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On the free amino acids in the haemolymph of a millipede
Comp. Biochem. Physiol., 1971, VoL 38B, pp. 1 to 4. Pergamon Press. Printed in Great Britain
O N T H E FREE A M I N O A C I D S IN T H E H A E M O L Y M P H OF A M I L L I P E D E V. S. KRISHNAN NAIR and V. K. K. PRABHU Department of Zoology, University of Kerala, Kariavattom, Trivandrum, India (Received 19 ffune 1970) A b s t r a c t - - 1 . Chromatographic studies on the free amino acids in the blood of the millipede~onespeltis splendidus revealed cystine, lysine, arginine, asparaglne,
serine, glycine, hydroxyproline, threonine, alanine, proline, tyrosine, methionine, valine, phenylalanine, leucine and histidine. 2. Serine, threonine and alanine occurred at a higher concentration in the blood of this species than the other amino acids. 3. All the above amino acids except cystine occurred at a higher concentration in the blood of the male. 4. Glycine and hydroxyproline were not detected in the blood of the female. 5. Total amino acid concentration in the blood of the female was found to be 51.7 _+9.6 rag/100 ml blood and that of the male, 108"3 +_6"1 rag/100 ml.
INTRODUCTION THE FREEamino acid concentration in the haemolymph of insects is known to be very high compared to that of other animals (Wyatt, 1961 ; Gilmour, 1965;Chen, 1966; Huggins & Munday, 1968). Sex-]imited differences with regard to the concentration of some amino acids have also been reported in insects (Duffy, 1964; Chen, 1966). W e have, however, very little information on the free amino acid p a t t e r n of the h a e m o l y m p h o f diplopods (Krishnan, 1968), a g r o u p p h y l o genetically closely related to the insects. T h e present r e p o r t is on the s t u d y o f free a m i n o acids in the blood of a m i l l i p e d e J o n e s p e l t i s splendidus. MATERIALS A N D METHODS Well-tanned males and females of the millipede Jonespeltis splendidus collected locally were used for the present study. Haemolymph from animals was collected from a dorsal puncture made on the "thoracic" segments, deproteinized by 80% ethanol and kept in a refrigerator overnight. It was centrifuged for 10 min at 1500 rev/min next morning. The protein-free supernatant containing free amino acids was used for the study. Supematant equivalent to 60 #1 haemolymph was evaporated to a residue at 60°C. This was assayed by the method of Lee & Takahashi (1966) as total ninhydrin-positive material against glutamic acid standard at 570 m/z using a Bausch & Lomb Spectronic 20 spectrophotometer. For chromatography supernatant containing amino acids equivalent to 0"1 ml blood evaporated to a residue and redissolved in a small quantity of 70% ethanol was used for spotting. Twodimensional thin-layer chromatography was carried out on plates coated with silica gel-G using chloroform-methanol-17% ammonia ( 2 : 2 : 1 v/v) in one direction and phenolwater (75 : 25 w/w) in the other. Spots were visualized by ninhydrin reagent (Randerath,
V. S. KRISHNAN NAIR AND V. K. K. PRABHU
1964). One-dimensional ascending paper chromatography was carried out overnight on Whatman No. 1 filter paper using butanol-acetic acid-water (450 : 50 : 125) after Banks & Randolph (1968). The chromatogram was dried at room temperature and run once more in the same solvent. After drying, the spots were visualized by ninhydrin reagent (Smith, 1960). The individual amino acids were identified by their RI values and by comparing the spots of standard amino acids in chromatograms run simultaneously. Paper chromatograms were scanned in a Densicord Densitometer (Photovolt Corporation), and the concentration of individual amino acids in the haemolymph was calculated from the total free amino acid concentration and the densitometric chart. RESULTS It was found by quantitative assay that the well-tanned female had a free amino acid concentration of only 51.7 + 9.6 mg ( M + S.D.) per 100 ml blood as against 108.3 +6-1 rag/100 ml in the male. T h i n layer chromatograms showed only a m a x i m u m of ten spots. On the other hand, there were a total of eighteen spots distinguishable in the paper chromatogram of which all except two were identified. T h e concentration of individual amino acids is given in Table 1. As the Table shows all amino acids except cystine were represented at a higher concentration in TABLE 1--FREE AMINOACIDCONCENTRATIONIN THE HAEMOLYMPHOF THE MILLIPEDE
ffonespeltis splendidus Concentration of amino acids (mg/100 ml blood) Amino acids
Male
Cystine 1.39 Lysine 4.55 Arginine 8"07 Unidentified (1) 1.61 Asparagine 7-70 Unidentified (2) 13.79 Serine 18"86 Glycine 1"98 Hydroxyproline 2"79 Threonine 4"99 Alanine 21 "64 Proline 2"42 Tyrosine 3'01 Methionine 5-36 Valine } Phenylalanine 10"13 Leucine Histidine detectable in traces only, Total 108"3
Female
Ratio of male/female
4"72 1"09 1"66 ND* 2-60 4.52 13"23 ND* ND* 4-15 10"64 1"14 0"83 1"92
0"30 4"18 4"86 --t 2-96 3.05 1-43 --t --t 1"20 2"03 2.12 3-63 2"79
5-19
1'95
at times 51-7
* Not detectable. Not found in one sex in measurable quantity; so the ratio not calculated.
FREE A M I N O ACIDS I N THE H A E M O L Y M P H OF A M I L L I P E D E
the male. Except glycine, hydroxyproline and an unidentified ninhydrin-positive material all the other amino acids present in the male were represented in the female though in a lesser concentration. Histidine was only occasionally detected as a faint spot. DISCUSSION We were unable to detect isoleucine, glutamic acid and aspartic acid reported to be present in Thyropygus, another diplopod (Krishnan, 1968), though asparagine, cystine and valine were additionally detected in Jonespeltis. Whereas in insect blood glutamine, proline, arginine, lysine and histidine were in higher concentration (Wyatt, 1961), and in the crustaceans it was arginine and glycine (Florkin, 1960; Huggins & Munday, 1968), in Jonespeltis serine, threonine and alanine were the more conspicuous amino acids in the blood. However, the present studies revealed that the free amino acids in the haemolymph of the millipede resembled the non-insect groups in that it showed low concentration (Florkin, 1960; Wyatt, 1961; Gilmour, 1965; Huggins & Munday, 1968). This is interesting in view of the fact that diplopods are supposed to be phylogenetically closely related to the insects. Studies on sex differences with regard to the free amino acids in insects have been largely limited to whole-body extracts (Chen, 1958, 1963; Kaplan et al., 1958; Duffy, 1964). These workers observed a high concentration of fl-alanine in male mosquitoes whereas methionine sulphoxide was in larger amounts in female mosquitoes and methionine in the female Drosophila. Duffy (1964) observed less conspicuous differences between the sexes with regard to the other amino acids, but the total concentration of amino acids was higher in the female. Though our studies were limited to the blood, they showed that the males had a high concentration of almost all amino acids except cystine which alone occurred in a higher concentration in the female. The total free amino acid concentration in the blood of the male millipede was twice that of the female. In the crab Paratelphusa also the males tended to have a higher free amino acid content in the blood than the females (Padmanabhanaidu & Ramamurthy, 1961). The significance of this could not be interpreted with accuracy with the present knowledge of the physiology of this group of animals. Possibly this was due to the fact that the free amino acids in the haemolymph were utilized for protein synthesis which in the female were deposited as yolk in the growing eggs. It may be noted that all the well-tanned females had developing oocytes in their body cavity containing plenty of protein yolk (unpublished observation).
Acknowledgements--We are grateful to Professor K. K. Nayar for critically going through the manuscript, and to the Ford Foundation for generous assistance. One of us (V. S. K. N.) thanks the University of Kerala for a Junior Research Fellowship. REFERENCES
BANKSW. M. & RANDOLPHE. F. (1968) Free amino acids in the cockroach Blaberusgiganteus. Ann. ent. Soc. Am. 61, 1027-1028.
V. S. KRISHNAN NAIR AND V. K. K. PRABHU C H ~ P. S. (1958) Studies on the protein metabolism of Culex pipiens.--II. Quantitative differences in free amino acids between male and female adult mosquitoes. )t. lnsect Physiol. 2, 128-136. CHEr~ P. S. (1963) Studies on protein metabolism of Culex pipiens L . - - I V . Separation of free aminoacids and peptides in adult mosquitoes by column chromatography, ft. Insect Physiol. 9, 453--462. CHEN P. S. (1966) Aminoacid and protein metabolism in insect development. In Advances in Insect Physiology (Edited by BEAMENT L. W. L., TREHERNE J. E. & WIGGLESWORTH V. B.), Vol. 3, pp. 53-132. Academic Press, New York. DUFFY J. P. (1964) Sex differences in the free aminoacids of three colonised mosquito species. Ann. ent. Soc. Am. 57, 24-28. FLORKIN M. (1960) Blood chemistry. In The Physiology of Crustacea (Edited by WATERMAN T. H.), Vol. I, pp. 141-159. Academic Press, New York. GILMOUR D. (1965) The Metabolism of Insects. Oliver & Boyd, London. HUGGINS A. K. & MUNDA¥ K. A. (1968) Crustacean metabolism. In Advances in Comparative Physiology and Biochemistry (Edited by LOWENSTEIN O.), Vol. 3, pp. 271-378. Academic Press, New York. KAPLAN W. C., HOLDEN J. T. & HOCHMAN B. (1958) Occurrence of unequal amounts of free methionine in male and female Drosophila melanogaster. Science 127, 473-474. KRISHNAN G. (1968) The millipede Thyropygus. C.S.I.R. Zoological Memoirs on Indian Animal Types, No. 1. LEE Y. P. & TAKAHASHIT. (1966) An improved colorimetric determination of aminoacids with the use of ninhydrin. Analyt. Biochem. 14, 71-77. PADMANABHANAIDUB. & RAMAMURTHYR. (1961) T h e influence of sex and size on the osmotic pressure, the chloride and the free aminoacids of the blood of the freshwater field crab, Paratelphusa sp. and the freshwater mussel, Lamellidens rnarginalis. J. exp. Biol. 38, 35-41. RaNDERATH K. (1964) Thin-layer Chromatography. Academic Press, New York & London. SMITH I. (1960) Chromatographic and Electrophoretic Techniques, Vol. I. Pp. 617. Interscience, New York. WYATT G. R. (1961) T h e biochemistry of insect haemolyrnph. A. Rev. Ent. 6, 75-100.
Key Word Index--Amino acids; haemolymph; Millipede; ffonespeltis splendidus.
O N T H E FREE A M I N O A C I D S IN T H E H A E M O L Y M P H OF A M I L L I P E D E V. S. KRISHNAN NAIR and V. K. K. PRABHU Department of Zoology, University of Kerala, Kariavattom, Trivandrum, India (Received 19 ffune 1970) A b s t r a c t - - 1 . Chromatographic studies on the free amino acids in the blood of the millipede~onespeltis splendidus revealed cystine, lysine, arginine, asparaglne,
serine, glycine, hydroxyproline, threonine, alanine, proline, tyrosine, methionine, valine, phenylalanine, leucine and histidine. 2. Serine, threonine and alanine occurred at a higher concentration in the blood of this species than the other amino acids. 3. All the above amino acids except cystine occurred at a higher concentration in the blood of the male. 4. Glycine and hydroxyproline were not detected in the blood of the female. 5. Total amino acid concentration in the blood of the female was found to be 51.7 _+9.6 rag/100 ml blood and that of the male, 108"3 +_6"1 rag/100 ml.
INTRODUCTION THE FREEamino acid concentration in the haemolymph of insects is known to be very high compared to that of other animals (Wyatt, 1961 ; Gilmour, 1965;Chen, 1966; Huggins & Munday, 1968). Sex-]imited differences with regard to the concentration of some amino acids have also been reported in insects (Duffy, 1964; Chen, 1966). W e have, however, very little information on the free amino acid p a t t e r n of the h a e m o l y m p h o f diplopods (Krishnan, 1968), a g r o u p p h y l o genetically closely related to the insects. T h e present r e p o r t is on the s t u d y o f free a m i n o acids in the blood of a m i l l i p e d e J o n e s p e l t i s splendidus. MATERIALS A N D METHODS Well-tanned males and females of the millipede Jonespeltis splendidus collected locally were used for the present study. Haemolymph from animals was collected from a dorsal puncture made on the "thoracic" segments, deproteinized by 80% ethanol and kept in a refrigerator overnight. It was centrifuged for 10 min at 1500 rev/min next morning. The protein-free supernatant containing free amino acids was used for the study. Supematant equivalent to 60 #1 haemolymph was evaporated to a residue at 60°C. This was assayed by the method of Lee & Takahashi (1966) as total ninhydrin-positive material against glutamic acid standard at 570 m/z using a Bausch & Lomb Spectronic 20 spectrophotometer. For chromatography supernatant containing amino acids equivalent to 0"1 ml blood evaporated to a residue and redissolved in a small quantity of 70% ethanol was used for spotting. Twodimensional thin-layer chromatography was carried out on plates coated with silica gel-G using chloroform-methanol-17% ammonia ( 2 : 2 : 1 v/v) in one direction and phenolwater (75 : 25 w/w) in the other. Spots were visualized by ninhydrin reagent (Randerath,
V. S. KRISHNAN NAIR AND V. K. K. PRABHU
1964). One-dimensional ascending paper chromatography was carried out overnight on Whatman No. 1 filter paper using butanol-acetic acid-water (450 : 50 : 125) after Banks & Randolph (1968). The chromatogram was dried at room temperature and run once more in the same solvent. After drying, the spots were visualized by ninhydrin reagent (Smith, 1960). The individual amino acids were identified by their RI values and by comparing the spots of standard amino acids in chromatograms run simultaneously. Paper chromatograms were scanned in a Densicord Densitometer (Photovolt Corporation), and the concentration of individual amino acids in the haemolymph was calculated from the total free amino acid concentration and the densitometric chart. RESULTS It was found by quantitative assay that the well-tanned female had a free amino acid concentration of only 51.7 + 9.6 mg ( M + S.D.) per 100 ml blood as against 108.3 +6-1 rag/100 ml in the male. T h i n layer chromatograms showed only a m a x i m u m of ten spots. On the other hand, there were a total of eighteen spots distinguishable in the paper chromatogram of which all except two were identified. T h e concentration of individual amino acids is given in Table 1. As the Table shows all amino acids except cystine were represented at a higher concentration in TABLE 1--FREE AMINOACIDCONCENTRATIONIN THE HAEMOLYMPHOF THE MILLIPEDE
ffonespeltis splendidus Concentration of amino acids (mg/100 ml blood) Amino acids
Male
Cystine 1.39 Lysine 4.55 Arginine 8"07 Unidentified (1) 1.61 Asparagine 7-70 Unidentified (2) 13.79 Serine 18"86 Glycine 1"98 Hydroxyproline 2"79 Threonine 4"99 Alanine 21 "64 Proline 2"42 Tyrosine 3'01 Methionine 5-36 Valine } Phenylalanine 10"13 Leucine Histidine detectable in traces only, Total 108"3
Female
Ratio of male/female
4"72 1"09 1"66 ND* 2-60 4.52 13"23 ND* ND* 4-15 10"64 1"14 0"83 1"92
0"30 4"18 4"86 --t 2-96 3.05 1-43 --t --t 1"20 2"03 2.12 3-63 2"79
5-19
1'95
at times 51-7
* Not detectable. Not found in one sex in measurable quantity; so the ratio not calculated.
FREE A M I N O ACIDS I N THE H A E M O L Y M P H OF A M I L L I P E D E
the male. Except glycine, hydroxyproline and an unidentified ninhydrin-positive material all the other amino acids present in the male were represented in the female though in a lesser concentration. Histidine was only occasionally detected as a faint spot. DISCUSSION We were unable to detect isoleucine, glutamic acid and aspartic acid reported to be present in Thyropygus, another diplopod (Krishnan, 1968), though asparagine, cystine and valine were additionally detected in Jonespeltis. Whereas in insect blood glutamine, proline, arginine, lysine and histidine were in higher concentration (Wyatt, 1961), and in the crustaceans it was arginine and glycine (Florkin, 1960; Huggins & Munday, 1968), in Jonespeltis serine, threonine and alanine were the more conspicuous amino acids in the blood. However, the present studies revealed that the free amino acids in the haemolymph of the millipede resembled the non-insect groups in that it showed low concentration (Florkin, 1960; Wyatt, 1961; Gilmour, 1965; Huggins & Munday, 1968). This is interesting in view of the fact that diplopods are supposed to be phylogenetically closely related to the insects. Studies on sex differences with regard to the free amino acids in insects have been largely limited to whole-body extracts (Chen, 1958, 1963; Kaplan et al., 1958; Duffy, 1964). These workers observed a high concentration of fl-alanine in male mosquitoes whereas methionine sulphoxide was in larger amounts in female mosquitoes and methionine in the female Drosophila. Duffy (1964) observed less conspicuous differences between the sexes with regard to the other amino acids, but the total concentration of amino acids was higher in the female. Though our studies were limited to the blood, they showed that the males had a high concentration of almost all amino acids except cystine which alone occurred in a higher concentration in the female. The total free amino acid concentration in the blood of the male millipede was twice that of the female. In the crab Paratelphusa also the males tended to have a higher free amino acid content in the blood than the females (Padmanabhanaidu & Ramamurthy, 1961). The significance of this could not be interpreted with accuracy with the present knowledge of the physiology of this group of animals. Possibly this was due to the fact that the free amino acids in the haemolymph were utilized for protein synthesis which in the female were deposited as yolk in the growing eggs. It may be noted that all the well-tanned females had developing oocytes in their body cavity containing plenty of protein yolk (unpublished observation).
Acknowledgements--We are grateful to Professor K. K. Nayar for critically going through the manuscript, and to the Ford Foundation for generous assistance. One of us (V. S. K. N.) thanks the University of Kerala for a Junior Research Fellowship. REFERENCES
BANKSW. M. & RANDOLPHE. F. (1968) Free amino acids in the cockroach Blaberusgiganteus. Ann. ent. Soc. Am. 61, 1027-1028.
V. S. KRISHNAN NAIR AND V. K. K. PRABHU C H ~ P. S. (1958) Studies on the protein metabolism of Culex pipiens.--II. Quantitative differences in free amino acids between male and female adult mosquitoes. )t. lnsect Physiol. 2, 128-136. CHEr~ P. S. (1963) Studies on protein metabolism of Culex pipiens L . - - I V . Separation of free aminoacids and peptides in adult mosquitoes by column chromatography, ft. Insect Physiol. 9, 453--462. CHEN P. S. (1966) Aminoacid and protein metabolism in insect development. In Advances in Insect Physiology (Edited by BEAMENT L. W. L., TREHERNE J. E. & WIGGLESWORTH V. B.), Vol. 3, pp. 53-132. Academic Press, New York. DUFFY J. P. (1964) Sex differences in the free aminoacids of three colonised mosquito species. Ann. ent. Soc. Am. 57, 24-28. FLORKIN M. (1960) Blood chemistry. In The Physiology of Crustacea (Edited by WATERMAN T. H.), Vol. I, pp. 141-159. Academic Press, New York. GILMOUR D. (1965) The Metabolism of Insects. Oliver & Boyd, London. HUGGINS A. K. & MUNDA¥ K. A. (1968) Crustacean metabolism. In Advances in Comparative Physiology and Biochemistry (Edited by LOWENSTEIN O.), Vol. 3, pp. 271-378. Academic Press, New York. KAPLAN W. C., HOLDEN J. T. & HOCHMAN B. (1958) Occurrence of unequal amounts of free methionine in male and female Drosophila melanogaster. Science 127, 473-474. KRISHNAN G. (1968) The millipede Thyropygus. C.S.I.R. Zoological Memoirs on Indian Animal Types, No. 1. LEE Y. P. & TAKAHASHIT. (1966) An improved colorimetric determination of aminoacids with the use of ninhydrin. Analyt. Biochem. 14, 71-77. PADMANABHANAIDUB. & RAMAMURTHYR. (1961) T h e influence of sex and size on the osmotic pressure, the chloride and the free aminoacids of the blood of the freshwater field crab, Paratelphusa sp. and the freshwater mussel, Lamellidens rnarginalis. J. exp. Biol. 38, 35-41. RaNDERATH K. (1964) Thin-layer Chromatography. Academic Press, New York & London. SMITH I. (1960) Chromatographic and Electrophoretic Techniques, Vol. I. Pp. 617. Interscience, New York. WYATT G. R. (1961) T h e biochemistry of insect haemolyrnph. A. Rev. Ent. 6, 75-100.
Key Word Index--Amino acids; haemolymph; Millipede; ffonespeltis splendidus.