Functions of the white and topaz loci of Lucilia cuprina in the production of the eye pigment xanthommatin

The white and topaz eye color mutants of L. cuprina are defective in the production of the brown screening pigment xanthommatin. Both white and topaz mutants were found to be unable to accumulate xanthommatin precursors in the larval malpighian tubules, correlating with their reduced early pupal level of this metabolite. In addition, white mutants showed reduced rates of accumulation of kynurenine and 3-hydroxykynurenine in the adult eyes. Another mutant strain, grape, was also defective in its ability to accumulate these xanthommatin precursors in the eyes, although accumulation was normal in the larval tubules. In contrast, the topaz mutants were found to be normal in eye accumulation, although tubule accumulation was markedly abnormal. These properties of the white and topaz mutants of L. cuprina are compared with those of the white and scarlet mutants of D. melanogaster, and it seems likely that in the two species these genes are involved with the uptake or storage of xanthommatin precursors in specific tissues.This work was supported by Grant D2 75/15248 from the Australian Research Grants Committee.     

Transport defects as the physiological basis for eye color mutants of Drosophila melanogaster

Kynurenine-H ³ transport and conversion to 3-hydroxykynurenine were studied in organ culture using the Malpighian tubules and developing eyes from wild type and the eye color mutants w, st, 1td, ca, and cn of Drosophila melanogaster. Malpighian tubules from wild type have the ability to concentrate kynurenine and convert it to 3-hydroxykynurenine. The tubules from w, st, 1td, and ca are deficient in the ability to transport kynurenine, as are the eyes of the mutants w, st, and 1td. This defect in kynurenine transport provides a physiological explanation for the phenotypic properties of the mutants. The relationship of these measurements to previous observations on these eye color mutants is discussed and the transport defect hypothesis is consistently supported. We have concluded that several of the eye color mutants in Drosophila are transport mutants.     

Ommochrome formation and kynurenine excretion in Pieris brassicae: Relation to tryptophan supply on an artificial diet

The brown-red pigment in the larval epidermis and in the testis of Pieris brassicae was identified as xanthommatin on the basis of solubility, redox behaviour, chromatography, degradation, visible and infrared spectra. In the epidermis, this pigment accumulates during the larval feeding period and disappears rapidly in the wandering stage. Larvae fed an artificial diet produce about half the amount of xanthommatin as larvae fed cabbage. This effect is caused by a lack of dietary tryptophan. Xanthommatin formation is increased by the addition of tryptophan which also increases body weight. At a tryptophan concentration of 0.2 mg per g, however, weight increase is lower than in controls and high mortality is observed. Pieris larvae excrete kynurenine in relation to dietary tryptophan. No measurable amounts are excreted in the last instar on the non-supplement diet. After feeding different quantities of tryptophan, different amounts of kynurenine are excreted only on the day following ecdysis.     

Developmental patterns of 3-hydroxykynurenine accumulation in white and various other eye color mutants of Drosophila melanogaster

Several points of biochemical similarity between white and scarlet mutants suggest that both are defective in the transport of xanthommatin precursors. In both, accumulation of 3-hydroxykynurenine is negligible during larval life and occurs at only a slow rate during adult development. Larvae of both mutants also excrete 3H-3-hydroxykynurenine and 3H-kynurenine rapidly, which probably accounts for the normal levels of kynurenine during larval life. 3-Hydroxykynurenine levels are abnormal in all white mutants which were studied, although in two alleles which are strongly pigmented (w(sat) and w(col)) accumulation is enhanced rather than diminished. In w(a), larval accumulation is normal but accumulation during adult development is greatly diminished, suggesting that this mutation has a tissue-specific effect. Similar levels were found in zeste females. Of the 11 other eye color mutants tested, abnormal levels of 3-hydroxykynurenine were found in eight. In four of these (claret, light, lightoid, and pink), larval accumulation is negligible, suggesting that these have defects in the kynurenine transport system like scarlet and white. In three others, however (brown, karmoisin, and rosy), accumulation during larval life is enhanced. In cardinal accumulation is normal during larval life but is excessive during adult development. This evidence supports the suggestion that the cd mutation blocks the final step of xanthommatin synthesis.     

Transient expression of the Drosophila melanogaster cinnabar gene rescues eye color in the white eye (WE) strain of Aedes aegypti

The lack of eye pigment in the Aedes aegypti WE (white eye) colony was confirmed to be due to a mutation in the kynurenine hydroxylase gene, which catalyzes one of the steps in the metabolic synthesis of ommochrome eye pigments. Partial restoration of eye color (orange to red phenotype) in pupae and adults occurred in both sexes when first or second instar larvae were reared in water containing 3-hydroxykynurenine, the metabolic product of the enzyme kynurenine hydroxylase. No eye color restoration was observed when larvae were reared in water containing kynurenine sulfate, the precursor of 3-hydroxykynurenine in the ommochrome synthesis pathway. In addition, a plasmid clone containing the wild type Drosophila melanogaster gene encoding kynurenine hydroxylase, cinnabar (cn), was also able to complement the kynurenine hydroxylase mutation when it was injected into embryos of the A. aegypti WE strain. The ability to complement this A. aegypti mutant with the transiently expressed D. melanogaster cinnabar gene supports the value of this gene as a transformation reporter for use with A. aegypti WE and possibly other Diptera with null mutations in the kynurenine hydroxylase gene.     

A biochemical study of the scarlet eye-color mutant of Drosophila melanogaster

3-Hydroxykynurenine is virtually absent from st larvae but accumulates during adult development in the puparium. Over the period of adult emergence, the accumulated 3-hydroxykynurenine is excreted so that st adults contain none. Larvae of st fed on tryptophan-C ¹⁴ medium produce labeled 3-hydroxykynurenine, at a reduced rate, perhaps, compared to wild type. Xanthurenic acid levels in st pupae are similar to those in wild type. Thus the failure of st larvae to accumulate 3-hydroxykynurenine does not seem to be due either to an inability to synthesize this compound or to an excessive rate of its conversion to xanthurenic acid. Rather, it appears that the mechanism of 3-hydroxykynurenine storage during larval life is defective, so that this compound is excreted at an abnormally high rate. The inability of the pigment cells of the eyes of st to synthesize xanthommatin may result from a similar defect in their ability to take up or store 3-hydroxykynurenine.     

Nonshivering thermogenesis and cold resistance during seasonal acclimatization in the Djungarian hamster

Summary The absence of juvenile hormone (JH) at the time of head capsule slippage during the molt to the fifth (final) instar of the tobacco hornworm was found to cause ommochrome (primarily dihydroxanthommatin) synthesis in the epidermis during the first two days after ecdysis. Then synthesis decreased until its transient reappearance during the wandering stage. Either JH-I (ED₅₀=8x10^–4 g) or methoprene (ED₅₀=1.4x10^–2 g) applied at this critical time during the molt prevented the first synthesis. A comparison of developmental profiles of tryptophan and its metabolites, kynurenine and 3-hydroxykynurenine, in normal and allatectomized wild type larvae showed that JH at this critical time prevented both the conversion of kynurenine to 3-hydroxykynurenine and 3-hydroxykynurenine to ommochromes. A similar study in normal and methoprene-treatedblack mutant larvae showed that only the latter conversion was inhibited by JH. The accumulation of 3-hydroxykynurenine in the epidermis of the JH-treatedblack mutant is thought to be due to the altered tryptophan metabolism in these mutants in previous instars due to lower JH levels. Neither starvation of theblack mutant nor injection of 3-hydroxykynurenine significantly affected ommochrome synthesis by the epidermis. Preliminary studies of the enzymes involved showed that JH at the critical period suppressed the later activity and/or production of kynurenine 3-hydroxylase in the wild type larva, but had little effect on the particulate ommochrome synthetase activity of the epidermis.Abbreviations CA corpora allata - JH juvenile hormone - PTTH prothoracicotropic hormone     

GENETIC CONTROL OF CYTODIFFERENTIATION

The cells of the anterior region of the larval fatbody of Drosophila melanogaster accumulate kynurenine at the end of the third larval instar, whereas the cells of the posterior region are involved in pteridine metabolism. Through a series of transplantation experiments it has been demonstrated that the anterior fat cells synthesize kynurenine. The mutant vermilion lacks kynurenine, and the anterior fat cells of this mutant strain lack the autofluorescence characteristic of kynurenine. When the non-allelic suppressor gene is combined with vermilion, the synthesis of kynurenine is restored in the anterior fat cells, and some of the cells of the posterior region contain kynurenine as well. A similar extension in the number of cells containing kynurenine can be induced in the normal Ore-R strain by feeding the precursor tryptophan. It has been concluded that the absence of a physiological process in a differentiated cell does not necessarily represent a loss of the genetic potential for that process. The normal allele at the suppressor locus inhibits the occurrence of kynurenine in the posterior fat cells, whereas the mutant allele su²-s allows the expression of this potential. An inducer such as tryptophan can overcome this inhibition in the normal strain, and as a result the cells which are normally differentiated as "isoxanthopterin cells" may produce kynurenine as well.     

Terminal synthesis of xanthommatin in Drosophila melanogaster. I. Roles of phenol oxidase and substrate availability

Eye color mutants of Drosophila melanogaster are known which block the conversion of 3-hydroxykynurenine to xanthommatin. It has been proposed that this reaction depends on the presence of 3-hydroxykynurenine and a redox system maintained by phenol oxidase activity. The mutants st and ltd lack throughout development detectable amounts of 3-hydroxykynurenine or its metabolic derivatives. When the substrate is fed or injected, these mutants fail to form xanthommatin even though phenol oxidase activity is normal. The mutant cd accummulates excessive amounts of 3-hydroxykynurenine, has normal phenol oxidase activity, but is also deficient in xanthommatin formation. Mutants are also known which lack phenol oxidase activity but nevertheless form xanthommatin. It is concluded that the proposed relationship between 3-hydroxy-kynurenine and phenol oxidase activity is not sufficient to explain the in vivo synthesis and regulation of synthesis of xanthommatin in Drosophila. The bearing of these findings on the actual mode of synthesis is discussed.Supported by PHS 1029 and NSF GB-4539.     

Genetic and functional analysis of tryptophan transport in Malpighian tubules of Drosophila

Dissected Malpighian tubules from wild type and the eye color mutant white of Drosophila were compared with respect to their abilities to transport tryptophan and kynurenine into tubule cells. It was determined that mutation at white greatly impairs the ability of Malpighian tubule cells to take up tryptophan. Functional studies on the extracellular spaces and ultrastructural observations indicated no differences in these respects between wild type and white tubules. It is consistent with several observations that much of the tryptophan associated with white exists in the intercellular spaces. Furthermore, the uptake of tryptophan by the w ⁺ system of wild type tubules is inhibited by the analogue 5-methyl-tryptophan. However, the incorporation of radioactive tryptophan into protein in tubule cells from wild type and white occurs at the same rates and is not affected by 5-methyl-tryptophan. Therefore, it is apparent that Malpighian tubules have a transport system that enables entry of tryptophan into a cellular pool and that this cellular pool is initially independent of the tryptophan pool used for protein synthesis. The mutant white lacks this transport system. From these studies and others it appears that compartmentalization of cellular pools may be brought about via the utilization of specific membrane transport systems.     

Tryptophan metabolism during development of the stick insect,Carausius morosus Br. tissue distribution and interrelationships of metabolites of the kynurenine pathway

Summary During larval development ofCarausius morosus kynurenic acid is the major end product of tryptophan metabolism. Tryptophan and kynurenic acid have been found in the fat body, haemolymph and gut contents but only traces of kynurenine have been detected. The ommochromes ommin and xanthommatin are formed in relatively small amounts in the epidermis during larval development. 3-hydroxykynurenine was found only in the epidermis, the site of ommochrome deposition.During larval development, the amount of free tryptophan increases with body dry weight. The amount of kynurenic acid excreted also corresponds to the increase of body weight but is significantly reduced in the faeces of adults. This is related to a high tryptophan content of yolk proteins. The concentration of tryptophan in the haemolymph decreases immediately before ecdysis, whereas that in the gut increases during this time and falls sharply at the start of ecdysis.     

MUTANT GENES REGULATING THE INDUCIBILITY OF KYNURENINE SYNTHESIS

Alterations in the cellular synthesis of kynurenine in the larval fatbody of Drosophila melanogaster may be obtained by feeding the precursor tryptophan or by changing the genotype. In the wild type Ore-R strain, autofluorescent kynurenine globules normally occur in the cells in the anterior regions of the fatbody designated as regions 1, 2, and 3. When tryptophan is included in the larval diet, kynurenine will develop throughout the entire fatbody, thus extending to the cells in regions 4, 5, and 6. In the fatbodies of both the sepia mutant strain and the mutant combinations of the suppressible vermilion alleles with the suppressor gene (su²-s, v¹ and su²-s, v²), kynurenine is found in the cells from region 1 through region 4. This involvement of additional cells in the synthesis of kynurenine occurs under the usual culture conditions for Drosophila. When sepia larvae are fed tryptophan, kynurenine appears in all of the cells of the fatbody. However, dietary tryptophan does not induce kynurenine production in cells in regions 5 and 6 in the mutant combination su²-s, v¹ or su²-s, v². In the latter strains, an increase in the quantity of kynurenine in the fatbody is detected, but this increase remains limited to the same cells in which kynurenine production is found under normal feeding conditions. When the v^36f allele is combined with the su²-s allele, an extremely faint autofluorescence characteristic of kynurenine is found in some of the anteriormost fat cells of regions 1 and 2. This autofluorescence becomes intensified when tryptophan is fed to su²-s, v^36f larvae. The genetic control of kynurenine synthesis in the cells of the fatbody of Drosophila melanogaster has been previously demonstrated. The present observations establish genetic regulation of the ability to induce kynurenine production within a cell through the administration of the inducer tryptophan. Kynurenine production has been considered as a unit function of the cell as a whole rather than of the enzyme alone, and it has been concluded that even though cells in different parts of the body perform this same function (kynurenine production), the gene loci regulating this function may be different for cells in different regions of the body. A phenomenon of overlapping domains of gene actions at the cellular level offers a genetic and cellular basis for developmental and physiological homeostasis.     

Oxidation of 3-hydroxykynurenine to produce xanthommatin for eye pigmentation: a major branch pathway of tryptophan catabolism during pupal development in the yellow fever mosquito, Aedes aegypti.

This study concerns the metabolic pathways of 3-hydroxykynurenine in Aedes aegypti mosquitoes during development with emphasis on its oxidation pathway to produce xanthommatin during eye pigmentation. Oxidation of tryptophan to 3-hydroxykynurenine is the major pathway of tryptophan catabolism in Aedes aegypti, but 3-hydroxykynurenine oxidizes easily under physiological conditions, which stimulate the production of reactive oxygen species. Our data show that in Aedes aegypti, the chemically reactive 3-hydroxykynurenine is converted to the chemically stable xanthurenic acid by a transaminase-catalyzed reaction during larval development, while 3-hydroxykynurenine is transported to the compound eyes for eye pigmentation during pupal development. Our data suggest that (1) the transamination pathway of 3-hydroxykynurenine is down-regulated during the pupal development, (2) 3-hydroxykynurenine produced in other body tissues is actively transported to the compound eyes during the pupal stage, (3) the compound eye is the place where ommochromes are produced, and (4) formation of ommochromes results from nonenzymatic oxidation of 3-hydroxykynurenine in the compound eyes.     

Terminal Synthesis of Xanthommatin in DROSOPHILA MELANOGASTER. III. Mutational Pleiotropy and Pigment Granule Association of Phenoxazinone Synthetase

Phenoxazinone synthetase, which catalyzes the condensation of 3-hydroxykynurenine to xanthommatin, the brown eye pigment of Drosophila, is shown to exist in association with a particle which resembles the cytologically defined Type I pigment granule. Several classical eye color mutants (v, cn, st, ltd, cd, w), including two which effect other enzymes in the xanthommatin pathway (v, cn), have low levels of phenoxazinone synthetase activity and disrupt the normal association of the enzyme with the pigment granule. A model is proposed depicting several structural and enzymatic interrelationships involved in the developmental control of xanthommatin synthesis in Drosophila.     

Disruption of kynurenine pathway reveals physiological importance of tryptophan catabolism in Henosepilachna vigintioctopunctata

Kynurenine pathway is critically important to catabolize tryptophan, to produce eye chromes, and to protect nervous system in insects. However, several issues related to tryptophan degradation remain to be clarified. In the present paper, we identified three genes (karmoisin, vermilion and cardinal) involved in kynurenine pathway in Henosepilachna vigintioctopunctata. The karmoisin and cardinal were highly expressed in the pupae and adults having compound eyes. Consistently, high-performance liquid chromatography result showed that three ommochrome peaks were present in adult heads rather than bodies (thoraces, legs, wings and abdomens). RNA interference (RNAi)-aided knockdown of vermilion caused accumulation of tryptophan in both adult heads and bodies, disappearance of ommochromes in the heads and a complete loss of eye color in both pupae and adults. Depletion of cardinal brought about excess of 3-hydroxykynurenine and insufficient ommochromes in the heads and decolored eyes. RNAi of karmoisin resulted in a decrease in ommochromes in the heads, and a partial loss of eye color. Moreover, a portion of karmoisin-, vermilion- or cardinal-silenced adults exhibited negative phototaxis, whereas control beetles showed positive phototaxis. Furthermore, dysfunctions of tryptophan catabolism impaired climbing ability. Our findings clearly illustrated several issues related to kynurenine pathway and provided a new insight into the physiological importance of tryptophan catabolism in H. vigintioctopunctata.

     

A specific and sensitive fluorometric assay for tryptophan oxygenase

A spectrophotofluorometric assay was used to measure tryptophan oxygenase activity in several species. The fluorescent assay depends on the conversion of the product of the reaction, N-formyl-l-kynurenine, to anthranilate by means of the coupling enzymes kynurenine formamidase and kynureninase. These enzymes are easily obtained from l-tryptophan-induced N. crassa; and the product, anthranilate, is readily separated by organic extraction from other tryptophan catabolites and easily identified fluorometrically. With this assay, tryptophan oxygenase has been demonstrated in vitro for the first time in N. crassa.     

Terminal synthesis of xanthommatin in drosophila melanogaster. IV. Enzymatic and nonenzymatic catalysis

Nonenzymatic and enzymatic catalysis of the oxidation of 3-hydroxykynurenine (and 3-hydroxyanthranilic acid) has been studied and characterized in Drosophila extracts, clearing up some of the confusion surrounding the synthesis of the brown eye pigment, xanthommatin. The genetic basis of the terminal steps in pigment synthesis remains obscure, since all mutants tested have full synthetase activity.     

The tryptophan oxidation pathway in mosquitoes with emphasis on xanthurenic acid biosynthesis

Oxidation of tryptophan to kynurenine and 3-hydroxykynurenine (3-HK) is the major catabolic pathway in mosquitoes. However, 3-HK is oxidized easily under physiological conditions, resulting in the production of reactive radical species. To overcome this problem, mosquitoes have developed an efficient mechanism to prevent 3-HK from accumulating by converting this chemically reactive compound to the chemically stable xanthurenic acid. Interestingly, 3-HK is a precursor for the production of compound eye pigments during the pupal and early adult stages; consequently, mosquitoes need to preserve and transport 3-HK for compound eye pigmentation in pupae and adults. This review summarizes the tryptophan oxidation pathway, compares and contrasts the mosquito tryptophan oxidation pathway with other model species, and discusses possible driving forces leading to the functional adaptation and evolution of enzymes involved in the mosquito tryptophan oxidation pathway.     

Precursors of pattern specific ommatin in red wing scales of the polyphenic butterfly Araschnia levana L.: Haemolymph tryptophan and 3-hydroxykynurenine

In the seasonally diphenic butterfly Araschnia levana¹⁴C-labelled tryptophan and 3-hydroxykynurenine, the principal precursors of ommochromes, injected into young pupae caused a pattern specific radiolabel of mature red scales. [¹⁴C]glucose and [³⁵S]methionine also labelled red scales but only when injected shortly before or during the time of pigment synthesis in the wing. In developing non-diapause pupae contents of 3-hydroxykynurenine increased until an abrupt decrease when pigments appeared in the wings. In diapausing pupae 3-hydroxykynurenine remained low but increased after injection of 20-hydroxyecdysone which induced pupal-adult development. Supply of wing scale cells with ommochrome precursors via the haemolymph was analysed after injection of [³H]tryptophan. In developing pupae haemolymph contents of [³H]tryptophan and [³H]3-hydroxykynurenine increased at the time of wing pigment formation and decreased shortly before adult emergence. In diapausing pupae haemolymph contents of [³H]tryptophan and [³H]3-hydroxykynurenine were low compared to non-diapause pupae but increased at the time of wing pigment formation after injection of 20-hydroxyecdysone. Isolated wings incubated in Grace's medium containing [¹⁴C]tryptophan and [¹⁴C]3-hydroxykynurenine incorporated radiolabel specifically into red portions of the wing colour pattern due to labelling of ommatin. Incorporation into red wing areas occurred specifically depending on different colour patterns of the spring- and the summer-morph.The results demonstrate that both tryptophan as well as 3-hydroxykynurenine are delivered via the haemolymph, and both can serve as precursors of ommatin formation in the scale cells. Therefore, the complete set of enzymes for the tryptophan-ommatin pathway is present in scale-forming cells.     

Synthesis,accumulation and excretion of kynurenine during the pupal and adult stages of Papilio xuthus

Changes of kynurenine levels in fat body, haemolymph and wings during the pupal stage of Papilio xuthus were determined. In fat body and haemolymph, free kynurenine begins to increase at the time of the onset of eye pigmentation, reaches a maximum at the stage of, or shortly after red spot appearance in the wings and then rapidly decreases. The bound form of kynurenine seems not to be present. In the wings, on the contrary, the bound form of kynurenine begins to increase shortly before the appearance of red spot and accumulates up to the time of emergence.Tryptophan oxygenase activity is greatest in the fat body, with less in the wings and haemolymph did not show any activity.At the emergence of the butterfly, a considerable amount of kynurenine is excreted in the meconium. Empty pupal cases also contain some kynurenine. At the same time in the adult, the yellow scales contain the largest amounts of kynurenine, and the adult body (minus yellow scales) also contain a considerable amount of kynurenine.All these results suggest the possibility that kynurenine is synthesized in the fat body, is transported through the haemolymph and accumulates as the bound form in the yellow scales. But the possibility cannot be ruled out that some kynurenine is synthesized in the wings.