Studies on the protein metabolism of Culex pipiens L.—I

J. ins. Physiol.,

1958, Vol. 2, pp. 38 to 51.

STUDIES

ON

METABOLIC

THE CULEX

CHANGES LARVAL

Pergamon Press Ltd., London

PROTEIN PIPIENS OF

AND

P. Institute

of Zoology

FREE

PUPAL

and Comparative (Received

S.

METABOLISM L.-I AMINO

ACIDS

OF DURING

DEVELOPMENT*

CHEN

Anatomy, 13 September

University

of Ziirich,

Switzerland

1957)

Abstract-Using paper partition chromatography, the free amino acids and related substances in the methanol extracts and in the blood from various larval and pupal stages of Culexpipiens (autogenous form) were studied. The ninhydrin-positive pattern is the same as that described earlier (LAVEN and CHEN, 1956), and no qualitative differences were found between the developmental periods. In the course of larval development the quantities of proline, tyrosine, and glutamine per individual increase rapidly. According to previous workers, these compounds are particularly involved in the synthesis of cuticular proteins in the fully grown larvae. However, the concentration as well as the amount of total free ninhydrin-positive components per unit body weight or total N remain essentially unchanged during the larval life. The free amino acid content in the developing pupae is low. At about 24 hr after pupation tyrosine, proline, glutamic acid, glutamine, and valine exhibit a slight increase. This fact suggests that the histolytic process is at its maximum at this period.

INTRODUCTION

to the biochemical studies of lethal factors in Drosophila melanogaster shows that the protein metabolism in the lethal mutants differs to a great extent from that in the wild types (for references, see HADORN, 1954 and 1956). The preliminary work of LAVEN and CHEN (1956) on the factor “mel” of Culexpipiens (autogenous form) has brought evidence that the blood of lethal larvae is very poor in free amino acids. The total concentration of free ninhydrin-positive substances in the affected individuals is reduced to about 70 per cent of that in the normals. Owing to shortage of material only a limited number of determinations was carried out at that time. For further analysis of this lethal factor it is desirable to have a more complete knowledge on the metabolic changes of the free amino acids during normal development. The variations in the free amino acid content of the insect blood have been followed in a number of species at various developmental stages. We need only refer to the more recent work on Galleria mellonella (AUCLAIR and DUBREUIL, 1952), Culex quinquefasciatus and Aedes aegypti (MICKS and ELLIS, 1952), Mamothylacea rubi (DRILHON, 1952), Bombyx mori (SARLET et al., 1952; AMANIEU et al., 1956), REFERENCE

* This study was supported by a grant from the “Karl Hescheler Stiftung”. I am greatly indebted to Prof. Dr. E. HADOFWfor his valuable criticism in the preparation of the manuscript. 38

STUDIESON THE PROTEINMETABOLISM OF CULEX

PIPIENS

L.

39

Drosophila :meZanogaster (HADORN and MITCHELL, 1951; HADO& and STUMMZOLLINGER, 1953 ; STUMM-ZOLLINGER, 1954; CHEN and HADORN, 1955 ; BENZ, 1957), Corethrapulmicornis (CHEN and HADORN, 1954), Cdiphora augur (HACKMAN, 1956), and Ephestia kiihniella (CHEN and K~~HN, 1956). In all these insects there are steady changes in blood composition as development proceeds. The mai.n phenomena in the development of holometabolic insects are growth during early larval life, followed by histolysis and histogenesis at metamorphosis. These processes involve either the synthesis of new proteins or the transformation of them from one form to another, and cause therefore the fluctuation of ninhydrinThe present study serves as a further example positive components in the blood. to illustrate the close relationship between protein metabolism and morphogenesis in developing insects. MATERIALS

AND METHODS

The CuZex pipiens stock (autogenous form) used in the present study was first reared in the Max-Planck Institute of Biology, Tubingen, and later in our laboratory ,for many generations .* Eggs were collected immediately after oviposition, and the time of hatching was observed. The larvae were kept in culture dishes of 12 cm diameter containing about 450 ml of water and were fed daily The feeding usually began on the third day after with powdered dog-biscuit. hatching. During larval development the water in the culture dishes was changed every 2-3 days. Since larvae from the same batch of eggs may not pupate at the same time, the culture dishes were inspected at short intervals during the period of pupation. Immediately after the beginning of pupal life the pupae were collected from the culture dishes and kept in separate bottles until the time of emergence. During both larval and pupal development the number of individuals per container (usually 20-30 individuals per dish) was controlled in order to avoid overcrowding. All cultures were maintained at 22.5” C. At this temperature the embryonic development lasts about 48 hr and the larval and pupal life 13-15 days and 2-3 days respectively. This is in close agreement with the observation reported by M~~LLRING (1956). The same author has also gi\,en a detailed account of the duration of the four larval stages. The free amino acids and related substances in the extracts of whole larvae and pupae, and their concentrations in the blood of corresponding developmental stages, wer’e investigated by the method of paper chromatography. During sampling tine specimens were first thoroughly washed in several changes of distilled water and the adhering water was removed with filter paper. After Accordcounting the number of individuals, the total fresh weight was estimated. ing to developmental stages, 12-100 larvae (Table 3) or 12 pupae were used for each determination. The extraction of free amino acids was carried out by crushing the specimens in a small glass tube followed by washing them in 80 per cent methanol. After centrifuging, the supernatant was quantitatively * The author wishes to thank Dr. H. LAVEN, Tiibingen,

for providing the Culex pipiem stock.

40

P. S. CHEN

transferred to a Whatman no. 1 filter paper (28 x 46 cm) for chromatographic separation. The separation of individual amino acids was carried out two-dimensionally, using as the first solvent 70 per cent n-propanol and as the second solvent watersaturated phenol. For qualitative analysis the chromatograms were sprayed with a 0.2 per cent ninhydrin solution and the colour developed at 90°C for 15-20 min. For quantitative determination the ninhydrin reaction was done in a buffer solution according to procedures worked out by BOISSONNAS (1950). The quantities of individual amino acids were calculated from correction factors given by STUMM-ZOLLINGER (1954) and CIIEN and HADORN (1954). each specimen was opened carefully under a For taking blood samples, binocular on a slide coated with paraffin, and the haemolymph collected in a For the determination of total concentration, 2 ~1 of calibrated micropipette. blood was obtained from 3-33 larvae (Table 2) or 5-9 pupae (Table 5). More individuals were used for early larval development: there is considerably less The blood sample was delivered immediately to a haemolymph in aged pupae. Whatman no. 1 sheet (15 x 24 cm) and chromatographed one-dimensionally The total concentration of ninhydrin-positive comin 70 per cent n-propanol. ponents was determined according to methods described earlier (HADORN and STUMM-ZOLLINGER, 1953; CHEN and HADORN, 1954). For estimating the concentration of individual amino acids, 20 ~1 haemolymph was collected from either fully grown larvae or pupae at ca. 24 hr of age. The blood was transferred into a glass tube and diluted with an equal volume of twice distilled water. The proteins were denatured by dipping the glass tube briefly into boiling water. The sample was then centrifuged and the upper clear layer transferred to a filter paper for two-dimensional chromatography, as described for the methanol extract. As a basis for comparison, the increase of both fresh weight and total N during larval and pupal development was followed. The weight values were obtained from 5 separate culture dishes started at the same time, and each contained about the same number of individuals. For the determination of total N a micro-Kjeldahl method was used (BOELL and SHEN, 1954). RESULTS As shown in Fig. 1, the curves of both fresh weight and total N are very similar during the larval development. Growth is rather slow until about the sixth day after hatching, then more rapid in the next 4-5 days, and again somewhat slower shortly before the beginning of pupation. Towards the end of larval life there is apparently a larger increase of total N. At pupal development, values of fresh weight and total N remain more or less constant, except that there seems to be a slight decrease of both approaching the time of emergence (Tables 4 and 6). Qualitative analyses of the ninhydrin-positive components in larval extracts confirmed the results reported earlier (LAVEN and CHEN, 1956, Fig. 2, p. 275).

STUDIESON THE PROTEINMETABOLISMOF CVLEX However,

spot

sulphoxide.

which

was

acid in the previous !?pecific

can be oxidised of ammonium stages

16,

No.

y-aminobutyric

tests showed

to methionine molybdate

exhibits

CHEN,

the

according

to

its

has now been identified

that it is a sulphur-containing

sulphone

(cf.

essentially

designated

study,

same

41

L.

PIPIENS

by hydrogen

1958).

The

blood

values

amino

peroxide

chromatographic

R,

as

as methionine acid and

in the presence

at corresponding pattern

except

50

larval that

the

5

UQN

mg 4

40-

3

2

FIG. 1. Successive changes in fresh weight (- - -) and total N (-) during larval development at 22.5”C. Ordinate: right, fresh weight in mg per larva; left, total N in pg per larva. Abscissa: larval age in days after hatching. Each point represents the average value of 3-6 determinations. The vertical lines indicate the extreme values. concentration higher

(cf.

During

of ct-alanine, Fig.

pupal

Glutamic

glutamine,

2a in LAVEN development

acid,

leucine,

the

and

lysine,

and CHEN,

1956

and arginine and

Fig.

ninhydrin-positive

possibly

seems

picture

phenylalanine

to be relatively

2 in the present

appear,

remains however,

study). similar.

in larger

quantities. TABLE I-AVE:MGE FRESHWEIGHTS AND TOTAL QUANTITIESOF FREE NINHYDRIN-POSITIVE SUBE~TANCES DURINGLARVALDEVELOPMENT OF Culex pipiens AT 22.5”C T

Larval age (days after hatching)

Fresh weight

Number of determinations

2 7

45 4

: 11

t 5

(mg/l=va)

0~11810~0012 0.37110.0246 0.633 10.0374 1.178*0.0491 1.653 *0.1054 3.474*0.1597

Extinction/larva

Extinction/mg

0.0144 *0.0024 0.039210.0052 0.0795 10.0045 0.145 *0.0074 0.196 &0.0171 0.461 10.0233

0.12210-0152 0~104&0~0091 0.12610.0039 0.124 ho.0094 0~118~0~0182 0.133 10.0041

1

Parallel

tcs growth,

per larva increases

with

the

total

amount

the advance

of

free

ninhydrin-positive

of development

(Table

1).

The

substances extinction

42

P. S. CHEN

value rises from 0.0144 for larvae aged 5 days to 0.461 for individuals ready for pupation. However, as shown in the last column in Table 1, the quantities per unit body weight remain unchanged. Similarly, no distinct variation in the blood concentration has been detected at this developmental period (Table 2). The values vary from 0.210 for larvae at 6-7 days of age to 0.235 for those aged TABLE

Z-TOTAL

CONCENTRATIONS

OF FREE NINHYDRIN-POSITIVE OF C&X ~i&?ZS LARVAE

SUBSTANCES IN THE BLOOD

-

Larval age (days after hatching)

Number of determinations

6-7 7-8 9-10 II-12

Number of larvae needed for 2 ~1 blood

4

Fresh

_-

--

33 15 9 3

: 16

Extinction per 2 ~1 blood

weight

(mg/lama)

0~210*0~0105 0~254~0~0111 0.218 f0.0132 0.235 +0.0081

0.305 &0.0356 0.848 +0.0016 1~950&0~3181 3~900+0~1577

-

-

11-12 days after hatching. During this period their fresh weights have increased more than ten times (see the fourth column in Table 2). 12

1

71

5r

5

6

7

0

9

10

11

12 days

FIO. 3. Changes in free amino acids during larval development. Each point represents the average value of 4 determinations. Ordinate: pg amino acid per larva. Abscissa: larval age in days after hatching. The upper part of the proline curve is shortened. 0 proline, 0 glutamine, 0 glutamic acid, @ serine, @ valine.

With regard to individual amino acids, the amount of substances per larva increases rapidly in proline, tyrosine, and glutamine, but only slowly in histidine, methionine sulphoxide, leucine, glutamic acid, serine, and valine (Figs. 3 and 4).

76

n

Fie,. 2. Free ninhydrin-positive substances in the haem~)lynaph (20 ,*1) of fully ~zro~n larvae. 1, aspartic acid; 2 glutamic acid; 3, cystine; 4, serine; 5, glycine; 6, threonine: 7, tyrosine; 8, :~-alanine; 9, glutamine; 10, (~-alanine; 11, arginine; 12, lysine; 13, xaline: 14, leucine (and/or isoleucine); 15, proline; 16, methionine sulphoxide; 17, histidine: I' 1---3, peptides.

STUDIES ON THE PROTEIN

METABOLISM

OF CVLEXPIPIENS

43

L.

The increase in proline is especially great. It reaches a value of 3.64 lJ.8 per larva on the ninth day and rises to 11.84 pg before pupation begins. At the last larval

A

5

6

7

9

10

11

:12 ‘S

FIG. 4. Changes in free amino acids during larval development. Each point represents the average value of 4 determinations. Ordinate: pg amino acid per larva. Abscissa: larval age in days after hatching. A tyrosine, LI a-alanine, B histidine, A methionine sulphoxide, /r, leucine.

stage studied, tyrosine and glutamine show values of 4.33 and 3.77 pg per larva respectively. Considering that both fresh weight and total N rise to high values during this period, the general increase of these substances appears self-evident. TABLE

~-QUANTITIES

_

PER UGTOTAL '

N

ATSUCCESSIVE

-

Larval Number of larvae age per deter(days after mination hatching) 6-7 7-8 8-9 9-10 II-12

OF FREE AMINO ACIDS IN G(G SUBSTANCE LARVALSTAGES AT 22.5"c

100 ;: 20 12

I

Total

:I%’ 4.594 7.403 13.513 19.388 42.183

I Glutamic acid ___-. 0*0488* 0.0569 0.0538 0*0401 0.0362

_. Serine

-. 0.0332 0.0398 0.0296 0*0355 0.0296

Amino acid (pg)/N( pg) Tyrosine

alanine

0.0761 0.0949 0.0847 0.0928 0.1025

0.0492 0.0549 0.0493 0.0410 0.0573

a-

Valine

Leucine

Proline

-~ 0.0192 0.0196 0.0191 0.0212 0.0171

0.0266 0.0278 0.0264 0.0273 0.0224

0.0963 0.1705 0.1661 0.1879 0.2806

* Each figure represents the average value of 4 determinations.

In fact, when the amount of amino acid is expressed per pg N, only in tyrosine, proline, and possibly cr-alanine is there a detectable increase (Table 3). For glutamic acid, serine, valine, and leucine the values are even lower at 11-12 days of age.

P. S. CHEN

44

At metamorphosis The total quantities per individual at the (Table 4). The same TABLE ~-AVERAGE

FRESH WEIGHTS AND

SUBSTANCES

Pupal age (hr after pupation) 12-24 24-36 36-55

there is a general decrease of the free amino acid content. of ninhydrin-positive components are reduced from 0.315 early pupal life, to 0.217 at the time of imaginal emergence is true of values per unit body weight, which are 0.103 and

DURING

Number of determinations

PUPAL

TOTAL QUANTITIES OF FREE NINHYDRIN-POSITIVE DEVELOPMENT OF Cllk?X~i&?nS AT 22.5”C

Fresh weight Extinction/pupa

(mgipupa)

0.315 ~0~0052 0.308 *0.0207 0.217 *0.0386

3.085 10.0688 3.237 *0.0723 2.787 *0.0546

: 4

Extinctionlmg

0.103 50.0028 0.095 10.0065 0.078 ho.0146

O-078 for younger and older pupae respectively. Comparing Tables 1 and 4, it can readily be seen that there is a distinct reduction of free amino acids during the time of pupation. The values are 0.461 per fully grown larvae and 0.315 per pupa at 12-24 hr of age. This decrease is even more marked in the total concentration of blood. For larvae aged 11-12 days the extinction value is 0.235 unit per 2 ~1 blood (Table 2), while for pupae aged 12-24 hr it amounts to only 0.174 unit (Table 5). It should be pointed out that it is almost impossible to TABLE

S-TOTAL

CONCENTRATIONS

Pupal age (hr after pupation) 12-24 24-36 36-60

OF FREE NINHYDRIN-POSITIVE OF &k?X &&?TIS PU+AE

Number of determinations

Number of pupae needed for 2 ~1 blood

10 6 6

:: 9

SUBSTANCES

IN THE

BLOOD

Extinction per 2 ~1 blood

0~174*0~0051 0.143 *o~olol 0.12410.0084

obtain pure haemolymph from pupae without contamination of fat body and other tissues. The pupal blood was thus possibly somewhat contaminated during sampling. However, as already described, this difference is also detectable for extinction values per whole larva and pupa. The figures presented in the last column of Table 5 indicate further that there is a general decrease of blood concentration in the course of pupal development. Further studies of the concentration of individual amino acids in the blood of fully grown larvae and pupae at 24 hr of age showed that, although the total ninhydrin-positive composition decreases during pupation, the individual components by no means behave in the same way. As illustrated by the diagram

STUDIES

ON

THE

PROTEIN

METABOLISM

OF

CULEX

PIPIENS

L.

4.5

in Fig. 5, the concentrations of glutamic acid and leucine are even higher in the pupae than in the larvae. That of valine is about the same in both. For the other amino acids the concentration is definitely lower in the pupal than in the larval blood. The difference is particularly marked for proline and glutamine. The values are 102.2 pg per 20 ~1 blood at the larval, and 21.3 pg per 20 ~1 blood

0.8

1.0

FIG. 5. Comparison of the free amino acid concentration in the blood between fully grown larvae (empty columns) and pupae at 24 hr of age (hatched columns). The heights of columns give the concentrations in pg substances per 20 ~1 haemolymph (for proline, 0.15 cm= 1 pg ; for other amino acids, 0.5 cm= 1 pg). Each value of the larval blood represents the average of 4 determinations and that of the pupal blood the average of 5 determinations. Spot; which are circled and not filled with columns indicate that the substances have been located on the two-dimensional chromatograms but not estimated quantitatively. (See Fig. 2 for description of spots.)

at the pupal, stage for proline. The corresponding figures for glutamine are 24.4 and 5.8. As will be discussed later, these substances are largely involved in the synthesis of cuticular proteins (see discussion). The estimations of various amino acids per pupa during 3 developmental stages revealed that at about 24 hr a slight increase is detectable for tyrosine, glutamic acid, glutamine, and valine (Fig. 6). The fluctuation is even more distinct for proline. But no such temporary rise has been observed for a-alanine, leucine, and serine (Fig. 7). These 3 amino acids decrease steadily with the advance of pupal age. In Table 6 values of free amino acid content estimated

46

P. S. CHEN

per unit total N are given. The total N for pupae aged 48 hr is relatively low. Similar to the results quoted for quantities per whole pupa, both serine and

FIG. 6. Changes in free amino acids during pupal development. per pupa. Abscissa: pupal age in hr after pupation. 0 tyrosine, acid, 0 glutamine, @ valine.

12

24

36

Ordinate : pg substance 0 glutamic

l proline,

48

hours

FIG. 7. Changes in free amino acids during pupal development. Ordinate: yg substance per pupa. Abscissa: pupal age in hr after pupation. h. leucine, n a-alanine, A serine. TABLE 6-QUANTITIES

Pupal age (hr after

;: 48

OF FREEAMINO ACIDS IN PG SUBSTANCEPER I*G TOTAL N DURING PUPAL DEVELOPMENTAT 22’5°C

I

~~

Number of determinations

Total N (pg/ pupa)

Glutamic acid

4

56.238 58.240 50.778

0.0243* 0.0269 0.0225

pupation) 1

:

-I

Amino acid (pg)/N (pg)

Serine -___ 0.0088 0.0077 0.0068

Glutamine

Valine

Leucine

Proline

0.0148 0.0185 0.0139

0.0128 0.0132 0.0124

0.0228 0.0214 0.0152

0.0334 0.0468 0.0376

* Each figure represents the average value of 4-5 measurements.

leucine show a successive reduction during this period. For glutamic acid, glutamine, valine, and proline a slight increase is again noticeable at 24 hr after pupation.

STUDIES ON THE PROTEIN

METABOLISM

OF CULEX

PIPIENS

L.

47

DISCUSSION

The metabolic changes in free amino acids during development are obviously different in different insects. In Drosophila melunogaster the larval life is characterized by a continuous decrease of the concentration of free amino acids in the haemolymph (HADORN and STUMM-ZOLLINGER, 1953; CHEN and HADORN, 1954). As already described, the Culex pipiens larvae show neither a decrease in their total quantities of free ninhydrin-positive substances per unit body weight or total N, nor a lowering of their blood concentration with the advance of age. The same is true of Ephestiu Kiihniellu larvae (CHEN and K~~HN, 1956). Apparently both feeding and the rate of protein synthesis play a part in the regulation of blood concentration. In addition, the amino acids are believed to play a part in adjusting the osmotic pressure and the buffering action of the body fluid (WIGGLESWOI~TH, 1947, p. 230; ZWICKY, 1954; HACKMAN, 1956). They may In the haemolymph of Drosophila also be directSy consumed through oxidation. larvae, an electrophoretic study .has shown that there is a reciprocal relationship between amino acid concentration and protein content (CHEN, 1956). The amount of soluble proteins per unit volume blood increases steadily as larval development proceeds. It would be of interest to investigate how the protein content of blood behaves in Ephestia and Culex larvae. As reported earlier (LAVEN and CHEN, 1956), and again observed in the present study, the content of proline in the blood of the mosquito larvae is especially high. At the earlier pupal life there is also a distinct increase of this amino acid (cf. Fig. 6). It seems that large quantities of proline are incorporated into the proteins of the larval tissues. This fact is in agreement with the findings of HACKMAN (1053) that there is a high content of proline in the proteins of insect cuticles. Proline also plays an active role in the overall protein metabolism. According to LANG (1952) it can be oxidized to glutamic acid by proline oxidase. There is also evidence to suggest that proline can be changed to ornithine and vice versa. The metabolic relations of glutamic acid, ornithine, and proline are given in the recent work of VOGEL (1955). Tyrosine is another amino acid which occurs in large quantities in the free state at later larval stages (Fig. 4). The biochemical studies of this substance indicate that it enters into important metabolic activities at metamorphosis: (1) a large amount of it is incorporated into the proteins of the larval cuticle for the shortly before pupation (HACKMAN, 1953); (2) it serves as the substrate tanning reaction of the larval cuticle at the time of puparium formation (FRAENKEL and RUDALL, 1940, 1947; DENNELL, 1946, 1947,1949; PRYOR, RUSSELL, and TODD, 1947; HACK&IAN, 1953b). Considering the key role of glutamine in protein synthesis the accumulation of this amide in aged larvae appears to be expected. Various experiments suggest that it part:.cipates in transamination (MEISTER, 1954) and transpeptidation (HANES et al., 1952; MEISTER, 1956; BARRY, 1956). There is also evidence which indicates that it works as a detoxicating agent for a number of aromatic acids and possibly ammonia (WEIL-MALHERBE, 1953). Particularly in the metabolism

48

P. S.

CHEN

of insects, glutamine is involved in the synthesis of uric acid (USSING, 1946) and n-glucosamine (LELOIR and CARDINI, 1953; LOWTHER and ROGERS, 1955). The latter substance plays a part in the formation of chitin. In general the content of free ninhydrin-positive materials is much lower in the pupae than in the larvae which are ready for pupation (compare Tables 1 and 4). The reduction of glutamine, proline, and tyrosine is particularly marked (Figs. 3, 4 and 6). Reference to the work on Sarcophaga falculata (DENNELL, 1946) and Calliphora augur (HACKMAN, 1953) indicates that new cuticular material is formed just before the beginning of pupal life, so that the larval cuticle increases in thickness at this period. In the foregoing sections we have seen that all these three compounds are especially concerned in the synthesis of cuticular proteins and their decrease would thus seem probable. Of course other factors like the stopping of feeding after pupation and the consumption of amino acids for energy may also lead to a general lowering of the blood concentration. There is no question but that the content of free amino acids at a particular pupal stage represents mainly the balance between the histolytic and histogenetic process, i.e. the production of amino acids through degradation of larval tissues on the one hand and on the other the utilization of them for the formation of imaginal organs. The fluctuation of the amino acids already quoted (pp. 4546) suggests that in the mosquito pupae the histolytic process is at its maximum at about 24 hr after the onset of pupal development. Similar observations have been reported for Calliphora erythrocephala (AGRELL, 1949) and Ephestia kiihniella (CHEN and K~~HN, 1956). LEA et al. (1956) succeeded in rearing Aedes aegypti larvae on a chemically defined medium which contained in addition to other components 17 amino acids. GOLBERG and DEMEILLON (1948) reported that for growth of mosquito larvae glycine, leucine, isoleucine, histidine, arginine, lysine, threonine, tryptophan, phenylalanine, and methionine are essential. The last three amino acids were not detected in extracts of the Culex pi&ens larvae. However, methionine sulphoxide is the oxidation product of methionine. Both substances can be changed from one into the other (LANG, 1952). According to earlier experience from studies on Ephestia and Drosophila, the quantities of free tryptophan in the insect haemolymph are usually very small except in those mutants which have a block in the metabolism of this amino acid (CHEN and K~~HN, 1956; GREEN, 1949). It would therefore be more difficult to locate this substance on the chromatogram. With the solvents used in the present study, the R, values of phenylalanine are very close to those of leucine and isoleucine. It is possible that a small amount of this amino acid is also present in the leucine spot. The above authors further noted that the mosquito larvae can equally use tyrosine Naturally it remains to be proved, if the list of essential instead of phenylalanine. amino acids given by GOLBERG and DEMEILLON (1948), who worked on Aedes aegypti, is also valid for Culex pipiens. Upon comparing the free amino acids reported by MICKS and ELLIS (1952) for Culex quinquefasciatus and Aedes aegypti, it is noticeable that they recorded

STUDIESON THE PROTEINMETABOLISMOF CULEX

PIPIENS

L.

49

taurine, whereas cystine was found in the present study. It is known that taurine arises from cysteine which is the reduction product of cystine. These authors also could not locate tryptophan on their chromatograms, though they used a different method to detect its presence. They found methionine but no methionine sulphoxide. CLARK and GORDON (1952) recorded in various species of Culicid larvae, acid.

in addition Further.

to other

investigation

free amino is needed

related to diffeerences in the technique,

acids,

tryptophan

and E-amino-n-butyric

to find out how far these

discrepancies

and to what extent they are species

are

specific.

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