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Autoradiographic study of glycine and tyrosine uptake by integument of different developmental stages of Galleria mellonella
J. InsectPhysiol.,1971, Vol. 17, pp. 261 to 267. Pergamon Press. Printed in Great Britain
AUTORADIOGRAPHIC STUDY OF GLYCINE AND TYROSINE UPTAKE BY INTEGUMENT OF DIFFERENT DEVELOPMENTAL STAGES OF GALLERIA Jk!XLONELLA R. P. SRIVASTAVA Zoologisches Institut, Abteilung Entwicklungsphysiologie, Germany
der Universitat Tubingen,
(Received 15 July 1970) Abstract-Labelled glycine and tyrosine were injected in the haemolymph of l- to 2-day-old mature larvae. Their subsequent appearance in the cuticle of different developmental stages of Galleria mellonella was studied. During the first 12 hr of injection the radioactivity was found to be very low, especially in the glycine 14C injected larvae. In the pharate pupae and pharate adults the new cuticles were comparatively more labelled. However, there was a decline in the radioactive labelling in the cuticle of adult moths. INTRODUCTION WHILE studying the amino acid compositions mellonella (SRIVASTAVA,1970,1971),
of the cuticular proteins of G&ha
it was observed that the percentage of glycine is
very high in most of the hydrolysates and the behaviour of tyrosine in all the protein fractions is consistent throughout. In view of these observations an attempt has now been made to investigate the turnover of labelled glycine and tyrosine from the haemolymph to the cuticle during the development of this moth. Among the earlier papers on the subject are those by KARLSON (1960), KARLSON and SEKERIS (1962), KARLSON et al. (1962), SEKERIS and KARLSON (1962), KARLSON and AMMON (1963), AMMON and KARLSON (1964), SCHLOSSBERGER-RAECKEand KARLSON (1964), and KARLSON and HERRLICH (1965) on the effects of labelled tyrosine on the cuticle of different insects. However, so far little is known regarding the turnover of labelled glycine from the haemolymph to the cuticle. Hence this account, besides providing a general description of the uptake of labelled glycine by the cuticular tissues, also presents a comparison between the turnovers of labelled glycine and tyrosine in the cuticle of different developmental stages of this moth. MATERIALS
AND METHODS
Culture of Galleria mellonella and selection of the Zarwze A culture of this moth was maintained in the laboratory. Mature larvae (head width: 1.8 mm) in sufficiently large numbers were isolated from a culture of known Present address : Entomology, Agricultural Experiment Udaipur (Rajastan), India. 261
Station, University of Udaipur,
R. P. SRIVASTAVA
262
age and kept in Petri dishes. Before starting the present investigation their development was studied in detail. The mature larva undergoes the larval-pupal apolysis after 4 to 5 days and the larval-pupal ecdysis occurs 4 to 5 days later. The pupaladult apolysis occurs 3 to 4 days after the larval-pupal ecdysis and 10 to 12 days later the pupal-adult ecdysis takes place. For autoradiographic studies three groups of 20 larvae were selected for each treatment. Every larva was weighed (average weight 0.085 g). Care was taken to select only l- to 2-day-old mature larvae for experimentation. Autoradiographic techniques The radioactive preparations, glycine -‘“C(U) specific activity 11.3 mCi/mM, and L-tyrosineJ4C(U) specific activity 12.9 mCi/mM, were purchased from the Radio Chemical Centre, Amersham. They were dissolved in Ringer’s solution for insects (Merck Darmstadt) ; 0.002 ml of this solution was injected for every 0.1 g weight of the larva. Injections were given at the base of the first proleg. In the control larvae only Ringer’s solution was injected. Twelve hours after the injection a few larvae were killed and fixed in Weaver and Thomas’s fixative (5 ml of 40% formaldehyde, 2.5 ml acetic acid, and 20 g chloral hydrate in 100 ml of water) for 48 hr. After fixation the tissues were washed in running water for 12 hr, dehydrated, and embedded in paraffin (Tissuemat) following the standard histological procedures. Eight to 10 p sections were cut and stained with Delafield’s haematoxylin and eosin (1% eosin in 95:/, ethanol). Sections were dried and coated with Kodak autoradiographic stripping films AR 10. After an exposure of 3 to 10 weeks the slides were developed in ultrathin (Kodak) for 10 min at 17°C rinsed in 1% acetic acid, fixed in Acidotix for 10 min, washed with running water for 15 min, and finally rinsed in distilled water before drying. Some of the slides were kept for 5 min in xylol and mounted with Canada balsam. However, unmounted slides also served the same purpose. For each experiment the duration and strength of the solutions for developing and fixing were kept the same. Similar autoradiographic preparations were processed from pharate pupae (72 hr after injection), freshly formed pupae, pharate adults (4 days after pupation), and freshly emerged moths. Grain counts and photographs were taken under x 500 magnification. Grain density was determined by counting the grains on three slides of the same stage. Background counts, wherever present, were subtracted from the counts made on different cuticles. It is worth noting that grain counts of endo- and exocuticles in most of the stages represent only an approximate number since it was difficult to ascertain the exact extents of these two sub-layers. Further, grain counts of epidermis have also been taken into consideration because of its intimate association with the cuticle (Table 1). RESULTS It was observed
larval and pupal
that with the injected radioactive material the durations of instars were invariably shortened. In tyrosine injected larvae the
GLYCINEAND TABLE
TYROSINEUPTAKEBY
GALLERIA
263
MELLONELLA
~-GRAIN COUNTS REPRESENTING RELATIVE UPTAKE OF GLYCINE l*C AND L-TYROSINE 14C BY THE TISSUES OF DIFFERENT DEVELOPMENTAL STAGES OF Galleria mellonella Pharate adult
Pharate pupa
Layers Glycine l*C Epicuticle Exocuticle Endocuticle Epidermis Tyrosine l*C Epicuticle Exocuticle Endocuticle Epidermis
Larva
-
Old cuticle
2 5
5 6 7
5 15
10 8 4
-
New cuticle
Pupa
Old cuticle
Adult
-
-
-
-
8 22 15
20 15 10
5 8 5
10 20 15
12 8 5
-
4 18 10 8
4 28 12 10
6 24 15 12
5 10 8 6
8 5 6
-
New cuticle
duration of the final instar was reduced to 5 to 7 days instead of 8 to 10 days, whereas in glycine injected caterpillars the duration was reduced to 6 to 8 days. The pupal durations were also shortened by 3 to 5 days in both. No such reduction in the durations of different developmental stages was noticed in control larvae. Uptake of glycine 14C The uptake of glycine 14C by the cuticle of mature larvae 12 hr after injection was very low; only the endocuticle and epidermal cells showed a few grains (Fig. 1). However, in the next stage (72 hr after injection) the label in the cuticle rose greatly. The latter stage in fact exhibited two cuticles: old cuticle of the mature larva and the newly formed cuticle of the pharate pupa (Fig. 2). The latter was found especially very rich in grain counts; its endocuticle showed a tremendous rise of radioactivity, and the epidermis was also flooded with numerous grains. In the freshly formed pupal cuticle (Fig. 3) the grains were also present in the exocuticle, endocuticle, and epidermis. Sections of 4-day-old pupae showed a heavy rise in grain counts in the newly formed cuticle of the pharate adult (Fig. 4). In the cuticle of adult moths the activity was found to be more in the exocuticle than in endocuticle and epidermal cells (Fig. 5). Uptake of tyrosine 14C It can be clearly seen (Fig. 7) that the uptake of tyrosine by the cuticle of mature larva 12 hr after injection is also low, but in the epidermis a large number of grains could be observed. There was an overall increase in grain counts 72 hr after the injection but the number of grains present in the old and new cuticles do not vary much (Fig. 8). However, in the latter the grain counts are a little more in the exocuticle than in the endocuticle. The cuticle of freshly formed pupae exhibited
264
R. P. SRIVASTAVA
radioactive grains in almost all of its sublayers including the epicuticle (Fig. 9). In the sections of 4-day-old pupae, higher radioactivity in both, new and old cuticles, are visible (Fig. 10). In the new cuticle (that is of the pharate adult) the epicuticle has also been found to be rich in grain counts. In the adult moth there is a general fall in the radioactivity in all the sublayers of its cuticle (Fig. 11).
DISCUSSION CLARKE and GILLOT (1967) found that incorporation of r4C-glycine into the protein of Locusta migratoria reaches a maximum after 7 hr of injection. However, in the present series of experiments the label of glycine 14C failed to appear in the cuticle of mature larva even after 12 hr of incubation. Almost parallel results were obtained in the tyrosine 14C injected series. A low level of radioactivity in the cuticle of mature larvae during the first 12 hr of injection is in conformity with the previous observations made by a number of workers (PRICE, 1966; LOCKE and COLLINS, 1967, 1968; TOBE and LOUGHTON, 1969). In the pharate pupae a great contrast between the labels of radioactivity in the cuticle was recorded. In glycine injected larvae the cuticle of pharate pupae showed a tremendous rise in the grain counts (Table 1). Contrary to this in tyrosine 14C injected larvae the newly formed cuticle of pharate pupae of the same age did not show such a remarkable rise. In the cuticle of freshly formed pupae the epicuticle showed for the first time a clear indication of the incorporation of radioactivity in the tyrosine 14C injected series. Such an observation could not be made in the pupal cuticles of glycine injected specimens. In 4-day-old pupae the label of tyrosine i4C activity in all the sublayers of old and new cuticles was much higher than the label showed by glycine 14C. In the cuticle of the adults there was a general fall in the grain counts in both the series of experiments. A comparison of the radioactivities as exhibited by glycine 14C and tyrosine r4C during the development of this moth suggests that as far as appearance of grains in the cuticle is concerned the trend in both is similar but their intensities differ in different sublayers of the cuticle. For example, in tyrosine injected specimens there is a tendency of increased activity in the procuticular portions of the cuticle and more so in the exocuticle (Table 1). Similar observations were also made by AMMON and KARLSON (1964) ; they inferred that with the increase in age of an instar the activity of tyrosine 14C is concentrated more and more in the exocuticle and to a lesser extent in the endocuticle. Whether under the same conditions glycine 14C is also incorporated to a greater extent in the exocuticle is difficult to judge on the basis of the present observations. However, it could at least safely be inferred that the incorporation of glycine 14C in the cuticle is affected in a regular sequence in much the same way as that of the tyrosine 14C but their functional significance and state of existence seem to differ considerably. As far as glycine is concerned it appears to exist in such a state of chemical bondage in cuticular proteins that it is easier to isolate it through the regular chemical processes of extraction and detection.
26.5
-
9 FIG. 1. Cuticle of larva, 12 hr after injection with glycine r4C. Scales on all figures equal 20 pm. FIG. 2. Cuticle of pharate pupa, 72 hr after injection with glycine r4C. (A) Cuticle of old larva; (B) cuticle of pharate pupa. FIG. 3. Cuticle of pupa (glycine injected series). FIG. 4. Cuticle of pharate adult (glycine injected series). FIG. 5. Cuticle of adult moth (glycine injected series). FIG. 6. Cuticle of larva (control). FIG. 7. Cuticle of larva, 12 hr after injection with tyrosine r4C. FIG. 8. Cuticle of pharate pupa, 72 hr after injection with tyrosine r4C, (A) Cuticle of old larva; (B) cuticle of pharate pupa. FIG. 9. Cuticle of pupa (tyrosine series). FIG. 10. Cuticle of pharate adult (tyrosine injected series). (A) Cuticle of old pupa; (B) cuticle of pharate adult.
.
GLYCINEAND TYROSINEUPTAKEBY GALLERIA
MELLONELLA
267
Acknowledgements-I am grateful to Professor V. SCHWARTZfor his constant help and guidance. My thanks are also due to Professor P. KARLSONand Dr. LEIBENGUTH for constructive suggestions. To Al exander von Humboldt Stiftung I express my thanks for providing a post-doctoral fellowship in Germany. REFERENCES AMMON H. and KARLSONP. (1964) Zum Tyrosinstoffwechsel der Insekten-XIV. Radioautographische Lokalisation der Sklerotisierungssubstanz im Puppentijmrchen von Calliphova erythrocephala. J. Insect Physiol. 10,525-527. CLARKEK. U. and GILLOT C. (1967) Studies on the effects of the removal of the frontal ganglion in Locusta migrutoria L.-I. The effect on protein metabolism. J. exp. Biol. 46,13-25. KARLSONP. (1960) uber den Einbau von Tyrosin-Umwandlugsproduktion in das Puppentijnnchen der Schmeissfliege Calliphora erythrocephala. Hoppe-Seyler’s 2. physiot. Chem. 318,194-200. KARLSONP. and AMMON H. (1963) Zum Tyrosinstoffwechsel der Insekten-XI. Biogenese und Schicksal der Acetylgruppe des N-Acetyldopamins. Hoppe-Seyler’s 2. physiol. Chem. 330,161-168. KARLSONP. and HERRLICHP. (1965) Z urn Tyrosinstoffwechsel der Insekten-XVI. Der Tyrosinstoffwechsel der Heuschrecke Schistocerca gregaria Forsk. J. Insect Physiol. 11, 79-89. KARLSONP. and SEKERISC. E. (1962) Kontrolle des Tyrosinstoffwechsels durch Ecdyson. Biochim. biophys. Acta 63,489+95. KARLSONP., SEKERISC. E. and SEKERISK. E. (1962) Identifizierung von N-Acetyl-3,4dihydroxy-phenylathylamin als Tyrosinmetabolit. Hoppe-Seyler’s 2. physiol. Chem. 327,86-94. LOCKEM. and COLLINSJ. V. (1967) Protein uptake into multivascular bodies in the moltintermolt cycle of an insect. Science, Wash. 155,467-469. LOCKEM. and COLLINSJ. V. (1968) Protein uptake into multivascular bodies and storage granules in the fat body of an insect. 2. Cell Biol. 36,453-483. PRICEG. M. (1966) The in vitro incorporation of (U-14C) valine into fat body protein of the larva of blowfly, Calliphora erythrocephala. J. Insect Physiol. 12,73 l-740. SCHLOSSBERGER-RAECKE I. and KARLSONP. (1964) Zum Tyrosinstoffwechsel der InsektenXI II. Radioautographische Lokalisation von Tyrosinmetaboliten in der Cuticula von Schistocercagregaria Forsk. J. Insect Physiol. 10,261-266. SEKERISC. E. and KARLSONP. (1962) Der ketabolische Abbau des Tyrosins und die Biogenese der Sklerotisierungssubstanz, N-Acetyl-dopamin. Biochim. biophys. Acta 62, 103-113. SRIVASTAVA R. P. (1970) Electrophoretic behaviour of cuticular proteins of different developmental stages of Galleria mellonella. J. Insect Physiol. 16, 2345-2351. SRIVASTAVA R. P. (1971) The amino acid compositions of cuticular proteins of different developmental stages of Galleria mellonella. J. Insect Physiol. 17,189-196. TOBE S. S. and LOUGHTONB. G. (1969) An autoradiographic study of haemolymph protein uptake by the tissues of the fifth instar locust. J. Insect Physiol. 15,1331-l 346. WEAVERN. and THOMASR. C. (1956) A fixative for use of dissecting insects. Stain Technol. 31,47.
AUTORADIOGRAPHIC STUDY OF GLYCINE AND TYROSINE UPTAKE BY INTEGUMENT OF DIFFERENT DEVELOPMENTAL STAGES OF GALLERIA Jk!XLONELLA R. P. SRIVASTAVA Zoologisches Institut, Abteilung Entwicklungsphysiologie, Germany
der Universitat Tubingen,
(Received 15 July 1970) Abstract-Labelled glycine and tyrosine were injected in the haemolymph of l- to 2-day-old mature larvae. Their subsequent appearance in the cuticle of different developmental stages of Galleria mellonella was studied. During the first 12 hr of injection the radioactivity was found to be very low, especially in the glycine 14C injected larvae. In the pharate pupae and pharate adults the new cuticles were comparatively more labelled. However, there was a decline in the radioactive labelling in the cuticle of adult moths. INTRODUCTION WHILE studying the amino acid compositions mellonella (SRIVASTAVA,1970,1971),
of the cuticular proteins of G&ha
it was observed that the percentage of glycine is
very high in most of the hydrolysates and the behaviour of tyrosine in all the protein fractions is consistent throughout. In view of these observations an attempt has now been made to investigate the turnover of labelled glycine and tyrosine from the haemolymph to the cuticle during the development of this moth. Among the earlier papers on the subject are those by KARLSON (1960), KARLSON and SEKERIS (1962), KARLSON et al. (1962), SEKERIS and KARLSON (1962), KARLSON and AMMON (1963), AMMON and KARLSON (1964), SCHLOSSBERGER-RAECKEand KARLSON (1964), and KARLSON and HERRLICH (1965) on the effects of labelled tyrosine on the cuticle of different insects. However, so far little is known regarding the turnover of labelled glycine from the haemolymph to the cuticle. Hence this account, besides providing a general description of the uptake of labelled glycine by the cuticular tissues, also presents a comparison between the turnovers of labelled glycine and tyrosine in the cuticle of different developmental stages of this moth. MATERIALS
AND METHODS
Culture of Galleria mellonella and selection of the Zarwze A culture of this moth was maintained in the laboratory. Mature larvae (head width: 1.8 mm) in sufficiently large numbers were isolated from a culture of known Present address : Entomology, Agricultural Experiment Udaipur (Rajastan), India. 261
Station, University of Udaipur,
R. P. SRIVASTAVA
262
age and kept in Petri dishes. Before starting the present investigation their development was studied in detail. The mature larva undergoes the larval-pupal apolysis after 4 to 5 days and the larval-pupal ecdysis occurs 4 to 5 days later. The pupaladult apolysis occurs 3 to 4 days after the larval-pupal ecdysis and 10 to 12 days later the pupal-adult ecdysis takes place. For autoradiographic studies three groups of 20 larvae were selected for each treatment. Every larva was weighed (average weight 0.085 g). Care was taken to select only l- to 2-day-old mature larvae for experimentation. Autoradiographic techniques The radioactive preparations, glycine -‘“C(U) specific activity 11.3 mCi/mM, and L-tyrosineJ4C(U) specific activity 12.9 mCi/mM, were purchased from the Radio Chemical Centre, Amersham. They were dissolved in Ringer’s solution for insects (Merck Darmstadt) ; 0.002 ml of this solution was injected for every 0.1 g weight of the larva. Injections were given at the base of the first proleg. In the control larvae only Ringer’s solution was injected. Twelve hours after the injection a few larvae were killed and fixed in Weaver and Thomas’s fixative (5 ml of 40% formaldehyde, 2.5 ml acetic acid, and 20 g chloral hydrate in 100 ml of water) for 48 hr. After fixation the tissues were washed in running water for 12 hr, dehydrated, and embedded in paraffin (Tissuemat) following the standard histological procedures. Eight to 10 p sections were cut and stained with Delafield’s haematoxylin and eosin (1% eosin in 95:/, ethanol). Sections were dried and coated with Kodak autoradiographic stripping films AR 10. After an exposure of 3 to 10 weeks the slides were developed in ultrathin (Kodak) for 10 min at 17°C rinsed in 1% acetic acid, fixed in Acidotix for 10 min, washed with running water for 15 min, and finally rinsed in distilled water before drying. Some of the slides were kept for 5 min in xylol and mounted with Canada balsam. However, unmounted slides also served the same purpose. For each experiment the duration and strength of the solutions for developing and fixing were kept the same. Similar autoradiographic preparations were processed from pharate pupae (72 hr after injection), freshly formed pupae, pharate adults (4 days after pupation), and freshly emerged moths. Grain counts and photographs were taken under x 500 magnification. Grain density was determined by counting the grains on three slides of the same stage. Background counts, wherever present, were subtracted from the counts made on different cuticles. It is worth noting that grain counts of endo- and exocuticles in most of the stages represent only an approximate number since it was difficult to ascertain the exact extents of these two sub-layers. Further, grain counts of epidermis have also been taken into consideration because of its intimate association with the cuticle (Table 1). RESULTS It was observed
larval and pupal
that with the injected radioactive material the durations of instars were invariably shortened. In tyrosine injected larvae the
GLYCINEAND TABLE
TYROSINEUPTAKEBY
GALLERIA
263
MELLONELLA
~-GRAIN COUNTS REPRESENTING RELATIVE UPTAKE OF GLYCINE l*C AND L-TYROSINE 14C BY THE TISSUES OF DIFFERENT DEVELOPMENTAL STAGES OF Galleria mellonella Pharate adult
Pharate pupa
Layers Glycine l*C Epicuticle Exocuticle Endocuticle Epidermis Tyrosine l*C Epicuticle Exocuticle Endocuticle Epidermis
Larva
-
Old cuticle
2 5
5 6 7
5 15
10 8 4
-
New cuticle
Pupa
Old cuticle
Adult
-
-
-
-
8 22 15
20 15 10
5 8 5
10 20 15
12 8 5
-
4 18 10 8
4 28 12 10
6 24 15 12
5 10 8 6
8 5 6
-
New cuticle
duration of the final instar was reduced to 5 to 7 days instead of 8 to 10 days, whereas in glycine injected caterpillars the duration was reduced to 6 to 8 days. The pupal durations were also shortened by 3 to 5 days in both. No such reduction in the durations of different developmental stages was noticed in control larvae. Uptake of glycine 14C The uptake of glycine 14C by the cuticle of mature larvae 12 hr after injection was very low; only the endocuticle and epidermal cells showed a few grains (Fig. 1). However, in the next stage (72 hr after injection) the label in the cuticle rose greatly. The latter stage in fact exhibited two cuticles: old cuticle of the mature larva and the newly formed cuticle of the pharate pupa (Fig. 2). The latter was found especially very rich in grain counts; its endocuticle showed a tremendous rise of radioactivity, and the epidermis was also flooded with numerous grains. In the freshly formed pupal cuticle (Fig. 3) the grains were also present in the exocuticle, endocuticle, and epidermis. Sections of 4-day-old pupae showed a heavy rise in grain counts in the newly formed cuticle of the pharate adult (Fig. 4). In the cuticle of adult moths the activity was found to be more in the exocuticle than in endocuticle and epidermal cells (Fig. 5). Uptake of tyrosine 14C It can be clearly seen (Fig. 7) that the uptake of tyrosine by the cuticle of mature larva 12 hr after injection is also low, but in the epidermis a large number of grains could be observed. There was an overall increase in grain counts 72 hr after the injection but the number of grains present in the old and new cuticles do not vary much (Fig. 8). However, in the latter the grain counts are a little more in the exocuticle than in the endocuticle. The cuticle of freshly formed pupae exhibited
264
R. P. SRIVASTAVA
radioactive grains in almost all of its sublayers including the epicuticle (Fig. 9). In the sections of 4-day-old pupae, higher radioactivity in both, new and old cuticles, are visible (Fig. 10). In the new cuticle (that is of the pharate adult) the epicuticle has also been found to be rich in grain counts. In the adult moth there is a general fall in the radioactivity in all the sublayers of its cuticle (Fig. 11).
DISCUSSION CLARKE and GILLOT (1967) found that incorporation of r4C-glycine into the protein of Locusta migratoria reaches a maximum after 7 hr of injection. However, in the present series of experiments the label of glycine 14C failed to appear in the cuticle of mature larva even after 12 hr of incubation. Almost parallel results were obtained in the tyrosine 14C injected series. A low level of radioactivity in the cuticle of mature larvae during the first 12 hr of injection is in conformity with the previous observations made by a number of workers (PRICE, 1966; LOCKE and COLLINS, 1967, 1968; TOBE and LOUGHTON, 1969). In the pharate pupae a great contrast between the labels of radioactivity in the cuticle was recorded. In glycine injected larvae the cuticle of pharate pupae showed a tremendous rise in the grain counts (Table 1). Contrary to this in tyrosine 14C injected larvae the newly formed cuticle of pharate pupae of the same age did not show such a remarkable rise. In the cuticle of freshly formed pupae the epicuticle showed for the first time a clear indication of the incorporation of radioactivity in the tyrosine 14C injected series. Such an observation could not be made in the pupal cuticles of glycine injected specimens. In 4-day-old pupae the label of tyrosine i4C activity in all the sublayers of old and new cuticles was much higher than the label showed by glycine 14C. In the cuticle of the adults there was a general fall in the grain counts in both the series of experiments. A comparison of the radioactivities as exhibited by glycine 14C and tyrosine r4C during the development of this moth suggests that as far as appearance of grains in the cuticle is concerned the trend in both is similar but their intensities differ in different sublayers of the cuticle. For example, in tyrosine injected specimens there is a tendency of increased activity in the procuticular portions of the cuticle and more so in the exocuticle (Table 1). Similar observations were also made by AMMON and KARLSON (1964) ; they inferred that with the increase in age of an instar the activity of tyrosine 14C is concentrated more and more in the exocuticle and to a lesser extent in the endocuticle. Whether under the same conditions glycine 14C is also incorporated to a greater extent in the exocuticle is difficult to judge on the basis of the present observations. However, it could at least safely be inferred that the incorporation of glycine 14C in the cuticle is affected in a regular sequence in much the same way as that of the tyrosine 14C but their functional significance and state of existence seem to differ considerably. As far as glycine is concerned it appears to exist in such a state of chemical bondage in cuticular proteins that it is easier to isolate it through the regular chemical processes of extraction and detection.
26.5
-
9 FIG. 1. Cuticle of larva, 12 hr after injection with glycine r4C. Scales on all figures equal 20 pm. FIG. 2. Cuticle of pharate pupa, 72 hr after injection with glycine r4C. (A) Cuticle of old larva; (B) cuticle of pharate pupa. FIG. 3. Cuticle of pupa (glycine injected series). FIG. 4. Cuticle of pharate adult (glycine injected series). FIG. 5. Cuticle of adult moth (glycine injected series). FIG. 6. Cuticle of larva (control). FIG. 7. Cuticle of larva, 12 hr after injection with tyrosine r4C. FIG. 8. Cuticle of pharate pupa, 72 hr after injection with tyrosine r4C, (A) Cuticle of old larva; (B) cuticle of pharate pupa. FIG. 9. Cuticle of pupa (tyrosine series). FIG. 10. Cuticle of pharate adult (tyrosine injected series). (A) Cuticle of old pupa; (B) cuticle of pharate adult.
.
GLYCINEAND TYROSINEUPTAKEBY GALLERIA
MELLONELLA
267
Acknowledgements-I am grateful to Professor V. SCHWARTZfor his constant help and guidance. My thanks are also due to Professor P. KARLSONand Dr. LEIBENGUTH for constructive suggestions. To Al exander von Humboldt Stiftung I express my thanks for providing a post-doctoral fellowship in Germany. REFERENCES AMMON H. and KARLSONP. (1964) Zum Tyrosinstoffwechsel der Insekten-XIV. Radioautographische Lokalisation der Sklerotisierungssubstanz im Puppentijmrchen von Calliphova erythrocephala. J. Insect Physiol. 10,525-527. CLARKEK. U. and GILLOT C. (1967) Studies on the effects of the removal of the frontal ganglion in Locusta migrutoria L.-I. The effect on protein metabolism. J. exp. Biol. 46,13-25. KARLSONP. (1960) uber den Einbau von Tyrosin-Umwandlugsproduktion in das Puppentijnnchen der Schmeissfliege Calliphora erythrocephala. Hoppe-Seyler’s 2. physiot. Chem. 318,194-200. KARLSONP. and AMMON H. (1963) Zum Tyrosinstoffwechsel der Insekten-XI. Biogenese und Schicksal der Acetylgruppe des N-Acetyldopamins. Hoppe-Seyler’s 2. physiol. Chem. 330,161-168. KARLSONP. and HERRLICHP. (1965) Z urn Tyrosinstoffwechsel der Insekten-XVI. Der Tyrosinstoffwechsel der Heuschrecke Schistocerca gregaria Forsk. J. Insect Physiol. 11, 79-89. KARLSONP. and SEKERISC. E. (1962) Kontrolle des Tyrosinstoffwechsels durch Ecdyson. Biochim. biophys. Acta 63,489+95. KARLSONP., SEKERISC. E. and SEKERISK. E. (1962) Identifizierung von N-Acetyl-3,4dihydroxy-phenylathylamin als Tyrosinmetabolit. Hoppe-Seyler’s 2. physiol. Chem. 327,86-94. LOCKEM. and COLLINSJ. V. (1967) Protein uptake into multivascular bodies in the moltintermolt cycle of an insect. Science, Wash. 155,467-469. LOCKEM. and COLLINSJ. V. (1968) Protein uptake into multivascular bodies and storage granules in the fat body of an insect. 2. Cell Biol. 36,453-483. PRICEG. M. (1966) The in vitro incorporation of (U-14C) valine into fat body protein of the larva of blowfly, Calliphora erythrocephala. J. Insect Physiol. 12,73 l-740. SCHLOSSBERGER-RAECKE I. and KARLSONP. (1964) Zum Tyrosinstoffwechsel der InsektenXI II. Radioautographische Lokalisation von Tyrosinmetaboliten in der Cuticula von Schistocercagregaria Forsk. J. Insect Physiol. 10,261-266. SEKERISC. E. and KARLSONP. (1962) Der ketabolische Abbau des Tyrosins und die Biogenese der Sklerotisierungssubstanz, N-Acetyl-dopamin. Biochim. biophys. Acta 62, 103-113. SRIVASTAVA R. P. (1970) Electrophoretic behaviour of cuticular proteins of different developmental stages of Galleria mellonella. J. Insect Physiol. 16, 2345-2351. SRIVASTAVA R. P. (1971) The amino acid compositions of cuticular proteins of different developmental stages of Galleria mellonella. J. Insect Physiol. 17,189-196. TOBE S. S. and LOUGHTONB. G. (1969) An autoradiographic study of haemolymph protein uptake by the tissues of the fifth instar locust. J. Insect Physiol. 15,1331-l 346. WEAVERN. and THOMASR. C. (1956) A fixative for use of dissecting insects. Stain Technol. 31,47.