Insect Biochem., Vol. 11, pp. 43 to 47. 0020-1790/81/0201--0043 $02.00/0 © Pergamon Press Ltd. 1981. Printed in Great Britain.

TEMPERATURE-DEPENDENT INTERCONVERSION BETWEEN GLYCOGEN A N D TREHALOSE IN DIAPAUSING

PUPAE OF PHILOSAMIA CYNTHIA RICINI A N D PRYERI

YOICHI HAYAKAWA and HARUO CHINO Biochemical Laboratory, Institute of Low Temperature Science, Hokkaido University, Sapporo, Japan

(Received 7 May 1980)

Abstraet--A temperature-dependent interconversion between fat body glycogen and haemolymph trehalose was demonstrated in diapausing pupae of the silkworm, Philosamia cynthia pryeri. When pupae at early- diapause stage were placed at 2°C for several weeks, the haemolymph trehalose content increased to about 35-50 mg/ml haemolymph, whereas the trehalose content of insects maintained at 20°C remained at 5-10 mg/ml. Concomitant with this change in haemolymph trehalose level, the glycogen content of the fat body dropped from 29-41 mg to 6.6-8.6 mg/g wet weight. This interconversion could be demonstrated repeatedly if the diapausing pupae were successively exposed to high and low temperatures, although the total amount of carbohydrates decreased slightly during repeated interconversions.

Non-diapausing pupae of the silkworm, Philosamia cynthia ricini, did not accumulate trehalose appreciably even when exposed to 2°C for a long period.

Key Word Index: Philosamia cynthia ricini and pryeri silkworms, pupae, fat body glycogen, haemolymph trehalose

I N T R O D U C T I O N

SINCE the finding (CHINO, 1957, 1958; WYATT and KALF, 1958) that glycogen is converted into such sugar alcohols as sorbitol and glycerol during the embryonic or pupal diapause of the silkworms Bombyx mori and Hyalophora cecropia, the accumulation of sugar alcohols in diapausing stages has been reported in many insects (see ASAHINA, 1969). In some diapausing insects (pupae of H. cecropia, prepupae of C. flavescence and adult carpenter ants Camponotus obscuripes), this conversion is temperature-dependent; the accumulation of glycerol occurs only when these insects are exposed to low temperatures (0-10°C) or the accumulation is considerably accelerated by such temperatures (ZIEGLER and WYATT, 1975; TAKEHARA, 1963; TANNO, 1962). In other minor groups of insects (for example, prepupae of Trichiocampuspopuli) sugar alcohols do not accumulate appreciably during diapause; instead, the amount of trehalose is considerably elevated (ASAHINA and TANNO, 1962; TANNO, 1965). However, the source of the trehalose has not been determined. The present report describes the build up of large concentrations of trehalose in the haemolymph of diapausing pupae of Philosamia cynthia silkworms, and provides evidence that during diapause in this insect almost all fat body glycogen is converted into trehalose. The conversion is largely temperature-dependent; trehalose is formed from glycogen only when the diapausing pupae are exposed to low temperature, whereas the reverse reaction proceeds when the pupae are exposed to high temperature.

M A T E R I A L S AND METHODS Animals

The pupae of Philosamia cynthia pryeri were collected from

43

the field late in September and used as diapausing pupae. The pupae of Philosamia cynthia ricini were kindly given by Dr. ISHIZAKl of Nagoya University and used as non-diapausing pupae. Haemolymph was collected from pupae by squeezing the fluid through a small cut on the head into a chilled centrifuge tube. Haemocytes were removed by centrifuging the haemolymph at 1500 g for 5 min.

Chemicals Anthrone and i-erythritol were purchased from Wako

Chemical Co., Japan, and Sigma Chemical Co., U.S.A., respectively. TRI-SIL 'Z' was obtained from Pierce Chemical Co., U.S.A., and used for trimethylsilylation prior to gas-liquid chromatography. D-( +)-Trehalose was purchased from Nakarai Chemical Co., Japan. All other chemicals were of analytical grade.

Gas-liquid chromatograph)' ( GLC) for haemolymph sugar To 0. I ml freshly collected haemolymph 0.1 ml standard

erythritol solution (10 mg/ml) was added, and the mixture was then evaporated at 60°C using a nitrogen evaporator. The residue was dried overnight under reduced pressure over P2Os. To the dried materials 0.4 ml TRI-SIL 'Z' was added and the solution was heated at 65°C for 2 hr to prepare the trimethylsilylated derivatives. The resulting derivatives were then applied to GLC (Shimadzu gas chromatograph, model GC-4CMPF) using a glass column, 3 m x 3 mm i.d. packed with 1.5% (w/w) OV-I on Chromosorb W.

Thin-layer chromatography ( TLC) Freshly collected haemolymph (0.8-1.0 ml) was subjected

to chromatography with a Sephadex G-50 column to separate low mol. wt. compounds from protein fractions. The column was eluted with distilled water, and the fraction eluting immediately after the protein fraction (monitored by absorption at 280 nm) was collected and submitted to TLC on silica gel 60 plate (Merck No. 5721). The plate was developed with isopropanol-acetone--0.1 M lactic acid (4:4:2 by vol), and was sprayed with anthrone/sulphuric acid reagent to locate the sugar spots. Standard trehalose and glucose were run as markers.

44 YOICH! HAYAKAWA AND HARUO CHINO

Determination o f fat body glycogen and haemolymph trehalose with anthrone/sulphuric acid reagent

Whole fat body dissected from a pupa was washed several times with saline (0.16 M KCI, 0.02 M NaCI), blotted with a filter paper and weighed. In most experiments, 200-500 mg fat body were homogenized with 10~ (w/v) trichloroacetic acid (20 ml/g tissue). The bomogenate was centrifuged at 10,000 g for 10 rain at 4°C and the glycogen content in an aliquot of the supernatant was determined by the anthrone/sulphuric acid method (DREYWOOD, 1946) using glucose as standard. In some experiments, prior to addition of the anthrone reagent, glycogen was precipitated by adding ethanol (final concentration 50~o v/v) to the extract. However, the amount of glycogen precipitated by ethanol was practically equal to the amount of glycogen contained in the original extract, therefore, the precipitation step by ethanol was omitted in most experiments.

Freshly collected haemolymph (0.1 ml) was mixed with 0.1 ml I0.% (w/v) trichloroacetic acid, and centrifuged at 10,000g for 10 min at 4°C. The amount of trehalose in the supernatant was determined by the anthrone/sulphuric acid method. In order to measure total haemolymph trehalose content of each animal, the following procedure was followed. After most of the haemolymph was collected in a small tube, the fat body and digestive tract were dissected and rinsed in a small beaker containing 50 ml saline. The remaining tissues (mainly cuticle) were also rinsed in the same beaker. Since a preliminary GLC analysis of trehalose in such (unwashed) tissues as fat body, gut and cuticular muscle indicated that the trehalose content in these tissues is almost negligible compared to that in haemolymph, it is unlikely that trehalose level in haemolymph is elevated appreciably with trehalose which may have leaked from the tissues during washing. The 'washing' was combined with the original haemolymph, and centrifuged at 1500 g for 5 min to remove small tissue

I: 1"

G

(e)

_h.___

G G

(b) (d)

"r ]-

Fig. 1. Gas-liquid chromatograms of the trimethylsilyl derivatives prepared from haemolymph of the diapausing pupae of P. cynthia pryeri. (a) Haemolymph of pupae maintained at 2°C for 2 months. (b) Haemolymph of pupae 9 days after transfer from 2°C to 24°C. (c) Haemolymph of pupae 3 days after transfer from 24 to 2°C. (d) Haemolymph of pupae maintained at 20°C after pupation. Column temperature was programmed from 130 to 270°C at 5°C/rain and held at 270°C. E: erythritol internal standard; G:

glucose; T: trehalose.

Glycogen and trehalose 45

fragments. An aliquot of the supernatant was then analysed to determine trehalose content according to the procedures described above.

RESULTS

Accumulation of trehalose in haemolymph at low temperature

Figures l(a) and (d) reveal that a considerable amount of trehalose accumulates in haemolymph of diapausing pupae of P. cynthia pryeri transferred to 2°C, whereas trehalose levels remain low when the pupa is maintained at 20°C after pupation. It is evident also that the glucose content is very low in either condition.

The accumulation of trehalose at 2°C was observed only when the pupae were transferred to 2°C from room temperature (20-25°C) at ten days or later after pupation, and did not occur if pupae were placed at 2°C immediately after pupation (data not shown). Figure l(b) demonstrates that if hypertrehalosemic pupae are transferred from 2 to 24°C, the accumulated trehalose falls to almost the same level observed in the haemolymph from pupae that were kept at room temperature after pupation. Figure l(c) indicates that the maximal trehalose level is regained only three days after returning the pupae from 24 to 2°C.

In order to demonstrate that the sugar accumulated in diapausing pupae at 2°C is solely trehalose, the

preparation from pupal haemolymph was analysed by TLC. The chromatogram obtained clearly reveals that trehalose is the major sugar in the haemolymph (chromatogram not shown).

Interconversion between fat body glycogen and haemolymph trehalose

The possibility that the trehalose which accumulates in cold-stressed pupae is derived from fat body glycogen- reserves was tested by measuring the quantities of haemolymph trehalose and of fat body glycogen in pupae exposed to low (2°C) and high temperature (24°C).

Figure 2 demonstrates that the trehalose which accumulates in haemolymph is steadily decreased when the pupa is transferred from 2 to 24°C; furthermore, concomitant with the decrease of trehalose, fat body glycogen levels become elevated. It is also evident from Fig. 2 that the reverse conversion occurs rapidly if pupae are transferred from 24 to 2°C. Although the results presented in Fig. 2 suggest an interconversion between haemolymph trehalose and fat body glycogen when the animals are exposed to low or high temperatures, the data indicate only the relative change in amounts of trehalose and glycogen. However, the total haemolymph trehalose and fat body glycogen contents were determined and given in Table 1. The total haemolymph trehalose content remain low (5.0--6.4 mg/g body weight) and the total fat body glycogen content remained high (13-15 mg/g

.3C

- ~ 2 C 09

c3

o xc

oc~

E

Z uJ t_o

1C t.J > -

Y

/

o o 2 c ~ c

i ~ § 4 5 ~ ~ 8 TIME [D]

9 10 I I 12

Fig. 2. Quantities of haemolymph trehalose and fat body glycogen of diapausing pupae of P. cynthia pryeri exposed to different temperatures. The pupae were kept at 2°C for three months before transferring to 24°C.

Abscissa: time (days) in different temperature conditions.

46 Yolcm HAYAKAWA AND HARUO CHINO

Table 1. Contents of total haemolymph trehalose and total fat body glycogen per individual*

Conditions Body Total

weight Total trehalose/Body weight Total glycogen/Body weight carbohydrate/Body weight (g) (mg/g) (mg/g) (mg/g)

At 20°C for 2.5 6.4 13 19.4 about 3 months after pupation 2.2 5.0 15 20.0

At 2°C for 3.8 19 2.7 21.7 about 3 months after pupation 3.5 20 2.7 22.7

Nine days after 3.3 9.7 8.8 18.5 transferring from 2 to 24°C 3.8 6.8 8.9 15.7

* Since it wag ~hown that within each group the total contents of trehalose and glycogen were almost proportional to body weight, the amounts of these carbohydrates were expressed as mg/g of body weight. Two pupae were tested for each condition.

body weight) when pupae were kept at 20°C after pupation; however, the trehalose level was elevated by three to four fold and glycogen was markedly reduced (2.7 mg/g body weight) when the pupae were exposed to 2°C. If pupae that had been kept at 2°C were shifted to 24°C, the total amount of trehalose was decreased considerably, being accompanied by a recovery of glycogen levels in fat body. Table 1 also indicates that total carbohydrate (trehalose plus glycogen) remains almost constant under different regimens, although repeated exposure of pupae to different temperatures causes some loss of carbohydrate. These data serve to substantiate the proposal that interconversion between haemolymph trehalose and fat body glycogen occurs in the diapausing pupae under the conditions defined in this study.

It was of obvious interest to determine if a similar interconversion occurs in non-diapausing pupae. To resolve this question, the developing pupae of P. cynthia ricini were exposed to 2°C and the trehalose content of haemolymph determined. As shown in Table 2, no accumulation of trehalose in haemolymph of the developing pupae was observed even after the pupae were exposed to 2°C for five days, thus suggesting that the interconversion between haemolymph trehalose and fat body glycogen is a phenomenon specific to diapausing pupae.

DISCUSSION

Two distinct patterns of carbohydrate metabolism

have been described in diapausing insects. In some species glycogen is converted to sugar alcohols such as sorbitol and glycerol (CHINO, 1957, 1958; WYATT and KALF, 1958; ASAHINA, 1969) whereas in other species there is no appreciable accumulation of sugar alcohols but instead large amounts of trehalose are formed (TANNO, 1965). The present study indicates that in diapausing pupae of P. cynthia pryeri, the large amount of trehalose that accumulates in the haemolymph is derived from fat body glycogen. The data presented in this report also demonstrate that this conversion between haemolymph trehalose and fat body glycogen is temperature-dependent; the conversion of glycogen to trehalose is induced by exposing pupae to low temperatures, and the reverse conversion occurs after returning the animals to a higher temperature (Fig. 1, 2; Table 1). This temperature-dependent interconversion appears to be a metabolic phenomenon analogous to the temperature-dependent interconversion between fat body glycogen and haemolymph glycerol observed in the diapausing pupae of H. cecropia or C. flavescence (WYATT, 1967; TAKEHARA, 1963).

The present finding, together with other reports concerning carbohydrate metabolism during diapause, support the suggestion that diapausing insects may be categorized into at least two types; those that accumulate sugar alcohol and those that accumulate trehalose. The former type seems more economical in terms of energy efficiency, because the conversion of glycogen to sugar alcohols can converse hydrogen atoms (CHINO, 1960), which subsequently

Table 2. Change in trehalose content of haemolymph of the pupae of P. cynthia ricini (non-diapause) and P. cynthia pryeri (diapause) when exposed to low temperature

Trehalose content* Pupae Conditions (mg/ml, +_ S.D.)

At 20°C for 2 months after pupation 6.5 + 1.9 Diapause

2 months after transferring to 2°C 46.0 _+ 15

At 20cC for 10 days after pupation 8.2 +_ 0.5

5 days after transferring to 2C at 10 days in developing 7.9 _+ 0.2 Non-diapause

* Five experiments for each value.

Glycogen and trehalose 47

serve to produce the ATP necessary for glycogen resynthesis; by contrast, the formation of trehalose from glycogen consumes one mole ATP for each molecule of trehalose produced and glycogen resynthesis from trehalose via trehalase, hexokinase, U D P G pyrophosphorylase and glycogen synthetase requires two moles of ATP for each addition of a glucose unit to the glycogen chain.

Preliminary experiments have revealed that a phosphorylase in the fat body of diapausing pupae of Philosamia species as well as that of diapausing pupae H. cecropia (ZIEGLER and WYATT, 1975), is considerably activated by exposure of the animals to cold temperatures (unpublished observation). An important question arises as to why, unlike diapausing pupae of H. cecropia the end product from glycogen in fat body is not glycerol but is trehalose. Investigations concerning the mechanism are currently in process.

Acknowledgements--The authors wish to thank Dr. R. G. H. DOWNER, University of Waterloo, for reading the manuscript. This study was supported in part by research grants from the Ministry of Education of Japan.

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ASAHINA E. and TANNO K. (1964) A large amount of trehalose in a frost resistant insect. Nature, 204, 1222.

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ZIEGLER R. and WYATT G. R. (1975) Phosphorylase and glycerol production activated by cold in diapausing silkmoth pupae. Nature, New Biol. 254, 622~i23.