative was puriWed on the aluminum oxide column, and subjected to analysis by GC/MS (QP5050, Shimadzu) equipped with a DB-35ms column (J&W, 0.25 mm £ 30 m, He Xow rate of 1.2 ml/ min). Ions m/z D 76 and m/z D 90 were monitored for JH III and for JH I, respectively. 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%. 2.5. Ecdysteroid analysis The hemolymph ecdysteroid titer was determined with a scintillation proximity assay (SPA) as described in Munyiri and Ishikawa (2004). In brief, 50 l of hemolymph sample was mixed with 50 l of [23,24-3H(N)]- ecdysone in assay buVer (ca. 12,000 cpm), 50 l of 1:1000 diluted anti-20-hydroxyecdysone rabbit antiserum (Research Plus, Bayonne, NJ) and 50 l of SPA reagent conjugated with antirabbit IgG (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, Aloka, Japan). 20Hydroxyecdysone (20HE, 1–100 ng/ml) was used as the standard, and the titer was expressed as nanograms of 20HE equivalent per milliliter since the antibody showed weak (<20-fold) cross-reaction to ecdysone. Each sample was assayed in duplicate. Under these conditions, the detection limit of ecdysteroids was 10 pg/ sample. 2.6. Data analysis Statistical analyses were conducted using a software package, Statview (SAS Institute, NC). Data were analyzed by the t test. In the Wgures, values are presented as means § standard deviations.
3. Results 3.1. JH titers in the normally fed fourth instar larvae We noticed the presence of two populations of the fourth instar larvae based on their hemolymph JH titers. One group exhibited high titers ranging from 1.2 to 2.1 ng/ml through to day 13 of the fourth instar; the other, similarly high titers in the early part of the instar, but low titers (reaching 0.12 ng/ml on day 13) in the later part of the instar (Fig. 1A).
3.2. EVect of starvation after 4 or 6 days of feeding on JH titers When fourth instar larvae were fed 4 days and thereafter starved, hemolymph JH titers rapidly decreased from 2.1 to 0.3 ng/ml (t D 5.40, df D 7; P < 0.001) in the next 24 h (Fig. 1B). This decline continued and JH became virtually undetectable on day 11, just before the prepupal stage in which the insect body softens considerably and acquires the characteristic curved body shape. It was of interest to Wnd out if starvation after any other period of feeding would result in a similar decrease in the JH titers. When larvae were starved after 6 days of feeding, the hemolymph JH titers decreased sharply within 24 h after the onset of starvation. The titers never recovered thereafter and were virtually undetectable by day 11 of the instar (Fig. 1B). 3.3. EVect of total starvation on JH titers When larvae were starved from day 0 of the fourth instar, although the JH titer on day 1 was comparable to that of the normally fed larvae (compare Figs. 1A and C), the titers of the starved larvae rose signiWcantly on day 3 (Fig. 1C). This increase in titers continued as starvation progressed, becoming 3.8-fold higher on day 10 than on day 1. To clarify whether dehydration of the starved larvae was responsible for this elevation of the JH titer due to passive concentration, we fed fourth instar larvae with water (2% agar) from day 0. Larvae fed on the agar for the Wrst 6 days but ceased feeding on the subsequent days. In parallel to the change in feeding, the hemolymph JH titer remained low (below 3 ng/ ml) for the Wrst 6 days, but then rose sharply reaching 8.8 ng/ml on day 10 (Fig. 1C). This Wnding suggests that dehydration can be a cause of elevation in the JH titer. However, as discussed below, since no signiWcant increase was observed in ecdysteroids in the same samples, other mechanisms should underlie this increase in JH. 3.4. Ecdysteroid titers in normally fed fourth instar larvae We recognized two populations of ecdysteroid titers in the fourth instar larvae (Fig. 1D). This result agrees well with the previous observation that while half of the normally fed fourth instar larvae pupate at the end of this instar, the other half molts to the Wfth instar on day 13 (Shintani et al., 1996). The hormone titers in the fed fourth instar larvae remained below 8 ng/ml for the Wrst 8 days of the instar. Then in one population, the titers increased to form a peak of 43.3 ng/ml on day 10, coinciding well with the occurrence of a molt to the Wfth instar in half of the population (day 13). In the second population, ecdysteroid titers did not increase until day 14 when a small peak of 13.8 ng/ml was observed. This
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Fig. 1. Hemolymph JH III (A–C) and ecdysteroid (D–F) titers of fourth instar Psacothea hilaris larvae. (A and D) Normally fed. (B and E) Starved after 4 and 6 days of feeding. (C and F) Starved or fed 2% agar from day 0 of fourth instar. P. hilaris larvae were maintained under the long day photoperiod (15L:9D). Each data point represents the mean § SD of four to six separate measurements. E5, ecdysis to the Wfth instar; S, onset of starvation; PP, prepupa. 20HE, 20-hydroxyecdysone; M 5th, molted to Wfth instar; P 4th, pupdated without a further larval molt. Asterisks in Fig. 1D and 1E indicate signiWcant increase of the titer from the previous day at the P<0.001 level.
peak was followed by a large peak of 69.5 ng/ml on day 17, coinciding well with the period the larvae entered the prepupa stage (Fig. 1D). Pupation in the larvae corresponding to this population normally occurs 5 days later. 3.5. EVect of starvation after 4 or 6 days of feeding on ecdysteroid titers When larvae were fed 4 days and thereafter starved, hemolymph ecdysteroid titers remained below 9 ng/ml
for the Wrst 5 days (Fig. 1E). On day 6, a temporary but signiWcant increase (to 20.5 ng/ml) in the hormone titer was observed. Titers began to rise again peaking at 63.1 ng/ml on day 11, just before larvae entered the prepupa stage (day 13). When larvae were starved after 6 days of feeding, a similar pattern of the hormone titers was observed (Fig. 1E); the titers remained below 7 ng/ ml for the Wrst 7 days. On day 8, a small peak in the hormone titers (12.2 ng/ml) occurred, followed by a large peak of 72.8 ng/ml on day 11, just before the prepupa stage.
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3.6. EVect of total starvation on ecdysteroid titers Starvation of larvae from day 0 of the fourth instar did not cause an elevation of hemolymph ecdysteroid titers; titers remained below 5.3 ng/ml throughout the period investigated. Neither did agar consumption have any stimulatory eVect on the levels of hemolymph ecdysteroid (Fig. 1F).
4. Discussion 4.1. Hemolymph JH titers and premature pupation Prolonged starvation after the Wrst few days of feeding in the fourth instar completely changes the developmental destiny of P. hilaris in favor of premature pupation (Munyiri et al., 2003; Shintani et al., 2003). The JH titer of the larvae starved after a few days of feeding shows a sharp decrease within 24 h (Fig. 1B). This sudden decline in the JH titer may possibly be attributed to either direct or indirect deactivation of the CA by some unidentiWed factor(s), resulting in a rapid decrease in synthesis and/or secretion of JH. When Wfth instar M. sexta larvae are starved for 3 days and thereafter refed, 90% undergo an extra larva molt to the sixth instar due to the activation of the CA (Bhaskaran and Jones, 1980) and the subsequent elevation of the JH titer (Cymborowski et al., 1982). Lee and Horodyski (2002) showed that in M. sexta, starvation resulted in an increase in allatotropin mRNA in the larval nerve cord. Allatotropins are neural peptides that stimulate the CA to synthesize JH, and are known to occur in several insect species (Elekonich and Horodyski, 2003). Since fourth instar P. hilaris that are starved after the Wrst few days of feeding pupate prematurely, a mechanism converse to that in M. sexta may be operational. It is possible that allatostatins are involved in the cascade of events resulting in the rapid decrease in the hemolymph JH (Aguilar et al., 2003; Li et al., 2004). Alternatively, a rapid activation of JH esterase, singly or in combination with CA suppression, may explain the clearance of JH from the hemolymph. Psacothea hilaris larvae must exceed a threshold weight of 180 mg for successful metamorphosis (Munyiri et al., 2003). Therefore, the activity of the CA and/or JH esterase will be dependent on whether or not they have exceeded this weight threshold. If the larvae have exceeded this threshold weight, the CA are rapidly shut oV and/or the degradation of JH is activated to clear JH from the system. In M. sexta, a critical weight of 5 g must be acquired for pupation, and if the larvae are starved after attaining this weight, the process that leads to the secretion of prothoracicotropic hormone cannot be inhibited (Nijhout and Williams, 1974a,b). These workers demonstrated that the CA became ‘turned oV’ once
the larvae attained this critical weight. The rapid disappearance of JH from the hemolymph may be attributed to this ‘turn oV’ of the CA. Regarding the increase in the JH titers in the larvae starved from the day of ecdysis, we Wrst presumed that passive concentration of the hormone in the hemolymph was a likely cause for this increase. However, in view of the gradual elevation of the JH titer in the larvae that had no access to water, the sudden increase in the hormone titer observed between day 7 and day 10 in the animals given 2% agar cannot be explained by mere dehydration. Even more surprising was the Wnding that ecdysteroid titers were similar regardless of the availability of water (Fig. 1F). Thus, an unknown factor must be responsible for this sharp increase in JH titers. 4.2. Hemolymph ecdysteroid titers and premature pupation The small ecdysteroid peak observed on day 14 of the fourth instar may be functionally similar to the small peak observed on day 8 of the Wfth instar larvae, which is presumed to commit larvae to pupal development (Munyiri and Ishikawa, 2004). A similar peak has been observed in several other species and has mainly been associated with pupal commitment (Aribi et al., 1997; Connat et al., 1991; Nijhout, 1976; Riddiford, 1978; Truman et al., 1974). The second peak, which was much larger than the Wrst, may be associated with the synthesis of a new cuticle, as has been observed in numerous insects (Delbecque et al., 1978; Mizoguchi et al., 2002). Starvation of the fourth instar larvae after the Wrst 4 or 6 days of feeding resulted in premature pupation (Munyiri et al., 2003; Shintani et al., 2003). Each of these treatments resulted in a sharp decrease in the JH titer within 24 h. On the other hand, the ecdysteroid titers did not change until the next 24 h when a small but signiWcant increase in the titers was observed (Fig. 1E). The starvation-induced premature termination of larval development in this species is closely associated with the early occurrence of this small ecdysteroid peak. In comparison to the normally fed fourth instar larvae where the peak occurs on day 14, starvation after 4 days of feeding quickens the appearance of this commitment peak by 8 days, and by 6 days in the case of the larvae fed 6 days prior to starvation. Taken together, the present study revealed the chronological order of the endocrine events leading to premature pupation in this beetle. The sharp decrease in the JH titers, accompanied by the small increase in the ecdysteroid titers one day later, ultimately lead to the early pupal commitment in this beetle. Once the pupal commitment occurs, the next surge of ecdysteroid in the absence of JH leads to the pupal molt.
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Acknowledgments We thank Mr. W. Asano for help with 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. This research was supported by a Grant-in-Aid for ScientiWc Research No. 16380039 from the Japan Society for the Promotion of Science (JSPS).
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Kamimura, M., Kiuchi, M., 2002. Applying fenoxycarb at the penultimate instar triggers an additional ecdysteroid surge and induces perfect extra larval molting in the silkworm. Gen. Comp. Endocrinol. 128, 231–237. Lee, K.Y., Horodyski, F.M., 2002. Restriction of nutrient intake results in the increase of a speciWc Manduca sexta allatotropin (ManseAT) mRNA in the larval nerve cord. Peptides 23, 653–661. Li, Y., Martinez, S.H., Noriega, F.G., 2004. Inhibition of juvenile hormone biosynthesis in mosquitoes: eVect of allatostatic head factors, PISCF- and YXFGL-amide-allatostatins. Regul. Pept. 118, 175– 182. Mizoguchi, A., Dedos, S.G., Fugo, H., Kataoka, H., 2002. Basic pattern of Xuctuation in hemolymph PTTH titers during larval–pupal and pupal–adult development of the silkworm, Bombyx mori. Gen. Comp. Endocrinol. 127, 181–189. Morita, M., Tojo, S., 1985. Relationship between starvation and supernumerary ecdysis and recognition of the penultimate-larval instar in the common cutworm, Spodoptera litura. J. Insect Physiol. 31, 307–313. Munyiri, F.N., Asano, W., Shintani, Y., Ishikawa, Y., 2003. Threshold weight for starvation-triggered metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae). Appl. Entomol. Zool. 38, 509–515. Munyiri, F.N., Ishikawa, Y., 2004. Endocrine changes associated with metamorphosis and diapause induction in the yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae). J. Insect Physiol. 50, 1075–1081. Nijhout, H.F., 1975. Threshold size for metamorphosis in the tobacco hornworm, Manduca sexta. Biol. Bull. 149, 214–225. Nijhout, H.F., 1976. Role of ecdysone in pupation in Manduca sexta. J. Insect Physiol. 22, 453–463. Nijhout, H.F., Williams, C.M., 1974a. Control of molting and metamorphosis in the tobacco hornworm, Manduca sexta L.: growth of the last larval instar and the decision to pupate. J. Exp. Biol. 61, 481–491. Nijhout, H.F., Williams, C.M., 1974b. Control of molting and metamorphosis in the tobacco hornworm, Manduca sexta L.: cessation of juvenile hormone secretion as a trigger for pupation. J. Exp. Biol. 61, 493–501. Riddiford, L.M., 1978. Ecdysone-induced change in cellular commitment of epidermis of tobacco hornworm Manduca sexta, at initiation of metamorphosis. Gen. Comp. Endocrinol. 34, 438–446. ShaWei, M., Moczek, A.P., Nijhout, H.F., 2001. Food availability controls the onset of metamorphosis in the dung beetle Onthophagus taurus (Coleoptera: Scarabaeidae). Physiol. Entomol. 26, 172–180. Shintani, Y., Ishikawa, Y., Tatsuki, S., 1996. Larval diapause in the yellow-spotted longicorn beetle, Psacothea hilaris Pascoe (Coleoptera: Cerambycidae). Appl. Entomol. Zool. 31, 489–494. Shintani, Y., Munyiri, F.N., Ishikawa, Y., 2003. Change in signiWcance of feeding during larval development in the yellow-spotted longicorn beetle, Psacothea hilaris. J. Insect Physiol. 49, 975–981. Truman, J.W., Riddiford, L.M., Safranek, L., 1974. Temporal patterns of response to ecdysone and juvenile-hormone in epidermis of tobacco hornworm, Manduca sexta. Dev. Biol. 39, 247–262.
Endocrine changes associated with the starvation-induced premature metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris Florence N. Munyiri, Yukio Ishikawa ¤ Laboratory of Applied Entomology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan Received 25 December 2004; revised 6 May 2005; accepted 23 May 2005 Available online 15 July 2005
Abstract Under 25 °C and a long day photoperiod, about half of the fourth instar Psacothea hilaris larvae molt to the Wfth instar on day 13 and pupate about 18 days later; the rest pupate without a further larval molt with a mean fourth instar period of 24 days. However, starvation of fourth instar larvae exceeding a threshold weight induces premature pupation, resulting in the formation of small but morphologically normal adults. To clarify the endocrine basis for this premature pupation, hemolymph juvenile hormone (JH) and ecdysteroid titers were quantiWed during the fed and the starved periods. Normally fed fourth instar larvae exhibited two populations with regard to JH and ecdysteroid titers, one having JH titers ranging from 1.2 to 2.1 ng/ml through to day 13, the other, similarly high titers in the early part of the instar but low titers reaching 0.1 ng/ml on day 13. One population had ecdysteroid titers with a peak of 43 ng/ml on day 10, coinciding well with the period when some larvae normally molt to the Wfth instar (day 13), the other, a small peak of 14 ng/ml on day 14 and a large peak of 70 ng/ml on day 17 coinciding well with the period just before the prepupa stage. When fourth instar larvae were starved after 4 days of feeding, JH titers decreased sharply in the next 24 h and never recovered, and a small but signiWcant increase (to 21 ng/ml) in ecdysteroid titers was observed on day 6, followed by a large peak of 63 ng/ml on day 11. Altogether, these results suggest that starvation induces a rapid decline in the JH titer, and this cues the early occurrence of a small ecdysteroid peak that commits larvae to early metamorphosis. 2005 Elsevier Inc. All rights reserved. Keywords: Psacothea hilaris; Starvation; Premature pupation; Juvenile hormone; Ecdysteroid; Threshold weight
1. Introduction In many insect species, the availability of food during larval development is critical for metamorphosis (Cymborowski et al., 1982; Jones et al., 1990; Morita and Tojo, 1985; Nijhout, 1975). Under normal rearing conditions, Manduca sexta larvae are committed to pupation in the early Wfth instar (Nijhout, 1975) but when newly ecdysed Wfth instar larvae are starved for 3 days and thereafter refed, 90% molt to the sixth instar
*
Corresponding author. Fax: +81 3 5841 5060. E-mail address: [email protected] (Y. Ishikawa).
0016-6480/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ygcen.2005.05.006
(Cymborowski et al., 1982; Jones et al., 1980; Nijhout, 1975). Allatectomy prior to starvation abolishes the supernumerary molt (Bhaskaran and Jones, 1980). Hemolymph juvenile hormone (JH) titers in the starved lepidopteran larvae appear to be maintained at a high level (see Cymborowski et al., 1982 for M. sexta; see Bogus and Szolajska, 1995 for Galleria mellonella; see Jones et al., 1990 for Trichoplusia ni), ensuring that a larval molt instead of a pupal one is induced (Cymborowski et al., 1982; Jones et al., 1980). Recently, Kamimura and Kiuchi (2002) showed that combining starvation and an application of fenoxycarb (a JH analog) consistently induced supernumerary molts in Bombyx mori larvae.
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These observations suggest that the program of metamorphosis in the last instar can be altered by starvation. Bhaskaran and Jones (1980) showed that the high JH titer in starved M. sexta larvae is due to activation of the corpora allata (CA) by allatotropin secreted from the brain medial neurosecretory cells. More recently, Lee and Horodyski (2002) showed that starvation of M. sexta results in an increase of allatotropin mRNA in the larval nerve cord. Under 25 °C and a long day photoperiod (15 h light:9 h dark, 15L:9D), about 50% of fourth instar Psacothea hilaris larvae (Coleoptera; Cerambycidae) molt to the Wfth instar on day 13 and pupate about 18 days thereafter; the rest pupate without a larval molt with a mean fourth instar period of 24 days (Shintani et al., 1996). However, Munyiri et al. (2003) found that starvation of fourth instar larvae not only strongly suppresses molting to the Wfth instar but also induces premature (»5 days earlier than normal) pupation in fourth instar larvae exceeding a threshold weight (180 mg), resulting in the formation of unusually small but morphologically normal adults, while larvae weighing less cannot pupate and eventually die. Induction of premature pupation upon starvation was also reported in the scarabaeid, Onthophagus taurus (ShaWei et al., 2001). Hence it is now emerging that insects respond to starvation in diverse ways. Although starvation induces a supernumerary molt in the above-mentioned lepidopteran species and premature metamorphosis in the coleopterans, P. hilaris and O. taurus, the physiological and the endocrine mechanisms initiating such divergent responses have not been reported in these coleopteran species. To understand the basis for this premature metamorphosis in P. hilaris, we investigated the changes in the hemolymph JH and ecdysteroid titers of fourth instar larvae that were subjected to a starvation regimen that induces 100% premature metamorphosis (feeding 4 or 6 days prior to starvation). For comparison, we also quantiWed hemolymph JH and ecdysteroid titers of fourth instar larvae starved from the day of ecdysis to the fourth instar, which neither molt to the Wfth instar nor pupate but eventually die (Munyiri et al., 2003; Shintani et al., 2003).
2. Materials and methods 2.1. Insect rearing and staging of larvae The colony of P. hilaris was established from adults collected from a mulberry Weld in Ino (33.5°N, 133.4°E), Kochi Prefecture, Japan in 1996. Insects were reared on artiWcial diets for silkworms (Silkmate 2S and Insecta LF, Nosan, Yokohama), following the method of Shintani et al. (1996). Newly hatched larvae were reared
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individually in Petri dishes (6 cm in diameter) at 25 °C, at 50–60% relative humidity, and under a 15L:9D photoperiod. Upon each molt, the date and the weight of the individual were recorded. The larvae that had ecdysed to the fourth instar were removed daily between the sixth and the eighth hour in the photophase, and those weighing 0.15–0.20 g were selected for the experiments and staged as day 0. These larvae were assigned at random to either the experimental or control group. Control larvae had unlimited access to food for the entire larval development. Experimental larvae were transferred into clean Petri dishes with no food or moisture source on day 0 or day 4 of the fourth instar. To assess the inXuence of water intake on the hemolymph JH and ecdysteroid titers, another group of larvae was isolated on the day of ecdysis and given access to water (in the form of 2% agar) but deprived of food throughout the experimental period. To conWrm our results on the induction of premature metamorphosis by removal of food, we set up another experimental group by isolation and starvation of larvae on day 6 of the fourth instar and then quantiWed their hemolymph JH and ecdysteroid titers. 2.2. Reagents and glassware JH I and JH III were purchased from SciTech (Praha, Czech Republic), and puriWed by thin-layer chromatography before use. CD3OD and 20-hydroxyecdysone were obtained from Sigma-Aldrich (St. Louis, MO). -[23,24-3H(N)]ecdysone was purchased from NEN Life Science Products (Boston, MA). TriXuoroacetic acid (TFA) was purchased from Nacalai Tesque (Kyoto, Japan). Aluminum oxide (activity grade III) was obtained from ICN Ecochrom (Germany). All solvents were of residual pesticide analysis grade purchased from Wako Pure Chemicals (Osaka, Japan). All glassware was sequentially rinsed with distilled water, acetone and hexane, and baked overnight at 230 °C before use. 2.3. Hemolymph collection Each of the larvae was tightly tied between the eighth and ninth 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 l of 100 nM EDTA. The tubes were centrifuged at 1000 rpm for 6 min, allowing the larvae to bleed. The amount of hemolymph collected from each larva under these conditions ranged from 60 to 80 l. We measured ecdysteroid in the same hemolymph samples for which JH measurements had been performed. 2.4. JH measurement The method for quantifying JH was described in detail by Munyiri and Ishikawa (2004). In brief, after
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addition of JH I (internal standard, 9 ng) to a hemolymph sample, JH was extracted using hexane and puriWed on a small aluminum oxide column. The JH sample was then derivatized to its methoxyhydrine using CD3OD and TFA. The JH derivative was puriWed on the aluminum oxide column, and subjected to analysis by GC/MS (QP5050, Shimadzu) equipped with a DB-35ms column (J&W, 0.25 mm £ 30 m, He Xow rate of 1.2 ml/ min). Ions m/z D 76 and m/z D 90 were monitored for JH III and for JH I, respectively. 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%. 2.5. Ecdysteroid analysis The hemolymph ecdysteroid titer was determined with a scintillation proximity assay (SPA) as described in Munyiri and Ishikawa (2004). In brief, 50 l of hemolymph sample was mixed with 50 l of [23,24-3H(N)]- ecdysone in assay buVer (ca. 12,000 cpm), 50 l of 1:1000 diluted anti-20-hydroxyecdysone rabbit antiserum (Research Plus, Bayonne, NJ) and 50 l of SPA reagent conjugated with antirabbit IgG (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, Aloka, Japan). 20Hydroxyecdysone (20HE, 1–100 ng/ml) was used as the standard, and the titer was expressed as nanograms of 20HE equivalent per milliliter since the antibody showed weak (<20-fold) cross-reaction to ecdysone. Each sample was assayed in duplicate. Under these conditions, the detection limit of ecdysteroids was 10 pg/ sample. 2.6. Data analysis Statistical analyses were conducted using a software package, Statview (SAS Institute, NC). Data were analyzed by the t test. In the Wgures, values are presented as means § standard deviations.
3. Results 3.1. JH titers in the normally fed fourth instar larvae We noticed the presence of two populations of the fourth instar larvae based on their hemolymph JH titers. One group exhibited high titers ranging from 1.2 to 2.1 ng/ml through to day 13 of the fourth instar; the other, similarly high titers in the early part of the instar, but low titers (reaching 0.12 ng/ml on day 13) in the later part of the instar (Fig. 1A).
3.2. EVect of starvation after 4 or 6 days of feeding on JH titers When fourth instar larvae were fed 4 days and thereafter starved, hemolymph JH titers rapidly decreased from 2.1 to 0.3 ng/ml (t D 5.40, df D 7; P < 0.001) in the next 24 h (Fig. 1B). This decline continued and JH became virtually undetectable on day 11, just before the prepupal stage in which the insect body softens considerably and acquires the characteristic curved body shape. It was of interest to Wnd out if starvation after any other period of feeding would result in a similar decrease in the JH titers. When larvae were starved after 6 days of feeding, the hemolymph JH titers decreased sharply within 24 h after the onset of starvation. The titers never recovered thereafter and were virtually undetectable by day 11 of the instar (Fig. 1B). 3.3. EVect of total starvation on JH titers When larvae were starved from day 0 of the fourth instar, although the JH titer on day 1 was comparable to that of the normally fed larvae (compare Figs. 1A and C), the titers of the starved larvae rose signiWcantly on day 3 (Fig. 1C). This increase in titers continued as starvation progressed, becoming 3.8-fold higher on day 10 than on day 1. To clarify whether dehydration of the starved larvae was responsible for this elevation of the JH titer due to passive concentration, we fed fourth instar larvae with water (2% agar) from day 0. Larvae fed on the agar for the Wrst 6 days but ceased feeding on the subsequent days. In parallel to the change in feeding, the hemolymph JH titer remained low (below 3 ng/ ml) for the Wrst 6 days, but then rose sharply reaching 8.8 ng/ml on day 10 (Fig. 1C). This Wnding suggests that dehydration can be a cause of elevation in the JH titer. However, as discussed below, since no signiWcant increase was observed in ecdysteroids in the same samples, other mechanisms should underlie this increase in JH. 3.4. Ecdysteroid titers in normally fed fourth instar larvae We recognized two populations of ecdysteroid titers in the fourth instar larvae (Fig. 1D). This result agrees well with the previous observation that while half of the normally fed fourth instar larvae pupate at the end of this instar, the other half molts to the Wfth instar on day 13 (Shintani et al., 1996). The hormone titers in the fed fourth instar larvae remained below 8 ng/ml for the Wrst 8 days of the instar. Then in one population, the titers increased to form a peak of 43.3 ng/ml on day 10, coinciding well with the occurrence of a molt to the Wfth instar in half of the population (day 13). In the second population, ecdysteroid titers did not increase until day 14 when a small peak of 13.8 ng/ml was observed. This
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Fig. 1. Hemolymph JH III (A–C) and ecdysteroid (D–F) titers of fourth instar Psacothea hilaris larvae. (A and D) Normally fed. (B and E) Starved after 4 and 6 days of feeding. (C and F) Starved or fed 2% agar from day 0 of fourth instar. P. hilaris larvae were maintained under the long day photoperiod (15L:9D). Each data point represents the mean § SD of four to six separate measurements. E5, ecdysis to the Wfth instar; S, onset of starvation; PP, prepupa. 20HE, 20-hydroxyecdysone; M 5th, molted to Wfth instar; P 4th, pupdated without a further larval molt. Asterisks in Fig. 1D and 1E indicate signiWcant increase of the titer from the previous day at the P<0.001 level.
peak was followed by a large peak of 69.5 ng/ml on day 17, coinciding well with the period the larvae entered the prepupa stage (Fig. 1D). Pupation in the larvae corresponding to this population normally occurs 5 days later. 3.5. EVect of starvation after 4 or 6 days of feeding on ecdysteroid titers When larvae were fed 4 days and thereafter starved, hemolymph ecdysteroid titers remained below 9 ng/ml
for the Wrst 5 days (Fig. 1E). On day 6, a temporary but signiWcant increase (to 20.5 ng/ml) in the hormone titer was observed. Titers began to rise again peaking at 63.1 ng/ml on day 11, just before larvae entered the prepupa stage (day 13). When larvae were starved after 6 days of feeding, a similar pattern of the hormone titers was observed (Fig. 1E); the titers remained below 7 ng/ ml for the Wrst 7 days. On day 8, a small peak in the hormone titers (12.2 ng/ml) occurred, followed by a large peak of 72.8 ng/ml on day 11, just before the prepupa stage.
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3.6. EVect of total starvation on ecdysteroid titers Starvation of larvae from day 0 of the fourth instar did not cause an elevation of hemolymph ecdysteroid titers; titers remained below 5.3 ng/ml throughout the period investigated. Neither did agar consumption have any stimulatory eVect on the levels of hemolymph ecdysteroid (Fig. 1F).
4. Discussion 4.1. Hemolymph JH titers and premature pupation Prolonged starvation after the Wrst few days of feeding in the fourth instar completely changes the developmental destiny of P. hilaris in favor of premature pupation (Munyiri et al., 2003; Shintani et al., 2003). The JH titer of the larvae starved after a few days of feeding shows a sharp decrease within 24 h (Fig. 1B). This sudden decline in the JH titer may possibly be attributed to either direct or indirect deactivation of the CA by some unidentiWed factor(s), resulting in a rapid decrease in synthesis and/or secretion of JH. When Wfth instar M. sexta larvae are starved for 3 days and thereafter refed, 90% undergo an extra larva molt to the sixth instar due to the activation of the CA (Bhaskaran and Jones, 1980) and the subsequent elevation of the JH titer (Cymborowski et al., 1982). Lee and Horodyski (2002) showed that in M. sexta, starvation resulted in an increase in allatotropin mRNA in the larval nerve cord. Allatotropins are neural peptides that stimulate the CA to synthesize JH, and are known to occur in several insect species (Elekonich and Horodyski, 2003). Since fourth instar P. hilaris that are starved after the Wrst few days of feeding pupate prematurely, a mechanism converse to that in M. sexta may be operational. It is possible that allatostatins are involved in the cascade of events resulting in the rapid decrease in the hemolymph JH (Aguilar et al., 2003; Li et al., 2004). Alternatively, a rapid activation of JH esterase, singly or in combination with CA suppression, may explain the clearance of JH from the hemolymph. Psacothea hilaris larvae must exceed a threshold weight of 180 mg for successful metamorphosis (Munyiri et al., 2003). Therefore, the activity of the CA and/or JH esterase will be dependent on whether or not they have exceeded this weight threshold. If the larvae have exceeded this threshold weight, the CA are rapidly shut oV and/or the degradation of JH is activated to clear JH from the system. In M. sexta, a critical weight of 5 g must be acquired for pupation, and if the larvae are starved after attaining this weight, the process that leads to the secretion of prothoracicotropic hormone cannot be inhibited (Nijhout and Williams, 1974a,b). These workers demonstrated that the CA became ‘turned oV’ once
the larvae attained this critical weight. The rapid disappearance of JH from the hemolymph may be attributed to this ‘turn oV’ of the CA. Regarding the increase in the JH titers in the larvae starved from the day of ecdysis, we Wrst presumed that passive concentration of the hormone in the hemolymph was a likely cause for this increase. However, in view of the gradual elevation of the JH titer in the larvae that had no access to water, the sudden increase in the hormone titer observed between day 7 and day 10 in the animals given 2% agar cannot be explained by mere dehydration. Even more surprising was the Wnding that ecdysteroid titers were similar regardless of the availability of water (Fig. 1F). Thus, an unknown factor must be responsible for this sharp increase in JH titers. 4.2. Hemolymph ecdysteroid titers and premature pupation The small ecdysteroid peak observed on day 14 of the fourth instar may be functionally similar to the small peak observed on day 8 of the Wfth instar larvae, which is presumed to commit larvae to pupal development (Munyiri and Ishikawa, 2004). A similar peak has been observed in several other species and has mainly been associated with pupal commitment (Aribi et al., 1997; Connat et al., 1991; Nijhout, 1976; Riddiford, 1978; Truman et al., 1974). The second peak, which was much larger than the Wrst, may be associated with the synthesis of a new cuticle, as has been observed in numerous insects (Delbecque et al., 1978; Mizoguchi et al., 2002). Starvation of the fourth instar larvae after the Wrst 4 or 6 days of feeding resulted in premature pupation (Munyiri et al., 2003; Shintani et al., 2003). Each of these treatments resulted in a sharp decrease in the JH titer within 24 h. On the other hand, the ecdysteroid titers did not change until the next 24 h when a small but signiWcant increase in the titers was observed (Fig. 1E). The starvation-induced premature termination of larval development in this species is closely associated with the early occurrence of this small ecdysteroid peak. In comparison to the normally fed fourth instar larvae where the peak occurs on day 14, starvation after 4 days of feeding quickens the appearance of this commitment peak by 8 days, and by 6 days in the case of the larvae fed 6 days prior to starvation. Taken together, the present study revealed the chronological order of the endocrine events leading to premature pupation in this beetle. The sharp decrease in the JH titers, accompanied by the small increase in the ecdysteroid titers one day later, ultimately lead to the early pupal commitment in this beetle. Once the pupal commitment occurs, the next surge of ecdysteroid in the absence of JH leads to the pupal molt.
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Acknowledgments We thank Mr. W. Asano for help with 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. This research was supported by a Grant-in-Aid for ScientiWc Research No. 16380039 from the Japan Society for the Promotion of Science (JSPS).
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