- Email: [email protected]
Ommochrome synthesis and kynurenic acid excretion in relation to metamorphosis and allatectomy in the stick insect, Carausius morosus Br.
J. Insect Physiol., Vol. 25, pp. 925 to 929. Pergamon Press Ltd. 1979. Printed in Great Britain.
OMMOCHROME SYNTHESIS AND KYNURENIC ACID EXCRETION IN RELATION TO METAMORPHOSIS ANC ALLATECTOMY IN THE STICK INSECT, CARAUSIUS MOROSUS BR. E. STRATAKIS Zoologisches Institut, Lehrstuhl fiir experimentelle Morphologie, Weyertal 119,D-500 KBln, W. Germany (Received 7 March 1979; revised 8 June 1979)
Abstract-End products oftryptophan metabolism in Carausiusmorosus are the ommochromes ommin and xanthommatin in the epidermis, and kynurenic acid in the faeces.During larval and adult life ommochromes and mainly kynurenic Bcidare formed. The concentration of kynurenic acid in the faeces of adult females is 2.5 times lower than in the larvae and in adult males. Allatectomy on the first day after a larval moult induces a much longer instar (10 days) than normal. After the following mot& the allatectomized animals are transformed into adultoids. The allatectomized and normal larvae produce similar amounts of kynurenic acid and ommochrome during the larval instar. Twenty days after last ecdysis, the ommochrome content in adult and adultoids is increased. In the faeces of adultoids, however, the concentration of kynurenic acid is higher than in normal female adults, but lower than in males and larvae. Key
Word Index:
Carausius
morosus,
tryptophan metabolism, kynurenic acid, ommochrome,
allatectomy, development
INTRODUCTION THE COLOURATION of the integument in some species of
insects is controlled by the corpora allata. JOLY(1952, 1958) and ROUSSEL(1967) have demonstrated that in migratoria and Cryllus bimaculatus, Locusta respectively, removal of the corpora allata causes a black colouration of the integument, whereas the implantation of additional corpora allata results in bright (yellow to green) colouration. In Carausius morosus, allatectomy leads to beige colouration, while implantation of extra corpora allata results in black colouration (RAABE, 1956, 1961). Biochemical analysis of morphological colour change in the stick insect has not clarified completely the influence of the corpora allata on ommochrome formation. According to DUSTMANN (1964), the implantation of additional corpora allata promotes ommochrome synthesis in the epidermis. After extirpation, on the other hand, there is no degradation of the ommochromes; once the ommochromes are present, they remain in the epidermis. BERTHOLD (1973) assumed that the approach to the adult stage brings about a reduction of the insects’ ability to form ommochrome. In addition, BERTHOLD and B&KMANN (1975) showed that the formation of ommochromes in the epidermis is correlated with a decrease in excretion of kynurenic acid. It is the major end product of tryptophan metabolism in the feeding of Carausius (STRATAKIS, unpublished larva observations). In this study, the problem posed is whether or not the two pathways of kynurenine degradation, namely kynurenic acid and ommochrome formation, are under developmental control. In most insects, larvae allatectomized in an earlyinstar undergo a precocious metamorphosis. In Carausius, ahatectomy causes a I.P. 25/12-n
partial reversal of metamorphosis in the moulting insect (KALUSCHE,1972). We therefore studied the ommochrome formation in the epidermis and kynurenic acid excretion of normal and allatectomized animals during the transitional period from the larval to the adult stage.
MATERIALS AND METHODS The conditions under which insects were reared have been described previously (STRATAKIS,1976a). Carausius morosus exhibits constant parthenogenesis and males are very rare. The operations were performed according to NEUGEBAUER (1961). Larvae of the 3rd, 4th and 5th instar, about 12 hr after ecdysis, were operated upon under CO, narcosis. Possible damage to the corpora cardiaca was ignored. The instruments used for the operation were kept sterile. Two to four larvae and one each of adults and adultoids were taken for each serial ommochrome determination. The separation and quantitative determination of ommin and xanthommatin were performed as described by STRATAKIS(1976b). To isolate kynurenic acid, 50 mg samples of dried faeces were extracted with 2 ml of 90% methanol and the extracts were dried on a rotary evaporator. The residue was dissolved in 1 ml of 5% trichloroacetic acid (TCA) and centrifuged. The supernatant was chromatographed on a Sephadex G-25 column (fine, 400 x 15 mm). Elution was performed with 1% TCA. Kynurenic acid appeared between the 48th and 54th fraction (each fraction 2 ml, Fig. 1). Quantitative determination of the kynurenic acid in the eluent was performed by measuring the absorption at 240 nm (Fig 2, El$, = 3100).
925
E. STRATAKIS
60
Fig. 1. Typical U.V.elution curve of faeces homogenate. The supernatant was applied to a column of Sephadex G25(400 x 15 mm) and eluted with 1% TCA. Fractions of 2 ml were collected. The positions of elution peaks were measured at 254 nm. Fig. 3. Comparison of ommochrome changes in allatectomized and normal animals. Filled plus open bars = ng ommochromes per animal + S.E.M. The amount of xanthommatin (open) and ommin (filled) were determined after separation by ion-exchange chromatography. 1 = Larvae after ecdysis (n = .20), 2 = adultoids and adults after last ecdysis (n = 15), 3 = adultoids and adults 20 days after last ecdysis (n = 15-35). 6
0.6
i a
0.4
0.2
I
160
240
300 WAVE LENGTH nm
360
Fig. 2. Ultraviolet absorption spectra of synthetic (-------) ) kynurenic acid, isolated from faeces and natural (--of Carausius morosus, in 1% TCA.
RESULTS
Effect of allactectomy on development Allatectomy had the same effect on the development of the insects in all three study series: at 18°C the larval instar of control insects lasts 18 + 3 days, while that of allatectomized insects lasts 28 + 3 days. After the subsequent moult, all insects display adult features: they are coloured red on the forelegs and the sternites of the mesothorax and metathorax. The red pigmentation in allatectomized insects is more pronounced the earlier they are allatectomized. After the premature last ecdysis, the adultoids grow at almost the same rate as normal insects. The external genitalia of the adultoids are less differentiated, and the subgenital plate does not extend to the end of the body. The insects which are operated upon in the 3rd and 4th instars fail to lay any eggs, although a few eggs without shells have been produced. 75% of the insects allatectomized in the 5th instar lay eggs just like normal adults, cu. 20 days after the last ecdysis.
Ommochrome content of the normal and allatectomized insects In the insects allatectomized in the 3rd and 4th instars, no ommochrome synthesis takes place in the epidermis during larval development. Twenty eight days after allatectomy, the ommochrome values of freshly-moulted adultoids’ does not differ from those of normal larvae of the 3rd and 4th instars (Fig. 3). In contrast, in freshly-moulted adultoids which develop after allatectomy in the 5th instar the ommochrome amount is greater than in normals of the 5th instar (P~O.001). This increase can be compared with that of ommochrome in the last larval instar of normals (Fig. 3). As adultoids, however, all three groups produce considerably more ommochrome. In normal adults, the ommochrome content of the insects increases by about 1.5 times (P
Metamorphosis
and allatectomy in the stick insect
927
-m-.- operated control allatectomized _=-.- normal
.
J’
/\
II .i
n \/ . \
l\.
‘1. ! 1 -.-.
/“--%
l
P0
.’
2
6
E
4th instar 10
14
18
22
5 th instar 4
8
12
16
20
days
Fig. 4. The average daily weight of farces per animal collected each day from the experimental groups. After the following ecdysis: c+&--o, adultoids; e, normal larvae in 5th instar. A: indicates the normal development in days (operated control, normal); B: indicates the prolonged instar (allatectomized).
than in the operated controls (in. these the glands were not removed). The operated controls moult as usual after 18 f 3 days and produce 42 mg/animal of faeces during the 4th instar. The curves for the daily amount of faeces produced during the 4th instar in normal, allatectomized and operated controls are shown with the curve for normal animals during the 5th instar and adultoids in Fig. 4. The concentration of kynurenic acid in the faeces of normal and allatectomized larvae is almost identical in the 3rd, 4th and 5th instar (Fig. 5). After the next moult, the daily faeces production in the adultoids is similar to that in the normals in the first 15 days of the 5th instar (Fig. 4). Eaeces-
greater
NA -iii-
NA -ivLARVAE
NA V ADULTOIIJS
ADULTS
Fig. 5. A comparison of kyrmreriic acid excretion in allatectomixed (A) and normal control (N) animals. Values are expressed in mg kynnrenic acid per gram dry weight faeces +S.E.M. (n = 8). III, IV, V = larval instar; A,, A,, A, = adultoids after 3rd 4th, and 5th ecdysis.
kynurenic acid concentration in the adult females is lower than in the larvae, adult males and adultoids
(PcO.001, PcO.001, P~0.01 respectively), but the difference in kynurenic acid concentration between the larvae and adult males is not significant (P>O.4). In the adultoids kynurenic acid concentration varies between the concentration of the adult females and males, but differences in kynurenic acid concentration between the adultoids (A,, A,, A, in Fig. 5) are not significant. DISCUSSION The changes in integument colour in some species of insects due to environmental conditions or to developmental stages are known to be controlled by the corpora allata (review FUZEAU-BRAEXH, 1972). Whereas in Gryllus bimaculatus the melanization of the cuticle largely determines the colour of the integument (ROUSSEL,1967), the epidermal pigments ommin and xanthommatin are responsible for the colour of Caraurius. Allatectomy, which is performed on the first day after a larval ecdysis, is followed by ecdysis into adultoids. According to BERTHOLD (1973), the premature approach to the adult stage induces a reduction of the insects’ ability to produce ommochrome. This is not so in allatectomized insects which, despite the approach to the adult stage, produce increased amounts of ommochrome after the lower halves of the eyes have been painted (BERTHOLD, 1973). B~~CKMANN (1977) found that this opticahy induced morphological change of colour was equally pronounced in all normal larval instars. Since ecdysis to the adult stage is considered to be of particular significance for ommochrome synthesis and
928
E.
STRATAKIS
colour adaptation, the results for larvae and adults are discussed separately. Larval development
In Carausius ommochromes must be fixed irreversibly in the epidermis, since no ommochromes or their break-down products have been demonstrated in the faeces (DUSTMANN, 1964). The difference between the amount of ommochromes in freshlyhatched larvae and freshly-moulted adults corresponds to the amount of ommochromes produced during larval development (duration 110 days for 6 larval instars). It amounts to about 15 pg and is produced mainly in the course of the last larval instar. During larval development, no ommochromes are produced in insects which are allatectomized in the 3rd or 4th instar, whereas an increase in can be observed in insects ommochromes allatectomized in the 5th instar (see Fig. 3). In normal larvae, kynurenic acid is excreted at a concentration of 1.3 mg/g dry weight faeces in the instars investigated. The amount of kynurenic acid excreted by normal larvae is about 450 pg in the course of larval development (STRATAKIS, unpublished obseivations). Although allatectomy prolongs the instar by 10 days, allatectomized and normal larvae excrete the same amount of kynurenic acid in the course of an instar. This is due to the reduced production of faeces in the allatectomized. The comparison of the allatectomized with the operated controls indicates that the reduction is due to the operation (see Fig. 4). The identical ommochrome and kynurenic acid values in allatectomized and normal larvae show that, in larvae, the two pathways of kynurenink degradation, namely kynurenic acid and ommochrome formation, are not controlled by the corpora allata. Adults
A comparison of the ommochrome content of freshly-moulted and of 20-day-old adults shows that the amount of ommochrome stored in the epidermis doubles during this period. A corresponding increase is observed also in adultoids. The results of the adults and adultoids do not agree with data published by DUSTMANN (1964), in which the ommochrome formation in the adults is low. This discrepancy might be due to differences in the methods used @ the separation of ommochromes. The synthesis of ommochrome in adults cannot be influenced by environmental factors during a period of time in which the morphogenetic processes of colouration are of no importance. The fact that ommochrome synthesis in adultoids is greater than in normal adults may be attributed to impairment of typtophan catabolism and premature ageing of the insects. In Carausius, kynurenic acid, the main product of tryptophan catabolism, is present in the faeces of larvae and adults. The reduction of kynurenic acid in the faeces of females is not associated with the synthesis of ommochrome in the epidermis, as BERTHOLDand BOCKMANN(1975) have established in larvae. Although males and adultoids synthesize increased amounts of ommochromes, the concentration of kynurenic acid in their faeces remains high at the same time.
The decreased excretion of kynurenic acid in the female adults may indicate a reduced pool of free tryptophan and kynurenine. Such a decrease reflects again an increased demand for the synthesis of protein. A high level of tryptophan occurs in the yolk protein during the embryonic development of Carausius (STRATAKIS,1976a). On the other hand, allatectomy or implantation of additional corpora allata into a larval instar of Carat&us impairs the production of eggs (PFLUGFJZLDER, 1969). In many female insects the corpora allata controls protein synthesis (ENGELMANN,1970) and oijcyte development (STRONG, 1965). Consequently, it can be assumed with some confidence that changes in the yolk protein synthesis reflect corresponding changes in the kynurenic acid excretion of adultoids. Acknowledgements-This work was supported by the Deutsche Forschungsgemeinschaft, SFB 87, Projekt A,, in the Department of Biology I, University of Wm. The author is indebted to Miss G. FRANK,University of Ulm, for technical assistance and Mr. J. JACOBI, University of Cologne,
for drawing
the figures.
REFERENCES BERTHOLD G. (1973) Der EinfluB der corpora allata auf die Pigmentierung von Carausius morosus Br. Wilhem Roux Arch. EntwMech. Ora. 173, 249-262. .BERTHOLD G. and BUCKMANN D. (1975) Morphologischer Farbwechsel und Kynurenslure-Exkretion bei der Stabheuschrecke Carausius morosus. J. camp. Physiol. 100, 347-350. B~~CKMANND. (1977) Morphological colour change: stage independent, optically induced ommochrome synthesis in larvae of the stick insect, Carausius morosus Br. J. camp. Physiol. 115, 185-193. DUSTMANN H. (1964) Die Redoxpigmente von Carausius morosus und ihre Bedeutung fiir den morphologischen Farbwechsel. Z. vergl. Physiol. 49, 28-57. ENGELMANN F. (1970) Physiology of Insect Reproduction. Pergamon Press, Oxford. FUZEAU-BRAESCH S. (1972) Pigments and colour changes. A. Rev. Ent. 17, 403-424. JOLY P. (1952) DCterminisme endocrine de la pigmentation chez Acrida turrita L. C.r. SPanc. Sot. Biol. 235, 1054-1056. JOLY P. (1958) Les corrtlations humorales chez les acridiens. Annls. Biol. 34, 97-118. KALUSCHE D. (1972) Wirkungen der Exstirpation und Transplantation der Corpora allata auf die Entwicklung des Geschlechtsapparates der Stabheuschrecke Carausius morosus Br. Zool. Jb. 89, 117-165. NEUGEBAUER W. (1961) Wirkungen der Exstirpation und Transplantation der corpora allata auf den Sauerstoffverbrauch, die Eibildung und den Fettkiirper von Carausius morosus Br. Wilhelm Roux Arch. EntwMech. Org. 153, 314-352. PFLUGFELDER 0. (1969) Wirkungen implantierter iiberz&hliger corpora allata auf die Oocytenentwicklung von Carausius morosus Br. Wilhelm Roux Arch. EntwMech. Org. 164, 182-198. RAABE M. (1956) Les mtcanismes de I’adaptation chromatique ch& les’ insectes. Annls. Biol. 32, 247-282. RAAE+EM. (196 1) Recherches sur le determinisme des genises de pigments chez le phasme, Carausius morosus. C.r. hebd. Sianc. Acad. Sci. Paris 252, 3663-3665. ROUSSELJ. (1967) Fonctions des corpora allata et contrBle de la pigmentation chez GryNus bimaculatus de Gcer. J. Insect physiol. 13, 113-l 30.
MetamOrplIOSlS
and allatectomy in
E. (1976a) Der Tryptophanstoffwechsel und die Ommochromsynthese wahrend der Embryonalentwicklung der Stabheuschrecke Carausius morosus Br. J. Insect
STRATAKIS
Physibl. 22, 1207-1212. E. (1976b) Quantitative
Analyse des Ommochrompigments der Augen von Mannchen und Weibchen von
STRATAKIS
the stuzkmsect
929
Pieris brassicae. J. Insect Biochem. 6, 29-34.
L. (1965) The relationships between the brain, corpora allata, and oocyte growth in the Central American locust, Schistocerca sp. I.-The cerebral neurosecretory system, the corpora allata, and oiicyte growth. J. Insect
STRONG
Physiol. 11, 135-146.
OMMOCHROME SYNTHESIS AND KYNURENIC ACID EXCRETION IN RELATION TO METAMORPHOSIS ANC ALLATECTOMY IN THE STICK INSECT, CARAUSIUS MOROSUS BR. E. STRATAKIS Zoologisches Institut, Lehrstuhl fiir experimentelle Morphologie, Weyertal 119,D-500 KBln, W. Germany (Received 7 March 1979; revised 8 June 1979)
Abstract-End products oftryptophan metabolism in Carausiusmorosus are the ommochromes ommin and xanthommatin in the epidermis, and kynurenic acid in the faeces.During larval and adult life ommochromes and mainly kynurenic Bcidare formed. The concentration of kynurenic acid in the faeces of adult females is 2.5 times lower than in the larvae and in adult males. Allatectomy on the first day after a larval moult induces a much longer instar (10 days) than normal. After the following mot& the allatectomized animals are transformed into adultoids. The allatectomized and normal larvae produce similar amounts of kynurenic acid and ommochrome during the larval instar. Twenty days after last ecdysis, the ommochrome content in adult and adultoids is increased. In the faeces of adultoids, however, the concentration of kynurenic acid is higher than in normal female adults, but lower than in males and larvae. Key
Word Index:
Carausius
morosus,
tryptophan metabolism, kynurenic acid, ommochrome,
allatectomy, development
INTRODUCTION THE COLOURATION of the integument in some species of
insects is controlled by the corpora allata. JOLY(1952, 1958) and ROUSSEL(1967) have demonstrated that in migratoria and Cryllus bimaculatus, Locusta respectively, removal of the corpora allata causes a black colouration of the integument, whereas the implantation of additional corpora allata results in bright (yellow to green) colouration. In Carausius morosus, allatectomy leads to beige colouration, while implantation of extra corpora allata results in black colouration (RAABE, 1956, 1961). Biochemical analysis of morphological colour change in the stick insect has not clarified completely the influence of the corpora allata on ommochrome formation. According to DUSTMANN (1964), the implantation of additional corpora allata promotes ommochrome synthesis in the epidermis. After extirpation, on the other hand, there is no degradation of the ommochromes; once the ommochromes are present, they remain in the epidermis. BERTHOLD (1973) assumed that the approach to the adult stage brings about a reduction of the insects’ ability to form ommochrome. In addition, BERTHOLD and B&KMANN (1975) showed that the formation of ommochromes in the epidermis is correlated with a decrease in excretion of kynurenic acid. It is the major end product of tryptophan metabolism in the feeding of Carausius (STRATAKIS, unpublished larva observations). In this study, the problem posed is whether or not the two pathways of kynurenine degradation, namely kynurenic acid and ommochrome formation, are under developmental control. In most insects, larvae allatectomized in an earlyinstar undergo a precocious metamorphosis. In Carausius, ahatectomy causes a I.P. 25/12-n
partial reversal of metamorphosis in the moulting insect (KALUSCHE,1972). We therefore studied the ommochrome formation in the epidermis and kynurenic acid excretion of normal and allatectomized animals during the transitional period from the larval to the adult stage.
MATERIALS AND METHODS The conditions under which insects were reared have been described previously (STRATAKIS,1976a). Carausius morosus exhibits constant parthenogenesis and males are very rare. The operations were performed according to NEUGEBAUER (1961). Larvae of the 3rd, 4th and 5th instar, about 12 hr after ecdysis, were operated upon under CO, narcosis. Possible damage to the corpora cardiaca was ignored. The instruments used for the operation were kept sterile. Two to four larvae and one each of adults and adultoids were taken for each serial ommochrome determination. The separation and quantitative determination of ommin and xanthommatin were performed as described by STRATAKIS(1976b). To isolate kynurenic acid, 50 mg samples of dried faeces were extracted with 2 ml of 90% methanol and the extracts were dried on a rotary evaporator. The residue was dissolved in 1 ml of 5% trichloroacetic acid (TCA) and centrifuged. The supernatant was chromatographed on a Sephadex G-25 column (fine, 400 x 15 mm). Elution was performed with 1% TCA. Kynurenic acid appeared between the 48th and 54th fraction (each fraction 2 ml, Fig. 1). Quantitative determination of the kynurenic acid in the eluent was performed by measuring the absorption at 240 nm (Fig 2, El$, = 3100).
925
E. STRATAKIS
60
Fig. 1. Typical U.V.elution curve of faeces homogenate. The supernatant was applied to a column of Sephadex G25(400 x 15 mm) and eluted with 1% TCA. Fractions of 2 ml were collected. The positions of elution peaks were measured at 254 nm. Fig. 3. Comparison of ommochrome changes in allatectomized and normal animals. Filled plus open bars = ng ommochromes per animal + S.E.M. The amount of xanthommatin (open) and ommin (filled) were determined after separation by ion-exchange chromatography. 1 = Larvae after ecdysis (n = .20), 2 = adultoids and adults after last ecdysis (n = 15), 3 = adultoids and adults 20 days after last ecdysis (n = 15-35). 6
0.6
i a
0.4
0.2
I
160
240
300 WAVE LENGTH nm
360
Fig. 2. Ultraviolet absorption spectra of synthetic (-------) ) kynurenic acid, isolated from faeces and natural (--of Carausius morosus, in 1% TCA.
RESULTS
Effect of allactectomy on development Allatectomy had the same effect on the development of the insects in all three study series: at 18°C the larval instar of control insects lasts 18 + 3 days, while that of allatectomized insects lasts 28 + 3 days. After the subsequent moult, all insects display adult features: they are coloured red on the forelegs and the sternites of the mesothorax and metathorax. The red pigmentation in allatectomized insects is more pronounced the earlier they are allatectomized. After the premature last ecdysis, the adultoids grow at almost the same rate as normal insects. The external genitalia of the adultoids are less differentiated, and the subgenital plate does not extend to the end of the body. The insects which are operated upon in the 3rd and 4th instars fail to lay any eggs, although a few eggs without shells have been produced. 75% of the insects allatectomized in the 5th instar lay eggs just like normal adults, cu. 20 days after the last ecdysis.
Ommochrome content of the normal and allatectomized insects In the insects allatectomized in the 3rd and 4th instars, no ommochrome synthesis takes place in the epidermis during larval development. Twenty eight days after allatectomy, the ommochrome values of freshly-moulted adultoids’ does not differ from those of normal larvae of the 3rd and 4th instars (Fig. 3). In contrast, in freshly-moulted adultoids which develop after allatectomy in the 5th instar the ommochrome amount is greater than in normals of the 5th instar (P~O.001). This increase can be compared with that of ommochrome in the last larval instar of normals (Fig. 3). As adultoids, however, all three groups produce considerably more ommochrome. In normal adults, the ommochrome content of the insects increases by about 1.5 times (P
Metamorphosis
and allatectomy in the stick insect
927
-m-.- operated control allatectomized _=-.- normal
.
J’
/\
II .i
n \/ . \
l\.
‘1. ! 1 -.-.
/“--%
l
P0
.’
2
6
E
4th instar 10
14
18
22
5 th instar 4
8
12
16
20
days
Fig. 4. The average daily weight of farces per animal collected each day from the experimental groups. After the following ecdysis: c+&--o, adultoids; e, normal larvae in 5th instar. A: indicates the normal development in days (operated control, normal); B: indicates the prolonged instar (allatectomized).
than in the operated controls (in. these the glands were not removed). The operated controls moult as usual after 18 f 3 days and produce 42 mg/animal of faeces during the 4th instar. The curves for the daily amount of faeces produced during the 4th instar in normal, allatectomized and operated controls are shown with the curve for normal animals during the 5th instar and adultoids in Fig. 4. The concentration of kynurenic acid in the faeces of normal and allatectomized larvae is almost identical in the 3rd, 4th and 5th instar (Fig. 5). After the next moult, the daily faeces production in the adultoids is similar to that in the normals in the first 15 days of the 5th instar (Fig. 4). Eaeces-
greater
NA -iii-
NA -ivLARVAE
NA V ADULTOIIJS
ADULTS
Fig. 5. A comparison of kyrmreriic acid excretion in allatectomixed (A) and normal control (N) animals. Values are expressed in mg kynnrenic acid per gram dry weight faeces +S.E.M. (n = 8). III, IV, V = larval instar; A,, A,, A, = adultoids after 3rd 4th, and 5th ecdysis.
kynurenic acid concentration in the adult females is lower than in the larvae, adult males and adultoids
(PcO.001, PcO.001, P~0.01 respectively), but the difference in kynurenic acid concentration between the larvae and adult males is not significant (P>O.4). In the adultoids kynurenic acid concentration varies between the concentration of the adult females and males, but differences in kynurenic acid concentration between the adultoids (A,, A,, A, in Fig. 5) are not significant. DISCUSSION The changes in integument colour in some species of insects due to environmental conditions or to developmental stages are known to be controlled by the corpora allata (review FUZEAU-BRAEXH, 1972). Whereas in Gryllus bimaculatus the melanization of the cuticle largely determines the colour of the integument (ROUSSEL,1967), the epidermal pigments ommin and xanthommatin are responsible for the colour of Caraurius. Allatectomy, which is performed on the first day after a larval ecdysis, is followed by ecdysis into adultoids. According to BERTHOLD (1973), the premature approach to the adult stage induces a reduction of the insects’ ability to produce ommochrome. This is not so in allatectomized insects which, despite the approach to the adult stage, produce increased amounts of ommochrome after the lower halves of the eyes have been painted (BERTHOLD, 1973). B~~CKMANN (1977) found that this opticahy induced morphological change of colour was equally pronounced in all normal larval instars. Since ecdysis to the adult stage is considered to be of particular significance for ommochrome synthesis and
928
E.
STRATAKIS
colour adaptation, the results for larvae and adults are discussed separately. Larval development
In Carausius ommochromes must be fixed irreversibly in the epidermis, since no ommochromes or their break-down products have been demonstrated in the faeces (DUSTMANN, 1964). The difference between the amount of ommochromes in freshlyhatched larvae and freshly-moulted adults corresponds to the amount of ommochromes produced during larval development (duration 110 days for 6 larval instars). It amounts to about 15 pg and is produced mainly in the course of the last larval instar. During larval development, no ommochromes are produced in insects which are allatectomized in the 3rd or 4th instar, whereas an increase in can be observed in insects ommochromes allatectomized in the 5th instar (see Fig. 3). In normal larvae, kynurenic acid is excreted at a concentration of 1.3 mg/g dry weight faeces in the instars investigated. The amount of kynurenic acid excreted by normal larvae is about 450 pg in the course of larval development (STRATAKIS, unpublished obseivations). Although allatectomy prolongs the instar by 10 days, allatectomized and normal larvae excrete the same amount of kynurenic acid in the course of an instar. This is due to the reduced production of faeces in the allatectomized. The comparison of the allatectomized with the operated controls indicates that the reduction is due to the operation (see Fig. 4). The identical ommochrome and kynurenic acid values in allatectomized and normal larvae show that, in larvae, the two pathways of kynurenink degradation, namely kynurenic acid and ommochrome formation, are not controlled by the corpora allata. Adults
A comparison of the ommochrome content of freshly-moulted and of 20-day-old adults shows that the amount of ommochrome stored in the epidermis doubles during this period. A corresponding increase is observed also in adultoids. The results of the adults and adultoids do not agree with data published by DUSTMANN (1964), in which the ommochrome formation in the adults is low. This discrepancy might be due to differences in the methods used @ the separation of ommochromes. The synthesis of ommochrome in adults cannot be influenced by environmental factors during a period of time in which the morphogenetic processes of colouration are of no importance. The fact that ommochrome synthesis in adultoids is greater than in normal adults may be attributed to impairment of typtophan catabolism and premature ageing of the insects. In Carausius, kynurenic acid, the main product of tryptophan catabolism, is present in the faeces of larvae and adults. The reduction of kynurenic acid in the faeces of females is not associated with the synthesis of ommochrome in the epidermis, as BERTHOLDand BOCKMANN(1975) have established in larvae. Although males and adultoids synthesize increased amounts of ommochromes, the concentration of kynurenic acid in their faeces remains high at the same time.
The decreased excretion of kynurenic acid in the female adults may indicate a reduced pool of free tryptophan and kynurenine. Such a decrease reflects again an increased demand for the synthesis of protein. A high level of tryptophan occurs in the yolk protein during the embryonic development of Carausius (STRATAKIS,1976a). On the other hand, allatectomy or implantation of additional corpora allata into a larval instar of Carat&us impairs the production of eggs (PFLUGFJZLDER, 1969). In many female insects the corpora allata controls protein synthesis (ENGELMANN,1970) and oijcyte development (STRONG, 1965). Consequently, it can be assumed with some confidence that changes in the yolk protein synthesis reflect corresponding changes in the kynurenic acid excretion of adultoids. Acknowledgements-This work was supported by the Deutsche Forschungsgemeinschaft, SFB 87, Projekt A,, in the Department of Biology I, University of Wm. The author is indebted to Miss G. FRANK,University of Ulm, for technical assistance and Mr. J. JACOBI, University of Cologne,
for drawing
the figures.
REFERENCES BERTHOLD G. (1973) Der EinfluB der corpora allata auf die Pigmentierung von Carausius morosus Br. Wilhem Roux Arch. EntwMech. Ora. 173, 249-262. .BERTHOLD G. and BUCKMANN D. (1975) Morphologischer Farbwechsel und Kynurenslure-Exkretion bei der Stabheuschrecke Carausius morosus. J. camp. Physiol. 100, 347-350. B~~CKMANND. (1977) Morphological colour change: stage independent, optically induced ommochrome synthesis in larvae of the stick insect, Carausius morosus Br. J. camp. Physiol. 115, 185-193. DUSTMANN H. (1964) Die Redoxpigmente von Carausius morosus und ihre Bedeutung fiir den morphologischen Farbwechsel. Z. vergl. Physiol. 49, 28-57. ENGELMANN F. (1970) Physiology of Insect Reproduction. Pergamon Press, Oxford. FUZEAU-BRAESCH S. (1972) Pigments and colour changes. A. Rev. Ent. 17, 403-424. JOLY P. (1952) DCterminisme endocrine de la pigmentation chez Acrida turrita L. C.r. SPanc. Sot. Biol. 235, 1054-1056. JOLY P. (1958) Les corrtlations humorales chez les acridiens. Annls. Biol. 34, 97-118. KALUSCHE D. (1972) Wirkungen der Exstirpation und Transplantation der Corpora allata auf die Entwicklung des Geschlechtsapparates der Stabheuschrecke Carausius morosus Br. Zool. Jb. 89, 117-165. NEUGEBAUER W. (1961) Wirkungen der Exstirpation und Transplantation der corpora allata auf den Sauerstoffverbrauch, die Eibildung und den Fettkiirper von Carausius morosus Br. Wilhelm Roux Arch. EntwMech. Org. 153, 314-352. PFLUGFELDER 0. (1969) Wirkungen implantierter iiberz&hliger corpora allata auf die Oocytenentwicklung von Carausius morosus Br. Wilhelm Roux Arch. EntwMech. Org. 164, 182-198. RAABE M. (1956) Les mtcanismes de I’adaptation chromatique ch& les’ insectes. Annls. Biol. 32, 247-282. RAAE+EM. (196 1) Recherches sur le determinisme des genises de pigments chez le phasme, Carausius morosus. C.r. hebd. Sianc. Acad. Sci. Paris 252, 3663-3665. ROUSSELJ. (1967) Fonctions des corpora allata et contrBle de la pigmentation chez GryNus bimaculatus de Gcer. J. Insect physiol. 13, 113-l 30.
MetamOrplIOSlS
and allatectomy in
E. (1976a) Der Tryptophanstoffwechsel und die Ommochromsynthese wahrend der Embryonalentwicklung der Stabheuschrecke Carausius morosus Br. J. Insect
STRATAKIS
Physibl. 22, 1207-1212. E. (1976b) Quantitative
Analyse des Ommochrompigments der Augen von Mannchen und Weibchen von
STRATAKIS
the stuzkmsect
929
Pieris brassicae. J. Insect Biochem. 6, 29-34.
L. (1965) The relationships between the brain, corpora allata, and oocyte growth in the Central American locust, Schistocerca sp. I.-The cerebral neurosecretory system, the corpora allata, and oiicyte growth. J. Insect
STRONG
Physiol. 11, 135-146.