Free amino acids and related compounds during metamorphosis of the blowfly Phormia regina

J. Insect Physiol., 1966, Vol. 12.pp. 1343to 1362. Pergamon Press Ltd.

Printed in Great Britain

FREE AMINO ACIDS AND RELATED COMPOUNDS DURING METAMORPHOSIS OF THE BLOWFLY PHORMIA

L. LEVENBOOK

REGINA

and MARIA

LUISA

DINAMARCA*

Laboratory of Physical Biology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, Public Health Service, Department of Health, Education, and Welfare, Bethesda, Maryland. (Received 3 December 1965) Abstract-Phormia reginalarvae raised on an aseptic semi-defined diet have been employed to determine the profiles of free amino acids and related ninhydrin-positive compounds during metamorphosis. Two-dimensional high-voltage ionophoresis, paper chromatography, enzymatic reactions, hydrolysis, and specific colour tests were used to identify all the major peaks obtained by running deproteinized extracts on an automatic amino acid analyser. Each developmental stage was examined before and after acid hydrolysis of the extracts, and profiles of the following substances recorded: cysteic+ homocysteic acids, phosphoserine + phosphothreonine, glycerophosphoethanolamine, phosphoethanolamine, taurine, urea, L-methionine-d-sulphoxide, aspartic acid, glutamine, threonine, serine, glutamic acid, proline, glycine, alanine, cc-amino-nbutyric acid, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, p-alanine, &uninoisobutyric acid, y-amino-n-butyric acid, ethanolamine, ammonia, lysine, histidine, and arginine. At certain stages of development omithine, tryptophan, and a number of peptide peaks were observed but these have not been estimated. No evidence was obtained for the presence of any D-an-h0 acids. Following acid hydrolysis aspartic acid, glutamic acid, and glycine increased the most, /?-alanine, ,Y-aminoisobutyric acid, y-aminobutyric acid, taurine, and tyrosine showed no increase in titre, while the remaining components increased to an intermediate extent. During adult development the concentration of the following amino acids showed the most marked changes: tyrosine, methionine sulphoxide and methionine, proline and glutamic acid, but the titres of most of the compounds examined varied to some degree. INTRODUCTION

numerous studies concerning free amino acids (FAA) in insects have recently been reviewed by FLORKIN (1959), GILMOUR (1961, 1965), WYATT (1961), and CHEN (1962). For the most part attention has been directed to the high titres of these substances in insect haemolymph, which has justifiably been proposed as a biochemical characteristic of the class (FLORKIN, 1959). It is becoming increasingly clear, however, that high levels of FAA occur also in insect tissues (e.g. LEVENBOOK, 1962; BURSELL,1963 ; KIRSTENet al. (1963), and, as pointed out by AGRELL THE

*Present address : Department of Parasitology, Biochemistry Section, School of Medicine, University of Chile, Santiago, Chile. 1343

1344

L. LEVENBOOK ANDMARIA LUISA DINAMARCA

(1964), analyses of isolated haemolymph, particularly with reference to changes during growth and development, present only an incomplete picture relative to the entire organism. Aside from the special case of cocoon formation (cf. WYATT, 1961), no satisfactory experimental evidence has yet been adduced to explain the function of the uniquely high titre of FAA in insects. In general, this titre is considerably higher in Holometabola than in Hemimetabola, suggesting that FAA may be of some special importance during metamorphosis. There is ample evidence, however, that in at least three orders the size of the FAA pool does not change materially during adult development (HELLER, 1924; EVANS, 1932 ; AGRELL, 1949 ; HADORN and STUMM-ZOLLINGER, 1953 ; PATTERSON, 1957; CHEN, 1958; LEVENBOOK, 1962). Nevertheless, the titres of certain individual amino acids vary widely during development (e.g. LEVENBOOK, 1962; CHEN and HANIMANN, 1965), and if such changes compensate each other, little or no change may be evident in the over-all a-amino N. Besides FAA, special interest also devolves on the characterization of low molecular weight substances in insects which, inasmuch as they yield an increase in FAA following acid hydrolysis, appear to be free peptides. MITCHELL and ion-exchange chromatographic procedures have SIMMONS (1962), employing provided evidence for the occurrence of over 600 free peptides in Drosophila, of peptides in two species and CHEN (1963) h as similarly found large numbers of mosquitoes. It is noteworthy that most of these are not detectable by paper chromatographic techniques (MITCHELL and SIMMONS, 1962). The present investigation has been undertaken as a necessary preliminary to subsequent metabolic experiments on the role of FAA during adult development of the blowfly Phormia regina. Our primary aim has been to obtain an accurate picture of the profiles of individual FAA and related compounds during this period of profound morphological change ; blowfly peptides will be discussed in a later publication.

MATERIAL

AND METHODS

Material P, regina was raised aseptically at 85°F and 75 per cent. r.h. on a chemically semi-defined diet slightly modified from that described for houseflies (MUNROE, 1962). About 250 fly eggs sterilized in commercial ‘Clorox’ diluted 1: 50 were placed in 250 ml Erlenmeyer flasks containing 75 ml of autoclaved medium. Fully grown third instars which had purged their guts and had reached the ‘wandering stage’ were selected for analysis. Larvae which had ‘rounded up’ at the so-called ‘white pupa’ stage were termed zero-time pupae. Under the above environmental conditions the duration for development from ‘white pupa’ to adult emergence was about 100 hr, and this time span is denoted as 100 per cent pupal and pharate adult duration in the figures.

FREE

AMINO

ACIDSANDRELATED COMPOUNDS OF THEBLOWFLY

1345

Methods Preparation of extracts. Tared groups of 10-12 insects of identical chronological age and presumably at the same stages of physiological development were homogenized and simultaneously deproteinized in 9 vol. of 3% (w/v) sulphosalicylic acid. Following centrifugation the supernatant was clarified by filtration through a bed of Hyflo-supercel. The precipitate was washed twice on the centrifuge with 1% sulphosalicylic acid, filtered as above, and the three extracts combined. A pilot experiment utilizing l*C-labelled amino acids added to the homogenate showed that 98-99 per cent of the added counts were recovered by this procedure. Picric (LEVENBOOK, 1962) and perchloric acid extracts were also examined, both for a-amino N and individual free amino acids, and the results obtained were essentially identical to those with sulphosalicylic acid. These former acids, however, were not as convenient to use as the latter. Extracts prepared from boiling 80% (v /v ) eth anol were not identical with those from the above deproteinizing agents. Hydrolysis. 1.0 ml portions of the extracts were hydrolysed with an equal volume of concentrated (N 12 N) HCl in sealed 2.0 ml ampoules at 112°C for 18 hr. The contents of the vials were subsequently transferred quantitatively to 50 ml flasks and lyophilised. The dried residues were made up to 2-O ml with pH 2.91 citrate buffer and centrifuged prior to analysis. Chemical determinations. Total a-amino N of the extracts before and after hydrolysis was measured according to ROSEN (1957), and phenylalanine by the procedure of LA Du and MICHAEL (1960). Ion-exchange chromatography. The ninhydrin-positive components of both unhydrolysed and hydrolysed extracts were analysed on a Phoenix Precision Instrument Co. automatic amino acid analyser employing a slight modification of the procedure of PIEZ and MORRIS (1960). Norleucine was frequently added to the samples as an internal standard (WALSH and BROWN, 1962). During the earlier phases of the work the ninhydrin reagent described by PIEZ and MORRIS was employed, but this was subsequently replaced by the KCN-ninhydrin reagent of ROSEN et al. (1962). In our experience the latter reagent was stable in air over several weeks, showed no tendency to clog the reaction coil, and for most amino acids gave higher colour constants. For those compounds present at concentrations of 1-O pmole/g or higher, the precision of the method as measured by replicate analyses was approximately 3 per cent. However, the accuracy was decreased by an order of magnitude for those substances occurring at titres below about 0.2 pmole/g and which yielded correspondingly small peaks. The concentrations of threonine, serine, and tyrosine have been corrected upwards by 5, 10 and 5 per cent, respectively, for losses due to acid hydrolysis (cf. HIRS et al., 1954). Identijication of chromatographic peaks. Chromatographic analysis of P. reginu extracts revealed the presence of some 34 ninhydrin-positive substances, and identification of these peaks was the first undertaking. For this purpose we have made extensive use of two-dimensional high-voltage ionophoresis (cf. GROSS,

1346

L. LEVENBOOK ANDMARIALUISADINAMARCA

1959), employing an apparatus similar to that of KATZ et al. (1959). Extracts were desalted by the method of DF&ZE et al. (1954), concentrated on a rotary evaporator, and applied to Whatman 3 MM paper for separation in the first direction in 8% (v/v) formic acid, pH 1.9, at 45 V/cm. The paper was then dried and a desired 18 in. length was extended laterally by stitching it to a second length of paper prior to ionophoresis at about 40 V/cm in a second tank containing 0.05 M carbonate-bicarbonate buffer, pH 9.2. Following separation in the second dimension the paper was dried in an oven at 8O”C, immersed in ninhydrin ‘colour dip’ (MERIGAN et al., 1962) and the different colours developed at 80°C. A map of 40 known compounds prepared by the above procedure was used for reference. Several peaks were easily identified ; their acid stability and relative elution volumes compared with published chromatograms (SPACKMAN et al., 1958 ; PIEZ and MORRIS, 1960 ; ZACHARIUSand TALLEY, 1962 ; HAMILTON, 1963) corresponded to the presence of the presumed compounds in the desalted extracts as judged by ninhydrin colour, ionophoretic mobilities, and paper chromatography. Peaks giving ambiguous results, or which could not be readily identified, were individually isolated from the column, desalted, and concentrated, and the unknown compound(s) subjected to ionophoresis and/or paper chromatography employing a number of solvent systems for amino acids and various developing reagents (e.g. ninhydrin ‘colour dip’, isatin, platinic iodide, dimethylaminobenzaldehyde, Pauly reagent, and vanillin (BLOCKet al., 1958) ). Th e reaction products after acid hydrolysis were also examined. ar-Monoamino acids could be distinguished from other ninhydrin-positive compounds on paper chromatograms by the copper treatment of LARSENand KJAER (1960). Most peaks could be identified by these additional procedures, while further criteria for characterizing certain unusual compounds are discussed below. Standards. A standard mixture of 18 amino acids was supplied by the Phoenix Precision Instrument Co. Other standard compounds were purchased either from Calbiochem or Mann Research Laboratories. Glycerophosphoethanolamine (GPE) was prepared from L-ar-cephalin according to the method of DAWSON (1960), and purified by paper chromatography. Following acid hydrolysis of the isolated material, analyses for inorganic P, glycerol (determined enzymatically according to WIELAND, 1963), and ethanolamine (by ionophoresis) yielded molar ratios of unity.

RESULTS A typical chromatogram of an unhydrolysed P. regina extract is shown in Fig. 1. It serves to show the resolution obtainable by our procedure, the wide range in concentration among the 35 compounds present, and the location of unidentified peaks. Following hydrolysis many peaks increase in area, several are no longer present (e.g. Nos. 2, 3, 5, 7, 10, 19, 20, and 26) and 3 new ones appear, as described below.

FREE AMINO

ZkQ

390

420

1347

ACIDS AND RELATED COMPOUNDS OF THE BLOWFLY

4kO

480 ml

510 of

540

5fO

600

630

61%

effluent

FIG. 1. Ninhydrin-positive constituents of unhydrolysed sulphosalicylic acid extract of P. regina pupae at 22 per cent (1 day) of pupal plus pharate adult duration. Identification of the numbered peaks is as follows: 1, phosphoserine f phosphothreonine; 2, glycerophosphoethanolamine; 3, phosphoethanolamine; 4, taurine; 5, peptide ( ?) 6, urea; 7, peptide ( ?); 8, methionine sulphoxide; 9, aspartic acid; 10, glutamine; 11, threonine; 12, serine; 13, glutamic acid; 14, proline (measured at 440 rnp) ; 15, glycine; 16, alanine; 17, a-aminobutyric acid; 18, valine; 19, ?; 20, ?; 21, methionine; 22, isoleucine; 23, leucine; 24, tyrosine; 25, phenylalanine + /?-alanine ; 26, ? ; 27, B-aminoisobutyric acid; 28, y-amino-nbutyric acid; 29, ethanolamine; 30, ammonia; 31, lysine; 32, histidine; 33, ? (possibly methyl histidine) ; 34, tryptophan ; 35 , arginine. For further details see text.

The following profiles of the various ninhydrin-positive components during adult development (Fig. 2) are presented in the approximate sequence in which they emerge off the column. Peak No. 1. In unhydrolysed extracts this peak is due to free phosphothreonine and phosphoserine. These two amino acids cannot be distinguished on the analyser, but are readily separable by ionophoresis at pH 1.9, yielding two approximately equal-sized spots. After acid hydrolysis of the isolated and desalted peak the expected hydrolysis products, serine, threonine, and inorganic P were obtained. However, hydrolysis of the entire extract results in a new peak at the same location, consisting now of cysteic and homocysteic acids. These two S-containing amino acids similarly could be separated by ionophoresis but not on the column. The source of the cysteic acid is unknown, particularly in the absence of any discernible cystine, cysteine, or glutathione, whereas the homocysteic acid is derived from hydrolysis of methionine sulphoxide and/or methionine (FLOYD et al., 1963).

L. LEVENBOOK AND MARIA LUISA DINAMARCA

1348

The concentration adult development mation (Fig. 2).*

4

of these pairs of amino acids remains relatively constant during except for a temporary decrease at the time of puparium for-

Cyste~f Homocysletc acids

,o----__O__

8

2 I\ w.

-

--

fhosphoinleonme

A

41

5

G/ycerophosphoethono/omine

I

Urea ---_O

,,’ I.p

*.._

‘.,/

_,A

f Phosphoseune

5

5

**’

y -NH2 -

Butyric

acid

250- L *

___o

Percent

_

pup01 plus phorote

adult dumtim

FIG. 2a. FIG. 2.

The variation

of P. regimz.

in individual

amino acid titre during adult development -0 after acid hydrolysis. L, larva; For further details see text.

0- - - 0 before hydrolysis; A, adult,

+ To conform with general biochemical practice and to permit comparison with other forms, our data are calculated on the basis of wet weight. However, in order to evaluate meaningful changes in concentration during adult development from Fig. 2 it is important to note that an approximately 20-25 per cent decrease in weight occurs during the transition from mature larva to ‘white pupa’ (L-O on the abscissa of Fig. 2), and an approximately 10 per cent further decrease at the time of adult emergence (100-A), attributable to the weight of the puparium. Thus a decrease in the indicated concentrations at these particular stages is actually greater, per indimid&, than appears to be the case, whereas an increase may be more apparent than real,

FREEAMINOACIDSANDRELATED COMPOuNDS OF THEBLOWFLY

L 0

25

50

1349

75

Percent

pupal pius phorate

adult

duration

FIG. 2b. Peak No. 2. Criteria for the identification of this peak as GPE were as follows : co-chromatography with known GPE resulted in a single peak on the analyser and a single spot on ionophoresis ; hydrolysis of the isolated peak with 6 N HCl yielded only an ethanolamine peak on the analyser ; hydrolysis according to DAWSON (1960) yielded only inorganic P, glycerol, and ethanolamine, in stoichiometric amounts. From about half-way along adult development the GPE peak becomes overlapped on its leading edge by a second peak due to an unknown compound, the presence of which precludes estimation of the former. Peak No. 3. Hydrolysis of this isolated peak yielded only inorganic P and ethanolamine, while the unhydrolysed material co-chromatographed with phosphoethanolamine (PE) both on the analyser and on ionophoresis. A pronounced increase in PE titre occurring at about 25 per cent of pupal plus pharate adult duration (P.D.) is followed by a gradual decline to the original low level. Peak No. 4. This sharp peak, frequently the highest on the chromatogram, is due to taurine. The concentration of this S-containing amino acid increased slightly throughout development, and remained unchanged by acid hydrolysis. Peak No. 5. In unhydrolysed extracts this small peak has not been identified. In hydrolysed extracts peak No. 5 is almost exactly overlapped by a higher new peak, with a 440/570 rnp absorption ratio of about 1.3. This ratio and its position

1350

L. LEVJNBOOK AND MARIA LUISA DINAMARCA

on the chromatogram (cf. ZACHARIUS and TALLEY, 1962) strongly suggested laevulinic acid. This was confirmed by co-chromatography with known material, Laevulinic acid (which contains and by identity of mobilities on ionophoresis. no N) is most probably an experimental artifact, produced by acid hydrolysis of sugars in the insect extracts.

z -

Tyrasine

24P D 8 ZO2 ._ 5 16% g 12i 84-

0’

ii ’ ’ L 0

I

25

I

50

,

75

Percent pupal

I

,

-___;;_____o_;2fLf

L 0 25 IOOA plus pharate adult dumfion

50

75

lOOA

FIG. 2c.

Peak No. 6. Identification of this peak as urea is@upported by several tests ; in addition to co-chromatography and ionophoresis with urea standards, the isolated material gave characteristic colours with dimethylaminobenzaldehyde (yellow) and ethanolic phenol+NaClO (green), and a positive reaction with urease. The small size of the peak belies its true titre on account of the low During adult development the initial urea colour yield of urea with ninhydrin. concentration more than doubles to over 8 pmoles/g at 25 per cent P.D., and then Following acid hydrolysis, which would be expected to result in slowly declines. extensive urea destruction, an unexplained pronounced increase in the size of the ‘urea’ peak is observed ; the nature of this post-hydrolysis material is unknown. Peak No. 7. This very small peak has not been identified, but since it is absent after acid hydrolysis, it is tentatively ascribed to a peptide. Peak No. 8. Ionophoresis, paper chromatography, and positive nitroprusside and platinic iodide reactions of this isolated and desalted peak material were

FREF.

AMINO

ACIDS ANDRELATED COMPOUNDS OFTHEBLOWFLY

1351

consistent with it being methionine sulphoxide. After acid hydrolysis this single peak was replaced by a smaller symmetrical double peak, typical of the hydrolysis product from methionine-containing proteins due to the incomplete resolution of the resulting two methionine sulphoxide diastereomers. The occurrence of only a single peak was therefore at first puzzling until the subsequent demonstration that it was in fact due to the single L&isomer (LEVENBOOK et al.,1965). Since methionine (peak No. 21) and its sulphoxide are so closely related, we have combined their titres for the present consideration. Thus, commencing with a relatively low concentration of about 1-Opmole/g in the larva, an almost sixfold increase occurs during the latter half of adult development. The apparent loss on acid hydrolysis is undoubtedly due to demethylation to homocysteic acid and other oxidation products (FLOYD et al.,1963). Peak No. 8a. Following acid hydrolysis of P. regina extracts a well-defined new peak is observed at an elution volume of about 120 ml. This unidentified ‘pre-aspartic’ peak has no acid-stable counterpart on any of the published Dowex-50 chromatograms, representing over 150 ninhydrin-positive compounds. Peak No. 9. Aspartic acid ; this amino acid was present in consistently low titre throughout development, and in fact was scarcely measurable in the imago. However, after acid hydrolysis the amount of free aspartate rose markedly; the average increase throughout development, about 10-12 times the unhydrolysed titre, was relatively higher than for any other component on the chromatogram. The hydrolysable aspartate is not derived from asparagine inasmuch as this amide could not be detected in blowfly extracts. Peak No. 9a. A second new peak, generally smaller than peak 8a, and with an elution volume of about 165 ml is observed after hydrolysis. It is apparently not methionine sulphone which has a comparable elution volume, and its identity remains unknown. Peak No. 10. This peak forms a marked shoulder on the leading edge of the subsequent peak No. 11 and has been identified as glutamine. This amide is partially hydrolysed to glutamic acid and NH, by passage through the ion-exchange column, but we have found that the extent of such hydrolysis is proportional to the time that glutamine is in contact with the resin, and suitable corrections could therefore be applied. A sharp increase in glutamine concentration occurs at the onset of metamorphosis, and this is followed by a gradual decrease until adult emergence, when the titre again increases. Glutamine is, of course, quantitatively converted to glutamic acid and NH, on acid hydrolysis. Peak No. 11. Threonine : the titre of this amino acid is remarkable for its constancy throughout adult development. The value after hydrolysis also shows minimal variation, and the increase in titre is relatively small. Peak No. 12. Serine: the concentration of serine is slightly higher in the larva and early pupa than during the remainder of adult development, but the overall variation is in any case small. The acid hydrolysed increment shows a steady decline throughout metamorphosis.

1352

L. LEVENBOOKANDMARIA LUISA DINAMARCA

Peak No. 13. Glutamic acid: this amino acid forms a major component* of the FAA pool, and undergoes unique variations during adult development. The onset of metamorphosis is associated with a small decrease in titre, which then progressively rises to attain a value of over 10 ymoles/g about half-way through development. The concentration then falls during the latter half of metamorphosis and is minimal in the adult. An approximately constant increment of about 4 pmoles/g over and above the combined glutamate +glutamine values is observed glutamate curve after hydrolysis ; however, the profile of the acid-hydrolysed does not precisely follow that of the free glutamate ; thus at the onset of pupation and at adult emergence the hydrolysable glutamate values do not decrease as do those of free glutamate, while the maximum titre of the former is attained somewhat earlier than that of the latter. Peak No. 14. Proline: this amino acid is characterized by a marked increase in titre shortly before adult emergence, such that the concentration in the imago is about three times that of the larva. The increase after hydrolysis is small. Peak No. 15. Glycine : the titre remains almost constant throughout development. After hydrolysis the amount of glycine at all stages increases approximately fourfold. Peak No. 16. Alanine: the concentration of free alanine follows a U-shaped course during metamorphosis, while the markedly augmented titre after hydrolysis follows a somewhat similar profile except the upward trend is less pronounced during the last third of adult development. acid (butyrine) : although present in barely Peak No. 17. c+Amino-n-butyric determinable amounts, the presence of this amino acid was confirmed by co-chromatography on the ion-exchange column, by paper chromatography, and The very low level persists throughout development, but the by ionophoresis. consistently small increase after acid hydrolysis is difficult to explain inasmuch as bound forms of the amino acid are apparently unknown. Peak No. 18. Valine : very little change in the valine titre, either before or after hydrolysis, occurs during adult development. Peaks Nos. 19 and 20. Two very small peaks, which have not been identified. Peak No. 21. Methionine : this amino acid has been discussed above under methionine sulphoxide. Peaks Nos. 22 and 23. Isoleucine and leucine, respectively: the concentrations and profiles of these two amino acids during metamorphosis are very similar. The titres are slightly higher towards the end of adult development than at the beginning, while there is some indication of utilization at puparium formation The increase in leucine after acid hydrolysis appears to and at adult emergence. be somewhat higher than for isoleucine. Peak No. 24. Tyrosine: this phenolic amino acid is the principal component of the FAA pool of the late third instar and white pupa. (The tyrosine peak of * The values for free giutamic acid prior to hydrolysis are slightly too high, due to a contribution from resin-hydrolysed glutamine; but the overall profile is not materially altered by this error.

FRFJIAMINO ACIDSANDRELATED COMPOUNDSOFTHEBLOWFLY

1353

Fig. 1 (l-day-old pupa) gives little indication of its actual magnitude at these earlier stages.) A very rapid synthesis of tyrosine is observed just prior to puparium formation, such that the concentration in the white pupa is about twice that of the late third instar. This is followed by a dramatic decrease from about 26 to 2 pmoles/g during the ensuing few hours. For the remainder of adult development a progressive slow increase in the tyrosine titre ensues. Virtually no change in the tyrosine values follows acid hydrolysis, but it should be noted in this connexion that tyrosine suffers an unpredictable but significant measure of destruction on acid hydrolysis, particularly in the presence of methionine sulphoxide (cf. NEUMANN et al., 1962). For this reason the somewhat arbitrary 5 per cent correction applied to the hydrolysed values may be too low. Peak No. 25. Phenylalanine plus ,f?-alanine: these two amino acids could not be resolved by the procedure employed, and peak No. 25 was frequently markedly asymmetric even though this is not evident from Fig. 1. In order to estimate both components, phenylalanine was determined separately on portions of the original sulphosalicylic acid extracts before and after acid hydrolysis according to LA Du and MICHAEL (1960).* The phenylalanine content having been determined, and its ninhydrin colour value on the analyser being known, the fractional area under the combined phenylalanine + fl-alanine curve attributable to the latter could be computed by difference. No @alanine could be detected in blowfly larvae, i.e. phenylalanine as calculated, from the entire chromatographic peak was in good agreement with that determined enzymatically; this was confirmed by a run on a Beckman analyser employing the Moore and Stein system, which clearly resolves phenylalanine and /3-alanine. At the white pupa stage, however, fi-alanine occurs at its highest concentration, whereas phenylalanine decreases compared to its larval level. The trend for both amino acids is subsequently the same during adult development-a gradual decrease in titre followed by a slight rise. The levels of p-alanine are unchanged on acid hydrolysis, whereas those of phenylalanine increase significantly. Peak No. 26. A very small peak, not always observed, and not identified. Peak No. 27. Identified by two-dimensional ionophoresis and paper chromatography of the isolated peak as /3-aminoisobutyric acid. The titre in the larva and during the first quarter of adult development is so low that the peak was barely discernible on the chromatograms; however, although present in relatively very low concentration throughout metamorphosis, a consistently higher titre is observed during the last half of adult development. No increase occurs after acid hydrolysis. Peak No. 28. y-Aminobutyric acid: the criteria for identification and the profile of this amino acid during adult development are essentially the same as those described for the preceding /3-arninoisobutyric acid. * The accuracy of these measurements in our hands was not as high as stated by LA DIJ and MICHAEL, possibly due to the widely varying amounts of free tyrosine and/ortryptophan

in the insect extracts. 81

1354

L. LEVENBOOK AND

MARIA LUISA DINAMARCA

Peak No. 29. Ethanolamine: a relatively minor component, the concentration of free ethanolamine is rather constant throughout adult development. In view of the presence of acid hydrolysable GPE and PE the expected increase in ethanolamine after hydrolysis is in fact apparent. Peak No. 29a. In certain runs, a generally small but occasionally mediumsized peak occurred just behind ethanolamine. This compound, whose presence was sporadic and seemingly unrelated to the stage of development, was identified by ionophoresis, paper chromatography, and its specific colour with vanillin as ornithine. No quantitative estimations have been made. Peak No. 30. Ammonia: frequently the largest peak on the chromatogram, NH, is present in surprisingly high titre in the blowfly throughout development. The levels indicated in Fig. 2 exaggerate the true NH, content of the living insect due to the contaminating NH, in the various reagents.* The extent of this blank NH, has been found to vary from one series of runs to another, and hence subtraction of a constant blank value from the experimentally determined titres is not possible. However, such blanks never exceeded the equivalent of 34 pmoles NH,/g, either before or after acid hydrolysis, suggesting that the free NH, titre during metamorphosis varies between 5-10 pmoles/g. After acid hydrolysis the concentration increases about 6 times, with a maximum at about 75 P.D. time. Peak No. 31. Lysine: no significant changes in lysine concentration occur during P. regina adult development, and the increase after acid hydrolysis is at all times relatively small. Peak No. 32. Histidine: a slight increase in histidine titre observed at about 2.5 per cent P.D. is shortly followed by a gradual decline in concentration to the original low larval level. Early adult development is accompanied by a progressively smaller increment in the amount of acid hydrolysable histidine until about half-way along development, at which time the histidine values before and after hydrolysis are practically identical. Peak No. 33. An unidentified, small, acid-stable peak which, from its chromamay be due to 3-methylhistidine is present throughout tographic position, development. Peak No. 34. Tryptophan: small amounts of free tryptophan were observed at most stages of adult development, but the concentrations could not be calculated due to the extreme flatness of the peak. Tryptophan was absent from hydrolysed extracts and was scarcely detectable in the adult fly. Peak No. 35. Arginine: the concentration of arginine follows a very slightly U-shaped curve during adult development, with some indication of utilization, i.e. a decrease in titre, at the onset of metamorphosis. Very little, if any, additional arginine is obtained after acid hydrolysis. + Cultures of P. regina larvae raised on meat were maintained in the vicinity of the analyser. Because of the arrangement of the laboratory it was frequently necessary to carry cultures past the analyser. As is well known, such cultures smell strongly of NH*, and hence under these conditions contamination by NH, of the acidic reagents was inevitable.

FREE AMINO ACIDS AND RELATED COMPOUNDS

Optical

OF THE BLOWFLY

135.5

conjiguration

In view of the reports by AUCLAIR and PATTON (1950) and SRINIVASANet al. (1962 ; 1965) on the occurrence of D-alanine in the milkweed bug and n-serine in certain lepidopterous larvae, it was of interest to ascertain whether amino acids of the D-configuration might exist also in P. regina. Two experimental procedures were employed ; (i) hog renal n-amino acid oxidase (Worthington Biochemical Corp.) was purified according to BOULANGERand OSTEUX (1963) A sulphosalicylic acid extract of about O-5 g l-Z-day-old pupae was desalted on a Dowex-2 column, and the amino acid eluate lyophilized. The residue, taken up in 1-O ml H,O, was assayed manometrically for 0, uptake in the presence of the oxidase preparation (BOULANGERand OSTEUX, 1963). No 0, consumption could be detected, whereas n-alanine added to a similar extract was quantitatively oxidized. In a second experiment 48 adult flies, containing the equivalent of about 100 pmoles FAA calculated as leucine, were extracted with boiling 80 per cent (v/v) ethanol. The ethanol was removed on a rotary evaporator and the residue examined manometrically as in the first experiment. Under these conditions 1 part of D-amino acid in 500 could have been detected, but the results were again negative. (ii) In the second procedure the larval FAA were separated by two-dimensional ionophoresis, the dried paper sprayed with D-amino acid oxidase, and tested for the formation of or-keto acid dinitrophenylhydrazine derivatives according to AUCLAIR and PATTON(1950). No evidence for ol-keto acid formation was obtained. Accordingly, we conclude that free n-amino acids are either absent from P. regina or are present in undetectably small amounts. DISCUSSION Identification

of ninhydrin-positive

compounds

Characterization of the FAA and related compounds in deproteinized P. regina extracts has depended principally on various chromatographic and related procedures which, although widely employed, are not unequivocal. The possibility thus exists that an erroneous identification might have been made, particularly in the cases of the less common amino acids present in low titre, such as cysteic and homocysteic acids, phosphothreonine and phosphoserine, 01- and y-NH,butyric acids and /I-NH,-isobutyric acid. Two exceptions to the above generalization may be noted: the identity of GPE has been confirmed by the identification and stoichiometry of its three hydrolysis products, while r.,d-methionine sulphoxide has been rigorously and unambiguously characterized (LEVENBOOKet al., 1965). Effects of acid hydrolysis Acid hydrolysis of deproteinized P. regina extracts results in an increase in the titre of several amino acids, and the question therefore arises as to their source. Thus ethanolamine is undoubtedly derived from the hydrolysis of GPE and,PE, homocysteic acid from methione sulphoxide, glutamate (in part) from glutamine, and NH, (in part) from urea and glutamine. However, the increase in most

1356

L. LEVENBOOK AND MARIA LUWAD~AMARCA

amino acids must result from the cleavage of some higher molecular weight material(s) ; the evidence for our belief that these are probably peptides is as follows : (1) the hydrolysable material is dialysable ; (2) the composition of the acid-released amino acids does not resemble that of bulk blowfly protein ; (3) some 5-6 peptides have been isolated from P. regina; (4) the increment in aspartic and glutamic acids is particularly marked, whereas that of the basic amino acids is slight; this is reminiscent of the composition of peptides isolated from Drosophila (MITCHELL and SIMMONS, 1962) and mosquitoes (CHEN, 1963). The possibility still remains, however, that some acidic, low molecular weight protein not precipitated by sulphosalicylic acid is present in the extracts, and traces of such material on hydrolysis could substantially augment the FAA titres. Quantitative

considerations

Data to be presented elsewhere indicate that when compared at identical stages of development, the titres of free alanine and lysine can vary as much as fivefold among individual blowflies of the same chronological age raised from the same batch of eggs on horse-meat. Accordingly, it is evident that with such wide biological variation the quantitative values of Fig. 2 must be considered as only approximate, inasmuch as they represent the mean of but three series of analyses on groups of 10-12 insects. Furthermore, no consideration has been made of any possible sex differences, although to judge from Drosophila (CHEN and HANIMANN, 1965), such differences are unlikely to be large. A second source of error arises from the possibility that a single peak might be due to exact overlapping of an amino acid and one or more peptides or other form of conjugated amino compound. The presence of such compounds in a ninhydrinnegative form is likely to become evident on acid hydrolysis (cf. CHEN, 1963). These possibilities were examined by performing two manual runs according to SPACKMANet al. (1958) on extracts of white and 2-day-old pupae, respectively. 1-O ml portions of the fractions collected on a fraction collector were reacted with ninhydrin directly to plot out the various peaks. A second 1-O ml portion was hydrolysed with HCl, evaporated to dryness, and the residue redissolved in 1.0 ml Ha0 before being in turn reacted with ninhydrin. In this manner the ninhydrin values of each fraction before and after hydrolysis could be directly compared. For the most part no new peaks nor any significant increases in prehydrolysis peak areas were obtained. However, eluate fractions in the vicinity of the peaks of phosphoserine, phosphothreonine, phosphoethanolamine, urea, methionine sulphoxide, and lysine indicated the presence of additional unidentified ninhydrin-positive material following hydrolysis. Perhaps of equal interest to the ninhydrin-positive compounds identified in the blowfly are those which have not been found. Thus, free asparagine, citrulline, cysteine, dopa, glucosamine, glutathione, hydroxyproline, and tyrosine-o-phosphate, one or more of which have been reported from various insects, are apparently absent from the P. regina. Trace amounts of a compound which has the elution volume of cystine has on occasion been observed on some chromatograms, but

FREEAMINO ACIDSANDRELATED COMPOUNDS OFTHEBLOwFLY

1357

virtually the entire amount of S-containing FAA is in the form of methionine sulphoxide and taurine. Comparison of the total a-amino N of the sulphosalycylic extracts in terms of the sum of the various ninhydrin-positive components is instructive. During metamorphosis the a-amino N values prior to hydrolysis vary only between a low of 65 pmoles leucine equivalents/g wet wt. at 25 per cent P.D. and 81 pmoles/g wet wt. in the adult. The sum of the individual components accounts for at least 90 per cent of the corresponding a-amino N values throughout development, indicating that no major fractions are likely to have been overlooked. Agreement is not as good for the hydrolysed extracts, with a-amino N titres about twice those mentioned above ; in these cases the sums of the individual components account for only 75-85 per cent of the total a-amino N. Biological szgniJicance of the changes in FAA content during metamorphosis

The present data support the findings of several investigators, e.g. AGRELL (1949), PATTERSON(1957), CHEN (1958), and JEUNIAUXet al. (1961) that, apart from some notable exceptions, most of the FAA do not change greatly in titre during insect metamorphosis. Since there is no pronounced increase in FAA associated with tissue histolysis during the earlier stages of metamorphosis, nor any concomitant decrease during subsequent histogenesis, it must be concluded that if in fact protein is degraded to FAA during histolysis-a hypothesis as yet lacking experimental verification-then such amino acids must be rapidly metabolized and/or incorporated into developing adult protein as they do not accumulate. Further, as shown in Table 1, there is actually little, if any, correlation between the amino acid composition of P. regina protein and that of the FAA pool, which would also contra-indicate the likelihood that protein amino acids materially contribute to the FAA pool. The biological significance of both the occurrence and qualitative variations in ninhydrin-positive substances in insects is largely conjectural. It appears likely that the present changes in free GPE (previously found in Drosophila (CHEN and HANIMANN, 1965) and silkworm haemolymph (KONDO, 1957) ), PE, phosphothreonine, and phosphoserine are related to breakdown and synthesis of phospholipids, possibly from cell membranes. It is relevant in this connexion that P. regina phospholipids are predominantly of the ethanolamine type (BIEBER et al., 1961), whereas PE is reported to be incorporated into lepidopteran cephalins in toto (CHOJNACKI,1961; CHOJNACKIand KORZYBSKI,1962). The threefold increase in urea titre during early adult development suggests that urea may be a true end-product of N catabolism. This is somewhat surprising, since urea is not generally considered to be an excretory product of blowflies (cf. GILMOUR, 1961). Free NH,, which is excreted by P. regina larvae, occurs in uniformly high titre throughout development. The unexplained large increment after acid hydrolysis has also been observed in aphid hydrolysates (STRONG, 1964). /3-Alanine is either absent or is present in extremely low titre in larvae, but appears abruptly in the white pupa. It is tempting to relate this to the

1358

L. LEVENBOOKANDMARIALUISA DINAMARCA

TABLE I-COMPARISON

OF AMINO

ACID

COMPOSITION

THEFREE

AMINO

OF

P. regina

PROTEIN

WITH

THAT

OF

ACID POOL

Protein amino acids

Free amino acids

Amino acid ymoles/g live wt. Arginine Histidine Lysine Tyrosine Phenylalanine Methionine Serine Threonine Leucine Isoleucine Valine Glutamic acid + glutamine Aspartic acid Glycine Alanine Proline P-Alanine Total, pmoles

28.6 11.7 41.5 26.9 23.0 13.2 27.1 23.9 37.9 25.7 33.1 67.5 69.3 41.0 37.3 22.2 4.9 570.8

o/0total

pmoles/g live wt.

y0 total

5.0

3.3 5.1 3.0 4.0 0.8 0.3 1.0 o-9 0.5 0,9 2.2 13.9 0.5 3.0 1.8 3.6 1.0

7.2 11.1 6.6 8.7 1.7 0.7 2.2 2.0 1.1 2.0 4.8 30.3 1.1 6.6 3.9 7.9 2.2

2.0 7.3 4.7 4.0 2.3 4.7 4.2 6.6 4.5 5.8 11.8 12.1 7.2 6.5 3.9 0.9

45.8

Values for the amino acid composition of blow fly pupa plus pharate adult protein are calculated from the data of HENRY and COOK (1963). Values for the FAA pool are taken from Fig. 2 for insects of 50 per cent pupal plus pharate adult duration time.

presence of /3-alanine in P. yegina cuticular protein (HENRY et al., 1964). The glutamic acid and glutamine changes during metamorphosis cannot yet be explained, but the profiles are to a varying extent similar to those observed in a number of other insects (cf. LEVENBOOK, 1962). Two amino acids, proline and methione sulphoxide, stand apart from the rest by virtue of their progressive increase in concentration during the latter part of metamorphosis, with maximal titres in the imago. Proline has been implicated as having a special function in insect flight metabolism (BURSELL, 1963 ; KIRSTEN et al., 1963 ; SACKTOR, 1965), and its higher concentration in the volant adult is thus understandable; the role of L,d-methionine sulphoxide, however, is entirely unknown save for the suggestion of DENT (1947) that it may somehow be involved in the redox potential of biological systems. The changes in tyrosine concentration merit special consideration. The titre in the ‘wandering stage’ larva amounts to about 4.7 mg/g, which is already about ten times the solubility of tyrosine in water (O*05°h w/w), and might indicate that in vivo tyrosine occurs in the solid state as appears to be the case for dopa in certain blood cells of Drosophila (RIZKI and RIZKI, 1959). Some 24 hr later, at the white pupa stage, the tyrosine titre approximately doubles, and this is consistent

FREE AMINOACIDSANDRELATEDCOMPOUNDS OF THE BLOWFLY

1359

with the findings of FRAENKELand RUDALL(1947) for Surcophuga, and OHNISHI (1954) for Drosophila, that the haemolymph tyrosine content rises to a maximum The origin of this increased tyrosine raises at about this time in development. an interesting question ; the most probable biosynthetic pathway, the hydroxylation of phenylalanine (FUKUDA,1956; BRICTEUX-GR~GOIRE et al., 1956) seems unlikely to be of major importance because of the consistently low titre of free phenylalanine. The release of tyrosine through protein breakdown appears equally unlikely, since no other FAA are concomitantly released. In view of the multiplicity of free phenolic compounds isolated from insects (cf. BRUNET’S (1963) review) search for a pool of ninhydrin-negative phenolic tyrosine precursors might prove fruitful. But if the biochemistry of the increased P. reginu tyrosine concentration obviously requires further work, the precipitous decrease upon puparium formation is not unexpected, and is consistent with the known function of tyrosine in insect cuticle sclerotization (BRUNET, 1963; HACKMAN,1964). It is of interest that in Drosophila similarly dramatic changes are observed not in free tyrosine, but in tyrosine-o-phosphate (CHEN and HANIMANN,1965), which is absent from Phormiu. Finally, it is worth recording that although free tryptophan has not been determined, nevertheless comparison of the tryptophan peaks during metamorphosis suggests a definite increase during the first 25 per cent of P.D. This is in accord with unpublished data of Dr. T. A. SCOTT (personal communication) whose quantitative determinations show a similar profile for Culliphmu erythrocephulu, with the apparent difference that in P. regina tryptophan is absent or present in very low titre in the imago, whereas in C. erythrocephulu the earlier high level is maintained. Acknowledgements-We would like to thank Mrs. ELLEN NELSON for competent technical help, and Dr. FRED LUCASfor the manual amino acid runs and valuable discussion

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