Journal of Chemical Ecology, Vol. 13, No. 8, 1987
PRESENCE OF PTERIN PIGMENTS IN WINGS OF LIBYTHEIDAE BUTTERFLIES
O A K L E Y SHIELDS I 4890 Old Highway Mariposa, California 95338
Abstract--A few pterin pigments were discovered in the wings of six of eight libytheid species tested, using thin-layer chromatography with 1% HCI in butanol as a solvent. A 10X sample of Libytheana bachmanii larvata (Strecker) produced xanthopterin, isoxanthopterin, erythropterin, and leucopterin. Leucopterin was absent in the other libytheids tested. Morphology and pterin pigment data from wings suggest a Pieridae ancestry for the Libytheidae from the region of the northern Neotropical realm (including the Greater Antilles). Key Words--Pterin pigments, Libythea, Libytheana, Libytheidae, Lepidoptera, thin-layer chromatography.
INTRODUCTION The isolated family Libytheidae (two genera: Libythea, Old World; Libytheana, New World) shows a morphological relationship to both the Nymphalidae (adults) and the Pieridae (immatures) but not to Riodinidae (Jordan, 1925; Kristensen, 1976; Shields, 1974). It was decided to test for the presence of pterin pigments in libytheid wing scales, since these pigments are universally distributed in pierid wings but have been shown to be lacking in the wings of all other butterfly families except erythropterin in the nymphalid Heliconius (Heliconiinae) and isoxanthopterin in Nymphalidae, some Lycaenidae, one Riodinidae, and one Papilionidae (Ford, 1947a,b; Baust, 1967; Rawson, 1968; Robinson, 1971). If Libytheidae is a phylogenetically transitional family between Pieridae (primitive) and Nymphalidae (advanced), as its morphology indicates, it might still contain an array of pterins, at least in trace amounts. Research Associate in Entomology, Los Angeles County Museum of Natural History. 1843 0098-0331/87/0800q843505.00/0
9 Plenum Publishing Corporation
1844
Sm~LDS
In Pieridae wings, pterins are deposited on the canal walls in the scales during the pupal and teneral adult stages and give the white, yellow, and red coloration to the wings (Wigglesworth, 1939; Ziegler-Gunder, 1956). Since pterins are chemically related to uric acid, they are probably excretory products transported by the hemolymph (Ford, 1947a; Ziegler-Gunder, 1956). Small amounts of uric acid and other nitrogenous breakdown products also occur in Pieridae wings (Gates, 1947; Hannsen, 1966). Pieridae differs dramatically in the origin and structure of its wing-scale pigments from the other butterfly families (Ford, 1947a). In insects, pterins are reported in the bodies of various Rhopalocera and Geometridae, Ascalaphus (Neuroptera), Gaema (Cicadidae), Tettigonia (jassid), various Hemiptera, Vespa (Hymenoptera), some Diptera, and Bombyx (Bombycidae) (Bartel et al., 1958; Rawson, 1968). Thus, within the Pterygota, pterins are present in some of the advanced infraclasses of Paraneoptera (hemipteroids) and Oligoneoptera (Coleoptera, Neuroptera, Lepidoptera, etc.) but are lacking in the more primitive infraclasses of Paleoptera (Ephemerida and Odonata) and Polyneoptera (orthopteroids) (see Fox and Fox, 1964, pp. 337339). METHODS AND MATERIALS An attempt to identify pterins from eight of the 12 species of libytheids was made by removing both wings from one side of an individual specimen of each and macerating them in 1% HC1. The mixture was centrifuged twice at 5000 rpm for 7 min. The decanted supematent was air-dried in test tubes, redissolved in 1% HC1, and 13-33/xl spotted on a Baker-Flex thin layer cellulose plate. The solvent of l-butanol-acetic acid-water (4 : 1 : 1) follows Watt (1964) and Pfeiler (1970). Two wings of Eurema nicippe (Cramer) were similarly extracted and 10/xl spotted as above, as the control on the same plate. The TLC plate was allowed to equilibrate against the butanol solvent vapor for 1 hr. Then the butanol solvent was added and allowed to migrate up the plate for 3-4~ hr in a chromatography tank. The 1% HC1 in butanol was found to yield a far greater number of pterins than when 1% NH4OH in butanol was used. Spots from the libytheid species were matched to the control in color and Rf values under UV light after drying. RT values and colors under UV follow Harmsen (1966) and Pfeiler (1970). The pterin spots were located under short wavelength UV light (2000 A) for xanthopterin and under long wavelength UV light (ca. 4000 A) for the other pterins. Because unusual amounts of xanthopterin occur in Libytheana bachmanii larvata Strecker from Ciudad Victoria, Mexico, it was decided to further isolate the pterins from this population by removing the wings from five specimens (22.1 mg), or approximately ten times the sample size of the other species
PTERIN PIGMENTS IN BUTTERFLIES
1845
tested. The same procedures were used (93/zl spotted), with Pieris rapae (Linnaeus) as a control using the RUvalues for its pterins in Pfeiler (1970), except this time propanol solvent (1-propanol-1% aqueous ammonia, 2: 1) was mn in one direction while the butanol solvent was run at 90 ~ on the same plate. Finally, the spots isolated under UV were scraped off and run separately on another TLC plate in the butanol solvent only.
RESULTS
Pterins were entirely absent in the western Old World species Libythea labdaca Westwood (Uganda), L. laius Trimen (South Africa), and L. celtis celtis Fuessly (Spain). However, xanthopterin was present in Libythea narina rohini Marshall (Thailand), which lacked other pterins, and in Libytheana bachmanii bachmanii, Libythea myrrha, and L. geoffroy. Erythropterin was faintly present in Libythea geoffroy orientalis Godman & Salvin (Guadalcanal) but appeared fairly prominently in L. celtisformosana Fruhstorfer (Taiwan), L. myrrha sanguinalis Fruhstorfer (Thailand), Libytheana carinenta Cramer (Brazil), and L. b. bachmanii Kirtland (Tennessee). Leucopterin was not detected in any of the libytheids, and isoxanthopterin was absent in all except L. celtis formosana for this sample size. The larger sample of Libytheana bachmanii larvata (three males, two females) collected in August 1937 at Ciudad Victoria, Mexico, contained a very prominent amount of xanthopterin (yellow green under UV of 2000 A , Rf = 0.42) which decomposed to uric acid after several weeks, prominent leucopterin (pale blue, RU = 0.06-0.08, 2000 A), and a faint spot of isoxanthopterin (blue under UV of ca. 4000 A , Rf = 0.27). Erythropterin (RI = 0.05) was also present but sepiapterin was absent. Xanthopterin and sepiapterin have identical Rf values in butanol but not in propanol. The Ciudad Victoria L. b. larvata wings had brown mottling on a whitish background on the undersurface, while L. b. bachmanii's underside contained much less white and more brown, also reflected in the pterin results.
DISCUSSION
Pterin pigments present in the wings of most libytheids strengthen a possible tie with Pieridae that is also suggested on morphological grounds. This ancestral type may have had a yellow (prominent), white, and red wing pattern judging by the pterin results for L. b. larvata. The Cuban Libytheana motya Hubner (not tested here) is likely the most primitive libytheid because of its extensive white coloration; it appears related to nearby L. bachmanii and L. terena Godart, but not to L. carinenta. The next
1846
SHIELDS
least advanced libytheids are probably the worldwide brown forms (L. bachmanii, L. terena, L. carinenta, L. fulvescens Lathy, L. laius, L. celtis, L. myrrha) with L. labdaca, L. narina, and L. geoffroy the most advanced black (and iridescent) forms. This scheme is substantiated by the pterin pigment results presented here and the phylogeny of their Celtis host plants (Shields, 1986). Thus L. motya is the most pierid-like libytheid with its white coloration, while at the opposite end of the spectrum L. geoffroy is the most nymphalid-like. This trend from primitive to advanced also included a corresponding shortening of their elongated labial palpi. New World Libytheana genitalia are closest to pierids, while Old World Libythea genitalia are closer to nymphalids (Shields, 1985, 1986). It is noteworthy that African Mylothris, one of the pierids probably close to libytheids in relationship, contains a very prominent amount of xanthopterin (Harmsen, 1970), as did L. b. larvata. Another is Melete (Neotropical, 13 species) which approaches Libytheana in genitalia and palpi. Both Mylothris and Melete use Loranthaceae as larval food plants, a family occasionally parasitic on Celtis (Celtidaceae), the food plant of libytheids. Earliest Celtis fossils date from the uppermost Cenomanian 92-94 million years ago (lower Upper Cretaceous time) (Shields, 1986).
CONCLUSION
Six of eight Libytheidae species tested by TLC contained pterin pigments in their wings: xanthopterin (four species), erythropterin (five species), and isoxanthopterin (one species). Few or no pterins were found in the most advanced species. A 10-fold sample of Libytheana bachmanii larvata wings from northeastern Mexico contained large amounts of xanthopterin and leucopterin, lesser amounts of erythropterin and isoxanthopterin, and no sepiapterin. Only the butterfly family Pieridae is known so far to contain such an array of pterin pigments in their wings. The findings help to strengthen the ties between the Pieridae and Libytheidae that had been suggested on morphological and behavioral grounds. The Libytheidae, a transitional family between the Pieridae and Nymphalidae, probably arose from Pieridae (e.g., Melete) in the northern Neotropical realm no earlier than lower Upper Cretaceous times, the earliest age of its Celtis host-plant fossils. Acknowledgments--Dr. C.L. Judson (University of Caiifomia, Davis) kindly provided taboratory equipment, research facilities, and helpful advice. J.P. Donahue (Los Angeles County Museum) donated six of the species used in these experiments. I also wish to thank Drs. A.M. Shapiro and S.R. Sims for lively discussions, Dr. J.A. Scott and the two reviewers for criticisms of the manuscript, and J. Lackner for laboratory assistance.
PTERIN PIGMENTS IN BUTTERFLIES
1847 REFERENCES
BARTEL, A.H., HUDSON,B.W., and CRAIG, R. 1958. Pteridines in the milkweed bug, Oncopeltus fasciatus (Dallas) I. Identification and localization. J. Insect. Physiol. 2:348-354. BAUST, J.G. 1967. Preliminary studies on the isolation of pterins from the wings of heliconiid butterflies. Zoologica (N. E) 52:15-20. FORD, E.B. 1947a. A murexide test for the recognition of pterins in intact insects. Proc. R. Entomol. Soc. London (A) 22:72-76. FORD, E.B. 1947b. Studies on the chemistry of pigments in the Lepidoptera, with reference to their bearing on systematics. 5. Pseudopontia paradoxa Felder. Proc. R. Entomol. Soc. London (A) 22:77-78. Fox, R.M., and Fox, J.W. 1964. Introduction to Comparative Entomology. Reinhold, New York. GATES, M. 1947. The chemistry of the pteridines. Chem. Rev. 41:63-95. HAR~SEN, R. 1966. Identification of fluorescing and UV absorbing substances in Pieris brassicae L. J. Insect Physiol. 12:23-30. HARMSEN,R. 1970. Biosynthesis of simple pterins in insects, pp. 405-411, in K. Iwai, A. Akino, M. Goto, and Y. Iwanami (eds.). Chemistry and Biology of Pteridines. International Academic Printing Co., Tokyo. JORDAN, K. 1925. Uber die Stellung der Tagfaltergattung Libythea. Proceedings of the Third International Entomology Congress, Zurich, pp. 361-366. KRISTENSEN,N.P. 1976. Remarks on the family-level phylogeny of butterflies. Z Zool. Syst. Evol.Forsch. 14:25-33. PFEILER, E.J., JR. 1970. The effect of pterin pigments on wing coloration of four species of Pieridae. J. Res. Lepid. 7:183-189. RAWSON, G.W. 1968. Study of fluorescent pigments in Lepidoptera by means of paper partition chromatography. J. Lepid Soc. 22:27-40. ROBINSON, R. 1971. Lepidoptera Genetics. Pergamon Press, Oxford. SHmLDS, O. 1974. Toward a theory of butterfly migration. J. Res. Lepid. 13:217-238. SHmLDS, O. 1985. Zoogeography of the Libytheidae (snouts or beaks). Tokurana 9:1-58. SHtELDS, O. 1986. Sequential evolution of Celtis (hackberries, Celtidaceae) and Libytheidae. Ann. Missouri Bot. Gard. In review. WATT, W.B. 1964. Pteridine components of wing pigmentation in the butterfly Colias eurytheme. Nature 201:1326-1327. W~GGLESWORTH, V.B. 1939. The Principles of Insect Physiology. E.P. Dutton, New York. ZIEGLER-GUNDER,I. 1956. Pterine: Pigmente und Wirkstoffe im Tierreich. Biol. Rev. 31:313-348.
PRESENCE OF PTERIN PIGMENTS IN WINGS OF LIBYTHEIDAE BUTTERFLIES
O A K L E Y SHIELDS I 4890 Old Highway Mariposa, California 95338
Abstract--A few pterin pigments were discovered in the wings of six of eight libytheid species tested, using thin-layer chromatography with 1% HCI in butanol as a solvent. A 10X sample of Libytheana bachmanii larvata (Strecker) produced xanthopterin, isoxanthopterin, erythropterin, and leucopterin. Leucopterin was absent in the other libytheids tested. Morphology and pterin pigment data from wings suggest a Pieridae ancestry for the Libytheidae from the region of the northern Neotropical realm (including the Greater Antilles). Key Words--Pterin pigments, Libythea, Libytheana, Libytheidae, Lepidoptera, thin-layer chromatography.
INTRODUCTION The isolated family Libytheidae (two genera: Libythea, Old World; Libytheana, New World) shows a morphological relationship to both the Nymphalidae (adults) and the Pieridae (immatures) but not to Riodinidae (Jordan, 1925; Kristensen, 1976; Shields, 1974). It was decided to test for the presence of pterin pigments in libytheid wing scales, since these pigments are universally distributed in pierid wings but have been shown to be lacking in the wings of all other butterfly families except erythropterin in the nymphalid Heliconius (Heliconiinae) and isoxanthopterin in Nymphalidae, some Lycaenidae, one Riodinidae, and one Papilionidae (Ford, 1947a,b; Baust, 1967; Rawson, 1968; Robinson, 1971). If Libytheidae is a phylogenetically transitional family between Pieridae (primitive) and Nymphalidae (advanced), as its morphology indicates, it might still contain an array of pterins, at least in trace amounts. Research Associate in Entomology, Los Angeles County Museum of Natural History. 1843 0098-0331/87/0800q843505.00/0
9 Plenum Publishing Corporation
1844
Sm~LDS
In Pieridae wings, pterins are deposited on the canal walls in the scales during the pupal and teneral adult stages and give the white, yellow, and red coloration to the wings (Wigglesworth, 1939; Ziegler-Gunder, 1956). Since pterins are chemically related to uric acid, they are probably excretory products transported by the hemolymph (Ford, 1947a; Ziegler-Gunder, 1956). Small amounts of uric acid and other nitrogenous breakdown products also occur in Pieridae wings (Gates, 1947; Hannsen, 1966). Pieridae differs dramatically in the origin and structure of its wing-scale pigments from the other butterfly families (Ford, 1947a). In insects, pterins are reported in the bodies of various Rhopalocera and Geometridae, Ascalaphus (Neuroptera), Gaema (Cicadidae), Tettigonia (jassid), various Hemiptera, Vespa (Hymenoptera), some Diptera, and Bombyx (Bombycidae) (Bartel et al., 1958; Rawson, 1968). Thus, within the Pterygota, pterins are present in some of the advanced infraclasses of Paraneoptera (hemipteroids) and Oligoneoptera (Coleoptera, Neuroptera, Lepidoptera, etc.) but are lacking in the more primitive infraclasses of Paleoptera (Ephemerida and Odonata) and Polyneoptera (orthopteroids) (see Fox and Fox, 1964, pp. 337339). METHODS AND MATERIALS An attempt to identify pterins from eight of the 12 species of libytheids was made by removing both wings from one side of an individual specimen of each and macerating them in 1% HC1. The mixture was centrifuged twice at 5000 rpm for 7 min. The decanted supematent was air-dried in test tubes, redissolved in 1% HC1, and 13-33/xl spotted on a Baker-Flex thin layer cellulose plate. The solvent of l-butanol-acetic acid-water (4 : 1 : 1) follows Watt (1964) and Pfeiler (1970). Two wings of Eurema nicippe (Cramer) were similarly extracted and 10/xl spotted as above, as the control on the same plate. The TLC plate was allowed to equilibrate against the butanol solvent vapor for 1 hr. Then the butanol solvent was added and allowed to migrate up the plate for 3-4~ hr in a chromatography tank. The 1% HC1 in butanol was found to yield a far greater number of pterins than when 1% NH4OH in butanol was used. Spots from the libytheid species were matched to the control in color and Rf values under UV light after drying. RT values and colors under UV follow Harmsen (1966) and Pfeiler (1970). The pterin spots were located under short wavelength UV light (2000 A) for xanthopterin and under long wavelength UV light (ca. 4000 A) for the other pterins. Because unusual amounts of xanthopterin occur in Libytheana bachmanii larvata Strecker from Ciudad Victoria, Mexico, it was decided to further isolate the pterins from this population by removing the wings from five specimens (22.1 mg), or approximately ten times the sample size of the other species
PTERIN PIGMENTS IN BUTTERFLIES
1845
tested. The same procedures were used (93/zl spotted), with Pieris rapae (Linnaeus) as a control using the RUvalues for its pterins in Pfeiler (1970), except this time propanol solvent (1-propanol-1% aqueous ammonia, 2: 1) was mn in one direction while the butanol solvent was run at 90 ~ on the same plate. Finally, the spots isolated under UV were scraped off and run separately on another TLC plate in the butanol solvent only.
RESULTS
Pterins were entirely absent in the western Old World species Libythea labdaca Westwood (Uganda), L. laius Trimen (South Africa), and L. celtis celtis Fuessly (Spain). However, xanthopterin was present in Libythea narina rohini Marshall (Thailand), which lacked other pterins, and in Libytheana bachmanii bachmanii, Libythea myrrha, and L. geoffroy. Erythropterin was faintly present in Libythea geoffroy orientalis Godman & Salvin (Guadalcanal) but appeared fairly prominently in L. celtisformosana Fruhstorfer (Taiwan), L. myrrha sanguinalis Fruhstorfer (Thailand), Libytheana carinenta Cramer (Brazil), and L. b. bachmanii Kirtland (Tennessee). Leucopterin was not detected in any of the libytheids, and isoxanthopterin was absent in all except L. celtis formosana for this sample size. The larger sample of Libytheana bachmanii larvata (three males, two females) collected in August 1937 at Ciudad Victoria, Mexico, contained a very prominent amount of xanthopterin (yellow green under UV of 2000 A , Rf = 0.42) which decomposed to uric acid after several weeks, prominent leucopterin (pale blue, RU = 0.06-0.08, 2000 A), and a faint spot of isoxanthopterin (blue under UV of ca. 4000 A , Rf = 0.27). Erythropterin (RI = 0.05) was also present but sepiapterin was absent. Xanthopterin and sepiapterin have identical Rf values in butanol but not in propanol. The Ciudad Victoria L. b. larvata wings had brown mottling on a whitish background on the undersurface, while L. b. bachmanii's underside contained much less white and more brown, also reflected in the pterin results.
DISCUSSION
Pterin pigments present in the wings of most libytheids strengthen a possible tie with Pieridae that is also suggested on morphological grounds. This ancestral type may have had a yellow (prominent), white, and red wing pattern judging by the pterin results for L. b. larvata. The Cuban Libytheana motya Hubner (not tested here) is likely the most primitive libytheid because of its extensive white coloration; it appears related to nearby L. bachmanii and L. terena Godart, but not to L. carinenta. The next
1846
SHIELDS
least advanced libytheids are probably the worldwide brown forms (L. bachmanii, L. terena, L. carinenta, L. fulvescens Lathy, L. laius, L. celtis, L. myrrha) with L. labdaca, L. narina, and L. geoffroy the most advanced black (and iridescent) forms. This scheme is substantiated by the pterin pigment results presented here and the phylogeny of their Celtis host plants (Shields, 1986). Thus L. motya is the most pierid-like libytheid with its white coloration, while at the opposite end of the spectrum L. geoffroy is the most nymphalid-like. This trend from primitive to advanced also included a corresponding shortening of their elongated labial palpi. New World Libytheana genitalia are closest to pierids, while Old World Libythea genitalia are closer to nymphalids (Shields, 1985, 1986). It is noteworthy that African Mylothris, one of the pierids probably close to libytheids in relationship, contains a very prominent amount of xanthopterin (Harmsen, 1970), as did L. b. larvata. Another is Melete (Neotropical, 13 species) which approaches Libytheana in genitalia and palpi. Both Mylothris and Melete use Loranthaceae as larval food plants, a family occasionally parasitic on Celtis (Celtidaceae), the food plant of libytheids. Earliest Celtis fossils date from the uppermost Cenomanian 92-94 million years ago (lower Upper Cretaceous time) (Shields, 1986).
CONCLUSION
Six of eight Libytheidae species tested by TLC contained pterin pigments in their wings: xanthopterin (four species), erythropterin (five species), and isoxanthopterin (one species). Few or no pterins were found in the most advanced species. A 10-fold sample of Libytheana bachmanii larvata wings from northeastern Mexico contained large amounts of xanthopterin and leucopterin, lesser amounts of erythropterin and isoxanthopterin, and no sepiapterin. Only the butterfly family Pieridae is known so far to contain such an array of pterin pigments in their wings. The findings help to strengthen the ties between the Pieridae and Libytheidae that had been suggested on morphological and behavioral grounds. The Libytheidae, a transitional family between the Pieridae and Nymphalidae, probably arose from Pieridae (e.g., Melete) in the northern Neotropical realm no earlier than lower Upper Cretaceous times, the earliest age of its Celtis host-plant fossils. Acknowledgments--Dr. C.L. Judson (University of Caiifomia, Davis) kindly provided taboratory equipment, research facilities, and helpful advice. J.P. Donahue (Los Angeles County Museum) donated six of the species used in these experiments. I also wish to thank Drs. A.M. Shapiro and S.R. Sims for lively discussions, Dr. J.A. Scott and the two reviewers for criticisms of the manuscript, and J. Lackner for laboratory assistance.
PTERIN PIGMENTS IN BUTTERFLIES
1847 REFERENCES
BARTEL, A.H., HUDSON,B.W., and CRAIG, R. 1958. Pteridines in the milkweed bug, Oncopeltus fasciatus (Dallas) I. Identification and localization. J. Insect. Physiol. 2:348-354. BAUST, J.G. 1967. Preliminary studies on the isolation of pterins from the wings of heliconiid butterflies. Zoologica (N. E) 52:15-20. FORD, E.B. 1947a. A murexide test for the recognition of pterins in intact insects. Proc. R. Entomol. Soc. London (A) 22:72-76. FORD, E.B. 1947b. Studies on the chemistry of pigments in the Lepidoptera, with reference to their bearing on systematics. 5. Pseudopontia paradoxa Felder. Proc. R. Entomol. Soc. London (A) 22:77-78. Fox, R.M., and Fox, J.W. 1964. Introduction to Comparative Entomology. Reinhold, New York. GATES, M. 1947. The chemistry of the pteridines. Chem. Rev. 41:63-95. HAR~SEN, R. 1966. Identification of fluorescing and UV absorbing substances in Pieris brassicae L. J. Insect Physiol. 12:23-30. HARMSEN,R. 1970. Biosynthesis of simple pterins in insects, pp. 405-411, in K. Iwai, A. Akino, M. Goto, and Y. Iwanami (eds.). Chemistry and Biology of Pteridines. International Academic Printing Co., Tokyo. JORDAN, K. 1925. Uber die Stellung der Tagfaltergattung Libythea. Proceedings of the Third International Entomology Congress, Zurich, pp. 361-366. KRISTENSEN,N.P. 1976. Remarks on the family-level phylogeny of butterflies. Z Zool. Syst. Evol.Forsch. 14:25-33. PFEILER, E.J., JR. 1970. The effect of pterin pigments on wing coloration of four species of Pieridae. J. Res. Lepid. 7:183-189. RAWSON, G.W. 1968. Study of fluorescent pigments in Lepidoptera by means of paper partition chromatography. J. Lepid Soc. 22:27-40. ROBINSON, R. 1971. Lepidoptera Genetics. Pergamon Press, Oxford. SHmLDS, O. 1974. Toward a theory of butterfly migration. J. Res. Lepid. 13:217-238. SHmLDS, O. 1985. Zoogeography of the Libytheidae (snouts or beaks). Tokurana 9:1-58. SHtELDS, O. 1986. Sequential evolution of Celtis (hackberries, Celtidaceae) and Libytheidae. Ann. Missouri Bot. Gard. In review. WATT, W.B. 1964. Pteridine components of wing pigmentation in the butterfly Colias eurytheme. Nature 201:1326-1327. W~GGLESWORTH, V.B. 1939. The Principles of Insect Physiology. E.P. Dutton, New York. ZIEGLER-GUNDER,I. 1956. Pterine: Pigmente und Wirkstoffe im Tierreich. Biol. Rev. 31:313-348.
