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A possible storage form of tyrosine in the haemolymph of Pieris brassicae L. and four other lepidoptera
r. Insect Physiol.,
1976, Vol. 22, pp. 95 to 100.
Pergamon
Press.
Printed
in Great Britain.
A POSSIBLE STORAGE FORM OF TYROSINE IN THE HAEMOLYMPH OF PIERIS BRASSICAE L. AND FOUR OTHER LEPIDOPTERA ERKKI JUNNIKKALA Department of Physiological Zoology, University of Helsinki, 00100 Helsinki 10, Finland (Received
27 June 1975)
Abstract-A ninhydrin-positive compound, called peptide I (PI), has been found in the haemolymph of Pieris brassicae. The substance has been partially purified and is presumed to be a peptide of low molecular weight and rich in tyrosine, or a derivative of tyrosine. Its phenolic nature is evident. PI is not present in fourth instar larvae but is first found in fifth instar larvae a few hours after ecdysis. Its concentration rises almost linearly and is 25 times higher at the end of feeding. The concentration falls to one-third of the peak value shortly after pupation. Some PI is still found in pharate adults and young emerged adults. Both sexes contain about equal amounts of PI. The content of tyrosine in the haemolymph was about 4 pmole/ml during the period studied (ecdysis was not examined). The content of tyrosine increases towards the end of the feeding period during both the fourth and fifth instars. This occurs also after pupation, when the concentration of
PI decreases. PI is distributed in the ratio of 1 : 5 between tissues and haemolymph at the end of the fifth instar. PI is not of dietary origin. Its occurrence during development suggests a r81ein pupation. It is possible that PI is a reserve of tyrosine, owing to its greatersolubilityin comparison to thatof tyrosine. A substance identical or very similar to the PI of P. brassicae was also found in four other randomly
chosen species, Smerinthus ocellatus L., Pergesa elpenor L., Celerio galii L., and, possibly, Dicranula vinula, L. This would suggest that such a compound occurs rather commonly in Lepidoptera.
INTRODUCTION
and LE~ENBOOK,1969), and gamma-L-glutamyl-Lphenylalanine in Musca domestica (BODNARYK, THE CENTRALrole of tyrosine as the precursor of 1970). In other insect orders only indirect evidence diphenols mediating the darkening and hardening of has been obtained of the storage forms of tyrosine. insect cuticles has been well documented (HACKMAN, JUNNIKKALA(1968) reported the presence of a 1964). Most of this work has been done with ninhydrin-positive compound in the haemolymph cyclorrhaphous Diptera during puparium formaof Pieris brassicae, identified tentatively as a peptide tion, which must be regarded as a rather specialized rich in tyrosine and called peptide I or PI. More form of sclerotization. Tyrosine, however, acts as a recently SIENKIEWICH and PIECHOWSKA(1973) precursor during the tanning of the larval and adult reported the presence of a dipeptide L-tyrosyl-Ofly cuticle also (SELIGMAN et al., 1969; PRICE, acetyldopamine (celerin) in the haemolymph of Other orders studied in this respect 1971). Celeyio euphorbiae and suggested a role for it as a show similar functions for tyrosine, i.e. Blattaria storage form of tyrosine. (KENNAUGH,1958; Fox and MILLS, 1971; WIRTZ This paper reports more information on PI in and HOPKINS, 1974), Orthoptera (KRISTENSEN, Pieris and the occurrence in several Lepidoptera of 1968), and Lepidoptera (POST and DEJONG, 1973). a compound remarkably similar to PI as well as to The low solubility limits the concentration of free celerin of C. euphovbiae. (LEVENBOOK, 1950), tyrosine in haemolymph especially during ecdysis when the requirement for tyrosine increases. However, since the concenMATERIALS AND MFZTHODS tration of tyrosine often tends to be high during Pieris brassicae were from a laboratory-reared ecdysis, storage forms for tyrosine have been postulated (LENNIE and BIRT, 1965; HENDERSON stock grown on a semi-synthetic diet as described previously (JUNNIKKALA,1969 ; TURLJNEN,1973). and GLASSMAN,1969; WIRTZ and HOPKINS, 1974) The age of the insect was determined with the and actually found in cyclorrhaphous Diptera, fourth to fifth larval ecdysis as the starting point in for example, tyrosine-O-phosphate in Drosophila melanogaster (MITCHELL et al., 1960; LUNAN counting larval age and the larval-pupal or pupaladult ecdysis as the starting point in counting pupal beta-alanyl-L-tyrosine and MITCHELL, 1969), (Sarcophagine) in Sarcophaga bullata (BODNARYK or adult age, respectively. 95
ERKI(IJUNNIKKALA
96
In order to prevent blackening of the haemolymph all samples were diluted with neutralized KCN and stored at - 18°C. The technique has been described in detail previously (JUNNIKKALA, 1966). Isolation of PI Preliminary tests with gel filtration on Sephadex G-25 fine revealed that PI was eluted among free amino acids with distilled water. For this reason separation was done by paper chromatography, starting with samples of 2 to 4 ml haemolymph. The samples were deproteinized for 2 min in a boiling water-bath, centrifuged, and the supernatants desalted in Dowex SOW x 8, mesh 200/400, H+ co1umns, eluted with 4 N ammonia, and evaporated to dryness in a vacuum at +SO”C. For chromatography the eluates were dissolved in 2 ml of 10% iso-propanol and pipetted as uniform lines across Whatman 3MM sheets (57 x 30 cm) 11 cm below the upper margin. The time of run in the descending direction was 24 hr in a solvent system of n-butanol-acetic acid-water (12 : 3 : 5). The sheets were dried for 1 hr in a ventilated oven at + 50°C. PI was located by cutting out three narrow vertical strips from each sheet, one through the middle and two from each margin and treating the strips with ninhydrin. Areas corresponding to PI were cut out and PI was eluted with water. The eluates were combined, evaporated to dryness, and the procedure repeated in a solvent system of n-propanol-1% ammonia (2 : 1). PI was again eluted with water and lyophilized. To achieve higher purity the sample was applied on the long column of a Beckman Unichrom automatic amino-acid analyzer @AA), in which the method of SPACKMANet al. (1958) and BENSONand PATTERSON(1965) was used. Beginning with the 133rd minute, the eluate was collected for 20 min, covering the elution time for PI. The eluate was desalted and evaporated to dryness in a rotary evaporator at +SO”C. Completeness of isolation was checked by paper (PC) and thin-layer chromatography (TLC). The U.V. spectrum of the compound was determined with a Beckman DB spectrophotometer with distilled water as the solvent. Hydrolysis
of PI
Hydrolysis was carried out in 1 M formic acid for 10 min at -I-7O”C, or in 3 N HCl at 112°C for 12 hr, or in 7 N HCI at IIO’C for 48 hr, in precooled, evacuated, and sealed ampoules. The results were checked by PC, TLC, and/or AAA. RESULTS
Characteristics
of PI
The U.V. spectrum of PI after isolation with PC is reproduced in Fig. 1. Distinct absorption maxima
Fig. 1. The U.V.spectrum of PI in distilled water after isolation with PC showing absorption peaks at 276 and 269 nm.
are shown at 276 and 269 mn. In the two-dimensional PC used in this study the Rj values for PI were 0.14 for n-butanol-acetic acid-water (4 : 1 : 5) and 0.45 for water-saturated phenol. With TLC on silica gel-G the Rf values for PI were 0.42 for n-propanol-water (70 : 30, v/v) and 0.18 for n-butanol-acetic acid-water (80 : 20 : 20, v/v/v) (cf. STAHL, 1969). With AAA PI is eluted from the long column beginning at the 130th minute and appears after glycine. It overlaps to some extent with alpha-alanine but is well separated from valine which appears next. Further details are provided in JUNNIKKALA (1969). Since the isolation of PI was carried out on a very small scale and complete purity was not obtained, the physical and chemical properties of the compound cannot be specifically outlined. However, the lyophilized preparations have a slightly yellowish tint and the product is at least moderately soluble in cold water. Isolation of PI by PC gave a compound of such purity that only one spot was obtained when rechecked by additional PC or TLC. If this spot is treated with the phenol reagent Fast Blue B salt (STAHL, 1969), a phenol reaction is obtained. If AAA is heavily loaded with the isolation product, a slight contamination with glycine, serine, or glutamine, and histidine is found. To eliminate these the isolation product was further purified by AAA (cf. Materials and Methods) which gave a compound free of ninhydrin-positive contaminants
Storage of tyrosine in haemolymph in Leptidoptera other than possibly glycine. After this the compound, when desalted and evaporated, was submitted to acid hydrolysis. The aliquots hydrolysed were of different sizes and usually so small that quantitation was not attempted. Mild hydrolysis with formic acid had no effect on PI. This was done to find out whether a protein complex might be involved (LINDER et al., 1959). A rather mild hydrolysis with 3 N HCI was capable of splitting PI completely, as shown by rechecking the hydrolysate by AAA; the long column gave only tyrosine instead of PI, and the short column gave ammonia but in a relatively greater quantity than before hydrolysis. Strong hydrolysis with 7 N HCI had a rather different effect on PI. In all chromatograms @AA) PI had disappeared and tyrosine dominated the pattern obtained, but additionally a great number of other amino acids were regularly found in small but easily detectable quantities. From the long column the following amino acids were eluted: Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr and Phe, and from the short column Lys, His, Try ?, and Arg. Changes in the concentration of PI during larvalgrowth andpupation With the molecular weight of PI unknown, the estimation of changes in the concentration of the compound has been based on a postulate that the compound is made up of tyrosine only. This of course is not the case, but it affords a convenient basis for the comparison of relative changes, shown in Fig. 2. No PI was demonstrated in fourth instar larvae. In samples taken 3 hr after the fourth to fifth larval PI
I
1 -2
A mixed A females
I
I
-I
E
I
Time,
I 3
2
G’
d,!ti
days
Fig. 2. Changes in PI and tyrosine concentration during larval growth and pupation. E, Fourth to fifth larval ecdysis; G, pharate pupa; P, pupa. Each sign represents one determination with AAA. The samples are composed of the pooled haemolymph of 5 to 30 insects.
97
ecdysis some PI is apparent. From le.5 pmole/mi haemolymph at 3 hr after ecdysis the concentration of PI increases almost linearly for 3 days to a value of 348 pmole/ml for females and 40.2 pmole/ml for males, about 25 times the concentration measured after ecdysis. In females the peak of 39.5 ,umole/ml is reached about 24 hr later, when the insects have attached themselves for pupation. From these values the concentration again falls in a nearly linear fashion to 17.1 pmole/ml in a l-day-old female and 11.3 pmole/ml in a 1-day-old male pupae. In 4-dayold pupae, approaching apolysis, the differences among the sexes have almost disappeared, the females having 6-5 pmole/ml and the males 7-8 pmole/ml of the compound. Collecting haemolymph samples that would be sufficiently large and clean from older pupae and adults was found to be impracticable. However, an idea of the concentration of PI in these developmental stages was considered important. Pharate adults 8 days after the larval-pupal ecdysis or adults 3 hr after emergence were used in these experiments, six insects per sample. The abdomens were removed from pupae to avoid including the meconium and the wings were removed from adults. The remaining tissues were homogenized in PotterElvehjem-type homogenizers in a neutralized solution of KCN, centrifuged, and the supernatant removed. The procedure was repeated and both supernatants were combined and prepared for AAA. The concentration of PI and tyrosine per g fresh weight were calculated. Owing to different sampling techniques the results from tissue samples are not strictly comparable with those from haemolymph. They do show, however, that detectable quantities of PI are present in pharate adults and newly emerged adults. Thus 2.5 pmole/g was found in 8-day-old females, 1.9 pmole/g in X-day-old males, and 4.8 pmole/g in 3 hr-old female adults and 2.7 pmole/g in 3 hr-old male adults. Tyrosine was determined from the same samples and the results are shown in Fig. 2. There are fairly small changes in the content of tyrosine from the basal value, about 4 E.cmoIe/mlof haemolymph. An increase occurs at the end of the feeding period during the fifth instar (12.5 pmole/ml in females, 7.2 pmole/ml in males), but the basal level is again reached in prepupae in both sexes. An additional increase occurs after pupation. In fourth instar larvae the content of tyrosine is also higher towards the end of the feeding period (6-l pmole/ml, sexes combined), but decreases to a value of 4.3 pmole/ml
after the fourth to fifth ecdysis. It is pointed out, however, that no samples were taken during the ecdysis itself. Distribution The
of PI between haemolymph and tissues
haemolymph
volume
of fifth instar larvae
ERKKZJUNNIKKALA
98
during the walking period was determined in a previous study (TURUNEN and JUNNIKKALA, 1974), and found to be 140 ,~l in females and 146 ~1 in males. The blood volume/larval weight ratios were 0.33 and 0.30, respectively. By measuring the concentration of PI in the haemolymph and the whole organism the distribution of PI among the haemolymph and tissues could be calculated. The content of PI was found to be 34.8 pmole/ml haemolymph in females and 40.2 pmole/ml haemolymph in males, or, counted per g larvae, 10.5 p/mole in the haemolymph of females and 13.2 p/mole in the haemolymph of males. The content of PI was 4.6 pmole per g whole female larvae and 4.2 pmole per g whole male larvae. Judging from these values, PI appears to occur mainly in the haemolymph, although some of it is also found in other tissues. The ratio would be approximately 1 : 5 between tissues and haemolymph. The occuwence
of PI in other Lepidoptera
To find out whether other Lepidoptera contain PI, larvae of a number of species were collected during the late summer of 1971 and reared up to the walking stage on plants on which they were found. The species examined as well as their food plants are listed in Table 1. Preliminary tests to locate PI from the haemolymph of these species were made by PC. PI was found in Smerinthus ocellatus, Celerio galii, and Pergesa elpenor but not in Diwanula vinula. Analysis by AAA, however, showed PI to be present in all four species. Isolation of PI with the method used for P. brassicae was found suitable for the location of the peptide in the haemolymph of S. ocellatus, C. galii, and P. elpenor but failed again to isolate PI from the haemolymph of D. vinula. The U.V. spectra of PI of the three former species were identical with that of P. brassicae, but the detection of such a spectrum from D. vimla failed. The difference between D. vinula and the three other
species may result from differences in timing and concentration, for the walking periods are not strictly comparable. D. vinula characteristically stays a long time in the cocoon before pupation. All samples, however, were taken from larvae that were moving freely. Hydrolyses of the extracts of these species gave a pattern of amino acids identical with that from P. brassicae. Apparently therefore PI is a compound which is found, in addition to Pieris, in S. ocellatus, C. galii, and P. elpenor, and possibly also in D. vinula. Its structure in all these species may be identical or strikingly similar. This would suggest that it is a rather common occurrence among Lepidoptera. DISCUSSION The extraction and isolation of a ninhydrinpositive compound called peptide I or PI in the haemolymph of P. brassicae were not sufficiently complete to allow physical and chemical characterization. A number of conclusions are nevertheless possible. The behaviour of the compound in PC and TLC, or in the ion-exchange column, from which it is eluted among free amino acids, all point to a compound with a rather small molecular weight. Elution among free amino acids in gel filtration also suggests this. The shape of the U.V. spectrum, as well as the phenolic reagent used, point to the phenolic character of PI, in agreement also with the results of acid hydrolysis, in which tyrosine is obtained. Hydrolysis with 3 N HCl, giving tyrosine and ammonia, suggests that the compound is a derivative of tyrosine instead of a peptide. For example, 3-amino-tyrosine, with Rt: values comparable to those of PI (SMITH, 195S), might well be studied in this respect. Hydrolysis with 7 N HCl, resulting in 80% tyrosine and a large number of other amino acids, failed to clarify the structure of the compound, especially in view of the results obtained with mild formic acid hydrolysis, indicating that a protein
Table 1. List of species studied for PI
Species
(Sphinges, Sphingidse) R3rgesa e1*lenor Celerio galii Smerinthus
ocellatus
Food plant
Fireweed (Esobium
angustifoliun]
_ I* _ Great sallow (Salix oaprea)
(Bombyces, Notodontidae) Dicranura vinulg
_n_
Storage of tyrosine in haemolymph in Lepidoptera complex might not be involved. This discussion is based on findings that indicate PI to be a derivative of tyrosine but whose exact nature is not clear. In the author’s opinion this compound is very nearly identical in all five insect species studied. This is suggested by the behaviour of the compound in chromatography, by its U.V. spectrum and the products of hydrolysis and, equally, by the stage at which it occurs in all species. In this respect comparison with the compound found in C. euphorbiae and identified as L-tyrosyl-O-acetyldopamine (celerin) by SIENKIEWICZand PIECHOWSKA (1973) is interesting. I would be inclined to believe in the identity of these two substances. The Rt values given to celerin match those of PI fairly well. It also seems from the report of these authors that celerin is eluted from the long column of AAA at the 130th minute, as does PI. An exact comparison with the elution time is not possible, for SIENKIEWICZ and PIECHOWSKA(1973), while they indicate that they used the resin Beckman PA-28, failed to indicate the specifications of the apparatus they used. The values of their U.V. spectra are ambiguous if they are interpreted in connexion with the figure that the authors refer to, but the spectrum does show peaks at 276 and 269 nm, which are characteristic also for PI. In 3 N hydrolysis SIENKIEWICZand PIECHOWSKA (1973) obtained tyrosine and dopamine, with the latter eluting from the short column of AAA. With this hydrolysis the present author was able to demonstrate tyrosine from the long column but only ammonia from the short column. However, in checking the hydrolysate with PC and TLC the above authors found only one spot, that of dopamine. The absence of tyrosine in this analysis is odd; on the other hand, the spot found by them may represent that of tyrosine. Ammonia is not shown by PC or TLC, which would explain the presence of one spot only. Given this ambiguity, the identity of PI and celerin cannot be determined. Further tests regarding the physiological role of PI have not been made. Judging from its time of occurrence and the variation in its content, as well as from its location, some hypotheses are possible. There are several criteria to argue that PI is not of dietary origin. The natural diet of Pi&s, namely the leaves of the Swede, Bras&z napus v. napobrassica, lacks PI (JUNNIKKALA,1966). The semi-synthetic diet also is lacking in PI. Larvae before the fifth instar have not been found to contain PI, although older larvae reared on identical diets begin to accumulate it. The peak values of PI in haemolymph are found after feeding has ceased. Further, assuming that PI and compounds found in the other Lepidoptera were identical, it would be odd if such diverse plants as the Swede, great sallow, and fireweed should contain an identical substance of this nature. It seems safe to assume that PI is synthesized by the insect.
99
The high content of tyrosine in PI, whether a derivative of tyrosine or a peptide, places the compound among those physiologically active substances that either contain tyrosine of phenylalanine (LEVENBOOKet al., 1969; BODNARYK,1970), in contrast to dipeptides, also frequently present in haemolymph, which lack tyrosine and which occur rather less recurrently during development (LEVENBOOK, 1966 ; BODNARYKand LEVE~TBOOK, 1968). The presence of PI during the fifth instar, its rapid and nearly linear increase up to the walking stage, and an equally rapid decrease at the time of pupation, all suggest a r8le for the compound in pupation. Analyses of tyrosine from these samples are in agreement with the results of POST and DEJONG (1973) in Pieris. They have shown that at the fourth to fifth ecdysis the content of tyrosine in larval haemolymph rapidly increases to about three times the basal value. Unfortunately their sampling does not cover pupation, but it is apparent from their results that an increase occurs at that period also. The present author has not found equally abrupt changes in the content of tyrosine, owing to less frequent sampling which omits the exact ecdysis. A comparable rapid increase of tyrosine during ecdysis has also been found in Periplaneta, (WIRTZ and HOPKINS, 1974), suggesting that it may be common in a larger number of insects. Owing to its slight solubility free tyrosine cannot be present in the haemolymph in sufficient quantity. In Calliphma PRICE (1971) states that the fat body is a storage and release site of tyrosine. Information is lacking on the role of the fat body af Pieris in this respect, but judging from the present and previous data PI could be assumed tu be synthesized in the fat body and released into the haemolymph. PI in haemolymph would thus be a depot of easily available tyrosine during pupation. AcknowZedgement-The author wishes to thank Dr. SEPPOTURUNENfor translating the manuscript as
well as for fruitful discussions during the course of this work.
REFERENCES
BENSON J. V. JR. and PATTERSON J. A. (1965) Accelerated automatic chromatographic analysis of amino acids on a spherical resin. Analyt. Chem. 37, 1108. BODNARYK R. P. (1970) Biosynthesis of gamma-nglutamyl-l-phenyialanine by the larva of the house fly Musca domestica. .Y. Insect Physiol. 16, 919-929. BO~NARYX R. P. and LEVENBOOK L. (1968) Naturally
occurring low-molecular-weight peptides from the blowfly Phormia regina. Biochem. -7. 110,771-773.
BODNARYKR. P. and LEWNBOOKL. (1969) The role of beta-alanyl-L-tyrosine (sarcophagine) in puparium formation in the fleshfly Sarcophaga bullata. Comp. Biochem. Physiol. 30, 909-921.
100
ERKKI JUNNIKKALA
FOX F. R. and MILJ_S R. R. (1971) Cuticle sclerotization by the American cockroach: differential incorporation of leucine and tyrosine during post-ecdysis. y. kwct Physiol. 17, 2363-2374. HACKMANR. H. (1964) Chemistry of the insect cuticle. In The Physiology of Insecta (Ed. by ROCKSTEINM.), 3, 471-506. Academic Press, New York. HENDERSONA. S. and GLASSMANE. (1969) A possible storage form for tyrosinase substrates in Drosophila melanogaster. J. Insect Physiol. 15, 2345-2355. JUNNIKKALAE. (1966) Effect of Braconid parasitization on the nitrogen metabolism of Pieris brassicae L. Ann. Acad. Sci. fenn. (A)4, 100, l-83. JUNNIKKALA E. (1968) A preliminary study of the character of peptide I (PI) in the hemolymph of Pievis brassicae L. Ann. Acad. Sci. fenn. (A)4, 132, l-11. JUNNIKKALAE. (1969) Effect of a semi-synthetic diet on the level of the main nitrogenous compounds in the hemolymph of larvae of Pieris bra&cue L. Ann. Acad. Sci. fenn. (A)4, 155, l-10. KENNAUGH J. H. (1958) Amino acid metabolism, polyphenol production, and the process of tanning in the cockroach Periplaneta americana. J. Insect Physiol. 2, 97-107. KRISTENSENB. I. (1968) Time course of incorporation of tyrosine into rubber-like cuticle of locusts. J. Insect Physiol. 14, 1135-l 140. LENNIE R. W. and BIRT L. M. (1965) The localization of a particle-bound tyrosine activating enzyme in Lucilia cuprina and the distribution of free amino acids during the life cycle. r. Insect Physiol. 11, 1213-1224. LEVENBOOKL. (1950) The composition of horse bot fly Gastrophilus intestinalis larva blood. Biochem. J. 47, 336-346. LEVENBOOK L. (1966) Hemolymph amino acids and peptides during larval growth of the blowfly Phovmia regina. Comp. Biochem. Physiol. 18, 341-351. LEVENBOOK L., BODNARYK R. P., and SPANDE T. F. (1969) Beta-alanyl-L-tyrosine, chemical synthesis, properties and occurrence in larvae of the fleshfly, Sarcophaga bullata Parker. Biochem. J. 113, 837-841. LINDER E. B., ELMQUIST A., and PORATH J. (1959) Gel filtration as a method for purification of protein-bound peptides exemplified by oxytocin and vasopressin. Nature, Lond. 184, 1565.
LUNAN K. D. and MITCHELL H. K. (1969) The metabolism of tyrosine-O-phosphate in Drosophila. Avchs. Biochem. Biophys. 132, 450-456. MITCHELL H. K., CHEN P. S., and HADORN E. (1960) Tyrosine phosphate on paper chromatograms of Drosophila melanogaster. Experientia 16, 410-411. POST L. C. and DEJONG B. J. (1973) Bursicon and the metabolism of tyrosine in the moulting cycle of Pieris larvae. r. Insect Physiol. 19, 1541-1546. PRICE G. M. (1971) Tyrosine metabolism in the blowfly larva, Calliphora erythrocephala. Biochem. J. 121, 28P-29P. SELIGMAN M., FRIEDMAN S., and FRAENKEL G. (1969) Bursicon mediation of tyrosine hydroxylation during tanning of the adult cuticle of the fly, Sarcophaga bullata. J. Insect Physiol. 15, 5.53-561. SIENKIEWICZZ. and PIECHOWSKAM. J. (1973) L-tyrosyl0-acetyldopamine, a new dipeptide found in the haemolymph of caterpillar of Celerio euphorbiae L. (Lepidoptera). Bull. Acad. Pol. Sci., Ser. Sri. Biol. 21, 797-802. SMITH I. (1958) Chvomatographic Techniques. Heinemann, London. SPACKMAND. H., STEIN W. H., and MOORE S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190-1206. STAVL E. (1969) Thin-layer Chromatography, 2nd ed. Springer, Berlin. TURUNEN S. (1973) R61e of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris bra&cue. J. Insect Physiol. 19, 2327-2340. TURUNEN S. and JUNNIKKALAE. (1974) Haemolymph fatty acids and lipid content in larval Pieris brasszkae L. (Lep., Pieridae). Ann. Ent. Fenn. 40, 145-149. WIRTZ R. A. and HOPKINS T. L. (1974) Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamophosis. J. Insect Physiol. 20, 1143-1154.
1976, Vol. 22, pp. 95 to 100.
Pergamon
Press.
Printed
in Great Britain.
A POSSIBLE STORAGE FORM OF TYROSINE IN THE HAEMOLYMPH OF PIERIS BRASSICAE L. AND FOUR OTHER LEPIDOPTERA ERKKI JUNNIKKALA Department of Physiological Zoology, University of Helsinki, 00100 Helsinki 10, Finland (Received
27 June 1975)
Abstract-A ninhydrin-positive compound, called peptide I (PI), has been found in the haemolymph of Pieris brassicae. The substance has been partially purified and is presumed to be a peptide of low molecular weight and rich in tyrosine, or a derivative of tyrosine. Its phenolic nature is evident. PI is not present in fourth instar larvae but is first found in fifth instar larvae a few hours after ecdysis. Its concentration rises almost linearly and is 25 times higher at the end of feeding. The concentration falls to one-third of the peak value shortly after pupation. Some PI is still found in pharate adults and young emerged adults. Both sexes contain about equal amounts of PI. The content of tyrosine in the haemolymph was about 4 pmole/ml during the period studied (ecdysis was not examined). The content of tyrosine increases towards the end of the feeding period during both the fourth and fifth instars. This occurs also after pupation, when the concentration of
PI decreases. PI is distributed in the ratio of 1 : 5 between tissues and haemolymph at the end of the fifth instar. PI is not of dietary origin. Its occurrence during development suggests a r81ein pupation. It is possible that PI is a reserve of tyrosine, owing to its greatersolubilityin comparison to thatof tyrosine. A substance identical or very similar to the PI of P. brassicae was also found in four other randomly
chosen species, Smerinthus ocellatus L., Pergesa elpenor L., Celerio galii L., and, possibly, Dicranula vinula, L. This would suggest that such a compound occurs rather commonly in Lepidoptera.
INTRODUCTION
and LE~ENBOOK,1969), and gamma-L-glutamyl-Lphenylalanine in Musca domestica (BODNARYK, THE CENTRALrole of tyrosine as the precursor of 1970). In other insect orders only indirect evidence diphenols mediating the darkening and hardening of has been obtained of the storage forms of tyrosine. insect cuticles has been well documented (HACKMAN, JUNNIKKALA(1968) reported the presence of a 1964). Most of this work has been done with ninhydrin-positive compound in the haemolymph cyclorrhaphous Diptera during puparium formaof Pieris brassicae, identified tentatively as a peptide tion, which must be regarded as a rather specialized rich in tyrosine and called peptide I or PI. More form of sclerotization. Tyrosine, however, acts as a recently SIENKIEWICH and PIECHOWSKA(1973) precursor during the tanning of the larval and adult reported the presence of a dipeptide L-tyrosyl-Ofly cuticle also (SELIGMAN et al., 1969; PRICE, acetyldopamine (celerin) in the haemolymph of Other orders studied in this respect 1971). Celeyio euphorbiae and suggested a role for it as a show similar functions for tyrosine, i.e. Blattaria storage form of tyrosine. (KENNAUGH,1958; Fox and MILLS, 1971; WIRTZ This paper reports more information on PI in and HOPKINS, 1974), Orthoptera (KRISTENSEN, Pieris and the occurrence in several Lepidoptera of 1968), and Lepidoptera (POST and DEJONG, 1973). a compound remarkably similar to PI as well as to The low solubility limits the concentration of free celerin of C. euphovbiae. (LEVENBOOK, 1950), tyrosine in haemolymph especially during ecdysis when the requirement for tyrosine increases. However, since the concenMATERIALS AND MFZTHODS tration of tyrosine often tends to be high during Pieris brassicae were from a laboratory-reared ecdysis, storage forms for tyrosine have been postulated (LENNIE and BIRT, 1965; HENDERSON stock grown on a semi-synthetic diet as described previously (JUNNIKKALA,1969 ; TURLJNEN,1973). and GLASSMAN,1969; WIRTZ and HOPKINS, 1974) The age of the insect was determined with the and actually found in cyclorrhaphous Diptera, fourth to fifth larval ecdysis as the starting point in for example, tyrosine-O-phosphate in Drosophila melanogaster (MITCHELL et al., 1960; LUNAN counting larval age and the larval-pupal or pupaladult ecdysis as the starting point in counting pupal beta-alanyl-L-tyrosine and MITCHELL, 1969), (Sarcophagine) in Sarcophaga bullata (BODNARYK or adult age, respectively. 95
ERKI(IJUNNIKKALA
96
In order to prevent blackening of the haemolymph all samples were diluted with neutralized KCN and stored at - 18°C. The technique has been described in detail previously (JUNNIKKALA, 1966). Isolation of PI Preliminary tests with gel filtration on Sephadex G-25 fine revealed that PI was eluted among free amino acids with distilled water. For this reason separation was done by paper chromatography, starting with samples of 2 to 4 ml haemolymph. The samples were deproteinized for 2 min in a boiling water-bath, centrifuged, and the supernatants desalted in Dowex SOW x 8, mesh 200/400, H+ co1umns, eluted with 4 N ammonia, and evaporated to dryness in a vacuum at +SO”C. For chromatography the eluates were dissolved in 2 ml of 10% iso-propanol and pipetted as uniform lines across Whatman 3MM sheets (57 x 30 cm) 11 cm below the upper margin. The time of run in the descending direction was 24 hr in a solvent system of n-butanol-acetic acid-water (12 : 3 : 5). The sheets were dried for 1 hr in a ventilated oven at + 50°C. PI was located by cutting out three narrow vertical strips from each sheet, one through the middle and two from each margin and treating the strips with ninhydrin. Areas corresponding to PI were cut out and PI was eluted with water. The eluates were combined, evaporated to dryness, and the procedure repeated in a solvent system of n-propanol-1% ammonia (2 : 1). PI was again eluted with water and lyophilized. To achieve higher purity the sample was applied on the long column of a Beckman Unichrom automatic amino-acid analyzer @AA), in which the method of SPACKMANet al. (1958) and BENSONand PATTERSON(1965) was used. Beginning with the 133rd minute, the eluate was collected for 20 min, covering the elution time for PI. The eluate was desalted and evaporated to dryness in a rotary evaporator at +SO”C. Completeness of isolation was checked by paper (PC) and thin-layer chromatography (TLC). The U.V. spectrum of the compound was determined with a Beckman DB spectrophotometer with distilled water as the solvent. Hydrolysis
of PI
Hydrolysis was carried out in 1 M formic acid for 10 min at -I-7O”C, or in 3 N HCl at 112°C for 12 hr, or in 7 N HCI at IIO’C for 48 hr, in precooled, evacuated, and sealed ampoules. The results were checked by PC, TLC, and/or AAA. RESULTS
Characteristics
of PI
The U.V. spectrum of PI after isolation with PC is reproduced in Fig. 1. Distinct absorption maxima
Fig. 1. The U.V.spectrum of PI in distilled water after isolation with PC showing absorption peaks at 276 and 269 nm.
are shown at 276 and 269 mn. In the two-dimensional PC used in this study the Rj values for PI were 0.14 for n-butanol-acetic acid-water (4 : 1 : 5) and 0.45 for water-saturated phenol. With TLC on silica gel-G the Rf values for PI were 0.42 for n-propanol-water (70 : 30, v/v) and 0.18 for n-butanol-acetic acid-water (80 : 20 : 20, v/v/v) (cf. STAHL, 1969). With AAA PI is eluted from the long column beginning at the 130th minute and appears after glycine. It overlaps to some extent with alpha-alanine but is well separated from valine which appears next. Further details are provided in JUNNIKKALA (1969). Since the isolation of PI was carried out on a very small scale and complete purity was not obtained, the physical and chemical properties of the compound cannot be specifically outlined. However, the lyophilized preparations have a slightly yellowish tint and the product is at least moderately soluble in cold water. Isolation of PI by PC gave a compound of such purity that only one spot was obtained when rechecked by additional PC or TLC. If this spot is treated with the phenol reagent Fast Blue B salt (STAHL, 1969), a phenol reaction is obtained. If AAA is heavily loaded with the isolation product, a slight contamination with glycine, serine, or glutamine, and histidine is found. To eliminate these the isolation product was further purified by AAA (cf. Materials and Methods) which gave a compound free of ninhydrin-positive contaminants
Storage of tyrosine in haemolymph in Leptidoptera other than possibly glycine. After this the compound, when desalted and evaporated, was submitted to acid hydrolysis. The aliquots hydrolysed were of different sizes and usually so small that quantitation was not attempted. Mild hydrolysis with formic acid had no effect on PI. This was done to find out whether a protein complex might be involved (LINDER et al., 1959). A rather mild hydrolysis with 3 N HCI was capable of splitting PI completely, as shown by rechecking the hydrolysate by AAA; the long column gave only tyrosine instead of PI, and the short column gave ammonia but in a relatively greater quantity than before hydrolysis. Strong hydrolysis with 7 N HCI had a rather different effect on PI. In all chromatograms @AA) PI had disappeared and tyrosine dominated the pattern obtained, but additionally a great number of other amino acids were regularly found in small but easily detectable quantities. From the long column the following amino acids were eluted: Asp, Thr, Ser, Glu, Gly, Ala, Val, Ile, Leu, Tyr and Phe, and from the short column Lys, His, Try ?, and Arg. Changes in the concentration of PI during larvalgrowth andpupation With the molecular weight of PI unknown, the estimation of changes in the concentration of the compound has been based on a postulate that the compound is made up of tyrosine only. This of course is not the case, but it affords a convenient basis for the comparison of relative changes, shown in Fig. 2. No PI was demonstrated in fourth instar larvae. In samples taken 3 hr after the fourth to fifth larval PI
I
1 -2
A mixed A females
I
I
-I
E
I
Time,
I 3
2
G’
d,!ti
days
Fig. 2. Changes in PI and tyrosine concentration during larval growth and pupation. E, Fourth to fifth larval ecdysis; G, pharate pupa; P, pupa. Each sign represents one determination with AAA. The samples are composed of the pooled haemolymph of 5 to 30 insects.
97
ecdysis some PI is apparent. From le.5 pmole/mi haemolymph at 3 hr after ecdysis the concentration of PI increases almost linearly for 3 days to a value of 348 pmole/ml for females and 40.2 pmole/ml for males, about 25 times the concentration measured after ecdysis. In females the peak of 39.5 ,umole/ml is reached about 24 hr later, when the insects have attached themselves for pupation. From these values the concentration again falls in a nearly linear fashion to 17.1 pmole/ml in a l-day-old female and 11.3 pmole/ml in a 1-day-old male pupae. In 4-dayold pupae, approaching apolysis, the differences among the sexes have almost disappeared, the females having 6-5 pmole/ml and the males 7-8 pmole/ml of the compound. Collecting haemolymph samples that would be sufficiently large and clean from older pupae and adults was found to be impracticable. However, an idea of the concentration of PI in these developmental stages was considered important. Pharate adults 8 days after the larval-pupal ecdysis or adults 3 hr after emergence were used in these experiments, six insects per sample. The abdomens were removed from pupae to avoid including the meconium and the wings were removed from adults. The remaining tissues were homogenized in PotterElvehjem-type homogenizers in a neutralized solution of KCN, centrifuged, and the supernatant removed. The procedure was repeated and both supernatants were combined and prepared for AAA. The concentration of PI and tyrosine per g fresh weight were calculated. Owing to different sampling techniques the results from tissue samples are not strictly comparable with those from haemolymph. They do show, however, that detectable quantities of PI are present in pharate adults and newly emerged adults. Thus 2.5 pmole/g was found in 8-day-old females, 1.9 pmole/g in X-day-old males, and 4.8 pmole/g in 3 hr-old female adults and 2.7 pmole/g in 3 hr-old male adults. Tyrosine was determined from the same samples and the results are shown in Fig. 2. There are fairly small changes in the content of tyrosine from the basal value, about 4 E.cmoIe/mlof haemolymph. An increase occurs at the end of the feeding period during the fifth instar (12.5 pmole/ml in females, 7.2 pmole/ml in males), but the basal level is again reached in prepupae in both sexes. An additional increase occurs after pupation. In fourth instar larvae the content of tyrosine is also higher towards the end of the feeding period (6-l pmole/ml, sexes combined), but decreases to a value of 4.3 pmole/ml
after the fourth to fifth ecdysis. It is pointed out, however, that no samples were taken during the ecdysis itself. Distribution The
of PI between haemolymph and tissues
haemolymph
volume
of fifth instar larvae
ERKKZJUNNIKKALA
98
during the walking period was determined in a previous study (TURUNEN and JUNNIKKALA, 1974), and found to be 140 ,~l in females and 146 ~1 in males. The blood volume/larval weight ratios were 0.33 and 0.30, respectively. By measuring the concentration of PI in the haemolymph and the whole organism the distribution of PI among the haemolymph and tissues could be calculated. The content of PI was found to be 34.8 pmole/ml haemolymph in females and 40.2 pmole/ml haemolymph in males, or, counted per g larvae, 10.5 p/mole in the haemolymph of females and 13.2 p/mole in the haemolymph of males. The content of PI was 4.6 pmole per g whole female larvae and 4.2 pmole per g whole male larvae. Judging from these values, PI appears to occur mainly in the haemolymph, although some of it is also found in other tissues. The ratio would be approximately 1 : 5 between tissues and haemolymph. The occuwence
of PI in other Lepidoptera
To find out whether other Lepidoptera contain PI, larvae of a number of species were collected during the late summer of 1971 and reared up to the walking stage on plants on which they were found. The species examined as well as their food plants are listed in Table 1. Preliminary tests to locate PI from the haemolymph of these species were made by PC. PI was found in Smerinthus ocellatus, Celerio galii, and Pergesa elpenor but not in Diwanula vinula. Analysis by AAA, however, showed PI to be present in all four species. Isolation of PI with the method used for P. brassicae was found suitable for the location of the peptide in the haemolymph of S. ocellatus, C. galii, and P. elpenor but failed again to isolate PI from the haemolymph of D. vinula. The U.V. spectra of PI of the three former species were identical with that of P. brassicae, but the detection of such a spectrum from D. vimla failed. The difference between D. vinula and the three other
species may result from differences in timing and concentration, for the walking periods are not strictly comparable. D. vinula characteristically stays a long time in the cocoon before pupation. All samples, however, were taken from larvae that were moving freely. Hydrolyses of the extracts of these species gave a pattern of amino acids identical with that from P. brassicae. Apparently therefore PI is a compound which is found, in addition to Pieris, in S. ocellatus, C. galii, and P. elpenor, and possibly also in D. vinula. Its structure in all these species may be identical or strikingly similar. This would suggest that it is a rather common occurrence among Lepidoptera. DISCUSSION The extraction and isolation of a ninhydrinpositive compound called peptide I or PI in the haemolymph of P. brassicae were not sufficiently complete to allow physical and chemical characterization. A number of conclusions are nevertheless possible. The behaviour of the compound in PC and TLC, or in the ion-exchange column, from which it is eluted among free amino acids, all point to a compound with a rather small molecular weight. Elution among free amino acids in gel filtration also suggests this. The shape of the U.V. spectrum, as well as the phenolic reagent used, point to the phenolic character of PI, in agreement also with the results of acid hydrolysis, in which tyrosine is obtained. Hydrolysis with 3 N HCl, giving tyrosine and ammonia, suggests that the compound is a derivative of tyrosine instead of a peptide. For example, 3-amino-tyrosine, with Rt: values comparable to those of PI (SMITH, 195S), might well be studied in this respect. Hydrolysis with 7 N HCl, resulting in 80% tyrosine and a large number of other amino acids, failed to clarify the structure of the compound, especially in view of the results obtained with mild formic acid hydrolysis, indicating that a protein
Table 1. List of species studied for PI
Species
(Sphinges, Sphingidse) R3rgesa e1*lenor Celerio galii Smerinthus
ocellatus
Food plant
Fireweed (Esobium
angustifoliun]
_ I* _ Great sallow (Salix oaprea)
(Bombyces, Notodontidae) Dicranura vinulg
_n_
Storage of tyrosine in haemolymph in Lepidoptera complex might not be involved. This discussion is based on findings that indicate PI to be a derivative of tyrosine but whose exact nature is not clear. In the author’s opinion this compound is very nearly identical in all five insect species studied. This is suggested by the behaviour of the compound in chromatography, by its U.V. spectrum and the products of hydrolysis and, equally, by the stage at which it occurs in all species. In this respect comparison with the compound found in C. euphorbiae and identified as L-tyrosyl-O-acetyldopamine (celerin) by SIENKIEWICZand PIECHOWSKA (1973) is interesting. I would be inclined to believe in the identity of these two substances. The Rt values given to celerin match those of PI fairly well. It also seems from the report of these authors that celerin is eluted from the long column of AAA at the 130th minute, as does PI. An exact comparison with the elution time is not possible, for SIENKIEWICZ and PIECHOWSKA(1973), while they indicate that they used the resin Beckman PA-28, failed to indicate the specifications of the apparatus they used. The values of their U.V. spectra are ambiguous if they are interpreted in connexion with the figure that the authors refer to, but the spectrum does show peaks at 276 and 269 nm, which are characteristic also for PI. In 3 N hydrolysis SIENKIEWICZand PIECHOWSKA (1973) obtained tyrosine and dopamine, with the latter eluting from the short column of AAA. With this hydrolysis the present author was able to demonstrate tyrosine from the long column but only ammonia from the short column. However, in checking the hydrolysate with PC and TLC the above authors found only one spot, that of dopamine. The absence of tyrosine in this analysis is odd; on the other hand, the spot found by them may represent that of tyrosine. Ammonia is not shown by PC or TLC, which would explain the presence of one spot only. Given this ambiguity, the identity of PI and celerin cannot be determined. Further tests regarding the physiological role of PI have not been made. Judging from its time of occurrence and the variation in its content, as well as from its location, some hypotheses are possible. There are several criteria to argue that PI is not of dietary origin. The natural diet of Pi&s, namely the leaves of the Swede, Bras&z napus v. napobrassica, lacks PI (JUNNIKKALA,1966). The semi-synthetic diet also is lacking in PI. Larvae before the fifth instar have not been found to contain PI, although older larvae reared on identical diets begin to accumulate it. The peak values of PI in haemolymph are found after feeding has ceased. Further, assuming that PI and compounds found in the other Lepidoptera were identical, it would be odd if such diverse plants as the Swede, great sallow, and fireweed should contain an identical substance of this nature. It seems safe to assume that PI is synthesized by the insect.
99
The high content of tyrosine in PI, whether a derivative of tyrosine or a peptide, places the compound among those physiologically active substances that either contain tyrosine of phenylalanine (LEVENBOOKet al., 1969; BODNARYK,1970), in contrast to dipeptides, also frequently present in haemolymph, which lack tyrosine and which occur rather less recurrently during development (LEVENBOOK, 1966 ; BODNARYKand LEVE~TBOOK, 1968). The presence of PI during the fifth instar, its rapid and nearly linear increase up to the walking stage, and an equally rapid decrease at the time of pupation, all suggest a r8le for the compound in pupation. Analyses of tyrosine from these samples are in agreement with the results of POST and DEJONG (1973) in Pieris. They have shown that at the fourth to fifth ecdysis the content of tyrosine in larval haemolymph rapidly increases to about three times the basal value. Unfortunately their sampling does not cover pupation, but it is apparent from their results that an increase occurs at that period also. The present author has not found equally abrupt changes in the content of tyrosine, owing to less frequent sampling which omits the exact ecdysis. A comparable rapid increase of tyrosine during ecdysis has also been found in Periplaneta, (WIRTZ and HOPKINS, 1974), suggesting that it may be common in a larger number of insects. Owing to its slight solubility free tyrosine cannot be present in the haemolymph in sufficient quantity. In Calliphma PRICE (1971) states that the fat body is a storage and release site of tyrosine. Information is lacking on the role of the fat body af Pieris in this respect, but judging from the present and previous data PI could be assumed tu be synthesized in the fat body and released into the haemolymph. PI in haemolymph would thus be a depot of easily available tyrosine during pupation. AcknowZedgement-The author wishes to thank Dr. SEPPOTURUNENfor translating the manuscript as
well as for fruitful discussions during the course of this work.
REFERENCES
BENSON J. V. JR. and PATTERSON J. A. (1965) Accelerated automatic chromatographic analysis of amino acids on a spherical resin. Analyt. Chem. 37, 1108. BODNARYK R. P. (1970) Biosynthesis of gamma-nglutamyl-l-phenyialanine by the larva of the house fly Musca domestica. .Y. Insect Physiol. 16, 919-929. BO~NARYX R. P. and LEVENBOOK L. (1968) Naturally
occurring low-molecular-weight peptides from the blowfly Phormia regina. Biochem. -7. 110,771-773.
BODNARYKR. P. and LEWNBOOKL. (1969) The role of beta-alanyl-L-tyrosine (sarcophagine) in puparium formation in the fleshfly Sarcophaga bullata. Comp. Biochem. Physiol. 30, 909-921.
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ERKKI JUNNIKKALA
FOX F. R. and MILJ_S R. R. (1971) Cuticle sclerotization by the American cockroach: differential incorporation of leucine and tyrosine during post-ecdysis. y. kwct Physiol. 17, 2363-2374. HACKMANR. H. (1964) Chemistry of the insect cuticle. In The Physiology of Insecta (Ed. by ROCKSTEINM.), 3, 471-506. Academic Press, New York. HENDERSONA. S. and GLASSMANE. (1969) A possible storage form for tyrosinase substrates in Drosophila melanogaster. J. Insect Physiol. 15, 2345-2355. JUNNIKKALAE. (1966) Effect of Braconid parasitization on the nitrogen metabolism of Pieris brassicae L. Ann. Acad. Sci. fenn. (A)4, 100, l-83. JUNNIKKALA E. (1968) A preliminary study of the character of peptide I (PI) in the hemolymph of Pievis brassicae L. Ann. Acad. Sci. fenn. (A)4, 132, l-11. JUNNIKKALAE. (1969) Effect of a semi-synthetic diet on the level of the main nitrogenous compounds in the hemolymph of larvae of Pieris bra&cue L. Ann. Acad. Sci. fenn. (A)4, 155, l-10. KENNAUGH J. H. (1958) Amino acid metabolism, polyphenol production, and the process of tanning in the cockroach Periplaneta americana. J. Insect Physiol. 2, 97-107. KRISTENSENB. I. (1968) Time course of incorporation of tyrosine into rubber-like cuticle of locusts. J. Insect Physiol. 14, 1135-l 140. LENNIE R. W. and BIRT L. M. (1965) The localization of a particle-bound tyrosine activating enzyme in Lucilia cuprina and the distribution of free amino acids during the life cycle. r. Insect Physiol. 11, 1213-1224. LEVENBOOKL. (1950) The composition of horse bot fly Gastrophilus intestinalis larva blood. Biochem. J. 47, 336-346. LEVENBOOK L. (1966) Hemolymph amino acids and peptides during larval growth of the blowfly Phovmia regina. Comp. Biochem. Physiol. 18, 341-351. LEVENBOOK L., BODNARYK R. P., and SPANDE T. F. (1969) Beta-alanyl-L-tyrosine, chemical synthesis, properties and occurrence in larvae of the fleshfly, Sarcophaga bullata Parker. Biochem. J. 113, 837-841. LINDER E. B., ELMQUIST A., and PORATH J. (1959) Gel filtration as a method for purification of protein-bound peptides exemplified by oxytocin and vasopressin. Nature, Lond. 184, 1565.
LUNAN K. D. and MITCHELL H. K. (1969) The metabolism of tyrosine-O-phosphate in Drosophila. Avchs. Biochem. Biophys. 132, 450-456. MITCHELL H. K., CHEN P. S., and HADORN E. (1960) Tyrosine phosphate on paper chromatograms of Drosophila melanogaster. Experientia 16, 410-411. POST L. C. and DEJONG B. J. (1973) Bursicon and the metabolism of tyrosine in the moulting cycle of Pieris larvae. r. Insect Physiol. 19, 1541-1546. PRICE G. M. (1971) Tyrosine metabolism in the blowfly larva, Calliphora erythrocephala. Biochem. J. 121, 28P-29P. SELIGMAN M., FRIEDMAN S., and FRAENKEL G. (1969) Bursicon mediation of tyrosine hydroxylation during tanning of the adult cuticle of the fly, Sarcophaga bullata. J. Insect Physiol. 15, 5.53-561. SIENKIEWICZZ. and PIECHOWSKAM. J. (1973) L-tyrosyl0-acetyldopamine, a new dipeptide found in the haemolymph of caterpillar of Celerio euphorbiae L. (Lepidoptera). Bull. Acad. Pol. Sci., Ser. Sri. Biol. 21, 797-802. SMITH I. (1958) Chvomatographic Techniques. Heinemann, London. SPACKMAND. H., STEIN W. H., and MOORE S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190-1206. STAVL E. (1969) Thin-layer Chromatography, 2nd ed. Springer, Berlin. TURUNEN S. (1973) R61e of labelled dietary fatty acids and acetate in phospholipids during the metamorphosis of Pieris bra&cue. J. Insect Physiol. 19, 2327-2340. TURUNEN S. and JUNNIKKALAE. (1974) Haemolymph fatty acids and lipid content in larval Pieris brasszkae L. (Lep., Pieridae). Ann. Ent. Fenn. 40, 145-149. WIRTZ R. A. and HOPKINS T. L. (1974) Tyrosine and phenylalanine concentrations in haemolymph and tissues of the American cockroach, Periplaneta americana, during metamophosis. J. Insect Physiol. 20, 1143-1154.