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Carbohydrates in the haemolymph of the cotton leaf-worm, Spodoptera littoralis Boisduval larvae (Lepidoptera: Noctuidae)
Comp. Biochem. Physiol., 1974, VoL 49B, pp. 79 to 82. Pergamon Press. Printed in Great Britain
CARBOHYDRATES IN THE HAEMOLYMPH OF THE COTTON LEAF-WORM, SPODOPTERA L I T T O R A L I S BOISDUVAL LARVAE (LEPIDOPTERA: NOCTUIDAE) I. Z. B O C T O R Laboratory of Plant Protection, National Research Centre, Dokki, Cairo, Egypt (Received 10 September 1973)
Abstract--1. Carbohydrates in the haemolymph of the second, third, fourth, fifth and sixth instar larvae of Spodoptera littoralis Boisduval were determined quantitatively by thin-layer chromatography. 2. Trehalose was shown to be the principal blood sugar in the haemolymph of all instars. A small amount of glucose spot was usually present. 3. Trehalose showed a continuous increase as development proceeded. INTRODUCTION THE NON-REDUCING disaccharide trehalose (1-[c~-D-glucopyranosyl]-a-D-glucopyranoside) is the principal sugar in the haemolymph of many different insects (Wyatt & Kalf, 1957; Wyatt, 1961). In most species of insects it seems to have a physiological role comparable to that of glucose in the blood of vertebrates (Gilmour, 1965). It has been demonstrated that trehalose is actively synthesized in the fat bodies of insects and decreases during active movement and starvation (Clegg & Evans, 1961; Saito, 1963). The haemolymph trehalose levels respond strikingly to the nutritional state, i.e. quantity and quality of food intake (Hansen, 1964); to developmental stages of insects (Howden & Kilby, 1960); and to physiological conditions (Nowosielski & Patton, 1964). The purpose of this study was to determine the carbohydrate components of the haemolymph of the cotton leaf-worm, Spodoptera littoralis, during its larval development. MATERIALS AND METHODS Spodoptera littoralis was reared on castor-oil leaves in the laboratory according to EI-
Ibrashy & Chenouda (1970). Haemolymph was collected according to the method described in an earlier paper (Boctor & Salem, 1973). The procedure of Pant & Agrawal (1964) was used for the preparation of sugar extracts from the haemolymph. Analar chemicals were employed for all solvents. Sugars in the haemolymph extracts were separated by thin-layer chromatography according to the following procedure. A slurry of silica gel-G (Stahl), prepared by mixing 25 g of the silica gel and 55 ml distilled water, was applied in a layer 250 pm thick, on 20 x 20 cm'glass plates. The plates were heated at 100-110°C for 2 hr and were stored in a desiccator. Individual sugars used as 79
80
I . Z . BOCTOR
reference standards were spotted on the chromatoplates as single components and mixtures. T h r e e solvents were used: (1) n-butanol-acetic acid-water ( 3 : 1 : 1 v/v); (2) n-butanolacetic acid-ether-water (9 : 6 : 3 : 1 v/v); (3) n-butanol-acetic acid-water (4 : 1 : 1 v/v). This last solvent proved very satisfactory. T h e position of the separated sugars was detected by spraying the plates with diphenylamine-aniline-phosphoric acid reagent prepared according to the procedure of Buchan & Savage (1952). Development was brought about by heating in an oven at 110°C for 30 min. For the quantitative separation of sugars in the haemolymph extracts, the extracts were applied to the chromatoplates in 20/zl aliquots using a micropipet until a total of six to seven 20-/zl aliquots were placed in discrete spots across the plate. An additional 20-/zl aliquot was applied to one side of the plate, slightly apart from the rest, to serve as a guide in marking the plates, and the chromatoplates were developed using the solvent system nbutanol-acetic acid-water (4 : 1 : 1 v/v). Only the guide sample was sprayed with diphenylamine-aniline-phosphoric acid and was used as a marker in locating the separated sugars. After the plates were marked, trehalose spots were removed by scraping the silica gel off across the plate with a razor blade and collecting it in small test-tubes. T h e sugar was thoroughly extracted with 1-2 ml of distilled water followed by filtration through a small sintered glass funnel. An aliquot (0"5 ml) of this sugar extract was mixed with 3 ml of anthrone reagent prepared according to Mokrasch (1954). Colour was developed by heating at 100°C for 15 min and read at 620 m/~ in the Unicam colorimeter. Total sugars in the haemolymph extracts were also determined quantitatively by reaction with anthrone. Trehalose served as the calibration standard, and the total sugar was expressed as mg trehalose/100 ml haemolymph. RESULTS T h i n - l a y e r c h r o m a t o g r a p h y of h a e m o l y m p h o f S. littoralis larvae r e v e a l e d large a m o u n t s o f t r e h a l o s e a n d a little glucose w h i c h c o u l d n o t b e d e t e r m i n e d q u a n t i t a t i v e l y . T r e h a l o s e was f o u n d on all c h r o m a t o p l a t e s of h a e m o l y m p h a n d s h o w e d t h e R t v a l u e s as a u t h e n t i c t r e h a l o s e . T h e v a l u e s o f t o t a l s u g a r s a n d t h a t of t r e h a l o s e in t h e h a e m o l y m p h o f t h e larval i n s t a r s so far s t u d i e d are r e p o r t e d in T a b l e 1. T A B L E 1 - - T R E H A L O S E AND TOTAL SUGARS IN THE H A E M O L Y M P H OF S .
Total sugars (mg/100 ml haemolymph) Second instar T h i r d instar Fourth instar Fifth instar Sixth instar
66-3 + 0"3 126"2 + 1-0 131 "1 + 1 "1 158"6 + 2"6 352"5 + 2"5
Trehalose (mg/100 ml haemolymph) 59"2 + 0"43 113"0 + 4'3 121 "6 + 2"3 145"6 + 2"9 288"0 + 1 "7
littoralis
LARVAE
Trehalose total sugars ratio (per cent) 89"3 89"5 92"7 91"8 81 "7
T r e h a l o s e s h o w e d a c o n t i n u o u s rise w i t h d e v e l o p m e n t ( f r o m 59.2 m g / 1 0 0 m l h a e m o l y m p h in t h e s e c o n d i n s t a r to 288.0 m g / 1 0 0 m l h a e m o l y m p h in t h e sixth instar). E a c h o f t h e t h i r d a n d s i x t h i n s t a r s c o n t a i n e d t w i c e as m u c h t r e h a l o s e as in t h e s e c o n d a n d fifth i n s t a r s r e s p e c t i v e l y . A l s o t h e t o t a l s u g a r s in t h e h a e m o l y m p h
C A R B O H Y D R A T E S I N H A E M O L Y M P H O F C O T T O N L E A F - W O R M LARVAE
81
increased continuously as development proceeded (from 66.3 mg/100 ml haemolymph in the second instar to 352-5 mg/100 ml haemolymph in the sixth instar). Trehalose was shown to be the principal blood sugar in the haemolymph of all instars; it comprises about 90 per cent of the total haemolymph sugars in the second, third, fourth and fifth instars and 81.7 per cent in the sixth instar. DISCUSSION In insect haemolymph, trehalose is the major and characteristic sugar although a small amount of glucose is also usually present (Wyatt, 1967). The state of high trehalose and low glucose in the haemolymph is not universal in insects. In Phormia regina larvae, for example, there is a high glucose concentration in comparison to that of the adult (Treherne, 1967). In some insect species, the fall in the blood trehalose level during muscular activity or starvation is due to the fact that trehalose acts as an immediately available carbohydrate reserve (Saito, 1960; Clegg & Evans, 1961). Therefore, blood glucose is maintained at a fixed level by a dynamic equilibrium between synthesis and breakdown of trehalose. Trehalose biosynthesis takes place in the fat body (Clegg & Evans, 1961 ; Saito, 1963) involving uridine diphosphate glucose (Kilby, 1965). It has been demonstrated in a number of insects that insect haemolymph often contains much trehalose so that the sugar can easily be obtained from it in crystalline form (Randall & Derr, 1965). Wyatt & Kalf (1956, 1957) reported that in ten types of insects, which they studied, trehalose was found in the larval and cocoon stage. They showed that trehalose comprises more than 90 per cent of the total carbohydrate in the haemolymph of four species of Lepidoptera and about 80 per cent in the Hymenoptera Diprion hercyniae. In the present investigation it is also found that trehalose comprises about 90 per cent of the total haemolymph sugars. Egorova (1963) reported that in Antheraea pernyi no trehalose was found in the wall of the gut; the fatty body and musculature had approximately equal amounts of trehalose and the haemolymph contained most trehalose (367.6-861.9 rag/100 g tissue). Glucose was found in the wall of gut, but almost none in the haemolymph. In the oak silkworm, trehalose is present throughout the life cycle, and undergoes a steady increase in its level from the egg (0.026 per cent of dry weight) to the pupa (up to 2.6 per cent) (Egorova & Smolin, 1962). DuchSteau-Bosson et al. (1963) working with Bombyx mori reported that the trehalose level in the larval blood rose to a maximum prior to the fourth and fifth instar moult. Our findings with Spodoptera show that there is a continuous rise of trehalose and total haemolymph sugars as the larval development proceeds. REFERENCES
BOCTORI. Z. & SALEMS. I. (1973) Free amino acids of the haemolymph of the c o t t o n leafworm, Spodopteralittoralis Boisduval larvae (Lepidoptera: Noctuidae). Comp.Biochem. Physiol. 45, 785-790. BUCHANJ. L. & SAVACER. I. (1952) Paper chromatography of some starch conversion products. Analyst 77, 401-406.
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CLEGG J. S. & EVANS D. R. (1961) Blood trehalose and flight metabolism in the blowfly. Science, Wash. 134, 54-55. DUCH~,TEAU-BossoN G., JEUNIAUX CH. & FLORKIN M. (1963) Contributions ~ la biochimie du ver {t s o i e - - X X V I I . Trehalose, trehalase et mue. Archs int. Physiol. Biochim. 71, 566-576. EGOROVAT. A. (1963) Trehalose in the tissues ofAntheraeapernyi. Nauch. Dokl. vyssh. Shk., Biol. Nauki 1963, 1, 88-91. (Cited from Chem. Abstr. 59, 924.) EGOROVA T. A. & SMOLIN A. N. (1962) Trehalose in the organism of the oak silkworm in different stages of its development. Biochemistry 27, 407-410. EL-IBRASHY M. T. & CHENOUDAM. S. (1970) Cholinesterase activity in Spodoptera littoralis Boisd., in relation to development and metamorphosis. Z. angew. Ent. 65, 146-156. GILMOUR D. (1965) The ~fetabolism of Insects. Oliver & Boyd, London. HANSEN O. (1964) Effect of diet on the amount and composition of locust blood carbohydrates. Biochem.J. 92, 333-337. HOWDEN G. F. & KILBY B. A. (1960) Biochemical studies on insect h a e m o l y m p h - - I . Variations in reducing power with age and the effect of diet. ft. Insect Physiol. 4, 258-269. KILBY B. A. (1965) Intermediary metabolism and the insect fat body. In Aspects of Insect Biochemistry (Edited by GOODWIN T. W.), pp. 39-48. Academic Press, New York. MOKRASCH L. C. (1954) Analysis of the hexose phosphates and sugar mixtures with the anthrone reagent. J. biol. Chem. 208, 55-59. NOWOSIELSKI J. W. & PATTON R. L. (1964) Variation in the haemolymph proteins, amino acids and lipid levels in adult house crickets, Acheta domesticus (L.) of different ages. J. Insect Physiol. 11,263-270. PANT R. & AGRAWALH. C. (1964) Free amino acids of the haemolymph of some insects. J. Insect Physiol. 10, 443-446. RANDALL D. D. & DERIR R. F. (1965) Trehalose: occurrence and relation to egg diapause and active transport in the differential grasshopper, Melanoplus differentialis, ft. Insect Physiol. 1, 329-335. SAITO S. (1960) Trehalose of the silkworm, Bombyx mori. Purification and properties of the enzyme. J. Biochem. Tokyo 48, 101-109. SAITO S. (1963) Trehalose in the body fluid of the silkworm, Bombyx mori L. J. Insect Physiol. 9, 509-519. TREHERNEJ. E. (1967) Gut absorption. A. Rev. Ent. 12, 43-58. WYATT G. R. (1961) T h e biochemistry of insect haemolymph. A. Rev. Ent. 6, 75-102. WYATT G. R. (1967) T h e biochemistry of sugars and polysaccharides in insects. In Advances in Insect Physiology (Edited by BEAMENTJ. W. L., TREHERNE J. E. & WIGGLESWORTH V. B.), Vol. 4, pp. 287-360. Academic Press, New York. WYATT G. R. & KALF G. F. (1956) Trehalose in insects. Fedn Proc. Fedn Am. Socs exp. Biol. 15, 388 (abstract 1269). WYATT G. R. & KALF G. F. (1957) T h e chemistry of insect h a e m o l y m p h - - I I . Trehalose and other carbohydrates. J. gen. Physiol. 40, 833-847.
Key Word Index--Trehalose ; haemolymph; Spodoptera Httoralis.
CARBOHYDRATES IN THE HAEMOLYMPH OF THE COTTON LEAF-WORM, SPODOPTERA L I T T O R A L I S BOISDUVAL LARVAE (LEPIDOPTERA: NOCTUIDAE) I. Z. B O C T O R Laboratory of Plant Protection, National Research Centre, Dokki, Cairo, Egypt (Received 10 September 1973)
Abstract--1. Carbohydrates in the haemolymph of the second, third, fourth, fifth and sixth instar larvae of Spodoptera littoralis Boisduval were determined quantitatively by thin-layer chromatography. 2. Trehalose was shown to be the principal blood sugar in the haemolymph of all instars. A small amount of glucose spot was usually present. 3. Trehalose showed a continuous increase as development proceeded. INTRODUCTION THE NON-REDUCING disaccharide trehalose (1-[c~-D-glucopyranosyl]-a-D-glucopyranoside) is the principal sugar in the haemolymph of many different insects (Wyatt & Kalf, 1957; Wyatt, 1961). In most species of insects it seems to have a physiological role comparable to that of glucose in the blood of vertebrates (Gilmour, 1965). It has been demonstrated that trehalose is actively synthesized in the fat bodies of insects and decreases during active movement and starvation (Clegg & Evans, 1961; Saito, 1963). The haemolymph trehalose levels respond strikingly to the nutritional state, i.e. quantity and quality of food intake (Hansen, 1964); to developmental stages of insects (Howden & Kilby, 1960); and to physiological conditions (Nowosielski & Patton, 1964). The purpose of this study was to determine the carbohydrate components of the haemolymph of the cotton leaf-worm, Spodoptera littoralis, during its larval development. MATERIALS AND METHODS Spodoptera littoralis was reared on castor-oil leaves in the laboratory according to EI-
Ibrashy & Chenouda (1970). Haemolymph was collected according to the method described in an earlier paper (Boctor & Salem, 1973). The procedure of Pant & Agrawal (1964) was used for the preparation of sugar extracts from the haemolymph. Analar chemicals were employed for all solvents. Sugars in the haemolymph extracts were separated by thin-layer chromatography according to the following procedure. A slurry of silica gel-G (Stahl), prepared by mixing 25 g of the silica gel and 55 ml distilled water, was applied in a layer 250 pm thick, on 20 x 20 cm'glass plates. The plates were heated at 100-110°C for 2 hr and were stored in a desiccator. Individual sugars used as 79
80
I . Z . BOCTOR
reference standards were spotted on the chromatoplates as single components and mixtures. T h r e e solvents were used: (1) n-butanol-acetic acid-water ( 3 : 1 : 1 v/v); (2) n-butanolacetic acid-ether-water (9 : 6 : 3 : 1 v/v); (3) n-butanol-acetic acid-water (4 : 1 : 1 v/v). This last solvent proved very satisfactory. T h e position of the separated sugars was detected by spraying the plates with diphenylamine-aniline-phosphoric acid reagent prepared according to the procedure of Buchan & Savage (1952). Development was brought about by heating in an oven at 110°C for 30 min. For the quantitative separation of sugars in the haemolymph extracts, the extracts were applied to the chromatoplates in 20/zl aliquots using a micropipet until a total of six to seven 20-/zl aliquots were placed in discrete spots across the plate. An additional 20-/zl aliquot was applied to one side of the plate, slightly apart from the rest, to serve as a guide in marking the plates, and the chromatoplates were developed using the solvent system nbutanol-acetic acid-water (4 : 1 : 1 v/v). Only the guide sample was sprayed with diphenylamine-aniline-phosphoric acid and was used as a marker in locating the separated sugars. After the plates were marked, trehalose spots were removed by scraping the silica gel off across the plate with a razor blade and collecting it in small test-tubes. T h e sugar was thoroughly extracted with 1-2 ml of distilled water followed by filtration through a small sintered glass funnel. An aliquot (0"5 ml) of this sugar extract was mixed with 3 ml of anthrone reagent prepared according to Mokrasch (1954). Colour was developed by heating at 100°C for 15 min and read at 620 m/~ in the Unicam colorimeter. Total sugars in the haemolymph extracts were also determined quantitatively by reaction with anthrone. Trehalose served as the calibration standard, and the total sugar was expressed as mg trehalose/100 ml haemolymph. RESULTS T h i n - l a y e r c h r o m a t o g r a p h y of h a e m o l y m p h o f S. littoralis larvae r e v e a l e d large a m o u n t s o f t r e h a l o s e a n d a little glucose w h i c h c o u l d n o t b e d e t e r m i n e d q u a n t i t a t i v e l y . T r e h a l o s e was f o u n d on all c h r o m a t o p l a t e s of h a e m o l y m p h a n d s h o w e d t h e R t v a l u e s as a u t h e n t i c t r e h a l o s e . T h e v a l u e s o f t o t a l s u g a r s a n d t h a t of t r e h a l o s e in t h e h a e m o l y m p h o f t h e larval i n s t a r s so far s t u d i e d are r e p o r t e d in T a b l e 1. T A B L E 1 - - T R E H A L O S E AND TOTAL SUGARS IN THE H A E M O L Y M P H OF S .
Total sugars (mg/100 ml haemolymph) Second instar T h i r d instar Fourth instar Fifth instar Sixth instar
66-3 + 0"3 126"2 + 1-0 131 "1 + 1 "1 158"6 + 2"6 352"5 + 2"5
Trehalose (mg/100 ml haemolymph) 59"2 + 0"43 113"0 + 4'3 121 "6 + 2"3 145"6 + 2"9 288"0 + 1 "7
littoralis
LARVAE
Trehalose total sugars ratio (per cent) 89"3 89"5 92"7 91"8 81 "7
T r e h a l o s e s h o w e d a c o n t i n u o u s rise w i t h d e v e l o p m e n t ( f r o m 59.2 m g / 1 0 0 m l h a e m o l y m p h in t h e s e c o n d i n s t a r to 288.0 m g / 1 0 0 m l h a e m o l y m p h in t h e sixth instar). E a c h o f t h e t h i r d a n d s i x t h i n s t a r s c o n t a i n e d t w i c e as m u c h t r e h a l o s e as in t h e s e c o n d a n d fifth i n s t a r s r e s p e c t i v e l y . A l s o t h e t o t a l s u g a r s in t h e h a e m o l y m p h
C A R B O H Y D R A T E S I N H A E M O L Y M P H O F C O T T O N L E A F - W O R M LARVAE
81
increased continuously as development proceeded (from 66.3 mg/100 ml haemolymph in the second instar to 352-5 mg/100 ml haemolymph in the sixth instar). Trehalose was shown to be the principal blood sugar in the haemolymph of all instars; it comprises about 90 per cent of the total haemolymph sugars in the second, third, fourth and fifth instars and 81.7 per cent in the sixth instar. DISCUSSION In insect haemolymph, trehalose is the major and characteristic sugar although a small amount of glucose is also usually present (Wyatt, 1967). The state of high trehalose and low glucose in the haemolymph is not universal in insects. In Phormia regina larvae, for example, there is a high glucose concentration in comparison to that of the adult (Treherne, 1967). In some insect species, the fall in the blood trehalose level during muscular activity or starvation is due to the fact that trehalose acts as an immediately available carbohydrate reserve (Saito, 1960; Clegg & Evans, 1961). Therefore, blood glucose is maintained at a fixed level by a dynamic equilibrium between synthesis and breakdown of trehalose. Trehalose biosynthesis takes place in the fat body (Clegg & Evans, 1961 ; Saito, 1963) involving uridine diphosphate glucose (Kilby, 1965). It has been demonstrated in a number of insects that insect haemolymph often contains much trehalose so that the sugar can easily be obtained from it in crystalline form (Randall & Derr, 1965). Wyatt & Kalf (1956, 1957) reported that in ten types of insects, which they studied, trehalose was found in the larval and cocoon stage. They showed that trehalose comprises more than 90 per cent of the total carbohydrate in the haemolymph of four species of Lepidoptera and about 80 per cent in the Hymenoptera Diprion hercyniae. In the present investigation it is also found that trehalose comprises about 90 per cent of the total haemolymph sugars. Egorova (1963) reported that in Antheraea pernyi no trehalose was found in the wall of the gut; the fatty body and musculature had approximately equal amounts of trehalose and the haemolymph contained most trehalose (367.6-861.9 rag/100 g tissue). Glucose was found in the wall of gut, but almost none in the haemolymph. In the oak silkworm, trehalose is present throughout the life cycle, and undergoes a steady increase in its level from the egg (0.026 per cent of dry weight) to the pupa (up to 2.6 per cent) (Egorova & Smolin, 1962). DuchSteau-Bosson et al. (1963) working with Bombyx mori reported that the trehalose level in the larval blood rose to a maximum prior to the fourth and fifth instar moult. Our findings with Spodoptera show that there is a continuous rise of trehalose and total haemolymph sugars as the larval development proceeds. REFERENCES
BOCTORI. Z. & SALEMS. I. (1973) Free amino acids of the haemolymph of the c o t t o n leafworm, Spodopteralittoralis Boisduval larvae (Lepidoptera: Noctuidae). Comp.Biochem. Physiol. 45, 785-790. BUCHANJ. L. & SAVACER. I. (1952) Paper chromatography of some starch conversion products. Analyst 77, 401-406.
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I . Z . BOCTOR
CLEGG J. S. & EVANS D. R. (1961) Blood trehalose and flight metabolism in the blowfly. Science, Wash. 134, 54-55. DUCH~,TEAU-BossoN G., JEUNIAUX CH. & FLORKIN M. (1963) Contributions ~ la biochimie du ver {t s o i e - - X X V I I . Trehalose, trehalase et mue. Archs int. Physiol. Biochim. 71, 566-576. EGOROVAT. A. (1963) Trehalose in the tissues ofAntheraeapernyi. Nauch. Dokl. vyssh. Shk., Biol. Nauki 1963, 1, 88-91. (Cited from Chem. Abstr. 59, 924.) EGOROVA T. A. & SMOLIN A. N. (1962) Trehalose in the organism of the oak silkworm in different stages of its development. Biochemistry 27, 407-410. EL-IBRASHY M. T. & CHENOUDAM. S. (1970) Cholinesterase activity in Spodoptera littoralis Boisd., in relation to development and metamorphosis. Z. angew. Ent. 65, 146-156. GILMOUR D. (1965) The ~fetabolism of Insects. Oliver & Boyd, London. HANSEN O. (1964) Effect of diet on the amount and composition of locust blood carbohydrates. Biochem.J. 92, 333-337. HOWDEN G. F. & KILBY B. A. (1960) Biochemical studies on insect h a e m o l y m p h - - I . Variations in reducing power with age and the effect of diet. ft. Insect Physiol. 4, 258-269. KILBY B. A. (1965) Intermediary metabolism and the insect fat body. In Aspects of Insect Biochemistry (Edited by GOODWIN T. W.), pp. 39-48. Academic Press, New York. MOKRASCH L. C. (1954) Analysis of the hexose phosphates and sugar mixtures with the anthrone reagent. J. biol. Chem. 208, 55-59. NOWOSIELSKI J. W. & PATTON R. L. (1964) Variation in the haemolymph proteins, amino acids and lipid levels in adult house crickets, Acheta domesticus (L.) of different ages. J. Insect Physiol. 11,263-270. PANT R. & AGRAWALH. C. (1964) Free amino acids of the haemolymph of some insects. J. Insect Physiol. 10, 443-446. RANDALL D. D. & DERIR R. F. (1965) Trehalose: occurrence and relation to egg diapause and active transport in the differential grasshopper, Melanoplus differentialis, ft. Insect Physiol. 1, 329-335. SAITO S. (1960) Trehalose of the silkworm, Bombyx mori. Purification and properties of the enzyme. J. Biochem. Tokyo 48, 101-109. SAITO S. (1963) Trehalose in the body fluid of the silkworm, Bombyx mori L. J. Insect Physiol. 9, 509-519. TREHERNEJ. E. (1967) Gut absorption. A. Rev. Ent. 12, 43-58. WYATT G. R. (1961) T h e biochemistry of insect haemolymph. A. Rev. Ent. 6, 75-102. WYATT G. R. (1967) T h e biochemistry of sugars and polysaccharides in insects. In Advances in Insect Physiology (Edited by BEAMENTJ. W. L., TREHERNE J. E. & WIGGLESWORTH V. B.), Vol. 4, pp. 287-360. Academic Press, New York. WYATT G. R. & KALF G. F. (1956) Trehalose in insects. Fedn Proc. Fedn Am. Socs exp. Biol. 15, 388 (abstract 1269). WYATT G. R. & KALF G. F. (1957) T h e chemistry of insect h a e m o l y m p h - - I I . Trehalose and other carbohydrates. J. gen. Physiol. 40, 833-847.
Key Word Index--Trehalose ; haemolymph; Spodoptera Httoralis.