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The free amino acids of the ageing female Aedes aegypti mosquito
J. Ins. Physiol., 1962, Vol. 8, pp. 133 to 143. Pergamon Press Ltd.
Printed in Great Britain
THE FREE AMINO ACIDS OF THE AGEING FEMALE AEDES AEGYPTI MOSQUITO” D. W. THAYER Naval
Medical
Research
Institute,
National
and L. A. TERZIAN Naval
(Received 6 October
Medical
Center,
Bethesda,
Maryland
1961)
Abstract-Quantitative
chromatographic methods were used to compare the free amino acid patterns of tissue extracts of ageing female Aedtu aegypti mosquitoes maintained on three different dietary regimens. Alanine, arginine, glutamic acid, glutamine, glycine, hiitidine, leucine and/or isoleucine, phenylalanine, proline, serine, taurine, threonine, tryptophan, and valine were consistently present in the tissue extracts. Of these alanine, histidme, arginine, glutamic acid, and lysine were present in the highest concentrations. Aspartic acid was found in only one age group, and tyrosine was found only in mosquitoes which had had a blood meal. In contrast to the mosquitoes maintained on sucrose, in which the level of methionine remained constant while glutamine and hi&dine decreased, the mosquitoes maintained on raisins or given blood meals showed significant increases in the levels of these three amino acids. A peptide which has been labelled X1 in the quantitative studies was present in large quantities at the spot occupied by @-alanine in the standard chromatograms. In all three groups this peptide decreased uniformly in amount as the mosquitoes aged.
INTRODUCTION IT HAS been shown that the resistance of the mosquito Cedes uegypti to infection with the malarial parasite Pbmodium gallinaceum increases markedly as it grows older (TERZIAN et al., 1956). It has also been shown that mosquitoes given a normal blood meal and those maintained on cooked raisins became again as susceptible to infection as newly emerged, young mosquitoes. The studies reported in this paper were undertaken in an effort to elucidate what changes, if any, occur in the free amino acids as the mosquito ages and what relationship, if any, these changes might have with the increase in the innate immunity of the ageing mosquito. CLARK and BALL (1952) found alanine, arginine, glutamic acid, glutamine, glycine, histidine, leucine and/or isoleucine, methionine, proline, serine, threonine, tryptophan, tyrosine, and cu-amino-n-butyric acid in Culex tars&s, Culex stigmatosoma, Aedes varipalpus, and Culiseta incidens. Aspartic acid, p-alanine, cysteic acid, lysine, and taurine were found in many but not all mosquitoes. MICKS and ELLIS (1952) reported finding alanine, arginine, ~-alanine, glutamic acid, glycine, histidine, tioleucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine in the pupae and adults of Culex quinquefasciatus and Aedes aegypti. Aspartic acid appeared in chromatograms of the eggs and larva, but not in * The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. 9
133
134
D. W. THAYERANDL. A. TERZIAN
those of the pupae and adults. Phenylalaninewas not found and the authors noted that the larvae seem equally capable of utilizing either phenylalanine or tyrosine. In 1957, MICKS and GIBSONreported that strains of Aedes aegypti as well as other strains and species could easily be distinguished on the basis of quantitative differences in several amino acids. MATERIALS AND METHODS A laboratory strain of Aedes aegypti mosquitoes maintained at a constant temperature of 26~5°C and a relative humidity of 75 per cent was used for these experiments. The larvae were grown in glass crocks and fed solely on guinea-pig food pellets added in appropriate amounts from time to time during the larval developmental period. The adult mosquitoes were placed in wire-screened cages, measuring 15 x 15 x 15 in. and were selected from the emerging pupae so as to give a uniform age grouping of not less than 24 nor more than 48 hr at the start of each experiment. The mosquitoes for each experimental series were obtained from the same colony. The adult mosquitoes were routinely fed 4% sucrose solutions which were made available after the 24-48 hr age group had been removed for analysis. In addition, a source of water was provided for all caged mosquitoes. Those mosquitoes permitted to have blood meals were removed from the screen cages at the time of the blood meal and placed in lucite cylinders in groups of 80-100 females. They were permitted to gorge themselves on young normal pullets and were then held in the cylinders on a routine diet of 4% sucrose for 1 week before they were analysed. The mosquitoes receiving a, dietary supplement of cooked raisinswere given the raisinsas soon as the 24-48 hr sample had been removed and thereafter were provided with fresh raisins as needed. Total ‘nitrogen and moisture Three groups of twenty and one of 100 females were routinely used for the analysis. The lightly anaesthetixedmosquitoes were weighed and then were dried in vacua over P,O, to a constant weight of plus or minus O-2 mg. After a constant weight had been obtained the dry weights were obtained The group of 100 was retainedfor the amino acid analysisand the three groups of twenty were used for the determination of total nitrogen by the micro-Kjeldahl procedure (Ass. OFF.AGRIC. CHEM., 1950). Ali samples were included in the calculation of the per cent moisture and of the dry weight per mosquito. Amino acid determination The 100 mosquito sample (approximately 50 mg) described above was pulverized within its weighing bottle, in this case a 10 x 75 mm glass-stopperedtest tube. Then O-2 ml of cold glass-distilled water was added to the tissue and the resulting slurry mixed thoroughly by tapping the sides of the tube. The sample was then allowed to stand overnight. The following day O-8ml of cold absolute ethanol was added and mixed with the slurry and the sample was again allowed to stand overnight. The precipitated protein was removed by centrifugation at 8000
THE FREE AMINO
ACIDS OF THE AGEING
FEMALE AEDES AEGYPTI
MOSQUITO
135
revjmin for at least 1 hr; the amino acid extract decanted and recentrifuged. The above operations were all carried out at 4°C and the sample tube was always sealed with a heavy coating of paraf%nfilm in order to prevent loss by evaporation. This provided a stable extract of the free amino acids in 80% ethanol. Chromatography was carried out by the procedure modified from REDFIELD (1953) which was described by WYATTet aZ. (1956). Whatman No. 1 filter paper was used in 15 x 15 in. sheets sprayed before use with 12 ml of O.O2o/oEDTA in 60% ethanol. The chromatograms were run without prior vapour equilibration by
METHANOL PYRIDINE HOH 20*1:5 t TIME 7HR
MILL
DIRECTION-=+
FIG. 1. Tracing of amino acid chromatogram of an extract of l-day-old Aedes aegypti. Development time, 16 hr. Stain, 2% ninhydrin. Paper sprayed before with 10 ml of EDTA (O@O2%)in 60% ethanoL
the ascending method in borosilicate glass cylinders 6 x 18 in., which were sealled with weighted plate-glass covers. The first solvent contained tert-butanol 10, methyl ethyl ketone 10, concentrated ammonium hydroxide 3, and water 5 parts by volume. The second dimension was run in Redfield’s solvent consisting of methanol 20, pyridine 1, and water 5 parts by volume. Typical runs were made in 16 hr in the first dimension and 7 hr in the second. Typical chromatograms are presented in Figs. 1 and 2. The amino acids were estimated using the 2% solution of ninhydrin in absolute ethanol recommended by WELLINGTON (1952). The technique used differed in that a rapid dipping procedure was used rather than the spray technique of WELLINGTON. The papers were dipped in the ninhydrin solution and allowed to
136
D. W. THAYEBAND L. A. TERZIAN
air-dry in the dark overnight before evaluation. In this laboratory, this technique gave a more uniform distribution of the dye without smearing so long as the absolute ethanol used was obtained from freshly opened bottles.
06
RF
04
0.2
co AR0
LYS
t #
i
I
FIG. 2. Tracing of amino acid chromatogram of an extract of l-day-old Aedes aegypti. Solvent I: tert-Btitanol-methyl ethyl ketone-concentrated ammonia-water (10 : 10 : 3 : 5). Solvent 11: Methanol-pyridine-water (20 : 1 : 5).
After development a permanent record of each chromatogram was made for future reference by tracing the locations of the amino acid spots on graph paper. Each coloured area was then cut out of the chromatogram, weighed and placed in a centrifuge tube for extraction with 5 ml of cold 50% aqueous n-propanol. Blanks of the same size were taken at four levels and used for correcting the amino acid spots at each level for background absorbance of dye. The corrections were made by comparison of the weight of the blank to that of the amino acid spot and using the resulting fraction to determine the background correction for the particular amino acid spot. The tubes were shaken for 15 min in a mechanical shaker and then centrifuged in the cold to settle the lint formed during the shaking.
THE FREE AMINO ACIDS OF THE AGEING FEMALE AEDES AEGYPTI
MOSQUITO
137
The optical absorbance for all areas was determined in 1 cm Corex cuvettes in the Beckman Model DU spectrophotometer at 570 my, except proline, which was determined at 350 mp. A calibration curve was prepared for each amino acid from chromatograms of standard mixtures of 2, 5, and 10 gamma concentrations, which were run at the same time as the samples. The equation for each curve was determined by the method of least squares and the unknown determined directly from the equation. Three replicas of each sample were run simultaneously for quantitativeevaluation, and in addition a varying number of other replicas were run for qualitative evaluations. Individual amino acids were determined by position and by specific chemical tests where possible. RESULTS
Total nitrogen, we+, and moisture The values for the total nitrogen, weight, and moisture determinations are summarized in Tables 1 and 2. An examination of the wet and dry weights in both tables shows that they parallel each other, and that the moisture content of the TABLE ~-THE
Sample
PERCENTAGE
NITROGRN AND WRT AND DRY WRIGHT OF ADULT FRMALE
de&s aegypti MOSQUITOES (maintainedon 4% sucrose)
Mosquito age (days)
Per cent nitrogen
Wet wt.fmosq. (mg)
DrY wt./mosq. (mid
7.84 6.15 6.87 7.35 5.86
l-64 1.68 1.47 1.57 1.51
O-48 0.62 0.52 0.53 0.56
7.30 7.51
l-41 1.34
::g
Per cent moisture
mosquitoes remains comparatively constant during the experimental period. Table 1 shows that there is an increase in weight during the first week following emergence; the weight then declines during the remaining $-week period of the experiment. On the other hand, the percentage of tissue nitrogen declines during the first week, but then begins to increase and reaches a maximum at the end of the 6-week period. A comparison of the data for the wet and dry weights and the percentage of tissue nitrogen of these mosquitoes over the 6-week period indicates a relation in which the values for one set of data appear to be the reciprocals of the other. It appears not unreasonable to assume, therefore, that the nitrogen content of the mosquitoes remains comparatively constant during this period of time. Free amino acid The identity of individual amino acid spots on the chromatograms was determined by position and/or specific chemical tests. In cases where the spot
138
D. W. THAYER
AND L. A. TERZIAN
was so faint as to be questionable, a larger sample was used for chromatography in order to illustrate the presence or absence of the particular amino acid. Arginine, which was invariably present, was tested for by a modification of the Sakaguchi TABLE ~-THE PEXEN!I'AGE WET ANDDRYWEIGHT OFADULT FEMALE Aedes aegypti
MOSQUITOES
Sample
wet wt./mosq. Cm&
Mosquito age (days)
Dry wt./mosq. (md
Per cent moisture
Maintained on 4% sucrose and blood meals*
A
:
Bc E F
f;’ 28 35
1-77
0.51
71.13
2.43 2.00
0.85 0.63
67.98 65.02
2.46 2.24 2-27
0.83 0.78 0.75
66.26 65.18 66.96
Maintained on cooked raisins*
A :
::
: F
;: 3”;
*
1.77 2.45 2.03
o-51 O-85 O-61
71.13 65.31 69.71
2.06 2.03 1.90
0.67 o-59 0.59
70.94 67.48 68.95
* Pairedsamples.
reaction (ACHER and CROCKER,1952). The presence of histidine was established by the method of CURZ~N and GILTROW (1954). The presence of proline and the absence of hydroxyproline was established by two methods, GIRI and NAGABHUSHANAM (1,952) and SMITH (1953). Tryptophan was consistently demonstrated by the use of Ehrlich’s reagent as described by SMITH (1953). Tyrosine was consistently absent in most of the chromatograms using the cu-nitroso-/3-naphthol reagent described by JEPSO~V and SMITH (1953). Tyrosine was found only after the mosquitoes had been given a blood meal. The sensitivity of this test was found to be at least O-1 gamma, which would seem to rule out the presence of tyrosine where no positive could be obtained. Since cysteic acid had been reported to be present in some species of mosquitoes by CLARK and BALL (1952), a qualitative test (TOENNIESand KOLB, 1951) was carried out for cysteine and/or cystine. Both were consistently absent in this strain of Aedes aegypti.
THE FREE AMINOACIDSOF THE AGEINGFEMALEAEDES
AEGYPTI
139
MOSQUITO
In addition to the above, a peptide, labelled X, in the quantitative studies, was present invariably in large quantities at the spot occupied by &ala&e in the standard chromatograms. This fact can be observed by examination of the values TABLE 3-F%EE
AMINOACIDDISTRIBUTION OF Aedes aegypti (maintained on 4%
sucrose)
Reported as per cent dry sample weight. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis.
Amino acid
I
Age (days) l-2
7
14
21
28
35
42
0.660 0.134 0 0.042 0.136 0.041 0.299
@614 0.098
0.561 0.066
O-946 0.137
0.525 0.063
0.718 0.150
0.504 0.097
zoo7 0.016 0.030 0.171
x*022 0.017 0.012 0.182
z-025 0.104 0.041 0.218
t-052 0~055 0.034 0.155
x.099 0.117 8:Z
k46 0.042 o+lo 0.157
0.040 O-070 trace 0.025 Pas 0.043 0.032 0.040 0.047
@004 o-01 3 trace trace pas 0.014 0.007 trace 0.007
0.004 0.026 trace trace Pas 0.011 0.009 0.014 0.013
0.023 0.032 trace 0.033 Pas 0.027 0.027 0.018 O*OlO
0.011 0.039 trace trace Pas 0.035 0@08 0.016 0.009
0.025 0.061 0.024 0.022 Pas O-o.54 0.036 0.006 0.036
@006 0.015 0.006 trace Pas 0.019 0.013 0.014 0.006
Total
1.609
0.981
0.937
1*641
1.002
1.634
0.965
Taurine X1
trace 0.345
trace 0.414
trace 0.336
trace 0.331
0.028 0440
0*055 0.114
WO26 0.248
Alanine Arginine
Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/o isoleucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryprophan Valine
given for it in Tables 3-5 even though the peptide values are based on the arbitrary assumption that its colour yield is the same per unit weight as that of glycine. Upon hydrolysis by 20% HCl at 100°C for 16 hr, it was apparent that fi-alanine was indeed part of the peptide. Spots also appeared on the chromatograms which corresponded with those of cysteine andtyrosine, which had not been present before the hydrolysis. An apparent increase in the amount of threonine also occurred. The peptide may, therefore, consist at least in part of fi-alanine, cysteine and/or cystine, threonine, and tyrosine. In summary, the following amino acids were found consistently in the extracts of Aedes aegypti tissue: alanine, arginine, glutamic acid, glutamine, glycine, hi&line, leucine and/or isoleucine, phenylalanine, proline, serine, threonine, tryptophan, and valine. Tyrosine was found only after a blood meal had been given, whereas aspartic acid was found in only one age group. Taurine was normally present
140
D. W. THAYER AND L. A. TERZIAN
in rather small quantities.
A peptide X, was present in considerable quantities in the extracts of the tissue. The results of the quantitative analysis of the amino acids are summarized in Tables 3-8. TABLB ~-FREE AMINO ACID DISTRIBUTION OF Cedes uegypti (maintained on 4% sucrose and blood meals) Reported as per cent dry sample weight. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis.
T Amino acid
Alanine Arginine Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/or isoleucine Lysine Methionine ~kx;x+$ke Serine Threonine Tryptophan Tyrosine Valine
Age (days) and, in parentheses, blood meal age (days)
l-2 (none)
7 (none)
14 (7)
0.914 O-487
0.240 0.125 0 0.009 0.241 0.033 0.171
0.527 0.088 0 0.018 0.095 0.016 0.118
0.678 0.122 0 0.023 0.058 0.040 0.166
0.617 0.138
:*409 0.269 0.257 0.291
0.792 0.224 0.060 0.130 O-180 0.066 0.276
0.113 0.390 0.047 0.117 Pas 0.106 0.058 0.013
o*oso 0.099 0.010 0@4 Pas 0.058 trace trace
00.193
00.068
0.014 0.043 0,017 .0.018 0.126 0.026 0.010 trace 0.015 0,026
0.016 0.083 0.015 0*045 Pas 0.020 trace trace 0,086 0.028
0.032 0.030 O-024 o*oss 0.185 o*oso 0.013 trace O-032 0.038
0.031 0.032 0.020 o*oso 0.114 O+lO 0.019 0.003 0.057 0.042
3.664
2.057
0.988
1.155
1.361
1.806
Pas Pas
0.255 0468
0.074 0.236
0.098 0.271
0.135 0.205
0.103 0.243
Total Taurine X1
z.037 0.216 O-042 0462
* Not included in total. DISCUSSION
By examination of the total amount of free amino acid in the ageing mosquito, it becomes apparent that the amount of free amino acid presents much the same pattern as does the total nitrogen for the same period (TERZIAN et al., 1957). That is, as the weight of the insect increases during the first 2 weeks following emergence the percentage of total free amino acid declines. When the weight declines during the next 3 weeks the percentage of total free ammo acid increases. A comparison of the data for the dry weights and of the data for the percentages of total amino acid over the S-week period thus indicates a relationship in which the values for one set
TABLE S-FREE
AMINO
ACID DISTRIBUTION
OF
(maintained on cooked raisins)
Aedes aegypti
Reported as per cent dry sample weight. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis.
Amino acid Alanine A&nine Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/or &Ieucine Lysine Methionine Phenylalanine Proline* Serine Threonine ;grphan Total Taurine XI
Age (days) l-2
7
14
21
28
35
0.914 O-487
0.615 0.459 0.152 0.142 0.254 0.006 0.390
0.640 0.152 0 0.028 0.159 0.029 0.175
0.237 0.103
0.407 0.165
0.411 0.119
i.014 0.260 0.019 0.152
i-020 0.150 0.038 0.226
t-029 0.367 0.040 0.211
0.062 0.152 0*04.5 0.048 Pas 0.507 trace trace 0.098
0.046 0.047 0.067 0.061 0.205 0.050 0.020 0.008 0.052
0.013 0.043 0.010 0.020 Pas 0.021 0.007 0.018 0.022
0.021 0.039 0.024 0.033 0.157 0.042 0.011 trace 0.032
0.022 0.042 0.020 0.027 0.116 0.036 0.019 0.003 0,033
t-409 0.269 0.257 0.291 0.113 0.390 0.047 0.117 Pas 0.106 ::::: 0.193 3.664
2.930
1.534
0.939
1.208
1.379
Pas Pas
0.277 0.547
0.220 0.281
0.076 0.246
0.016 0.109
::::.
* Not included in total. TABLE ~-FREE AMINOACID DISTRIBUTION OF Aedes aegypti (maintained on 4% sucrose) Reported as per cent total free ammo acid. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis.
Amino acid Alanine A&nine Aspartic acid Glutamic acid Glutamine Glycine Hi&dine Leucine and/or isoleucine Lysine Methionine Pheny&nine Proline Serine Threonine Tophan . e Total
l-2
7
14
21
g-7
59.9 7.0 0 2.4
57.6 8.4 0
1E
;:: 17.4
::; 19.4
;:i trace 1.6 Pas
0.4 l-3 trace trace Pas
0.4 2.8 trace trace Pas
A:$ trace 0.7
41.0 8.3 0 3:;
;:g ;:; 100.0
62a6 10.0
99.9
28
42
;:; 1Zd ;:; trace trace Pas
::;
;:; trace 2.0 Pas 1.6 1.6
:::
;:;
A:;
100.1
35
99.9
0.6 ;:z trace Pas 2.0
;:;
loo-2
::; 0.6 99.9
100.1
TABLE ~-FREE AMINO ACID DISTRIBUTIONOF Aedes uegypti (maintained on 4% sucrose and blood meals) Reported as per cent total free ammo acid. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis. Age (days) and, in parentheses, blood meal age (days). Amino acid
Alanine Arginine Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/or tioleucine Lysine Methionine Phenylalanine Proline Serine Threonine
l-2 (none)
7 (none)
24.9 13.3
38.5 10.9
24.3 12.6
lY.2 7.3 7.0 7.9
;:;
i.9 24.4
. 12
1.3 3.2 Pas 2.9 A:; 0 5.3
Z!~Ze!han Vahne Total
100.0
f :; 13.4 ff 0.5 21 Pas 2.8 trace trace 0 3.3 99.9
(:z)
(Z) 45-6 7.6 0
49-8 9-o 0
;:;
::; . *is
.
14.4
1A.Z
::::
::;
::;
::;
Pas 2.6 1.0 trace ::; 99.8
7:; trace trace 7.4 2.4 99.9
;:;
::p
::;
;:;
Pas
Pas 2.2
::;: trace ;::: 100.0
;:; i:f 100.0
TABLE 8-I?‘REE AMINO ACID DISTRIBUTIONOF Aedes aegy$ti (maintained on cooked raisins) Reported as per cent total free amino acid. 0, no visible spot; pas, present but not estimated; trace, spot present but below limits of analysis.
Amino acid Alanine Arginine Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/or isoleucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Valine Total
I
Age (days) l-2
7
14
21
28
35
17.2
21.0 15.7 5.2 4.8
41.7 8.9 0
25.2 11.0 0
33.7 13.6 0
29.8 8-6 0
;:; 7.9
;:; 13.3
1i.f: 1.9 11.4
2:*: 2.0 16.2
1:.: 3.1 18.7
2% 2.9 15.3
3.0
1.7 ;:; 2.7 Pas
1.6 3.0
;:; trace 2.6 99.7
24.9 13.3
1%
2.1 5.2
::;
;:;
Pas 2.9 A:: 5.3
Pas 17.3 trace trace 3.3
;:; 4.0 Pas 3.2 1.3 0.5 3.4
100.0
99.9
100.0
;:; Pas 2.6 A:; 2.4 99.9
THE FREE AMINOACIDSOF THE AGEINGFEMALEAEDES
AEGYPTI
MOSQUITO
143
of data appear to be the reciprocals of the other. Therefore, it is not unreasonable to assumethat the amount of free amino acid actuallypresent remainsapproximately at the same level during this S-week period. Although the relative amount of the individual amino acids changed during the experimental period (Tables 6-S), nevertheless, except in the case of the amino acids noted below, the changes were the same on all three regimens. On the other hand, in contrast to those maintained on sucrose alone, in which the level of methionine remained constant while glutamine and histidine decreased, the mosquitoes maintained on raisins, and ,particularly those given blood meals, showed a significant increase in glutamine, histidine, and methionine, while, as indicated before, tyrosine appeared only in those allowed to take blood. REFERENCES ACHER R. and CROCKERC. (1952) Reactions color&es specifiques de I-arginine et de la tyrosine r&lisbes apres chromatographie sur papier. Biochim. biophys. Acta 9, 704-705. ASSOCIATIONOF OFFICIAL AGRICULTURALCHEMISTS (1950) Oficial Methodr of Analysis (7th ed.), pp. 745-747. Monasha, Wisconsin. CLARK E. W. and BALL G. H. (1952) The free amino acids in the whole bodies of Culicid mosquitoes. Exp. Parasit. 1,339-346. CURZONG. and GILTROW J. (1954) Aromatic aldehydes as specific chromatographic colour reagents for amino-acids. Nature, Land. 173, 314-315. GIRI K. V. and NAGABHUSHANAM A. (1952) Sodium 1,2-naphthoquinone-4-sulfonate as a reagent for identification of amino-acids and peptides and for determination of proline and hydroxyproline separated on paper chromatograms. Naturz&enschaften 39,
548549. JEPSON J. B. and SI~IITIII. (1953) Multiple dipping procedures in paper chromatography: a specific test for hydroxyproline. Nature, Land. 172, 1100-1101. MICK~ D. W. and ELLIS J. P. (1952) Free amino acids in adult mosquitoes. Proc. Sot. exp.
Biol., N. Y. 79, 191-193. Micas D. W. and GIBBON F. J. (1957) The characterization of insect ticks by their free amino acid patterns. Amt. ent. Sot. Amer. 50, 500-505. REDFIELD R. R. (1953) Two-dimensional paper chromatographic systems with high resolving power for amino acids. Biochim. biophys. Acta 10,344-345. SMITH I. (1953) Colour reactions on paper chromatograms by a dipping technique. Nature, Lond. 171,43-X TERZIAN L. A., IRRE~ERREF., and STAHLZRN. (1957) A study of nitrogen and uric acid patterns in the excreta and body tissues of adult Aedes aegypti. J. Ins. Physiol- 1,221-228. TERZIAN L. A., STAHLERN., and IRREVERREF. (1956) The effects of ageing, and the modifications of these effects, on the immumty of mosquitoes to malarial infection. 3. Imntunol.
76,308-313. TOENNIESG. and KOLB J. J. (1951) Techniques and reagents for paper chromatography. Analyt. Chem. 23, 823-826. WELLINGTONE. F. (1952) An ultramicro method for quantitative determination of amino acids. Canad.J. Chem. 30,823-826. WYATT G. R., LOUGHED T. C., and WYATT S. S. (1956) The chemistryof insect hemolymph. r. gen. Physiol. 39, 853-868.
Printed in Great Britain
THE FREE AMINO ACIDS OF THE AGEING FEMALE AEDES AEGYPTI MOSQUITO” D. W. THAYER Naval
Medical
Research
Institute,
National
and L. A. TERZIAN Naval
(Received 6 October
Medical
Center,
Bethesda,
Maryland
1961)
Abstract-Quantitative
chromatographic methods were used to compare the free amino acid patterns of tissue extracts of ageing female Aedtu aegypti mosquitoes maintained on three different dietary regimens. Alanine, arginine, glutamic acid, glutamine, glycine, hiitidine, leucine and/or isoleucine, phenylalanine, proline, serine, taurine, threonine, tryptophan, and valine were consistently present in the tissue extracts. Of these alanine, histidme, arginine, glutamic acid, and lysine were present in the highest concentrations. Aspartic acid was found in only one age group, and tyrosine was found only in mosquitoes which had had a blood meal. In contrast to the mosquitoes maintained on sucrose, in which the level of methionine remained constant while glutamine and hi&dine decreased, the mosquitoes maintained on raisins or given blood meals showed significant increases in the levels of these three amino acids. A peptide which has been labelled X1 in the quantitative studies was present in large quantities at the spot occupied by @-alanine in the standard chromatograms. In all three groups this peptide decreased uniformly in amount as the mosquitoes aged.
INTRODUCTION IT HAS been shown that the resistance of the mosquito Cedes uegypti to infection with the malarial parasite Pbmodium gallinaceum increases markedly as it grows older (TERZIAN et al., 1956). It has also been shown that mosquitoes given a normal blood meal and those maintained on cooked raisins became again as susceptible to infection as newly emerged, young mosquitoes. The studies reported in this paper were undertaken in an effort to elucidate what changes, if any, occur in the free amino acids as the mosquito ages and what relationship, if any, these changes might have with the increase in the innate immunity of the ageing mosquito. CLARK and BALL (1952) found alanine, arginine, glutamic acid, glutamine, glycine, histidine, leucine and/or isoleucine, methionine, proline, serine, threonine, tryptophan, tyrosine, and cu-amino-n-butyric acid in Culex tars&s, Culex stigmatosoma, Aedes varipalpus, and Culiseta incidens. Aspartic acid, p-alanine, cysteic acid, lysine, and taurine were found in many but not all mosquitoes. MICKS and ELLIS (1952) reported finding alanine, arginine, ~-alanine, glutamic acid, glycine, histidine, tioleucine, lysine, methionine, proline, serine, threonine, tryptophan, tyrosine, and valine in the pupae and adults of Culex quinquefasciatus and Aedes aegypti. Aspartic acid appeared in chromatograms of the eggs and larva, but not in * The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. 9
133
134
D. W. THAYERANDL. A. TERZIAN
those of the pupae and adults. Phenylalaninewas not found and the authors noted that the larvae seem equally capable of utilizing either phenylalanine or tyrosine. In 1957, MICKS and GIBSONreported that strains of Aedes aegypti as well as other strains and species could easily be distinguished on the basis of quantitative differences in several amino acids. MATERIALS AND METHODS A laboratory strain of Aedes aegypti mosquitoes maintained at a constant temperature of 26~5°C and a relative humidity of 75 per cent was used for these experiments. The larvae were grown in glass crocks and fed solely on guinea-pig food pellets added in appropriate amounts from time to time during the larval developmental period. The adult mosquitoes were placed in wire-screened cages, measuring 15 x 15 x 15 in. and were selected from the emerging pupae so as to give a uniform age grouping of not less than 24 nor more than 48 hr at the start of each experiment. The mosquitoes for each experimental series were obtained from the same colony. The adult mosquitoes were routinely fed 4% sucrose solutions which were made available after the 24-48 hr age group had been removed for analysis. In addition, a source of water was provided for all caged mosquitoes. Those mosquitoes permitted to have blood meals were removed from the screen cages at the time of the blood meal and placed in lucite cylinders in groups of 80-100 females. They were permitted to gorge themselves on young normal pullets and were then held in the cylinders on a routine diet of 4% sucrose for 1 week before they were analysed. The mosquitoes receiving a, dietary supplement of cooked raisinswere given the raisinsas soon as the 24-48 hr sample had been removed and thereafter were provided with fresh raisins as needed. Total ‘nitrogen and moisture Three groups of twenty and one of 100 females were routinely used for the analysis. The lightly anaesthetixedmosquitoes were weighed and then were dried in vacua over P,O, to a constant weight of plus or minus O-2 mg. After a constant weight had been obtained the dry weights were obtained The group of 100 was retainedfor the amino acid analysisand the three groups of twenty were used for the determination of total nitrogen by the micro-Kjeldahl procedure (Ass. OFF.AGRIC. CHEM., 1950). Ali samples were included in the calculation of the per cent moisture and of the dry weight per mosquito. Amino acid determination The 100 mosquito sample (approximately 50 mg) described above was pulverized within its weighing bottle, in this case a 10 x 75 mm glass-stopperedtest tube. Then O-2 ml of cold glass-distilled water was added to the tissue and the resulting slurry mixed thoroughly by tapping the sides of the tube. The sample was then allowed to stand overnight. The following day O-8ml of cold absolute ethanol was added and mixed with the slurry and the sample was again allowed to stand overnight. The precipitated protein was removed by centrifugation at 8000
THE FREE AMINO
ACIDS OF THE AGEING
FEMALE AEDES AEGYPTI
MOSQUITO
135
revjmin for at least 1 hr; the amino acid extract decanted and recentrifuged. The above operations were all carried out at 4°C and the sample tube was always sealed with a heavy coating of paraf%nfilm in order to prevent loss by evaporation. This provided a stable extract of the free amino acids in 80% ethanol. Chromatography was carried out by the procedure modified from REDFIELD (1953) which was described by WYATTet aZ. (1956). Whatman No. 1 filter paper was used in 15 x 15 in. sheets sprayed before use with 12 ml of O.O2o/oEDTA in 60% ethanol. The chromatograms were run without prior vapour equilibration by
METHANOL PYRIDINE HOH 20*1:5 t TIME 7HR
MILL
DIRECTION-=+
FIG. 1. Tracing of amino acid chromatogram of an extract of l-day-old Aedes aegypti. Development time, 16 hr. Stain, 2% ninhydrin. Paper sprayed before with 10 ml of EDTA (O@O2%)in 60% ethanoL
the ascending method in borosilicate glass cylinders 6 x 18 in., which were sealled with weighted plate-glass covers. The first solvent contained tert-butanol 10, methyl ethyl ketone 10, concentrated ammonium hydroxide 3, and water 5 parts by volume. The second dimension was run in Redfield’s solvent consisting of methanol 20, pyridine 1, and water 5 parts by volume. Typical runs were made in 16 hr in the first dimension and 7 hr in the second. Typical chromatograms are presented in Figs. 1 and 2. The amino acids were estimated using the 2% solution of ninhydrin in absolute ethanol recommended by WELLINGTON (1952). The technique used differed in that a rapid dipping procedure was used rather than the spray technique of WELLINGTON. The papers were dipped in the ninhydrin solution and allowed to
136
D. W. THAYEBAND L. A. TERZIAN
air-dry in the dark overnight before evaluation. In this laboratory, this technique gave a more uniform distribution of the dye without smearing so long as the absolute ethanol used was obtained from freshly opened bottles.
06
RF
04
0.2
co AR0
LYS
t #
i
I
FIG. 2. Tracing of amino acid chromatogram of an extract of l-day-old Aedes aegypti. Solvent I: tert-Btitanol-methyl ethyl ketone-concentrated ammonia-water (10 : 10 : 3 : 5). Solvent 11: Methanol-pyridine-water (20 : 1 : 5).
After development a permanent record of each chromatogram was made for future reference by tracing the locations of the amino acid spots on graph paper. Each coloured area was then cut out of the chromatogram, weighed and placed in a centrifuge tube for extraction with 5 ml of cold 50% aqueous n-propanol. Blanks of the same size were taken at four levels and used for correcting the amino acid spots at each level for background absorbance of dye. The corrections were made by comparison of the weight of the blank to that of the amino acid spot and using the resulting fraction to determine the background correction for the particular amino acid spot. The tubes were shaken for 15 min in a mechanical shaker and then centrifuged in the cold to settle the lint formed during the shaking.
THE FREE AMINO ACIDS OF THE AGEING FEMALE AEDES AEGYPTI
MOSQUITO
137
The optical absorbance for all areas was determined in 1 cm Corex cuvettes in the Beckman Model DU spectrophotometer at 570 my, except proline, which was determined at 350 mp. A calibration curve was prepared for each amino acid from chromatograms of standard mixtures of 2, 5, and 10 gamma concentrations, which were run at the same time as the samples. The equation for each curve was determined by the method of least squares and the unknown determined directly from the equation. Three replicas of each sample were run simultaneously for quantitativeevaluation, and in addition a varying number of other replicas were run for qualitative evaluations. Individual amino acids were determined by position and by specific chemical tests where possible. RESULTS
Total nitrogen, we+, and moisture The values for the total nitrogen, weight, and moisture determinations are summarized in Tables 1 and 2. An examination of the wet and dry weights in both tables shows that they parallel each other, and that the moisture content of the TABLE ~-THE
Sample
PERCENTAGE
NITROGRN AND WRT AND DRY WRIGHT OF ADULT FRMALE
de&s aegypti MOSQUITOES (maintainedon 4% sucrose)
Mosquito age (days)
Per cent nitrogen
Wet wt.fmosq. (mg)
DrY wt./mosq. (mid
7.84 6.15 6.87 7.35 5.86
l-64 1.68 1.47 1.57 1.51
O-48 0.62 0.52 0.53 0.56
7.30 7.51
l-41 1.34
::g
Per cent moisture
mosquitoes remains comparatively constant during the experimental period. Table 1 shows that there is an increase in weight during the first week following emergence; the weight then declines during the remaining $-week period of the experiment. On the other hand, the percentage of tissue nitrogen declines during the first week, but then begins to increase and reaches a maximum at the end of the 6-week period. A comparison of the data for the wet and dry weights and the percentage of tissue nitrogen of these mosquitoes over the 6-week period indicates a relation in which the values for one set of data appear to be the reciprocals of the other. It appears not unreasonable to assume, therefore, that the nitrogen content of the mosquitoes remains comparatively constant during this period of time. Free amino acid The identity of individual amino acid spots on the chromatograms was determined by position and/or specific chemical tests. In cases where the spot
138
D. W. THAYER
AND L. A. TERZIAN
was so faint as to be questionable, a larger sample was used for chromatography in order to illustrate the presence or absence of the particular amino acid. Arginine, which was invariably present, was tested for by a modification of the Sakaguchi TABLE ~-THE PEXEN!I'AGE WET ANDDRYWEIGHT OFADULT FEMALE Aedes aegypti
MOSQUITOES
Sample
wet wt./mosq. Cm&
Mosquito age (days)
Dry wt./mosq. (md
Per cent moisture
Maintained on 4% sucrose and blood meals*
A
:
Bc E F
f;’ 28 35
1-77
0.51
71.13
2.43 2.00
0.85 0.63
67.98 65.02
2.46 2.24 2-27
0.83 0.78 0.75
66.26 65.18 66.96
Maintained on cooked raisins*
A :
::
: F
;: 3”;
*
1.77 2.45 2.03
o-51 O-85 O-61
71.13 65.31 69.71
2.06 2.03 1.90
0.67 o-59 0.59
70.94 67.48 68.95
* Pairedsamples.
reaction (ACHER and CROCKER,1952). The presence of histidine was established by the method of CURZ~N and GILTROW (1954). The presence of proline and the absence of hydroxyproline was established by two methods, GIRI and NAGABHUSHANAM (1,952) and SMITH (1953). Tryptophan was consistently demonstrated by the use of Ehrlich’s reagent as described by SMITH (1953). Tyrosine was consistently absent in most of the chromatograms using the cu-nitroso-/3-naphthol reagent described by JEPSO~V and SMITH (1953). Tyrosine was found only after the mosquitoes had been given a blood meal. The sensitivity of this test was found to be at least O-1 gamma, which would seem to rule out the presence of tyrosine where no positive could be obtained. Since cysteic acid had been reported to be present in some species of mosquitoes by CLARK and BALL (1952), a qualitative test (TOENNIESand KOLB, 1951) was carried out for cysteine and/or cystine. Both were consistently absent in this strain of Aedes aegypti.
THE FREE AMINOACIDSOF THE AGEINGFEMALEAEDES
AEGYPTI
139
MOSQUITO
In addition to the above, a peptide, labelled X, in the quantitative studies, was present invariably in large quantities at the spot occupied by &ala&e in the standard chromatograms. This fact can be observed by examination of the values TABLE 3-F%EE
AMINOACIDDISTRIBUTION OF Aedes aegypti (maintained on 4%
sucrose)
Reported as per cent dry sample weight. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis.
Amino acid
I
Age (days) l-2
7
14
21
28
35
42
0.660 0.134 0 0.042 0.136 0.041 0.299
@614 0.098
0.561 0.066
O-946 0.137
0.525 0.063
0.718 0.150
0.504 0.097
zoo7 0.016 0.030 0.171
x*022 0.017 0.012 0.182
z-025 0.104 0.041 0.218
t-052 0~055 0.034 0.155
x.099 0.117 8:Z
k46 0.042 o+lo 0.157
0.040 O-070 trace 0.025 Pas 0.043 0.032 0.040 0.047
@004 o-01 3 trace trace pas 0.014 0.007 trace 0.007
0.004 0.026 trace trace Pas 0.011 0.009 0.014 0.013
0.023 0.032 trace 0.033 Pas 0.027 0.027 0.018 O*OlO
0.011 0.039 trace trace Pas 0.035 0@08 0.016 0.009
0.025 0.061 0.024 0.022 Pas O-o.54 0.036 0.006 0.036
@006 0.015 0.006 trace Pas 0.019 0.013 0.014 0.006
Total
1.609
0.981
0.937
1*641
1.002
1.634
0.965
Taurine X1
trace 0.345
trace 0.414
trace 0.336
trace 0.331
0.028 0440
0*055 0.114
WO26 0.248
Alanine Arginine
Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/o isoleucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryprophan Valine
given for it in Tables 3-5 even though the peptide values are based on the arbitrary assumption that its colour yield is the same per unit weight as that of glycine. Upon hydrolysis by 20% HCl at 100°C for 16 hr, it was apparent that fi-alanine was indeed part of the peptide. Spots also appeared on the chromatograms which corresponded with those of cysteine andtyrosine, which had not been present before the hydrolysis. An apparent increase in the amount of threonine also occurred. The peptide may, therefore, consist at least in part of fi-alanine, cysteine and/or cystine, threonine, and tyrosine. In summary, the following amino acids were found consistently in the extracts of Aedes aegypti tissue: alanine, arginine, glutamic acid, glutamine, glycine, hi&line, leucine and/or isoleucine, phenylalanine, proline, serine, threonine, tryptophan, and valine. Tyrosine was found only after a blood meal had been given, whereas aspartic acid was found in only one age group. Taurine was normally present
140
D. W. THAYER AND L. A. TERZIAN
in rather small quantities.
A peptide X, was present in considerable quantities in the extracts of the tissue. The results of the quantitative analysis of the amino acids are summarized in Tables 3-8. TABLB ~-FREE AMINO ACID DISTRIBUTION OF Cedes uegypti (maintained on 4% sucrose and blood meals) Reported as per cent dry sample weight. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis.
T Amino acid
Alanine Arginine Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/or isoleucine Lysine Methionine ~kx;x+$ke Serine Threonine Tryptophan Tyrosine Valine
Age (days) and, in parentheses, blood meal age (days)
l-2 (none)
7 (none)
14 (7)
0.914 O-487
0.240 0.125 0 0.009 0.241 0.033 0.171
0.527 0.088 0 0.018 0.095 0.016 0.118
0.678 0.122 0 0.023 0.058 0.040 0.166
0.617 0.138
:*409 0.269 0.257 0.291
0.792 0.224 0.060 0.130 O-180 0.066 0.276
0.113 0.390 0.047 0.117 Pas 0.106 0.058 0.013
o*oso 0.099 0.010 0@4 Pas 0.058 trace trace
00.193
00.068
0.014 0.043 0,017 .0.018 0.126 0.026 0.010 trace 0.015 0,026
0.016 0.083 0.015 0*045 Pas 0.020 trace trace 0,086 0.028
0.032 0.030 O-024 o*oss 0.185 o*oso 0.013 trace O-032 0.038
0.031 0.032 0.020 o*oso 0.114 O+lO 0.019 0.003 0.057 0.042
3.664
2.057
0.988
1.155
1.361
1.806
Pas Pas
0.255 0468
0.074 0.236
0.098 0.271
0.135 0.205
0.103 0.243
Total Taurine X1
z.037 0.216 O-042 0462
* Not included in total. DISCUSSION
By examination of the total amount of free amino acid in the ageing mosquito, it becomes apparent that the amount of free amino acid presents much the same pattern as does the total nitrogen for the same period (TERZIAN et al., 1957). That is, as the weight of the insect increases during the first 2 weeks following emergence the percentage of total free amino acid declines. When the weight declines during the next 3 weeks the percentage of total free ammo acid increases. A comparison of the data for the dry weights and of the data for the percentages of total amino acid over the S-week period thus indicates a relationship in which the values for one set
TABLE S-FREE
AMINO
ACID DISTRIBUTION
OF
(maintained on cooked raisins)
Aedes aegypti
Reported as per cent dry sample weight. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis.
Amino acid Alanine A&nine Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/or &Ieucine Lysine Methionine Phenylalanine Proline* Serine Threonine ;grphan Total Taurine XI
Age (days) l-2
7
14
21
28
35
0.914 O-487
0.615 0.459 0.152 0.142 0.254 0.006 0.390
0.640 0.152 0 0.028 0.159 0.029 0.175
0.237 0.103
0.407 0.165
0.411 0.119
i.014 0.260 0.019 0.152
i-020 0.150 0.038 0.226
t-029 0.367 0.040 0.211
0.062 0.152 0*04.5 0.048 Pas 0.507 trace trace 0.098
0.046 0.047 0.067 0.061 0.205 0.050 0.020 0.008 0.052
0.013 0.043 0.010 0.020 Pas 0.021 0.007 0.018 0.022
0.021 0.039 0.024 0.033 0.157 0.042 0.011 trace 0.032
0.022 0.042 0.020 0.027 0.116 0.036 0.019 0.003 0,033
t-409 0.269 0.257 0.291 0.113 0.390 0.047 0.117 Pas 0.106 ::::: 0.193 3.664
2.930
1.534
0.939
1.208
1.379
Pas Pas
0.277 0.547
0.220 0.281
0.076 0.246
0.016 0.109
::::.
* Not included in total. TABLE ~-FREE AMINOACID DISTRIBUTION OF Aedes aegypti (maintained on 4% sucrose) Reported as per cent total free ammo acid. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis.
Amino acid Alanine A&nine Aspartic acid Glutamic acid Glutamine Glycine Hi&dine Leucine and/or isoleucine Lysine Methionine Pheny&nine Proline Serine Threonine Tophan . e Total
l-2
7
14
21
g-7
59.9 7.0 0 2.4
57.6 8.4 0
1E
;:: 17.4
::; 19.4
;:i trace 1.6 Pas
0.4 l-3 trace trace Pas
0.4 2.8 trace trace Pas
A:$ trace 0.7
41.0 8.3 0 3:;
;:g ;:; 100.0
62a6 10.0
99.9
28
42
;:; 1Zd ;:; trace trace Pas
::;
;:; trace 2.0 Pas 1.6 1.6
:::
;:;
A:;
100.1
35
99.9
0.6 ;:z trace Pas 2.0
;:;
loo-2
::; 0.6 99.9
100.1
TABLE ~-FREE AMINO ACID DISTRIBUTIONOF Aedes uegypti (maintained on 4% sucrose and blood meals) Reported as per cent total free ammo acid. 0, no visible spot; pos, present but not estimated; trace, spot present but below limits of analysis. Age (days) and, in parentheses, blood meal age (days). Amino acid
Alanine Arginine Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/or tioleucine Lysine Methionine Phenylalanine Proline Serine Threonine
l-2 (none)
7 (none)
24.9 13.3
38.5 10.9
24.3 12.6
lY.2 7.3 7.0 7.9
;:;
i.9 24.4
. 12
1.3 3.2 Pas 2.9 A:; 0 5.3
Z!~Ze!han Vahne Total
100.0
f :; 13.4 ff 0.5 21 Pas 2.8 trace trace 0 3.3 99.9
(:z)
(Z) 45-6 7.6 0
49-8 9-o 0
;:;
::; . *is
.
14.4
1A.Z
::::
::;
::;
::;
Pas 2.6 1.0 trace ::; 99.8
7:; trace trace 7.4 2.4 99.9
;:;
::p
::;
;:;
Pas
Pas 2.2
::;: trace ;::: 100.0
;:; i:f 100.0
TABLE 8-I?‘REE AMINO ACID DISTRIBUTIONOF Aedes aegy$ti (maintained on cooked raisins) Reported as per cent total free amino acid. 0, no visible spot; pas, present but not estimated; trace, spot present but below limits of analysis.
Amino acid Alanine Arginine Aspartic acid Glutamic acid Glutamine Glycine Histidine Leucine and/or isoleucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Valine Total
I
Age (days) l-2
7
14
21
28
35
17.2
21.0 15.7 5.2 4.8
41.7 8.9 0
25.2 11.0 0
33.7 13.6 0
29.8 8-6 0
;:; 7.9
;:; 13.3
1i.f: 1.9 11.4
2:*: 2.0 16.2
1:.: 3.1 18.7
2% 2.9 15.3
3.0
1.7 ;:; 2.7 Pas
1.6 3.0
;:; trace 2.6 99.7
24.9 13.3
1%
2.1 5.2
::;
;:;
Pas 2.9 A:: 5.3
Pas 17.3 trace trace 3.3
;:; 4.0 Pas 3.2 1.3 0.5 3.4
100.0
99.9
100.0
;:; Pas 2.6 A:; 2.4 99.9
THE FREE AMINOACIDSOF THE AGEINGFEMALEAEDES
AEGYPTI
MOSQUITO
143
of data appear to be the reciprocals of the other. Therefore, it is not unreasonable to assumethat the amount of free amino acid actuallypresent remainsapproximately at the same level during this S-week period. Although the relative amount of the individual amino acids changed during the experimental period (Tables 6-S), nevertheless, except in the case of the amino acids noted below, the changes were the same on all three regimens. On the other hand, in contrast to those maintained on sucrose alone, in which the level of methionine remained constant while glutamine and histidine decreased, the mosquitoes maintained on raisins, and ,particularly those given blood meals, showed a significant increase in glutamine, histidine, and methionine, while, as indicated before, tyrosine appeared only in those allowed to take blood. REFERENCES ACHER R. and CROCKERC. (1952) Reactions color&es specifiques de I-arginine et de la tyrosine r&lisbes apres chromatographie sur papier. Biochim. biophys. Acta 9, 704-705. ASSOCIATIONOF OFFICIAL AGRICULTURALCHEMISTS (1950) Oficial Methodr of Analysis (7th ed.), pp. 745-747. Monasha, Wisconsin. CLARK E. W. and BALL G. H. (1952) The free amino acids in the whole bodies of Culicid mosquitoes. Exp. Parasit. 1,339-346. CURZONG. and GILTROW J. (1954) Aromatic aldehydes as specific chromatographic colour reagents for amino-acids. Nature, Land. 173, 314-315. GIRI K. V. and NAGABHUSHANAM A. (1952) Sodium 1,2-naphthoquinone-4-sulfonate as a reagent for identification of amino-acids and peptides and for determination of proline and hydroxyproline separated on paper chromatograms. Naturz&enschaften 39,
548549. JEPSON J. B. and SI~IITIII. (1953) Multiple dipping procedures in paper chromatography: a specific test for hydroxyproline. Nature, Land. 172, 1100-1101. MICK~ D. W. and ELLIS J. P. (1952) Free amino acids in adult mosquitoes. Proc. Sot. exp.
Biol., N. Y. 79, 191-193. Micas D. W. and GIBBON F. J. (1957) The characterization of insect ticks by their free amino acid patterns. Amt. ent. Sot. Amer. 50, 500-505. REDFIELD R. R. (1953) Two-dimensional paper chromatographic systems with high resolving power for amino acids. Biochim. biophys. Acta 10,344-345. SMITH I. (1953) Colour reactions on paper chromatograms by a dipping technique. Nature, Lond. 171,43-X TERZIAN L. A., IRRE~ERREF., and STAHLZRN. (1957) A study of nitrogen and uric acid patterns in the excreta and body tissues of adult Aedes aegypti. J. Ins. Physiol- 1,221-228. TERZIAN L. A., STAHLERN., and IRREVERREF. (1956) The effects of ageing, and the modifications of these effects, on the immumty of mosquitoes to malarial infection. 3. Imntunol.
76,308-313. TOENNIESG. and KOLB J. J. (1951) Techniques and reagents for paper chromatography. Analyt. Chem. 23, 823-826. WELLINGTONE. F. (1952) An ultramicro method for quantitative determination of amino acids. Canad.J. Chem. 30,823-826. WYATT G. R., LOUGHED T. C., and WYATT S. S. (1956) The chemistryof insect hemolymph. r. gen. Physiol. 39, 853-868.