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The free amino acids in the haemolymph of the maturing adult boll weevil, Anthonomus grandis boheman
Comp. Biochem. Physiol., 1968, Vol. 25, pp. 139 to 148. Pergamon Press. Printed in Great Britain
T H E FREE A M I N O ACIDS IN T H E HAEMOLYMPH OF T H E M A T U R I N G A D U L T BOLL WEEVIL, A N T H O N O M U S G R A N D I S BOHEMAN N O R M A N M I T L I N , G L E N N W I Y G U L and JOE K. M A U L D I N * Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, State College, Mississippi 39762, U.S.A.
(Received 11 September 1967) A b s t r a c t w l . The free and hydrolyzed free amino acids in the haemolymph
of the boll weevil, Anthonoraus grandis Boheman, were identified, and the amounts present during the first 5 days of adulthood were estimated. 2. In all, thirty ninhydrin-positive compounds were found, and of these, twenty-six were identified and, in most cases, quantitated. 3. Differences were found in both the titer of the total amino acids on a daily basis and in the amounts of individual amino acids, particularly tyrosine. 4. Total nitrogen levels varied with both sex and age, but amino nitrogen levels remained constant. INTRODUCTION THE mGn titer of free amino acids in the haemolymph of insects has drawn the attention of many investigators (see reviews by Wyatt, 1961 ; Florkin & Jeuniaux, 1964; Gilmour, 1965; Chen, 1966). However, little quantitative information is available on the dynamics of the free amino acids in the haemolymph as the insect matures and ages. In earlier reports, we examined the tissues of the boll weevil, Anthonomus grandis Boheman, during metamorphosis (Mitlin et al., 1966) and investigated the amino acids as end-products (Mitlin et aL, 1964). To further expand our knowledge of the free amino acids of the boll weevil, we have now undertaken a quantitative determination of these acids in the haemolymph of maturing adult boll weevils of both sexes during the first 5 days of adulthood. This particular period was chosen because the female needs at least 4 full days to reach sexual maturity, and we were particularly interested in the effects gonadal growth and its accompanying protein synthesis had upon the amino acids. Also, we wished to see whether the pattern in the male paralleled that in the female because the testicular growth and maturity have been completed at eclosion, and we wanted to determine whether the amino acids in the haemolymph of the boll weevil differed significantly from those of other insects as reported by other researchers. * Present address: Wood Products Insect Laboratory, Forest Service, U.S. Department of Agriculture, Gulfport, Mississippi. 139
140
NORMAN MITLIN, GLENNWIYGULANDJOE K. MAULDIN
MATERIALS AND METHODS
Biological materials The boll weevils used in the study were from the colony reared at this laboratory by the methods of Gast (1966). The insects were separated from the larval medium immediately after eclosion just before adult pigmentation occurred and were held until the requisite age level was attained. Throughout the holding period, they were fed the artificial diet of Gast (1966). We obtained the haemolymph by centrifuging whole weevils (held in a screen basket) at - 1 0 ° C at 2500 RCF since this procedure inhibited both melanization and clotting. Preliminary tests had shown that there was little contamination by other body fluids. The amino acids were extracted and hydrolyzed by the procedure reported earlier (Mitlin et al., 1966).
Chromatographic analysis Identifications of the amino acids were made by using thin-layer, electrophoretic and column chromatographic methods. For the thin-layer chromatography, we used isopropanol-formic acid-water ( 4 0 : 2 : 1 0 v / v ) in the first dimension and tert-butanol-methyl ethyl ketone-ammonia (0.88)-water ( 5 0 : 3 0 : 10: 10v/v) in the second dimension (Jones & Heathcote, 1966). Chromatograms were run for each age for both the unhydrolyzed and hydrolyzed amino acids. For the electrophoretic determination, we used an Electrophorator* with No. 4 Whatman chromatography paper as the substrate and a formic acid-acetic acid buffer (pH 1.9) in the first dimension. The extracts were subjected to 4000 V for 25 min. Either butanol-acetic acid-water ( 1 2 0 : 3 0 : 5 0 ) or lutidine-water (2 : 2 : 1) (Efron, 1960) was used as the second-dimension solvent system. Standard amino acids obtained from Calbiochem, Los Angeles, California, were used as markers, and identification maps were made by using ninhydrin as a general location agent. Also Ehrlich's reagent was used to identify indoles, sulfanilic acid reagent was used to detect imidazole and phenolic compounds, and ammonium molybdate reagent was used to detect phosphorus compounds (Smith, 1960). For column chromatography, to achieve good estimates of quantity we used an amino acid analyzer (Technicon Corp., Chauncey, New York) and employed the Piez & Morris modification (1960) of the method of Spackman et al. (1958). At least two different samples were extracted for each age, and at least two replicates were made for each sample. A standard mixture of amino acids was used to establish elution times and color reactivities. Doubtful identifications were resolved on the analyzer by co-chromatography and on the thin-layer system with standard materials. Since the elution times of the phosphorylated ninhydrin-positive compounds differed from each other only slightly on the analyzer, these were captured from the analyzer by using the * Mention of a proprietary product does not necessarily imply endorsement of this product by the U.S.D.A.
FREE A M I N O A C I D S I N H A E M O L Y M P H
OF THE BOLL WEEVIL
141
stream-splitting device and were hydrolyzed and identified from the positions of their hydrolytic products on thin-layer plates and on paper.
Nitrogen analysis Determinations of the total nitrogen of the haemolymph were made by the micro-Kjeldahl method of Kabat & Mayer (1961). T h e data thus obtained were subjected to an analysis of variance, and the significance was determined by a t-test. RESULTS
We were able to detect and identify twenty amino acids by using the combination of electrophoresis and paper chromatography; the thin-layer system was more effective: its ability to produce small definitive spots allowed us to detect as many as twenty-three ninhydrin-positive spots, of which we could identify twenty-two. With the column procedure we detected thirty and identified twenty-five ninhydHn-positive materials, and could usually estimate the quantities; however, two phosphorylated compounds, because they were eluted along with a large peptide, could not be quantitated. A (composite) chromatogram that is typical of the accelerated system we used is shown in Fig. 1. T h e run was completed in 6½ hr. This speed had the effect of decreasing resolution somewhat from longer runs of 22 hr; however, we found little difference between chromatograms made by the more lengthy procedure, either qualitatively or quantitatively. Table 1 shows the data obtained for the free amino acids from male and female weevils, respectively. Any amino acids that appeared in amounts too low for a meaningful figure are noted as "trace". T h e total amounts of amino acids (Fig. 2) in the male insect were highest upon eclosion, dropped rather sharply until the second day of adulthood, rose to a peak the third day and then dropped gently to the lowest level. T h e titer in the female followed a similar pattern, but the total quantity did not increase the third day; instead a slight drop occurred. Hydrolysis caused a change in the pattern. Again, the total quantity of amino acids was highest at eclosion for both sexes and, again, the titer receded for both sexes, but this time it reached a low point the first day after eclosion in the male and the second day after eclosion in the female. Both values rose again the third day and dropped rather sharply the fourth and fifth days. In addition, hydrolysis caused two new peaks to appear in the basic range, peaks 28 and 29 (Fig. 1) which could not be identified by our procedures. Several ninhydrin-positive materials that usually appeared in that region of the chromatogram (3-methyl histidine, 2,4-diaminobutyric acid, ethylamine and 2-thiohistidine) were co-chromatogrammed, but no confirming identifications could be made. Also, we were unable to identify peak 24 (Fig. 1) which appeared in both unhydrolyzed and hydrolyzed extracts. The increase in quantities of the amino acids shown in Table 2 reflects the addition of the individual amino acids that resulted from hydrolysis. Aspartic
142
NORMAN ~IITLIN, GLENNWIYGULAND JoE K. MAULDIN
and glutamic acids particularly were increased greatly by their conversions from asparagine and glutamine, respectively. Proline, in the unhydrolyzed extract, was found at typically high levels, but the pattern differed somewhat from that of the
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other amino acids since the highest level occurred at day 1 rather than at emergence. Also, the changes were not so marked from day to day. The rise in titer of ethanolamine in the hydrolyzed extracts was probably caused partly by the hydrolysis of
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phosphoethanolamine, which, though not quantitated, appeared from the thinlayer chromatograms to be present in substantial quantities. The most striking differences between sexes appeared to be the amounts of free tyrosine at eclosion. We found twenty-five times as much in the female as in the male; however, the amount was depleted rapidly to trace quantities, at day 1 in the male and at the second day in the female. Such a difference was not found in bound tyrosine; the quantities present were similar in the two sexes though the amounts dropped as the insects aged. ~ M a l e - ~ - ~ - - Female ............... Male, H y d r o I. [+10 ] ..... Female,Hydrol. [+10]
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haemolymph, but we can offer no explanation for a similar drop and subsequent rise in the male. Certainly, the change was not nutritionally inspired, because the diet was uniform throughout the study. O.8
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FIG. 3. Levels of nitrogen in the haemolymph a s indicated. TABLE 3~PERCENTAGES
OF N I T R O G E N ACCOUNTED FOR B Y A M I N O N I T R O O I ~
Age (day)
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The large amounts of peptides reflected in the large increase of values shown in Table 2 seem to be unusual. Florkin & Jeuniaux (1964) reported that, except in Drosophila, the level of peptides in the haemolymph of insects is generally low. Whether. this condition is unique to the boll weevil must be determined by examination of other insects. We cannot readily explain the large difference between sexes in the quantities of tyrosine found at eclosion other than to speculate that possibly the rate of sclerotization differs. By way of comparison, Chen (1958) found twice as much tyrosine in newly emerged whole female mosquitoes, Culex pipiens, as in males, and, as in our test, found that the tyrosine rapidly dropped to barely discernible
FREE A M I N O A C I D S I N H A E M O L Y M P H
OF THE BOLL WEEVIL
147
levels as the insect aged. Also, in our earlier tests (Mitlin et al., 1966) we found, as have other researchers with other insects, that tyrosine has the highest titer of all free amino acids in the last pupal stage of the boll weevil. On the other hand, our figures for tyrosine may be open to question. Although all samples of haemolymph were treated uniformly, some samples darkened as they aged. When the samples were tested on the analyzer immediately after processing, the amount of tyrosine was relatively high; when the same samples were tested after they had darkened somewhat, the titers were low or non-existent. Possibly a powerful tyrosinase is present in the haemolymph of the boll weevil that is resistant to denaturation and carries over into the final extract. Needless to say, all results reported here are for fresh extracts; however, the tyrosine was unique in its apparent lability. The biological significance of the high titer of proline and its build-up during the initial maturation compared with the other amino acids is speculative. However, the possibility is good that in the boll weevil, as in Glossina (Burseil, 1966) and in the house fly, Musca domestics L. (Sacktor, 1965), proline acts as an energy source for flight metabolism. Our results cannot be used to verify this hypothesis since our weevils were kept confined, but it is curious that the titer of free proline is higher in the male when the female, after maturity, is the more active flyer (W. H. Cross, this laboratory, personal communication). As we found earlier with pupae (Mitlin et al., 1966), several amino acids found in the feces of adult weevils (Mitlin et al., 1964) did not occur in the haemolymph. These were a-amino-n-butyric acid, 7-amino butyric acid and ornithine. However, hydroxylysine, which was not detected in pupal tissues, was found in both the haemolymph and the feces. We believe that our earlier assumption that the missing compounds were of dietary origin is still valid. The paucity of the free sulfur amino acids noted in our earlier studies (Mitlin et aL, 1964, 1966) was again apparent in the haemolymph. Seemingly, this lack is fairly consistent in insects (Gilmour, 1961 ; Florkin & Jeuniaux, 1964). However, we did not find the difference in the amount of methionine or methionine sulfoxide (Chen, 1958; Kaplan et al., 1958). We are inclined to believe that the two unknown ninhydrin-positive peaks found in the basic range as a result of hydrolysis are artifacts. According to Crestfield et al. (1963), artifacts sometimes occur as the result of acid hydrolysis. However, the question remains open, to be resolved by further investigation. Interestingly, the relatively large amounts of at least one basic amino acid, histidine, that was found accords with Wyatt's (1961) observation that in insects large concentrations of at least one basic acid are usual. REFERENCES BURSRLLJ. (1966) Aspects of the flight metabolism of tsetse flies, Glossina. Comp. Biochem. Physiol. 19, 809-818. Crn~r P. S. (1958) Studies on the protein metabolism of Culex pipiens L.--II. Quantitative differences in free amino acids between male and female adult mosquitoes, ft. Insect Physiol. 2, 128-136.
148
NORMAN MITLIN, GLENN WIYGULAND JoB K. MAUI.DIN
Cn~_~ P. S. (1966) Amino acid and protein metabolism in insect development. Adv. Insect Physiol. 3, 53-132. CRES~IELD A., MOORS S. & ST~L~ W. H. (1963) The preparation and enzymatic hydrolysis of reduced and s-carboxymethylated proteins. 3. biol. Chem. 230, 622-627. EVRON M. (1960) High voltage paper electrophoresis. In Chromatographic and Electrophoretic Techniques (Edited by SMITH I.) Vol. II, pp. 158-189. Heinemarm, London. FLORKL~ M. & JE~IAUX C. H. (1964) Haemolymph composition. In The Physiology of Insecta (Edited by ROCKS~IN M.) Vol. III, Chapter 21. Academic Press, New York and London. GAST R. T. (1966) Oviposition and fecundity of boll weevils in mass laboratory cultures. 3. econ. Ent. 59, 173-176. GILMOUR D. (1961) The Metabolism of Insects. p. 236. 0liver & Boyd, Edinburgh. Joins K. & I-IEATHCOTEJ. G. (1966) The rapid resolution of naturally occurring amino acids by thin-layer chromatography. 3. Chromatogr. 24, 106-111. KABAT E. A. & MAYER M. M. (1961) Experimental Immunochemistry. Charles C. Thomas, Springfield, Illinois. KAPLAN W. D., Hota~F2¢ J. T. & HocnM~,Xr B. (1958) Occurrence of unequal amounts of free methionine in male and female Drosophila melanogaster. Science AT. Y. 127, 473-474. MITLIN N., MAULDINJ. K. & I-I~Ir¢ P. A. (1966) Free and protein-bound amino acids in the tissue of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae) during metamorphosis. Comp. Biochem. Physiol. 19, 35-43. MITr~IN N., VIemms D. H. & Hm~tN P. A. (1964) End products of metabolism in the boll weevil, Anthonomus grandis Boheman: non-protein amino acids in the feces. J. Insect Physiol. 10, 393-397. Pmz K. A. & MogaIs L. (1960) A modified procedure for automatic analysis of amino acid. ..qnalyt. Biochem. 1, 167-201. SACrCrOR B. (1965) Energetics and respiratory metabolism of muscular contraction. In The Physiology of Insecta (Edited by ROCKSTmNM.) Vol. 2, pp. 483-580. Academic Press, New York. SMITH I. (1960) In Chromatographic and Electrophoretic Techniques (Edited by SMITH I.) Vol. I, pp. 82-103. Heinemann, London. SPACKM~ D. H., STEIN W. H. & Moom~ S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190-1206. WYATT G. R. (1961) The biochemistry of insect haemolymph. Ann. Rev. Ent. 6, 75-102.
T H E FREE A M I N O ACIDS IN T H E HAEMOLYMPH OF T H E M A T U R I N G A D U L T BOLL WEEVIL, A N T H O N O M U S G R A N D I S BOHEMAN N O R M A N M I T L I N , G L E N N W I Y G U L and JOE K. M A U L D I N * Entomology Research Division, Agricultural Research Service, U.S. Department of Agriculture, State College, Mississippi 39762, U.S.A.
(Received 11 September 1967) A b s t r a c t w l . The free and hydrolyzed free amino acids in the haemolymph
of the boll weevil, Anthonoraus grandis Boheman, were identified, and the amounts present during the first 5 days of adulthood were estimated. 2. In all, thirty ninhydrin-positive compounds were found, and of these, twenty-six were identified and, in most cases, quantitated. 3. Differences were found in both the titer of the total amino acids on a daily basis and in the amounts of individual amino acids, particularly tyrosine. 4. Total nitrogen levels varied with both sex and age, but amino nitrogen levels remained constant. INTRODUCTION THE mGn titer of free amino acids in the haemolymph of insects has drawn the attention of many investigators (see reviews by Wyatt, 1961 ; Florkin & Jeuniaux, 1964; Gilmour, 1965; Chen, 1966). However, little quantitative information is available on the dynamics of the free amino acids in the haemolymph as the insect matures and ages. In earlier reports, we examined the tissues of the boll weevil, Anthonomus grandis Boheman, during metamorphosis (Mitlin et al., 1966) and investigated the amino acids as end-products (Mitlin et aL, 1964). To further expand our knowledge of the free amino acids of the boll weevil, we have now undertaken a quantitative determination of these acids in the haemolymph of maturing adult boll weevils of both sexes during the first 5 days of adulthood. This particular period was chosen because the female needs at least 4 full days to reach sexual maturity, and we were particularly interested in the effects gonadal growth and its accompanying protein synthesis had upon the amino acids. Also, we wished to see whether the pattern in the male paralleled that in the female because the testicular growth and maturity have been completed at eclosion, and we wanted to determine whether the amino acids in the haemolymph of the boll weevil differed significantly from those of other insects as reported by other researchers. * Present address: Wood Products Insect Laboratory, Forest Service, U.S. Department of Agriculture, Gulfport, Mississippi. 139
140
NORMAN MITLIN, GLENNWIYGULANDJOE K. MAULDIN
MATERIALS AND METHODS
Biological materials The boll weevils used in the study were from the colony reared at this laboratory by the methods of Gast (1966). The insects were separated from the larval medium immediately after eclosion just before adult pigmentation occurred and were held until the requisite age level was attained. Throughout the holding period, they were fed the artificial diet of Gast (1966). We obtained the haemolymph by centrifuging whole weevils (held in a screen basket) at - 1 0 ° C at 2500 RCF since this procedure inhibited both melanization and clotting. Preliminary tests had shown that there was little contamination by other body fluids. The amino acids were extracted and hydrolyzed by the procedure reported earlier (Mitlin et al., 1966).
Chromatographic analysis Identifications of the amino acids were made by using thin-layer, electrophoretic and column chromatographic methods. For the thin-layer chromatography, we used isopropanol-formic acid-water ( 4 0 : 2 : 1 0 v / v ) in the first dimension and tert-butanol-methyl ethyl ketone-ammonia (0.88)-water ( 5 0 : 3 0 : 10: 10v/v) in the second dimension (Jones & Heathcote, 1966). Chromatograms were run for each age for both the unhydrolyzed and hydrolyzed amino acids. For the electrophoretic determination, we used an Electrophorator* with No. 4 Whatman chromatography paper as the substrate and a formic acid-acetic acid buffer (pH 1.9) in the first dimension. The extracts were subjected to 4000 V for 25 min. Either butanol-acetic acid-water ( 1 2 0 : 3 0 : 5 0 ) or lutidine-water (2 : 2 : 1) (Efron, 1960) was used as the second-dimension solvent system. Standard amino acids obtained from Calbiochem, Los Angeles, California, were used as markers, and identification maps were made by using ninhydrin as a general location agent. Also Ehrlich's reagent was used to identify indoles, sulfanilic acid reagent was used to detect imidazole and phenolic compounds, and ammonium molybdate reagent was used to detect phosphorus compounds (Smith, 1960). For column chromatography, to achieve good estimates of quantity we used an amino acid analyzer (Technicon Corp., Chauncey, New York) and employed the Piez & Morris modification (1960) of the method of Spackman et al. (1958). At least two different samples were extracted for each age, and at least two replicates were made for each sample. A standard mixture of amino acids was used to establish elution times and color reactivities. Doubtful identifications were resolved on the analyzer by co-chromatography and on the thin-layer system with standard materials. Since the elution times of the phosphorylated ninhydrin-positive compounds differed from each other only slightly on the analyzer, these were captured from the analyzer by using the * Mention of a proprietary product does not necessarily imply endorsement of this product by the U.S.D.A.
FREE A M I N O A C I D S I N H A E M O L Y M P H
OF THE BOLL WEEVIL
141
stream-splitting device and were hydrolyzed and identified from the positions of their hydrolytic products on thin-layer plates and on paper.
Nitrogen analysis Determinations of the total nitrogen of the haemolymph were made by the micro-Kjeldahl method of Kabat & Mayer (1961). T h e data thus obtained were subjected to an analysis of variance, and the significance was determined by a t-test. RESULTS
We were able to detect and identify twenty amino acids by using the combination of electrophoresis and paper chromatography; the thin-layer system was more effective: its ability to produce small definitive spots allowed us to detect as many as twenty-three ninhydrin-positive spots, of which we could identify twenty-two. With the column procedure we detected thirty and identified twenty-five ninhydHn-positive materials, and could usually estimate the quantities; however, two phosphorylated compounds, because they were eluted along with a large peptide, could not be quantitated. A (composite) chromatogram that is typical of the accelerated system we used is shown in Fig. 1. T h e run was completed in 6½ hr. This speed had the effect of decreasing resolution somewhat from longer runs of 22 hr; however, we found little difference between chromatograms made by the more lengthy procedure, either qualitatively or quantitatively. Table 1 shows the data obtained for the free amino acids from male and female weevils, respectively. Any amino acids that appeared in amounts too low for a meaningful figure are noted as "trace". T h e total amounts of amino acids (Fig. 2) in the male insect were highest upon eclosion, dropped rather sharply until the second day of adulthood, rose to a peak the third day and then dropped gently to the lowest level. T h e titer in the female followed a similar pattern, but the total quantity did not increase the third day; instead a slight drop occurred. Hydrolysis caused a change in the pattern. Again, the total quantity of amino acids was highest at eclosion for both sexes and, again, the titer receded for both sexes, but this time it reached a low point the first day after eclosion in the male and the second day after eclosion in the female. Both values rose again the third day and dropped rather sharply the fourth and fifth days. In addition, hydrolysis caused two new peaks to appear in the basic range, peaks 28 and 29 (Fig. 1) which could not be identified by our procedures. Several ninhydrin-positive materials that usually appeared in that region of the chromatogram (3-methyl histidine, 2,4-diaminobutyric acid, ethylamine and 2-thiohistidine) were co-chromatogrammed, but no confirming identifications could be made. Also, we were unable to identify peak 24 (Fig. 1) which appeared in both unhydrolyzed and hydrolyzed extracts. The increase in quantities of the amino acids shown in Table 2 reflects the addition of the individual amino acids that resulted from hydrolysis. Aspartic
142
NORMAN ~IITLIN, GLENNWIYGULAND JoE K. MAULDIN
and glutamic acids particularly were increased greatly by their conversions from asparagine and glutamine, respectively. Proline, in the unhydrolyzed extract, was found at typically high levels, but the pattern differed somewhat from that of the
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other amino acids since the highest level occurred at day 1 rather than at emergence. Also, the changes were not so marked from day to day. The rise in titer of ethanolamine in the hydrolyzed extracts was probably caused partly by the hydrolysis of
143
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phosphoethanolamine, which, though not quantitated, appeared from the thinlayer chromatograms to be present in substantial quantities. The most striking differences between sexes appeared to be the amounts of free tyrosine at eclosion. We found twenty-five times as much in the female as in the male; however, the amount was depleted rapidly to trace quantities, at day 1 in the male and at the second day in the female. Such a difference was not found in bound tyrosine; the quantities present were similar in the two sexes though the amounts dropped as the insects aged. ~ M a l e - ~ - ~ - - Female ............... Male, H y d r o I. [+10 ] ..... Female,Hydrol. [+10]
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NORMANMITLIN, GLENNWIYGULANDJog K. MAULDIN
haemolymph, but we can offer no explanation for a similar drop and subsequent rise in the male. Certainly, the change was not nutritionally inspired, because the diet was uniform throughout the study. O.8
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FIG. 3. Levels of nitrogen in the haemolymph a s indicated. TABLE 3~PERCENTAGES
OF N I T R O G E N ACCOUNTED FOR B Y A M I N O N I T R O O I ~
Age (day)
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Females
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16.7 16.8 14"1 18"6 19"7 12"8
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The large amounts of peptides reflected in the large increase of values shown in Table 2 seem to be unusual. Florkin & Jeuniaux (1964) reported that, except in Drosophila, the level of peptides in the haemolymph of insects is generally low. Whether. this condition is unique to the boll weevil must be determined by examination of other insects. We cannot readily explain the large difference between sexes in the quantities of tyrosine found at eclosion other than to speculate that possibly the rate of sclerotization differs. By way of comparison, Chen (1958) found twice as much tyrosine in newly emerged whole female mosquitoes, Culex pipiens, as in males, and, as in our test, found that the tyrosine rapidly dropped to barely discernible
FREE A M I N O A C I D S I N H A E M O L Y M P H
OF THE BOLL WEEVIL
147
levels as the insect aged. Also, in our earlier tests (Mitlin et al., 1966) we found, as have other researchers with other insects, that tyrosine has the highest titer of all free amino acids in the last pupal stage of the boll weevil. On the other hand, our figures for tyrosine may be open to question. Although all samples of haemolymph were treated uniformly, some samples darkened as they aged. When the samples were tested on the analyzer immediately after processing, the amount of tyrosine was relatively high; when the same samples were tested after they had darkened somewhat, the titers were low or non-existent. Possibly a powerful tyrosinase is present in the haemolymph of the boll weevil that is resistant to denaturation and carries over into the final extract. Needless to say, all results reported here are for fresh extracts; however, the tyrosine was unique in its apparent lability. The biological significance of the high titer of proline and its build-up during the initial maturation compared with the other amino acids is speculative. However, the possibility is good that in the boll weevil, as in Glossina (Burseil, 1966) and in the house fly, Musca domestics L. (Sacktor, 1965), proline acts as an energy source for flight metabolism. Our results cannot be used to verify this hypothesis since our weevils were kept confined, but it is curious that the titer of free proline is higher in the male when the female, after maturity, is the more active flyer (W. H. Cross, this laboratory, personal communication). As we found earlier with pupae (Mitlin et al., 1966), several amino acids found in the feces of adult weevils (Mitlin et al., 1964) did not occur in the haemolymph. These were a-amino-n-butyric acid, 7-amino butyric acid and ornithine. However, hydroxylysine, which was not detected in pupal tissues, was found in both the haemolymph and the feces. We believe that our earlier assumption that the missing compounds were of dietary origin is still valid. The paucity of the free sulfur amino acids noted in our earlier studies (Mitlin et aL, 1964, 1966) was again apparent in the haemolymph. Seemingly, this lack is fairly consistent in insects (Gilmour, 1961 ; Florkin & Jeuniaux, 1964). However, we did not find the difference in the amount of methionine or methionine sulfoxide (Chen, 1958; Kaplan et al., 1958). We are inclined to believe that the two unknown ninhydrin-positive peaks found in the basic range as a result of hydrolysis are artifacts. According to Crestfield et al. (1963), artifacts sometimes occur as the result of acid hydrolysis. However, the question remains open, to be resolved by further investigation. Interestingly, the relatively large amounts of at least one basic amino acid, histidine, that was found accords with Wyatt's (1961) observation that in insects large concentrations of at least one basic acid are usual. REFERENCES BURSRLLJ. (1966) Aspects of the flight metabolism of tsetse flies, Glossina. Comp. Biochem. Physiol. 19, 809-818. Crn~r P. S. (1958) Studies on the protein metabolism of Culex pipiens L.--II. Quantitative differences in free amino acids between male and female adult mosquitoes, ft. Insect Physiol. 2, 128-136.
148
NORMAN MITLIN, GLENN WIYGULAND JoB K. MAUI.DIN
Cn~_~ P. S. (1966) Amino acid and protein metabolism in insect development. Adv. Insect Physiol. 3, 53-132. CRES~IELD A., MOORS S. & ST~L~ W. H. (1963) The preparation and enzymatic hydrolysis of reduced and s-carboxymethylated proteins. 3. biol. Chem. 230, 622-627. EVRON M. (1960) High voltage paper electrophoresis. In Chromatographic and Electrophoretic Techniques (Edited by SMITH I.) Vol. II, pp. 158-189. Heinemarm, London. FLORKL~ M. & JE~IAUX C. H. (1964) Haemolymph composition. In The Physiology of Insecta (Edited by ROCKS~IN M.) Vol. III, Chapter 21. Academic Press, New York and London. GAST R. T. (1966) Oviposition and fecundity of boll weevils in mass laboratory cultures. 3. econ. Ent. 59, 173-176. GILMOUR D. (1961) The Metabolism of Insects. p. 236. 0liver & Boyd, Edinburgh. Joins K. & I-IEATHCOTEJ. G. (1966) The rapid resolution of naturally occurring amino acids by thin-layer chromatography. 3. Chromatogr. 24, 106-111. KABAT E. A. & MAYER M. M. (1961) Experimental Immunochemistry. Charles C. Thomas, Springfield, Illinois. KAPLAN W. D., Hota~F2¢ J. T. & HocnM~,Xr B. (1958) Occurrence of unequal amounts of free methionine in male and female Drosophila melanogaster. Science AT. Y. 127, 473-474. MITLIN N., MAULDINJ. K. & I-I~Ir¢ P. A. (1966) Free and protein-bound amino acids in the tissue of the boll weevil, Anthonomus grandis Boheman (Coleoptera: Curculionidae) during metamorphosis. Comp. Biochem. Physiol. 19, 35-43. MITr~IN N., VIemms D. H. & Hm~tN P. A. (1964) End products of metabolism in the boll weevil, Anthonomus grandis Boheman: non-protein amino acids in the feces. J. Insect Physiol. 10, 393-397. Pmz K. A. & MogaIs L. (1960) A modified procedure for automatic analysis of amino acid. ..qnalyt. Biochem. 1, 167-201. SACrCrOR B. (1965) Energetics and respiratory metabolism of muscular contraction. In The Physiology of Insecta (Edited by ROCKSTmNM.) Vol. 2, pp. 483-580. Academic Press, New York. SMITH I. (1960) In Chromatographic and Electrophoretic Techniques (Edited by SMITH I.) Vol. I, pp. 82-103. Heinemann, London. SPACKM~ D. H., STEIN W. H. & Moom~ S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190-1206. WYATT G. R. (1961) The biochemistry of insect haemolymph. Ann. Rev. Ent. 6, 75-102.