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Free amino acids of the haemolymph of the cotton leaf-worm, Spodoptera littoralis boisduval larvae (Lepidoptera: Noctuidae)
Comp. Biochem. Physiol., 1973, Vol. 45B, pp. 785 to 790. Pergamon Press. Printed in Great Britain
FREE AMINO ACIDS OF THE HAEMOLYMPH OF THE COTTON LEAF-WORM, SPODOPTERA LITTORALIS BOISDUVAL LARVAE (LEPIDOPTERA: NOCTUIDAE) I. Z. B O C T O R
and S. I. S A L E M
Laboratory of Plant Protection, National Research Centre, Dokki, Cairo, Egypt (Received 6 November 1972)
A b s t r a c t - - 1 . Free amino acids of the haemolymph of the second, third, fourth, fifth and sixth instar larvae of Spodoptera littoralis Boisduval, were
determined quantitatively by two-dimensional paper chromatography. 2. Nineteen amino acids were detected in the haemolymph of all instars. 3. Glutamine, threonine and lysine occurred in high concentrations and represent the largest part of free amino acids in all instars, while glutamic acid and citrulline were found in smaller quantities. 4. Glutamine and tyrosine showed a continuous decrease but histidine, citrulline and ?-aminobutyric acid increased steadily as development proceeded. 5. Other amino acids showed quantitative variations.
INTRODUCTION FLORKIN (1959), Gilmour (1961, 1965), Wyatt (1961), Chen (1962, 1966) and
Clements (1963) reviewed the numerous studies of free amino acids in insects. Most of these studies were undertaken to determine the titres of these substances in insect haemolymph, since high titres have been said to be characteristic of insects by Florkin (1959). Various authors have suggested that the amino acids may be concerned with osmotic homeostasis or protein synthesis (of. Buck, 1953), energy production for flight (Sacktor, 1961) or, as most dearly indicated by experimental evidence, cocoon construction (Wyatt, 1961). Also, many authors have demonstrated that the amino acids, in addition to their function as protein constituents enter into diverse metabolic pathways and participate in many other physiological activities. The primary aim of the present investigation was to determine the concentration of the free amino acids in 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 E1Ibrashy & Chenouda (1970). Haemolymph of the second, third, fourth, fifth and sixth instar larvae was obtained by cutting one or two of the abdominal legs. Haemolymph wells up at the wound site and was collected by microcapiUary tubes. The pooled haemolymph (0.5-1 ml) of each instar was kept immersed in a freezing mixture to prevent clotting and darkening. 785
786
I . Z . BOCTOR AND S, I. SALEM
+1+1 +1 ~
+1 +1 + 1 + 1 + 1 ~
+1+1 +1 +1 +1-4-1 +1+1 ~
+1+1-t-1+1+1+1+1+1+1+1+1+1+14-1+1-t-I+1+1+1
~,~
-H +1 +1 +1 +1+1 +1 + l + / + l
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+1 +1 +1 ~
+1+1 +1+1 +1
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~.
S~ O
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+1 -4-1 +1 -4-1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +~ ©
oL~ ",:l- t'-,I u'~ oO "~- ¢~ t'NI OO
'¢1- F'- ~
t---I v..~ ~
I-~¢~ c~
"O
_= "o ~ .~
~
"z ~
¢J
787
FREE AMINO ACIDS OF THE H A E M O L Y M P H OF COTTON LEAF-WORM LARVAE
All chemicals used wet'e of analytical reagent grade. For the preparation of amino acid extracts, 0.5-1 ml of the haemolymph was deproteinized and freed from fats according to the method of Pant & Agrawal (1964). Free amino acids in the haemolyrnph extracts were separated by two-dimensional paper chromatography according to the method of Levy et al. (1953). Thus, 20/~1 of the extract were applied as compact spots on Whatman No. 1 filter paper sheets (40 x 40 cm). The chromatograms were first developed with butanol-acetic acid-water (4 : 1 : 5) for 24 hr. The second development was made with m-cresol-phenolpH 8.3 borate buffer (30 g : 15 g : 7.5 ml). T h e method of Giri et al. (1952) was used for the quantitative estimation of the amino acids by eluting the ninhydrin-coloured dotted areas in 3 ml of 75 per cent ethanol containing 50 mg copper nitrate per 1. The tubes were then left in the dark for about 1-2 hr with occasional shaking. The optical density of the colours were measured on a Spekol photoelectric colorimeter at 510 n m against a blank extract prepared from a piece of the same filter paper of the same areas. The concentration of the separated amino acids were calculated from standard curves previously prepared using standard amino acids solutions and chromatographed for the same period using the same solvents. Proline was sprayed with isatin 0"2 per cent in acetone and eluted with acetone-water mixture (2 : 1) (Boctor, 1971). For each sample five to eight chromatographic separations were carried out, and the average and the experimental error were calculated (Table la). TABLE l b - - F r u m AMINOACIDSOF THE HAEMOLYMPHOF S. littoralis LARVAE
Amino acids Glycine Alanine Serine Threonine Valine Leucine Aspartic acid Asparagine Glutamic acid Glutamine Proline Lysine Arginine Histidine Tyrosine Citrulline Ornithine Cystine ~,-Amino butyric acid Totals
Second instar
Third instar
Fourth instar
Fifth instar
Sixth instar
5-01 2.75 5"78 8"97 4'82 3"54 2 "49 8-52 0"81 14"38 7"42 9'11 2"78 1"12 10"79 0.61 7"13 3"08 0.809
7"37 1"97 5"63 10-37 4"55 2"98 2 "49 5"66 1"30 19"62 3"02 11"60 2"58 4"25 3"32 1-22 7"10 4"39 0-504
4"65 2.94 3.97 10.94 4"02 2"51 4"14 9.74 2.11 19.11 1.93 5"64 2"68 5.56 4-61 1.58 6.75 5-07 1.97
4"13 2"96 3"74 12.84 6-61 3"34 2"67 4.68 1"11 16"79 5"24 7"98 3-83 6"88 4"35 1"67 5"29 3"24 2.45
4"22 2.53 6"04 10-20 3"88 2"75 6"49 5-63 0"97 14"58 3"38 11"41 3"78 7"79 2"35 1"33 7"24 2-40 2.94
99.92
99"92
99"92
99"80
99"91
Values are expressed as percentages by weight of the total content.
788
I . Z . BOCTOR AND S. I. SALEM
RESULTS During the larval development of S. littoralis, a total of seventeen amino acids and two amides have been identified on the two-dimensional paper chromatogram. The values of amino acids in the haemolymph of the larval instars so far studied are reported in Table la. To facilitate a comparison of the relative concentrations of amino acids at different larval instars the percentages of each amino acid in the total content are given in Table lb. Glutamine is the most abundant component, representing about 14-19 per cent of the total content of amino acids at all instars. Threonine and lysine also occur in high concentration, more than 8 per cent. Other amino acids namely glycine, serine, valine, asparagine, histidine, tyrosine and ornithine occur almost in the same proportions (4-8 per cent of the total amino acids content). On the other hand, alanine, leucine, aspartic acid, proline, arginine, cystine and 7-aminobutyric acid represent about 2-4 per cent of the total amino acids while citrulline and glutamic acid are present in smaller quantity, each representing about 1 per cent of the total amino acids. The percent quantity of glutamine and tyrosine decline as development proceeds (glutamine from 19 to 14.7 per cent and tyrosine from 10.3 to 2.3 per cent). On the other hand, the percentage of histidine, citrulline and y-aminobutyric acid showed a continuous rise with development. Other amino acids show quantitative variations at different instars. Tryptophan, methionine, cysteine, hydroxyproline and phenylalanine are not detected in the blood of S. littoralis larvae. DISCUSSION All of the amino acids commonly found in proteins have been identified in insect's blood, although some, such as methionine, cysteine, serine, hydroxyproline and tryptophan are found only in a few insects, or in low concentration (Gilmour, 1965). A complete assemblage of free protein forming amino acids with the exception of methionine, tryptophan, phenylalanine, cysteine and hydroxyproline was observed in the haemolymph of S. littoral#. It has been demonstrated in a number of insects that free glutamine is a major and in some insects the predominant component of the amino acid pool of haemolymph and certain tissues (Florkin, 1959; Kermack & Stein, 1959). In the present investigation it is also found that glutamine is the most abundant amino acid in the haemolymph of S. littoralis. Winteringham (1959) has speculated that glutamine in insects, by analogy with its physiological function in mammals is also concerned with ammonia transport and amide transfer. Gilmour (1965) and Chen (1966) verified that transamination occupies a central role in insect metabolism. Levenbook (1962) reported that glutamine plus glutamate in Prodenia eridania amounts to 10-20 per cent of the total concentration in the fat body and haemolymph whereas the value is closer to 50 per cent in the gut. Levenbook also stated that a large excess of glutamine over glutamate is atypical of insect haemolymph as of mammalian blood, but that in insect tissues the relative concentrations are considerably
FREE A M I N O ACIDS OF THE H A E M O L Y M P H OF COTTON LEAF-WORM LARVAE
789
more variable. The ratio of glutamine over glutamate for locust haemolymph is 9 (Kilby, in BeUamy, 1958) but 4"8 for haemolymph of the housefly (Price 1961). T h e corresponding ratio for S. littoralis ranges from 9.0 to 17"6 in the different instars. The concentration of tyrosine decreases, whereas proline shows slight changes during the larval development of S)odoptera. These amino acids are known to be involved in the synthesis of insect cuticle (Hackman, 1958). Gilmour (1965) reported that tyrosine builds up during larval feeding until the time of moulting, when it is used as the source of a phenolic tanning agent in the formation of the new cuticle. Our findings with S)odoptera show that there is a continuous increase of histidine as the larval development proceeds. Florkin (1959) found that the most remarkable change of the amino acid pattern during the fifth larval intar of Bombyx raori is due to a continuous increase in the concentration of histidine, and it remains at a high level during the whole pupal life. Florkin also reported that histidine not being used to make silk in B. raori increases in the haemolymph in spite of an excretion of some of the histidine introduced with the food. REFERENCES
BELLAMYD. (1958) The structure and metabolic properties of tissue preparations from Schistocerca gregaria (desert locust). Biochem..7. 70, 580-589. BOCTOR F. N. (1971) An improved method for colorimetric determination of proline with isatin. Analyt. Biochem. 43, 66-70. Buck J. B. (1953) The internal environment in regulation and metamorphosis. In Insect Physiology (Edited by ROEDERK.), pp. 191-217. Wiley, New York. CHEN P. S. (1962) Free amino acids in insects. In Amino Acid Pools (Edited by HOLDEN J. T.), pp. 115-135. Elsevier, Amsterdam. CHEN P. S. (1966) Amino acid and protein metabolism in insect development. In Advances in Insect Physiology (Edited by BEAMENTJ. W L., TREHE~E J. E. & WIGGLESWORTH V. B.), Vol. 3, pp. 53-132. Academic Press, New York. CLEMENTSA. M. (1963) The Physiology of Mosquitoes. Pergamon Press, Oxford. EL-IBRASHYM. T. & CrlENOUDAM. S. (1970) Cholinesterase activity in Spodoptera littoralis Boisd., in relation to development and metamorphosis. Z. angew. Entomol. 65, 146-156. FLORKINM. (1959) The free amino acids of insect haemolymph. In Biochemistry of Insects (Edited by LEVENBOOKL.), pp. 63-77. Pergamon Press, London. GILMOURD. (1961) The Biochemistry of Insects. Academic Press, New York. GILMOURD. (1965) The Metabolism of Insects. Oliver & Boyd, London. GIRI K. V., RADHAKRISHNANA. N. & VAIDYANATHANC. S. (1952) Some factors influencing the quantitative determination of amino acids separated by circular paper chromatography. Analyt. Chem. 24, 1677-1678. HACKMANR. H. (1958) Biochemistry of the insect cuticle. Proc. I V Int. Congr. Biochem. Vienna 12, 48-62. KERMACKW. O. & STEINJ. M. (1959) Nitrogenous constituents of the thoracic muscle of the African migratory locust (Locusta migratoria migratorioides). Biochem. ~. 71, 648-654. LE~'~-BOOKL. (1962) The distribution of free amino acids glutamine and glutamate in the southern armyworm Prodenia eridania. J. Insect Physiol. 8, 559-567. LEvYA. L. & CHUNGD. (1953) Two-dimensional chromatography of amino acids on buffered papers. Analyt. Chem. 25, 396-399.
790
I. Z. BOCTOR AND S. I. SALEM
PANT R. & AGRAWALH. C. (1964) Free amino acids of the haemolymph of some insects. J. Insect Physiol. 10, 443--446. PRICE G. (1961) Some aspects of amino acid metabolism in the adult housefly, Musca domestica. Biochem. J. 80, 420-428. SACKTOR B. (1961) The role of mitochondria in respiratory metabolism of flight muscle. 2t. Rev. Ent. 6, 103-130. WINTERINGHAMF. P. W. (1959) Comparative aspects of insect biochemistry with particular reference to insecticidal action. In Biochemistry of Insects (Edited by LEVENBOOKL.), pp. 201-215. Pergamon Press, London. WYATT J. R. (1961) The biochemistry of insect haemolymph. A. Rev. Ent. 6, 75-102. Key Word Index--Amino acids; insect amino acids; Spodoptera littoralis.
FREE AMINO ACIDS OF THE HAEMOLYMPH OF THE COTTON LEAF-WORM, SPODOPTERA LITTORALIS BOISDUVAL LARVAE (LEPIDOPTERA: NOCTUIDAE) I. Z. B O C T O R
and S. I. S A L E M
Laboratory of Plant Protection, National Research Centre, Dokki, Cairo, Egypt (Received 6 November 1972)
A b s t r a c t - - 1 . Free amino acids of the haemolymph of the second, third, fourth, fifth and sixth instar larvae of Spodoptera littoralis Boisduval, were
determined quantitatively by two-dimensional paper chromatography. 2. Nineteen amino acids were detected in the haemolymph of all instars. 3. Glutamine, threonine and lysine occurred in high concentrations and represent the largest part of free amino acids in all instars, while glutamic acid and citrulline were found in smaller quantities. 4. Glutamine and tyrosine showed a continuous decrease but histidine, citrulline and ?-aminobutyric acid increased steadily as development proceeded. 5. Other amino acids showed quantitative variations.
INTRODUCTION FLORKIN (1959), Gilmour (1961, 1965), Wyatt (1961), Chen (1962, 1966) and
Clements (1963) reviewed the numerous studies of free amino acids in insects. Most of these studies were undertaken to determine the titres of these substances in insect haemolymph, since high titres have been said to be characteristic of insects by Florkin (1959). Various authors have suggested that the amino acids may be concerned with osmotic homeostasis or protein synthesis (of. Buck, 1953), energy production for flight (Sacktor, 1961) or, as most dearly indicated by experimental evidence, cocoon construction (Wyatt, 1961). Also, many authors have demonstrated that the amino acids, in addition to their function as protein constituents enter into diverse metabolic pathways and participate in many other physiological activities. The primary aim of the present investigation was to determine the concentration of the free amino acids in 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 E1Ibrashy & Chenouda (1970). Haemolymph of the second, third, fourth, fifth and sixth instar larvae was obtained by cutting one or two of the abdominal legs. Haemolymph wells up at the wound site and was collected by microcapiUary tubes. The pooled haemolymph (0.5-1 ml) of each instar was kept immersed in a freezing mixture to prevent clotting and darkening. 785
786
I . Z . BOCTOR AND S, I. SALEM
+1+1 +1 ~
+1 +1 + 1 + 1 + 1 ~
+1+1 +1 +1 +1-4-1 +1+1 ~
+1+1-t-1+1+1+1+1+1+1+1+1+1+14-1+1-t-I+1+1+1
~,~
-H +1 +1 +1 +1+1 +1 + l + / + l
~_~
O t~
+1 +1 +1 ~
+1+1 +1+1 +1
t~
© m c~ O + I - H +1 + 1 + 1 + 1 +1 +1 +1 +1 +t +1 +1 +1 + l + l
+1 +1 +1
~.
S~ O
z
o
r-. "O
+1 -4-1 +1 -4-1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +1 +~ ©
oL~ ",:l- t'-,I u'~ oO "~- ¢~ t'NI OO
'¢1- F'- ~
t---I v..~ ~
I-~¢~ c~
"O
_= "o ~ .~
~
"z ~
¢J
787
FREE AMINO ACIDS OF THE H A E M O L Y M P H OF COTTON LEAF-WORM LARVAE
All chemicals used wet'e of analytical reagent grade. For the preparation of amino acid extracts, 0.5-1 ml of the haemolymph was deproteinized and freed from fats according to the method of Pant & Agrawal (1964). Free amino acids in the haemolyrnph extracts were separated by two-dimensional paper chromatography according to the method of Levy et al. (1953). Thus, 20/~1 of the extract were applied as compact spots on Whatman No. 1 filter paper sheets (40 x 40 cm). The chromatograms were first developed with butanol-acetic acid-water (4 : 1 : 5) for 24 hr. The second development was made with m-cresol-phenolpH 8.3 borate buffer (30 g : 15 g : 7.5 ml). T h e method of Giri et al. (1952) was used for the quantitative estimation of the amino acids by eluting the ninhydrin-coloured dotted areas in 3 ml of 75 per cent ethanol containing 50 mg copper nitrate per 1. The tubes were then left in the dark for about 1-2 hr with occasional shaking. The optical density of the colours were measured on a Spekol photoelectric colorimeter at 510 n m against a blank extract prepared from a piece of the same filter paper of the same areas. The concentration of the separated amino acids were calculated from standard curves previously prepared using standard amino acids solutions and chromatographed for the same period using the same solvents. Proline was sprayed with isatin 0"2 per cent in acetone and eluted with acetone-water mixture (2 : 1) (Boctor, 1971). For each sample five to eight chromatographic separations were carried out, and the average and the experimental error were calculated (Table la). TABLE l b - - F r u m AMINOACIDSOF THE HAEMOLYMPHOF S. littoralis LARVAE
Amino acids Glycine Alanine Serine Threonine Valine Leucine Aspartic acid Asparagine Glutamic acid Glutamine Proline Lysine Arginine Histidine Tyrosine Citrulline Ornithine Cystine ~,-Amino butyric acid Totals
Second instar
Third instar
Fourth instar
Fifth instar
Sixth instar
5-01 2.75 5"78 8"97 4'82 3"54 2 "49 8-52 0"81 14"38 7"42 9'11 2"78 1"12 10"79 0.61 7"13 3"08 0.809
7"37 1"97 5"63 10-37 4"55 2"98 2 "49 5"66 1"30 19"62 3"02 11"60 2"58 4"25 3"32 1-22 7"10 4"39 0-504
4"65 2.94 3.97 10.94 4"02 2"51 4"14 9.74 2.11 19.11 1.93 5"64 2"68 5.56 4-61 1.58 6.75 5-07 1.97
4"13 2"96 3"74 12.84 6-61 3"34 2"67 4.68 1"11 16"79 5"24 7"98 3-83 6"88 4"35 1"67 5"29 3"24 2.45
4"22 2.53 6"04 10-20 3"88 2"75 6"49 5-63 0"97 14"58 3"38 11"41 3"78 7"79 2"35 1"33 7"24 2-40 2.94
99.92
99"92
99"92
99"80
99"91
Values are expressed as percentages by weight of the total content.
788
I . Z . BOCTOR AND S. I. SALEM
RESULTS During the larval development of S. littoralis, a total of seventeen amino acids and two amides have been identified on the two-dimensional paper chromatogram. The values of amino acids in the haemolymph of the larval instars so far studied are reported in Table la. To facilitate a comparison of the relative concentrations of amino acids at different larval instars the percentages of each amino acid in the total content are given in Table lb. Glutamine is the most abundant component, representing about 14-19 per cent of the total content of amino acids at all instars. Threonine and lysine also occur in high concentration, more than 8 per cent. Other amino acids namely glycine, serine, valine, asparagine, histidine, tyrosine and ornithine occur almost in the same proportions (4-8 per cent of the total amino acids content). On the other hand, alanine, leucine, aspartic acid, proline, arginine, cystine and 7-aminobutyric acid represent about 2-4 per cent of the total amino acids while citrulline and glutamic acid are present in smaller quantity, each representing about 1 per cent of the total amino acids. The percent quantity of glutamine and tyrosine decline as development proceeds (glutamine from 19 to 14.7 per cent and tyrosine from 10.3 to 2.3 per cent). On the other hand, the percentage of histidine, citrulline and y-aminobutyric acid showed a continuous rise with development. Other amino acids show quantitative variations at different instars. Tryptophan, methionine, cysteine, hydroxyproline and phenylalanine are not detected in the blood of S. littoralis larvae. DISCUSSION All of the amino acids commonly found in proteins have been identified in insect's blood, although some, such as methionine, cysteine, serine, hydroxyproline and tryptophan are found only in a few insects, or in low concentration (Gilmour, 1965). A complete assemblage of free protein forming amino acids with the exception of methionine, tryptophan, phenylalanine, cysteine and hydroxyproline was observed in the haemolymph of S. littoral#. It has been demonstrated in a number of insects that free glutamine is a major and in some insects the predominant component of the amino acid pool of haemolymph and certain tissues (Florkin, 1959; Kermack & Stein, 1959). In the present investigation it is also found that glutamine is the most abundant amino acid in the haemolymph of S. littoralis. Winteringham (1959) has speculated that glutamine in insects, by analogy with its physiological function in mammals is also concerned with ammonia transport and amide transfer. Gilmour (1965) and Chen (1966) verified that transamination occupies a central role in insect metabolism. Levenbook (1962) reported that glutamine plus glutamate in Prodenia eridania amounts to 10-20 per cent of the total concentration in the fat body and haemolymph whereas the value is closer to 50 per cent in the gut. Levenbook also stated that a large excess of glutamine over glutamate is atypical of insect haemolymph as of mammalian blood, but that in insect tissues the relative concentrations are considerably
FREE A M I N O ACIDS OF THE H A E M O L Y M P H OF COTTON LEAF-WORM LARVAE
789
more variable. The ratio of glutamine over glutamate for locust haemolymph is 9 (Kilby, in BeUamy, 1958) but 4"8 for haemolymph of the housefly (Price 1961). T h e corresponding ratio for S. littoralis ranges from 9.0 to 17"6 in the different instars. The concentration of tyrosine decreases, whereas proline shows slight changes during the larval development of S)odoptera. These amino acids are known to be involved in the synthesis of insect cuticle (Hackman, 1958). Gilmour (1965) reported that tyrosine builds up during larval feeding until the time of moulting, when it is used as the source of a phenolic tanning agent in the formation of the new cuticle. Our findings with S)odoptera show that there is a continuous increase of histidine as the larval development proceeds. Florkin (1959) found that the most remarkable change of the amino acid pattern during the fifth larval intar of Bombyx raori is due to a continuous increase in the concentration of histidine, and it remains at a high level during the whole pupal life. Florkin also reported that histidine not being used to make silk in B. raori increases in the haemolymph in spite of an excretion of some of the histidine introduced with the food. REFERENCES
BELLAMYD. (1958) The structure and metabolic properties of tissue preparations from Schistocerca gregaria (desert locust). Biochem..7. 70, 580-589. BOCTOR F. N. (1971) An improved method for colorimetric determination of proline with isatin. Analyt. Biochem. 43, 66-70. Buck J. B. (1953) The internal environment in regulation and metamorphosis. In Insect Physiology (Edited by ROEDERK.), pp. 191-217. Wiley, New York. CHEN P. S. (1962) Free amino acids in insects. In Amino Acid Pools (Edited by HOLDEN J. T.), pp. 115-135. Elsevier, Amsterdam. CHEN P. S. (1966) Amino acid and protein metabolism in insect development. In Advances in Insect Physiology (Edited by BEAMENTJ. W L., TREHE~E J. E. & WIGGLESWORTH V. B.), Vol. 3, pp. 53-132. Academic Press, New York. CLEMENTSA. M. (1963) The Physiology of Mosquitoes. Pergamon Press, Oxford. EL-IBRASHYM. T. & CrlENOUDAM. S. (1970) Cholinesterase activity in Spodoptera littoralis Boisd., in relation to development and metamorphosis. Z. angew. Entomol. 65, 146-156. FLORKINM. (1959) The free amino acids of insect haemolymph. In Biochemistry of Insects (Edited by LEVENBOOKL.), pp. 63-77. Pergamon Press, London. GILMOURD. (1961) The Biochemistry of Insects. Academic Press, New York. GILMOURD. (1965) The Metabolism of Insects. Oliver & Boyd, London. GIRI K. V., RADHAKRISHNANA. N. & VAIDYANATHANC. S. (1952) Some factors influencing the quantitative determination of amino acids separated by circular paper chromatography. Analyt. Chem. 24, 1677-1678. HACKMANR. H. (1958) Biochemistry of the insect cuticle. Proc. I V Int. Congr. Biochem. Vienna 12, 48-62. KERMACKW. O. & STEINJ. M. (1959) Nitrogenous constituents of the thoracic muscle of the African migratory locust (Locusta migratoria migratorioides). Biochem. ~. 71, 648-654. LE~'~-BOOKL. (1962) The distribution of free amino acids glutamine and glutamate in the southern armyworm Prodenia eridania. J. Insect Physiol. 8, 559-567. LEvYA. L. & CHUNGD. (1953) Two-dimensional chromatography of amino acids on buffered papers. Analyt. Chem. 25, 396-399.
790
I. Z. BOCTOR AND S. I. SALEM
PANT R. & AGRAWALH. C. (1964) Free amino acids of the haemolymph of some insects. J. Insect Physiol. 10, 443--446. PRICE G. (1961) Some aspects of amino acid metabolism in the adult housefly, Musca domestica. Biochem. J. 80, 420-428. SACKTOR B. (1961) The role of mitochondria in respiratory metabolism of flight muscle. 2t. Rev. Ent. 6, 103-130. WINTERINGHAMF. P. W. (1959) Comparative aspects of insect biochemistry with particular reference to insecticidal action. In Biochemistry of Insects (Edited by LEVENBOOKL.), pp. 201-215. Pergamon Press, London. WYATT J. R. (1961) The biochemistry of insect haemolymph. A. Rev. Ent. 6, 75-102. Key Word Index--Amino acids; insect amino acids; Spodoptera littoralis.