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The effect of atmospheric oxygen pressure on the biosynthesis of simple pterines in pierid butterflies
J. Insect Physiol.,1969, Vol. 15, pp. 2239 to 2244. Pergamon Press. P&ted
in Great Britain
THE EFFECT OF ATMOSPHERIC OXYGEN PRESSURE ON THE BIOSYNTHESIS OF SIMPLE PTERINES IN PIERID BUTTERFLIES R. HARMSEN* Zoology Department, University College, Nairobi, Kenya (Received 16 June 1969) Abstract-The very abundant simple pterines of some groups of insects are metabolic end-products of C-6 substituted, hydrogenated pterines. Two alternate synthetic pathways exist, both starting from 7,8-dihydropterin. At high oxygen pressure this substance is dehydrogenated to pterin and oxidized to isoxanthopterin. At low oxygen pressure, dihydropterin is hydroxylated enzymically to dihydroxanthopterin and subsequently gives rise to xanthopterin, leucopterin, and the C-7 substituted pterines such as erythropterin. The pierid butterfly, Mylothris chloris, appears not to have an excretion barrier against pterin (an unnatural substance). This results in the appearance of large quantities of this material in the excreta of animals reared at very high oxygen pressure. Simultaneously the quantity of pterines deposited in the wings is much reduced. INTRODUCTION BECKER (1937) investigated the effect of oxygen pressure on the deposition of xanthopterin in the wings of the pierid butterfly, Gonapteryx rhamni, and reported a progressively reduced deposition of the yellow pigment with higher oxygen pressure. At 100 per cent 0, no yellow pigment is deposited, and the normally yellow butterfly is off-white (the author has recently seen some of Becker’s original specimens, now in the possession of Professor Schopf). It appears as if the total pterine content of the wings is much reduced, but a certain transition from yellow to white pigment is possible. If indeed the xanthopterin is not or only partly replaced by a white substance in the wings, it is not known if the xanthopterin (or other pterine) is stored in the fat body or excreted with the meconium, or if a reduced quantity of pterines is synthesized at high 0, pressure. The quantitative aspects of this situation have a direct bearing on recent work by the present author (HARMSEN, 1966a, b), while the qualitative aspects may throw some light on the biosynthetic relationships of the simple pterines. MATERIALS
AND METHODS
All the oxygen pressure work was done with Mylothris chloris agathina Cram. ; two other species of this genus were cursorily examined: M. yulei and M. poppea * Present Canada.
address:
Biology
Department, 2239
Queen’s
University,
Kingston,
Ontario,
2240
R. HARMSEN
tirikensis. All specimens were collected as eggs or caterpillars on epiphytic species of Loranthus in avocado pear trees in the vicinity of Nairobi. 0,-N, mixtures of known ratio were commercially obtained in pressurized form. The development chambers consisted of large (4-5 ft”) polythene bags filled under slight pressure with the desired gas mixture and kept taut throughout the experiment. The pupae were placed in the chambers within 12 hr of the larval-pupal ecdysis (before pterine synthesis commenced) and kept in the chambers until adult emergence. Wings were extracted in 1 ml 1 y0 HCl at approximately 80°C for a few seconds. Whole bodies were homogenized in a 0.75 ml chloroform-O.75 ml 1% HCI mixture as described earlier (HARMSEN,1966a). Meconium was dissolved by boiling in 1 ml HCl for about 1 min. Wing extracts, aqueous supernatant parts of body homogenates, and meconium solutions were chromatographed on Whatman No. 1 paper descending in two directions. The first-dimension solvent system was ethanol-ammonia 1% (7 : 3), and the second-dimension solvent was 4% sodium citrate in distilled water. The chromatograms were viewed in U.V. light of 365 rnp, and the intensity of fluorescence of the various substances was taken as a rough quantitative measure. RESULTS The three species of Mylothris are qualitatively similar in pterine content; they differ only in relative quantities and pigment distribution. The dominant orange pigment is erythropterin, although smaller quantities of unidentified yellow and orange fluorescing substances with low R, values are present. These may be di-pterines. Also one or two red ommochromes are found in minute quantities in the wings. The dominant yellow pterine is xanthopterin. A second yellow (yellow fluorescing) substance which may be a pterine is present; this substance is particularly noticeable in M. chloris and will be referred to as Y-pterin. Three white pterines are abundantly present in the wings: pterin (2-amino-4-hydroxy-pteridine), isoxanthopterin, and leucopterine. There are no measurable amounts of purines in the wings. In the bodies (mainly fat body) pterin, isoxanthopterin, leucopterin, and xanthopterin are present, as well as uric acid, xanthine, and hypoxanthine. Several minor u.v.-absorbing or fluorescing substances are present as well in trace quantities. Freshly pupated males and females of M. chloris were allowed to develop in O-5 per cent, 3 per cent, 20 per cent, 40 per cent, 80 per cent, and 99.5 per cent 0, respectively. All these animals developed into complete adults. The results were immediately recognizable as identical to Becker’s. At 80 per cent 0, an obvious reduction in orange and yellow pigmentation can be seen; at 99.5 per cent 0, the entire animal is off-white. Rough quantitative estimates of the fluorescing substances shows a possible overall reduction of total pterine content, but this cannot be established with certainty. It is, however, in the relative quantities of the different pterines that
BIOSYNTHESIS
OF SIMPLE
PTBRINBS
2241
IN PIBRID BUTTERFLIES
great differences are found (see Table 1). The dominant substances at normal or low 0, levels: xanthopterin, leucopterin, erythopterin, and Y-pterin are all but absent in animals reared at 99.5% 0,. In animals reared at 80% they are found in reduced quantities. Isoxanthopterin, and especially pterin, both minor constituents at normal 0, levels and virtually absent at reduced 0, levels, become the dominant wing pterines at very high 0, levels. TABLE
~--RELATIVE
ABUNDANCEOF
M.
MAJOR
ChlorisREARED
Percentage 0, in gas mixture
0 0 -_ -
3 20 40 80 99.5
PTBRINES
traces; 0, Not present; -, + + + , very large amount.
+++ +++ +++ +++ +-l-
+ ++ ++ -,
0,-N,
Xan
-
++ +++
INTHEWINGS
DIFFERENT
Isox
Pterin
0.5
IN
small amount;
OF
OFTHEADULTFEMALE
MIXTURES
Leuc
Ery
Y-pt
++ ++ + + + -
++ ++ + + +
+ + + + +
+ , medium
amount;
+ + , large amount;
In the bodies the same trend can be seen, but less distinctly. The purines seem to be affected little, or not at all. It is, however, interesting to note the differences in the meconium (see Table 2). The three pterines that are excreted TABLE
2-RELATIVE
ABUNDANCE REARED
Percentage 0 e in gas mixture
OF
-,
IN
THE
02-N,
MECONIUM
OF
FEMALE
Isox
Xan
Leuc
0 0
-
-
-
++
-
-
small amount;
+ , medium
M. chloris
MIXTURES
Pterin
1.5 3 40 80 0, Not present;
PTBRINES
IN DIFFERENT
0 amount;
0
+ + , large amount.
in traces at normal 0, levels: leucopterin, xanthopterin, and isoxanthopterin are absent at high 0, levels, while pterin, not present at normal 0, levels, becomes a dominant excretory product at high 0, levels. DISCUSSION
AND
CONCLUSIONS
It has been clearly established that pterines are formed in the insect from purine precursors in such a way that the initial pterines are hydrogenated in the
2242
R. HARMSEN
5, 6, 7, and 8 positions and carry a 3-C side chain in position 6 (WATT, 1967; There is little doubt that all oxidized, nonsubstituted pterines are metabolic end-products of the C-6 substituted pterines, and that C-7 substituted pterines (such as erythropterin) are secondarily supplied with a side-chain (WATT, 1967; ZIEGLERand HARMSEN,1969). It has been shown long ago that several xanthine dehydrogenase (or oxidase) preparations act on the C-7 position of the pterine-ring (BERGMANand KWIETNY, 1969). In other words, pterin is oxidized to isoxanthopterin and xanthopterin to leucopterin in reactions catalysed by this enzyme. Only recently, REMBOLDand GUTENSOHN(1968) have shown that at least some xanthine oxidases act on C-6 of 7,8-dihydrogenated pteridines. REMBOLDand GUTENSOHN(1968) postulate a complete C-6 side-chain breakdown, and the production of 7, Sdihydropterin as a ‘mother substance’ for all simple pterines and 7-substituted pterines. In Mylothris, under normal conditions, xanthopterin and leucopterin are synthesized in relatively large quantities. On the basis of the above-mentioned Rembold and Gutensohn model, 6-substituted pterines of the biopterin group would be broken down to 7, 8-dihydropterin. This latter substance is subsequently either oxidized to pterin or hydroxylated (xanthine oxidase!) to 7,8_dihydroxanthopterin. Any pterin produced (normally only a very small quantity) is subsequently hydroxylated enzymically to isoxanthopterin, while the relatively stable dihydroxanthopterin will be oxidized eventually to xanthopterin. If this final oxidation takes place in the presence of xanthine oxidase, leucopterin will be formed, otherwise xanthopterin may persist as such. Erythropterin is probably formed from either dihydroxanthopterin or from xanthopterin itself. Fig. 1 represents the various chemical biosynthetic relationships as postulated above. The present work supports this postulation and furnishes evidence for considering these reactions normal steps in the biosynthesis of simple pterines. Increased 0, pressure appears to favour a probably non-enzymic-oxidation of the very labile dihydropterin to pterin. Xanthine oxidase activity will transfer a certain amount of this pterin into isoxanthopterin. At very high 0s levels virtually no dihydroxanthopterin will be formed, and thus no xanthopterin, no leucopterin, and no erythropterin. It is not fully understood why males show the above-discussed trend less distinctly than females, nor why the fat body content is less affected by 0, pressure than the wing pigments. However, it was observed that gas exchange (i.e. atmospheric 0, consumption) is high in the first hours after pupation and during the last 1.5 to 2 days of pharate adult development. In the period in between, pupae will live and develop at a normal rate in pure N,, any gas exchange during this period seems to consist of CO, expulsion only. It is suggested that only after the breakdown of the pupal cuticle has commenced (i.e. during the period of maximal pterine deposition and/or synthesis in the wing) does external 0, pressure have a physiological effect on the animal, and especially in the external tissues. The reduced effect of 0, in the male may be related to a lower gas exchange in an animal which does not build an egg mass. cf. ZIEGLER and HARMSEN,1969).
BIOSYNTHESIS OF SIMPLEPTERINESIN PIERIDBUTTERFLIES
2243
The abnormally high amount of pterin in the meconium of animals reared high 0, concentrations may reflect the lack of a selective excretion threshold barrier in the Malpighian tubules. Such an excretion barrier was described leucopterin and isoxanthopterin in Pieris by HARMSEN (1966a) and appears C-6 Substituted
in or for to
Pterines
part dehydrogenation
and side chain elimination
OH J
7,8-Dihydroxanthopterin
Pterin
Pterines OH
xanthine
dehydrogenase
H H xanthine
dehydrogenase \
Isoxanthopterin \
OH
\
OH \ Leucopterin
FIG. 1. Biosynthetic pathways of simple pterines, as postulated for insects; based on in vitro studies of GUTENSOHN (1968), and oxygen pressure effects of pterine synthesis in M. chloris.
exist for the natural pterines in Myloth~is as well. Pterin, not a naturally occurring substance, appears to be freely excretable; an excretion barrier, favouring storage over excretion, has not developed for this substance. The apparent loss of pterine content of the wings of Becker’s oxygen-reared Gonapteryx is probably also based on an extensive pterin excretion as well as deposition of isoxanthopterin and pterin instead of xanthopterin. Acknowledgements-I wish to record my gratitude to Mr. R. C. CARCASSON, Director of the Nairobi Museum, for identifying the species of Mylotkris and to Miss I. JABBALfor her excellent technical assistance. The work was supported by a Rockefeller Foundation grant to the Science Faculty, University College, Nairobi.
2244
R. HARMSEN REFERENCES
BECKER E. (1937) Uber das Pterinpigment
bei Insekten und die Farbung und Zeichnung von Vespa im Besonderen. 2. Morph. c)kol. Tiere 32, 672-751. BERGMAN F. and KWIETNY H. (1969) Pteridines as substrates of mammalian xanthine oxidase-II. Pathways and rates of oxidation. Biochim. biophys. Acta 33, 2946. GUTENSOHN W. (1968) Chemische und biochemische Untersuchungen zum Abbau von Tetrahydroneopterin. Thesis, Ludwig-Max. University, Miinchen. HARMSEN R. (1966a) Identification of fluorescing and ultra-violet-absorbing substances in Pieris brassicae L. J. Insect Physiol. 12, 23-30. HARMSEN R. (1966b) The excretory role of pteridines in insects. J. exp. Biol. 45, 1-13. REMBOLD H. and GUTENSOHN W. (1968) 6-Hydroxylation of the pteridine ring by xanthine oxidase. Biochem. biophys. Res. Comm. 31, 837-841. WATT W. B. (1967) Pteridine biosynthesis in the butterfly Co&as eurythe-me. J. biol. Chem. 242, 565-572. ZIEGLER I. and HARMSEN R. (1969) The biology of pteridines in insects. Adw. Insect Physiol. 6. 139-203.
in Great Britain
THE EFFECT OF ATMOSPHERIC OXYGEN PRESSURE ON THE BIOSYNTHESIS OF SIMPLE PTERINES IN PIERID BUTTERFLIES R. HARMSEN* Zoology Department, University College, Nairobi, Kenya (Received 16 June 1969) Abstract-The very abundant simple pterines of some groups of insects are metabolic end-products of C-6 substituted, hydrogenated pterines. Two alternate synthetic pathways exist, both starting from 7,8-dihydropterin. At high oxygen pressure this substance is dehydrogenated to pterin and oxidized to isoxanthopterin. At low oxygen pressure, dihydropterin is hydroxylated enzymically to dihydroxanthopterin and subsequently gives rise to xanthopterin, leucopterin, and the C-7 substituted pterines such as erythropterin. The pierid butterfly, Mylothris chloris, appears not to have an excretion barrier against pterin (an unnatural substance). This results in the appearance of large quantities of this material in the excreta of animals reared at very high oxygen pressure. Simultaneously the quantity of pterines deposited in the wings is much reduced. INTRODUCTION BECKER (1937) investigated the effect of oxygen pressure on the deposition of xanthopterin in the wings of the pierid butterfly, Gonapteryx rhamni, and reported a progressively reduced deposition of the yellow pigment with higher oxygen pressure. At 100 per cent 0, no yellow pigment is deposited, and the normally yellow butterfly is off-white (the author has recently seen some of Becker’s original specimens, now in the possession of Professor Schopf). It appears as if the total pterine content of the wings is much reduced, but a certain transition from yellow to white pigment is possible. If indeed the xanthopterin is not or only partly replaced by a white substance in the wings, it is not known if the xanthopterin (or other pterine) is stored in the fat body or excreted with the meconium, or if a reduced quantity of pterines is synthesized at high 0, pressure. The quantitative aspects of this situation have a direct bearing on recent work by the present author (HARMSEN, 1966a, b), while the qualitative aspects may throw some light on the biosynthetic relationships of the simple pterines. MATERIALS
AND METHODS
All the oxygen pressure work was done with Mylothris chloris agathina Cram. ; two other species of this genus were cursorily examined: M. yulei and M. poppea * Present Canada.
address:
Biology
Department, 2239
Queen’s
University,
Kingston,
Ontario,
2240
R. HARMSEN
tirikensis. All specimens were collected as eggs or caterpillars on epiphytic species of Loranthus in avocado pear trees in the vicinity of Nairobi. 0,-N, mixtures of known ratio were commercially obtained in pressurized form. The development chambers consisted of large (4-5 ft”) polythene bags filled under slight pressure with the desired gas mixture and kept taut throughout the experiment. The pupae were placed in the chambers within 12 hr of the larval-pupal ecdysis (before pterine synthesis commenced) and kept in the chambers until adult emergence. Wings were extracted in 1 ml 1 y0 HCl at approximately 80°C for a few seconds. Whole bodies were homogenized in a 0.75 ml chloroform-O.75 ml 1% HCI mixture as described earlier (HARMSEN,1966a). Meconium was dissolved by boiling in 1 ml HCl for about 1 min. Wing extracts, aqueous supernatant parts of body homogenates, and meconium solutions were chromatographed on Whatman No. 1 paper descending in two directions. The first-dimension solvent system was ethanol-ammonia 1% (7 : 3), and the second-dimension solvent was 4% sodium citrate in distilled water. The chromatograms were viewed in U.V. light of 365 rnp, and the intensity of fluorescence of the various substances was taken as a rough quantitative measure. RESULTS The three species of Mylothris are qualitatively similar in pterine content; they differ only in relative quantities and pigment distribution. The dominant orange pigment is erythropterin, although smaller quantities of unidentified yellow and orange fluorescing substances with low R, values are present. These may be di-pterines. Also one or two red ommochromes are found in minute quantities in the wings. The dominant yellow pterine is xanthopterin. A second yellow (yellow fluorescing) substance which may be a pterine is present; this substance is particularly noticeable in M. chloris and will be referred to as Y-pterin. Three white pterines are abundantly present in the wings: pterin (2-amino-4-hydroxy-pteridine), isoxanthopterin, and leucopterine. There are no measurable amounts of purines in the wings. In the bodies (mainly fat body) pterin, isoxanthopterin, leucopterin, and xanthopterin are present, as well as uric acid, xanthine, and hypoxanthine. Several minor u.v.-absorbing or fluorescing substances are present as well in trace quantities. Freshly pupated males and females of M. chloris were allowed to develop in O-5 per cent, 3 per cent, 20 per cent, 40 per cent, 80 per cent, and 99.5 per cent 0, respectively. All these animals developed into complete adults. The results were immediately recognizable as identical to Becker’s. At 80 per cent 0, an obvious reduction in orange and yellow pigmentation can be seen; at 99.5 per cent 0, the entire animal is off-white. Rough quantitative estimates of the fluorescing substances shows a possible overall reduction of total pterine content, but this cannot be established with certainty. It is, however, in the relative quantities of the different pterines that
BIOSYNTHESIS
OF SIMPLE
PTBRINBS
2241
IN PIBRID BUTTERFLIES
great differences are found (see Table 1). The dominant substances at normal or low 0, levels: xanthopterin, leucopterin, erythopterin, and Y-pterin are all but absent in animals reared at 99.5% 0,. In animals reared at 80% they are found in reduced quantities. Isoxanthopterin, and especially pterin, both minor constituents at normal 0, levels and virtually absent at reduced 0, levels, become the dominant wing pterines at very high 0, levels. TABLE
~--RELATIVE
ABUNDANCEOF
M.
MAJOR
ChlorisREARED
Percentage 0, in gas mixture
0 0 -_ -
3 20 40 80 99.5
PTBRINES
traces; 0, Not present; -, + + + , very large amount.
+++ +++ +++ +++ +-l-
+ ++ ++ -,
0,-N,
Xan
-
++ +++
INTHEWINGS
DIFFERENT
Isox
Pterin
0.5
IN
small amount;
OF
OFTHEADULTFEMALE
MIXTURES
Leuc
Ery
Y-pt
++ ++ + + + -
++ ++ + + +
+ + + + +
+ , medium
amount;
+ + , large amount;
In the bodies the same trend can be seen, but less distinctly. The purines seem to be affected little, or not at all. It is, however, interesting to note the differences in the meconium (see Table 2). The three pterines that are excreted TABLE
2-RELATIVE
ABUNDANCE REARED
Percentage 0 e in gas mixture
OF
-,
IN
THE
02-N,
MECONIUM
OF
FEMALE
Isox
Xan
Leuc
0 0
-
-
-
++
-
-
small amount;
+ , medium
M. chloris
MIXTURES
Pterin
1.5 3 40 80 0, Not present;
PTBRINES
IN DIFFERENT
0 amount;
0
+ + , large amount.
in traces at normal 0, levels: leucopterin, xanthopterin, and isoxanthopterin are absent at high 0, levels, while pterin, not present at normal 0, levels, becomes a dominant excretory product at high 0, levels. DISCUSSION
AND
CONCLUSIONS
It has been clearly established that pterines are formed in the insect from purine precursors in such a way that the initial pterines are hydrogenated in the
2242
R. HARMSEN
5, 6, 7, and 8 positions and carry a 3-C side chain in position 6 (WATT, 1967; There is little doubt that all oxidized, nonsubstituted pterines are metabolic end-products of the C-6 substituted pterines, and that C-7 substituted pterines (such as erythropterin) are secondarily supplied with a side-chain (WATT, 1967; ZIEGLERand HARMSEN,1969). It has been shown long ago that several xanthine dehydrogenase (or oxidase) preparations act on the C-7 position of the pterine-ring (BERGMANand KWIETNY, 1969). In other words, pterin is oxidized to isoxanthopterin and xanthopterin to leucopterin in reactions catalysed by this enzyme. Only recently, REMBOLDand GUTENSOHN(1968) have shown that at least some xanthine oxidases act on C-6 of 7,8-dihydrogenated pteridines. REMBOLDand GUTENSOHN(1968) postulate a complete C-6 side-chain breakdown, and the production of 7, Sdihydropterin as a ‘mother substance’ for all simple pterines and 7-substituted pterines. In Mylothris, under normal conditions, xanthopterin and leucopterin are synthesized in relatively large quantities. On the basis of the above-mentioned Rembold and Gutensohn model, 6-substituted pterines of the biopterin group would be broken down to 7, 8-dihydropterin. This latter substance is subsequently either oxidized to pterin or hydroxylated (xanthine oxidase!) to 7,8_dihydroxanthopterin. Any pterin produced (normally only a very small quantity) is subsequently hydroxylated enzymically to isoxanthopterin, while the relatively stable dihydroxanthopterin will be oxidized eventually to xanthopterin. If this final oxidation takes place in the presence of xanthine oxidase, leucopterin will be formed, otherwise xanthopterin may persist as such. Erythropterin is probably formed from either dihydroxanthopterin or from xanthopterin itself. Fig. 1 represents the various chemical biosynthetic relationships as postulated above. The present work supports this postulation and furnishes evidence for considering these reactions normal steps in the biosynthesis of simple pterines. Increased 0, pressure appears to favour a probably non-enzymic-oxidation of the very labile dihydropterin to pterin. Xanthine oxidase activity will transfer a certain amount of this pterin into isoxanthopterin. At very high 0s levels virtually no dihydroxanthopterin will be formed, and thus no xanthopterin, no leucopterin, and no erythropterin. It is not fully understood why males show the above-discussed trend less distinctly than females, nor why the fat body content is less affected by 0, pressure than the wing pigments. However, it was observed that gas exchange (i.e. atmospheric 0, consumption) is high in the first hours after pupation and during the last 1.5 to 2 days of pharate adult development. In the period in between, pupae will live and develop at a normal rate in pure N,, any gas exchange during this period seems to consist of CO, expulsion only. It is suggested that only after the breakdown of the pupal cuticle has commenced (i.e. during the period of maximal pterine deposition and/or synthesis in the wing) does external 0, pressure have a physiological effect on the animal, and especially in the external tissues. The reduced effect of 0, in the male may be related to a lower gas exchange in an animal which does not build an egg mass. cf. ZIEGLER and HARMSEN,1969).
BIOSYNTHESIS OF SIMPLEPTERINESIN PIERIDBUTTERFLIES
2243
The abnormally high amount of pterin in the meconium of animals reared high 0, concentrations may reflect the lack of a selective excretion threshold barrier in the Malpighian tubules. Such an excretion barrier was described leucopterin and isoxanthopterin in Pieris by HARMSEN (1966a) and appears C-6 Substituted
in or for to
Pterines
part dehydrogenation
and side chain elimination
OH J
7,8-Dihydroxanthopterin
Pterin
Pterines OH
xanthine
dehydrogenase
H H xanthine
dehydrogenase \
Isoxanthopterin \
OH
\
OH \ Leucopterin
FIG. 1. Biosynthetic pathways of simple pterines, as postulated for insects; based on in vitro studies of GUTENSOHN (1968), and oxygen pressure effects of pterine synthesis in M. chloris.
exist for the natural pterines in Myloth~is as well. Pterin, not a naturally occurring substance, appears to be freely excretable; an excretion barrier, favouring storage over excretion, has not developed for this substance. The apparent loss of pterine content of the wings of Becker’s oxygen-reared Gonapteryx is probably also based on an extensive pterin excretion as well as deposition of isoxanthopterin and pterin instead of xanthopterin. Acknowledgements-I wish to record my gratitude to Mr. R. C. CARCASSON, Director of the Nairobi Museum, for identifying the species of Mylotkris and to Miss I. JABBALfor her excellent technical assistance. The work was supported by a Rockefeller Foundation grant to the Science Faculty, University College, Nairobi.
2244
R. HARMSEN REFERENCES
BECKER E. (1937) Uber das Pterinpigment
bei Insekten und die Farbung und Zeichnung von Vespa im Besonderen. 2. Morph. c)kol. Tiere 32, 672-751. BERGMAN F. and KWIETNY H. (1969) Pteridines as substrates of mammalian xanthine oxidase-II. Pathways and rates of oxidation. Biochim. biophys. Acta 33, 2946. GUTENSOHN W. (1968) Chemische und biochemische Untersuchungen zum Abbau von Tetrahydroneopterin. Thesis, Ludwig-Max. University, Miinchen. HARMSEN R. (1966a) Identification of fluorescing and ultra-violet-absorbing substances in Pieris brassicae L. J. Insect Physiol. 12, 23-30. HARMSEN R. (1966b) The excretory role of pteridines in insects. J. exp. Biol. 45, 1-13. REMBOLD H. and GUTENSOHN W. (1968) 6-Hydroxylation of the pteridine ring by xanthine oxidase. Biochem. biophys. Res. Comm. 31, 837-841. WATT W. B. (1967) Pteridine biosynthesis in the butterfly Co&as eurythe-me. J. biol. Chem. 242, 565-572. ZIEGLER I. and HARMSEN R. (1969) The biology of pteridines in insects. Adw. Insect Physiol. 6. 139-203.