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Feeding glucose or sucrose, but not trehalose, suppresses the starvation-induced premature pupation in the yellow-spotted longicorn beetle, Psacothea hilaris
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Journal of Insect Physiology 51 (2005) 1005–1012 www.elsevier.com/locate/jinsphys
Feeding glucose or sucrose, but not trehalose, suppresses the starvation-induced premature pupation 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; received in revised form 27 April 2005; accepted 27 April 2005
Abstract Under 25 1C and a long-day photoperiod, starvation induces premature pupation in 4th instar Psacothea hilaris larvae exceeding a threshold weight of 180 mg, resulting in the formation of small but morphologically normal adults. To investigate possible mechanisms underlying this phenomenon, we first measured the hemolymph trehalose and glucose levels of starved larvae. When larvae were starved after 4 days of feeding (attaining the threshold weight), glucose levels decreased 4-fold within the next 24 h, while trehalose levels, after a temporary slight decrease, increased remarkably to reach a peak just before the prepupa stage. The effects of ingesting various nutrients on the developmental fate and the hemolymph sugar titers of starving larvae were then examined. Feeding on agar blocks containing sucrose or glucose totally suppressed the occurrence of premature pupation, while trehalose, fructose, casein and starch were ineffective. Feeding on glucose or trehalose resulted in a 6-fold decrease in hemolymph glucose levels and remarkably elevated trehalose levels. Since feeding on glucose and trehalose induced similar changes in hemolymph sugar titers but trehalose was not effective in suppressing premature pupation, glucose may have exhibited its effects via gustatory mechanisms. r 2005 Elsevier Ltd. All rights reserved. Keywords: Psacothea hilaris; Starvation; Premature pupation; Hemolymph glucose; Trehalose
1. Introduction The availability of food during larval development is critical for metamorphosis (Cymborowski et al., 1982; Morita and Tojo, 1985; Jones et al., 1980). One of the most immediate changes that occur when insects are confronted with starvation is a change in the hemolymph carbohydrate levels; starvation during larval development as well as in the adult stage results in a rapid decrease in the hemolymph glucose and trehalose levels (Dalman, 1973; Jones et al., 1981; Siegert, 1987a,b; Ziegler, 1991; Satake et al., 2000). Trehalose Corresponding author. Tel.: +81 3 5841 5061; fax: +81 3 5841 5060. E-mail address: [email protected] (Y. Ishikawa).
0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.04.015
is the principal sugar circulating in the hemolymph of most insects, and serves as an immediate source of glucose for tissue metabolism. Trehalose is synthesized in the fat body following digestion of dietary carbohydrates (see Thompson (2003) for a review). The hydrolysis of trehalose into glucose is catalyzed by the enzyme trehalase. Studies show that the trehalose concentration in the blood is not finely regulated but highly variable, depending on environmental conditions, physiological state and nutrition (Thompson, 2003). It has been suggested that although glucose occurs in the insect hemolymph in much lower quantities than trehalose, this carbohydrate is the signal for the nutritional status of the animal (Fernandes et al., 2001). In the cerambycid beetle Psacothea hilaris, photoperiod, nutritional conditions and larval weight are key
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factors in defining the larval developmental destiny (Shintani et al., 1996, 2003; Munyiri et al., 2003, 2004). Under 25 1C and a long-day photoperiod (15 h light: 9 h dark, 15L:9D), about 50% of 4th instar P. hilaris larvae molt to the 5th instar on day 13 and pupate about 18 days later; the rest pupate without a further larval molt with a mean 4th instar period of 24 days (Shintani et al., 1996). Munyiri et al. (2003) found that starvation of 4th instar larvae not only strongly suppresses molting to the 5th instar but also induces premature (5 days earlier than normal) pupation in 4th instar larvae exceeding a threshold weight of 180 mg, resulting in the formation of small but morphologically normal adults, while larvae weighing less cannot pupate and eventually die. The starvation-induced premature pupation in P. hilaris is associated with a rapid decrease in the larval hemolymph juvenile hormone (JH) titers within 24 h of starvation (Munyiri and Ishikawa, 2005). This decrease is accompanied by a small but significant increase in ecdysteroid titers 24 h later, which presumably leads to the early pupal commitment in this beetle (Munyiri and Ishikawa, 2005). Nutritional signals are known to affect the release of allatotropins and allatostatins by the brain resulting in the activation or inhibition of JH synthesis (Noriega, 2004). In view of the findings obtained to date, we hypothesized that the premature pupation in starved P. hilaris larvae is cued by changes in the hemolymph carbohydrate levels. To test this hypothesis, we first quantified the hemolymph trehalose and glucose levels of larvae that had been exposed to various feeding, starvation, and neck-ligation regimens. We then examined the effect of the ingestion of glucose and other nutrients on the developmental destiny of starving larvae.
2. Materials and methods 2.1. Insect rearing and staging The colony of P. hilaris was established from adults collected from a mulberry field in Ino (33.51N, 133.41E), Kochi Prefecture in 1996. Insects were reared on artificial diets for silkworms (Silkmate 2STM, Nosan, Corporation, Yokohama), following the method of Shintani et al. (1996). According to the supplier, this diet contains mulberry leaf powder, soyabean protein, starch, sucrose, cereals powder, minerals, vitamins, citric acid, agar and food antiseptics (water 72–76%, fiber o3.9%, protein 46.0%, ash o3.9%, fat o1.1%). Neonate larvae were individually placed in Petri dishes (6 cm in diameter) containing fresh diet and reared continually at 50–60% humidity, 25 1C and 15L:9D. Upon each molt, the date and the weight of the individual were recorded. The larvae that molted to
the 4th instar between the 6th and 8th h in the photophase and weighing 0.15–0.20 g were selected daily and staged as day 0. The larvae were assigned at random to either the experimental or the control group. Under normal conditions, these staged larvae started feeding nearly 8 h after ecdysis and gained weight progressively as they continued feeding. The majority of these larvae attained their maximum weight by day 10 of the 4th instar after which a substantial decline in weight was evident. About 50% of the individuals molted to the 5th instar by day 13 while the rest entered the prepupal stage by day 19 and pupated 5 days thereafter. 2.2. Starvation and neck-ligation Control larvae were given 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 4th instar. To test the effect of cephalic factors on larval hemolymph carbohydrates, one group of larvae was neck-ligated on day 0, and the other, on day 4. 2.3. Effect of nutrients on suppression of premature pupation We first examined whether feeding larvae 8% glucose from the day of ecdysis would lead to successful metamorphosis. One group of the experimental larvae was given a block of 2% agar (2.0 g) containing 8% glucose in place of their normal food from day 0. The other groups of larvae were allowed to feed on the normal diet until day 4 when they became substantially heavier than the threshold weight and acquired the competence for metamorphosis, and then either starved throughout the 4th instar, or given a plain 2% agar block or a block containing 2% glucose, 8% glucose, 8% fructose, 8% trehalose, 8% sucrose, 8% casein or 8% starch. The agar block was replaced with a fresh piece every 2 days. Larvae were monitored daily and upon each molt, their weight and the date of the molt were recorded. 2.4. Hemolymph collection The larvae were ligated at the neck with a piece of thread. A small incision was made at the posterior segment using a sharp pair of scissors. Hemolymph (10 ml) was collected and immediately mixed with 50 ml of 100 nM EDTA in a microtube. The tube was heated to 90 1C for 3 min to denature proteins, and centrifuged at 1600 g for 5 min. The supernatant was transferred into another clean microtube, and stored at –20 1C until use.
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2.5. Trehalose and glucose assay
8
2.6. Chemicals The Glucose B-test TM kit and casein were obtained from Wako Pure Chemicals, Japan. Trehalose dehydrate, EDTA, D-()-fructose, soluble starch and agar powder were purchased from Nacalai Tesque, Inc. Kyoto, Japan. D-(+)-glucose and sucrose were obtained from Kanto Chemical Co., Japan. Trehalase was obtained from Sigma Chemicals, St. Louis, MO.
Glucose (mg/ml)
6
Statistical analyses were conducted using a software package, Statviews (SAS Institute Inc., NC, USA). Data were analyzed by ANOVA, and the significance of differences among means was determined with Tukey–Kramer’s multiple range test. In the tables and figures, values are presented as means standard deviations.
3. Results 3.1. Effect of starvation on glucose levels Fig. 1A shows the changes in the hemolymph glucose levels during the 4th instar of larvae fed throughout the instar, of those starved on the day of ecdysis, and of those starved after 4 days of feeding. For all the periods of time investigated, glucose levels in the normally fed larvae exhibited considerable variation. The hemolymph glucose level was low (2.2 mg/ml) on the day of ecdysis to the 4th instar, but increased as the larvae started feeding on the normal diet, reaching 3.9 mg/ml on day 4, and was then maintained at around this value for most of the time. When larvae were starved on day 0, glucose
4 3
1 0 0
2
(A)
4
6
8
10
12
14
Days in the 4th instar 40 Fed throughout Starved from day 0 Starved from day 4
30
20
10
0 0 (B)
2.7. Data analysis
5
2
Trehalose (mg/ml)
A Glucose B-test kit was used to determine the concentration of glucose. The hemolymph sample was thawed, vortexed for 30 s and centrifuged at 1600 g for 5 min. Aliquots (25 ml) of hemolymph were transferred to new microtubes. Twenty microliters of either phosphate buffer or trehalase solution (0.011 units) in phosphate buffer was placed into each tube and incubated at 37 1C for 1 h. Two microliters of either glucose or trehalose standard (0.5–4.0 mg/ml), or the test sample was placed in each well of a microplate, and 200 ml of coloring reagent (containing glucose oxidase, peroxidase and o-dianisidine) was added. After incubation at room temperature for 1 h, the absorbance of the wells at 490 nm was measured using a Bio-Rad 550 microplate reader. The amount of trehalose was estimated from the glucose concentrations in the trehalase-treated and untreated samples.
Fed throughout Starved from day 0 Starved from day 4
7
TM
1007
2
4 6 8 10 Days in the 4th instar
12
14
Fig. 1. Changes in hemolymph glucose (A) and trehalose (B) levels of the 4th instar P. hilaris larvae, which were either normally fed, or starved from the day of ecdysis, or starved after 4 days of feeding. Larvae were reared under a long-day photoperiod (15 h light and 9 h dark). Each data point represents the mean7SD of 4–6 separate measurements.
levels declined 1.6-fold in the next 24 h, and continued to decrease further as starvation progressed. When larvae were starved after 4 days of feeding, glucose levels decreased sharply (4-fold within the next 24 h), and with a slight fluctuation, finally declined to 0.2 mg/ml on day 13, the day the larvae entered the prepupa stage. Individual variations of the glucose titer in the starved larvae were small when compared with those in the fed larvae. 3.2. Effect of starvation on trehalose levels Hemolymph trehalose levels were 5-fold higher than glucose levels on the day of ecdysis to the 4th instar (compare Fig. 1A and B). As the larvae continued feeding on the normal diet, trehalose levels fluctuated between 12 and 16 mg/ml, but they started decreasing after day 10 and finally declined to 7 mg/ml on day 13 (Fig. 1B). When larvae were starved from day 0,
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trehalose levels started decreasing after 2 days, and were nearly undetectable by day 13. When larvae were starved after 4 days of feeding, trehalose levels increased greatly in the subsequent days of starvation reaching a peak of 23.5 mg/ml on day 12, one day before the larvae entered the prepupal stage.
3.3. Effect of neck ligation on glucose and trehalose levels When larvae were neck-ligated from day 0 of the 4th instar, hemolymph glucose levels decreased 7-fold in 2 days while trehalose levels decreased 2-fold (Table 1). When larvae were ligated after the first 4 days of feeding, hemolymph glucose levels declined 7-fold in 2 days while trehalose levels did not decline significantly.
Table 1 Effect of neck ligation on 4th instar Psacothea hilaris hemolymph glucose and trehalose levelsa Treatment
Glucose (mg/ml)b
Trehalose (mg/ml)b
Fed for 2 days Ligated for 2 days from day 0 Fed for 6 days Fed for 4 days and ligated for 2 days
3.371.1b 0.570.4a
13.772.4b 7.371.6a
3.671.9b 0.570.0a
17.277.1b 13.373.5b
a Larvae were either neck-ligated on the day of ecdysis or fed 4 days prior to ligation. b Values are the mean7SD of a minimum of 4 separate determinations. Means in the same column followed by the same letter are not significantly different at the 5% level.
3.4. Effect of feeding 8 % glucose from day 0
3.6. Ecdysis to the 5th instar
When 4th instar larvae were starved from day 0, most of them eventually died without pupating (Table 2). Feeding on 8% glucose was sufficient to promote metamorphosis in 78% (18 23) of the larvae. Interestingly, the larvae that pupated did so within a similar period of time as those fed the normal diet, suggesting that no premature pupation was induced. However, 13% (3 larvae) died before achieving metamorphosis. The individuals that pupated gave rise to pupae whose weight was 3-fold lower than that of pupae from larvae fed the normal diet (Table 2).
When fed the normal artificial diet, 56% of larvae molted to the 5th instar while the rest pupated with a mean larval period of 24 days (Table 3). Except for one individual, none of the starved larvae molted to the 5th instar. None of the larvae undertook another molt when fed plain agar either. When larvae were fed on different concentrations of glucose, they responded in a dosedependent manner, with 2% glucose eliciting 25% molt to the 5th instar while 8% glucose elicited a larval molting in as many as 84% of individuals (Table 3). Interestingly, feeding on 8% sucrose stimulated 52% of the larvae to molt to the 5th instar, which is similar to that elicited by the normal artificial diet. This result is reasonable because sucrose is included in the normal artificial diet as an ingredient. Of the sugars tested, fructose was the least effective in eliciting a molt to the 5th instar, followed by trehalose. Starch and casein, however, were the least effective of the substances tested, eliciting ecdysis to the 5th instar in only 12% of the larvae (Table 3).
3.5. Suppression of premature pupation When larvae were starved after 4 days of feeding, they all pupated prematurely with a mean duration of 19 days as opposed to those fed the normal diet that took 24 days (Table 3). However, feeding on glucose at a concentration as low as 2% was sufficient to prevent premature metamorphosis in P. hilaris; larvae pupated with a mean period of 25 days, which is similar to the pre-pupation period of the normally fed larvae. A higher concentration of glucose (8%) had a similar effect although in this case, only two individuals pupated from the 4th instar. Feeding on plain agar did not suppress this premature pupation; larvae pupated with a mean larval period of 21 days suggesting that the mechanical stimulus associated with swallowing is not responsible for the suppression. Among the nutrients tested, sucrose also suppressed premature pupation, but trehalose, fructose, starch and casein were ineffective (Table 3). Taken together, it appears that ingestion of specific sugars is a prerequisite for suppression of the starvationinduced premature pupation.
3.7. Pupal weight All larvae exhibited a similar weight gain when they were fed the normal diet for the first 4 days after ecdysis to the 4th instar (Table 3, F ¼ 1:3, df7,200; P40:5). However, after the consumption of the various test substances, the weight of the resulting pupae varied greatly (Table 3). Larvae fed on the normal diet throughout the instar produced the heaviest pupae, weighing 392 mg. Of all the substances tested, 8% glucose gave rise to the heaviest pupae though they weighed only 250 mg. Larvae fed 8% casein gave rise to pupae of the least weight (174 mg, Table 3).
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Table 2 Effect of feeding 8% glucose from the day of ecdysis on development of Psacothea hilaris under 15L:9Da Test substanceb
N
Weight on ecdysis to 4th instar (mg)c
% molt to 5th instar
% pupated from Duration of 4th 4th instar instar (days)c
Weight of pupae % died before (mg)c pupation
Starved 8% glucose Normal diet
30 23 27
189754a 174720a 205763a
0 (0) 4 (1) 56 (15)
7 (2) 78 (18) 44 (12)
13577a 143725a 392795b
19.572.1a 23.372.7b 23.872.1b
93 (28) 13 (3) 0
a
Numbers in parentheses indicate the numbers of individuals. Larvae were either starved or given agar blocks containing 8% glucose from the day of ecdysis. Control larvae were fed a normal diet throughout the 4th instar. c Values are means7SD. Duration of the 4th instar and weight of pupae refer to the individuals that pupated at the 4th instar. Means followed by the same letter in the same column are not significantly different at the 5% level. b
Table 3 Effect of feeding various dietary substances on premature pupation in Psacothea hilaris under 15L:9Da Test substanceb
N
Weight after 4d feeding normal dietc
% molt to 5th instar
% pupated from Duration of 4th 4th instar instarc
Weight of pupaec
% Permanent larvaed
Normal diet Starved 2% agar 2% glucose 8% glucose 8% sucrose 8% trehalose 8% fructose 8% starch 8% casein
27 32 25 24 25 23 28 24 24 26
374777a 369773a 346739a 354766a 340746a 344768a 350760a 349763a 342756a 350745a
56 3 0 25 84 52 14 29 13 12
44 97 100 75 8 48 86 71 88 88
392795d 221756b 211734b 221757b 250728c 234755bc 240754bc 239746bc 224744b 174742a
0 0 0 0 38 25 25 0 100 100
(15) (1) (0) (6) (21) (12) (4) (7) (3) (3)
(12) (31) (25) (18) (2) (11) (24) (17) (21) (23)
23.872.1c 19.071.9a 21.071.6b 24.671.7c 23.570.7c 23.572.1c 19.271.0a 21.972.5b 22.171.8b 20.771.4b
(8) (3) (1) (3) (3)
a
Numbers in parentheses indicate the numbers of individuals. Larvae were fed a normal artificial diet for the first 4 days after ecdysis to the 4th instar, then given an agar block containing a test substance. Control larvae were fed the normal diet throughout the 4th instar. c Values are means7SD. Means followed by the same letter in the same column are not significantly different at the 5% level. d The permanent larvae did not pupate even after 60 days in the 5th instar and are expressed as the % of larvae that became 5th instars. b
3.8. Permanent larvae Interestingly, some of the larvae that molted to the 5th instar after feeding on the various test substances survived as larvae for more than 60 days (Table 3). These larvae fed normally for the early part of the 5th instar but ceased feeding later in the instar, and they never started wandering nor became prepupae. We therefore considered these larvae to have become permanent larvae. Particularly interesting in the present experiment is the finding that all the larvae in the starch and casein treatments that molted to the 5th instar ended up as permanent larvae (Table 3). 3.9. Effect of feeding glucose on the hemolymph sugar levels When larvae were fed 8% glucose after 4 days of feeding on the normal diet, hemolymph glucose levels
fell 6-fold within the following 24 h, after which the low sugar levels were maintained throughout the experimental period (Fig. 2A). In contrast, hemolymph trehalose levels increased by 4% within the next 24 h of feeding (Fig. 2B). This was followed by a very large increase in the subsequent days as larvae consumed the glucose, reaching 50 mg/ml on day 13, when the majority of the larvae were found to ecdyse to the 5th instar. 3.10. Effect of feeding trehalose on the hemolymph sugar levels Overall, feeding on trehalose induced similar changes in the hemolymph glucose and trehalose titers as feeding on glucose. When larvae were fed on 8% trehalose after 4 days of feeding on the normal diet, hemolymph glucose levels fell 6-fold within the following 24 h (Fig. 2C). The sugar remained at around this level for
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8
8 Fed normal diet Fed 8%glucose
6 5 4 3 2
6 5 4 3 2
1
1
0
0
0
2
(A)
4 6 8 10 12 Days in the 4th instar
14
0
4 6 8 10 12 Days in the 4th instar
14
70
Fed normal diet Fed 8%glucose
60
2
(C)
Trehalose (mg/ml)
Trehalose (mg/ml)
70
50 40 30 20
Fed normal diet Fed 8%trehalose
60 50 40 30 20 10
10
0
0
0 (B)
Fed normal diet Fed 8%trehalose
7 Glucose (mg/ml)
Glucose (mg/ml)
7
2
4 6 8 10 12 Days in the 4th instar
0
14 (D)
2
4
6
8
10
12
14
Days in the 4th instar
Fig. 2. Hemolymph glucose (A, C) and trehalose (B, D) levels during the 4th instar of normally fed P. hilaris and of those fed 2% agar containing 8% glucose (A, B) or trehalose (C, D) after feeding on a normal diet for the first 4 days after ecdysis. All larvae were reared under the long-day photoperiod (15 h light and 9 h dark). Each data point represents the mean7SD of 4–6 separate measurements.
the rest of the time investigated. Hemolymph trehalose levels doubled within 24 h of feeding on 8% trehalose (Fig. 2D). The trehalose levels continued to increase in the subsequent days as the larvae fed on the trehalose, reaching 38.7 mg/ml on day 13 of the instar.
4. Discussion 4.1. Effect of starvation on glucose and trehalose titers One of the most intriguing findings in the present study is the differential response of the hemolymph trehalose levels induced by starvation initiated at different times. Starvation on day 0 of the 4th instar results in a rapid decrease in the trehalose levels, while starvation on day 4, when the larvae become substantially heavier than the threshold weight and acquire the competence for metamorphosis, results in a marked increase in the hemolymph trehalose levels. In contrast, glucose levels showed a rapid decrease regardless of the timing of starvation. The effect of neck-ligation on the trehalose levels was also dependent on the timing of the operation: neck-ligation on day 0 resulted in a decrease in the hemolymph trehalose levels while ligation on day 4 had no significant effect on its level. These findings suggest that the differences in response are likely to be attributable to the change
in the nature of the fat body (the site of trehalose synthesis), which has occurred during the 4 days of feeding, and that if neurohormones are involved in the change, they are not likely to be released from the cephalic part. In Bombyx mori, starvation after 3 days of feeding in the 4th instar larvae led to a decrease in the hemolymph glucose level and a significant increase in the trehalose level (Satake et al., 2000). Satake et al. (2000) observed an increase in the active form of glycogen phosphorylase and a decrease in the glycogen content in the fat body of the starved larvae, suggesting that trehalose production from glycogen was enhanced during starvation. Thus, the observed increase in the hemolymph trehalose level in P. hilaris larvae starved after 4 days of feeding may be due to the breakdown of glycogen to produce trehalose to meet the energy demands for metamorphosis. On the other hand, the low glucose levels in the starved larvae indicate that the glucose derived from glycogen was used for the synthesis of trehalose within the fat body, and was not released into the hemolymph (Siegert, 1987b). We noted considerable variation in the trehalose and glucose levels of the normally fed larvae (Fig. 1A and B). This variation was particularly evident after day 4 of the 4th instar. Under normally fed conditions, 50% of 4th instar larvae pupate at the end of this instar; the rest molt to the 5th instar on day 13 (Shintani et al., 1996). Thus, it is reasonable to attribute this variation to the
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two different populations of individuals having distinct developmental fates.
4.2. Effect of feeding glucose or trehalose on their hemolymph titers When larvae were fed on 8% glucose from day 4 of the 4th instar, hemolymph glucose levels declined more than 6-fold while trehalose levels increased remarkably. This observation is similar to that of Evans and Dethier (1957) who found that when the adult blowfly Phormia regina was fed glucose, hemolymph trehalose levels more than doubled while glucose levels declined more than 2-fold within the next 48 h. A similar increase in the hemolymph trehalose levels was noted in Manduca sexta 5th instar larvae after 3 days of feeding on a diet with increasing amounts of sucrose (Thompson et al., 2003), and also in Locusta migratoria fed on a diet containing high carbohydrate levels (Zanotto et al., 1996). These results suggest that trehalose was synthesized from the ingested sugars or carbohydrates. We suspect that a similar mechanism is operational in the case of P. hilaris; all the glucose ingested by the larvae was used for trehalose synthesis and therefore, this explains the greatly elevated trehalose levels (Fig. 2B and D). We also suspect that all the ingested trehalose is converted to glucose in the midgut cells before it is released into the hemolymph, and this is why ingestion of either glucose or trehalose induces almost the same response in their hemolymph titers.
4.3. Hemolymph glucose and trehalose levels as the cue for premature pupation Feeding either glucose or trehalose to starved P. hilaris 4th instar larvae resulted in entirely different developmental consequences. To our surprise, however, both glucose and trehalose induced similar responses in their hemolymph titers. These findings suggest that neither the hemolymph glucose nor the trehalose levels are the direct cue for the control of the CA activity, which is supposed to be shut off within 24 h of starvation (Munyiri and Ishikawa, 2005). Rather, ingestion of sugars (glucose and sucrose) may be constantly monitored, for instance, by epipharyngeal gustatory organs, and this information may be used to modulate the neuroendocrine system. Alternatively, the sugar levels might change on a much finer time scale, probably hourly, and this may be important in controlling the destiny of the starving larvae. We may have overlooked these fine changes because we examined only the daily changes in sugar levels.
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Acknowledgements We are 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 Scientific Research No. 16380039 from the Japan Society for the Promotion of Science (JSPS).
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icorn beetle, Psacothea hilaris. Journal of Insect Physiology 49, 975–981. Siegert, K.L., 1987a. Carbohydrate metabolism in Manduca sexta during late larval development. Journal of Insect physiology 33, 421–427. Siegert, K.L., 1987b. Carbohydrate metabolism in starved fifth instar larvae of Manduca sexta. Archives of Insect Biochemistry and Physiology 4, 151–160. Thompson, N.S., 2003. Trehalose—the insect blood sugar. In: Simpson, S.J. (Ed.), Advances in Insect Physiology. Elsevier Academic Press, Amsterdam, vol. 31. pp. 205–285.
Thompson, S.N., Borchardt, D.B., Wang, L.W., 2003. Dietary nutrient levels regulate protein and carbohydrate intake, gluconeogenic/glycolytic flux and blood trehalose level in the insect Manduca sexta L. Journal of Comparative Physiology B 173, 149–163. Zanotto, F.P., Raubenheimer, D., Simpson, S.J., 1996. Hemolymph amino acids and sugar levels in locusts fed nutritionally unbalanced diets. Journal of Comparative Physiology B 166, 223–229. Ziegler, R., 1991. Changes in lipid and carbohydrate metabolism during starvation in adult Manduca sexta. Journal of Comparative Physiology 161, 125–131.
Journal of Insect Physiology 51 (2005) 1005–1012 www.elsevier.com/locate/jinsphys
Feeding glucose or sucrose, but not trehalose, suppresses the starvation-induced premature pupation 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; received in revised form 27 April 2005; accepted 27 April 2005
Abstract Under 25 1C and a long-day photoperiod, starvation induces premature pupation in 4th instar Psacothea hilaris larvae exceeding a threshold weight of 180 mg, resulting in the formation of small but morphologically normal adults. To investigate possible mechanisms underlying this phenomenon, we first measured the hemolymph trehalose and glucose levels of starved larvae. When larvae were starved after 4 days of feeding (attaining the threshold weight), glucose levels decreased 4-fold within the next 24 h, while trehalose levels, after a temporary slight decrease, increased remarkably to reach a peak just before the prepupa stage. The effects of ingesting various nutrients on the developmental fate and the hemolymph sugar titers of starving larvae were then examined. Feeding on agar blocks containing sucrose or glucose totally suppressed the occurrence of premature pupation, while trehalose, fructose, casein and starch were ineffective. Feeding on glucose or trehalose resulted in a 6-fold decrease in hemolymph glucose levels and remarkably elevated trehalose levels. Since feeding on glucose and trehalose induced similar changes in hemolymph sugar titers but trehalose was not effective in suppressing premature pupation, glucose may have exhibited its effects via gustatory mechanisms. r 2005 Elsevier Ltd. All rights reserved. Keywords: Psacothea hilaris; Starvation; Premature pupation; Hemolymph glucose; Trehalose
1. Introduction The availability of food during larval development is critical for metamorphosis (Cymborowski et al., 1982; Morita and Tojo, 1985; Jones et al., 1980). One of the most immediate changes that occur when insects are confronted with starvation is a change in the hemolymph carbohydrate levels; starvation during larval development as well as in the adult stage results in a rapid decrease in the hemolymph glucose and trehalose levels (Dalman, 1973; Jones et al., 1981; Siegert, 1987a,b; Ziegler, 1991; Satake et al., 2000). Trehalose Corresponding author. Tel.: +81 3 5841 5061; fax: +81 3 5841 5060. E-mail address: [email protected] (Y. Ishikawa).
0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.04.015
is the principal sugar circulating in the hemolymph of most insects, and serves as an immediate source of glucose for tissue metabolism. Trehalose is synthesized in the fat body following digestion of dietary carbohydrates (see Thompson (2003) for a review). The hydrolysis of trehalose into glucose is catalyzed by the enzyme trehalase. Studies show that the trehalose concentration in the blood is not finely regulated but highly variable, depending on environmental conditions, physiological state and nutrition (Thompson, 2003). It has been suggested that although glucose occurs in the insect hemolymph in much lower quantities than trehalose, this carbohydrate is the signal for the nutritional status of the animal (Fernandes et al., 2001). In the cerambycid beetle Psacothea hilaris, photoperiod, nutritional conditions and larval weight are key
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factors in defining the larval developmental destiny (Shintani et al., 1996, 2003; Munyiri et al., 2003, 2004). Under 25 1C and a long-day photoperiod (15 h light: 9 h dark, 15L:9D), about 50% of 4th instar P. hilaris larvae molt to the 5th instar on day 13 and pupate about 18 days later; the rest pupate without a further larval molt with a mean 4th instar period of 24 days (Shintani et al., 1996). Munyiri et al. (2003) found that starvation of 4th instar larvae not only strongly suppresses molting to the 5th instar but also induces premature (5 days earlier than normal) pupation in 4th instar larvae exceeding a threshold weight of 180 mg, resulting in the formation of small but morphologically normal adults, while larvae weighing less cannot pupate and eventually die. The starvation-induced premature pupation in P. hilaris is associated with a rapid decrease in the larval hemolymph juvenile hormone (JH) titers within 24 h of starvation (Munyiri and Ishikawa, 2005). This decrease is accompanied by a small but significant increase in ecdysteroid titers 24 h later, which presumably leads to the early pupal commitment in this beetle (Munyiri and Ishikawa, 2005). Nutritional signals are known to affect the release of allatotropins and allatostatins by the brain resulting in the activation or inhibition of JH synthesis (Noriega, 2004). In view of the findings obtained to date, we hypothesized that the premature pupation in starved P. hilaris larvae is cued by changes in the hemolymph carbohydrate levels. To test this hypothesis, we first quantified the hemolymph trehalose and glucose levels of larvae that had been exposed to various feeding, starvation, and neck-ligation regimens. We then examined the effect of the ingestion of glucose and other nutrients on the developmental destiny of starving larvae.
2. Materials and methods 2.1. Insect rearing and staging The colony of P. hilaris was established from adults collected from a mulberry field in Ino (33.51N, 133.41E), Kochi Prefecture in 1996. Insects were reared on artificial diets for silkworms (Silkmate 2STM, Nosan, Corporation, Yokohama), following the method of Shintani et al. (1996). According to the supplier, this diet contains mulberry leaf powder, soyabean protein, starch, sucrose, cereals powder, minerals, vitamins, citric acid, agar and food antiseptics (water 72–76%, fiber o3.9%, protein 46.0%, ash o3.9%, fat o1.1%). Neonate larvae were individually placed in Petri dishes (6 cm in diameter) containing fresh diet and reared continually at 50–60% humidity, 25 1C and 15L:9D. Upon each molt, the date and the weight of the individual were recorded. The larvae that molted to
the 4th instar between the 6th and 8th h in the photophase and weighing 0.15–0.20 g were selected daily and staged as day 0. The larvae were assigned at random to either the experimental or the control group. Under normal conditions, these staged larvae started feeding nearly 8 h after ecdysis and gained weight progressively as they continued feeding. The majority of these larvae attained their maximum weight by day 10 of the 4th instar after which a substantial decline in weight was evident. About 50% of the individuals molted to the 5th instar by day 13 while the rest entered the prepupal stage by day 19 and pupated 5 days thereafter. 2.2. Starvation and neck-ligation Control larvae were given 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 4th instar. To test the effect of cephalic factors on larval hemolymph carbohydrates, one group of larvae was neck-ligated on day 0, and the other, on day 4. 2.3. Effect of nutrients on suppression of premature pupation We first examined whether feeding larvae 8% glucose from the day of ecdysis would lead to successful metamorphosis. One group of the experimental larvae was given a block of 2% agar (2.0 g) containing 8% glucose in place of their normal food from day 0. The other groups of larvae were allowed to feed on the normal diet until day 4 when they became substantially heavier than the threshold weight and acquired the competence for metamorphosis, and then either starved throughout the 4th instar, or given a plain 2% agar block or a block containing 2% glucose, 8% glucose, 8% fructose, 8% trehalose, 8% sucrose, 8% casein or 8% starch. The agar block was replaced with a fresh piece every 2 days. Larvae were monitored daily and upon each molt, their weight and the date of the molt were recorded. 2.4. Hemolymph collection The larvae were ligated at the neck with a piece of thread. A small incision was made at the posterior segment using a sharp pair of scissors. Hemolymph (10 ml) was collected and immediately mixed with 50 ml of 100 nM EDTA in a microtube. The tube was heated to 90 1C for 3 min to denature proteins, and centrifuged at 1600 g for 5 min. The supernatant was transferred into another clean microtube, and stored at –20 1C until use.
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2.5. Trehalose and glucose assay
8
2.6. Chemicals The Glucose B-test TM kit and casein were obtained from Wako Pure Chemicals, Japan. Trehalose dehydrate, EDTA, D-()-fructose, soluble starch and agar powder were purchased from Nacalai Tesque, Inc. Kyoto, Japan. D-(+)-glucose and sucrose were obtained from Kanto Chemical Co., Japan. Trehalase was obtained from Sigma Chemicals, St. Louis, MO.
Glucose (mg/ml)
6
Statistical analyses were conducted using a software package, Statviews (SAS Institute Inc., NC, USA). Data were analyzed by ANOVA, and the significance of differences among means was determined with Tukey–Kramer’s multiple range test. In the tables and figures, values are presented as means standard deviations.
3. Results 3.1. Effect of starvation on glucose levels Fig. 1A shows the changes in the hemolymph glucose levels during the 4th instar of larvae fed throughout the instar, of those starved on the day of ecdysis, and of those starved after 4 days of feeding. For all the periods of time investigated, glucose levels in the normally fed larvae exhibited considerable variation. The hemolymph glucose level was low (2.2 mg/ml) on the day of ecdysis to the 4th instar, but increased as the larvae started feeding on the normal diet, reaching 3.9 mg/ml on day 4, and was then maintained at around this value for most of the time. When larvae were starved on day 0, glucose
4 3
1 0 0
2
(A)
4
6
8
10
12
14
Days in the 4th instar 40 Fed throughout Starved from day 0 Starved from day 4
30
20
10
0 0 (B)
2.7. Data analysis
5
2
Trehalose (mg/ml)
A Glucose B-test kit was used to determine the concentration of glucose. The hemolymph sample was thawed, vortexed for 30 s and centrifuged at 1600 g for 5 min. Aliquots (25 ml) of hemolymph were transferred to new microtubes. Twenty microliters of either phosphate buffer or trehalase solution (0.011 units) in phosphate buffer was placed into each tube and incubated at 37 1C for 1 h. Two microliters of either glucose or trehalose standard (0.5–4.0 mg/ml), or the test sample was placed in each well of a microplate, and 200 ml of coloring reagent (containing glucose oxidase, peroxidase and o-dianisidine) was added. After incubation at room temperature for 1 h, the absorbance of the wells at 490 nm was measured using a Bio-Rad 550 microplate reader. The amount of trehalose was estimated from the glucose concentrations in the trehalase-treated and untreated samples.
Fed throughout Starved from day 0 Starved from day 4
7
TM
1007
2
4 6 8 10 Days in the 4th instar
12
14
Fig. 1. Changes in hemolymph glucose (A) and trehalose (B) levels of the 4th instar P. hilaris larvae, which were either normally fed, or starved from the day of ecdysis, or starved after 4 days of feeding. Larvae were reared under a long-day photoperiod (15 h light and 9 h dark). Each data point represents the mean7SD of 4–6 separate measurements.
levels declined 1.6-fold in the next 24 h, and continued to decrease further as starvation progressed. When larvae were starved after 4 days of feeding, glucose levels decreased sharply (4-fold within the next 24 h), and with a slight fluctuation, finally declined to 0.2 mg/ml on day 13, the day the larvae entered the prepupa stage. Individual variations of the glucose titer in the starved larvae were small when compared with those in the fed larvae. 3.2. Effect of starvation on trehalose levels Hemolymph trehalose levels were 5-fold higher than glucose levels on the day of ecdysis to the 4th instar (compare Fig. 1A and B). As the larvae continued feeding on the normal diet, trehalose levels fluctuated between 12 and 16 mg/ml, but they started decreasing after day 10 and finally declined to 7 mg/ml on day 13 (Fig. 1B). When larvae were starved from day 0,
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trehalose levels started decreasing after 2 days, and were nearly undetectable by day 13. When larvae were starved after 4 days of feeding, trehalose levels increased greatly in the subsequent days of starvation reaching a peak of 23.5 mg/ml on day 12, one day before the larvae entered the prepupal stage.
3.3. Effect of neck ligation on glucose and trehalose levels When larvae were neck-ligated from day 0 of the 4th instar, hemolymph glucose levels decreased 7-fold in 2 days while trehalose levels decreased 2-fold (Table 1). When larvae were ligated after the first 4 days of feeding, hemolymph glucose levels declined 7-fold in 2 days while trehalose levels did not decline significantly.
Table 1 Effect of neck ligation on 4th instar Psacothea hilaris hemolymph glucose and trehalose levelsa Treatment
Glucose (mg/ml)b
Trehalose (mg/ml)b
Fed for 2 days Ligated for 2 days from day 0 Fed for 6 days Fed for 4 days and ligated for 2 days
3.371.1b 0.570.4a
13.772.4b 7.371.6a
3.671.9b 0.570.0a
17.277.1b 13.373.5b
a Larvae were either neck-ligated on the day of ecdysis or fed 4 days prior to ligation. b Values are the mean7SD of a minimum of 4 separate determinations. Means in the same column followed by the same letter are not significantly different at the 5% level.
3.4. Effect of feeding 8 % glucose from day 0
3.6. Ecdysis to the 5th instar
When 4th instar larvae were starved from day 0, most of them eventually died without pupating (Table 2). Feeding on 8% glucose was sufficient to promote metamorphosis in 78% (18 23) of the larvae. Interestingly, the larvae that pupated did so within a similar period of time as those fed the normal diet, suggesting that no premature pupation was induced. However, 13% (3 larvae) died before achieving metamorphosis. The individuals that pupated gave rise to pupae whose weight was 3-fold lower than that of pupae from larvae fed the normal diet (Table 2).
When fed the normal artificial diet, 56% of larvae molted to the 5th instar while the rest pupated with a mean larval period of 24 days (Table 3). Except for one individual, none of the starved larvae molted to the 5th instar. None of the larvae undertook another molt when fed plain agar either. When larvae were fed on different concentrations of glucose, they responded in a dosedependent manner, with 2% glucose eliciting 25% molt to the 5th instar while 8% glucose elicited a larval molting in as many as 84% of individuals (Table 3). Interestingly, feeding on 8% sucrose stimulated 52% of the larvae to molt to the 5th instar, which is similar to that elicited by the normal artificial diet. This result is reasonable because sucrose is included in the normal artificial diet as an ingredient. Of the sugars tested, fructose was the least effective in eliciting a molt to the 5th instar, followed by trehalose. Starch and casein, however, were the least effective of the substances tested, eliciting ecdysis to the 5th instar in only 12% of the larvae (Table 3).
3.5. Suppression of premature pupation When larvae were starved after 4 days of feeding, they all pupated prematurely with a mean duration of 19 days as opposed to those fed the normal diet that took 24 days (Table 3). However, feeding on glucose at a concentration as low as 2% was sufficient to prevent premature metamorphosis in P. hilaris; larvae pupated with a mean period of 25 days, which is similar to the pre-pupation period of the normally fed larvae. A higher concentration of glucose (8%) had a similar effect although in this case, only two individuals pupated from the 4th instar. Feeding on plain agar did not suppress this premature pupation; larvae pupated with a mean larval period of 21 days suggesting that the mechanical stimulus associated with swallowing is not responsible for the suppression. Among the nutrients tested, sucrose also suppressed premature pupation, but trehalose, fructose, starch and casein were ineffective (Table 3). Taken together, it appears that ingestion of specific sugars is a prerequisite for suppression of the starvationinduced premature pupation.
3.7. Pupal weight All larvae exhibited a similar weight gain when they were fed the normal diet for the first 4 days after ecdysis to the 4th instar (Table 3, F ¼ 1:3, df7,200; P40:5). However, after the consumption of the various test substances, the weight of the resulting pupae varied greatly (Table 3). Larvae fed on the normal diet throughout the instar produced the heaviest pupae, weighing 392 mg. Of all the substances tested, 8% glucose gave rise to the heaviest pupae though they weighed only 250 mg. Larvae fed 8% casein gave rise to pupae of the least weight (174 mg, Table 3).
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Table 2 Effect of feeding 8% glucose from the day of ecdysis on development of Psacothea hilaris under 15L:9Da Test substanceb
N
Weight on ecdysis to 4th instar (mg)c
% molt to 5th instar
% pupated from Duration of 4th 4th instar instar (days)c
Weight of pupae % died before (mg)c pupation
Starved 8% glucose Normal diet
30 23 27
189754a 174720a 205763a
0 (0) 4 (1) 56 (15)
7 (2) 78 (18) 44 (12)
13577a 143725a 392795b
19.572.1a 23.372.7b 23.872.1b
93 (28) 13 (3) 0
a
Numbers in parentheses indicate the numbers of individuals. Larvae were either starved or given agar blocks containing 8% glucose from the day of ecdysis. Control larvae were fed a normal diet throughout the 4th instar. c Values are means7SD. Duration of the 4th instar and weight of pupae refer to the individuals that pupated at the 4th instar. Means followed by the same letter in the same column are not significantly different at the 5% level. b
Table 3 Effect of feeding various dietary substances on premature pupation in Psacothea hilaris under 15L:9Da Test substanceb
N
Weight after 4d feeding normal dietc
% molt to 5th instar
% pupated from Duration of 4th 4th instar instarc
Weight of pupaec
% Permanent larvaed
Normal diet Starved 2% agar 2% glucose 8% glucose 8% sucrose 8% trehalose 8% fructose 8% starch 8% casein
27 32 25 24 25 23 28 24 24 26
374777a 369773a 346739a 354766a 340746a 344768a 350760a 349763a 342756a 350745a
56 3 0 25 84 52 14 29 13 12
44 97 100 75 8 48 86 71 88 88
392795d 221756b 211734b 221757b 250728c 234755bc 240754bc 239746bc 224744b 174742a
0 0 0 0 38 25 25 0 100 100
(15) (1) (0) (6) (21) (12) (4) (7) (3) (3)
(12) (31) (25) (18) (2) (11) (24) (17) (21) (23)
23.872.1c 19.071.9a 21.071.6b 24.671.7c 23.570.7c 23.572.1c 19.271.0a 21.972.5b 22.171.8b 20.771.4b
(8) (3) (1) (3) (3)
a
Numbers in parentheses indicate the numbers of individuals. Larvae were fed a normal artificial diet for the first 4 days after ecdysis to the 4th instar, then given an agar block containing a test substance. Control larvae were fed the normal diet throughout the 4th instar. c Values are means7SD. Means followed by the same letter in the same column are not significantly different at the 5% level. d The permanent larvae did not pupate even after 60 days in the 5th instar and are expressed as the % of larvae that became 5th instars. b
3.8. Permanent larvae Interestingly, some of the larvae that molted to the 5th instar after feeding on the various test substances survived as larvae for more than 60 days (Table 3). These larvae fed normally for the early part of the 5th instar but ceased feeding later in the instar, and they never started wandering nor became prepupae. We therefore considered these larvae to have become permanent larvae. Particularly interesting in the present experiment is the finding that all the larvae in the starch and casein treatments that molted to the 5th instar ended up as permanent larvae (Table 3). 3.9. Effect of feeding glucose on the hemolymph sugar levels When larvae were fed 8% glucose after 4 days of feeding on the normal diet, hemolymph glucose levels
fell 6-fold within the following 24 h, after which the low sugar levels were maintained throughout the experimental period (Fig. 2A). In contrast, hemolymph trehalose levels increased by 4% within the next 24 h of feeding (Fig. 2B). This was followed by a very large increase in the subsequent days as larvae consumed the glucose, reaching 50 mg/ml on day 13, when the majority of the larvae were found to ecdyse to the 5th instar. 3.10. Effect of feeding trehalose on the hemolymph sugar levels Overall, feeding on trehalose induced similar changes in the hemolymph glucose and trehalose titers as feeding on glucose. When larvae were fed on 8% trehalose after 4 days of feeding on the normal diet, hemolymph glucose levels fell 6-fold within the following 24 h (Fig. 2C). The sugar remained at around this level for
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8
8 Fed normal diet Fed 8%glucose
6 5 4 3 2
6 5 4 3 2
1
1
0
0
0
2
(A)
4 6 8 10 12 Days in the 4th instar
14
0
4 6 8 10 12 Days in the 4th instar
14
70
Fed normal diet Fed 8%glucose
60
2
(C)
Trehalose (mg/ml)
Trehalose (mg/ml)
70
50 40 30 20
Fed normal diet Fed 8%trehalose
60 50 40 30 20 10
10
0
0
0 (B)
Fed normal diet Fed 8%trehalose
7 Glucose (mg/ml)
Glucose (mg/ml)
7
2
4 6 8 10 12 Days in the 4th instar
0
14 (D)
2
4
6
8
10
12
14
Days in the 4th instar
Fig. 2. Hemolymph glucose (A, C) and trehalose (B, D) levels during the 4th instar of normally fed P. hilaris and of those fed 2% agar containing 8% glucose (A, B) or trehalose (C, D) after feeding on a normal diet for the first 4 days after ecdysis. All larvae were reared under the long-day photoperiod (15 h light and 9 h dark). Each data point represents the mean7SD of 4–6 separate measurements.
the rest of the time investigated. Hemolymph trehalose levels doubled within 24 h of feeding on 8% trehalose (Fig. 2D). The trehalose levels continued to increase in the subsequent days as the larvae fed on the trehalose, reaching 38.7 mg/ml on day 13 of the instar.
4. Discussion 4.1. Effect of starvation on glucose and trehalose titers One of the most intriguing findings in the present study is the differential response of the hemolymph trehalose levels induced by starvation initiated at different times. Starvation on day 0 of the 4th instar results in a rapid decrease in the trehalose levels, while starvation on day 4, when the larvae become substantially heavier than the threshold weight and acquire the competence for metamorphosis, results in a marked increase in the hemolymph trehalose levels. In contrast, glucose levels showed a rapid decrease regardless of the timing of starvation. The effect of neck-ligation on the trehalose levels was also dependent on the timing of the operation: neck-ligation on day 0 resulted in a decrease in the hemolymph trehalose levels while ligation on day 4 had no significant effect on its level. These findings suggest that the differences in response are likely to be attributable to the change
in the nature of the fat body (the site of trehalose synthesis), which has occurred during the 4 days of feeding, and that if neurohormones are involved in the change, they are not likely to be released from the cephalic part. In Bombyx mori, starvation after 3 days of feeding in the 4th instar larvae led to a decrease in the hemolymph glucose level and a significant increase in the trehalose level (Satake et al., 2000). Satake et al. (2000) observed an increase in the active form of glycogen phosphorylase and a decrease in the glycogen content in the fat body of the starved larvae, suggesting that trehalose production from glycogen was enhanced during starvation. Thus, the observed increase in the hemolymph trehalose level in P. hilaris larvae starved after 4 days of feeding may be due to the breakdown of glycogen to produce trehalose to meet the energy demands for metamorphosis. On the other hand, the low glucose levels in the starved larvae indicate that the glucose derived from glycogen was used for the synthesis of trehalose within the fat body, and was not released into the hemolymph (Siegert, 1987b). We noted considerable variation in the trehalose and glucose levels of the normally fed larvae (Fig. 1A and B). This variation was particularly evident after day 4 of the 4th instar. Under normally fed conditions, 50% of 4th instar larvae pupate at the end of this instar; the rest molt to the 5th instar on day 13 (Shintani et al., 1996). Thus, it is reasonable to attribute this variation to the
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two different populations of individuals having distinct developmental fates.
4.2. Effect of feeding glucose or trehalose on their hemolymph titers When larvae were fed on 8% glucose from day 4 of the 4th instar, hemolymph glucose levels declined more than 6-fold while trehalose levels increased remarkably. This observation is similar to that of Evans and Dethier (1957) who found that when the adult blowfly Phormia regina was fed glucose, hemolymph trehalose levels more than doubled while glucose levels declined more than 2-fold within the next 48 h. A similar increase in the hemolymph trehalose levels was noted in Manduca sexta 5th instar larvae after 3 days of feeding on a diet with increasing amounts of sucrose (Thompson et al., 2003), and also in Locusta migratoria fed on a diet containing high carbohydrate levels (Zanotto et al., 1996). These results suggest that trehalose was synthesized from the ingested sugars or carbohydrates. We suspect that a similar mechanism is operational in the case of P. hilaris; all the glucose ingested by the larvae was used for trehalose synthesis and therefore, this explains the greatly elevated trehalose levels (Fig. 2B and D). We also suspect that all the ingested trehalose is converted to glucose in the midgut cells before it is released into the hemolymph, and this is why ingestion of either glucose or trehalose induces almost the same response in their hemolymph titers.
4.3. Hemolymph glucose and trehalose levels as the cue for premature pupation Feeding either glucose or trehalose to starved P. hilaris 4th instar larvae resulted in entirely different developmental consequences. To our surprise, however, both glucose and trehalose induced similar responses in their hemolymph titers. These findings suggest that neither the hemolymph glucose nor the trehalose levels are the direct cue for the control of the CA activity, which is supposed to be shut off within 24 h of starvation (Munyiri and Ishikawa, 2005). Rather, ingestion of sugars (glucose and sucrose) may be constantly monitored, for instance, by epipharyngeal gustatory organs, and this information may be used to modulate the neuroendocrine system. Alternatively, the sugar levels might change on a much finer time scale, probably hourly, and this may be important in controlling the destiny of the starving larvae. We may have overlooked these fine changes because we examined only the daily changes in sugar levels.
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Acknowledgements We are 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 Scientific Research No. 16380039 from the Japan Society for the Promotion of Science (JSPS).
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