[advances in insect physiology] volume 10 || the tryptophan → omrnochrome pathway in insects

The Tryptophan- Ommochrome Pathway in Insects' Bernt Linzen

Zoological Institute, University of Munich, Germany

1 Introduction 2 Fluorescent tryptophan metabolites found in insects 2.1. Notes on methodology 2.2. 'Tryptophan and its fluorescent metabolites

3 The absence of the glutarate pathway 4 Ommochromes

. 4.1 Notes on nomenclature 4.2 Isolation . 4.3 Properties 4.4 Distribution and tissue localization . 4.5 Deposition of ommochromes: the pigment granule!; . 4.6 Binding of ommochromes to proteins

5.1 Ommochromes as screening pigments 5 Functions of ommochromes

5.2 Ommochromes as pattern pigments. Relation to other pigments 5.3 Ommochromes in morphological colour change 5.4 Ommochromes as waste products

6.1 Tryptophan oxygenase (EC 1.13.1.12) .

. 6 Enzymes involved in the kynurenine pathway

. 118

. 120

. 120

. 122

. 132

. 134

. 134

. 135

. 138

. 150

. 162

. 164

. 165

. 166

. 169

. 173

. 176

. 179

. 180 6.2 Kynurenine formamidase (aryl-formylamine amidohydrolase EC 3.5.1.9) 189 6.3 Kynurenine-3-hydroxylase (EC 1.14.1.2) . 189 6.4 Kynureninase and kynurenine transaminase (EC 3.7.1.3; 2.6.1.7) . . 193

7 Ommochrome biosynthesis . 193 8 Tryptophan metabolism in insect development . 197 8.1 Eggs and embryonal development . 197 8.2 Larval development. Hemimetabola . 199

8.4 Ontogeny of enzyme activities . 212

8.3 Accumulation of tryptophan metabolites during metamorphosis of holometabolous insects . 201

I This paper is dedicated to Professor Adolf Butenandl. on thc occasion of his 70th birthday, in token of my gratitude and admiration for his inspiring leadership, his fervent devotion to the chemistry of life, and his lucid manner of conveying thought and scholarship.

117

118 BERNT LINZEN

8.5 Attempts to establish a tryptophan balance . 218 . 220

10 Concluding remarks . 223 Acknowledgements . 224 References . 225

9 Detrimental effects of tryptophan and of tryptophan metabolites

1 Introduction

'Tryptophan is an outstanding amino acid. It has the highest molecular weight of all amino acids occurring in proteins, and comprises, in its indolyl moiety, a system capable of donating electrons and, therefore, liable to form complexes with a range of other molecules. Molecular interactions have been observed between tryptophan and nucleic acids, and a variety of other molecules of biological importance (details will be given in section 9). It is obvious that this property is both beneficial (allowing recognition and association of reacting systems, e.g. macromolecules) and detrimental (by interference with such associations). Thus, tryptophan may be viewed as a valuable ingredient of living matter; an ingredient, however, which must be utilized with some precaution.

It can accordingly be readily appreciated why tryptophan is less common than other amino acids. 'The average tryptophan content of animal proteins is about 1 per cent on a weight basis (Block and Weiss, 1956), corresponding to little more than ?4 per cent on a molar basis-only one tenth of what could be expected if the amino acids were uniformly distributed. In vertebrate blood plasma the level of free tryptophan is extremely low (0.01 1 mg ml-' in human plasma) and efficient mechanisms exist to prevent it from rising. In insects, which are characterized by extraordinarily high concentrations of free amino acids, the level of tryptophan is not proportionally higher, often being below the level of detection. In insects too, the tryptophan level seldom rises, and if it does, then only for short periods.

On the other hand, organisms have taken advantage of the potentialities of this molecule and have transformed it into a wide range of biologically active compounds. In these either the indole ring or its benzene nucleus are retained, the latter also being recast into the pyridine ring. Among these are a plant growth hormone (indole acetic acid), an enzyme cofactor (nicotinic acid), a hormone (melatonin), a neurotransmitter substance (serotonin), and such powerful drugs as the ergot and Rauwolfia alcaloids. Of the amino acids, only phenylalanine is comparable with regard to the highly branched metabolic pathways and great variety of compounds formed.

In insects, a major product of tryptophan degradation is a group of 10 to 15 pigments, the ommochromes. Being brownish-yellow, bright red or dark violet-purple, these pigments produce the deep tinge of insect eyes and

THE TRYPTOPHAN + OMMOCHROME PATHWAY IN INSECTS 119

contribute to the brilliant colouration of many species. Pigmentation itself has always attracted investigators. But the principal stimulus for investigations in this field was the discovery of pigment mutants which were widely used for study by geneticists and deve1opment;d physiologists. The search for the principle mediating the pigmentation of insect eyes represented the first important step into the field of biochemical genetics.

The metabolic pathway from tryptophan tci ommochromes forms the subject of the present review. Its aim is to assemble data which contribute to a physiological understanding of this pathway in insects, to appraise the role of ommochromes in insect life and the relation of tryptophan metabolism to insect development. It is intended to describe ommochromes in some detail, but not to present ommochrome chemistry proper, as this would require a good deal of phenoxazine chemistry in general. Further- more, the genetic aspects of ommochromes will not be covered, although, since Ziegler’s (1961) article, a great many new mutants have been described. However, work on mutants will be called upon whenever it contributes to the understanding of the normal function. Finally, the histology of ommochrome pigmentation will be omitted, except for a brief treatment of the pigment granules and a short chapter on ommochromes as pattern pigments, indicating their role in morphological colour change. Chromatophores and their hormonal control will, however, be completely omitted.

The earliest phase of ommochrome biochemistry was characterized by the search for the factors linking gene and phenotype. This was initiated by the transplantation experiments on Ephestia (Kiihn, 1932; Caspari, 1933) and on Drosophila (Beadle and Ephrussi, 1935; Ephrussi and Beadle, 1935). These led to the recognition of the diffusible, colour inducing substances (the alleged “gene-hormones”) as pigment precursors (v’ = a’-substance = kynurenine; cn’substance = 3-hydroxy-kynurenine (Butenandt et al., 1940a, 1949)), and to the establishment of a scheme in which the genes were proposed to govern a sequence of biochemical reactions by providing specific enzymes. Details and references of this early period may be found in the reviews of Becker (1938, 1942), Plagge (1939), Kiihn (1941), Ephrussi (1942a, 1942b), Caspari (1949), and Ziegler (1961).

Ommochromes themselves were first studied broadly and recognized as a separate and hitherto unknown pigment class by Erich Becker‘ in Kiihn’s laboratory (Becker, 1939, 1941a, 1942). Hi!; work was resumed in Butenandt’s laboratory in the early fifties, and was crowned by the

Becker not only initiated the study of ommochromes, but also the search for ecdysone; he took part in the identification of the ac(v+)-substance, and published papers on pteridines and other subjects. He was a most gifted insect biochemist, and his death (August 1941) in the Russian campaign was one of the losses to German science experienced in the Third Reich and Second World War.

AIP-5

120 BERNT LINZEN

elucidation and chemical synthesis of xanthommatin, the first natural phenoxazinone pigment to be identified (Butenandt et al., 1954a, 1954b, 1954c, 1954d). Progress in the post-war period is reflected in the “ommochrome series” (see references) of Butenandt’s group and in a series of review papers by Butenandt (1957, 1959, 1960), Cromartie (1959), Stamm and Galarza (1961), Butenandt and Schaefer (1962), Schaefer (1964; chemistry of phenoxazinones), Linzen (1967), and Fuzeau-Braesch (1972; pigments and colour changes). In recent years, the interest in the enzymatic basis of ommochrome formation, and in the physiological function of ommochromes has grown continuously. Proper consideration will be given to these developing areas.

2 Fluorescent tryptophan metabolites found in insects

2.1 NOTES ON METHODOLOGY

Since all tryptophan metabolites retaining the aromatic ring fluoresce, they can be easily detected under ultraviolet light. However, as in the case of kynurenine, fluorescence may not appear in solution but only in the dry state. In insect work the amount of material is in any case restricted and paper or thin-layer chromatography must be employed for separation. Fluorescent spots may then be measured by fluorimetry. Several commercial instruments are available for this purpose, but cheap and simple instruments-nevertheless efficient-can be easily constructed (Semm and Fried, 1952; Kiihn, 1955; Egelhaaf, 1963a; Linzen, 1971a). As the quantity of light emitted from a fluorescent spot on a chromatogram is subject to many variables (e.g. spot size, thickness and quality of paper, time after drying the chromatogram) it is advisable to run several standards on the same sheet of paper. Standard curves are not linear; the variation of the data is much greater than in photometry, but can be reduced with some care and experience.

Paper chromatography and paper electrophoresis, followed by fluorimetry, often allow the determination of fluorescent metabolites in individual animals or even small portions of tissue. Tryptophan which emits only light of short wavelengths can be measured with high specificity at the nanomole level by a method first described by Egelhaaf (1957) which is based on the formation of “tryptochrome” and “iodotryptochrome” by spraying with potassium iodate (Fearon and Boggust, 1950). The method was modified by Linzen (Biickmann et al., 1966). Interference may arise from overlapping spots; if practicable this can be overcome by two- dimensional separation. Suitable solvent systems are given by Hadorn and Kiihn (1953), Egelhaaf (1963a), Pinamonti et al. (1964), Linzen and Ishiguro (1966), and Wessing and Eichelberg (1968), to cite only a few.


Column chromatogaphy has been used for isolation of trace compounds from large quantities of material and for preparation of pure compounds in quantity. Benassi et al. (1964) studied chroinatography of tryptophan metabolites on Amberlite IR-120 using various buffers and achieved good separation with a pyridine-formic acid system. This has been applied to insect material (Pinamonti ct al., 1964). Chromatography on DOWEX 50, similar to the procedure of Brown and Price (1956), was employed to isolate 3-hydroxy-kynurenine at the preparative scale (Ishiguro and Linzen, 1965) and the glucosides of 3-hydroxy-kynurmine (Linzen and Ishiguro, 1966) and of 3-hydroxy-anthranilic acid (Ishiguro and Linzen, 1966). For purification of 3-hydroxy-kynurenine, particularly for separation from kynurenine, chromatography on Sephadex G-;!5 is very efficient (Hiraga, 1964; Ishiguro and Linzen, 1965).

A range of colour reactions is available for the determination of individual compounds in mixtures and even raw extracts. Tryptophan is usually determined by the Spies and Chambers (1948) method; a material to which this method was not applicable tiecause of the reaction of coloured impurities with nitrite was encountered by Linzen (1971b). Kynurenine may be determined by the Brattcm-Marshall (1939) reaction, but this is not entirely specific. Tryptophan yields some colour and the degradation of ommochromes produces Bratton-Marshall positive material (Pinamonti and Petris, 1966). The reaction has been standardized to give highly reproducible results (Schartau and Linzeri, to be published). Another compound suitable for kynurenine determ (nation is Tsudas reagent (Mochizuki, 1953, cited by Koga and Osanai 1967); tryptophan and anthranilic acid will also produce some colour which, however can be removed by washing with butanol. This method was successfully applied to the analysis of silkworm eggs (Koga and Osanai, 1967).

3-Hydroxy-kynurenine may be determined by either of two photometric methods: Inagami (1954a) obtained a yellow compound with maximal absorption at 390 nm by treatment with n trow acid. Linzen (1963) oxidized 3-hydroxy-kynurenine to give xanthommatin and extracted the latter after reduction into butanol. Exactly neutral p h in the oxidation step and sufficient amounts of oxidant and reductant to cope with possible impurities are critical in this method. Both inethods have been used in various laboratories. Inagami’s procedure requires less manipulation but is not entirely specific (3-hydroxy-anthranilic itcid and “products of the tyrosine-tyrosinase reaction” will also react), and the yellow product is photosensitive. Linzeri’s method is somewhat more laborious but very specific; the red colour obtained is stable for a1 least 24 h and less likely to be interfered with by “background absorption”.

Recently a fluorimetric method for 3-hydroxy-kynurenine determination was published by Watanabe et al. (1970a, 1970b); the procedure involves a

122 BERNT LINZEN

number of steps, including thin layer chromatography. Insect material has not been examined by this method as yet.

2.2 TRYPTOPHAN AND ITS FLUORESCENT METABOLITES

The scheme on page 123 depicts the metabolic conversions of the kynurenine pathway as far as they have been demonstrated in insects. 3-Hydroxy-kynurenine is a key compound in this scheme, as would be expected from the variety of reactive sites of this molecule. It may be anticipated that a number of other metabolites will be found in the course of time, namely compounds derived from the peripheral metabolites of this scheme, by further substitution. A multitude of such compounds- glucuronides, methylated, or acetylated compounds-have been discovered in mammals (for review, see Henderson et al., 1962). The primary and some of the secondary metabolites of tryptophan are rather easily demonstrated in most insects; a systematic listing of fluorescent metabolites is, therefore, not given. Instead, probable basic concentration levels will be given and examples provided in which particular compounds are of special significance. These findings are in most cases related to insect development in some way or another, yet it is hardly possible to separate the mere observation of a metabolite from its developmental aspect. The following paragraphs should therefore be viewed in conjunction with section 8, in which the developmental relations of tryptophan metabolism are described in detail.

2.2.1 Tryptophan

As in other animals, both protein-bound and free tryptophan are at low concentrations. The determination of protein-bound tryptophan can be troublesome, especially if whole tissues or animals are to be analysed (cf. Friedman and Finley, 1971). Thus, in some of the earlier studies extremely low values are reported which appear doubtful in the light of present day experience (e.g. -0.3 per cent in proteins of Saturnia pyr i and several other species: Stamm and Aguirre, 1955a, 1955b, 1 9 5 5 ~ ) . Green (1949) estimated the tryptophan content of Drosophila melanogaster protein at 0.84 mol per cent; that of D. virilis was slightly higher. Data on other Diptera reported by Jezewska (1926) and Grassmader (1968) are not comparable as they are given on a floating basis (per animal or percentage of a certain stage). Linzen and Schartau (to be published) determined protein tryptophan in Phormia and obtained values between 1.1 and 1.3 per cent. In Bombyx mori rb the average tryptophan content ranged between 0.96 per cent (in larvae with filled spinning glands) and 1.38 per cent (young pupa). In the pharate adult and in moths just after emergence 1.16 per cent were found (Linzen, 1971a). In Ephestia the total tryptophan

THE TRYPTOPHAN -+ OMMOCHROME PATHWAY I N INSECTS 123

Glutorote pothwoy

&)lfDY H2N CH (Glucoside)

COOH COOH I d H H CH, - .

COOH 5: H2 $ H2

I Cysteine

Methionine t &&@H (12)

0 - SO3H

The metabolism of tryptophan in insects. Thick itmows indicate well-established transformations; thin arrows indicate reactions involving other metabolic pathways or reactions of unknown significance. (1) tryptophan, (2) formyl-kynurenine, (3) kynurenine, (4a) kynurenic acid, (4bj kynurine, (5a) anthranilic acid, (5b) anthranilyl- glycine, (6) 3-hydroxy-kynurenine, (7a) xanthurenic acid, (7b) 4,8-dihydroxyquinoline, (7c) methyl ester of 8-hydroxy-quinaldic acid, (8a) .3-hydroxy-anthranilic acid, (8b) 3-hydroxy-anthranilyI-glycine, (9) xanthommatin, (10) cinnabarinic acid, (1 1 ) rhodommatin, (12) ommatin D. All 4-hydroxy derivatives of i;he quinoline ring are written in their tautomeric form. "L", "I" and "F" are unknown derivatives isolated by Inagami (1958) from silkworm pupae. The glucoside of 3-hydroxy-anthranilic acid has been detected only after injection of the parent compound into locusts.

124 BERNT LINZEN

was estimated at 61 (moths) to 105 (larvae) pg mg-’ nitrogen (Butenandt and Albrecht, 1952), protein-bound tryptophan at 1 to 1.5 per cent (w/w) (Egelhaaf, 1963a).

Some representative data on free tryptophan content are combined in Table 1. In spite of the uncertainties implied with the term “concentration”, the larval tryptophan level is probably between 0.2 and 0.3 mM, while in mature insects it may fall below 0.1 mM. Some of the higher values (Ephestia, last stage; freshly emerged Diptera) are related to the accumulation of tryptophan during metamorphosis. The generally low concentration of free tryptophan is in sharp contrast to the extremely high levels of other amino acids present in the blood of many insects; it is in fact comparable to the low concentration found in human plasma (-0.05 mM). The true concentration of tryptophan in solution may be much lower than estimated above, since some tissues may be able to concentrate tryptophan. This is true of the Malpighian tubules in Drosophilu (Wessing and Bonse, 1962),

TABLE 1

Representative data for concentration of free tryptophan in various insects

Species Experimental data Approximate

mM kg-‘ “concentration” Reference?

Ephestia kiihniella

Bombyx mori rb

Cerura uinula

Calliphora erythrocephala

Phormia terraenouae

Dosophila melano. gaster

Eggs: -4 pg mg-’ N Larvae, 3 weeks: -2 pg mg-’ N Larvae, last stage: -18 pg mg-’ N Larvae: -40 pg g-’ fresh weight Moths: <20 pgg-’ fresh weight Larvae, last stage (haemolymph):

Larvae off food: 2.6 pg per

Fly, just emerged:

Late larvae, feeding: 65 pg g-’ Flies, just emerged: -50 pg g-’ Flies, aged: -10 pg g-’ Flies, just emerged:

60 pg ml-’

animal

-4 pg per animal

0.215 mg g-’ dry weight

0.38 a

0.19 1.69 0.2 b

<o. 1

0.3

0.14

0.4 0.32 0.25 0.05

c

d

e

0.35 f

“Concentration” assumes equal distribution; it is calculated on the basis of average relations of fresh to dry weight, and of fresh weight to total protein.

t a. Egelhaaf (1963a); b. Linzen (1971a); c. Biickmann et al. (1966);d. Langer and Grassmader (1965); e. Linzen and Schartau (to be published); f. Green (1949); similar values obtained by Shapard (1960).


but probably also in other Diptera. Bonse (1969) noticed that the white strain of Drosophila was unable to accumulate tryptophan in the Malpighian tubules, an interesting pleiotropic effect of this mutation.

2.2.2 Formyl-kynurenine

Because of the high activity of kynurenine-formamidase and the spontaneous hydrolysis, which occurs even at neutral pH, this compound will probabIy not be observed in extracts. However, it is well established that formyl-kynurenine is the product of the tryptophan-oxygenase catalysed reaction. Green (1952) fed formyl-kynurenine to vermilion mutants of Drosophila and observed formation of brown eye pigment. With this very labile compound it was not possible to distinguish between a block in the oxygenase or the formamidase reaction for the same effect would have been obtained with kynurenine. A related compound, a-hydroxy- tryptophan (isolated from hydrolyzates of phalloidin), also appeared to be effective as pigment precursor in the feeding test (Butenandt et al . , 1940b), although some doubt has been subsequently. cast on these results (Kikkawa, 1953).

2.2.3 Kynurenine (the “a+-”= “v+-substance ”) Kynurenine was identified as an ommochrome precursor by Butenandt et al. (1940a) shortly after the discovery by Tatum (1939) that certain bacteria produced a substance from tryptophan which induced eye colour formation in the Drosophila mutant vermilion. Tatum and Haagen-Smit (1941) also identified the bacterial product with kynurenine, and Kikkawa (1941) was able to isolate the compound from eggs of B. mori white-1. The correct structure of kynurenine was given by Butenandt et al. (1942, 1943).

-Kynurenine is ubiquitous in insects, despite its extremely low concentration. In Phormia terraenovae the basic level is 12 pg g-’ (50 p M ) (Linzen and Schartau, t o be published); in EphestM (Egelhaaf, 1963a) and Bombyx prior to spinning (Linzen, unpublished) similar levels have been determined. In the cricket, Gryllus bimaculatus, the concentration is between 5 and 30 pg per animal (50 to 150 p M ) during the last larval stage, the higher values being attained only in the last two days before the final moult (Tiedt, 1971). Extremely low values were reported for individual tissues of Schistocerca (Pinamonti et al . , 1964) although the recovery might have been low too, due to the method used.

Again, “concentration” is not an exactly appropriate term, as the kynurenine determined in whole animals may be confined to individual tissues. This is certainly true in Drosophilu, where the anterior region of the larval fat body accumulates kynurenine near the time of pupation, and exhibits a light blue fluorescence (Rizki, 1961). This is in accord with the

126 BEANT LINZEN

transplantation experiments of Beadle (1937) which showed the fat body to be a source of the v+-substance. Since the fluorescence of kynurenine in solution is extremely weak (Udenfriend, 1969; this corrected earlier reports of strong fluorescence), it must be in a bound form in the fat body of Drosophilu. Rizki (1 96 1 ) described the appearance of fluorescent spherical granules around the nuclei of the fat cells, which later were scattered over the cytoplasm while increasing in size. Rizki and Rizki (1963) explicitly state that there is no diffusion of kynurenine between fat cells, although removal via the haemolymph is not excluded (it may be noted that the results of the transplantation experiments can not (be related to this particular problem, since the fat body is subject to histolysis early in metamorphosis).

In addition to the fat body, the Malpighian tubules could possibly accumulate kynurenine, although the evidence is not as convincing as in the case of tryptophan and 3-hydroxy-kynurenine storage. Kynurenine can be regularly detected by paper chromatography (Ursprung et d., 1958; Wessing and Eichelberg, 1968; Eichelberg, 1968; Bonse. 1969) and is said to be excreted “at a high rate” (Bonse). Malpighian tubules also contain the enzyme, tryptophan oxygenase (see below), and exhibit the highest known specific activity of this enzyme in Phormia larvae. Finally. kynurenine storage is also observed in eggs of Drosophilu (but missing in v/v and v/+ eggs) (Graf, 1957). Here also, brightly fluorescent inclusions. are observed which are assumed to be yolk granules (Muckenthaler, 1971). Although it was observed that the kynurenine disappeared during embryonal development, no physiological significance could be envisaged, for v/v eggs are fully viable. In Ephestiu kynurenine is absent from the eggs (Egelhaaf 1957) while in Bombyx eggs the concentration is 0.3 mM soon after completion of pigment synthesis (Koga and Osanai, 1967).

Kynurenine is a major factor in the pigmentation of the wings of Pupilionid butterflies (complete reference list in Umebachi and Yoshida, 1970). From the yellow scales of these wings a large amount of kynurenine can be extracted (Umebachi and Nakamura, 1954); only a small part of this is originally in a free state. Most of it is bound in the form of a yellow pigment called “papiliochrome”. This can be resolved by paper chromatography into five components; two of these, Y-IIa and Y-IIb, have been examined in detail (Umebachi and Yoshida, 1970). From uv, ir, ord, and cd spectra it was inferred that they have the same chemical structure but are optical isomers. Kynurenine is split off even under extremely mild conditions. The other half of the molecule is a phenolic nitrogen-containing compound, derived from dopamine. The biosynthesis of these pigments occurs in discrete areas of the wing epithelium, which can be beautifully demonstrated by radioautography of wings after administration of labelled precursors (Umebachi, 1959; Umebachi and Yoshida 1970).


2.2.4 3-Hydroxy-kynurenine (the “cn+-substance”)

This compound, comprising five functional groups and an aromatic nucleus, occupies the key position in tryptophan metabolism. It was first isolated from flies and simultaneously synthesized by Butenandt’s group (Butenandt, 1949; Butenandt et af . 1949; Schlossberger, 1949), but independently obtained by Kikkawa from Bonzbyx eggs (Hirata et af . , 1949, 1950) and Musajo (chemical synthesis; Musajo et af., 1950). Possibly, Kikkawa had a minute .quantity of the natural compound in his hands as early as 1943 (cf. Hirata et af . , 1950). Today, the synthetic (racemic) compound is commercially available (synthesis: Butenandt and Hallmann, 1950; Musajo et af., 1950; Hirata and Nakanishi, 1950; Butenandt et al., 1957f), as well as the natural L-isomer which can be isolated with good yields from Cafliphora pupae (Ishiguro and Linzen, 1965) and from the Bombyx mutant rb (Iwata and Ogata, 1966). The latter, which accumulates 3-hydroxy-kynurenine to abnormal levels (Makino et af . , 1954), is the source of the commercial product.

Although 3-hydroxy-kynurenine has been idlmtified in many species, there are relatively few data which would allow the definition of a basic concentration level in either the larval or adult stages of insects. Most quantitative determinations are concerned with alterations occurring during metamorphosis. Inagami (1958), in his study of tryptophan metabolism in the rb (red blood, jap. “aka-aka”) mutant of BGmbyx mori, has obtained many data on 3-hydroxy-kynurenine concentration in haemolymph and tissues of various strains (Table 2). Data on larval and imaginal 3-hydroxy- kynurenine in whole rb animals (0.06 and 0.03 p M g-’ , respectively; Linzen and Ishiguro, 1966) and the level observed in last stage Cerura Vinula larvae (-0.05 mM) agree with Inagami’s values in the order of magnitude. The high concentration in Malpighian tubules in Inagami’s

TABLE 2

Concentration of 3-hydroxy-kynureNne in haemolymph and various tissues of Bombyx mori larvae* (Inagami, 1958)

pg ml-’ mM

Haemolymph 30 0.13 Integument 90 0.4

Anterior gut 70 0.3 Posterior gut 40 0.18

* Normal strain, middle of 5th stage; all values have been

Malpighian tubules 230 1

rounded off by the author.

128 BERNT LINZEN

experiments is striking. It may be related to the high activity of kynurenine-3-hydroxylase in this tissue (see below), but the possibility of an accumulation mechanism cannot be eliminated at this stage. In blowflies the basic concentration varies with species. In Phormia it is about 0.15 mM both in larvae and in adult animals (Linzen and Schartau, to be published), while Calliphora larvae (off the food) contain 20 pg per animal, corresponding roughly to a 1 mM concentration; yet nearly all the substance is bound or precipitated in the Malpighian tubules (Hendrichs-Hertel and Linzen, 1969).

Of the Hemimetabola, only Gryllus and Schistocerca have been examined. In G . bimaculatus whole larvae the level is 0.1 to 0.2 mM (Tiedt, 1971), while in Schistocerca the compound is barely detectable, except for the integument (Pinamonti et al., 1964) and eggs (Colombo and Pinamonti, 1965).

Beadle had demonstrated (1937) that Malpighian tubules of Drosophila melanogaster are rich in cn+-substance. Since then, 3-hydroxy-kynurenine turned out to be a typical constituent of this tissue in Drosophila, Calliphora, and Bombyx (Inagami, 1958; Wessing and Danneel, 1961; Eichelberg, 1968; Hertel, 1968; Wessing and Eichelberg 1968. Eichelberg and Wessing, 1971). In Calliphora it is restricted to the Malpighian tubules, according to paper chromatographic analysis (Henning, 1957; Hertel, 1968). It is of interest to note that isolated Malpighian tubules of the stick insect, Carausius rnorosus, can be maintained active for a prolonged period of time if 3-hydroxy-kynurenine is added to the Ringer solution (Ramsay, 1956). 3-Hydroxy-kynurenine is concentrated by the Malpighian tubules of Drosophila and forms bright yellow concretions which gradually' fill out ampulla structures of the endoplasmic reticulum. Good pictures of these are published by Wessing and Danneel (1961) and Wessing and Eichelberg ( 19 72). The 3-hydroxy-kynurenine concretions appear within minutes after injecting the substance into larvae. They may be viewed as temporary deposits, since in the course of adult development in the pupa-during eye pigmentation-the contents of the ampullae disappear gradually. In the white mutant of Drosophila, the Malpighian tubules have lost the capacity to accumulate (and possibly t o synthesize) 3-hydroxy-kynurenine (Bonse, 1969).

While it has been suggested by Wessing that the Malpighian tubules of Drosophila are capable of both storing and synthesizing 3-hydroxy- kynurenine, no evidence has been advanced to support this hypothesis for any dipterans. Yet the ovary of Bombyx mori provides an example in which both preferential binding and biosynthesis from kynurenine co- operate in the establishment of a 3-hydroxy-kynurenine reserve. The ovaries show kynurenine-3-hydroxylase activity throughout their growth (Linzen and Hendrichs-Hertel, 1970), but at the same time they take up the

THE TRYPTOPHAN -* OMMOCHROME PATHWAY IN INSECTS 129

compound from the haemolymph and accumulate it in the oocytes. This process of 3-hydroxy-kynurenine accumulation is under the control of the “diapause hormone” secreted by the suboesophageal ganglion (Yamashita and Hasegawa, 1966, and earlier papers). A.s in the case of’ 3-hydroxy- kynurenine accumulation in eggs of Drosoohila (Muckenthaler, 197 1 ), 3-hydroxy-kynurenine is bound by the yolk spheres of the oocyte. This was beautifully demonstrated by injecting tritium-labelled 3-hydroxy- kynurenine into white-l pupae (where kynurenine hydroxylation is blocked) and fixing the ovarian follicles 12 h later for preparation of autoradiographs. Tritium label was confined to the surface of the yolk spheres, but absent from the rest of the oocyte cytoplasm, from the follicle, and nurse cells (Sonobe and Ohnishi, 1970). While the function of accumulated kynurenine in Drosophila e!ggs is obscure, 3-hydroxy- kynurenine in diapause eggs of Bombyx is consumed during synthesis of ommochromes in the “serosa” (Kikkawa, 1953; Koga and Goda, 1962; Koga and Osanai, 1967). It is said that the viability of pigmented diapause eggs is increased in comparison to unpigmented eggs.

With respect to eye pigmentation in Droso,bhzla, it is by no means clear whether the deposits of 3-hydroxy-kynureniiie in the Malpighian tubules are an indispensable requirement or merely a supplementary supply of pigment precursor. Eye pigmentation is essentially autonomous (Danneel, 1941; Horikawa, 1958). It is of special interesf, therefore, that in the honey bee apparently a major proportion of tryptophan transformation occurs in the developing eyes. This is deduced from the fact that eyeless mutants accumulate tryptophan, rather than its metabolites, which is later excreted by the adult bee. There are a number of Apis mutants (cf. Dustmann, 1969, for a listing) which are blocked at diffcrent steps along the tryptophan --* ommochrome pathway. A mutant type which is not known from other laboratory insects is chartreuse. The group of chartreuse mutants i s easily distinguished by a strong green fluorescence of the eyes, if viewed under ultraviolet light. In most chartreuse mutants, also the general appearance of the eyes is greenish after emergence, but may turn red or brown after some time. These mutants accumulate 3-hydroxy-kynurenine in a granular form in the pigment cells of the compound eyes (maximally 140 pg per animal), so that the pigment precursor actually assumes pigment function. Binding 3-hydroxy-kynurenine in this way is evidently a distinctive step in ommochrome formation; it is blocked in another series of mutants, viz. cream (cr), pearl (p) , and brick (bk, partially blocked) which accordingly accumulate 3-hydroxy-kynurenine, but e vcrete i t upon emergence (Dustmann, 1968, 1969, and personal commu~iication).

The function of a pigment is performed by 3-hydmxy-kynurenine also in butterflies of the subfamilies Heliconiime and Zthomiime. At first the yellow compound isolated from the wings and bodies of these species was

130 BERNT LINZEN

considered to have the structure of 2.3-dihydro-xanthurenic acid, which is analogous to “kynurenine yellow” described by Kotake and Shichiri (1 93 1). A reexamination, supplemented by mass spectral and nmr data, later revealed the identity of the yellow compound with 3-hydroxy- kynurenine (Brown, 1965; Brown and Becher, 1967; Tokuyama et al., 1967). In this connection, it may be mentioned that in the Papilionids which make use of kynurenine in producing a light yellow pigment, the level of 3-hydroxy-kynurenine remains low at all stages (Umebachi and Katayama, 1966).

As a rule, binding of a compound produces a shift of the absorption maximum which is related to binding energy; in the case of ommochrome binding this shift may amount to 60 nm. It is desirable, therefore, to examine the spectra of 3-hydroxy-kynurenine in the different states in which it is present in insects; that is, dissolved in the presence of proteins, in the state of amorphous concretions, in the adsorbed condition in wing scales and yolk spheres and in the granular form of chartreuse bees. Probably, some conclusions may be drawn From such measurements concerning the availability of 3-hydroxy-kynurenine at a given stage of development and concerning the specificity-and thus physiological significance-of 3-hydroxy-kynurenine binding.

3-Hydroxy-kynurenine may be formed in excess of the demand and, being a phenolic compound, is subject to known detoxication mechanisms. Thus, the sulphuric acid ester (Inagami, 1958) and the P-glucoside (Linzen and Ishiguro, 1966) are formed in Bombyx mori rb pupae; the sulphate also occurs in the bug, Rhodnius prolixus (Viscontini and Schmid 1963). Inagami also mentioned a “glucuronide” of 3-hydroxy-kynurenine, but it is surmised that the glucoside (which has been identified more rigorously) was mistaken for the glucuronide.

Pryor (1955), in a short communication, has suggested the participation of 3-hydroxy-kynurenine in the cuticular tanning of insects. Since then, to the author’s knowledge, no experiments have appeared to support this conjecture. Neither is there convincing evidence of formation of “mixed melanins” from 3-hydroxy-kynurenine and tyrosine metabolites, which had been proposed by Inagami (1954b).

2.2.5 Quinoline derivatives

Kynurenine and 3-hydroxy-kynurenine may be transaminated, the resulting keto acids spontaneously undergo cyclization to form kynurenic acid. and xanthurenic acid respectively. Kynurenic and xanthurenic acids have been identified in a variety of insects: Schistocerca gregaria (Pinamonti et al., 1964), Ephestia kiihniella (xanthurenic acid only after tryptophan injection, kynurenic acid excluded: Egelhaaf, 1963a), Plodia interpunctella (xanthurenic acid in wings; Mohlmann, 1958), Bombyx mori (both acids:


Inagami, 1958), Habrobracon juglandis (both acids: Leibenguth, 1965, 1967a), Drosophila melanogaster (kynurenic acid: Danneel and Zimmer- mann, 1954; Wessing and Eichelberg, 1968; xanthurenic acid: Umebachi and Tsuchitani, 1955; Wessing and Eichelberg, 1968), Musca domestica (both: Colombo and Pinamonti, 1967; Laudani and Grigolo, 1968). In most species only minute amounts of both compounds are found, and usually kynurenic acid is even less abundant than xanthurenic acid.

Xanthurenic acid is the major end product of tryptophan metabolism in the spinning larva of the parasitic wasp, Habrobracon juglandis: it is excreted by the prepupa (Leibenguth, 1965). This explains a paradoxical observation first made by Beadle et al. (1938): Larvae of the Habrobracon mutant o which are blocked in the hydroxylation of kynurenine and therefore devoid of ommochrome pigments should ingest a sufficient supply of 3-hydroxy-kynurenine and become pigmented if feeding on wild type Ephestia. This is, however, not the case, even if the level of 3-hydroxy-kynurenine in the host is elevated by injection. Under these circumstances, the Habrobracon larva will even excrete some 3-hydroxy- kynurenine. Nevertheless in the prepupa not ,i trace of the substance can be detected. Leibenguth (1967, 1970) explained this convincingly by the fact that all larval 3-hydroxy-kynurenine is removed via transamination. Eye pigmentation in Habrobracon thus depends on pupal production of 3-hydroxy-kynurenine, which of course is not any more influenced by the supply of host material.

Three compounds related to kynurenic and xanthurenic acids, respectively, have been isolated from insects. One is kynurin (4-hydroxy- quinoline), 605 mg of which were obtained l’rom 200 kg of dried silkworm pupae (Butenandt et al., 1951). Formally, kynurine could originate by decarboxylation of kynurenic acid, yet no experiments have been performed to check this. Correspondingly, 4,8-dihydroxy-quinoline was isolated by Inagami (1958) from silkworm pupae, where it appeared late in the pupal stage and more abundantly in males than in females. Finally the methyl ester of 8-hydroxy-quinaldic acid was identified by Schildknecht et al. (1969) in Ilybius fenestratus. This is a most intriguing finding, since it implies a dehydroxylation reaction, which is rather uncommon in nature. The compound is elaborated by defence glands, the opening of which are located in the prothorax of this water beetle and produces convulsions if the beetle is ingested by a frog or a mouse.

2.2.6 Anthranilic and 3-hydroxy-anthranilic acids

The specificity of the enzyme, kynureninaje is low with respect to the benzoyl half of its substrate (Weber and Wiss, 1966), for even 5-hydroxy- kynurenine (Butenandt et al., 1953) and the ommatins (Butenandt et al., 1954b) will be attacked. One should, therefore, expect that whenever this

132 BERNT LINZEN

enzyme is present in insects, both anthranilic and 3-hydroxy-anthranilic acids are formed. This is true in Bombyx mori, where both acids and their glycine conjugates were indentified (Kikkawa, 1951, 1953). Inagami (1958) found another conjugate in white-1 meconia, which he considered to be a glucuronide (today one would rather expect a glucoside). Inagami concluded that the anthranilic acids and their conjugates were the main end products of tryptophan metabolism during larval development of the silkworm, while in the pupa the formation of 3-hydroxy-kynurenine and of the pigments was favoured. Inagami also described three unknown fluorescent compounds (“F”, “L”, and “I”) which, according to their presence or absence in the normal strain and the white-1 and rb-mutants, must be .considered to be metabolites of 3-hydroxy-anthranilic acid. If injected into locusts, 3-hydroxy-anthranilic acid is rapidly converted into its fl-glucoside (Ishiguro and Linzen, 1966).

In flies, the anthranilic acids occur at best in trace amounts. Wessing and Eichelberg, in their list of fluorescent substances in the Malpighian tubules of Drosophila (1968), do not mention either compound. While Laudani and Grigolo (1968) did not detect either compound in Musca dornestica strains, Colombo and Pinamonti (1967) reported “trace amounts”. By injecting trypt~phan-methylene-’~C and searching for labelled alanine after variable lengths of time and in each developmental stage, Linzen and Schartau (to be published) proved the virtual absence of kynureninase activity in the blowfly, Phormia tmaenovae.

Egelhaaf (1963a) drew attention to a pair of fluorescent compounds, “r” and “s”, which appeared on two-dimensional paper chromatograms of Ephestia extracts, but were absent in extracts of the a mutant. After administration of tryptophan, labelled in the methylene carbon, these compounds were not labelled. This experiment should be repeated using tryptophan labelled in the benzene nucleus to clarify the biogenetic relation.

In passing, it should be mentioned that Calam (1972) isolated indole acetic acid as a major constituent from the salivary glands of Dasyneura species. This compound is probably responsible for the induction of galls in various plants by these insects.

3 The absence of the glutarate pathway

In mammals, the major part of administered tryptophan is metabolized via 3-hydroxy-anthranilic acid, its unstable oxidation product cr-amino- fl-carboxy-muconic-e-semialdehyde, and glutaryl-CoA (Henderson et al., 1962). Oxidation of the latter gives rise to CO, and acetate. The open chain intermediate provides a branching point, since reaction between the amino group and the aldehyde group leads to ring closure and formation of


pyridine carboxylic acids, notably nicotinic acld. Thus, the oxidation of 3-hydroxy-anthranilic acid has two important functions: to form a building block for the most important cofactors in oxidative metabolism and to provide a means of breaking down the benzene nucleus of tryptophan and to remove potentially detrimental intermediates. The function of the glutarate pathway may easily be tested by injecting tryptophan labelled with l4 C in the aromatic nucleus and testing for expired *4 CO, .

That this pathway is not operative in inslxts was indicated by the accumulation of metabolites prior to the branching point and by the cognition that all insects so far examined require nicotinic acid in their diet (Dadd, 1970). Schultz and Rudkin (1948) have specifically tested for a possible sparing action of tryptophan added to the growth medium of Drosophila larvae and found that there was none. Accordingly, no difference in the nicotinic acid content of normal, white-1, and white-2 strains of Bombyx (150-180 gg g-' dried material) was detected, and injections of tryptophan, 3-hydroxy-kynurenine, or 3-hydroxy-anthranilic acid failed to raise this level (Kikkawa and Kuwma, 1952).

The clue to these observations is provided in a most important paper by Lan and Gholson (1965). The authors injected 1),~-tryptophan-5- '~C into a variety of invertebrates, and poikilothermic and homoiothermic vertebrates, and collected the %O, produced during the subsequent 4.5 to 24 h. While all vertebrates produced radioactive CO,, the three insects (a grasshopper, Dissosteira longipennis, a cockroach, Periplaneta americana, and a cricket, Gryllus assimilis) did not. Assays for the three enzymes, 3-hydroxy-anthranilic acid oxidase, picolinic carboxylase, and a-amino- muconic semialdehyde dehydrogenase, yielded negative results in each case. Although the latter tests which were cosducted on whole organisms might not be fully conclusive by themselves (the authors report some contradictory results with snails and earthworms), it is clear that none of the arthropods tested are able to degrade the benzene nucleus of tryptophan. In a later study which included also three hcJometabolous insects (&is mellifera, Tenebrio molitor, Phormia terraenovae) Schartau and Linzen (1 969) furnished supplementary isotopic data.

With the usual proviso one might state that in insects the degradation of tryptophan is blocked at the step of 3-hydroxy-anthranilic oxidation. This is the key to understanding the functional significance of the accumulation of tryptophan metabolites and of the formation of ommochromes. Without this block, ommochromes would probably 'lot be as characteristic of insects as they are in fact. It is difficult to evaluate advantages and disadvantages of this metabolic block. In the author's opinion this block poses problems (e.g. the absolute need for niootinic acid, or the accumulation of highly reactive compounds) rather than meeting requirements evolved with the arthropod structure. In contrast, Brunet (1965)

134 BERNT LINZEN

speculates that ommochrome formation has been of such a high selective value for insects that loss of the ability to oxidize 3-hydroxy-anthranilic acid was nothing less than a logical consequence. This problem will be alluded to in later parts of this review.

4 Ornrnochrornes

4.1 NOTES ON NOMENCLATURE

Erich Becker, in his first papers on ommochromes (1939, 1941a), had distinguished between two groups of pigments: the ommatins (of rather low molecular weight) and the ommins (of a higher degree of polymerization). His major criteria to distinguish between the two were the degree of stability in alkaline media (the ommatins being extremely labile) and the ability to pass dialysis membranes. Only in his posthumous paper (1942) did Becker propose the term “ommochromes” to cover all related pigments. He was aware of the fact that some of these had been given different names which might have deserved priority, such as Chauvin’s (1938) “acridioxanthin” and “acridioerythrin” of the migratory locust. However, it may be argued-as Becker contended-that many of the pigment preparations thus denoted were not satisfactorily defined chemically, and that closer examination could justify a revision of nomenclature. For similar reasons the term “insectorubin” (Goodwin and Srisukh, 1950) will not be used in this paper, since it designates a preparation which by modern methods can be shown to be composed of different pigments, and since it suggests a widespread distribution of the locust ommochromes, which in fact is not the case.

Linzen (1967) proposed a subdivision of the ommochromes based on structural criteria. Those ommochromes containing a 1,P-pyridino-3H- phenoxazine moiety were designated to be ommatins, and those which yielded 3-hydroxy-kynurenine plus “pigment IV” upon acid hydrolysis, were called ommins. For the highly acid- and alkali-resistant eye-pigments of the migratory locust, new names, ommidin and cryptommidin, were introduced (Linzen, 19 66) which replaced “acridioxanthin” and “insectorubin”. It may be left open to further discussion whether other pigments containing only a simple 3H-phenoxazine nucleus (such as cinnabarinic acid) should be included in the ommatin group, which then would not be defined by its substituent but rather by its basic structure, or should be grouped separately.

Typical ommatins are xanthommatin (and its reduced form, dihydro- or “hydro-”xanthommatin), rhodommatin (dihydro-xanthommatin-0-fl-D glucoside), and ommatin D (dihydro-xanthommatin sulphate); the latter two are excreted by nymphalid butterflies in their meconia. Ommatin C is


an artifact obtained by Butenandt et al. (1954a). Bouthier (personal communication) has observed recently that one of the two “labile redox pigments” described by Linzen (1966) in locust integument actually decomposes into xanthommatin and an unknown compound. Bouthier proposed to name these two pigments “acridi ommatins” (acridiommatin I and 11). Needham (1970) has described an “oniscoid ommatin” from isopods which awaits further chemical study. Becker’s (1942) “phaeom- matin” from Calliphora eyes is undoubtedly xanthommatin.

Becker’s best studied ommin, “skotommin” of Ephestia kuhniella, is a mixture of closely related pigments; the name is no longer in use. Butenandt et al. (1959) used letters (in the order of motility in a particular paper chromatographic system) to distinguish between these components; ommin A is the most abundant in most cases. Linzen (1967) suggested an index determined by the wavelength of maximal absorption of visible light in Naz HPO, solution (e.g. ‘‘ommin537 ”). Kiihn’s “ommochrome I” and “ommochrome 11” (Kiihn and Egelhaaf, 1959) are ommins in all probability, as are Kawase’s (1955) “+-chrome 11” and “+-chrome 111” (“+-chrome I” is identical to xanthommatin). Lacciferic acid and laccaic acid which are obtained from stick lac, behave in a similar way to ommins (Singh et al., 1966). On the other hand, Becker (1939) had described a yellow water-soluble pigment as “xanthommin”. Although the latter has never been reinvestigated, it is doubtful whether it really belongs to the ommin group.

Table 3 lists the ommochromes and some of the features characteristic of each subgroup.

4.2 ISOLATION

As ommochromes are generally of low solubility, it is advantageous to extract other low molecular weight compounds first, usually by repeated treatment with ether (or benzene, acetone etc.) and with methyl alcohol. The pigments themselves will then be extracted by methyl alcohol containing 1-5 per cent of concentrated HCI and preferably gassed with SOz (in particular if xanthommatin is to be extracted), or by 0.1 M NazHPO, (if rhodommatin and ommatin D are involved). The best composition of the solvent for extraction is determined by the properties of the starting material and should be checked first. For example, for extraction of rhodommatin and ommatin D from butterfly wings it was found to be advantageous to employ dilute ammonia.

Purification may proceed, via repeated precipjtation, from slightly alkaline buffers, via preparative paper or column chromatography. The latter is especially efficient for separation of rhodommatin and ommatin D which always occur in association. On the analytical or micro-preparative

TABLE 3

A classification of ommochromes in the order of (presumably) increasing complexity

Compound

1 Ommatins Cinnabarinic acid

Xanthommatin

Rhodommatin, Ommatin D

Acridiommatin I Acridiommatin I1

2 Ommidins Ommidin Cryp tommidin

3. Ommins Ommin A (skotommin, ommin52,) and at least four related compounds

Characteristics Precursors Distribution in insects

Decoburized by reduction; autoxidation a t alkaline pH; alkali-labile

Yellow in oxidized, red in reduced state, autoxidizable, very labile

Red, stable against oxidation in air; alkali-labile

Red, readily oxidized, extremely labile; acridiommatin I1 decomposed to xanthommatin (?)

Yellow; red in acid; very stable against hydrolysis; sulphur-containing; readily dialysed

No autoxidation in alkaline media; violet to purple, yellow after oxidation by nitrite: relatively stable in alkaline media; slowly dialysed; acid hydrolysis yields 3-hydroxy-kynurenine and “pigment IV”

3-Hydroxy-anthranilic acid

3-Hydroxy-kynurenine

3 -Hydroxy- kynurenine , xanthommatin? Glucose and sulphate respectively Tryptophan ria 3-hydroxy-kynurenine and xanthommatin?

3-Hydroxy-kynurenine, methionin (proved for ommidin only)

3-Hydroxy-kynurenine, mrthionine via cysteine

In haemolymph and excreta of Bombyx mori, normal strain (also a pigment in mushrooms) Ubiquitous, mixed with other ommochromes: in Diptera only eye pigment Excretory and wing pigments in Lepidoptera

Many tissues and excreta of Orthoptera; Odonata (integument)

Eye pigments in Orthoptera, occasionally in integument

Nearly ubiquitous in insect eyes, except for some Diptera and Orthoptera; frequently in hypodermis and around internal organs; not in excreta

Data on spectral properties are listed in Table 4; details of distribution among.insects are given in Table 5. The listing of cinnabarinic -id md of +he Urridiommathm in the ommatin group,u regnrded as proviaional and subject to further diacussion.

THE TRYPTOPHAN + OMMOCHROME PATHWAY IN INSECTS 1 37

scale, Ecteola is most convenient, since the ommatins are adsorbed in a very narrow zone even from extremely dilute buffer (:pH 7 ) solutions.' Elution is achieved by pyridine-acetate buffers (Butenandt et al., 1960b). Reduced xanthommatin may be isolated by the same method but yields of only about 65 per cent are obtained. On the preparative scale, circular paper chromatography was employed in the earlier work of the Butenandt group (Butenandt et al., 1954a). Later, polyamide powder proved to be a suitable adsorbent for column chromatography (Kiibler, 1960), and conditions have been worked out which allow the isolation of rhodommatin and ommatin D in milligram to gram amounts (Butenandt et al., 1960c; Traub 1962).

Xanthommatin is normally a companion of ommins. It may be separated by taking advantage of its solubility-in the oxidized state-in dilute acids (Butenandt et al . . 1954a; Dustmann, 1964), 01- by chromatography on SE-Sephadex (Osanai and Koga, 1966). Wend (1969) has devised a chromatographic method for xanthommatin determination which results in rather broad peaks, but which might be expected to retain ommins.

The ommins are still the most refractory group in every respect. Their separation is best achieved by circular paper chromatography. Two solvent systems are available: collidin-water (3 : 1) (Butenandt et al., 1959) or formic acid-methyl alcohol-water-concentrated. HCl (68 : 15 : 12 : 2) (Linzen, 1968). The latter has been developed lrom similar systems used earlier in Butenandt's laboratory. Success of separation depends on the amounts of pigment detected and on the type and amount of impurities present. Again there are variables in this system which must first be determined. Butenandt et al. (1967) have also succeeded in purifying ommin A by treatment with phenol-water, followed by chromatography on polyamide. In the latter step, rapid elution is critical, as prolonged contact of the pigments with the column material leads 1.0 irreversible adsorption. Apparently the separation of the various ommiris is not very sharp. Even the most painstakingly purified ommin preparations usually contain small amounts of protein (Butenandt and Neubert, 1958; Butenandt et al., 1967) which may amount to 1 or 2 per cent. In the cour:je of biosynthetic studies, Linzen (1970) has examined a variety of methods (treatment with TCA, phenol, proteinases) to remove this residual contamination but has not been successful.

Finally, the ommidins may be obtained in a pure state by a small number of steps, comprising circular paper chromatography and ion-exchange chromatography (Linzen, 1966; Bouthier, 1969).

A few ommochromes have been obtained in crystalline state: dihydro- xanthommatin (Butenandt et al., 1954a), rhodommatin (as a pyridine complex: Butenandt et al., 1963), ommidin (Bouthief, 1969), cryptom-

I The properties of Ecteola vary and each batch must first be checked.

1 38 BERNT LINZEN

midin, and acridiommatin I (Bouthier, 1972). In all these cases, clusters of needle-shaped crystals are obtained. Becker (1942) also described crystallized ommatins, but according to Butenandt et al. (1954a) his preparations were of low purity.

For details the original literature should be consulted; many helpful suggestions will be found, together with descriptions of unsuccessful approaches, in the dissertations of Beckmann (1956), Neubert (1956), Linzen (1957), Baumann (1957), Traub (1962), and Kiibler (1960). Furthermore, the series of ommochrome papers by the Butenandt school will provide a broader background on phenoxazinones.

4.3 PROPERTIES

4.3.1 Solubility, aggregation and adsorption

Ommochromes have, generally speaking, many adverse properties: limited solubility, instability in both alkaline and acid media, together with a strong tendency towards aggregation and adsorption. These fa.ctors contributed to their late discovery, in spite of their wide distribution, as well as to the slow progress of ommochrome chemistry and biochemistry.

In their red, reduced state ommochromes are insoluble in all common neutral organic solvents, water (sometimes colloidal solutions are obtained), and dilute acids. To varying degrees they are soluble in alkaline and strongly acid solutions, in buffers around and above neutrality, in formamide, phenol-water, ethylene glycol, glycerol, methyl cellosolve, acetic anhydride and in solutions of urea. Exact quantitative data on solubility are not given in any case. Estimates for the solubility of ommidin in formic acid and trifluoroacetic acid are from 2 to 5 per cent, in dimethyl sulphoxide, 2 per cent, and in sodium hydroxide, more than 10 per cent Whether a pigment preparation will dissolve or not is highly dependent on its degree of dryness. Preparations which have been rigorously dried and stored for long periods require drastic treatment for dissolution. Ommochromes may decompose relatively rapidly in many of the solvents listed above. Again a systematic study has not been undertaken, but the splitting of xanthommatin in buffer solution of pH 8 (Butenandt et al., 1960b), and of ommatin D in 5 N HCl (Butenandt et al., 1960c) may serve as examples.

The insolubility of ommochromes represents one of their important physiological properties (see below); solubilization of xanthommatin by formation of the P-glucoside (rhodommatin) and the sulphate (ommatin D) is of significance in Lepidoptera.

A phenomenon no less annoying than the low solubility of ommochromes is their tendency for strong aggregation. This is evident in


the formation of colloidal solutions and the migration of ommins as “mixed” bands in paper chromatography. Elution of pigment bands and rechromatography yields a main band plus at leist the two adjoining ones. The degree of association of xanthommatiri and of three catalins (xanthommatin-like phenoxazinones employed in ophthalmology) was measured by Nakagaki et al. (1962). From the diffusion constant it was calculated that xanthommatin formed aggregates (micelles) of 2 to 5 molecules (in neutral buffer) depending on temperature (1 8 to 37” C). It may be noted that the size of the micelles is independent of concentration once a critical concentration has been passed, but depends on the properties of the solvent (e.g. ion concentration) (Scholtan, 1955). Micelle formation is a common phenomenon in the physical chemistry of pigments; it is typical of compounds with hydrophobic ini.eraction and a fairly rigid structure (Worz and Scheibe, 1969, and Brooker et al., cited by them). Both these conditions pertain in ommochromes. Certainly they are not only of theoretical significance, but also of physiological importance (e.g. in relation to ommochromes being excrei ory products deposited permanently in a living cell).

The strength of adsorption is related to the tendency to aggregate (Worz and Scheibe, 1969). It is not surprising, therefore, that ommochromes are strongly adsorbed to all kinds of material, the ommins in particular. An outstanding example is the adsorption of ommins to ordinary laboratory glass. This can be shown when a raw extract of ommins in methyl alcohol-HC1 is brought to complete dryness in a rotary evaporator, cooled, and ice-cold buffer (pH 7.5-9) carefully poured over the residue. A light brown, fluffy proteinaceous material is then brought into suspension, while the dark violet ommin mixture remains stuck tci the glass wall. In ommin isolation this step has been routinely employed.

4.3.2 Chromatography

The chromatography of ommochromes is hampered by adsorption. For elution, either strong acids, or solvents based on pyridine or its methyl derivatives have to be employed. A few of these have been mentioned above. Methods for column chromatography are described by Butenandt et al. (1960b, 1960c), Linzen (1966), Butenandt el al. (1967), Wenzl (1969), Bouthier (1969), Osanai and Koga (1966), Umebachi and Uchida (1970); for convenience the list comprises both primary and secondary sources.

For paper chromatographic identification extremely powerful eluants must also be employed. The most useful systems for ommatins (Butenandt et al., 1954a) are: (1) collidine-lutidine-water (1 : 1 : 2; upper phase) which gives a neat separation of rhodommatin, ommatin D, and ommidin, while xanthommatin is decomposed; (2) collidine-0.5 M KH2P04 (2 : 3; upper phase) by which the substituted ommatins are again fairly well separated,

140 BERNT LINZEN

while xanthommatin is found at the salt front (this is best seen under ultraviolet light; its position depends on the temperature and age of the system) and ommidin is found at about RF 0.1. There is no migration of ommins in either system, although migration of some ill-defined cherry-red materid is observed.

Ommins and ommidins are, in the author’s experience, best separated by circular paper chromatography, using formic acid-methyl alcohol-water- concentrated HCl in the ratio of 68 : 15 : 12 : 2 (50 : 15 : 20 : 1 was also employed for ommidins) (Linzen, 1966, 1967). A further system is collidin-water (3 : 1) (Butenandt et al., 1959) in which ommin A-the slowest moving in all systems-is not removed completely from the starting line (but is in the formic acid systems). Kuhn and Egelhaaf (1959) obtained good separation of the Ephestia ommins by ascending paper chromatography with a formic acid system. Even well isolated ommin bands must be subjected to rechromatography if they are to be considered pure.

The only two pigments which can be chromatographed under milder conditions are the “labile ommochromes” (acridiommatins) of locust integument. Butanol-acetic acid-water (4 : 1 : 1) and propanol-formic acid-water (1 : 1 : 4) are suitable solvent systems for these pigments.

A number of solvent systems for thin-layer chromatography have been reported by Ajami and Riddiford (1971a). However, only one or two spots are obtained from the ommin fraction, instead of the three to six expected according to experience with other material.

4.3.3 Redox properties; spectra

The most conspicuous property of ommochromes is the change from red to yellow colour observed upon oxidation. This is very uncommon, as the oxidized states of pigments are usually more deeply coloured while a lightening is brought about by reduction. This unusual behaviour of ommochromes is very useful for analytical purposes, enabling them to be easily observed in histological sections or on paper chromatograms; oxidation and reduction can be repeated many times. Spontaneous oxidation of dihydro-xanthommatin occurs if it is left in contact with air at pH values around or above neutrality, and also if a diluted solution of ommins in acid methyl alcohol is left to stand for a prolonged time.

In contrast, rhodommatin and ommatin D are stable against autoxidation, as the reduced state is protected by the substituents. If, on the other hand, neutral solutions of these two pigments are treated with ferricyanide they turn yellow immediately. Dihydro-xanthommatin is liberated by this reaction, but, surprisingly, after reduction of the solution sizable quantities of rhodommatin and ommatin D can be recovered. These observations have been interpreted in terms of the primary formation


of a phenoxazonium cation which is stabilized by mesomerization and hydrolysed in a secondary, much slower reaction (Fig. 1 ; Traub, 1962).

The oxidation-reduction potential of an omvnin preparation from ganglia and optic lobes of Cecropia silkworms was estimated to lie between 123 and 217 mV (Ajami and Riddiford, 1971a).

YOOH H2N-C-H

1 - 2 e , - H +

COOH FOOH H,N - + -H HZN-C-H

COOH

NH

0 + t i * + Glucose

Fig. 1. Oxidative splitting of rhodommatin. The intbermediary cation is stabilized by mesomerization and hydrolysed in a secondary, sbw reaction. (Proposed by Traub, 1962.)

The explanation for the peculiar spectral behaviour of reduced ommochromes has recently been given by Schafer and Geyer (1972). By comparing the spectra of numerous and. carefully selected model compounds they concluded that upon reduction a new extended resonance system is formed (Fig. 2). This system is characterized by the polar structures involving the phenoxazine nitrogen and the carbonyl groups in its neighbourhood (the 4-hydroxy-pyridine ring being in its tautomeric

142

COOH

BERNT LINZEN

Fig. 2. Structures of xanthommatin and dihydro-xanthommatin. Upon reduction a new extended resonance system is formed which is responsible for the deep red colour obtained. The location of the “central” hydrogen is not known precisely. (From Schafer and Geyer, 1972.)

pyridone form) and by the complete ring system lying in a plane. It is conceded that the hydrogen attached to the phenoxazine nitrogen might form a bridge to either carbonyl oxygen and thus contribute to the shift of the absorption band, but it is argued that this contribution is relatively small. In fact, Pfleiderer as well as Schafer (both personal communica- tions) discuss the possibility that the hydrogen atom is not attached primarily to the phenoxazine nitrogen but to the carbonyl oxygen of the pyridone ring.

Spectral data of ommochromes are presented in Table 4 and a few spectra are shown in Figs 3-8. In the publications from Butenandt’s laboratory frequently an a-value is used as a measure of specific absorbance; this is equal to absorbance of a 0.1 per cent solution in a 1 cm cell.

Phenoxazinones usually exhibit four absorption bands in the uv and visible range. These are designated A, By C, and D in the studies of Butenandt et al. (1954d, 1960a) and Schafer and Geyer (1972). Under certain conditions C and D merge, as in the case of xanthommatin in neutral solution. If ommochromes are dissolved in concentrated sulphuric acid, a violet halochromic effect is observed. Generally, the spectra of the ommins are rather poorly defined, and small shifts in wavelength of the maxima are often observed in apparently identical pigments. Linzen


TABLE 4

Spectral data of ommochromes*

log E Solvent E Amax 0.1%

Compound

Rhodommatin

Acridiommatin I

Xanthommatin 235 440 243 375 475

380 495

308 445 492 210 377 49 7

Dihydro-xanthommatin -

-

-

(265) 306 43 7

Omrnatin D -

370 490 215 305 440 230 360 443 234 3 70 47 2 424 484

Acridiommatin I1 230 368 455 448 360 465

(285)

31 000 4.49 13200 4.12 29 400 4.47

7 350 3.87 11 730 4.07 - -

4680? 3.67? 7 200 3.86 - -

8 500 3.93 4470 3.65 6400* * 3.81

34 900 3 280 7 100

(15 700) 14 300

7 200

(15 000) 3 160 7 500

33 300 13 000 6 620

-

-

-

-

-

-

-

- -

-

-

-

- -

4.54 3.52 3.85

(4.19) 4.16 3.86

(4.18) 3.50 3.87 4.52 4.11 3.82

-

-

-

-

- -

-

- - -

-

pH 7, 0.067 M

phosphate 5 N HC1

pH 7, 0.067 M phosphate ascorbate added 5 N HC1

butanol/HCl pH 7.4, 0.067 M phosphate

5 N HCI

pH 7.4, 0.067 M phosphate

5 N HCI

0.1 M.Na2HPO4

5 N HCI

HCOOH

0.1 M NazHP04

- 5 N H C I - HCOOA -

Referencet

a

a, b

C

C

d a

a

a, e

a

1

I

1

1

1

I

144

TABLE 4-continued

BERNT LINZEN

E log E Solvent Referencet xmax 0.1%

Compound

Ommidin

Cryptommidin

Ommin mixture* ,

(Crangon vulg.)

“Pigment IV”

Cinnabarinic acid

230 320 430 403 240

-43 3 48 5

426 224 438 460 228 408 5 20

(510)

530 245 375 49 7 315 435 440 45 5

8.0 - - 4.35 - 3.4 - 3.76 - 4.12 - 3.72 - 4.35 - 4.33

pH ,.. 9.5

N NaOH 5 N HCI

0.1 M NazHPOe 5 N HC1

1 N NaOH

pH 7.5, 0.067 M phosphate 5 N HCI pH 7.1

5 N HCI

pyridine

f f

i

s h

k

Data in brackets refer to inflexions; if an absorption band is expected but has not been recorded, a dash has been set.

* See text for further details. ** A value about half as high had been obtained in earlier studies (Butenandt et al.,

1960b); correspondingly, all measurements calculated with the old absorbance value must be doubled.

t a . Butenandt et al. (1960b); b. Butenandt et al. (1954d); c. Butenandt et al. (1963); d. Dustmann (1969); e. Butenandt et al. (1960~); f. Linzen (1966); g. Butenandt et aL (1958a); h. Butenandt et al. (1967); i. Bouthier (1972); k. Gripenberg et al. (1957).

(unpublished) has measured absorption spectra of several ommins isolated by circular paper chromatography and obtained the following wavelengths of the maxima: 527 nm (ommin A of Apis) , 536 nm (ommin B), 516-517 nm (ommin D); 530 nm (ommin A of Ephestiu), 537 nm (ommin B); 525 nm (ommin A of Gryllus birnaculutus), 534 nm (ommin B), 512 nm (ommin D). Ommin A and B are apparently the best defined members of the group, at present.

THE TRYPTOPHAN -* OMMOCHROME PATHWAY IN INSECTS 145

nrn

Fig. 3. Ultraviolet-visible spectra of xanthommatin in buffer solution of pH 7.0 to 7.3 '-) and 5 N HCI (- - -).

k3.4 Degradation reactions 3mmochromes are, as a rule, unstable in both alkaline and acid media. This is especially true of the ommatins which bleach rapidly. In 0.5 N sodium hydroxide at 90°C, for example, destruction is complete within 2 h, while in bicarbonate solution at room temperature two days are required. From the reaction mixture a number of fluorescent or ninhydrin-positive compounds may be isolated, of which xanthurenic acid (Butenandt et al., 1954b) and 2-amino-3-hydroxy-acetophenone (Butenandt et al., 1957e) have been identified. Ommins yield the same products, albeit under somewhat more drastic conditions (Butenandt ct al., 1958a). By acidifying md extracting into butanol the two compounlds may be readily prepared For paper chromatography.

146 BERNT LINZEN

30 OOC

20 000 u,

10 000

I I I I I I I I I I I L I I I I I

nrn

Fig. 4. Ultraviolet-visible spectra of rhodommatin in buffer solution of pH 7.0 to 7.3 +) and 5 N HCl (- - -).

These two compounds are not primary degradation products but are formed from the reaction intermediate, 3-hydroxy-kynurenine. 3- Hydroxy-kynurenine itself can be isolated only if the decomposition of xanthommatin is performed under very mild conditions (pH 8 at 37” C for 4 to 5 h). Under these circumstances also the “right-hand” part of the xanthommatin molecule is obtained (Butenandt et al., 1960b).

3-Hydroxy-kynurenine will also be split off if ommochromes are heated in a 1 : 1 mixture of formic acid and 6 N HCI for 10 to 24 h (Butenandt et al., 1958a). In the case of the ommins a brick-red pigment is also formed which has been isolated and well characterized (Butenandt and Neubert, 1958; Butenandt et al.. 1967). This has been provisionally designated

THE TRYPTOPHAN -, OMMOCHROME PATHWAY ird INSECTS

30000 -

147

200 300 400 500

nm

F i g . 5. Llltraviolet-visible spectra of ommatin D in buffer solution of pH 7.0 to 7.3 (-) and 5 N HCI (- - -).

“pigment IV”. It is at present not clear whether “pigment IV” is an integral constituent of ommins, or whether it is an artifact formed during the hydrolysis. Attempts to reconstitute an ommin from “pigment IV” and 3-hydroxy-kynurenine have failed. Linzen (1967) has shown (by paper chromatographic comparison only) that each of the individual ommins of G y l lus bimaculatus yield “pigment IV” and 3. hydroxy-kynurenine. He concluded that the difference between the ommins must be due to a third, still unknown portion of the pigment molecule. This conclusion must be viewed with some reservation, as other interpretations q e also conceivable. Yet a paper of de Almeida (1968) lends support to this hypothesis. Thus, if a solution of ommin A in formic acid is left to stand for a few days, ommin

148

20 000

10 000

2 I I I I I I I I I I I I I I I I I

1 300 400 500

nm

Fig. 6. Ultraviolet-visible spectrum of pigment “IV” in buffer solution of pH 7.0 to 7.3. (Courtesy of Dr Schaefer.)

A gradually disappears and “ommochrome I” (probably identical to ommin B) is formed; at the same time a blue fluorescing compound appears.

“Pigment IV” is a rather stable compound, but can be further broken down into another molecule of 3-hydroxy-kynurenine and a sulphur- containing hydroxy-quinone, once it has been oxidized. Butenandt et al. (1 964) synthesized 4,5,8-trihydroxy-6-mercapto-quinoline-P-carboxylic acid which they conceived could arise from degradation of “pigment IV”. However, the synthetic compound and the degradation product behaved differently on paper chromatograms and had different uv spectra.

The exception to the rule of ommochrome lability is provided by ommidin which is stable against both boiling acid and hot sodium hydroxide solution. Oxidative degradation of ommochromes has been attempted but has not met with success (Butenandt et nl., 1957d).

A remarkable reaction (in terms of enzyme specificity) is the splitting of

nm

Fig. 7. Ultraviolet-visible spectra of ommidin isolated from the migratory locust. a= E!;-.(From Linzen, 1966.)

nm

Fig. 8. Ultraviolet-visible spectra of ommin in phosphate buffer,’pH 7.5 (-, left), 5 N HCI (- - -) and concentrated sulphuric acid (--, right). a! = A$;$. (From Butenandt et al., 1958a.)

150 BERNT LINZEN

the ommatin amino acid side chain by kynureninase. Both xanthommatin and rhodommatin yield alanine, but other ommochromes have apparently not been tested.

4.4 DISTRIBUTION AND TISSUE LOCALIZATION

Ommochromes are ubiquitous in the Arthropoda and occur in a number of other invertebrates, such as Anthomedusae (Yoshida et al., 1967), Polychaeta (Dales, 1962), Echiurida (Linzen, 1959), and Cephalopoda (Schwinck, 1953; Butenandt et al., 1958b). The data relating to insects are summarized in Table 5.

The following principles have been employed in compiling Table 5. 1. No material earlier than Becker’s work has been included. Becker’s

findings are classified as a “pre-war study” to indicate lack of chromatographic separation and identification with presently known individual ommochromes.

2. Any implied criticism is meant to stimulate reinvestigation whenever appropriate. This survey revealed that virtually no species has been analysed completely with regard to the qualitative and quantitative occurrence of ommochromes. In particular the mixture of ommins has usually not been separated but has been treated as a single pigment. Certainly, much of the paper chromatographic work was intended only as a quick check on the distribution of a particular pigment and should be supplemented by detailed studies.

3. The listing includes only positive statements about the presence or absence of ommochromes (e.g. if for a particular species the occurrence of ommins in the eyes is documented, it is understood that no other tissue, or stage, has been investigated properly). Quantitative data have been included wherever appropriate. Data for “isolated” ommins are presented to give at least the order of magnitude. However, losses during purification might have been compensated by the impurities still present.

4. Statements about the redox-states of ommochromes have been avoided, as they can be assessed only by microspectrophotometry with the exception of those cases in which a striking colour change is macroscopically visible.

5. Not all references available for a particular species have been cited and often not the earliest ones in cases where later authors have provided identification in modern nomenclature. Frequent reference has been made to Becker’s work to demonstrate its broad approach and high impact. In many cases work on mutants has merely been hinted at.

From this Table it is possible to derive the following generalizations. 1 . The most widely distributed ommochrome is xanthommatin. It may

be expected, at least in trace amounts, whenever other ommochromes are observed (with one exception described under point 2). As ommochrome

TABLE 5

Distribution of ommochromes among insects

Order, species Details of ommochrome pigmentation Criticism* Reference?

ODONATA Anax imperator LEACH

Aeschnn cyanea Aeschnn juncea L. Aeschna mixta Sympetrum flaueolum L.

Sympetrum sanguineum 0 . - F .

Sympetrum wlgatum M ~ ~ L L E R

“Pooled Sympetsum species”

ORTHOPTERA Gryllus bimaculatus DE GEER

Gryllus domesticus L. Gryllotalpa vulgaris L.

Eyes: xanthommatin

“Ommatin in eggs” Ommin mixture in eyes “Ommatin” and “ommin” in eyes Xanthommatin (? ) in eyes

Xanthommatin in eyes

“Ommatin” and “ommin” in eyes; “ommatin” as hypodermal pigment in diaphragm and fat body Acridiommatin I as hypodermal pigment

Eyes: ommidin as main pigment, accompanied by xanthommatin and ommins. Integument: ommin mixture, large quantity in abdomen, source for micropreparative isolation. Approximate ratio of the four slowest moving ommins 3 : 5 : 4 : 3 (Linzen, unpublished). Traces of xanthommatin Eyes: wnthommatin Ommin mixture in eyes

PC only, superficial, ommins expected. Integument? Prewar study PC only. Integument? Re-war study PC only; ommins expected. Integument ? PC only; ommins expected. Integument? Re-war study

Preliminary study

Qualitative data well documented; no quantitative data

Preliminary PC. Other pigments? Preliminary PC. Xanthommatin?

C

b a b C

C

b, d

PP

c, e , f

C

a

TABLE 5-continued

Order, species Details of ommochrome pigmentation Giticism* Referencet

ORTHOPTERA

Rornalea rnicroptera BEAUV.

Tylotropidius speciosus WALK. Acanthacris ruficornis fulva

Anacridiurn aegyptiurn L. No mada cris sep te rn fascia ta

Ornithacris cyanea STOLL. Schistocerca gregaria FORSK.

SJOST.

SERV.

Locusta m e a t o r i a L.

Oedipoda coerulescens L. Dociostaurus maroccanus

Further species

Eyes: ommidin main pigment, cryptommidin, some xanthommatin. Integument: same pigments Eyes: ommidin, xanthommatin Eyes: ommidin, xanthommatin

“Insectorubin” (acridiommatins) in integument Eyes: ommidin

Eyes: ommidin Eyes: ommidin (main pigment), xanthommatin. Integument: acridiommatins I and 11. Quantitative data on total ommochrome (“insectorubin”) content under various conditions given by Goodwin and Srisukh (1950b) Eyes: ommidin (main pigment), cryptommidin, xanthommatin (?), acridiommatins I and 11. Integument: acridiommatins I and 11, xanthommatin (artifact?). Faeces: acridiommatins I and 11. Ommochrome quantity reduced in integument of albino mutant, not in eyes Eyes: ommidin. Whole animals: xanthommatin Eyes: ommin-like pigment, “yellow pigment in great quantity” (ommidin?) “Ommatins” as hypodermal pigments and a t inacrtion of musculature

Only qualitative data

PC only; superficial PC only; superficial

Pigments not separated PC only; superficial

PC only; superficial Not as well documented as for Locusta

Qualitative data well documented; quantitative data desirable

Superficially studied No identification by modem techniques Prewar study

f

f f

nn

f

f c. f

b

PHASMIDA Carausius morosus BRUNNER

DICI’YOPTERA Blatta orientalis L.

Mantis religwsa L.

HEMIPTERA LaccifL.r lacca

Rhodnius prolixus

Gerris lacustris L.

NEUROPTERA Chrysopa carnea

Chrysopa vulgaris SCHN.

LEPIDOPTERA cossus cossus L.

Eyes: ommins, xanthommatin, no ommidin. Integument: ommins and xanthommatin; quantity dependent on environmental conditions. Extremes: 5 pg of ommins, trace of xanthommatin in light animals; >540 pg of ommins, 260 pg of xanthommatin after dark adaptation

Eyes: ommin mixture, 3.2 mg isolated from 3500 animals, including larvae. No ommochrome isolated from rest of body Integument: xanthommatin, ommins

Lacciferic acid and laccaic acid isolated from whole larvae; assumed to be ommin-like Eyes: ommins, traces of xanthommatin. No ommochromes in body, wings, eggs, or excreta Eyes: ommins

Whole animals: 3.1 and 6.1 & per animal of xanthommatin in green and brown specimens, respectively “Ommatin” in integument

Larvae: xanthommatin in hypodermis

Careful quantitative analysis; ommins not separated

Ommins not separated. Xanthom- a

matin?

Ommins not separated. Eye 00

pigments?

Further identification required n

PC only c, m, d

PC only; superficial a

Eye pigments? 0

Pre-war study b

Well documented; other stages not investigated

ff

TABLE 5’-continued

Order, species Details of ommochrome pigmentation Criticism* Ref erencet

LEPIDOPTERA

Galleria melonella F.

Ephestia kiihniella 2.

Ptychopoda seriata SCHRK.

Plodia mterpunctella

Antheraea pernyi Hyalophora cecropia

Bombyx mori L.

Eyes: ommins and xanthommatin. No ommochromes in wings Larvae: ommins in ocelli, ommatin (xanthommatin?) in hypodermis. Moths: 3 ommins and xanthommatin in eyes and testis sheaths; ommochromes associated with nervous tissue; a number of mutants studied Larvae: ommatin (xanthommatin?) in hypodermis. Moths: same ommochromes in eyes as in Ephestia; ommochromes surrounding nervous system; “ommatin” in excreta Eyes: 3 ommins (ommin A, ommochromes I and I1 in Kiihns nomenclature) and xanthommatin. No ommochromes in wing scales. In mutant ra only xanthommatin As in Hyalophora cecropia below Eyes: ommins, xanthommatin. Ganglia and optic lobes: ommins (no xanthommatin?). No ommochromes in red areas of wings, nor in meconium Eggs: xanthommatin (0.22 mgg-’), 3 ommins (1.7 mg g-’ 1. Larval hypodermis: xanthommatin (= + - chrome “I”). Excreta: cinnabarinic acid. Adult eyes: ommin mixture (-4 pg per animal isolated). Several mutants investigated by Kawase (1955) and by Inagami (1958).

PC only a. c

Qualitatively well documented; a, b, c. d, s quantitative data desirable

Partly pre-war study, but eye pigments separated by PC

Qualitative study. Other stages?

Qualitatively well documented; no separation of ommins

Many qualified experiments by various workers; ommins might be better defined

b, m m

Vanessa (Pyrameis) atalanta L.

Vanessa (Pyrameu) cardui L.

Aglais (Vanessa) urticae L.

Inachis (Vanessa) io L.

Araschnia laevana L. Argynnis paphia L.

Hestina japonica

Sasakia charonda

Xanthommatin (protein-bound) in haemolymph of rb mutant Meconia of imago: rhodommatin and ommatin D (cf. Table 6 ) . Same pigments in wings. Eyes: ommins and xanthommatin As in V. atalanta (cf. Table 6 for quantitative data on excreted ommochromes) Larval and pupal hypodermis: wnthommatin. Meconium: rhodommatin and ommatin D, no xanthommatin. Wings: rhodommatin, ommatin D (-20 and -5 pg per animal, respectively), no xanthommatin. Eyes: ommins (9 pg per animal isolated), xanthommatin Larval hypodermis: xanthommatin (?). Meconia, wings, and eyes of imago: same ommochromes as in Aglais urticae Cut contents of pupa: rhodommatin and ommatin D Larvae and pupae: xanthommatin in hypodermis of aii stages. ivieconium of imago: rhodommatm and ommatin D (cf. Table 6). no xanthommatin. Wings of both sexes: rhodommatin and ommatin D, no xanthommatin. Eyes: xanthommatin (3 pg per eye isolated), ommins. In valesina strain only traces of ommatins in wings Xanthommatin in hypodermis of hibernating larvae; rhodommatin in gut contents at end of &pause Identical results as in Hestina above

Well documented, ommins not a, c, m separated. Larvae?

Well documented, ommins not separated. Larvae? Well documented a. c, m

c, m

Well documented, but no quantitative data

PC only Well documented

a, c. m

m

c, m

Quantitative study might reveal ee further interesting results. NO ommatin D ?

ee

TABLE 5-continued

Order, species Details of ommochrome pigmentation Criticism* Referencet

LEPIDOPTERA

Heliconius spec.

Aporia crataegi L.

Pieris brassicae L.

Colias edusa F. Papilio machaon L.

Papilio xuthus

Parmssius apollo L.

Biston cetularia Bupalus piniarius L.

Sphinx ligustri L.

Sphinx pimstr i L.

Deilephila (Pergesa) elpenor

Wings: xanthommatin (possibly artifact). Eyes: ommin mixture Larvae: xanthommatin in hypodermis. Meconium: rhodommatin and ommatin D (cf. Table 6). Eyes: ommins and xanthommatin Traces of rhodommatin and ommatin D in meconium Eyes: ommin mixture Eyes: ommins and xanthommatin. No ommochrome in wings Testes: 0.18 pmol of xanthommatin per animal. Eyes: 0.6 pmol of xanthommatin per animal, plus ommin mixture. Wings: yellow pigments derived from kynurenine belong to different pigment class Eyes: ommin mixture. Wings: red spots not containing ommochromes Eyes: ommins Eyes: ommins and xanthommatin. No ommochromes in wings, whole pupae, meconia Larval hypodermis: xanthommatin. Meconia: rhodommatin and ommatin D. Eyes: ommins Eyes: ommins and xanthommatin. No ommochromes in wings or meconia Eyes: ommins and xanthommatin. Pink pigmentation of body and wings not based on ommochromes

a, c PC only

PC only. Eye pigment?

PC only. Xanthommatin? PC only

Well documented; estimate of xanthommatin in eyes appears high

PC only; superficial

PC only; superficial PC only

Limited qualitative study

PC only

PC only

C

a

a. c

dd

a

a

c , m

Cerura uinuh L.

Agrotis comes Scoliopteryx libatrix L.

DIPTERA

Tipula oleracea L.

Culex pipiens L. Phryne fenestralis

Tabanus spec. (bovinus?) Luphria gibbosa Dwctria atricapilla Syrphus pyrastri L. Tubifera pendula L. Ceratitis capitata WIED.

Drosophila rnelanogaster MEIG.

Musca domestica I..

Larvae: xanthommatin in “saddle-patch”. dihydroxanthommatin in neck fold; spectacular colour change due to ommochrome synthesis a t end of feeding period. Peak quantities: xanthommatin 0.6 mg; rhodommatin 0.5 mg; ommatin D 0.5 mg. Rhodommatin and ommatin D in meconia. (See page 175) Eyes: ommins Eyes: ommins and xanthommatin. No ommochromes in wings or meconia

Eyes: ommins (9 pg per animal isolated), xanthommatin Eyes: unidentified ommochromes Eyes: ommins, “ommatin”. An ommatin in portions of fat body underlying hypodermis, and in testis sheath, Eyes: ommins Eyes: ommins, “ommatin” Eyes: ommins Eyes: xanthommatin only ommochrome “Ommatin” in abdominal pigment tissue (fat body?) Eyes: xanthommatin only ommochrome (-6 pg per animal; original estimate -13 pg) Eyes: xanthommatin (“brown pigment”). In Malpighian tubules of red mutant xanthommatin and ommin Eyes: xanthommatin only ommochrome. Several mutants studied quantitatively

Very detailed analysis, but adult animals not investigated

PC only; superficial PC only

Not adequately studied Prewar study

PC only; xanthommatin? Prewar study PC only; xanthommatin? PC only Prewar study

U

a

c, m

a, c

gg b

a

b a C

b 11

c, hh

c, ii

TABLE 5-continued

Order, species Details of ommochrome pigmentation Criticism* Referencet

DIPTERA

Calliphora erythrocephala MEIGEN

Phormin terraenovae

4 related species

HYMENOPTERA Ha bro bra con jugla nd is

Phobocampe unicincta ASHMEAD

Eyes: xanthommatin only ommochrome (-50 pg per animal; original estimate -80 pg). “Ommatin” in testes Eyes: xanthommatin only ommochrome (-35 pg per animal) Eyes: xanthommatin still detectable in 50-year- old museum specimens

Ommochromes (xanthommatin and “ommochrome 11”) in eyes only Whole pupae (early stage) “filled with dihydro- xanthommatin”; in late pupae reduced amount of

Well documented b, k k , rr

ss

C

Mixture of all ommins more likely than “ommochrome 11” alone Localization of pigment in pupal tissues?

r

c, m

oxidized xanthommatin and no other ommochromes Eyes: xanthommatin, ommins

Atta sexdens Eyes: ommin mixture PC only a Apis mellifica L. Ommin and xanthommatin content in eyes (pg per Age not given by Dustmann a, c, P, q

animal): 25 and 22 (drones); 14 and 13 (worker bees); 9 and 7 (queens); 11.6 and 27 (worker bees, age 10 days, data of Neese). Ommochrome synthesis continues after ecdysis; many mutants investigated by Dustmann 11968, 1969). Ommin mixture consists of four components, ratio 4 : 17 : 42 : 37 (ommin A = 4) (Linzen, unpublished)

COLEOPTERA Cicindela spec. Eyes: ommin, “yellow component” Pre-war study d Melolontha uulgaris L. Eyes: ommin mixture PC only Tenebrio molitor Pigment of Malpighian tubules resembles ommins Prewar study

a b

* PC, paper chromatography. “Pre-war study” designates lack of identification according to present standards (no chromatography, spectra missing or poor).

f a . Butenandt e ta l . (1958b); b. Becker (1942);~. Butenandt etal. (1960b); d. Becker (1939); e. Fuzeau-Braesch (1957);f. Linzen (1966); g. Fuzeau-Braesch (1968); h. Bouthier (1966); i Bouthier (1969); k. Calarza and Stamm (1959); 1. Dustmann (1964); m. Kiibler (1960); n. Singh et al. (1966); 0. Riidiger and Klose (1970); p. Dustmann (1966); q. Neese (1972); r. Leibenguth (1967a); s. Kiihn and Egelhaaf (1959); t. Mohlmann (1958); u. Linzen and Buckmann (1961); u. Kuhn and de Almeida (1961); w . Neubert (1956); x. Ajami and Riddiford (1971a);y. Kawase (1955); z. Inagami (1958); an. Koga and Osanai (1967); bb. Ishiguro and Nagamura (1971a. 1971b); cc. Butenandt et al. (1959); dd. Umebachi and Uchida (1970); ee. Osanai (1966a);ff. Merlini and Nasini (1968);gg. Dennhofer (1971); hh. Wessing and Bonse (1966); ii. Hiraga (1964); kk. Butenandt and Neubert (1955); 11. Ziegler and Feron (1965); mm. Kuhn (1963);nn. Colombo et al. (1955); 00. Vuilleaume (1968); pp. Bouthier (personal communication); IT. Linzen (1963); 5s. Linzen and Schartau (to be published).

1 60 BERNT LINZEN

synthesis is an oxidation process, and as 3-hydroxy-kynurenine is very reactive, the formation of xanthommatin as a by-product seems to be almost unavoidable. Xanthommatin is a regular companion of ommins. One might speculate (though there is yet no evidence to support such a speculation) that the varying proportion of xanthommatin to ommins depends mainly on the amount of the other ommin precursors which are derived from methionine. While in most cases of joint occurrence, ommins (as a group) appear to be predominant, they are at least equalled b y the amount of xanthommatin in bees.

On the other hand, there are a few species, all belonging to the Diptera, Cyclorrapha, in which xanthommatin appears to be the only ommochrome present in the eyes: Calliphora, Musca, Drosophila, Syrphus. For some time it was thought that these species had abandoned ommins completely, and that this represented a trait useful for chemotaxonomy. In 1966, however, Wessing and Bonse demonstrated ommins in Malpighian tubules of the Drosophila red mutant. Further chemical proof is desirable to strengthen this important finding.

Xanthommatin may be the only ommochrome in the hypodermis of Lepidopteran larvae (Ceruru, Ephestia, Vanessa), but is certainly not the only ommochrome in internal pigment tissues. Thus, ommins have been found in ganglia of Hyalophora cecropia (Ajami and Riddiford, 1971a), while the testis sheath of Ephestia contains the whole series of Ephestk ommochromes (Kuhn and Egelhaaf, 1959). Conversely, in Papilio xuthus xanthommatin is the only ommochrome in the testes (Umebachi and Uchida, 1970).

2. Xanthommatin is unlikely to be a native pigment if it is found in conjunction with rhodommatin and ommatin D (as in butterfly wings and meconia), since it is a degradation product of these pigments. Ommatin D liberates sulphuric acid upon hydrolysis, thereby initiating an autocatalytic reaction. Kubler (1960) and Butenandt et ul. (1960b) examined this problem very carefully by paper and column chromatography of freshly deposited Nymphalid meconia and were unable to detect even traces of xanthommatin if the meconia were processed immediately. Similarly, xanthommatin is absent from fresh wing material of Pyrumeis atalunta and Vanessa urticae, although it assumes an increasing proportion of the total ommatins in aged material (see also Linzen and Biickmann 1961). In retrospect it can be seen that it was fortunate that in the early phase of ommochrome chemistry xanthommatin should arise as an artifact by decomposition of the two other ommatins. It was thus isolated in high yield from Vanessa meconia and, being the simplest of the ommatins, the first of which the chemical structure could be established.

Rhodommatin and ommatin D apparently always occur jointly, although the instability of ommatin D might present some difficulties in its

THE TRYPTOPHAN + OMMOGHROME PATHWAY IN INSECTS 7 61

detection. These two ommatins have only been ohserved in Lepidoptera, an astonishing fact in view of the widespread ability of insects to make fl-glucosides and sulphuric acid esters. They haw been found only in fat body, haemolymph, Malpighian tubules, gut, excreta and wings. and are in all probability excretory products. Rhodommatin and ommatin D are never in the form of granules, being either diffusely distributed or dissolved in vacuoles. While in meconia rhodommatin and ommatin D have been found in approximately equal amounts (Butenandt et al. , 1960b), there is some indication that ommatin D predominates in Rhopaloceran wings.

3. Ommidin and cryptommidin are characteristic of the Orthoptera and have not been described in other insect orders. Ommidin also appears to be present in small amounts in Limulus. The ommidins are associated with xanthommatin (locust eyes), but may be also associated with ommins (Gryllus eyes). They are probably closely related to the latter by their common derivation from both tryptophan and methionin. Ommidins are bound to small granules as are the ommins. They are typical eye pigments, but in one instance, in Romalea microptera, they have been extracted from hypodermis (Linzen, 1966). 4. The ommins never occur singly, but always as a group of at least

three, five or six (“at least six” in the case of Sepia; Butenandt et al., 1959). The quantitative relations of these have been determined by the author only in two cases (Gryllus bimaculutus, Apis) and only by making gross simplifications (“amount” =A5mnm x vo ume). Even in these two cases there appeared major quantitative differences which show that the composition of a given mixture of ommins is species specific.

Next to xanthommatin, ommins are the most widespread ommochromes, being absent only from part of the Orthoptera and from a number of Diptera. In many, if not in most, insect species they are the dominant ommochromes. They occur in eyes, integument, pigment sheaths of testes, ganglia, and as egg pigments. They have, however, not been demonstrated in Lepidopteran wings, nor excreta; the substii.uted ommatins and the ommins appear to exclude each other.

A simple phenoxazinone has recently been identified in the silkworm: cinnabarinic acid (2-amino-phenoxazin-3-one-l,9dicarboxylic acid; Ishiguro and Nagamura, 1971a), which had previously been isolated from tropical mushroom species (Gripenberg et al.. 1957; Gripenberg, 1958). Cinnabarinic acid originates from 3-hydroxy-an1 hranilic acid which is a major metabolite of tryptophan in the silkworm. In the mutant rb (which is deficient in kynureninase and accumulates 3-hydroxy-kynurenine) xanthommatin is formed instead.

As a whole, ommochrome pigmentation might still be expected to yield a number of interesting results. For example, the xridiommatins formed in the hypodermis of the migratory locust have been little studied. From the

166 BERNT LINZEN

synthesized in insect eyes are yellowish brown in early stages, to mention only a few examples. Biickmann (1964, 1965) has pursued this problem in the case of epidermal xanthommatin in Cerura, and has denied a respiratory function, though he surmised that in periods of anoxia xanthommatin might play a minor function as an electron acceptor.

Ommochromes are thus unlikely to have a primary function as redox system. On the other hand, the evidence for a function as screening and pattern pigments, and for the deposition of ommochromes as metabolic end products is convincing and will be considered in some detail.

5.1 OMMOCHROMES AS SCREENING PIGMENTS

The light receptors of arthropod compound eyes are surrounded by the pigment cells in such a way that the screening function of ommochromes appears self evident. Screening affects light perception in two ways: first, the total light energy impinging on the receptors is reduced (so as to lower the sensitivity) and, secondly, the largely unidirectional light impinging on the receptors results in an increase in acuity. The first of these two’ principles is critical in marine or nocturnal animals and is counteracted by mechanisms which move the screening pigment in response to light intensity. It is also relevant in the perception of light of long wavelengths, where absorption by visual pigments is low.

A comparison of the absorption spectra of ommochromes in solution and of ommochromes in situ reveals some instructive differences and shows the degree of adaptation of these screening pigments at every level of organization. Microspectrophotometry has been applied to the study of ommochromes in Musca dornestica by Strother (1966). A detailed investigation of Calliphora screening pigments (xanthommatin, pteridines) has been carried out by Langer (1967) and of ommin granules by Hoglund et al. (1970) and by Langer and Struwe (1972).

Both the oxidized and the reduced forms of xanthommatin occur in vivo. This was first demonstrated in Calliphora by conventional light microscopy by Hanser (1959). However, Langer’s measurements on individual and small groups of granules suggest that there might be two different states of xanthommatin. In one type of granule it appears to be partially oxidized (to an unknown and certainly variable percentage) and possibly to be in the free state; in the other it is mainly reduced and bound to protein. The comparison of Calliphora mutants has been helpful in this deduction. In the white mutant of Calliphora, bright yellow granules are observed which exhibit absorption spectra very close to the spectrum of (oxidized) xanthommatin in solution (Ama = 435 nm). In the wild type, brownish-yellow granules are found. Their spectra may be imitated by a solution of fully reduced xanthommatin which has been left in contact

THE TRYPTOPHAN + OMMOCH ROME PATHWAY IN INSECTS 163

of colour, depending on the state of pigment (see p. 166). The distribution of pigment granules in Drosophila wild-type eyes and in various mutants has been described by Zeutschel (1958) and try Nolte (1950). Nolte also disputes earlier histological literature. Two detailed electron microscopic studies have been published by Shoup (1966) and Fuge (1967) who arrive at similar conclusions, dissenting only in the origin of the granules. Although pigment deposition might conceivably be preceded by the formation of colourless “matrix” granules, it is clear from their observations that both occur simultaneously. Granule formation com- mences abruptly at 44 to 48 h of pupal age. Shoup frequently observed small ommochrome granules (“Type I” granules in her nomenclature) adjacent to Golgi regions. Fuge, however, denied this and considered small cisternae of the endoplasmic reticulum to be precursors of the granules. These, it was postulated, were then filled with pigment and thereby increased in size. Fuge, on the other hand, suggested a derivation of drosopterin granules from Golgi vesicles, for which Shoup does not find any evidence. The problem remains unsettled m d justifies a further close investigation of the initial phases of granule biogenesis.

Growth of the granules is characterized by a linear increase of the diameter which doubles about every 24 h. Schwabl and Linzen (1972) measured granule growth in basal and approximately mid-ommatidial regions of the secondary pigment cells in Drosophila.They found significant size differences, the basal granules being larger from the initiation of pigmentation.

The above work does not provide evidence of any “matrix”, “ground substance”, or “carrier” of the pigment in Drosophila. Although microspectrophotometry might provide an answer by determining the position of maximal pigment absorption, direct positive evidence is provided by Schneider’s electron micrographs of Calliphora wild-type and chalky eyes (Langer, 1967). The chalky mutant which is completely devoid of all pigments, nevertheless, contains granules of appropriate size and shape which are filled by very fine granular, opaque material. In Ephestio Hanser (1946, 1948) demonstrated “carrier granules”, which lightly stained with haematoxilin after removal of the pigment. She also described “pre- granules” which subsequently took on reddish pigmentation. These granules which carry ommins are again surrounded by a membrane and are probably derived from vesicles formed by the Golgi apparatus and which actually decrease to reach their final size (0.5 to 0.6pm) while they are gradually filled with electron-dense material (Horstmann, 1971). Maier’s (1965) earlier findings of “structural complexes” as granule precursors appear to be a misinterpretation.

It appears then that the existence of a carrier protein in the pigment granules is beyond doubt, even if this is not apparent in the v and cn

164 BERNT LINZEN

mutants of Drosophila. The absence of “empty” granules in these mutants may be explained by the assumption that ommochrome precursors are required for the formation of the granules. In fact by feeding kynurenine to u larvae, and 3-hydroxy-kynurenine to cn larvae, normal growth of pigment granules is induced (Schwabl and Linzen, 1972). These granules also show a membrane.

One might suppose that the growing granules carry the complete pigment synthesizing apparatus which is gradually covered by pigment (and probably inactivated). This would correspond with the development of the melanosome. In Ephestiu, Muth (1967, 1968 1969) has shown that the capacity for ommochrome synthesis in the eye is influenced by the amount of precursor (administered kynurenine or 3-hydroxy-kynurenine). Muth developed a mathematical model which was able to describe pigment synthesis and comprised an induction of the biosynthetic apparatus.

Langer and Struwe (1972) attempted to estimate the pigment concentration within the granules from microspectrophotometric measurements. They arrived at a value of about 0.5 M , corresponding to roughly 20 per cent of the fresh weight.

It might be added that Fuzeau-Braesch (1971) observed using the scanning electron microscope, “nicely spherical’’ ommochrome and pteridine granules of 2 to 5 pm in diameter, in two Orthoptera.

Virtually no biochemistry has been done on ommochrome granules. The author is also extremely sceptical about the few papers published because of his doubts about the purity of the preparations employed. It is, in fact, not easy to obtain pure pigment granules, and a small contamination by other particles or membrane fractions may simulate metabolic properties actually not present. On the basis of histological stiining the granules were assumed to contain ribonucleic acid (Caspari and Richards, 1947/1948). Bellafny (1958) prepared “insectorubin particles” from locust tissues and measured a number of mitochondrial activities; he cautiously concluded that insectorubin particles were a species of mitochondria. Likewise, Ziegler and Jaenicke (1959) determined a number of mitochondrial enzymes in preparations of Drosophila pigment granules which, according to a re-examination in the author’s laboratory, could not have been more than 50 per cent pure. Currently, the xanthommatin granules of Culliphoru are being studied in the author’s laboratory. They are separated by density gradient centrifugation; their density was estimated at about 1.30. Purity was checked by the ratio of xanthommatin-residual nitrogen and by assay for succinic dehydrogenase. The best preparations were completely devoid of the latter enzyme.

4.6 BINDING OF OMMOCHROMES TO PROTEINS

Although it had been generally appreciated, since Becker’s work, that ommochromes were bound to proteins few attempts have been made to


isolate such chromoproteids. Bowness and Violken (1959) reported on a pigment from housefly heads (identification of the chromophore is lacking) which was bleached by strong light and also in the dark at pH 8. Yoshida et al. (1967) compared light-sensitive pigments from Anthomedusan ocelli and Lucilia caesar and stated that a macromo1ec:ular pigment extracted with cetyltrimethylammonium bromide behaved identically in chromatography on Sephadex G-100. The Japanese workers identified xanthommatin as a chromophoric group of this material. More recently, Ishiguro and Nagamura (1971b) and Ajami and Riddiford (1971b) isolated ommochrome-proteids. By extraction with Ringer solution, precipitation with ammonium sulphate, filtration through Sephadex G-200 and chromatography on DEAE-cellulose, the Japanese workers isolated a xanthommatin-carrying protein from the haeinolymph of the rb mutant of Bombyx mori which appeared homogenous in disc electrophoresis. From the xanthommatin content of 15-30 pg mg-’ protein, the molecular weight may be calculated to be above 140 000 Daltons. The Harvard group separated a xanthommatin-proteid from an ommin-proteid by gel filtration. The latter had a molecular weight -of about 24 000 Daltons; the redox potential at pH 7 was found to be 196 f 7 mV. The absorption maxima of both chromoproteids are situated at surprisingly short wavelengths and are not at all comparable to the microspectrophotometric data (Langer 1967; Hoglund et al., 1370) of whole granules :in v i m . The ommin-proteid actually absorbs at shorter wavelengths than purified ommin in solution, so that some doubts may arise concerning denaturation of the chromoproteids during isolation.

5 Functions of ommochromes An understanding of the functions of ommothromes requires a knowledge of the localization of these pigments and their characteristic properties: redox behaviour, absorption of ultraviolet and visible light, and low solubility. These properties could enable omrnochromes to act as electron accepting or donating systems, as authentic functional pigments and as metabolic end products. Of these possible roles, the participation in electron transfer has intrigued many worker!;, and continues to do so. A respiratory function of ommochromes was suggested by Horowitz (1940), Horowitz and Baumberger (1941), Bellamy (1958), and Harano and Chino (1971) and has been implied by other workers. Such a function might appear most likely, for a change from the oxidized to the reduced state is in fact observed in some species. The eggs of Ure(zhis caupo, for example. turn from yellow to red during ripening (Horowitz, ” 1940; Horowitz and Baumberger, 1941), while the larva of Ceruru vinula displays a spectacular colour change prior to pupation. This starts by a reduction of xanthommatin (Biickmann, papers to be cited below). The ommochromes

166 BERNT LINZEN

synthesized in insect eyes are yellowish brown in early stages, to mention only a few examples. Biickmann (1964, 1965) has pursued this problem in the case of epidermal xanthommatin in Cerura, and has denied a respiratory function, though he surmised that in periods of anoxia xanthommatin might play a minor function as an electron acceptor.

Ommochromes are thus unlikely to have a primary function as redox system. On the other hand, the evidence for a function as screening and pattern pigments, and for the deposition of ommochromes as metabolic end products is convincing and will be considered in some detail.

5.1 OMMOCHROMES AS SCREENING PIGMENTS

The light receptors of arthropod compound eyes are surrounded by the pigment cells in such a way that the screening function of ommochromes appears self evident. Screening affects light perception in two ways: first, the total light energy impinging on the receptors is reduced (so as to lower the sensitivity) and, secondly, the largely unidirectional light impinging on the receptors results in an increase in acuity. The first of these two’ principles is critical in marine or nocturnal animals and is counteracted by mechanisms which move the screening pigment in response to light intensity. It is also relevant in the perception of light of long wavelengths, where absorption by visual pigments is low.

A comparison of the absorption spectra of ommochromes in solution and of ommochromes in situ reveals some instructive differences and shows the degree of adaptation of these screening pigments at every level of organization. Microspectrophotometry has been applied to the study of ommochromes in Musca dornestica by Strother (1966). A detailed investigation of Calliphora screening pigments (xanthommatin, pteridines) has been carried out by Langer (1967) and of ommin granules by Hoglund et al. (1970) and by Langer and Struwe (1972).

Both the oxidized and the reduced forms of xanthommatin occur in vivo. This was first demonstrated in Calliphora by conventional light microscopy by Hanser (1959). However, Langer’s measurements on individual and small groups of granules suggest that there might be two different states of xanthommatin. In one type of granule it appears to be partially oxidized (to an unknown and certainly variable percentage) and possibly to be in the free state; in the other it is mainly reduced and bound to protein. The comparison of Calliphora mutants has been helpful in this deduction. In the white mutant of Calliphora, bright yellow granules are observed which exhibit absorption spectra very close to the spectrum of (oxidized) xanthommatin in solution (Ama = 435 nm). In the wild type, brownish-yellow granules are found. Their spectra may be imitated by a solution of fully reduced xanthommatin which has been left in contact

THE TRYPTOPHAN + OMMOCH ROME PATHWAY ird INSECTS 167

with air for a prolonged period (Amax = 490 nm I. Granules which appear bright red in the light microscope (the most numerous in the wild type) absorb maximally between 520 and 560 nm. If these are extracted with glycerol, so as to remove unbound ommochrome, maximal absorption occurs at 560 nm. The large bathochromic shift. from 492 nm (dihydro- xanthommatin in solution) to 560 nm is due to the binding of the pigment to protein. A bathochromic shift was also measured by reflection spectrometry of the integument of Hestina japonica (Osanai. 1966b).

To appreciate the natural situation it is necessary to superimpose the various spectra. Making some reasonable assumptions, and taking into consideration the absorption of blue light by the pteridines also present, a more or less even level of absorption is obtained which extends from 300 to 590 nm. This corresponds to the situation revealed by microspectrophotometry in a radially illuminated eye in which a bleached rhabdome is used as reference spot (Fig. 9). The total absorbance can be estimated to exceed 6. The absorbance of single granules lies tietween 0.25 (Langer and Struwe, 1972) and 1.0 (Langer, personal communication). Thus, the ommochrome screen constitutes a homogenous gTey filter with extremely low transmission and a sharp cut-off at 600 to 640 nm.

When ommins constitute the major screening pigment the situation is relatively simple, as the oxidized state is not involved. The absorption maximum of pigment granules of the moth, Cderio euphorbiae, lies at

nm

Fig. 9. Microspectrophotometry of Culliphoru pigment cells, demonstrating neutral grey filter function and cut-off at 600 nm in wild-type individuals (a) and effect of mutation to white (b). (Traced from an original recording in Langer, 1967.)

168 BERNT LINZEN

547 nm (Hoglund et al., 1970); the bathochromic shift caused by binding of the ommins to protein is therefore only 20 to 30 nm. In Heliconius the shift is a little greater (Langer and Struwe, 1972). Measurement of individual granules has revealed variations in light absorption between 350 and 540 nm. Difference spectra suggest that this might be due to a variable admixture of oxidized xanthommatin. Again, the general shape of the spectrum is that of a neutral grey filter with a cut-off just below 600 nrn.

The high absorbance values of this filter results in a substantial decrease in sensitivity in comparison to mutants devoid of screening pigment. Autrum (1955) compared electroretinograms of Calliphora wild-type and white eyes and found that the sensitivity of the mutant eyes was 100 times higher for red light, but l o 3 to l o 4 times higher for light of shorter wavelengths. If the amplitude of the “on-effect” (in mV) is plotted against wavelength a single maximum near 510 nm is obtained in the mutant, whereas a second maximum around 640 nm appears in the wild-type, following elevation of the quantum level. This means that stray light becomes effective (by exciting a greater number of receptors) in the region of high transmission of the pigment screen in spite of the low sensitivity of the receptors at this wavelength. The higher proportion of stray light was also demonstrated by measuring single cell potentials (Washizu et al. , 1964). The mutant chalky, which is not only devoid of ommochromes but also of pteridines, shows still higher “on-effects” in the short wavelength region. The carbohydrate utilization, measured in isolated irradiated heads, was also found to be highest in the eyes of the chalky mutant (Langer and Hoffmann, 1966).

The decrease in visual acuity associated with the absence of ommochromes has been studied by several workers (Drosophila: Kalmus, 1943; Fingerman, 1952; Gotz, 1964; Hengstenberg and Gotz, 1967; Burnet etaf., 1968; Wehner et al., 1969. Calliphora: Autrum, 1961. Apis: Neese, 1968). In Calliphora the optomotor responses of wild-type and white flies were identical as long as bright illumination prevailed. At low light intensities, however, acuity was reduced in the mutant (Autrum, 1961). Similarly contrast perception is lowered in Drosophila mutants wa and w , due to the higher proportion of stray light relative to total light intensity. The relative light intensities reaching the photoreceptors of +, se, wa , and w eyes were estimated at 1 : 1 : 7 : 19 (Gotz, 1964; Hengstenberg and G6tz, 1967), similar proportions also being obtained by independent experiments (Wehner et al., 1969). In Drosophila u bw the visual acuity increased i f the larvae were fed increasing amounts of kynurenine, causing biosynthesis of ommochromes (Burnet et al., 1968).

In the chartreuse mutant of the honey bee, which lacks all n m m o chromes at the time of emergence and which forms only xanthommatin and traces of ommins during imaginal life, orientation is severely impeded.

THE TRYPTOPHAN -+ OMMOCHROME PATHWAY IN INSECTS 169

Chartreuse worker bees are inferior collectors in comparison to the wild-type individuals. They show lowered contrast perception in training experiments, and reduced flight speed in field experiments, and this becomes more marked in areas which offer few orientation marks (Neese, 1968).

In conclusion, it would appear that visual acuity of the compound eye is reduced by the lack of ommochromes. This does not result from an increase in the visual field of a’ single ommatidium, but from the increased “background illumination” of the receptors, which makes it more difficult to distinguish between areas of different light intensity. For demonstration at the level of single receptor cells and relevant discussion, see Washizu et al. (1964) and Streck (1972).

In a number of insects, ommochromes form pigment sheaths around internal organs, such as testes (Ephestia, Papilio) and ganglia (Ptychopoda seriata, Fig. 10, Hyalophora cecropia). Since no experimental data concerning the function of these pigment sheaths are available we are presently left with blind speculation; for a few hints see Ajami and Riddiford (1971b).

Fig. 10. Young caterpillar of Ptychopodo seriuta made transparent to demonstrate pigmentation of ganglia. (From Kuhn, 1940.)

5.2 OMMOCHROMES AS PATTERN PIGMENTS. RELATb3N TO OTHER PIGMENTS

In no other phylum of animals is pigmentation ;is varied and as complex as in the Arthropoda, notably in insects. Ommochxomes represent one of half a dozen kinds of pigments, each comprising a variety of compounds occurring in different tissues and patterns and characterized by its own ontogeny. Ommochromes-besides their universal distribution in arthropod eyes-are frequently found in the hypodermis O F inseits (cf. Table 5 ) , and will, if they are not occluded by cuticular melanin, contribute to the outward appearance of the animals. Thus the brownish or reddish

170 BERNT LINZEN

colouration of many Orthoptera and Odonata (cf. also Becker, 1941; Krieger, 1954) are due to hypodermal ommochrome granules. The striped pattern of Schistocerca eyes is produced by ommochrome granules filling the tips of secondary pigment cells in distinct areas (Nolte, 1965). In the Neuroptera, hibernating Chrysopa females attain their light brown tint by formation of xanthommatin (Becker, 1942; Riidiger and Klose 1970). In the Lepidoptera many larvae have ommochrome granules in their hypodermis. These participate both in diffuse patterns and in those rich in contrast (Cerura vinula, Sphinx ligustri). The most brilliant appearance of ommochromes is in the wings of many Rhopaloceran butterflies (e.g. Argynnis paphia, Vanessa species, Heliconius species).

The function of insect pigment patterns will not be discussed in detail. A particularly good example of the importance of ommochromes in this respect is provided by the wings of a butterfly, Argynnis paphia (Magnus, 1958). The primary stimulus for mating behaviour of the male is provided by the rhythmical flashing of orange-yellow in the fluttering flight of the female. This induces the male to aim directly at the female and. in the presence of a specific odour, courtship will ensue. Magnus analysed the parameters of the innate optical releaser, which arouses the male to fly into target by placing dummies on a merry-go-round set up in a wood clearing and observing the males’ behaviour. He discovered that the quality of the natural colouration is optimal. However, neither the pattern of black specks nor the natural contour of the wings form part of the releasing signal. On the other hand, increasing the stimulus quantity (by increasing the coloured area and the frequency of its appearance) rendered dummy insects more attractive. This occurred whether the dummy was a rotating cylinder or a more natural device with flapping wings. Thus, “superoptimal releasers” of the first stage in mating behaviour can be easily constructed. In natural populations a mutant strain, A. paphia, forma valesina, is found in low percentage (0.3-0.4 per cent in the Stuttgart area) which lacks the ommochrome pigmentation of the wings of the female. These greyish, inconspicuous females usually remain unnoticed by the males (except for direct olfactory stimuli at short distance) and would have little chance to mate. On the other hand, they are less likely to fall prey to birds. The advantage gained by cryptic colouration is thus a counterweight main- taining a delicate balance between the two strains.

The brightness of reduced ommochromes may also serve in warning colouration. In the larva of Cerura vinula most of the hypodermal xanthommatin appears to be in the oxidized state. However, the intersegmental skin between the head capsule and prothorax is brilliantly red, due to its content of dihydro-xanthommatin. This part is deeply infolded and can suddenly be displayed if the animal is disturbed. At the same time 30 per cent formic acid is ejected from a gland in the ventral part


of the neck (Schildknecht and Schmidt, 1963), while at the hind end of the animal two filaments can be extruded and weved vigorously. These again are coloured red by reduced xanthommatin (Linzen and Biickmann, 1961; Biickmann, 1965, and other papers).

Cerura is also a good example to demonstrate the concealing effect which ommochromes may produce against a suitable background. The feeding larva exhibits a disruptive pattern, wich a countershading in both components of this pattern (lateral and kentral green areas, dorsal “saddle-patch”). At the time when the fully grown larva is about to leave its food and to crawl down the trunk of the food tree, the green colour (due to a bilin in the haemolymph) is covered by new ommochrome synthesized in the hypodermis. The animal attains a dark red shade which. fits well into the new background met with.

In Orthoptera, ommochromes are at least in part responsible for adaptive colouration (see below). Cott, in his book on adaptive colouration (1940-1 966), presents several examples (Mantis religiosa, Lepidopterous pupae), which document the survival value of this principle. other examples may be found in the more recent literature (e.g. de Ruiter, 1955).

Very little is known about the relation o f ommochromes to other pigments. I t appears that hypodermal ominochromes are frequently associated with melanin, localized in the overlying cuticle. Dustmann (1964) nicely demonstrated this correlation in the stick insect (Fig. l l ) , but stated that ommochrome was found also in areas where the overlying cuticle was devoid of melanin. Biickmann (1965) concluded that in Cerura all areas of the integument covered by cornpletely black cuticle also contained ommochrome granules, but that these never contribute to external colouration. In other areas ommoclirome pigmentation might occur per se, the cuticle being devoid of melanin. A linkage between cyticular melanin and hypodermai ommochrorne is also observed in other species (Nickerson, 1956; also, cf. Fig. 3 in Kiihn, 1940). The causal relation of this is not clear, for as both pigments are derived from essential

Fig. 11. Correspondence between cuticular melanin spots (Me) and epidermal ommochrome in the integument of Carmrsius morosus. (From Dustmann, 1964.)

172 BERNT LINZEN

amino acids which might be expected to accumulate simultaneously. A loose metabolic coupling of their biosynthesis is plausible, but could not explain the graduated differences observed.

Ommochrome pigmentation may supplement melanin pigmentation to make a species look entirely dark. This is the case in Gryllus bimaculatus. In the abdomen the ventral and lateral parts of the integument are intensely coloured by ommin granules, so that the obscure aspect is maintained even if the deeply melanized sclerites move apart during expansion of the abdomen (as in mature females full of eggs). In Gryllus, in contrast to Cerura and Carausius, melanin and ommochrome production do not appear to be metabolically interdependent.

There are several mutants in which both ommochrome and pteridine biosynthesis are affected. This is the case in the white mutants of Drosophila and in the mutant chalky of Calliphora. Possibly, in these mutants the structural protein or “matrix” of the pigment granules is blocked. However, the relation must be highly complex, for in Drosophila basic differences of structure and origin of the two types of granules have been shown (Shoup, 1966; Fuge, 1967). Both granule types are separated from the cytoplasm by a membrane, and one may speculate about a possible interference of the mutant genes with the proper and timely synthesis of this structural element. Another type of metabolic link between ommochromes and pteridines seems to exist in Ephestia. The mutation to a (which in biochemical terms results in an inactivation of tryptophan oxygenase) brings about not only the accumulation of free tryptophan and the absence of ommochromes, but also a striking change in the pattern of fluorescent compounds. This is shown in both paper chromatograms and in histological sections (Hadorn and Kiihn, 1953; Kiihn and Egelhaaf, 1955; Egelhaaf, 1956a, 1956b, 1963a; Reisener-Glasewald, 1956). The increase in fluorescence is due to dihydro-ekapterin (which during isolation is oxidized to ekapterin) (Viscontini and Stierlin, 1961, 1963). Injection of kynurenine will not only result in normal ommochrome synthesis, but also restore the normal pattern of fluorescent pteridines (Kiihn and Egelhaaf, 1955). No plausible biochemical explanation is available to the author. Ghosh and Forrest (1967a, 1967b) who discussed the involvement of pteridines in the tryptophan + ommochrome pathway, do not discuss this problem. Kiihn (1956) advanced the hypothesis that ommochromes and pteridines (and possibly their precursors) might compete for reactive sites on the pigment granules. In view of the specificity of most enzymes and, in Drosophila, of the deposition of ommochromes and pteridines on distinct types of granules, the hypothesis has lost its attractiveness. Moreover, Reisener-Glasewald (1956) found the most striking increase of fluorescence in the a eye in the crystalline cones. Ziegler and Harmsen (1969) consider cone fluorescence to be an artifact


resulting from secondary adsorption of dissolved pteridines during fixation of the tissue. It is not clear to the author whether there is experimental evidence to support this argument.

Since tetrahydro-pteridines may act as cofactors in hydroxylation, their involvement in ommochrome biosynthesis has been considered (Ghosh and Forrest, 1967a, 1967b). From recent work it appears unlikely that the enzyme, kynurenine-3-hydroxylase (see p. 191), has a pteridine cofactor. The possible inhibition of tryptophan oxygenase by preridines should, however, be considered.

With regard to pigment function, pteridines and ommochromes may, of course, supplement each other both in patterns o external colouration and in the screening of the ommatidia in the compound eye (see pp. 166-167).

5.3 OMMOCHROMES IN MORPHOLOGICAL COLOUR CHANGE

Morphological colour change has been dealt with in recent reviews by Rowel1 (1971) and Fuzeau-Braesch (1972), with valuable bibliographies. Morphological colour change is related to the aspects of pattern, of adaptation, and of development. The latter aspect gains much in importance when pigmentation is essentially a one-way process (i.e. when the pigment once deposited is not further meta.bolized). This is the case both with melanins and with ommochromes. But, while melanins may be shed along with the cuticle at each moult, ommochromes, being hypodermal, may merely be diluted by further growth and cell multiplication.

The role of ommochromes was quantitatively analysed in two species, namely Carausius morosus (Biickmann and Dustmann, 1962; Dustmann, 1964) and Cerura vinula (Linzen and Biickmann, 1961; Biickmann et al., 1966). In the author’s view, the most completi: biochemical analysis of morphological colour change in any species is that carried out in the stick insect, Carausius, by Buckmann and his students. The stick insect is able to adopt a great variety of colour shades. While the amount of pigments present sets the range of environmental stimuli to which the animal is able to respond, fine control is achieved by means of thromatophores which are controlled by neurohormones. Together with cuticular melanin, ommochromes are chiefly responsible for the light or sombre appearance. Dustmann (1964) identified xanthommatin and the usual mixture of ommins. If the animals are under normal daylight conditions, the hypodermal ommochrome content remains low (4.7 pg per animal). However, if the lower halves of the eyes are covered by black lacquer, a dramatic increase of nearly a hundredfold is observed within a few moults. Ommochrome synthesis is also enhanced at high temperature. Under these conditions it is much less dependent on the pattern and intensity of illumination. The total ommochrome content may amount to 0.8 mg per

174 BERNT LINZEN

animal which is equivalent to 0.6 per cent of the dry weight. The percentage of xanthommatin is increased under conditions of induced ommochrome synthesis. I t is important to notice that ommochrome pigmentation is irreversible. For example, if during the 4th larval stage the conditions were changed to induce light colouration, there was no change in ommochrome content up to the adult stage. Similarly, if conditions causing light pigmentation prevail throughout from the day of hatching, a constancy of ommochrome content is demonstrated. Approximately 5 pg of ommochromes are synthesized during embryonal development and no ommochrome at all thereafter. According to Berthold (1971) the formation of ommochromes is correlated with a decrease in excretion of kynurenic acid which appears to be the normal end product of tryptophan metabolism in the stick insect.

The induction of ommochrome synthesis may become effective within any given larval stage; but conditions must be changed immediately after a moult. This may reflect either a critical stage in the responsiveness of the integument, or a periodical sensitivity of the brain-corpora allata system which mediates between environmental change and integumental reaction. Berthold (1971) has critically examined the role of the corpora allata in induced ommochrome synthesis and found it to be at best indirect.

Willig (1969) analysed the carotenoids and bile pigment (biliverdin) of this species and found the quantitative changes to be much less spectacular. While the ommochrome content can vary by two orders of magnitude, the changes in these two other pigment groups do not exceed a factor of two. Interestingly, the carotenoid changes also involve pigment transport from the fat body to the hypodermis. In a recent paper (Berthold and Henze, 1971), the participation of pteridines in the colour change of Carausius is also shown, dark animals containing less leukopterin and isoxanthopterin than light green ones. Thus, at least 16 pigments are involved in colour adaptation of Carausius: melanin, xanthommatin, the ommin mixture (at least four pigments), biliverdin, five carotenoids, four pteridines (other fluorescent compounds are present in trace amounts). All of these, with the exception of ommochromes and melanin, play a minor role.

From the observations of Krieger (1954), it may be surmised that similar principles may be responsible for adaptive colour change in the larvae of Odonata. Krieger assigned the major role to melanin without, however, having performed quantitative measurements. In the case of the praying mantis and of the migratory locust there is some controversy in the literature (cf. Rowell, 1971, and Fuzeau-Braesch, 1972) resulting from the conflicting findings of Vuilleaume (1968) and of other workers in this field. Vuilleaume contends that the major factor responsible ‘for the colour varieties in these species is a bile pigment which may be oxidized to various degrees under irradiation. Although Vuilleaume had identified ommo-


chromes in the integument of Mantis, she denied the functional significance of these pigments. Susef-Michieli (1965), on the contrary, found a “much greater quantity of ommochromes” in brown individuals, while finding no evidence for the presence of bile pigment. From Goodwin’s (1952) discussion of locust pigmentation it is evident that the importance to be assigned to each pigment critically depends 011 the developmental stages. While “insectorubin” (ommochromes) is present in different amounts in gregariu and solitaria hoppers, these differences are not crucial in the outward appearance of the two phases, w’iich is, on the contrary, determined by the amounts of bile pigment a i d of melanin. However, in the immature and mature adult animals the ommochromes are at least partly responsible for the external aspect. As the methods available for quantitative pigment determination are now much refined, chemical analysis should be able to solve these current problems.

A quite different case of colour change is observed in Lepidopterous caterpillars (e.g. Cerura vinufa) in which it represents an obligate step in development. This type of colour change, although brought about by hormone action, is not mediated by neurohuinoral response to changing environmental stimuli. In Ceruru the synthesis of large quantities of ommochromes appears to be a secondary, metabolic effect, which, however, is precisely correlated with the abandonment of the leaves and the move to the trunk of the food tree. Buckmann (main papers: 1953, 1959a, 1959b, 1963, 1965; Karlson and Buckmann, 1956) has analysed the morphological and hormonal basis of this striking phenomenon and has found a number of interesting effects of temperature and atmospheric composition. The biochemical basis has been studied in collaboration with the present author (Linzen and Buckmann, 1961; Biickmann et ul., 1966). The larva of Ceruru is marked by a dark brown rhomboid pattern, the “saddle-patch” which contains xanthommatin. At the end of feeding, about 10 days prior to pupation, the pigment is reduced to its red state. At the same time synthesis of dihydro-xanthomniatin starts all over the integument. This causes the larva to turn almost black (because the haemolymph is dark green). Shortly afterwards the fat-body turns red, due to formation of rhodommatin and ommatin D, while the green haemolymph pigment gradually disappears. While the larva is contained within its cocoon it is bright red for a short period. The dihydro-xanthommatin then begins to disappear from the hypodermis, causing the anterior part of the larva to turn greenish again. Finally all of the ommochrome is found in the fat body and in the excretory organs, while the hypodermis retains only small amounts of red pigment. Colour photographs of these dramatic changes, with others demonstrating the effects of ”ligatures, have been published by Buckmann (1959). In Ceruru, rhcidommatin and ommatin D are found in many parts of the body: integument, fat body, gut, Malpighian

176 BERNT LINZEN

tubules (where they are formed at a very early stage), haemolymph (transiently). It is a tempting thought that rhodommatin and ommatin D in the haemolymph should represent the transport form of dihydro- xanthommatin removed from the hypodermis. This suggestion has, however, not been proved. The total amount of ommochromes per animal is about 1 mg equivalent to 0.2 per cent of the total dry weight.

Chemical analysis of the haemolymph during the period of colour change (Biickmann et al., 1966) has revealed significant changes in the contents of protein, carbohydrate, and individual amino acids, notably, proline, phenylalanine. tyrosine, and the aliphatic amino acids. Tryptophan and 3-hydroxy-kynurenine undergo a dramatic and transient increase. The chemical results are in accord with the histological findings. Thus immediately after termination of larval growth a profound rearrangement and partial degradation of tissues is initiated which leads to the liberation of proteins. Simultaneously the contents of the spinning gland are built up. It may be assumed that this secretion is poor in tryptophan, as are most silk proteins, so that there is an excess of tryptophan which is metabolized to yield ommochromes. Colour change thus appears to be secondary to the metabolic processes which are primarily governed by the necessity of preparing the organism for pupation and of providing a specialized secretion for protection.

Similar but less spectacular colour changes have been observed in many Lepidoptera, but biochemical investigations are scarce. In Hestina japonica and Sasakia charonda there is a larval diapause. When entering into diapause these larvae turn brown by formation of xanthommatin in the hypodermis. Again, at the end of hibernation xanthommatin has disappeared from the integument, while rhodommatin can be detected in the gut contents (Osanai and Arai, 1962a, 1962b; Osanai, 1966a). In the Neuropteran, Chrysopa, the xanthommatin content doubles at the onset of hibernation, while biliverdin drops by about 70 per cent (Riidiger and Klose, 1970).

5.4 OMMOCHROMES AS WASTE PRODUCTS

In a number of Lepidoptera, the meconia produced at pupal emergence have a vivid carmine colour. This is due to the presence of rhodommatin and ommatin D. Large-scale isolation of ommatins was in fact first carried out with meconia of Vanessa urticae both by Becker (1942) and Butenandt et al. (1954a). Some quantitative data are given in Table 6.

These ommochromes are formed early in metamorphosis. They make their first appearance at the time when the larvae leave the food and crawl about to find a suitable place for pupation. In Vanessa urticae, orange pigment appears in the midgut wall at the time when the larva is spinning the small web to hook itself up. Twelve hours later, at the time of


TABLE 6

The quantity of ommatins in meconia of some Lepidoptera Gug per animal)

Species Rhodommatin Ommatin D

Aporia crataegi 244 225 Pyrameis atalanta 130 115 Pyrameis cardui 27 52 Argynnis paphia 38 36

Taken from Butenandt et al. (1960b).

pupation, the gut is filled with red fluid containing about 50 pg each of rhodommatin and ommatin D (Kiibler, 1960). The histology of this process is described in detail for Ptychopoda serzata (Wolfram, 1949) and for Cerura vinula (Linzen and Buckmann, 1961). In both species, ommochromes appear first in the Malpighian tubules; they are then excreted and thus contribute to the red colour of the last faeces of the larva. Some time afterwards (but prior to any change in the hypodermis) the epithelium of the midgut is replaced. Ommochromes are formed in vacuoles of the new cells and are later released into the gut lumen. From the histological description of this process it appears that the ornmochromes are really synthesized locally within the gut cells and not taken up from the haemolymph. Yet, it is possible that precursors are absorbed from some other tissues. In Cerura, for example, pigment production is linked to profound histological changes occurring in other tissues: notably rejuven- ation of the midgut and histolysis of the fat body.

It is not surprising, therefore, that ommochromes are also formed when animals or tissues are maltreated SO as to disrupt normal metabolic function. In fourth-stage larvae of Cerura, the formation of ommochromes in the Malpighian tubules and gut can be induced by ligation or simply by starving the animals (Biickmann, 1959a). It is assumed that under these circumstances the animals draw on their protein reserves as an energy source, thus leading to an excess of tryptophan.

This assumption is also supported by the observation that locusts produce brick-red faeces in the periods of moulting (Chauvin, 1939). Chauvin related the red colour to “acridioxanthin”. Bouthier (19 72), using chromatographic methods, identified the “acridionimatins” I and 11, and succeeded in purifying some pigment from this starting material. Locust droppings are also red, when the animals are starved.

It has been noticed by several authors that dama!ge to insect tissues may result in ommochrome formation. In Drosophila, I he Malpighian tubules

178 BERNT LINZEN

are coloured red after non-specific experimental injury such as uv irradiation and osmotic shocks (Ursprung et al., 1958; Hertweck, 1960; cf. also Wessing and Bonse, 1962). In all probability this pigment is an ommochrome. Berthold (1971) noted that regenerated legs of Carausius morosus are more reddish than normal legs. She also observed more ommochrome granules in histological sections. Furthermore, if allatectomy, or a corresponding sham operation, is performed on the stick insect, a reddening of the integument anterior to the wound (in the head, and the antennae) is observed. Changes in at least three types of pigment are responsible for this effect and an increase in both xanthommatin and ommin was demonstrated by quantitative chemical analysis. In this connection, Berthold recalls a general statement by Giersberg (1928), namely that exposure of the stick insect to extreme environmental conditions, whatever these are, results in preferential synthesis of dark pigments, whereas specimens kept under optimal rearing conditions remain green. Berthold relates the incidence of ommochrome formation in the stick insect, whenever it is not under neurohormonal control, to a failure in the removal of kynurenine (e.g. by an impairment of circulation). She assumes that normally kynurenine is transported to and transaminated in the fat body. The typical excretory metabolite of tryptophan in the stick insect is kynurenic acid.

In the light of the foregoing discussion, the effects of high temperature on the ommochrome content of Cerura larvae (Biickmann, 1963, 1965) are quite unexpected. Raising the temperature to 35" C, which severely harms the larvae at prolonged exposure, does not result in an increase but in a decline in hypodermal ommochrome content. The most conspicuous event is the turning red of previously brown regions of the integument. Thir effect can also be produced topically by applying small heating elements. This colour change is slowly reversed, if the animals are returned to a temperature of 20°C. In the course of further growth such larvae synthesize much more xanthommatin in their integument than control animals continuously kept at the lower temperature. Evidently, we are concerned here with both short-term and long-term effects of high temperature, which are brought about by different mechanisms.

A change in the oxidation state of the hypodermal xanthommatin can also be brought about by placing Cerura larvae intermittently into an atmosphere of nitrogen or by plugging part of the spiracles. This treatment causes a slight, though not statistically significant, increase in the amount of xanthommatin (Buckmann, 1965). Changes in temperature and atmospheric composition have been known for a long time to affect development of insect pigments (cf. Biedermann, 1914), but very little is known about the physiological and biochemical mechanisms involved.

In conclusion, ommochrome formation is frequently a consequence of


cellular injury or impairment of normal metabolism. It is without doubt a means of “metabolic excretion” of tryptophan and is comparable in this respect to the production of alkaloids in plant metabolism. One might ask whether there is any selective value in forming an end product without really removing it along with the excreta. Harmsen (1966), in his discussion of the excretory role of pteridines in insects, requires that two criteria (among others) should be fulfilled for a pigment to be regarded as an excretory or “dry storage” product: ( 1 ) synthesis of the pigment should be continual during development and not take place shortly before it functions as a colouring matter; (2) the pigment should be produced in excess of the demand (if colouration alone is considered). The first requirement does not appear to be justified in insects as development itself is not continual (i.e. not a gradual change of size and structure). The excretory role of pigment synthesis may appear orily in periods of altered protein metabolism, as in the larval moults or during pupal metamorphosis. This condition would certainly apply to ommochrorne synthesis. Harmsen’s second requirement is met by the observation that hypodermal ommochromes are frequently occluded by melanin in the overlying cuticle, and thus never required for pigmentation.

A topic not considered in Harmsen’s paper is the possible advantage of “local excretion”. Two aspects are pertinent to ihis problem. First, the absolute amount of tryptophan to be excreted is usually low, as a result of the low percentage of tryptophan in proteins. Secondly, as reported above, most ommochromes are insoluble at physiological pH and may form concretions of high density. In contrast, all precursors of ommochromes are more or less readily soluble. In addition, the function of tryptophan both as an essential amino acid (which i s conserved for protein synthesis within the cell) and as a possibly harmful substance, must be contemplated. It can thus be conceived that “excretion in situ” of tryptophan, by transforming it into an insoluble metabolite, could be more efficient than removal by diffusion. This would be especially the case if circulation of the haemolymph and its clearance by Malpighian tubules Gr other excretory tissues were slow. This reasoning could also apply to cells not in direct contact with streaming haemolymph because they are tucked away in tissue folds or because of other interposing tissues.

6 Enzymes involved in the kynurenine pathway

The study of enzymes involved in the de,gradation of tryptophan has lagged behind the work on corresponding mammalian or microbial enzymes by five to ten years. The first to be detected in insect h6mogenates were kynurenine formamidase (Glassman, 1956) and lrynureninase (Inagami, 1955, 1958). While these have not been studied in detail in insects, more

180 BERNT LINZEN

attention has been paid to tryptophan oxygenase because of the distinction between suppressible and unsuppressible vermilion mutants in Drosophila melanogaster.

6.1 TRYPTOPHAN OXYCENASE (EC 1.13.1.12)

This enzyme, already well known from vertebrate and microbial sources, was first detected by Egelhaaf (1958) in Ephestia kiihniella and by Baglioni (1959) in Drosophila. It is a soluble enzyme which may have a Fe porphyrin prosthetic group. While Marzluf (1965a) could not restore the activity of dialysed tryptophan oxygenase nor increase the activity at any stage of purification by adding haematin, Baillie and Chovnick (1971) and Schartau (unpublished) were able to stimulate enzyme activity several-fold by the addition of methemoglobin. Nawa (1970) observed dependence of the activity of the purified enzyme on added methylene blue.

In insect extracts, tryptophan oxygenase activity is usually measured by means of the Bratton-Marshall reaction of the product, kynurenine (Bratton and Marshall, 1939). I t is tacitly assumed that the formylkynurenine formed during the incubation is hydrolysed by excess kynurenine formamidase or spontaneously during the incubation or after addition of trichloroacetic acid. The Bratton-Marshall reaction, however, is not entirely specific. Pinamonti and Petris (1966) pointed out that the degradation of ommochromes in the weakly alkaline incubation medium (Butenandt et al., 1960b) leads to Bratton-Marshall positive products. This is a serious interference in the assay of tryptophan oxygenase in many arthropod tissues, but can be overcome by suitable controls. In addition, other reactions might contribute to errors. This has been observed in Bombyx tissues by using two different assays (Linzen, 1971b). In such cases the kynurenine formed may be separated by paper chromatography or high-voltage electrophoresis and measured by paper fluorimetry (Egelhaaf, 1958, 1963a; Linzen, 1971b). By incubating directly on chromatography paper, Egelhaaf (1963a) could demonstrate the activity of the enzyme in single fat body lobules or ovarioles.

Partial purification of the enzyme has been achieved by a number of workers. Hiraga (1 964) separated tryptophan oxygenase from kynurenine formamidase by gradient elution from DEAE-cellulose. Chromatography on DEAE-cellulose was also the most efficient step in the work of Marzluf (1965a), Baillie and Chovnick (1971), Nawa (1970), and Schartau (unpublished), who purified tryptophan oxygenase about 16-, 65-, 80- and 200-fold, respectively. In Baillie’s and Chovnick’s study, the activity of the purified enzyme became almost entirely dependent on added methemoglobin.

The molecular weight of the Drosophila enzyme was estimated at

THE TRYPTOPHAN + OMMOCHROME PATHWAY I IV INSECTS 181

150 000 Daltons (Baillie and Chovnick, 1971) and that of the Phormia enzyme at 120 000 Daltons (Schartau, unpublished). This is probably an oligomer composed of several subunits and which would be expected to exhibit allosteric properties. Baillie and Chovnick indeed observed a sigmoidal dependence of reaction velocity on substrate concentration which was abolished by preincubation with cu-methyl-tryptophan. The same phenomenon had been described for microbial tryptophan oxygenase (Feigelson and Maeno, 1967).

Probably the “superadditivity effect”, observed in Ephestia a’/a by Egelhaaf and Caspari (1960) and clearly brought into focus by Tartof‘s (1969) investigation of the vermilion suppressor i n Drosophila, is related to subunit interaction. If wild-type and mutant extracts are mixed, the reaction rate is higher than the sum of the rates in each single extract. In the vermilion mutants superadditivity is correlated with suppressibility. Furthermore, the material responsible for the effect is thermolabile; this is precipitated by ammonium sulphate and, when chromatographed;eluted at the same position as the wild-type enzyme (Baillie and Chovnick, 1971). Tartof‘s conclusion is that the subunit of the mutant enzyme contains an intact catalytic site but is unable to associate, association supposedly being a prerequisite for activity. Formation of hybrid oligomers, however, which would occur after mixing wild-type and mutant extracts, should result in activation of the catalytic sites of both wild-type and mutant enzyme subunits.

Some data on tryptophan oxygenase are summarized in Table 7. The pH-optimum is usually sharp and on the alkaline side. Substrate inhibition has been observed several times but is not mentioned when higher substrate concentrations have not been tested. The enzyme is inhibited by a number of agents which are listed in Table 8. Of particular interest is inhibition by some naturally occurring pteridines (Ghosh and Forrest, 1967b). Though only two compounds are listed in the Table, it may be assumed that other pteridines (folic acid, biopterin, isosepiapterin and isoxanthopterin) will also affect the insect enzyme, since they exert strong inhibition on the rat liver enzyme. It was argued that in a group of white mutants of Drosophila, failure to reduce a pteridine precursor could lead to a double effect on tryptophan degradation: accumulation of a pteridine inhibitory on tryptophan oxygenase, and lack of a reduced cofactor supposedly required for kynurenine hydroxylation. Although up to now thLere is not much evidence to prove this hypothesis, some sort of interaction between tryptophan and pteridine metabolism clearly exists as shown by the observations of the Rizkis in Drosophila (Rizki, 1964; Rizki and Rizlki, 1964: altered pattern of kynurenine formation in larval fat bodies of the rosy ind sepia mutants) and of Kiihn and his collaborators in Ephestia (Kiihn, 1956).

The specific activity of the enzyme in total hoinogenates of insects is of

182 BERNT LINZEN

TABLE Tryptophan oxygenase in insects-selected data on

pH optimum Kln Substrate in uitro mM litre-' inhibition

Species Strain and stage

Drosophila Oregon-R, melanogaster flies

Oregon-R, all stages Oregon-R, larvae Oregon-R, flies Oregon-R, flies

Wild-type, flies

Ephestia kiihniella

Various stages

Sch isto cerca Adults gregaria Habro bracon Wild-type 33, jugla nd is various stages Bombyx mori rb mutant

Phormia All stages terraenouae

- - -

- 7.4 1 7.4 1.5 8.0*

-

- (3) * * -

7.4

-

8.5

8.25

Gryllus Ultimate 8.5-9 .O bimaculatus larval

0.6

2.6

-

2.6 Yes

3.5 Yes

* For suppressed uk. * * Sigmoidal velocity curve, value given for half-maximal velocity; after preincubation

with a-methyl-tryptophan the K , is about 1.5 x lo-' M litre-'. 05 It has to be assumed that optimal assay conditions were frequently not attained.

Only a small part of the available data are included in this column; most values have been rounded or are given as order of magnitude. Activities are based on different entities.

THE TRYPTOPHAN + OMMOCIiROME PATHWAY IN INSECTS

7 properties and occurrence of the enzyme

183

Optimal

mM litre-'

Stimulated Activity in whole homogeniites Inducibility Reference? by hematin? or tissues5 5

- - 133 nmol g-' h-' ,whole flies - a

b - - 600 nmol g-' h-' ,whole flies -

- - 2.3 x lo-* nmol per larva per hour yes C

- no (arbitrary units given) Yes d - - 300 nmol g-' h-", whole animals - e

6

2.4

f 10 nmol mg-' protein h-' ,

- - 30 pmol mg-' protein min-' , no g

- Yes -

whole animals

whole animal 140 pmol mg-' protein min-' , larva fat body 40 pmol mg-' protein min-' , spin gland

h 8 nmol mg-' protein h-' , fat body 4 nmol mg-' protein h-', no I

whole animal

-

60 nmol mg-' protein h-' , fat body$ - i 3 nmol mg-' protein h-' , gut

12 nmol mg-' protein h-' , ovaries 12 nmol mg-' protein h-' , testes 30 nmol mg-' protein h-' , developing wing

medium: gut, flight muscle low: ovaries, larval "residual body"

8 .O yes high: fat body, testes, Malpighian tubes no k

2.5-5 - 1 nmol mg-' protein min-' , fat body - I

$ Much depending on stage, maximal activities are given. In almost all studies the incubation temperature was 37OC.

f a . Baglioni (1960); 6. Kaufman (1962); c. Rizki and Rizki (1963); d. Marzluf (1965a. 1965b); e. Tartof (1969); f. Baillie and Chovnick (1971); g. Egelhaaf (1958, 1963a); h. Pinamonti and Petris (1966); i Leibenguth (1967a)pj. Linzen (1971b); k. Schartau (unpublished); I. Tiedt (1971).

AIP-7

184 BERNT LINZEN

TABLE 8

Effects of various inhibitors on insect tryptophan oxygenase

Inhibitor Concentration Inhibition

Species Reference? M litre-' %

c u + + Cyanide Fluoride Azide Hydroxylamine

Phenylthiourea Sodium diethyldithiocarbaminate

D-tryptophan 5-Methyl-D &tryptophan 2-Amino-4-hydroxy-6-hydroxy

2-Amino-4-hydroxy-6-carboxy-

methyl-pteridine

pt eridine

2.5 x 1 0 - ~ 4 x 10-5 5 x 1 0 - ~ 2 x ~ o - ~ 5 x10-4 4 x ~ o - ~ 3 x 1 0 - ~ 7.5 x 1 0 - ~ 2 x 5 x 1 0 - ~ 5 x 1 0 - ~ 6.5 x 1 0 - ~

6 x ~ O - ~

68 * 47 21 32 49 70 25 24 50 39 51 73

6

Drosophila b Ephestio a

a a a

Drosophila b Ephestia a

a Drosophila b

b b c

c

? a . Egelhaaf (1963a); b. Marzluf (1965a, 1965b); c. Ghosh and Forrest (1967b). (From the data provided by these authors inhibitor concentrations were selected for which the reported inhibition was nearest to 50 per cent. The species are Drosophila melanogaster and Ephestia kiihniella.)

* At a protein concentration of 6.6 mg ml-' .

the same order of magnitude as the activity in vertebrate liver. A precise comparison is impeded by variations of incubation conditions and of calculating the data. Within a given insect species, considerable differences exist between various strains: in Drosophilu melanoguster the Sevelen strain has higher activity than the Oregon-R and the Pavia strains; the ratios are about 100 : 77 : 48 (Baglioni, 1960; Kaufman, 1962). If tissues are dissected and assayed separately, the specific activity in some of them is distinctly higher than in vertebrate liver.

A striking difference of localization is provided by the occurrence of tryptophan oxygenase in a variety of insect tissues, while it is restricted to liver in vertebrates. While Rizki (1964) working with Drosophilu and Pinamonti and Petris (1966) studying Schistocercu, both believed that tryptophan oxygenase was restricted to fat bodies, it has been reported by Kaufman (1962) that Malpighian tubules of Drosophilu also contained the enzyme. Egelhaaf's meticulous studies on Ephestia (1958, 196Sa) extended the domain of the enzyme to gonads, spinning gland, gut, and epidermis.


Work in the author’s laboratory (Linzen, 1971b; Tiedt, 1971; Linzen and Schartau, in preparation) leads to the conclusion that the distribution of the enzyme among tissues is almost random. It may be found in almost any tissue, and there is no evident rule as to the specific activity characteristic of any particular tissue, or of specific activity proportions. For example, in Phormia the specific activity is highest in testes, and there is high activity in both larval and imaginal Malpighian tubules, w’hile in Bombyx mori rb it is at best “medium” in the testes and absent fr3m the Malpighian tubules. The ontogenetic pattern of tryptophan oxygenase in Bombyx will be discussed below.

Evidence on inducibility of tryptophan oxygimase in insects is scarce and conflicting. The Rizkis (Rizki, 1963, 1964; Rizki and Rizki, 1963, 1964) have shown that upon addition of tryptophan to the growth medium of Drosophila larvae a large amount of kynurenine is formed in the fat body. Kynurenine is normally localized in the anterior lobes, but extends throughout the entire fat body after tryptophan feeding. The activity of tryptophan oxygenase was markedly raised iii extracts of those larvae. These results were confirmed by Marzluf (19155b) for a wild-type and a suppressed vermilion strain. On the other hand, in Ephestia (Egelhaaf, 1963), in the parasitic wasp, Habrobracon (Leibenguth, 1967), and in Phormia flies (Schartau, unpublished), tryptophan oxygenase is not inducible by feeding or by injecting tryptophan

In this connection one should also focus attention on the relation of enzyme activity to gene dosage. In Drosophila the gene v + responsible for tryptophan oxygenase is located on the X chrornosome so that in males one would expect half the activity in females, if there were no dosage compensation. However, in males the activity equals the activity in females (Kaufman, 1962). If gene dosage is increased, the response of males is about twice the response of females (Baillie and Chovnick, 1971), suggesting a rather complicated regulatory mechanism (for discussion compare Seecof et al., 1969). In v+/v heterozygotes the activity is always higher than 50 per cent of v+/v+ activity (depending on the particular v mutant) but does not exceed 80 per cent. This, may be interpreted as an in vivo superadditivity effect. In Ephestia it was demonstrated both by direct enzyme assay (Egalhaaf and Caspari, 1960) and in vivo by tryptophan loading (Egelhaaf, 1963a) that tryptophan oxygenase activity depends on the number of a+ alleles present. In vitro there is a marked superadditivity effect; however, no data are available to relate in vivo and in vitro activities, as in Drosophila.

Investigations of Drosophila tryptophan oxygenase were usually stimulated by a search for the mechanism cif suppression of vermilion mutants. Vermilion mutants are practically indistinguishable by their phenotypes but fall into two classes when two sets of experiments are

186 BERNT LINZEN

employed. In the presence of a suppressor mutant part of the u mutants ( u s ) are suppressed, while the others are unaffected (u”) (Green, 1952). Furthermore, i f us larvae are subjected to partial starvation the adults will have pigmented eyes (‘I’atum and Beadle, 1939; Green, 1954). Suppressible mutants are u ( u l ), u’ , u k , unsuppressible mutants u36f, u 4 & , to u s “ . There is a series of suppressor mutations affecting vermilion, abbreviated su(s), the mutant most widely used for study being su(s)’. For details see Lindsley and Grell (1968) and references cited therein, and Baillie and Chovnick (1971).

’Tryptophan oxygenase activity in various mutants is compared in Table 9. Activity is only partially restored upon introduction of su(s) but will suffice ,for normal production of pigment precursor. The data also indicate that the “unsuppressible” mutant u36f will produce a small but detectable amount of additional activity if combined with su(s) ’ . This is substantiated by Kizki (1 964) who observed the appearance of kynurenine fluorescence in fat body regions 1 and 2 of u36f .w(s)’ larvae after feeding tryptophan. In u 1 su(s)’ the fluorescence extends further back to regions 3 and 4. Rizki states that “the difference between the unsuppressible u36f allele and the suppressible vermilion alleles is one of a quantitative nature rather than absolute”.

Early hypotheses implied that suppressible mutants produced functional tryptophan oxygenase which, however, was blocked by an inhibitor present

TABLE 9

Relative activities of tryptophan oxygenase in various combinations of vermilion and s u ( s ) alleles

Percentage enzyme activity as estimated by Strain

Baglioni (1960) Kaufman (1962) Tartof (1969)

Ore-R bw

u ’ b w u 3 6 f b ~ u ‘ x U 3 6 f

u ‘su (s) = u Isu (s) 3

u ISU (S)* -v-pr u 36 f su (s) 2

100 100 100 100 66,180*

1 1 2 1 7 1 1 7

20 20 9 17 10 14

6 1.5 4

It is emphasized that enzymatic activity is highly dependent on genetic background

* Two different strains. and therefore varies in different strains.

THE TRYPTOPHAN + OMMOCHROME PATHWAY IN INSECTS I a7

in the mutant, or else had to be activated in some way. Marzluf (1965a, 1965b) compared the properties of tryptophan oxygenase from wild type and suppressed vermilion flies, but did not find any difference in K,, pH dependence, energy of activation, percentage of inhibition by varying Cu++ concentration, and rate of inactivation. Neithcr did he find evidence for any easily removable inhibitor, and suggested that su(s) was a regulatory gene which induced the us mutants to produce tryptophan oxygenase at a low rate.

Tartof (1969) later discovered that there was a difference between u+ and suppressed u’ tryptophan oxygenase on the one hand, and oxygenase of uk on the other (the pH of maximal activity being shifted from 7.4 to 8.0 in the latter). He also found a strong correlation between suppressibility of the u alleles and the superadditivity effect exerted by their products on normal tryptophan oxygenase. Thus, us mutants are assumed to produce a potentially functional enzyme which, in a given cellular environment, does not attain the conformation necessary for function. Tartof concluded that su(s) is not an informational suppressor, bu: an indirect or metabolic suppressor (see Gorini and Beckwith, 1966). su(S) should be responsible for an altered cellular environment which would allow the us enzyme to become active (e.g. by dimerization). This is consistent with the recessive character of su(s). Similar conclusions have been drawn by Baillie and Chovnick (1971).

Direct evidence for such a type of mechanism was recently brought forward in a most exciting paper (Jacobson, 15171). Jacobson showed that if a u homogenate was treated with ribonuckase T I , a high increase in tryptophan oxygenase activity was observed. In wild-type homogenates the activity was not affected. Conversely, if ‘‘activated” (i.e. RNAase T , digested) tryptophan oxygenase was mixed with tRNA prepared from wild-type flies, strong inhibition resulted. The tRNA was further fractionated and tested against activated u 3xygenase. The inhibitory fraction was identified as a specific iso-accepting form of tyrosine tRNA (Fig. 12). Jacobson assumed that tryptophan oxygenase could associate with tRNATyr, but that in the case of the vermilion enzyme such association led to inhibition. Logically su(s) is expected to affect the inhibitory tRNA.

This has been shown in recent work by Twardzik et al. (1971). The tRNA’Y‘ of wild-type Drosophila could be resolved into three (two major and one minor) peaks; if homozygous su(s)’ flies were worked up in the same manner the second of these was missing. Three other species of tRNA were also examined and found unaffected by JU(S)’. The authors showed that both su(s)’ and the gene responsible for the altexd elution profile are located at the same position on the left end of the X chromosome. The fact that su(s)’ is recessive, and that it does not alter the total amdunt of

188 BERNT LINZEN

I

- NaCl concentration ( M I

0.4 4 8 ( b) p"

0.3

6 -

0.2

4 -

2 -

0.2 0.3 0.4 0.5

NaH, PO, concentrotion (M)

Fig. 12. (a) Fractionation of Drorophilu tRNh by reverse-phase chromatography and effect of fractions on tryptophan oxygenase activity (0-0-0). (b) Resolution of the active tRNA peaks by chromatography on hydroxylapatite. Three peaks are obtained, two of which can be charged with tyrosine ( 0 . . . . . . 0). The tRNA eluted last strongly inhibits tryptophan oxygenase. (From Jacobson, 1971 . )

tRNATY', but only alters the quantitative distribution among the two major iso-accepting forms, is a strong argument for the view that the suppressor mutation does not affect the structural gene of rRNATY' (of which a dozen copies might be present) but the gene coding for an enzyme (e.g. a methylase) by which tRNATY' is altered secondarily.

Genetically modified tRNA has been shown in many cases to be the cause of suppression in bacteria, acting by altered coding specificity. It is intriguing, therefore, t o find tRNA as a mediator of suppression in Urosophifa. However, the mechanism is entirely different for the effect is indirect, and occurs through the removal of a species of molecules (tRNATy') which would otherwise react with a cytoplasmic enzyme (try oxygenase). The activity of this enzyme is not affected in the wild type, but it is impaired by the interaction if it is itself altered by the us mutation.


A number of puzzling observations still await clarification. Tartof (1969), for example, found that su(s)’ influenced the measurable tryptophan oxygenase activity in v’/v heterozygotes. In two of these crosses the activity was rendered less than additive (only 35 per cent) by the presence of the suppressor. This indicates that interaction with tRNATY‘ is not restricted to the suppressible vermilion enzyme, but that it is a normal event determining the physical state of tryptophan oxygenase.

6.2 KYNURENINE FORMAM~DASE (ARY L-FORMY LAMINE

AMIDOHYDROLASE EC 3.5.1.9)

The earliest report on this enzyme in insects appears to be the one by. Glassman (1956). Later, Hiraga (1964) showed that the enzyme could be chromatographed on DEAE-cellulose. Recently some additional data have been gathered in the author’s laboratory. The hydrolysis of formyl- kynurenine is conveniently measured by recording absorbance at 360 nm (Mehler and Knox, 1950). Table 10 presents some of the results. In addition, it must be mentioned that the enzyme fiom Drosophila does not hydrolyse formylanthranilic acid (Glassman, 1956). There are two characteristic observations: the activity is found in almost any tissue and exceeds the activity of tryptophan oxygenase by one or two orders of magnitude. The latter fact is also known for vertebrate liver, and one wonders whether the enzyme might not perform some other function in addition to splitting formyl-kynurenine. In passing, it should be noted that kynurenine formamidase extracted from rat liver or rat skin is strongly inhibited by organophosphorus compounds (JansCri et al., 1969). While the enzyme has recently been purified 1800-fold from rat liver and obtained in an apparently homogenous state (Shinohara and Ishiguro, 1970), no corresponding attempt has been made with insect material.

To the author’s knowledge no mutant exists which lacks kynurenine formamidase activity. Such mutants are, however, riot likely to be detected by phenotype, because formylkynurenine undergoes spontaneous hydrolysis.

6.3 KYNURENINE-3-HYDROXYLASE (EC 1.14.1.2)

Attempts to demonstrate insect kynurenine hydroxylase in uitro were for a long time unsuccessful, in spite of a marked accuinulation of 3-hydroxy- kynurenine in certain developmental stages. In retrospect it appears that the main reason for failure was the fact that hynurenine hydroxylase is inactivated by light, a phenomenon first detected in Staudhger’s laboratory (Mayer et al., 1968) and probably due to the participation of FAD in the reaction.

TABLE 10

Kynurenine formamidase in insects: data on properties and occurrence of the enzyme*

Species Inhibitors Activity and localization** Stage Optimal Km

investigated pH M litre-' Reference+

D. melanogaster

D. uwilis Cry Ilus b ima cula tu s

Phormia terraenovae

Bombyx mori

Flies 7.3

Flies 7.3 Last larva1 9.0

All stages 7.3

All stages 9.25

3.1 x 1 0 - ~

3.1 x 1 0 - ~ 1.5 x 1 0 - ~

1.3 x 1 0 - ~

NaHS03

NaHSO 3

Substrate

Substrate L-kynurenine Substrate

Arbitrary units, no striking differences between strains

a

a

b Fat body: 4 nmol min-' mg-' protein Malpighian tubes: 15 nmol min-' mg-' protein Gut: 1-2 nmol min-' mg-' protein Testes: 4 nmol min-' mg-' protein Ovaries: no activity Larvae: 30 nmol min-' Flies: 4 nmol min-' mgYgprotein High: testes Medium: ovaries, Malpighian tubes, eyes, gut. fat body Low: silk gland, developing wings All stage dependent

-' protein c

d

* See also Hiraga (1 9 64). ** In homogenates of whole animals or isolated tissues. t a. Glassman (1956); b. Tiedt (1971); c. Linzen and Schartau (unpublished); d.Linzen (unpublished).


Some indirect observations on thc activity of the enzyme were made by Egelhaaf (1963a, 1963b), by injecting kynurenine into Ephestia moths and measuring the appearance of the product. A linear increase of about 3 pg h-’mg-’N was observed during at least 90 min. Ovaries (mainly the upper parts) and fat body were considered to be especially active.

In 1967 Ghosh and Forrest and Linzen and Hertel simultaneously published in uitro assays of kynurenine hydroxylase in Drosophila and Calliphora respectively. Later investigations were concerned with Bombyx mori rb (Linzen and Hendrichs-Hertel, 1970), Schistocerca gregaria (Pinamonti et al., 1970-71), and Apis mellifica (Dustmann, personal communication). 3-Hydroxy-kynurenine was measured either by Inagami’s nitrous acid method (Ghosh and Forrest), by oxidation to xanthommatin (author’s laboratory) or by two-dimensional chi-omatographic isolation. Hendrichs-Hertel and Linzen (1 969) proved enzymatic formation of 3-hydroxy-kynurenine independently by incubating tritiated kynurenine and isolating the labelled reaction product.

Evidently the optimal conditions for assay of the enzyme have not yet been awertained. Both in the author’s laboratory and that of the Italian group a preparation given by Ginoulhiac et al. ( 962) has been adopted with slight modifications, while Ghosh and Forrest (1967a) developed a medium of their own. NADPH is required for normal activity, but there is also a slight effect when NADH is added. Ghosh and Forrest (1967) also speculate on the participation of a pteridine cofactor. In the author’s view this is doubtful; in mammalian preparations a pteridine has not been found as part of the system, while the participaticn of FAD is now a well-established fact (Okamoto and Hayaishi, 1967, Horn et al.. 1971). The optimal substrate concentration was 4 mM in Ghosh and Forrest’s work, while in Calliphora preparations there appeared strong substrate inhibition at this level (Hendrichs-Hertel and Linzen, 1969). The K , has been determined only for the enzyme of Schistocerca (Pinamonti et al., 1970-71) and comes to 4 x lo-’ mol litre-’. The optimal pH is slightly above neutrality. Cysteine and azide ions appear to be stimulatory, except for the bee enzyme which is not stimulated by azide, and even inhibited by cysteine (Dustmann, personal communication). While cyanide inhibits oxidation of NADPH by the fraction of “light” mitochondria (Mayer and Staudinger, 1967), and might therefore increase the observed activity of kynurenine hydroxylase indirectly, it was shown to be inhibitory in recent experiments by Dustmann (personal communication) and Linzen (unpublished). Rat liver kynurenine hydroxylase is inhibited by cyanide only in a purified state (Horn et al., 1971). Dustmann reported strong inhibition of the bee enzyme by xanthommatin, so that wild-type eye extracts become completely inactive. Evidently compartmeritation is an important factor in pigment biosynthesis.

192 BERNT LINZEN

In mammals (Okamoto ~t al., 1967) and in Neurosporu (Cassady and Wagner, 1971) kynurenine hydroxylase is clearly localized in the outer mitochondrial membrane and for this reason is a useful marker enzyme of this fraction. It is probably also restricted to mitochondria in insects (Ghosh and Forrest, 1967, and Pinamonti et al., 1971, traced the activity to the mitochondrial membrane fraction), despite the contradictory findings of Hendrichs-Hertel and Linzen (1969) who consistently detected part of the activity in the soluble fraction. It may be argued that in insects even very careful homogenization (with a teflon pestle) might damage mitochondria because of the grinding action of cuticular particles. It might be further supposed that in some species the outer mitochondrial membrane could be extremely labile. Such a situation has been encountered by Graszynski (1970) in crayfish. Attention should also be drawn to electron microscopic studies by Wessing (1 962, 1963) on the fine structure of Drosophila Malipighian tubules. The endoplasmic reticulum of these cells forms a system of channels, which occasionally widen to form “storage ampoules”. These ampoules, in certain stages, are crammed with solid 3-hydroxy-kynurenine. Wessing proposed that these structures, which are typically found in the neighbourhood of Golgi apparatus, might also be a site of kynurenine hydroxylation. Direct evidence to support this hypothesis is, however, still lacking.

As in the case of tryptophan oxygenase, kynurenine hydraxylase cannot be assigned to a particular organ or tissue. The enzyme has been found in many types of tissue (nervous tissue has not been examined). In Rombyx the Malpighian tubules show a high peak of activity two days prior to spinning, but fat body, ovaries, and eyes are also able to hydroxylate kynurenine (see also p. 214 for more detailed treatment). In Schistocerca the eyes are the main source of the enzyme besides some activity in the integument (Pinamonti et al.. 1970-71) in bees the eyes are the only tissue active (Dustmann, personal Eommunication). In Calliphora the Malpighian tubules are particularly active, while the data obtained for fat body are at the limit of detection. In eye discs, kynurenine hydroxylase should certainly be present, since Danneel had demonstrated in 1941 that explanted heads of Drosophila u would readily form ommochrome if kept in a solution containing kynurenine. Horikawa (1958) has made similar, more elaborate experiments with eye-antenna1 discs in culture. Hendrichs- Hertel and Linzen (1969) failed to detect the enzyme in Calliphora eye tissue, possibly due to the inhibition of the enzyme by xanthommatin mentioned above.

Both in Ephestia and in Drosophila, kynurenine hydroxylase is a constitutive enzyme, as evident from the experiments of Egelhaaf, Danneel, and Horikawa (all cited above), with mutants blocked at the tryptophan


oxygenase step. The same holds for the bee mutant snow (Dustmann, personal communication).

6.4 KYNURENINASE AND KYNURENINE TRANSAMINASE (EC 3.7.1.3; 2.6.1.7)

Kynureninase (EC 3.7.1.3) is situated in the main pathway of tryptophan degradation in vertebrates, while kynurenine transaminase (EC 2.6.1.7) catalyses a side reaction. To the knowledge of the author, the only study relating to the former enzyme in insect material is Inagami's (1958). In Bombyx mori, the major metabolites of tryptophan are anthranilic acid and its conjugates. These are lacking in the mutant rb where 3-hydroxy- kynurenine is accumulated. Inagami proved that the mutation to rb is associated with a loss of kynureninase activity. With wild-type homogenates he could demonstrate the enzyme in gut wall (larvae) and in whole pupae where both kynurenine and 3-hydroxy-kynurenine were cleaved. The enzyme precipitated between 35 and 55 per cent saturation with ammonium sulphate and remained active after prolonged dialysis even if pyridoxal phosphate was omitted from the redction mixture. Inagami concluded that the cofactor is tightly bound to the enzyme.

Kynurenine transaminasr has been studied during development of the parasitic wasp, Habrobracon juglandis (Leibenguth, 1967a), and more closely in Schistocerca gregaria (Pinamonti et al., 1970). In both cases distinct enzymes, specific for either kynurenine or 3-hydroxy-kynurenine, were not found. For the locust enzyme the K, for the two substrates is 5.4 x and 1.5 x l o d 3 mol litre-' respectively. Pyruvate and oxalo- acetate are better amino group acceptors than a-kstoglutarate. The K, for pyridoxal phosphate was found to be 1.6 x mol litre-'. Optimal activity occurs at pH 8. The only tissue active is fat body.

7 Ommochrome biosynthesis

The oxidation of ortho-aminophenols can be rasily performed in the laboratory. Nevertheless, phenoxazinones will not result in every case; extensive studies of model reactions (Butenandt et al.. 1954d, 1957a, 1957b, 1957c) have shown that different ring systems can be formed from 3-hydroxy-anthranilic acid or 2-amino-3-hydroxy-acetophenone, depending on the conditions of oxidation. Enzymes catalysing the formation of phenoxazinones should, therefore, be specific not only for a particular substrate, but also for the type of reaction. Such enzymes were first demonstrated in microbial and plant material (Weissbach and Katz, 1961; Katz and Weissbach, 1962; Nair and Vaidyanathan, 1964; Nair and Vining,

194 BERNT LINZEN

1964-to cite a small selection of papers). FMN was reported to act as a cofactor in some of these systems. Systems oxidizing 3-hydroxy-anthranilic acid to cinnabarinic acid were also obtained from vertebrate liver (Joshi and Brown, 1959; Gutmann and Nagasawa, 1959; Morgan and Weimorts, 1964; Subba Rao et al., 1965), but there is no evidence that these systems have a biological role.

Since ortho-aminophenols are so easily oxidized, reports on enzymatic formation of xanthommatin should be viewed with some caution. The first of these studies (Butenandt et al., 1956) was started as a re-examination of Inagami’s (1954b) finding of a “red melanin” which arose during the incubation of a mixture of DOPA and 3-hydroxy-kynurenine with tyrosinase. The formation of a “mixed” red melanin was not confirmed. However, it turned out that Calliphora tyrosinase does not oxidize 3-hydroxy-kynurenine at neutral pH, but brings about the formation of xanthommatin, in the presence of DOPA in the incubation medium. Thus, DOPA-quinone as the primary oxidation product serves in lieu of a cofactor of phenoxazinone synthesis. Although the frequent association of cuticular ,

melanin with hypodermal ommochromes (Dustmann, 1964) is suggestive of a similar mechanism in vivo, there are many arguments against this possibility. First, while ommochromes and melanins are often found in close proximity, they do not really occur jointly; neither are they intermixed. Secondly, a correlation between tyrosinase activity and ommochrome synthesis could not be established in Hestina larvae or in silkworm diapause eggs (Osanai, 1968a, 1968b). Thirdly, mutants devoid of xanthommatin still have an active tyrosinase (Phillips et al., 1970). The oxidation of 3-hydroxy-kynurenine by tyrosinase from three sources other than insects has recently been employed for micropreparative synthesis of xanthommatin (de Antoni et al., 1970). These enzymes acted directly on the substrate, without mediation by DOPA.

Similar objections may be raised against the direct participation of the cytochrome system in xanthommatin synthesis. The formation of phenoxazinones, as observed by Joshi and Brown (1959), and Gutmann and Nagasawa (1959), must again be regarded (as in the case of the tyrosinase-DOPA system) as an unspecific oxidation by a system of high oxidation potential. It was also reasoned by Osanai (1967) that diapausing eggs of the silkworm are practically devoid of cytochrome c. A model system of a different type, by which phenoxazinones may be generated, was recently described by Ishiguro et al. (1971a). They showed that 3-hydroxy-anthranilic acid is oxidized to cinnabarinic acid by haemoglobin in the presence of Mg++. Interestingly, the magnesium-stimulated oxidation is fairly specific, as 3-hydroxy-kynurenine and DOPA are not oxidized at all, and o-aminophenol is oxidized at a reduced rate. Mention must also be made of the work of Rohner and Wolsky (Rohner, 1959; Wolsky, 1960)


who observed inhibition of pigment synthesis after exposing developing insects to an oxygen-carbon monoxide atmosphere.

Phillips and Forrest (1970) have recently reported the formation of phenoxazinones by an extract of Drosophila pupae. 3-Hydroxy-anthranilic acid was converted three times as fast as 3-hydroxy-kynurenine. The enzyme could be separated from tyrosinase activity and activated by heat treatment and passage over Sephadex G-50. It would be interesting to know whether this enzyme is localized in the eye anlagen. Surprisingly, the active principle is also present in mutants unablc to convert 3-hydroxy- kynurenine to xanthommatin.

Rhodommatin and ommatin D might arise from xanthommatin by direct glycosylation and esterification, respectively. In vitro evidence of these transformations is still lacking. Kubler (1960) injected labelled xanthommatin (prepared by biosynthesis in Callzphora) into late larvae o f Argynnis paphia and isolated the two substituted ommatins by repeated precipitation and two-dimensional paper chromatography. Ilhodommatin was clearly labelled, but an unexpected result was the complete absence of radioactivity from ommatin D. Actually, this supports the assumption that xanthommatin is glucosylated directly, for if the radioactivity in rhodommatin originated from 3-hydroxy-kynurenine (formed by decomposition of xanthommatin) one would also have expected radioactive labelling of ommatin D. It is curious, however, that neither at the onset of excretory pigment formation in Malpighian tubules and gut wall, nor later, can xanthommatin be detected among the ommatins present (Kubler, 1960).

Very little is known about the biosynthesis of ommins and ommidins. Since both contain a sulphur atom, the origin of this posed the first problem. Attempts to clarify this by incorporation of labelled sulphur are complicated by the strong tendency of ommins to retain small amounts of protein which might label more strongly. 1,in:cen ( 1 970) undertook the isolation and purification of “pigment IV” which still contains the sulphur atom. He demonstrated the incorporation of 35S from methionine and cysteine, sulphate and sulphide giving negative results. As the elementary analysis of “pigment IV” and dihydro-xanthonimatin differ by one atom each of carbon, nitrogen, and sulphur (in favour of the former) the incorporation of thiocyanate was tested recently. In these experiments no radioactivity was recovered in “pigment IV”. Unless it is established whether “pigment IV” is an artifact produced during the vigorous hydrolysis of ommins, or part of the original ommin molecule, all incorporation studies will be subject to some uncertainty.

In developing insect eyes, ommochromes appear in sequence. In Ephestia and Ptychopoda a striking observation has been made-(Kuhn, 1960, 1963; see also Muth, 1967): xanthommatin is the first ommochrome to be detected, followed by “ommochrome 11” and “ommochrome I” and,

196 BERNT LINZEN

finally, by ommin A (Fig. 13). This is exactly the series of decreasing mobility of the ommochromes in paper chromatography which is thought to correspond to the increasing complexity (in former terms, increasing degree of polymerization) of the ommochromes. The finding of this order is highly suggestive of a precursor role played by the “simpler” ommochromes in the biosynthesis of the more complex ones. De Almeida (1968), on the basis of extracting ommochromes for different lengths of time, speculated that ommin A was deposited later on the matrix of the pigment granule than the other ommochromes. Direct evidence of the conversion of one ommochrome into another is nevertheless still not available.

. . . ,

: : . : ( 1 . .

. . . :

. . . . . .

i. ( . ) .., ; J

. . I

(i ,)-Br~ck red

Carmine

Bluish violet

( a ) ( b ) ( C ) ( d )

Fig. 13. Sequence of appearance of ommochromes during eye pigmentation in Ptychopodn serinta. X, xanthommatin; 0, ommin (probably ommin A); I and I1 are Kiihn’s ommochromes “I” and “11”-probably ommins. (From Kiihn, 1963.)

Finally, in relation to ommochrome biosynthesis, we may ask whether there is any turnover of ommochromes. According to orthodox opinion ommochromes are regarded as end products of tryptophan metabolism which are either excreted or stored until the death of the insect. A drastic decrease of ommatin content in the gut of diapausing Ceruru pupae (Linzen and Biickmann, 196l) , originally viewed as metabolic breakdown, is in all probability due to spontaneous decomposition. Dustmann’s (1964) examination of the chemical basis for morphological colour change in the stick insect, Curuusius morosus, has revealed that during the life of the insect the ommochrome content only increases. Thus, most results do not favour the hypothesis of a metabolic degradation of ommochromes. Recently Hehl and Linzen (to be published) measured the incorporation of radioactivity from labelled tryptophan or 3-hydroxy-kynurenine into xanthommatin in the compound eye of the blowfly, Calliphoru. They found that even in the aged fly, when the amount of xanthommatin is at a


constant level, there is still some incorporation. This might be indicative of a very slow turnover. There is some histologica.1 evidence of degradation of pigment granules. In the eyes of the cricket, Pteronemobius heydeni, autophagous vacuoles have been observed, which enclose material of diverse origin, including membrane fragments and pigment granules (Wachmann, 1969). “Granulolysis” is also documented by electron micrographs of the stomatopod crustacean, Squilla mantis (Perrelet et al., 1971). Still, the turnover of ommochromes remains a matter for debate.

8 Tryptophan metabolism in insect development

The most important fact with regard to tryptophan metabolism in insects is the failure to degrade 3-hydroxy-anthranilic acid to water, carbon dioxide and ammonia (i.e. to easily permeable products). As a consequence, metabolites produced prior to this metabolic block will accumulate whenever the exchange of chemical compounds between the insect and its environment is impaired or rendered impossi‘ble. This is the case during embryonal development in the egg in dormant and pupal stages and during moults. Accumulated metabolites may be subsequently excreted or used for some secondary function. Examples have been given in section 5 .

8.1 EGGS AND EMBRYONAL DEVELOPMENT

Accumulation of tryptophan metabolites in the eggs is known in a number of species. In Ephestia it causes the pigmentation of the ocelli in larvae of the a/a genotype. It might be expected that such larvae would be devoid of pigment, but if they originate from heterozygous (a +/a) mothers they can draw on a supply of 3-hydroxy-kynurenine laid down during growth of the oocyte (Kiihn and Plagge, 1937; Egelhaaf, 1963a). If kynurenine is injected into Ephestia females it is transiently accumulated in the ovarioles, its concentration subsequently decreasing slowly while 3-hydroxy-kynurenine increases. It is assumed, therefore, that the enzyme, kynurenine-3- hydroxylase, is present in this tissue. In ovaries of the silkworm the activity of this enzyme is relatively high. In addition, there is active uptake of 3-hydroxy-kynurenine from the haemolymph. The selective absorption of this metabolite is under control of the “diapiiuse hormone”. This can be best demonstrated by use of the white-l mutant which is unable to hydroxylate kynurenine (Yamashita and Hasegawa, 1966, and earlier papers; Sonobe and Ohnishi, 1970). The level of 3-hydroxy-kynurenine in diapause eggs continues to rise during the lirst 12 h after oviposition, suggesting that the hydroxylase is located wi :hin the egg cytoplasm. The disappearance of 3-hydroxy-kynurenine in silkworm eggs and its conversion into ommochromes (Fig. 14) has been measured several times. Kikkawa

198 BERNT LINZEN

I 2 3 4 5 6 7

Days after oriposition

Fig. 14. Concentrations of free tryptophan, kynurenine, 3-hydroxy-kynurenine, xanthommatin and ommins in eggs of the silkworm, Eombyx mori The eggs are entering diapause. (From Koga and Osanai, 1967.)

(1941 j first measured disappearance of the “+-chromogen”, its identity being established later (Kikkawa, 1953; Inagami, 1958; Koga and Goda, 1962; Koga and Osanai, 1967; further work cited by Inagami). Inagami compared a number of mutants and found that the level of 3-hydroxy- kynurenine remains constant if ommochrome synthesis is blocked genetically. This shows that 3-hydroxy-kynurenine is neither transaminated nor cleaved by kynureninase at this stage of the life cycle. If the eggs are not fertilized and pigment synthesis does not ensue, the level of J-hydroxy- kynurenine, as measured by Ehrlich’s diazo reaction (not specific) remains


constant even 40 days after oviposition (Kikkawa, 1941). In fertilized diapause eggs about 95 per cent of the 3-hydroxy-kynurenine content is utilized for ommochrome synthesis by the serosa (Fig. 14), the remainder being conserved through the months of diapause. During the period of embryonic development there is also no decrease in level, the final stage examined being one day before hatching (Inagami, 1958). The function of the ommochromes in the serosa is not clear. !screening against light could be a plausible role. If white-1 eggs are supplied with S-hydroxy- kynurenine, they will synthesize ommochromes in the normal manner; such eggs will hatch at a higher percentage than unsupplemented white-1 eggs, and embryonal development will be more rapid (Kikkawa, 1948, cited by Kikkawa, 1953). However, a re-examination of the causal relations is desirable, since secondary metabolic effects might also be implied.

It is most intriguing, therefore, that in the Noi.odontid moth, Cerura vinula, a copious amount of an ommochrome (omniatin D) is produced by the endothelium of the ovariole stalk, a proces:; which later extends through the efferent ducts. Prior to oviposition, this pigment is dissolved and precipitated on the surface of the eggs, the exocliorion. The eggs appear reddish-brown. Thus the same postulated effect of protection from light could be achieved as in the silkworm, but by completely different means (Geiger, personal communication). According to Geiger, the viability of 'the eggs is not influenced by the pigmentation.

In spite of the examples just cited, the accumulation of tryptophan or of its metabolites within eggs does not appear to be the rule. In eggs of the migratory locust, Schistocerca gregaria. only traces clf kynurenine could be detected immediately after oviposition, other metabolites being completely absent. 3-Hydroxy-kynurenine appeared after ten days of incubation and rose gradually to about 0.1 pmol g-' fresh weight at thk time of hatching. During the last third of incubation, small quantities of kynurenic and xanthurenic acids were also found (Colombo and Pinamonti, 1965).

Apparently, embryonic development itself does not lead to an excess of tryptophan or of its metabolites. This can be understood if it is assumed that the pattern of embryonic metabolism is such that the composition of the yolk proteins is optimal. In Schistocerca, the appearance of S-hydroxy- kynurenine is clearly linked to the onset of ommochrome synthesis in the developing eyes.

An account of the accumulation of kynurenine in eggs of Drosophila melanogaster is provided on page 126.

8.2 LARVAL DEVELOPMENT. HEMIMETABOLA Most studies devoted to the developmental aspects of metabolism in insects were stimulated by the dramatic changes observed or expected during

200 BERNT LINZEN

metamorphosis. Relatively little is known, therefore, about the regulation of tryptophan metabolism during larval development. However, since the initiation of each moult requires some time (of the order of one day in the large holometabolous insects), and since further time elapses until feeding is resumed after a moult, one may anticipate that similar though quantitatively less important alterations in the pattern of tryptophan metabolism occur during larval moults.

Buckmann (1959a) noticed that ommochromcs appeared in the excreta in the early larval stages of Cerzira vinula, i f the animals were subjected to starvation or ligation. Evidently the potential to synthesize ommochromes (and to perform the prior degradative steps) is always present in the excretory system. In the green areas of the integument of the larvae, ommochromes are not formed after experimental injury o f this kind. In contrast, large quantities of xanthommatin are synthesized in the integument during the preparation for t.he larval-pupal moult. Possibly the juvenile hormone, in conjunction with other factors, controls pigment synthesis in the hypodermis, but not in the excretory organs. Wolfram (1949) observed ommochrome excretion in young larvae of Ptychopoda only in 2 out of 40 specimens; these two were examined “immediately prior t o moulting”.

The foregoing observations are indicative of a stimulation of tryptophan breakdown under abnormal conditions, such as injury or impairment of food supply. Further insight can be gained from some data on tryptophan metabolism in Hemimetabola. Pinamonti et ul. (1964) determined several metabolites in tissues of the last larval stage and in the young imago of Schistocerca greguria. In most samples the levels were too low’ for quantitative estimation, but the level of kynurenic acid was found to rise in the integument and in the Malpighian tubules, as the larvae approached the final moult. 3-Hydroxy-kynurenine is chiefly found in the integument and is not subject to major variations. In the cricket, Gryllus bimaculutus, the levels of free tryptophan, kynurenine, and 3-hydroxy-kynurenine have been determined in whole last stage larvae (Tiedt, 1971). Only the tryptophan and kynurenine levels change significantly (Fig. 15). 3-Hydroxy-kynurenine is strongly incorporated into ommins one day after the last larval moult (and possibly earlier ones), and subsequently at a decreasing rate (Linzen, 1968). Finally, the activity of the enzyme, tryptophan oxygenase, is elevated during the periods of moulting.

On the basis of these data and the observation that locusts excrete ommochromes during each moult, it can be tentatively concluded that during the periods of moulting tryptophan is likely to be liberated from proteins (which are broken down in the course of structural rearrangement or as a source of energy) and to rise to levels at which immediate degradation becomes critical. In the intermoult periods it is probably

THE TRYPTOPHAN + OMMOCHROME PATHWAY IF1 INSECTS 201

- 0

c 0

0

E .- L

20 i

0 50 100

Per cent of stage

Fig. 15. Concentrations of tryptophan (- - -) and kynurenine (-) in Cryllus bimuculatus during the final larval stage. (Adapted from Tiedt, 1971.)

consumed in protein synthesis. Similar considerations should apply to the larval development of the Holometabola. An observation, not mentioned before, to support this has been made by Inagami (1958): in the haemolymph of silkworm larvae there is a doubling of 3-hydroxy- kynurenine concentration (from 0.25 to 0.5 c(M m1-I) at the time of moul ting.

8.3 ACCUMULATION OF TRYPTOPHAN METABOLITES DUWNG METAMORPHOSIS OF

HOLOMETABOLOUS INSECTS

The metamorphosis of holometabolous insects presents serious metabolic problems. The morphological transformation is so radical that the animal must withdraw for a while from its activities and from its hitherto prevailing environment, discontinuing excretion and thus changing into a nearly closed system. Furthermore, all Holometabola attain their maximal weight prior to metamorphosis; the subsequent reduction of biomass, which accompanies development towards the adult animal, may amount to 80 per cent. This figure presumably marks an extreme, resulting, if part of the accumulated protein of the larva is consumed, in the construction of cocoons or in the generation of other devices which protect the metamorphosing insect.

As reported above, ommochromes are formed in many species of Lepidoptera at the onset of metamorphosis and are excreted upon eclosion from the pupa. From what we know about tryptophan degradation, this burst of pigment production indicates a massive Iireakdbwn of proteins. Such a breakdown should also be reflected by alterations in the levels of other metabolites. There is ample information to confirm this.

202 BERNT LINZEN

8.3.1 Bombyx mori The silkworm loses about 80 per cent of its fresh weight during metamorphosis: Yamafuji (1937) gives 78 per cent and Linzen (1971a) 85 per cent for the mutant 7-6. The varying water content of different developmental stages makes such data difficult to interpret, but measurement of total DNA has produced very similar figures (Linzen, 1971a; Chinzei and Tojo, 1972). The most spectacular decline is seen during the two days immediately preceding and following the onset .of cocoon spinning. In the rb mutant more than 40 per cent o f the total DNA loss occurred during this period. This recalls the statement of Kellner et al. (1884) that more than half of the silkworm’s total body protein is consumed during cocoon spinning. Since silk is poor in tryptophan (Lucas et al., 1958) but must be synthesized from protein of average composition, an excess of tryptophan should ensue. A steep rise and a peak of free tryptophan concentration is in fact observed at this stage (cf. upper diagram of Fig. 20), but the absolute amount is only a fraction of what must be expected (Linzen, 1971a; see also Fukuda et nl.. 1955, for tryptophan concentration in haemolymph). The remainder must be sought in the last larval excreta and in the form of various metabolites.

Two-dimensional fractionation of extracts on paper (Lirizen and Ishiguro, 1966) has indeed revealed, in the mutant rb, about ten fluorescent compounds which either make their first appearance at this stage or increase significantly in quantity. Some of these might belong to other metabolic pathways, and some might be accumulated only in this particular mutant because of the metabolic block at the kynureninase step. Kynurenine and 3-hydroxy-kynurenine are both accumulated, and both the glucoside and the sulphate (Inagami, 1958) of 3-hydroxy-kynurenine can be identified. In the normal strain a group of metabolites which are derived from anthranilic and 3-hydroxy-anthranilic acids are found. Some of these, such as Inagami’s compounds “F”, “I”, and “L”, still await identification. An appreciable, though not measured amount of tryptophan, is converted into pigment, thus causing the larvae to change colour.

Quantitative data on metabolite concentrations have been obtained by Kikkawa (1953), Inagami (1958), Linzen and Ishiguro (1966), Ishiguro et al. (1971b), Ishiguro and Nagamura (1971), and Linzen (unpublished). Starting on the first day of spinning, the level of kynurenine rises to 0.3-0.5 mg g-’ in white-l pupae, and to 0.28 mg g-’ in male pupae of the mutant rb. In the mutant rb the kynurenine concentration was found to diverge in males and females, beginning on day 5 after pupation (Fig. 16). Just before eclosion there is only half as much kynurenine in the female as in the male. Ishiguro et nl. (1971) described a reciprocal divergence of the 3-hydroxy-kynurenine level at the same time of development. The pronounced conversion of kynurenine to 3-hydroxy-kynurenine in the


1 . 4 r I I I 1 I 1 1 I 1 I 1 1 I L

. . * .

* *

. .

O O

. 0

0

0

0 . .

female is without doubt due to the activity of kynurenine-3-hydroxylase in the ovaries.

The level of 3-hydroxy-kynurenine rises immediately upon cessation of feeding, reaches 0.4 mg g-' in young pupae and a peak of 1 mg g-' two days prior to eclosion. The compound is not evenly distributed. It is found dissolved in the haemolymph and occurs at greater concentrations in hypodermis and in Malpighian tubules (0.15, 0.4, and 1 PM ml-' or g, respectively, in the middle of the 5th larval stage). This distribution may change. During the period of cocoon spinning the capacity of the Malpighian tubules to store 3-hydroxy-kynurenine is reduced (Hertel, 1968) At the same time, more 3-hydroxy-kynurenine goes into fat body and gut. In the late pupa, part of the compound is oxidized to ommochromes. Another portion is conserved within oocytes in the female: if the 3-hydroxy-kynurenine content of the ovaries is subtracted from the total, identical quantities are obtained in females and males (Ishiguro et al., 1971b).

In Bombyx mori, ommochromes are synthesized in the fat body, in the gut, and in Malpighian tubules mainly during the very e a l y and the late stages of metamorphosi:. This is deduced from visual inspection. A quantitative analysis of the xanthommatin content in pupae of the mutant

204 BERNT LINZEN

rb (Ishiguro et al., 1971b) revealed only a single burst of xanthommatin synthesis which occurred shortly before eclosion. If the appearance of red pigment and the gross morphological changes are compared from day to day, there seems to exist an overall parallelism in the intensity of both. In fat body, the stages of intensive pigment synthesis coincide with periods of declining DNA-RNA ratio, each time following a peak of this ratio (cf. Chinzei and Tojo, 1972).

8.3.2 Crrura vinula

Pupae o f this Notodontid moth undergo a diapause, which separates the two periods of interest with respect t o tryptophan breakdown and ommochromc synthesis (i.e. the periods of greatest morphogenetic activity). The early phase, from cessation of feeding until pupation, was investigated by Linzen and Biickmann (1961) and by Biickmann et al. (1966). This period is more extended than in Bonzbyx morz (9.5 versus 3-4 days) and can be easily divided into successive stages on the basis of the spectacular colour change described above (p. 175). In the course of one week, the larva loses 50 per cent in weight (Fig. 17(a)). Much of this represents water loss although a considerable amount of protein is spent in construction of the cocoon. Total haemolymph proteins show an absolute decrease in quantity. The protein concentration fluctuates strongly, however, as a consequence of fluctuations in haemolymph volume. In contrast, the concentration of ninhydrin-positive material shows a surprising constancy, while individual amino acids are subject to profound changes. A peak in tryptophan and 3-hydroxy-kynurenine concentration coincides with the stage of maximal dark-red colouration (Buckmann’s stage 11) (Fig. 17(b)); by this time the larva is actively building its cocoon. The level of kynurenine has not been determined, but, according to Geiger (personal communication), “large quantities” are excreted at the last defecation.

Only at the beginning of the metamorphosis of the Cerura larva are ommochromes synthesized in the integument. From stage I1 onwards they are deposited in fat body and gut. By injecting tryptophan, J-hydroxy- kynurenine, and ecdysone (either alone or in combination) into intact feeding animals or into isolated abdomina, it was demonstrated (Biickmann, personal communication) that the larvae are capable of degrading tryptophan to 3-hydroxy-kynurenine at any time. Integumental xanthommatin is, however, formed only under the influence of ecdysone. Further experiments are necessary to discover whether ecdysone directly induces individual enzymes along the ommochrome biosynthetic pathway or whether the synthesis of ommochromes is a more remote effect.

At the termination of pupal diapause there is a further shift of the ommochromes (Geiger, personal communication). The total ommochrome

5 10 15

Days U

I I I I I I I I I O 2 0 , I I I m r n P P2

Stages ( a )

50

10

1.6 -

I 4 -

1.0

08-

-

.d 0.2 -

q'j.o., I I I , I I I I I I 5 10 15

Days

Stages (b)

Fig. 17 (a and b). Total body weight, haemolymph volume, haemolymph as per cent of body weight, and concentrations of free tryptophan and 3-hydroxy-kynurenine in the haemolymph of Cerura vinula L. at the onset of metamorphosis. Abscissa in days and in stages: 0 2 and 0 4 are feeding larvae, I to I11 stages of morphological colour change, IV prepupa, P pupa, U marks beginning of integumental colour change. (From Biickmann et al., 1966.)

206 BERNT LINZEN

in fat body decreases by 70 per cent, while at the same time there is a strong increase in the gut. I t is highly probable that the pigment is transported via the haemolymph but in contrast to the stage prior to diapause (when ommatins appear in solution) no ommatins could be demonstrated in the haemolymph of the pharate adult.

8.3.3 Phormia terraenovae

Less complicated metabolic problems than those dealt with in the above two species can be expected in insects which do not spin cocoons but rely on other mechanisms for protection. As an example, the blowfly, Phormia terraenovae, was analysed with respect to the number and quantitative changes of tryptophan metabolites (Linzen and Schartau, to be published). It was found that the inventory of metabolites is restricted to the pathway leading directly to the ommochromes; traces of kynurenic and xanthurenic acids but neither of the anthranilic acids were found. The concentration levels of tryptophan and of two of its metabolites are shown in Fig. 18. Again, the primary event at the onset of metamorphosis is the liberation of tryptophan prior to pupation. I t is puzzling that its degradation is not noticeable three days later, although, as will be reported below; tryptophan oxygenase is always present. Spatial separation of enzyme and substrate, or temporary inhibition of the enzyme must be postulated. The rise of the kynurenine level coincides with the beginning of eye pigmentation. Although the rate of ommochrome synthesis is maximal during the day before eclosion, synthesis continues until the third day of the ffy’s life.

In Phormia also the course of total protein-bound tryptophan was determined. Although the method applied (Roth, 1939) is debatable (cf. Linzen, 1971a), the values obtained appear to be meaningful if compared with other data: from the day of pupation to eclosion, the fresh weight decreases by 20 mg, the “extracted dry weight” by 6 mg, and protein- bound tryptophan by 30-35 pg (all data, per animal). A balance will be presented below, which shows that practically all tryptophan liberated is converted into xanthommatin, which is used as screening pigment in the large compound eyes.

8.3.4 Other species

During studies dealing with particular aspects of tryptophan metabolism in insects, many results have been obtained which fit into the frame defined by the above three examples.

In Ephestia (Egelhaaf, 1957, 1963a) the tryptophan level begins to rise in the prepupa and reaches a peak of about 120 pg g-’ fresh weight on day 3 (20 per cent) of pupal development. In the strain BK 14 (wild type) the rise of the kynurenine level is roughly parallel. After the 4th day in the


Fig. 18. Levels of tryptophan, kynurenine and 3-hydroxy-kynurenine during development of Phormia terraenovae. Abscissa in per cent of larval development and in days. Figures along curves give amount in pg per animal. Figures at right margin give amount on 11th and 15th day after eclosion. P,, and F, are days of puparium formation and eclosion, respectively.

pupa there is a sharp drop. This could be a consequence of continued kynurenine hydroxylation at a moment when liberation of tryptophan from protein slows down. The metabolite levels dif6-r with strains and an influence of rearing conditions must be expected also, but has not been so far examined. The level of 3-hydroxy-kynurenine reaches its maximal value (-1 mM) in the pupal stage. In the mutant a which lacks kynurenine and all subsequent metabolites, the level of free tryptophan is elevated correspondingly (0.75 mM at the time of pupation, -1.5 mM at peak concentration) and remains high after emergence of the moth, although a

208 BEANT LINZEN

fraction of the tryptophan content is taken up by the eggs. In Plodia, the course of tryptophan concentration has not been followed but kynurenine and 3-hydroxy-kynurenine rise 10-20-fold, and about 4-fold, respectively (Mohlmann, 1958; de Almeida, 1961).

The data obtained in Calliphora erythrocephala compare well with those of Phormia (free tryptophan: Langer and Grassmader, 1965; Grassmader, 1968; 3-hydroxy-kynurenine and xanthommatin: Linzen, 1963). After a sharp rise, the tryptophan level declines slowly. A comparison of the wild type with the mutant chalky (pigmentless) reveals that about two thirds of the tryptophan stored must be excreted upon emergence and may therefore reside within the Malpighian tubules or hindgut. Analysis of the meconia for their tryptophan content has demonstrated this. 3-Hydroxy-kynurenine rises from the egg through the larval stages until the middle of the pupal stage (-35pg per animal, -2 mM) and is later consumed during eye pigment synthesis. The amount of xanthommatin in wild-type eyes given by Linzen (1963) must be reduced by a half, as it is based on an extinction coefficient which has now been shown to be incorrect (EY.fZ = 7.32; true value, 15.1 or higher). It is clearly seen that 3-hydroxy-kynurenine is quantitatively converted into xanthommatin, further 3-hydroxy- kynurenine (15-18 pg per animal) being required for completion of eye pigmentation. This is synthesized during the second half of pupal development. Again, this is seen in the 3-hydroxy-kynurenine level of the mutant chalky. In the mutant, however, the excess of 3-hydroxy- kynurenine is not accumulated within the eyes but is excreted. The meconia contain 16.4 pg per animal (Grassmader, 1968), but it is almost certain that excretion of 3-hydroxy-kynurenine continues beyond this time.

In Drosophila, which has been the subject of so many early investigations, the metabolite and enzyme levels have not been as well studied. Danneel and Zimmermann (1954) compared various mutant strains with respect to the presence or absence of kynurenine and reported that kynurenine disappeared from head extracts 60 h after pupation, but persisted in the remaining body throughout the adult stage. The role played in tryptophan metabolism by the Malpighian tubules was analysed in detail by measuring the fluorescence of tryptophan and the two kynurenines after paper chromatographic separation. Wessing and Bonse (1962) discovered that the Malpighian tubules of Drosophila accumulate free tryptophan (Fig. 19). This is most obvious in the vermilion mutant where 0.1 pg per animal is found after hatching from the pupa. This corresponds to some 25 per cent of the total free tryptophan, if Green’s (1949) figures are converted to a per animal basis by assuming 1 mg fresh weight per fly and 30 per cent dry matter. The storage function of the Malpighian tubules is even more pronounced in the adult fly (Eichelberg, 1968); the fluorescence

THE TRYPTOPHAN + OMMOCHROME PATHWAY IN INSECTS

I I

IO-

9 -

- 0 - .- ; 5 7 - &

209

- 4 ' \ I \ I I I I I I I

' I I I I I ,

I I

I /

I I

\ \ \ \ \ \ \ \ \ \

I -2d 5 - 6 d 10--14d

Fig. 19. Free tryptophan content of the Malpighian tubules in Drosophila melanogaster during metamorphosis. (From Wessing and Bonse, 1962.) Wild type (-), vermilion mutant (- - -).

intensities obtained are much higher (by more than an order of magnitude in the case of kynurenine) than in the larval and pupal stages. Only 3-hydroxy-kynurenine is highest in the larval stages. Unfortunately, only relative values were given.

In the parasitic wasp, Habrobrucon juglandis, there is a switch in tryptophan catabolism from transamination of the kynurenines during the preparatory phase of metamorphosis to ommochrome synthesis during the pupal stage (Leibenguth, 1965, 1967a, 1967b, 1970). Transamination in the spinning larva is so efficient that at the prepupal stage no J-hydroxy- kynurenine is left, even if the endogenous supply iir supplemented (cf. p. 131). The products, kynurenic and xanthurenic acids, are excreted. Kynurenine is always at a low level, while 3-hydroxy-k ynurenine reaches a maximum a day before eclosion. It is not excreted i n the meconia, but presumably consumed in continuing eye pigment synthcsis.

A selection of the quantitative data relating to tryptophan and metabolite concentrations is presented in Table 11. It is beyond doubt that

TABLE 11

Accumulation of tryptophan and tryptophan metabolites during metamorphosis of holometabolous insects (selected data, converted to approximate concentration)

Species

~ ~~~~~

Concentration

Larvae At peak Compound* (mMg-' fresh weight) Stage of peak** Remarks Reference

Ephestia kiihniella (mutant a ) TRY (wild type) KYN

Bombyx mori, mutant rb TRY KY N

3-HO-KYN

3-HO-KYN 3-HO-KYNglucoside

3-HO-KY N TRY KY N 3-HO-KYN

(wild type) Papilio xuthus

0.5 1.8 80% PD 0.05 0.2 50% PD Depends on strain 0.25 1 .o 75% PD 0.15 0.75 One day in cocoon 0.02 1.3 80% PD Males, less in females 1 .o 3 .O 80% PD Females, less in males 0.06 0.3 70% PD 0.1 3.0 80% PD Females, less in males

1.6 60% PD <u.1 1.7 90% PD Level almost constant at 0.1 mM

a

a

a b C

d d d e

e e

Cerura vinula TRY 3-HO-KYN

Calliphora ery throcephala TRY

3-HO-KY N

Phormia terraenovae Habrobracon juglandis KYN

(wild type) 3-HO-KY N KA XA

(mutant 0 ) KY N

0.3 0.1 0.1

0.8

See Fig. 19 0.12

0.75 0.0 0.1 0.3

1.8 Larva in cocoon 1 .o 0.5 20% PD

2.5 30% PD

0.6 20% PD

3.0 70% PD 0.06 Spinning larva 0.8 4.6

Haemolymph concentration f f

Crude estimate from original g

per animal values Concentrated in Malpighian h tubes

I

* TRY, tryptophan; KYN, kynurenine; 3-HO-KYN, 3-hydroxy-kynurenine; KA, kynurenic acid; XA, xanthurenic acid. ** PD, pupal and pharate adult development. t a. Egelhaaf (1963a); b. Linzen (1971a); c. Linzen (unpublished); d. lshiguro et al. (1971b); e. Umebachi and Katayama (1966);

f. Biickmann et al. (1966);g. Langer and Grassmader (1965); h. Linzen (1963); i. Leibenguth (1967a);j. Leibenguth (1965).

21 2 BERNT LINZEN

liberation and accumulation of free tryptophan at the onset of metamorphosis (usually evident when the larva ceases to feed) and its conversion into fluorescent metabolites or ommochromes is a phenomenon common to holometabolous insects. In each case, tryptophan accumulation is transitory. It appears that the metabolite most likely to persist at elevated levels is 3-hydroxy-kynurenine. In the majority of cases, kynurenine is held at very low concentration. It may be argued that the transitory rise of metabolite levels is a consequence of limited capacity of degradation. Alternatively, i t might be assumed that different steps of the degradative pathway are performed in different tissues (e.g. tryptophan oxygenation in fat body, and kynurenine hydroxylation in Malpighian tubules), so that diffusion of the intermediates could become the limiting factor.

In several instances the measurement of metabolite levels and of enzyme activities in vitro have led to conflicting results. Spatial separation is but one explanation, while others are conceivable. Certainly a point to be considered is the physical state of the metabolites. They may be accumulated in certain tissues, either in the form of solid concrements (as 3-hydroxy-kynurenine in Malpighian tubules) or adsorbed (as in oocyte yolk spheres). Even if a metabolite is found dissolved in the haemolymph, it is by no means established whether it is in true solution or whether it is partly or wholly adsorbed by proteins. In the case of mammalian plasma it has been shown that tryptophan is the only amino acid which is bound by protein to a significant extent (McMenamy and Oncley 1958; McArthur and Dawkins, 1969). No comparable data are available for kynurenine or 3-hydroxy-kynurenine, nor has the binding of tryptophan to proteins been studied in insect blood.

8.4 ONTOGENY OF ENZYME ACTIVITIES

It is obvious that the activities of the enzymes degrading tryptophan are not exempt from the profound reorganization of the holometabolous organism and its functions. Kaufman (1962) found that in Drosophila melanogaster the activity of tryptophan oxygenase increases in larvae, remains at an elevated level in pupae, and doubles its activity in adult flies. The first increase could serve in the synthesis of eye pigments (which starts on the second day after puparium formation) by providing the required precursor. No particular physiological role could be assigned to the enzyme in the adult stage. From data obtained by Wessing and Bonse (1962) it is evident that the presence of the enzyme counteracts an accumulation of free tryptophan. There is practically no rise during the pupal stage in the Oregon wild-type strain, while there is a tremendous increase in the mutant vermilion. This excess is gradually excreted after eclosion.


While Drosophila kynurenine formamidase was not studied during development (according to Glassman, 1956. the activity is constant in flies), marked changes have been observed in the activity of kynurenine-3- hydroxylase (Ghosh and Forrest, 1967a). The American authors did not find any activity in larvae, but they reported pronounced activity in pupae (150 mpmol g-' h- ' at 37'C) and low actkity in flies. Today, a re-examination taking the precautions realized in the meantime might possibly yield higher values.

Quite different results were obtained during development of blowflies (Hendrichs-Hertel and Linzen, 1968; Linzen and Schartau, to be published). Both in Calliphora erythrocephala and in Phormia terraenovae the specific activjty of tryptophan oxygenase is hiF,h in larvae and in flies, so that a U-curve is obtained. This is quite unexpected if compared to the metabolite levels (Fig. 18). These would have suggested a rise in tryptophan oxygenase activity in the middle of adult development. However, the measurement of the enzyme activity in crude homogenates is subject to errors, as homogenization is an artifact per se. Inhibitors as well as activators could be liberated during this process and could combine with the enzyme.

In Phormia too, the significance of high oxygenase activity in the adult fly is obscure. Most activity resides in fat body, in Malpighian tubules, and in the testes, there being only medium activity in the ovaries.

In blowflies, kynurenine formamidase and kynurenine-3-hydroxylase exhibit activity curves which are quite different from each other and from the curve of tryptophan oxygenase. The former enzyme is especially active in larval extracts; the activity declines during the pupal stage and reaches a low but constant level in the adult fly. Even at this stage it surpasses the activity of tryptophan oxygenase by a factor o f ten. Kynurenine-3- hydroxylase (Calliphora) increases during larval development, culminates the time of pupation, and declines to zero at the time of eclosion. This would accord with the observed 3-hydroxy-kynurenine level, which rises after pupation and declines in the second half of the pupal stage. From the data available for Phormia, a similar ontogeny of specific activity is apparent. The low activity of kynurenine-3-hydroxylase in pharate adult and adult flies might be due, in part at least, to the inhibitory action of xanthommatin liberated in the course of homogenizing the animals.

Although it is possible that each of the enzymes under consideration might be activated or inhibited independently (by factors present in the homogenates but not present in vivo at the site of the particular enzyme), the mere recognition of very different activity curves is striking. An argument to support the notion of true differential actimtion in vivo is Ghosh and Forrest's (1967a) finding that the course of kynurenine-3- hydroxylase activity is almost idcntical in crude extracts and in a

21 4 BERNT LINZEN

(presumably mitochondrial) particle fraction. It is important to recognize that the three enzymes are not in the least coordinated, in the sense of a programmed, concerted pattern of activity. Nor is there much evidence for a relation between substrate levels and enzyme activity. Only in Drosophilu has it been observed that tryptophan oxygenase in fat body cells can be induced by feeding tryptophan (cf. p. 185). Not only in blowflies, but also in other species, conflicting results were obtained when metabolite levels and enzyme activities were compared. In the parasitic wasp, Hubrobrucon juglandis, Leibenguth ( 1 967a, 196713) observed a sharp rise of kynurenine transaminase activity during the time from the spinning larva until eclosion of the imago, there being no significant formation of kynurenic and xanthurenic acids in vivo. Leibenguth suggested that pyridoxalphosphate and a-ketoglutarate concentrations might become limiting in the pupa. In Habrobracon also, tryptophan oxygenase activity is low in the pupa. Evidence was obtained, using mixed homogenates, that pupal extracts contain an inhibitor, which reduced the activity of imaginal extracts by 50 per cent. The inhibitor is inactivated by heat. In mosquito pupae, the activity of tryptophan oxygenase is only 40 per cent of the activity in larvae or in adult animals (Prasad and French, 19 7 1 ).

More detailed information came out of studies with Ephestiu kri’hniellu and Bombyx mori. Egelhaaf (1963a) discovered that tryptophan oxygenase activity is located within a number of tissues and that the specific activity within any tissue differs with developmental stage. Thus, in the fat body there is a sharp drop from the feeding larva to the prepupa, while in the hypodermis the activity rises. Activity present in larval and pupal testes disappeared completely after eclosion. Egelhaaf stated that the activity which he had measured in whole animals was subject to much less variation than the activity in individual tissues, and emphasized the importance of a separate assay.

In the silkworm, tryptophan oxygenase and kynurenine-3-hydroxylase were measured in a number of tissues in daily intervals (Linzen and Hendrichs-Hertel, 1970; Linzen, 1971b). The results of these studies, which are presented in idealized form in Fig. 20, lead to the following conclusions.

1. The in vivo transformations of the respective substrates can be easily accounted for even when the in uitro activities of both enzymes are low (i.e. in the pU range).

Fig. 20. Specfic activities in vitro (in picomoles product per min per mg protein) of tryptophan oxygenase (-) and kynurenine-3-hydroxylase (- - -) in various tiggues of Bombyx mori (mutant r b ) during metamorphosis. The inset in the upper diagram shows concentration of free tryptophan. S, first day inside cocoon (larva still visible); P, pupation; E, eclosion.


100

1000

AIP-8

21 6 BERNT LINZEN

2. The enzymes of tryptophan catabolism are not characteristic of any particular tissue. While both of the enzymes studied occur in fat body (i.e. in cells specialized for intermediary metabolism) either one or both are also found in excretory, reproductive, or organs of locomotion. This is in sharp contrast to the restricted localization observed in the vertebrate.

3. The activities of the two enzymes are not linked, in spite of their rather closely related function. Thus, either both may be detected in a particular tissue or may occur singly.

4. The ontogenetic pattern of enzyme activity differs with each tissue and is at the same time a characteristic of each of the two enzymes. Thus, in fat body, both enzymes fit into the common U-pattern. In the Malpighian tubules a most striking feature is the sharp peak of the hydroxylase just before the onset of spinning. Testes and ovaries both demonstrate gradually decreasing oxygenase activity, while on the other hand there is a transitory appearance of the hydroxylase in ovaries.

The notion of differential enzyme activity patterns in the development of insects has thus been amply confirmed. While it cannot be ruled out that extraneous factors (such as contamination by other tissues) may have influenced the results, it appears extremely unlikely that the differences observed could have exclusively originated from such artifacts. The changing levels of activity must to some extent be linked to other events in the tissue under study. In other metabolic pathways, researchers have encountered similar situations (e.g. Rechsteiner, 1970; Barnes and Goodfellow, 1971), although in no case have the separate tissues been studied in so much detail.

The question thus arises as to the means by which such enzyme activities are regulated. The central problem is then whether the observed changes are related to alterations in the concentration of enzyme protein, or to changes in the state of activation of existent enzyme. It is difficult to give an unequivocal answer, since sequential changes in enzyme protein in insects have not been measured. However, if the state of activation of a particular enzyme should change with development, the causes of such differences would be partially offset by the incubating conditions which provide constancy of pH, ionic strength, substrate, and cofactor concentrations. One is on somewhat safer ground, if, as has been done in the case of fructose diphosphate aldolase (Bauer and Levenbook, 1969), the enzyme is partially purified by electrophoresis or other procedures. It would appear, therefore, that it is most likely that it is the levels of apoenzyme which change.

Several principles can be envisaged which might cause sequential changes of enzyme concentration.

1. Enzyme induction by substrate. According to our present knowledge,

THE TRYPTOPHAN + OMMOCHROME PATHWAY I N INSECTS 21 7

the metabolite and enzyme levels are rather poorly related, and give no indication of probable induction. In several insect species there is evidence to show that tryptophan oxygenase and kynurenine-3-hydroxylase are constitutive enzymes with the exception of larval Drosophila tryptophan oxygenase.

2 . Differential enzyme synthesis in response to hormones. At present, no evidence is at hand. Biickmann (1959a) and Karlson and Biickmann (1956) have shown that colour change in the larva of Cerura is induced by ecdysone. Yet, it must be stressed that the kynurenine pathway is of secondary importance in metabolism and, therefore, not likely to be under direct control of morphogenetic hormones.

3. Cell multiplication, turnover and cell death. Some of the changes are evidently related to the formation or breakdown of tissues: tryptophan oxygenase activity in the wings of Bombyx increases during cell multiplication and growth, but drops when differentiation comes to completion and when the hypodermal cells die away. Also, the appearance of kynurenine- 3-hydroxylase in silkworm ovaries is clearly correlated with growth. In contrast, the rise in kynurenine-3-hydroxylase activity in the Malpighian tubules occurs during a period of cell constancy, and the drop of both enzymes in fat body during a period in which rxpid growth of fat body masses is manifest and total soluble protein is still increasing.

4. Differential activation of transcription and/or translation, according to a developmental program. This would most easily explain the changing levels of specific enzyme activity. However, in view of the results obtained in Bombyx, an explanation must be found for the rather accidental appearance of tryptophan oxygenase and kynurenine-3-hydroxylase activities. A hypothesis proposed by Linzen (1971b) implies that the corresponding genes are not linked (neither in the usual meaning nor in any other functional way), and are transcribed soldy according to tissue specific developmental programs. Reference is made to the theory of Britten and Davidson (1969), and it is recalled that in Drosophila the vermilion and cinnabar genes are located on (different chromosomes. Random (though programmed) gene expression ir different tissues would be compatible with normal function and development if the metabolite controlled were of minor importance (both by its quantity and by its position on the metabolic map) and if diffusion and transport as possibly limiting factors could be accounted for. The only requirement in such cases would be that the metabolite should be supplied at a minimum rate or be kept below a certain concentration. Such conditions might hold for the kynurenine pathway.

5 . Intracellular protein turnover with chunging ratios of enzyme synthesis and degradation. This would shift the problem of regulation as

21 8 BERNT LINZEN

outlined under (4) to an extrinsic function. While some work has been published on the turnover of haemolymph proteins (cf. Wyatt, 1968), our knowledge of intracellular protein and enzyme turnover in insects is apparently nil.

At present, it is impossible to decide which alternative is of greater importance. It is anticipated that each one will eventually be found to contribute to the accomplishment of ordered and responsive transformation of the metabolic intermediates.

Finally, the relation of enzyme activity determined in vitro to the rate of product appearance in vzvo might be examined. Most attempts of this kind have shown that the measured enzyme activity is greatly in excess of the demands of the organism. However, in vitro activity is by definition measured under optimal conditions of substrate concentration, pH etc., which may not necessarily prevail within the cell. For the very reason of the adaptive significance of the K, value, most enzymes must operate in vivo at substrate concentrations which are far from optimal (cf. Atkinson, 1969). These considerations can also be applied to the enzymes of the kynurenine pathway in insects. In most instances the in vitro activity (at optimal conditions) is far in excess of the in vivo reaction rates. Only at the onset of metamorphosis it appears that saturation of the enzymes could result from rapid liberation of tryptophan. In Bombyx mori it was estimated (Linzen, 1971b) that the rate of free tryptophan decrease at the time of pupation and in the young pupa is equal to and therefore limited by the total activity of tryptophan oxygenase in the organism This is low at this stage as the larval enzyme (mainly in fat body) has gone down by this time, while the enzyme of the pharate adult (mainly in ovaries and wings) has not yet been synthesized. This situation is indicated in the top diagram of Fig. 20.

8.5 AlTEMPTS TO ESTABLISH A TRYPTOPHAN BALANCE

While it is generally understood that in vertebrates all dietary tryptophan is eventually broken down via the kynurenine and glutarate pathways, ii is nevertheless difficult to substantiate this statement by straightforward experiments (cf. LeMem, 1971). Yet, since in vertebrates only two pathways of tryptophan degradation areL known, and, as the amount of 5-hydroxy-indoleacetic acid excreted .per day accounts for less than 1 per cent of the total amount of tryptophan administered (under conditions of tryptophan load), the only question which remains to be answered is which fraction of the remaining 99 per cent is metabolized via the kynurenine pathway and which by intestinal microorganisms. (The latter factor, incidentally, indicates the necessity of quantitating intestinal tryptophan

THE TRYPTOPHAN + OMMOCH ROME PATHWAY IN INSECTS 21 9

resorption and of tryptophan loss caused by secretion of digestive enzymes and by turnover of the intestinal lining.)

The metamorphosis of the Holometabola provides an opportunity to assess the scope of the kynurenine pathway in insects since with respect to tryptophan metabolism one is dealing with a closed system (cf. pp. 133 and 197). It is feasible to set up a balance of tryptophan metabolism by determining total protein-bound tryptophan and the sum of free tryptophan plus all detectable metabolites at two points of development. This has been tried in the Bombyx mori mutant rb (Linzen, 1971a), and in the blowfly, Phormia terraenovae (Linzen and Schartau, to be published).

In Bombyx, the period studied extends from the end of spinning (S + 2 days) to 48 h before eclosion (S + 11 days). The total decrease in protein-bound tryptophan during this period is about 5 pmol (if, in Fig. 4 of Linzen, 1971a, a line is drawn from S + 1 to S + 12 days). Free tryptophan decreases by 0.7 pmol. During the same period about 1 pmol of kynurenine, 2.7 pmol of 3-hydroxy-kynurenine 0.5 pmol of xanthommatin (corresponding to 1 pmol of tryptophan), and about 0.3 pmol of 3-hydroxy-kynurenine glucoside are formed (Linzen and Ishiguro, 1966; Ishiguro et a/. , 1971 b; Linzen, unpublished). Taken together, these metabolites make up for 5 pmol of tryptophan. The synthesis of small amounts of 3-hydroxy-kynurenine sulphate, of xanthurenic acid, 4,8- dihydroxy-quinoline, ommins, and possibly other compounds of minor importance must also be considered.

In Phormia the situation is simpler, since the tryptophan metabolism is less diverse. Between the 3rd and 5th day after puparium formation there is a decrease of 28 nmol of free and 46 nmol of bound tryptophan per animal. These are almost stoichiometrically accounted for by the metabolites: 20 nmol of kynurenine, 9.6 nmol of 3-hydroxy-kynurenine, and about 23 nmol of xanthommatin (to be multiplied by 2). There is also fair agreement between the decline in protein-bound t,ryptophan from the day of puparium formation to the day of eclosion (0.15-0.1 7 pmol), and the amount of xanthommatin synthesized in the eye!; (0.066-0.071 pmol per animal, multiply by 2). Difficulties arise, however, if estimates are made on a day-to-day basis due to sampling error and to the fact that the analyses of the different compounds were partly performed on different batches of animals. fn view of the relatively crude method uscd for the determination of total protein-bound tryptophan (the xanthoprotein reaction) it would be highly desirable that the results were checked by independent methods.

In spite of such reservation it is tentatively concluded, at least in these two species, that most, if not all, tryptophan is degraded via the kynurenine pathway. It may be argued that protein breakdown achiives an extreme at the beginning of metamorphosis, and that in o h r developmental stages different routes of tryptophan catabolism might b'e favoured. However, at

220 BERNT LINZEN

present there is no indication that other metabolites of tryptophan than those listed above are formed to a greater extent in any stage of insect development.

9 Detrimental effects of tryptophan and tryptophan metabolites

Many of the conclusions and interpretations in this review have emphasized that removal of free tryptophan from the cellular environment is of vital importance. The crucial question (the Cretchenfrage) is whether an elevated level of tryptophan can, in fact, produce toxic or harmful effects. Such a possibility was actually implied as early as 1952 by Nolte in a discussion of the role of the eye colour genes in Drosophila. Such effects might be difficult to detect, as they should be of a quantitative rather than of a qualitative nature. However, a survey of the literature (Grober and Linzen, unpublished) has revealed a wide range of negative effects cauacd by tryptophan or some of its metabolites. It appears that many of thew are related to developmental processes. Normal growth may be retartied or deflected to a pathological condition, such as the induction of bladder tumours and of leukemias by ortho-aminophenols (3-hydroxy-kynurenine, 3-hydroxy-anthranilic acid).

In insects, there are two lines of evidence relevant to this problem: retarded development in various species and increased penetrance of tumour promoting genes in Drosophila. A retardation of development by about 2 per cent was reported in the first paper about the a mutant of Ephestia (Kuhn and Henke, 1930).' In Drosophila, Wilson (1945) studied the effects of tryptophan added to sterile culture media at increasing concentration. She observed a significant prolongation of the pupal stage at concentrations from 10 mM down to 0.4 mM. At concentrations above 20 mM also the size of the animais was reduced. Hinton et al. (1951) reported a retardation of larval development in a chemically defined medium by L-tryptophan at concentrations above 15 mM, with an approximate doubling of the time necessary until pupation at 45 mM. Interestingly, addition of ribonucleic acid at least partly abolished this effect. Parsons and Green (1959) studied the effect of the v mutations on fitness. Under conditions of low competition there was no difference in the fitness of males (obtained by crossing homozygous 6w males to 6w females homozygous for various vermilion and vermilion suppressor genes), but in crowded cultures the suppressed vermilion males were fitter than unsuppressed (the sex ratio was used as a criterion). Since unsuppressed vermilion flies contain a higher level of free tryptophan, this would be in line with the hypothesis of tryptophan toxicity, but surprisingly the same

' According to personal communication from L. Caspari to A. Egelhaaf this effect has not been reproduced.


improvement of fitness as brought about by the suppressor can also be achieved by feeding kynurenine. This result is pexplexing: the authors state that “competition was almost completely larval”, competition between adults being negligible. As ommochrome synthesis induced by vermilion suppression or kynurenine supplementation is not a larval but a pupal process, the effect of kynurenine on fitness mighi be mediated by another, possibly roundabout effect.

The grain beetle, Oryraephilus surinamensis, grows best on a diet containing 0.2 per cent tryptophan, while increasing the concentration to 0.5 and 1 per cent results in a reduced rate of development (Davis, 1968).

The formation of tumours represents quitc a different aspect of tryptophan toxicity. In their attempt to establish a chemically defined medium for raising Drosophila larvae, Hinton eta ! . (1951) observed that at concentrations of 30 mM tryptophan “all the flier showed abnormalities of various types-for example, tumours throughout the body, deformed heads, wavy bristles” and more. These observations were made in a wild-type strain. In Drosophila, there exist strains with a hi:gh tumour incidence (e.g. tu and v t A ). The tumours formed in thcse strains are often of the benign type and arise from transformation and accumulation of cells rather than from uncontrolled cell division (cf. Rizki, 1957, and papers cited by him; a possibly important paper, but not obtained by the author, is that by Ghelelovich, 1969). Plaine and Glass (1955) studied the effects of various agents on the formation of melanotic tumours and “erupt” eyes, and found that tryptophan, along with indole, was most effective (see also Mittler, 1952). Deleterious concentrations of tryptophan and of tryptophan metabolites may apparently arise under natural conditions, tumour incidence being raised significantly if tu is crossed with v, cn, or st (Kanehisa, 1956a). Feeding supplementary tryptophan to such crosses enhances the effect, while kynurenine promotes tumour incidence to a lesser extent (Kanehisa, 1956b; see also Kanehisa and Fujii, 1967).

In a search for a metabolite which might be madc directly responsible for promoting melanotic tumours, Burnet and Sang (1968) found that anthranilic acid had a strong effect, while kynurlsnine was not effective. This could agree with Kanehisa’s (1956b) observations. It is noteworthy that L-methionine counteracted the tumorigenic effect. In contrast to the results just described, which are related to the tu and tu-er strains, the penetrance of tu-h (tumorous head) is significantly lowered if the larvae are raised on a medium containing 0.5 per cent L-tryptophan (Simmons and Gardner, 1958). With regard to all of these studies it must be emphasized strongly that the active agent (a particular constituent of the diet) and its effect (a malfunction of a particular type of cell in .3 complex organism) are as loosely linked as possible, so that speculation on possible biochemical mechanisms is without any basis.

222 BERNT LINZEN

From the physicochemical viewpoint tryptophan can be shown to have properties which make it prone to both desirable and undesirable interactions. In short, these properties reside in the indole nucleus which is capable of donating negative electrical charge to suitable acceptor molecules. Charge-transfer complexes of tryptophan are observed most readily when solutions of the two reaction partners are frozen and when by the freezing process the solutes are concentrated and forced to form aggregates. In dilute aqueous solution, solvation will prevent complex formation to varying degrees. A number of interactions have, however, been observed even under these unfavourable conditions. It must be noted that the cellular milieu cannot be looked at as a simple solution but that the solvent capacity of a cell is very limited and that the space available for diffusion may often present dimensions which are not much larger than the diameter of a single hydrated molecule (cf. Sols and Marco, 1971). It has also occasionally been suggested that water within a living cell may locally be in the state of an ordered lattice and therefore not participate in solvation. Thus, the probability of in uivo interaction between tryptophan and other molecules might correspond more closely to the condition of the “frozen solution” than expected by the orthodox view.

’The association of tryptophan with other molecules has been demonstrated by a variety of methods. Isenberg and Szent-Gyorgyi (1 958) examined the red product obtained by mixing 1 mM tryptophan and riboflavin-5-phosphate solutions and concluded that by transfer of one electron from tryptophan, riboflavin is reduced to its semi-quinoid state which in turn is stabilized by complex formation. Serotonin complexes seven times more strongly than tryptophan. Similar complexes are formed between tryptophan and pteridines, thereby shifting the absorption peak of the pteridines from about 350-390 nm to about 400-430 nm (Fujimori, 1959). Protein-bound tryptophan may react as well as free tryptophan.

Particularly interesting is the association of tryptophan with nucleic acids and their constituents. If DNA (1 mM) is heated in the presence of L-tryptophan (1 mM) the normal hyperchromic effect is abolished (Pieber et al.. 1969). This must be caused by association of the amino acid with the base moiety of the nucleotides, as revealed by nmr spectroscopy of appropriate mixtures. The solubility of ribonucleotides is also increased in these mixtures as a linear function of tryptophan concentration, the purines responding more strongly than the pyrimidines. The uv spectra did not change, but the cd spectra were clearly different from the calculated mean spectra of each pair of compounds. The viscosity of DNA-tryptophan mixtures did not change. The interpretation of these results (Arcaya et al., 1971) is that tryptophan interacts with DNA by intercalation between the nucleotide bases without changing much the tertiary structure of the macromolecule. This is supported by Raszka and Mandel (1971) who


demonstrated interaction between aromatic amino acids and polyadenylic acid by nmr spectroscopy and alluded to the possibility of “local melting” of DNA during replication and transcription, by this principle.

HCline and collaborators (Montenay-Garestier and Helene, 19 7 1 ; Heline, 19 7 1 ; Dimicoli and Hiline, 19 7 1) save conducted extensive physicochemical studies on tryptophan-nucleic acid interactions. They demonstrate a stoichiometric interaction between the amino acid and the bases and give a detailed account of the spectroscopic changes. It is emphasized by them that stacking of the two-ring systems (i.e. close sidewise orientation of planar structures) is necessary for complex formation. They also contend that in the ground state of the molecules charge transfer plays a minor role in stabilizing the. complex, in comparison to van der Waals forces, although charge transfer occurs upon excitation by light.

Heline et al. (1971) emphasize that tryptophan-nucleotide interaction may be of great importance in the binding of proteins to nucleic acids, implying that recognition of specific regions of nucleic acids could be made possible by this type of interaction. We might now envisage the potential hazards which may arise if tryptophan accumulates to levels high enough to provoke frequent interaction with nucleic acids. Probably nucleic acids which are bound in nucleoproteins would be protected, but this would concern only part of the total nucleic aci+ of a cell. The juxtaposition of detrimental effects in growth and development caused by tryptophan, and of the molecular interaction of tryptophan with nucleic acids is highly suggestive, but a warning must be expressed against premature conclusions. These two lines of observation are still too distant to be connected, a variety of experimental approaches being necessary to test this hypothesis. It must, for example, be determined whether tryptophan interferes with nucleic acid function and whether it could ever reach effective concentrations within cells. It must also be demonstrated that the observed inhibition of growth is directly related to impaired protein synthesis. Growth is subject to so many variables that none but the most careful and circumspect analysis will reveal the mechanism of tryptophan action.

10 Concluding remarks

It has been the intention of the author to show that the transformation of tryptophan into ommochromes, a biochemical pathway of seemingly minor importance, is implicated in a variety of physiological functions. In particular the necessity has been emphasized for the organism to limit the concentration of free tryptophan to a level compatible with the requirements of the complex biochemical machinery and its manifold and delicate interactions of macromolecules. In insects this necessity is correlated with the inability to cleave the aromatic nucleus of 3-hydroxy-anthranilic acid.

224 BERNT LINZEN

In spite of the survey made by Lan and Gholson (1 965) it cannot be stated whether this failure represents an “oversight of evolution” or whether it is a feature acquired secondarily (as Brunet, 1965, has speculated). Whatever it is, the outlet of ommochrome synthesis has endowed insects (and the rest of the arthropods) with a most valuable and variable means of handling light, by screening and by selective ,reflexion in pigment patterns. At the same time ommochromes have properties well suited to the function of storage excretion.

It is opportune to emphasize some outstanding problems. While most of the intermediates in tryptophan catabolism might have been identified, the chemical structure of only the minority of the ommochromes is currently known. It is expected that the structure of the ommidins and of the acridiommatins will become known within a few years, but the ommins might continue to pose problems to the chemist for some time. On the other hand, the enzymology of tryptophan degradation in insects, including the terminal steps of pigment synthesis, must be investigated further. Finally, the mechanisms governing each single reaction at different sites and at different developmental stages of the insect organism should become the subject of future research.

Throughout this review little attention has been paid to historical aspects: the great findings of the decade between 1930 and 1940 have been passed over. While the research along the tryptophan + ommochrome pathway in insects arose from the basic problem of the mechanism of gene expression, it has since contributed results which have gained importance in various fields of interest far beyond the domain of a single animal phylum. Filling the gaps in our knowledge was, however, not the only outcome of this research. While considerable insight has been gained it has also been realized that many new and basic questions have emerged. Without doubt, the discovery of facts has been paralleled by the discovery of problems.

Acknowledgements

I should like to express my gratitude to M. Alain Bouthier, Professor D. Biickmann, and Drs J. Dustmann, R. Geiger, and W. Schaefer for freely making available unpublished results or pending manuscripts, to Mr Nobuo Kita for his help with Japanese papers, to Professor A. Egelhaaf, Dr H. Kress, and Mrs M. Linzen for reading parts or the whole of the manuscript, and to those colleagues who provided me with reprints and copies of research papers. Most of the research in the author’s laboratory, including work reported here for the first time, was supported generously by the Deutsche Forschungsgemeinschaft.


References

Ajami, A. M. and Riddiford, L. M. (1971a). Identification of an ommochrome in the eyes and nervous systems of saturniid moths. Biochemistry (Washington), 10, 1451-1455.

Ajami, A. M. and Riddiford, L. M. (1971b). Purification and characterization of an ommochrome-protein from the eyes of saturniid moths. Biochemistry (Washington), 10, 1455-1460.

Almeida, F. F. de (1961). h e r die Konzentration von Kynurenin und Hydroxy- Kynurenin wahrend der Entwicklung bei den Genolypen ra’ und ra von Plodia interpunctella. Naturwissenschaften, 48, 27-28.

Almeida, F. F. de (1968). Das Ommochrom I als wahrscheinliche Vorstufe des Ommins in der Ommochromsynthese von Plodia interpunctella L.nd Ephestia kiihniella. Molec. gem Genetics, 102, 307-315.

Arcaya, G., Pantoja, M. E.. Pieber, M., Romero, C. and Tohi, J. C. (1971). Molecular interaction of L-tryptophan with bases, ribonucleosides and DNA. 2. Naturf: 26b,

Atkinson, D. E. (1969). Limitation of metabolite concentrations and the conservation of solvent capacity in the living cell. In “Current Topic:i in Cellular Regulation” (Eds B. L. Horecker and E. R. Stadtman), Vol. 1; pp. 29-43. Academic Press, New York and London.

Autrum, H. (1955). Die spektrale Empfindlichkeit der Augenmutation white-apricot von Calliphora erythrocephala. Biol. Zbl. 74, 51 5-524.

Autrum, H. (1961). Die Sehschtirfe pigmentfreier Fazettenaugen von Calliphora erythrocephala Biol. Zbl. 80, 1-4.

Baglioni, C. (1 959). Genetic control of tryptophan peroxidase-oxidase in Drosophila melanogaster. Nature, Lond. 184, 1084-1085.

Baglioni, C. (1960). Genetic control of tryptophan pyrrolase in Drosophila melanogaster and D. uirilis. Heredity, 15, 87-96.

Baillie, D. L. and Chovnick, A. (1971). Studies on the genetic control of tryptophan pyrrolase in Drosophila melanogaster. Molec. gen. Cenetcs , 112, 341-353.

Barnes, F. J. and Goodfellow, R. D. (1971). Mevalonate kinase: localization and variation in activity during the development of Sarcophaga bullata. J. Insect PhysioL

Bauer, A. C . and Levenbook, L. (1969). Fructose diphosphate aldolase during growth and development of the blowfly, Phormia regina (Meigen). Comp. Biochem. Physiol.

Baumann, U. (1957). Untersuchungen iiber Ommochrome. h e r Bombyx-Ommin und iiber den oxydativen Abbau substituierter Phenoxazone. Doctoral Thesis, University of Tiibingen.

Beadle, G. W. (1937). Development of eye colors in Drosophila: fat bodies and Malpighian tubes as sources of diffusible substances. Proc. natn. Acad. S c i U.S.A. 23.

Beadle, G. W. and Ephrussi, B. (1935). Diffkrenciation de la couleur de I’oeil cinnabar chez la Drosophile (Drosophila melanogaster). C.7. hebd Sianc. Acad. Sci, Paris,

1026-1 030.

17, 1415-1427.

28, 619-632.

146-1 52.

201. 620-622.

226 BERNT LINZEN

Beadle, G. W., Anderson, R. L. and Maxwell, J. (1938). A comparison of the diffusible substances concerned with eye color development in Drosophila, Ephestia and Habrobracon. Proc. natn. Acad. Sci U.S.A. 24,80-85.

Becker, E. (1938). Die Gen-Wirkstoff-Systeme der Augenausfirbung bei Insekten. Naturwissenschaften, 26,433441.

Becker, E. (1939). d e r die Natur des Augenpigments von Ephestia kiihniella und seinen Vergleich mit den Augenpigmenten anderer Insekten. Biol. ZbL 59, 597-627.

Becker, E. (1941a). Die Pigmente der Ommin- und Ommatingruppe, eine neue Klasse von Naturfarbstoffen. Natunuissenschaften, 29,237-238.

Becker, E. (1941b). Ein Beitrag zur Kenntnis der Libellenpigmente. Biol. Zbl. 61,

Becker, E. (1942). Uber Eigenschaften. Verbreitung und die genetisch-entwicklungsphysiologische Bedeutung der Pigmente der Ommatin- und Ommingruppe (Ommo- chrome) bei den Arthropoden. Z. VererbLehre, 80, 157-204.

Beckmann, R. (1 956). h e r Rhodommatin, ein natiirlich vorkommendes Ommochrom. Doctoral Thesis, University of Tiibingen.

Bellamy, D. (1958). The structure and metabolic properties of tissue preparations from Schistocerca gregaria (Desert Locust). Biochem. J. 70, 580-589.

Benassi, C. A., Veronese, F. M. and Scoffone, E. (1964). Separazione cromatografica di derivati biologici del triptofano su resine a scambio ionico. Ann. Chim. 54, 627-638.

Berthold, G. (1971). h e r die hormonale Steuerung von Wachstum und Pigmentierung der Indischen Stabheuschrecke Carausius morosus Br. Doctoral Thesis. University of Giessen.

Berthold, G. und Henze, M. (1971). h e r die Beteiligung der Pterine an den Farbmodifikationen der Stabheuschrecke, Carausius morosus. J. Insect Physiol. 17,

Biedermann, W. (1914). Farbe und Zeichnung der Insekten. In “HandbuCh der vergleichenden Physiologie” (Ed. H. Winterstein), Vol. 3/1/11, pp. 1657-1994. Fischer-Verlag, Jena.

Block, R. J. and Weiss, K. W. (1956). “Amino Acid Handbook”. Charles C. Thomas, Springfield.

Bonse, A. (1969). Uber d a s Auftreten von Pterinen, Tryptophan und dessen Derivate in verschiedenen Organen der Mutante white von Drosophila melanogaster. Z . Nature

Bouthier, A. (1966). Modifications des pigments (ommochromes et pterines) en relation avec la mutation albinos chez Locusta mipatoria cinerascens Fabr. (Orthoptkres, Aaididae). C.r. hebd. Sianc. Acad. Sci., Paris, Sir. D. 262, 1480-1483.

partir d’yeux de Locusta migratoria L. (Orthopdres, Acrididae). C.r. hebd. S*anc. Acad. S c i . Paris, 268,

Bouthier, A. (1972). The ommochromes of the Migratory Locust. NATO A d a Stud

Bowness, J. M. and Wolken, J. J. (1959). A light-sensitive yellow pigment from the

Bratton, A. C. and Marshall, E. K. (1939). A new coupling component for sulfanil-amide

588-602.

2375-238 1.

24b, 128-131.

Bouthier, A. (1969). Isolement d’ommidine i 1’Ctat cristallisk

141 0-14 12.

fnst. Chemistry of Insects, Varenna (Unpublished.)

house-fly. J. gen. Physiol. 42, 779-792.

determination. J. biol. C h e m 128,537-550.


Britten, R. J. and Davidson, E. H. (1969). Gene regulation for higher cells: a theory. Science (WashinKton), 165, 349-357.

Brown, K. S. (1965). A new L-a-amino acid from Lepidoptera. J. Am. chem. SOC. 87, 4202-4203.

Brown, K. S. and Becher, D. (1967). The mass spectra of’the kynurenines. Tetrahedron Lett. 1967, 1721-1726.

Brown, R. R. and Price, J. M. (1956). Quantitative studies. on metabolites of tryptophan in the urine of the dog, cat, rat, and man. J biol. Chem. 219, 985-997.

Brunet, P. C. J. (1965). The metabolism of aromatic conipounds. In “Aspects of Insect Biochemistry” (Ed. T. W. Goodwin), pp. 49-77. Biochem. SOC. Symp. No. 25. Academic Press, London and New York.

Biickmann, D. (1953). Uber den Verlauf und die Auslosiing von Verhaltensanderungen und Urnfirbungen erwachsener Schmetterlingsraupen. IlioL Zbl. 72, 276-3 1 1.

Biickmann, D. (1959a). Die Auslosung der Umfarbung durch das Hautungshormon bei Cerura uinula L. (Lepidoptera, Notodontidae). J. Insect Physiol. 3, 159-189.

Biickmann, D. (1959b). Farbwechsel als Teilvorgang tler Metamorphose und seine Hemmung in jungen Schmetterlingsraupen. Zool. A m . SuppL 22, 137-144.

Biickmann, D. (1963). Der Einfluss der Temperatur auf das Epidermispigment der Raupen von Cerura uinula L. Z . Naturf. 18b, 255-264.

Biickmann. D. (1964). Zur Frage einer respiratorischen Funktion von Ommochromen. Nuturwissenschaften, 51, 344.

Biickmann, D. (1965). Das Redoxverhalten von Ommochrom in uiuo. Untersuchungen an Cerura uinula L. J. Insect Physiol. 11, 1427-1462.

Biickmann, D. and Dustmann, J. H. (1962). Biochemische Untersuchungen iiber den morphologischen Farbwechsel von Carausius morosus. Natunuissenschaften, 49,379.

Biickmann. D., Willig, A. and Linzen, B. (1966). Verindelungen der Hamolymphe vor der Verpuppung von Cerura uinuia L.: Der Gehalt an Eiweiss, Aminosauren, Ommochrom-Vorstufen und Ommochromen. Z. Nuturf. 21b, 1184-1 195.

Burnet, B. and Sang, J. H. (1968). Physiological genetics of melanotic tumoun in Drosophila melanogaster. V. Amino acid metabolism and tumour formation in the tu bw; st su-tu strain. Genetics, 59, 211-235.

Burnet, B., Connolly, K. and Beck, J. (1968). Phenogenetic studies on visual acuity in Drosophila rnelanogaster. J. Inscct Physiol. 14, 855-866.

Butenandt, A. (1949). Biochemische Untersuchungen zur Wirkungsweise der Erbfak- toren. Angew. C h e m 61, 262-263.

Butenandt. A. (195 7). Uber Ommochrome, eine Klasse natiirlicher Phenoxazon- Farbstoffe. Angew. C h e m 69, 16-23.

Butenandt, A. (1959). Wirkstoffe des Insektenreiches. Natunuissenschaften, 46,

Butenandt, A. (1960). Uber neue Naturfarbstoffe. ihre Biogenese und physiologische Bedeutung. XVII. Int. Congr. Pure Appl. Chem. Munich, ‘Vol. 11, 11-31.

Butenandt, A. and Albrecht, W. (1952). Bestimmungen des Tryptophangehaltes verschiedener Rassen der Mehlmotte Ephestia kiihniella a ls Beitrag zur Analyse der Genwirkungen. Z . Nuturf. 7b, 287-290.

Butenandt, A. and Hdlmann, G. (1950). Neue Synthesen des d,l-Kynurenins und d,l-3-Oxy-Kynurenins. 2. Nuturf 5b. 444-446.

461-47 1.

228 BERNT LINZEN

Butenandt, A. and Neubert, G. (1955). Uber Ommochrome, V. Mitteilung. Xanthom- matin, ein Augenfarbstoff der Schmeissfliege Calliphora erythrocephala. Hoppe- Seyler’s Z . physiol. C h e m 301, 109-114.

Butenandt, A. and Neubert, G. (1958). Uber Ommochrome, XVII. Zur Konstitution der Ommine I. Liebig’s Ann. Chem 618, 167-173.

Butenandt, A. and Schafer, W. (1962). Ommochromes. In “Recent Progress in the Chemistry of Natural and Synthetic Colouring Matters and Related Fields” (Eds T. S. Gore, B. S. Joshi, S. V. Sunthankar and B. D. Tilak), pp. 13-33. Academic Press, New York and London.

Butenandt, A., Weidel, W. and Becker, E. (1940a) Kynurenin als Augenpigmentbildung ausliisendes Agens bei Insekten. Naturzuissenschaften, 28, 63-64.

Butenandt, A., Weidel, W. and Becker, E. (1940b). a-Oxytryptophan als “Prokyn- urenin” in der zur Augenpigmentbildung fiihrenden Reaktionskette bei Insekten. Natunuissenschaften, 28,447-448.

Butenandt, A., Weidel, W. and Derjugin, W. von (1942). Zur Konstitution des Kynurenins. Naturwissenschaften, 30, 51.

Butenandt, A., Weidel, W., Weichert, R. and Derjugin, W. von (1943). Uber Kynurenin. Physiologie, Kostitutionsermittlung und Synthese. Hoppe-Seyler’s Z . physiol. Chem.

Butenandt, A., Weidel, W. and Schlossberger, H. (1949). 3-Oxy-Kynurenin als

cn+-Gen-abhingiges Glied im intermediiren Tryptophan-Stoffwechsel. Z. Naturf. 4b, 242-244.

Butenandt, A., Karlson, P. and Zillig, W. (1951). Uber das Vorkommen von Kynurin in Seidenspinnerpuppen. Hoppe-Seyler’s Z. physiol. Chem. 288, 125-1 29.

Butenandt, A., Schulz, G. and Hanser, G. (1953). Uber 5-Oxy-kynurenin, seine Synthese und physiologische Bedeutung. Hoppe-Seyler’s Z. physiol. Chem. 295, 404-41 0.

Butenandt, A., Schiedt, U., Biekert, E. and Kornmann, P. (1954a). Uber Ommochrome, I. Mitteilung: Isolierung von Xanthommatin, Rhodommatin und Ommatin C aus den Schlupfsekreten von Vanessa urticae. Liebig’s Ann. Chem. 586, 21 7-228.

Butenandt, A., Schiedt, U. and Biekert, E. (1954b). Uber Ommochrome, 11. Mitteilung: Alkalischer und fermentativer Abbau von Xanthommatin und Rhodommatin. Alkalischer Abbau der Kynurenin-Seitenkette. Liebigs Ann. Chem. 586, 229-239.

Butenandt, A.. Schiedt, U. and Biekert. E. (1954~) . h e r Ommochrome, HI. Mitteilung: Synthese des Xanthommatins. Liebig’s Ann. Chem. 588, 106-1 16.

Butenandt, A., Schiedt, U.. Biekert, E. and Cromartie, R. J. T. (1954d). Uber Ommochrome, IV. Mitteilung: Konstitution des Xanthommatins. Liebig’s A n n C h e m 590. 75-90;

Butenandt, A., Biekert, E. and Linzen, B. (1956). Uber Ommochrome, VII. Mitteilung. Modellversuche zur Bildung des Xanthommatins in uiuo. Hoppe-Seyler’s Z . physioL C h e m 305, 284-289.

Butenandt, A., Keck, J. and Neubert, G. (1957a). h e r Ommochrome VIII. Modell- Versuche zur Konstitution der Ommochrome: Uber Oxydationsprodukte der 3-Hydroxy-anthranilsiure. Liebigs Ann. Chem. 602, 61-72.

Butenandt, A., Biekert, E. and Neubert, C. (1957b). h e r Ommochrome IX. Modell-Versuche zur Konstitution der Ommochrome: h e r 3-Hydroxy-5-acetyl- phenoxazon-(2) und 1.6-Diacetyl-triphendioxazin. Ein neuer Weg zur Darstellung von Phenoxazonen. Liebigs A n n Chem. 602, 72-80.

279, 27-43.


Butenandt, A., Biekert, E. and Neubert, G. ( 1 9 5 7 ~ ) . Uber Ommochrome X. Modellver- suche zur Konstitution der Ommochrome: 5-Dibi:nz(b.f)azepinchinonirnine als Reaktionsprodukte der 5-Acetyl-phenoxazone mit Alkali und als Oxydations- produkte des 2-Amino-3-hydroxy-acetophenons. Liebig’s Ann. Chem. 603, 200-206.

Butenandt, A., Biekert, E. and Baumann, U. (1957d). 13mmochrome. XI. Modellver- suche zur Konstitution der Ommochrome: Oxydativer Abbau des 3-Amino-4,5- diacetylphenoxazons-(2). Arch. Biochem Biophys. 69, 100-1 05.

Butenandt, A., Biekert, E. and Beckmann, R. (1957’:). Uber Ommochrome, XII. Reindarstellung von Rhodommatin und Ommatin D. Zur Struktur des Rhodom- matins. Liebig’s Ann. C h e m 607, 207-215.

Butenandt, A., Hallmann, G. and Beckmann, R. (1957:‘). Synthesen des 3-Hydroxy- kynurenins. Chem. Ber. 90, 1120-1 124.

Butenandt, A., Biekert, E. and Linzen, B. (1958a). Gber Ommochrome, XIII. Isolierung und Charakterisierung von Omminen. Ifoppe-Seyler’s Z. physiol. Chem. 31 2,

Butenandt, A., Biekert, E. and Linzen, B. (1958b). Uber Ommochrome, XIV. Zur Verbreitung der Ommine im Tierreich. Hoppe-Seylttr’s Z. physiol. Chem. 313, 251-258.

Butenandt, A., Neubert, G. and Baumann, U. (1959). h e r Ommochrome, XVI. Versuche zur Reindarstellung eines Ommins. Hoppe-StylerS Z. physiol. Chem. 3 14,

Butenandt, A., Biekert, E. and Schafer, W. (1960a). Uber Ommochrome, XVIII. Modellversuche zur Konstitution der Ommochronie: Die Kondensation von Hydroxy-chinonen mit o-Amino-phenolen. Liebig’s Ann. Chem. 632 , 134-143.

Butenandt, A., Biekert, E., Kiibler, H. and Linzen, B. (19611b). Uber Ommochrome, XX. Zur Verbreitung der Ommatine im Tierreich. Neue Methoden zu ihrer Identifizierung und quantitativen Bestimmung. Hoppe-Seyler’s Z. physisl. Chem. 3 19, 238-256.

Butenandt, A., Biekert, E., Koga, N. and Traub, P. (19601:). Uber Ommochrome, XXI. Konstitution und Synthese des Ommatins D. Hoppe-Seyler’s Z. physiol. Chem. 321. 258-275.

Butenandt, A., Biekert, E., Kubler, H., Linzen, B. and Traub, P. (1963). Uber Ommochrome, XXII. Konstitution und Synthese des Rhodommatins. Hoppe-Seyler’s Z. physiol. Chem. 334, 71-83.

Butenandt, A., Biekert, E. and Hark, E. (1964). Darstellung und Umwandlungen der 4,5,8-Trihydroxy-6-mercapto-chinolincarbonsaure-(2). Chem. Ber. 97, 285-294.

Butenandt, A., Schafer, W. and Neubert, G. (1967). uber Ommochrome, 24. Mitt. Zur Konstitution der Ommine 11. Monatsh. Chem. 98, 946-935.

Calam, D. H. (1972). Indolylacetic acid in the salivary glands of Dasyneura larvae. NATO Adu. Stud. Inst. Chemistry of Insects, Varenna, 1972. (Unpublished.)

Caspari, E. (1933). Uber die Wirkung eines pleiotropen Gens bei der Mehlmotte Ephestia kiihniella Zeller. Roux’ Arch. EntwMech. Org. :L30, 353-381.

Caspari, E. (1949). Physiological action of eye color mucants in the moths Ephestia kiihniella and Ptychopoda seriata. Q. Rev. Biol. 24, 185-199.

Caspari, E. and Richards, J. (1947/1948). Genic action. Carnegie fnst. Washington Year

Cassady, W. E. and Wagner, R. P. (1971). Separation of mitochondria1 membranes of Neurospora crassa I. Localization of L-kynurenine-3-hydroxylase. J. Cell B i d . 49,

227-236.

15-21.

Book, 47, 183-189.

536-541.

230 BERNT LINZEN

Chauvin, R. (1938). Sur le rougissement du criquet pelerin. C.r. hebd. Sianc. Acad. Sc i , Paris, 207, 1018-1020.

Chauvin, R. (1 939). Sur le pigment rose rejete dans les excrkmknts par le Criquet pelerin au moment de la mue. C.r. Sianc. SOC. Biol. 130. 1194-1195.

Chinzei, Y. and Tojo, S. (1972). Nucleic acid changes in the whole body and several organs of the silkworm, Bombyx mori, during metamorphosis. J. Insect Physiol. 18,

Colombo, G. and Pinamonti, S. (1965). Metaboliti del triptofano “via chinurenina” in Schistocerca gregaria Forsk durante lo sviluppo embrionale. Rend. Classe Sci fis. mat. nut. Acc. Naz. Lincei, Ser. VIII. 38, 728-731.

Colombo, G. and Pinamonti, S. (1967). Analisi biochimica di alcuni mutanti di Musca domestica L. Att iAss . genet. Ital. 12, 341-344.

Colombo, G., Franco, P. and Moccellin, E. (1955). Sulla pigmentazione degli Acridoidei. Ricerche sulla pigmentazione delle larve di Anacridium aegyptium L. Boll. Zool. Turin, 22. 309-322.

1683-1698.

Cott, H. B. (1940/66). “Adaptive Coloration in Animals”. Methuen, London. Cromartie, R. J. T. (1959). Insect pigments. A. Rev. Ent. 4, 59-76. Dadd, R. H. (1970). Arthropod nutrition. In “Chemical Zoology” (Eds M. Florkin and

B. T. Scheer), Vol. V/A, pp. 35-95. Academic Press, New York and London. Dales, R. P. (1962). T h e nature of the pigments in the crowns of sabellid and serpulid

polychaetes. J. mar. biol. Ass. U.K. 42. 259-274. Danneel, R. (1941). Die Ausfarbung uberlebender v- und cn-Drosophila-Augen mit

Produkten des Tryptophanstoffwechsels. Biol. Zbl. 61, 388-399. Danneel, R. and Zimmermann, B. (1954). Uber das Vorkommen und Schicksal des

Kynurenins bei verschiedenen Drosophilarassen. Z. Naturf. 9b, 788-792. Davis, G. R. F. (1968). Quantitative requirements of the saw-toothed grain beetle,

Oryzaephilus surinumensis, for dietary L-tryptophan. J. Insect Physiol. 14,

De Antoni, A., Allegri, G. and Costa, C. (1970). Xantommatina per via enzimatica. Gazz. chim Ital. 100. 1050-1055.

Dennhofer, U. (197 1). Augenfarbmutationen bei der Stechmiicke Culex pipiens L.-ihre chemische und genetische Ursache. Z. Nuturf. 26b, 599-603.

Dimicoli, J.-L. and Htlkne, C1. (1971). Interactions entre acides aminb et acides nuclkiques. 111-Etude par absorption et resonance magnetique nuclkaire de la formation de complexes entre le tryptophane et les constituants des acides nuclkiques. Biochimie, 53, 331-345.

Dustmann, J. H. (1964). Die Redoxpigmente von Carausius morosus und ihre Bedeutung fur den morphologischen Farbwechsel. Z. uergl. Physiol. 49. 28-57.

Dustmann. J. H. (1966). Uber Pigmentuntersuchungen an den Augen der Honigbiene Apis mellifica. Naturwissenschaften, 53, 208.

Dustmann, J. H. (1968). Pigment studies on several eye-colour mutants of the honey bee, Apis mellifera. Nature, Lond. 219, 950-952.

Dustmann, J. H. (1969). Eine chemische Analyse der Augenfarbmutanten von Apis mellifica. J. Insect Physiol. 15, 2225-2238.

Egelhaaf, A. (1 956a). Quantitativer Vergleich der Fluoreszenzmuster der Eier dreier Genotypen von Ephestin kiihniella Natunuirsenschaften, 43, 165-166.

Egelhaaf, A. (1956b). Die fluoreszierenden Stoffe in den Organen dreier Genotypen von

1693-1696.

THE TRYPTOPHAN --* OMMOCHROME PATHWAY IN INSECTS 231

Ephestia kiihniella. Beitrag zur Analyse von Genwirkungen in Pterin- und Trypto- phanstoffwechsel. Z . VererbLehre, 8 7, 769-78 3.

Egelhaaf, A. (1957). Der Gehalt an freiem Tryptophan und Kynurenin bei den Genotypen a+ und a von Ephestia kiihniella w a r e n d der Entwicklung. Z. Naturf.

Egelhaaf, A. (1958). Nachweis eines genabhkgigen L-Tryptophanoxydase-Systems bei Ephestia kiihniella. Z . Naturf. 13b, 275-279.

Egelhaaf, A. (1963a). Uber das Wirkungsmuster des a-Locus von Ephestia kiihniella. Z . VererbLehre, 94, 348-384.

Egelhaaf, A. (1963b). Biochemische Wirkungen des a-Locus von Ephestia kiihniella in der Ei- und Larvenentwicklung. Zool. Anr. Suppl. 26. 79-84.

Egelhaaf, A. and Caspari, E. (1960). h e r die Wirkungswe:.se und genetische Kontrolle des Tryptophanoxydasesystems bei Ephestia kiihniella. 2 . VererbLehre, 91, 373-379.

Eichelberg, D. (1 968). Quantitative Untersuchungen uber die Ommochromvorstufen in den Malpighischen Gefassen verschiedener Drosophila-Mutanten. Z Naturf. 23b, 547-554.

Eichelberg, D. and Wessing, A. (1971). Elektronenoptische Untersuchungen an den Nierentubuli (Malpighische Gefasse) von Drosophila melanogaster XI. Transzelluliire membrangebundene Stofftransportmechanisnien. Z . i’ellforsch. mikrosk. Anat.

Ephrussi, B. (1942a). Analysis of eye color differentiation in Drosophila. Cold Spring

Ephrussi, B. (1942b). Chemistry of “eye color hormones” of Drosophila. Q. Rev. Biol.

Ephrussi, B. and Beadle, G. W. (1935). La transplantation dcs disques imaginaux chez la Drosophile. C.r. hebd. Sdanc. Acad. Sci.. Paris, 201.98-100.

Fearon, W. R and Boggust, W. A. (1950). Pigments derived from tryptophan: (1) urorosein, (2) tryptochrome. Biochem. J. 46, 62-67.

Feigelson, P. and Maeno, H. (1967). Studies on entyme-substrate interactions in the regulation of tryptophan oxygenase activity. Biochem. biophys. Res. Commun 28.

Fingerman, M. (1952). The role of the eye pigments of L)rosophila melanogaster in photic orientation. J. exp. Zool. 120, 131-164.

Friedman, M. and Finley, J. W. (1971). Methods of tryptophan analysis. J. agr. Fd C h e m 19.626-631.

Fuge, H. (1967). Die Pigmentbildung im Auge von Drosopkila melanogaster und ihre Beeinflussung durch den white+-Locus. Z . Zellforsch. mikrosk. Anat. 83,468-507.

Fujimori, E. (1959). Interaction between pteridines and tryptophan. Proc. natn. Acad. Sci. U.S.A. 45, 133-136.

Fukuda, T., Kirimura, J., Matuda, M. and Suzuki, T. (1955). Biochemical studies on the formation of the silk protein-I. ?he kinds of free amino acids concerned in the biosynthesis of the silk protein. J. Biochem., Tokyo. 42, 341-346.

Fuzeau-Braesch, S. (1957). Fractionnement des pigments o:cydort$ucteurs de I’hypo- derme et des yeux d’un grillon. C.r. hebd. Sdanc. Acad. S 8 i , Paris, 245, 2401-2404.

Fuzeau-Braesch, S. (1968). Recherche de la xanthommatine chez deux insectes: Locusta migratoria L. et. Oedipoda coerulesens L. C.Y. hebd. Sdanc. Acad. S c i , Paris, 267,

12b. 465-472.

121, 127-152.

Harb. Symp. quant. Biol. 10, 40-48.

17, 327-353.

289-293.

2030-2033.

232 BERNT LINZEN

Fuzeau-Braesch, S. (1971). Observation de granules pigmentaires (ommochromes et ptkidines) du ttgument d'insectes au microscope klectronique i balayage. C.r. hebd SPanc. Acad. Sci., Paris, Sir. D. 272, 3322-3324.

Fuzeau-Braesch, S. (1972). Pigments and color changes. A . Rev. Ent. 17, 403-424. Galarza, A. M. and Stamm, M. D. (1959). Estudios sobre omocromes: 11.-datos sobre

omocromes en insectos. Act. V. Reunwn An. SOC. Espan. Cienc. Fisiol. Madrid, 1959. Ghelelovich, S . (1969). Melanotic tumors in Drosophila melanogaster. Natl. Cancer Inst.

Monogr. No. 31, p. 263. Ghosh, D. and Forrest, H. S. (1967a). Enzymatic studies on the hydroxylation of

kynurenine in Drosophila melanogaster. Genetics, Princeton, 55, 42343 1. Ghosh, D. and Forrest, H. S. (1967b). Inhibition of tryptophan pyrrolase by some

naturally occurring pteridines. Arch. Biochem. Biophys. 120, 578-582. Giersberg, H. (1 928). h r den morphologischen und physiologischen Farbwechsel der

Stabheuschrecke Dixippus morosus. Z. vergl. Physiol. 7, 65 7-695. Ginoulhiac, E., Tenconi, L. T. and Fabro, S. (1962). Ricerche sulla chinurenina-

idrossilasi: (1) Metodo di dosaggio e determinazione in diversi organi del ratto. Boll. SOC. ital. Biol. sper. 38. 1803-1807.

Glassman, E. (1 956). Kynurenine formamidase in mutants of Drosophila Genetics, Princeton, 41, 566-574.

Goldsmith, T. H. and Philpott, D. E. (1957). The microstructure of the compound eyes of insects. J. biophys. biochem Cytol. 3, 429-440.

Goodwin, T. W. (1952). The biochemistry of locust pigmentation. Biol. Rev. (Cambridge), 27, 439-460.

Goodwin, T. W. and Srisukh, S. (1950). Biochemistry of locusts. 3. Insectorubin: the redox pigment present in the integument and eyes of the Desert Locust (Schistocerca gregaria Forsk.), the African Migratory Locust (Locusta mipatoria migratorioides R. and F.) and other insects. Biochem. 1. 47, 549-554.

Gorini, L. and Beckwith, J. R. (1966). Suppression. A . Rev. Microbiol. 20,401-422. Gotz, K. G. (1964). Optomotorische Untersuchung des visuellen Systems einiger

Graf, G . E. (1957). Biochemical predetermination in Drosophila. Experientia, 13,

Grassmader, H.-H. (1 968). Untersuchungen iiber die Ommochromsynthese in Wildform und Mutante c von Calliphora erythrocephala. Ph.D. Thesis, University of Wiirzburg.

Graszynski. K. (1 970). Intrazellulire Lokalisation von Enzymen des Flusskrebses. Enzyme der Fettsaureoxydation und des Ketonkorperstoffwechsels, Dehydrogenasen des energieliefernden Stoffwechsels und Proteasen der Mitteldanndriise. Z. vergl. Physiol. 66, 107-122.

Green, M. M. (1949). A study of tryptophane in eye color mutants of Drosophila Genetics 34, 564-572.

Green, M. M. (1952). Mutant isoalleles at the vermilion locus in Drosophila melanogaster. Proc. natn. Acad. Sci. U.S.A. 38, 300-305.

Green, M. M. (1954). Pseudo-allelism at the vermilion locus in Drosophila melanogaster. Proc. natn. Acad. Sci. U.S.A. 40, 92-99.

Gripenberg, J. (1958). Fungus pigments VIII. The structure of cinnabarin and cinnabarinic acid. Acta c h e m scand. 12, 603-610.

Augenmutanten der Fruchtfliege Drosophila Kybemetik, 2, 77-92.

404-405.


Gripenberg, J., Honkanen, E. and Patoharju, 0. (1957). Fungus pigments V. Degradations of cinnabarin. Acta chem. scand. 11, 1485-1492.

Gutmann, H. R. and Nagasawa, H. T. (1959). The oxidation of o-aminophenols by cytochrome c and cytochrome oxidase. J. biol. Chem. :!34, 1593-1 599.

Hadorn, E. and Kiihn, A. (1953). Chromatographische und fluorometrische Unter- suchungen zur biochemischen Polyphanie von Ac.genfarb-Genen bei Ephesticr kiihniella. Z. Naturf. 8b, 582-589.

Hanser, G. (1946). Tragergranula und Ommochroinpigmente in den Augen verschiedener Rassen und Transplantat-Trager von Ephestia kiihniella Z. und Ptychopoda seriata Schrk. Z . Naturf. 1, 396-399.

Hanser. G. (1948). h e r die Histogenese der Augenpigmentgranula bei verschiedenen Rassen von Ephestia kiihniella Z. und Ptychopoda seriata Schrk. 2. VererbLehre. 82, 74-97.

Hanser, G. (1 959). Genphysiologische Untersuchungen an tier w-Mutante von Calliphora erythrocephala. 2. Naturf. 14b, 194-201.

Harano, T. and Chino, H. (1971). A new diaphorase from Bombyx silkworm eggs-cytochrome c reductase activity mediated with xanthommatin. Archs Biochem.

Harmsen, R. (1966). The excretory role of pteridines in insects. J . exp. Biol. 45, 1-13. HtlPne, C1. (1971). Role of aromatic amino-acid residues in the binding of enzymes and

proteins to nucleic acids. Nature, N e w Biol. 234, 120-1211. Htlkne, Cl., Dimicoli, J.-L. and Brun, P. (1971). Binding of tryptamine and

5-hydroxytryptamine (serotonin) to nucleic acids. Fluorescence and proton magnetic resonance studies. Biochemistry. N. Y . 10, 3802-3809.

Henderson, L. M., Gholson, R. K. and Dalgliesh, C. E. (1962). Metabolism of aromatic amino acids. In “Comparative Biochemistry” (Eds M. Florkin and H. S. Mason), Vol. 4, pp. 245-342. Academic Press, New York and London.

Hendrichs-Hertel, U. and Linzen, B. (1969). Uber die Kynurenin-3-Hydroxylase bei Calliphora erythrocephala. Z. vergl. Physiol. 64,411-431.

Hengstenberg, R. and Gotz, K. G. (1967). Der Einfluss des Schirmpigmentgehalts auf die Helligkeits- und Kontrastwahrnehmung bei Drosophila-Augenmutanten. Kybernet ik , 3,276-285.

Henning, H. D. (1957). Unpublished experiments at the Max Planck lnstitute of Biochemistry, Munich.

Hertel, U. (1968). Untersuchungen iiber die Kynurenin-3-Hydroxylase bei Insekten. Ph.D. Thesis, University of Munich.

Hertweck, H. (1960). Notes on the formation of a red eye pigment in the Malpighian tubules and fat body in Drosophila melanogaster. Folia. biol., Praha, 6 , 293-296.

Hinton, T., Noyes, D. T. and Ellis, J. (1951). Amino acids and growth factors in a chemically defined medium for Drosophila. Physiol. 2001. 24, 335-353.

Hiraga, S. (1964). Tryptophan metabolism in eye-color muta.nts of the housefly. Jap. J. Genet. 39,240-253.

Hirata, Y. and Nakanishi, K. (1950). The synthesis of 3-hydroxykynurenine (+ chromogen). Bull. chem. SOC., Japan, 23, 134-137.

Hirata, Y., Nakanishi, K. and Kikkawa, H. (1949). Crystals.of + chromogen obtained in silkworms. Jap. J. Genet. 24, 190. (In Japanese.)

Biophys. 146, 467-476.

234 BERNT LINZEN

Hirata, Y., Nakanishi, K. and Kikkawa, H. (1950). Crystalline + chromogen obtained from Bombyx mori. Science, 112, 307-308.

Hoglund, G., Langer, H., Struwe, G. and Thorell, B. (1970). Spectral absorption by screening pigment granules in the compound eyes of a moth and a wasp. Z. uergl.

Horikawa, M. (1958). Developmental-genetic studies of tissue cultured eye discs of D. nielanogaster I. Growth, differentiation and tryptophan metabolism. Cytologia (Tokyo) , 23,468-477.

Horn, U., Ullrich, V. and Staudinger, H. (1971). Purification and characterization of L-kynurenine 3-hydroxylase (EC 1.14.1.2) from rat liver. Hoppe-Seyler’s Z. physiol. C h e m 352, 837-842.

Horowitz, N. H. (1940). A respiratory pigment from the eggs of a marine worm. R o c . natn. Acad. Sci. U.S.A. 26, 161-163.

Horowitz. N. H. and Baumberger, J. P. (1941). Studies on the respiratory pigment of Urechis eggs.]. biol. C h e m 141,407-415.

Horstmann, G. (1971). Uber die Pigmentgranulogenese im Auge von Ephestin kiihniella Z. Z . Naturf. 26b, 484-485.

Inagami, K. (1954a). Chemical and genetical studies on the formation bf the pigment in the silkworm-111. On the microanalysis of 3-hydroxy-kynurenine. J. Sericult. Sci Japan, 23,299-303. (In Japanese.) (1955). Chem. Abstr. 4 9 , 1 0 5 3 6 ~ .

Inagami, K. (1954b). Mechanism of the formation of red melanin in the silkworm. Nature, Lond. 174, 1105.

Inagami, K (1955). Chemical and genetical studies on the formation of pigment and metabolism of tryptophan in the silkworm-X. Biochemical studies on the “Red Blood-rb”gene in the silkworm.]. agric. chem. SOC. Japan, 29, 12. (In Japanese.)

Inagami, K. (1958). The mechanism of formation of tryptophan pigments and the metabolism of tryptophan in silkworm. Bull. Kumamoto sericult. Exp. Stat. 6 , 5-58. (In Japanese.)

Isenberg, I. and Szent-Gyorgyi, A. (1958). Free radical formation in riboflavin complexes. R o c . natn. Acad. Sci U.S.A. 44, 857-862.

Ishiguro, I. and Linzen. B. (1965). Praparative Isolierung und Tritium-Markierung von 3-Hydroxy-L-kynurenin. Hoppe-Seyler’s Z. physiol. Chem. 340. 286-288.

Ishiguro, I. and Linzen, B. (1966). Bildung von Glucosiden bei zwei Insektenarten, Locusta migratoria und Bombyx mori J. Insect Physiol. 12, 267-273.

Ishiguro, I. and Nagamura, Y. (1971a). The red pigments in feces urine of the silkworm, Bombyx mori Medicine and Biology, 82, 149-153.

Ishiguro, I. and Nagamura, Y. (1971b). Identification of xanthommatin and isolation of red-protein in the body-fluid of rb-mutant of the silkworm. Medicine and Biology,

Ishiguro, I., Nagamura, Y. and Hara, A. (1971a). Studies on the formation of phenoxazine pigment from o-aminophenol derivatives by hemoglobin. 1. Conversion of 3-OH-anthranilic acid into cinnabarinic acid in the presence of MnZ+. Yukugaku Zasshi, 91,760-765.

Ishiguro, I., Nagamura, Y. and Yamamoto, T. (1971b). Formation of phenoxazine- pigment and conversion of 3-hydroxy-kynurenine in the silkworm with metamorphosis. Seikagaku, 43, 401-407.

Physiol. 67. 238-242.

82, 123-126.


Iwata, S. and Ogata, K. (1966). Large scale isolation of L-3-hydroxy-kynurenine from mutant strain (aka-aka) of silkworm larva. Yakugaku Zasshi, 86,249-250.

Jacobson, K. B. (1971). Role of an isoacceptor transfer ribonucleic acid as an enzyme inhibitor: effect on tryptophan pyrrolase of Drosophila. Nature, New Biol. 23 1,

JansCn, C. T., Santti, R. S. and Hopsu-Havu, V. K. (1969). Kynurenine formamidase in rat skin. J. invest. Derm. 52, 430-436.

Jezewska, M. (1926). Les changements de la teneur en tryptophane au cours de dkveloppement des chrysalides des mouches. C.r. Siunc SOC. Biol. 95, 910-912.

Joshi, S. and Brown, R. R. (1959). Pigment formation from hydroxy-kynurenine by a rat liver system. Proc. Fedn. A m . SOCS exp. Biol. 18,255.

Kalmus, H. (1943). The optomotor responses of some eye mutants of Drosophila. J. Genet. 45, 206-213.

Kanehisa, T. (1956a). Eye-colour genes and tumour incidence. Jap. J. Genet. 31, 144- 1 46.

Kanehisa, T. (1956b). Relation between the formation of melanotic tumours and tryptophane metabolism involving eye-colour in Drosophila. Annotnes Z O O L jap. 29,

Kanehisa, T. and Fujii, S. (1967). Further biochemical studies on “melanotic tumour” and “freckled-type melanotic tumor” in D. melanogaster. Jap. J. Genet. 42, 299.

Karlson, P. and Buckmann, D. (1956). Experimentelle Auslosung der Umfikbung bei Ceruru-Raupen durch Prothorakaldriisenhormon. Nuturulissenschaften, 43,4445.

Katz, E. and Weissbach, H. (1962). Biosynthesis of the actinomycin chromophore; enzymatic conversion of 4-methyl-3-hydroxyanthranilic acid to actinocin. J. biol. Chem. 237, 882-886.

Kaufman, S. (1962). Studies on tryptophan pyrrolase in Drosophila melunogaster. Genetics, Princeton, 47, 807-817.

Kawase, S. (1955). Studies on the pigments found in the integument of Bombyx mori L. I. Red pigment and some other epidermal pigments. /. sericult. S c i /upan, 24,

Kellner, O., Sako, T. and Sawano, J. (1884). Chemische Untersuchungen uber die Entwicklung und ErnZhrung des Seidenspinners (Bombyx mori). Lundw. Vers.-Stat.

Kikkawa, H. (1941). Mechanism of pigment formation in Bombyx and Drosophila.

Kikkawa, H. (1951). On fluorescent substances involved in silkworm eggs. J. sericult.

Kikkawa, H. (1953). Biochemical genetics of Bombyx mori (silkworm). Adu. Genet. 5,

Kikkawa, H. and Kuwana, H. (1952). Origin of nicotinic acid in Eombyx mori (silkworms). Annotncs zool. /up. 25, 30-33.

Koga, N. and Coda, Y. (1962). 3-Hydroxy-kynwenin in frisch gelegten Bombyx-Eiern. Hoppe-Scyler’s Z . physiol. Chem. 328 272-274.

Koga, N. and Osanai, M. (1967). Der Gehalt an Tryptophan, Kynurcnin, 3-Hydroxy- kynurenin und Ommochromen bci den iiberwinternden Eiern des Seidenspinners Eombyx mori wiihrend der Entwicklung. Hoppe-Scyler’s 2. physwl . Chem. 348, 979-982.

17-19.

97-100.

69-76.

30. 59-86.

Genetics, Princeton, 28, 587-607.

S c i Japan, 20,66. (In Japanese, cited after Kikkawa, 1953.)

107-140.

236 BERNT LINZEN

Kolb, G. and Autrum, H. (1972). Die Feinstruktur im Auge der Biene bei Hell- und Dunkeladaptation. J. comp. Physiol. 77, 113-125.

Kotake, Y. and Shichiri, G. (1931). h e r die Spaltung des Kynurenins mittels Natriumbicarbonatlosung. Hoppe-Seyler's Z . physiol. Chem. 195,152-158.

Krieger, F. (1954). Untersuchungen iiber den Farbwechsel der Libellenlarven Z. vergl. Physiol. 36, 352-366.

Kiibler, H. ( 1 960). Untersuchungen zur Biogenese und Verbreitung der Ommochrome. Doctoral Thesis, University of Munich.

Kiihn, A. (1932). Entwicklungsphysiologische Wirkung einiger Gene bei Ephestia kiihniella. Naturwissenschaften, 20, 974-97 7.

Kiihn, A. (1940). Eine Mutation der Augen- und Korperpigmentierung (dec) bei Ptychopoda seriata Schrk. Z. VerebLehre, 78, 1-13.

Kiihn, A. (1941). Uber eine Gen-Wirkkette der Pigmentbildung bei Insekten. Nachr. Akad. Wiss. Cattingen, Math.Phys. Kl. 1941, 231-261.

Kiihn, A. (1955). Ein Fluorometer fiir Papierchromatogramme. Natunuissenschaften 42,

Kiihn, A. (1956). Versuche zur Entwicklung eines Modells der Genwirkung. Natur- wissenschaften, 43,25-28.

Kiihn, A. (1960). h e r den Ausfirbungsverlauf der Imaginalaugen von Ephestia- Genotypen in der Puppe. Biol. Zbl. 79, 385-392.

Kiihn, A. (1963). Die zeitliche Folge der Bildung der Ommochrome in den Imaginalaugen von Ephestia und Ptychopada. Z . Naturf. 18b, 252-254.

Kiihn, A. and Almeida, F. F. de (1961). Fluoreszierende Stoffe und Ommochrome bei Genotypen von Plodia interpunctella. Z. VererbLehre 92, 126-132'.

Kiihn, A. and Egelhaaf, A. (1955). Zur chemischen Polyphanie bei Ephestia kiihniella. Naturwissenschaften, 14, 634-635.

Kiihn, A. and Egelhaaf, A. (1959). h e r die Mutation br (braunaugig) bei Ephestia kuhniella. Z. VererbLehre, 90, 244-250.

Kiihn, A. and Henke, K. (1930). Eine Mutation der Augenfarbe und Entwicklungs- geschwindigkeit bei der Mehlmotte Ephestia kiihniella. Wilhelm Roux Arch. EntwMech. Org. 122, 204-212.

Kiihn, A. and Plagge, E. (1937). Radetermination der Raupenpigmentierung bei Ephestia kiihniella durch den Genotypus der Mutter und durch arteigene und artfremde Implantate. Biol. Zbl. 57, 113-126.

Lan, S. J. and Gholson, R. K. (1965). A comparative study of tryptophan catabolism. J. biol. Chem. 240,3934-3937.

Langer, H . (1967). Uber die Pigmentgranula im Facettenaugc von Calliphora erythrocephala. Z . vergl. Physiol. 55, 354377.

Langer, H. and Crassmader, H. (1965). Freies Tryptophan in Wildform und Augen- farbmutanten von Calliphora erythrocephula wiihrend der Imaginalentwicklung. Z. Naturf. 20b, 172-177.

Langer, H. and Hoffmann, Ch. (1966). Elektro- und stoffwechselphysiologische Untersuchungen iiber den Einfluss von Ommochromen und Pteridinen auf die Funktion des Facettenauges von Calliphora erythrocephah J. Insect Physiol. 12, 35 7-38 7.

Langer, H. and Struwe, C. (1972). Spectral absorption by screening pigment granules in the compound eye of butterflies (Heliconius). J. comp. Physiol. 79 203-212.

529-531.


Laudani, U. and Grigolo, A. (1968). Sulla presenza di alcuni metaboliti del triptofano, pterine ed altre sostanze U.V. fluorescenti in individui di Musca domestica L., normali e mutanti peril colore degli occhi. Att iAss . genet. Ital. 13, 180-182.

Leibenguth, F. (1965). Kynurensaure und Xanthurensaure bei Habrobracon juglandis Ashmead. Z . Naturf. 20b, 315-317.

Leibenguth, F. (1 967a). Entwicklungsphysiologisch-genetische Untersuchungen am Tryptophanstoffwechsel zweier Genotypen von Habrobracon juglandis Ashmead. Wilhelm Roux A r c h EntwMech. Org. 158, 246-300.

Leibenguth, F. (1967b). Regulation of tryptophan metabolism in the parasitic wasp, Habro bracon juglandis. Experien tia, 23, 1069-107 1.

Leibenguth, F. (1970). Concerning non-darkening of mutant Habrobracon (Bracon hebetor) eyes as consequence of new chromogen-reducing mechanism in insect larvae. Experientia, 26, 659.

Leklem, J. E. (1971). Quantitative aspects of tryptophan metabolism in humans and other species: a review. A m . J . clin. Nub. 24, 659-672.

Lindsley, D. L. and Grell, E. H. (1968). Genetic variations of Drosophila melanogaster. Carnegie Institute, Washington, Public. No. 627.

Linzen, B. (1957). h e r Ommine und iiber das Auftreten von Ommochromen im Tierreich. Doctoral Thesis, University of Tubingen.

Linzen, B. (1959). h e r Ommochrome, XV. a e r die Identifizierung des “Urecho- chroms” als Xanthommatin. Hoppe-Seyler’s Z, physiol. Chem. 314, 12-14.

Linzen, B. (1 963). Eine spezifische quantitative Bestimmung des 3-Hydroxy-kynurenins. 3-Hydroxy-kynurenin und Xanthommatin in der Imaginalentwicklung von Call:- phora. Hoppe-Seyler’s 2. physiol. Chem. 333, 145-148.

Linzen, B. (1966). aer Ommochrome, XXIII. Ommidine-ein neuer Typus von Ommochromen aus Orthopteren. Z. Naturf. 21b, 1038-1047.

Linzen, B. (1967). Zur Biochemie der Ommochrome. Unterteilung, Vorkommen, Biosynthese und physiologische Zusammenhkge. Natunuissenschaften, 54. 259-267.

Linzen, B. (1968). Versuche zur Biosynthese der Ommochrome. ZooL Anz. Suppl. 31,

Linzen, B. (1970). Zur Biosynthese von Ommochromen, I. Einbau ”S-markierter Vorstufen in Ommine. Hoppe-Seyler’s Z. physiol. Chem. 351, 622-628.

Linzen, B. (1971a). Tryptophan catabolism in silkworm metamorphosis: changes in total matter, DNA, free, and protein-bound tryptophan. J. Insect Physiol. 17.

Linzen, B. (1971b). Autonomes Verhalten Tryptophan-abbauender Enzyme wahrend der Metamorphose von Bombyx mori rb: Lokalisation und zeitlicher Verlauf der Tryptophanpyrrolase. Wilhelm Roux Arch. EntwMech. Osg. 168, 320-331.

Lmzen, B. and Buckmann, D. (1961). Biochemische und histologische Untersuchungen zur Umfirbung der Raupe von Cerura Dinula L. Z. Nuturf. 16b, 6-18.

Linzen, B. and Ishiguro, I. (1966). 3-Hydroxy-kynurenin bei Bombyx mori rb.-Ein neuer Tryptophanmetabolit: 3-Hydroxy-kynurenin-glucosid. Z. Naturf. 21b,

Linzen, B. and Hendrichs-Hertel, U. (1970). Kynurenin-3-Hydroxylase in der Meta- morphose von Bombyx mori rb: Organ- und stadienspezifische Wechsel der Enzymaktivitat. Wilhelm Roux Arch. EntwMech. Org. 165, 26-34.

’

148-153.

2005-2016.

132-137.

238 BERNT LINZEN

Linzen, B. and Hertel, U. (1967). In uitro assay of insect kynurenine-3-hydroxylase.

Lucas, F., Shaw, J. T. B. and Smith, S. G . (1958). The silk fibroins. Adv. Protein Chem.

Magnus, D. (1958). Experimentelle Untersuchungen zur Bionomie und Ethologie des Kaisermantels Argynnis paphin L. (Lep. Nymph.). I. Uber optische Ausloser von Anfliegereaktionen und ihre Bedeutung fiir das Sichfiiden der Geschlechter. Z . Tierpsychol. 15, 397-426.

Maier, W. (1 965). Elektronenmikroskopischer Vergleich von Retinula-Pigmentzellen’der Wildform wa+ und der Mutante wa von Ephestia kiihniella Z. Z . Naturf. 20b,

Makino, K., Takahashi, H., Satoh, K. and Inagami, K. (1954). Abnormal accumulation of 3-hydroxy-kynurenine in the mutant “aka-aka” of silkworm. Nature, Lond. 179,

Marzluf, G . A. (1965a). Tryptophan pyrrolase of Drosophila: partial purification and properties. Z VererbLehre, 97, 10-17.

Marzluf, G. A. (1965b). Enzymatic studies with the suppressor of vermilion of Drosophiln melanogaster. Genetics, Princeton, 52. 503-512.

Mayer, G. and Staudinger, H. (1967). Zur Frage der Aktivierung von L-Kynurenin-3- Hydroxylase in Rattenlebermitochondrien durch Cyanid. Hoppe-Seyler ‘s Z . physiol. Chem. 348, 733-734.

Mayer, G., Ullrich, V. and Staudinger, H. (1968). Possible involvement of cytochrorne b5 in L-kynurenine-3-hydroxylase of rat liver mitochondria. Hoppe-Seyler’s Z . physiol. Chem. 349, 459-464.

McArthur, J. N. and Dawkins, P. D. (1969). The effect of sodium salicylate on the binding of L-tryptophan to serum proteins. J. Pharm. Pharmac. 21, 744750.

McMenamy, R. H. and Oncley, J. L. (1958). The specific binding of L-tryptophaq to serum albumin. J. biol. Chem. 233, 1436-1447.

Mehler, A. H. and Knox, W. E. (1950). The conversion of tryptophan to kynurenine in liver. 11. The. enzymatic hydrolysis of formylkynurenine. 1. biol. Chem. 187,

Merlini, L. and Nasini, G. (1968). Pigmenti degli insetti V.-Xantommatina nel tegument0 di Cossus cossus L. (Lepidoptera Cossidae). Ist. Lombardo, Rendic. Cl. Sci. (A), 102,890-892.

Mittler, S. (1952). Influence of amino acids upon incidence of tumors in TuS0j stock of D. melanogaster. Science, 116, 657-659.

Mohlmann, E. (1 958). Chromatographische Untersuchungen an verschiedenen Ceno- typen von Plodin interpunctella im Vergleich mit anderen Schmetterlingsarten. Z . VererbLehre, 89, 651-674.

Montenay-Garestier, Th. and Htlkne, C1. (1971). Reflectance and luminiscence studies of molecular complex formation between tryptophan and nucleic acid components in frozen aqueous solutions. Biochemistry (Washington), 10, 300-306.

Morgan, L. R. and Weimorts, D. M. (1964). The conversion of 3-hydroxyanthranilic acid to 2-aminophenoxazin-3-one-l,8-dicarboxylic acid by extracts of acetone powders of poikilothermic animal livers. Biochim. biophys. Acta, 82, 645-647.

Mnckenthaler, F. A. (1971). Kynurenine localization in the egg of Drosophila rnelanogaster Experientia, 27,828-830.

Natunuissenschaften, 54,21.

13. 108-242.

31 2-3 14.

586-587.

431-439.

THE TRYPTOPHAN --* OMMOCHROME PATHWAY itv INSECTS 239

Musajo, L., Spada, A. and Casini, E. (1950). Sulla sintesi della d,l-3-ossichinurenina. Gmz. chim. Ital. 80, 171-176.

Muth, F. W. (1967). Quantitative Untersuchungen zum Pigmentierungsgeschehen in den Augen der Mehlmotte Ephestia kiihniella. I. Grundlagen. Wilhelm Roux Arch. EntwMech. Org. 159, 379-411.

Muth, F. W. (1968). Quantitative Untersuchungen zum Pigmentierungsgeschehen in den Augen der Mehlmotte Ephestiu kiihniella. 11. Die Pigmentbildung im a-Stamm nach Injektion kleiner Mengen von 3-Hydroxykynurenin. Wilhelm Roux Arch. EntwMech

Muth, F. W. (1969). Quantitative Untersuchungen zum Pigmentierungsgeschehen in den Augen der Mehlmotte Ephestiu kiihniella 111. Die Pigmentbildung im a-Stamm nach Injektion hoher Dosen von Kynurenin und der Versuch einer Deutung. Wilhelm Roux Arch. EntwMech. Org. 162, 56-77.

Nair, P. M. and Vaidyanathan, C. S. (1964). Isophenoxazine synthase. Biochim. biophys. Acta, 81, 507-516.

Nair, P. M. and Vining, L. C. (1964). An isophenoxazine synthase from Pycnoporus coccineus (Fr.) Bond. and Sing. Can. J. Biochem. 42, 1515-1526.

Nakagaki, M., Koga, N. and Iwata, S. (1962). Bio-physicochemical studies on phenoxazone compounds. I. Diffusion constant and degree of association. Yakugaku Zasshi, 82, 1134-1138.

Nawa, S. (1970). Purification and properties of tryptophan pyrrolase in Ephestia. A . Rep. Nut. Inst. Genetics (Japan), 21, 24-25.

Needham, A. E. (1970). The integumental pigments of some isopod crustacea. Comp. Biochem. Physiol. 35, 509-534.

Neese, V. (1968). Zur optischen Orientierung der Augenmutante “Chartreuse” von Apis mellifica L. Z . uergl. Physiol. 60, 41-62.

Neese, V. (1972). Die Altersabhangigkeit des Ommochromgehalts im Komplexauge der Bienen. Ein quantitativer Vergleich zwischen Wildtyp und der Augenmutante chartreuse. J . Insect Physiol. 18, 229-236.

Neubert, G. (1956). Modellversuche zur Bildung der Ommochrome. Doctoral Thesis, University of Tiibingen.

Nickerson, B. (1956). Pigmentation of hoppers of the Desert Locust (Schistocerca gregaria Forskal) in relation to phase coloration. Anti-Locust Bull No. 24.

Nolte, D. J. (1950). The eye-pigmentary system of Drosophila: the pigment cells. J. Genet. 50, 79-99.

Nolte, D. J. (1952). The eye-pigmentary system of Drosophila 111. The action of eye-colour genes. J. Genet. 51, 142-186.

Nolte, D. J. (1961). The pigment granules in the compound eye of Drosophila Heredity, Lond. 16, 25-38.

Nolte, D. J. (1965). The pigmentation of Locusts. S. Afr. J. S i i 61, 173-178. Okamoto, H. and Hayaishi, 0. (1967). Flavin adenine dinucleotide requirement for

kynurenine hydroxylase of rat liver mitochondria. Biocheri. biophys. Res. Commun. 29, 394399.

Okamoto, H., Yamamoto, S., Nozaki, M. and Hayaishi, 0. ”(1967). On the submitochondrial localization of L-kynurenine-3-hydrox) lase. Biochem. Biophys. Res. Commun. 26, 309-314.

Osanai, M. (1966a). a e r die Umfirbung der Raupen von Hestina japonica zu Beginn

Org. 161, 336-350.

240 BERNT LINZEN

der Uberwinterung, 11. Pigmente der Raupen von Hestina japonica und Sasakia charonda. Hoppe-Seyler’s Z. physiol. Chem. 347, 145-155.

Osanai, M. (1966b). h e r die Umfiirbung der Raupen von Hestina japonica zu Beginn der Uberwinterung, 111. Ein Ommochromoprotein mit Redoxverhalten in der Haut der Hestina-Raupe. J. Insect Physiol. 12, 1295-1301.

Osanai, M. (1967). Mange1 an Cytochrom c im iiberwinternden Ei des Seidenspinners, Bom byx mori Hoppe-Seyler’s Z . physiol. Chem. 348, 469-470.

Osanai, M. (1968a). Tyrosinaseaktivitat bei der Hestinn-Raupe zu Beginn der h e r - winterung. J. Insect Physiol. 14. 261-267.

Osanai, M. (1 968b). Tyrosinaseaktivitat in den Eiern von Ommochrom-Mangelmutanten des Seidenspinners, Bombyx mori J. Insect Physiol. 14, 403-408.

Osanai, M. and Arai, Y. (1962a). Effect of ligation at different levels on the change of body colour in. the larvae of the nymphalid butterfly, Hestinn japonica. at the beginning of hibernation. Zool. Mag. (Dobotsugaku Zasshi), 71, 202-205. (In Japanese with English summary and colour plates.)

Osanai, M. and Arai, Y. (1962b). Uber die Umfiirbung der Raupen von Hestinajaponica zu Beginn der Uberwinterung I. Durchschniirungsversuche an der Umfiirbung der Hestina-Raupe. Cen. comp. Endocr. 2, 31 1-316.

Osanai, M. and Koga, N. (1966). Isolierung des Xanthommatins und der Ommine aus Eiem des Seidenspinners mit SE-Sephadex. Eine neue Methode zur Abtrennung und quantitativen Bestimmung der Ommochrome. Hoppe-Seyler’s Z. physiol. Chem. 347,

Parsons, P. A. and Green, M. M. (1959). Pleiotropy and competition at the vermilion locus in Drosophila melanogaster. Proc. nntn. Acad. Sci U.S.A. 45,993-996.

Perrelet, A., Orci, L. and Baumann, F. (1971). Evidence for granulolysis in the retinula cells of a stomatopod crustacean, Squilla mantis. J. Cell Biol. 48, 684-688.

Phillips, J. P. and Forrest, H. S. (1970). Terminal synthesis of xanthommatin in Drosophila melanogaster. 11. Enzymatic formation of the phenoxazinone nucleus. Biochem. Genetics 4, 489-498.

Phillips, J. P., Simmons, J. R. and Bowman, J. T. (1970). Terminal synthesis of xanthommatin in Drosophila melanogaster. I. Roles of phenol oxidase and substrate availability. Biochem. Genetics, 4, 481487.

Pieber, M., Romero, C., Tirapegui, C. and Tohi, J. (1969). Absence of hyperchromic effect in DNA heated above Tm. in the presence of tryptophan. Z. Naturf. 24b,

Pinamonti, S . and Petris, A. (1966). Studies on tryptophan pyrrolase of Schistocerca gregaria Forsk. (Orthoptera). Comp. Biochem. Physiol. 17, 1079-1087.

Pinamonti, S., D’Orazio, G. and Veronese, F. M. (1964). Ricerche s u l metabolismo del triptofano via chinurenina in Schistocerca gregaria Forsk. (Ortotteri). Boll. Zool. 31,

Pinamonti, S . , Petris, A. and Miliani, M. (1970). Studies on the kynurenine transaminare of Schistocerca gregaria Forsk. (Orthoptera, Acrididae). Comp. Biochem. Physiol. 37,

156-1 60.

508-5 10.

567-5 7 1.

311-320. Pinamonti, S., Petris, A. and Ottaviani, F. (1970/71). Studio sulla

chinurenina-3-idrossilasi in Schistocerca gregurie Forsk. (Orthoptera, Arcrididae). Att iAccad. S c i Ferrara, 48, 1-12.

Plagge, E. (1939). Genabhangige Wirkstoffe bei Tieren. Ergebn. Biol. 17. 105-150.


Plaine, H. L. and Glass, B. (1955). Influence of tryptophan and related compounds upon the action of a specific gene and the induction of melanotic tumors in Drosophih melanogaster. J. Genet. 53, 244261.

Prasad, C. and French, W. L. (1971). Tryptophan pyrrolase activity during development of the mosquito (Culex pipiens). Comp. Biochem. Physiol. 38b 627-629.

Ramsay, J. A. (1956). Excretion by the Malpighian tubules of the stick insect, Dixippus morosus (Orthoptera, Phasmidae) : calcium, magnesium, chloride, phosphate and hydrogen ions. J. exp. Biol. 33, 697-708.

Raszka, M. and Mandel, M. (1971). Interaction of aromatic amino acids with neutral polyadenylic acid. Proc. natn. Acad S c i U.S.A. 68, 1190-1191.

Rechsteiner, M. C. (1970). Drosophila lactate dehydrogenase and a-glycerolphosphate dehydrogenase: distribution and change in activity during development. J. Insect Physiol. 16, 1179-1192.

Reisener-Glasewald, E. (1956). Uber die Entwicklung des Bestandes an fluoreszierenden Stoffen in den Kopfen von Ephestia kiihniella in Abhangigkeit von verschiedenen Augenfarbgenen. Z. VererbLehre, 87,668-693.

Rizki, M. T. M. (1957). Tumor formation in relation to metamorphosis in Drosophila melanogaster. J. Morph. 100, 459-472.

Rizki, M. T. M. (1961). Intracellular localization of kynurenine in the fat body of Drosophila J. biophys. biochem. Cytol. 9, 567-572.

Rizki, T. M. (1963). Genetic control of cytodifferentiation. .I. CellBiol. 16, 513-520. Rizki, T. M. (1964). Mutant genes regulating the inducibility of kynurenine synthesis. J.

CellBiol. 21, 203-211. Rizki, T. M. and Rizki, R. hi. (1963). An inducible enzyme system in the larval cells of

Drosophila melanogaster. J . Cell Biol. 17, 87-92. Rizki, T. M. and Rizki, R. M. (1964). Factors affecting the intracellular synthesis of

kynurenine. J. Cell Biol. 21, 27-33. Rohner, Sr. M. C. (1959). The influence of depression of respiratory metabolism by

carbon monoxide on the pigment development in the insect eye. Z. VererbLehre, 90,

Roth, H. (1939). Die Bestimmung des freien und gebundenen Tryptophans in Pflanzen. Angew. Chem. 52.149-151.

Rowell, C. H. F. (1971). The variable coloration of the acridoid grasshoppers. In “Advances in Insect Physiology” (Eds J. W. L. Beament, J. E. Treherne and V. B. Wigglesworth), Vol. 8, pp. 145-198. Academic Press, London and New York.

Riidiger, W. and Klose, W. (1970). h e r die Pigmente der E’lorfliege Chrysopa carnea Experientia, 26,498.

Ruiter, L. de (1955). Countershading in caterpillars. Archs n k i d Zool. 11, 1-57. Schiifer, W. (1964). The chemistry of the phenoxazones. Prog. Org. Chem. 6, 135-163. Schafer, W. and Geyer, I. (1972). fher das Redoxverhalten der Ommochrome.

UV/S-Spektren von 3H-Phenoxazinonen-( 3) und Phenoxazinen. Tetrahedron, 28,

Schartau, W . and Linzen, B. (1969). Tryptophan wird von Insekten nicht veratmet. Naturw issenschaften, 56, 329.

Schildknecht, H. and Schmidt, H. (1963). Die chemische Zusammensetzung des Wehrsekretes von Dicranura uinula. XVII. Mitteilung iiber Insektenabwehrstoffe. Z. Naturf. 18b, 585-587.

257-262.

526 1-5279.

242 BERNT LINZEN

Schildknecht, H., Birringer, H. and Krauss. D. (1969). Uber Arthropoden-Abwehrstoffe XXXVI. Aufklirung des gelben Prothorakalwehrdriisen-Farbstoffes von Ilybius fenestratus. Z . Naturf. 24b, 38-47.

Schlossberger, H. G. (1949). Synthese und physiologische Bedeutung des 3-Oxy- kynurenin, eines Zwischenproduktes bei der Bildung der Ommochrome aus Trypto- phan im Insektenorganismus. Ph.D. Thesis, University of Tubingen.

Scholtan, W. (1956). Kolloidchemische Eigenschaften von Phenothiazinderivaten, insbesondere von Megaphen. Medizin und Chemie (reports of Farbenfabriken Bayer A.G.) 5. 295-313. Verlag Chemie, Weinheim.

Schultz, J. and Rudkin, G. T. (1948). Absence of a sparing action of tryptophane on nicotinamide requirements of the fly, Drosophila melanogaster. Fedn Proc. Fedn Am. Socs exp. Biol. 7, 185.

Schwabl, G. and Linzen, B. (1972). ijber die Bildung der Augenpigmentgranula bei Drosophila v und cn nach Verfiitterung von Ommochromvorstufen. Wilhelm Roux Arch. EntwMech. Org. 171, 223-227.

Schwinck, I. (1953). iiber den Nachweis eines Redox-Pigmentes (Ommochrom) in der Haut von Sepia officinalis. Naturwissenschaften, 40, 365.

Seecof, R. L., Kaplan, W. D. and Futch, D. G. (1969). Dosage compensation for enzyme activities in Drosophila melanogaster. Proc. natn. Acad. Sci. U.S.A. 62. 528-535.

Semm, K. and Fried, R. (1952). Einfache Apparatur zur Fluoreszenzmessung auf Filtrierpapierstreifen. Naturwissenschaften, 39, 326-327.

Shapard. P. B. (1960). A physiological study of the vermilion eye color mutants of Drosophila melanogaster. Genetics, Princeton, 45, 359-3 7 6.

Shinohara, R. and Ishiguro, I. (1970). The purification and properties of formamidase from rat liver. Biochim. biophys. Acta, 198. 324-331.

Shoup, J. R. (1966). The development of pigment granules in the eyes of wild type and mutant Drosophila melanogaster. f. Cell Biol. 29. 223-249.

Simmons, J. R. and Gardner, E. J. (1958). The effect of tryptophan on the effect of tumorous head in crosses among selected stocks of Drosophila melanogaster. Genetics, Princeton, 43, 164-171.

Singh, H.. Seshadri, T. R. and Subramanian, G. B. V. (1966). A note on the colouring matter of lac larvae. Tetrahedron lett. 1966, 1101-1108.

Sols, A. and Marco, R. (1970). Concentrations of metabolites and binding sites. Implications in metabolic regulation. In “Current topics in cellular regulation” (Eds B. L. Horecker and E. R. Stadtman), Vol. 2, pp. 227-273. Academic Press, New York and London.

Sonobe, H. and Ohnishi, E. (1970). Accumulation of 3-hydroxy-kynurenine in ovarian follicles in relation to diapause in the silkworm, Bombyx mori L. Devl Growth Different. 12,41-52.

Spies, J. R. and Chambers, D. C. (1948). Tryptophan determination. Color-forming reactions with p-dimethyl-aminobenzaldehyde and sodium nitrite in sulfuric acid. Analyt. Chem. 20, 30-39.

Stamm, M. D. and Aguirre, L. (1955a). Estudios sobre bioquimica de insectos I. Aminoacidos aromiticos y triptbfano en la metamorfosis de Saturnia Pyr i Rev. Espan. Fisiol. 11, 63-68.

Stamm, M. D. and Aguirre, L. (1955b). Estudios sobre bioquimica de insectos 11.


Aminoicidos aromiticos y tript6fano en la metamorfosis de Pieris Brassicae y Ocnogyna Baetica Rev. Espan. Fisiol. 11, 69-74.

Stamm, M. D. and Aguirre, L. (1955~) . Estudios sobrc bioquimica de insectos 111. Aminoicidos aromiticos y tript6fano en dos cole6pteros. un hemiptero y un ortbptero. Rev. Espan. Fisiol. 11, 75-81.

Stamm, M. D. and Galarza, A. M. (1961). Bioquimica de 10s omocromos. An. Real. Acad. Farm. 1961, 115-140.

Streck, P. (1972). Der Einfluss des Schirmpigmentes auf dz s Sehfeld einzelner Sehzellen der Fliege Calliphora erythrocephala Meig. Z. uergl. Physiol. 76, 372-402.

Strother, G. K. (1966). Absorption of Musca domestictr screening pigment. J. gen. Physiol. 49, 1087-1088.

Subba Rao, P. V., Jegannathan, N. S. and Vaidyanathan, C. S. (1965). Enzymic conversion of 3-hydroxyanthranilic acid into cinnabarinic acid by the nuclear fraction of rat liver. Biochem. J. 95, 628-632.

Suiec-Michieli, S. (1965). Biochemical investigations of the morphological colour changes in Mantis religiosa (Dictyoptera). PTOC. XII. Int . Congr. Ent. London, 1964, Sect. 3, pp. 208-209.

Tartof, K. D. (1969). Interacting gene systems: I. Thc regulation of tryptophan pyrrolase by the vermilion-suppressor of vermilion system in Drosophila Genetics, Princeton, 62, 781-795.

Tatum, E. L. (1939). Development of eye-colors in Drosophila: Bacterial synthesis of u+

hormone. Proc. nutn. Acad. Scr U.S.A. 25, 486-490. Tatum, E. L. and Beadle, G. W. (1939). Effect of diet O’L eye-color development in

Drosophila melanogaster. Biol. Bull. mm. biol Lab.. Woods Hole, 77, 415-422. Tatum, E. L. and Haagen-Smit, A. J. (1941). Identification of Drosophila u’ hormone

of bacterial origin. J. biol. Chem. 140, 575-580. Tiedt, S. (1971). Untersuchungen zum Tryptophanabbaii in der Entwicklung von

Cryllus bimaculatus de Geer. Dipl. Thesis, University of Munich. Tokuyama, T., Senoh, S., Sakan, T., Brown, K. S. and Witkop, B. (1967). The

photoreduction of kynurenic acid to kynurenine yellow and the occurrence of 3-hydroxy-L-kynurenine in butterflies. J. Amer. chem. S O C . 89, 101 7-1021.

Traub, P. (1962). Die Struktur und Synthese des Rhodommatins und Ommatins D. Ein Beitrag zur Chemie der Ommochrome. Doctoral Thesis, University of Munich.

Twardzik, D. R., Grell, E. H. and Jacobson, K. B. (1971). Mechanism of suppression in Drosophila: a change in tyrosine transfer RNA. J. molec Lliol. 57, 231-245.

Udenfriend, S. (1969). “Fluorescence Assay in Biology and Medicine”, Vol. 11, pp. 235-237. Academic Press, London and New York.

Umebachi. Y. (1959). Yellow pigments in the wings of the papilionid butterflies. 111. The radioautographs of the wings of five species of Papiliu injected with CI4-labelled tryptophan. Annotnes zool. jap. 32, 112-116.

Umebachi, Y. and Katayama, M. (1966). Tryptophan and tyrosine metabolism in the pupa of papilionid butterflies. 11. The general pattern of tryptophan metabolism during the pupal stage of Papilio xuthus. J . Insect Physwl. 12, 1539-1547.

Umebachi, Y. and Nakamura, A. (1954). The presence of kynurenine in the wings of the papilionid butterflies. Zool. Mag. Tokyo, 63, 57-61. (In Japanese with English summary.)

244 BERNT LINZEN

Umebachi, Y. and Tsuchitani, K. (1955). The presence of xanthurenic acid in the fruit-fly, Drosophila melanogaster. J . Biochem., Tokyo, 42, 81 7-824.

Umebachi, Y. and Uchida, T. (1970). Ommochromes of the testis and eye of Papilio xuthus. /. Insect Physiol. 16, 1797-1812.

Umebachi, Y. and Yoshida, K. (1970). Some chemical and physical properties of papiliochrome I1 in the wings of Papdio xuthus. J . Insect Physiol. 16, 1203-1 228.

Ursprung, H., Graf, C. E. and Anders, G. (1958). Experimentell ausgeloste Bildungvon rotem Pigment in den Malpighischen Gefassen von Drosophila melanogaster. Rev. Suisse Zool. 65, 449-460.

Viscontini, M. and Schmid, H. (1963). Fluoreszierende Stoffe aus Rhodnius prolixus Stal (Hemiptera, Heteroptera). Ein neues Kynureninderivat: 3-Hydroxy-kynurenin- schwefelsaure-ester = Rhodnitin. Helv. chim. Acta, 46, 2509-2516.

Viscontini, M. and Stierlin, E. (1961). Isolierung, Struktur und Synthese von Pterinen aus Ephestia kiihniella ZELLER. Helv. chim. Acta, 44, 1783-1785.

Viscontini, M. and Stierlin, E. (1963). Synthese von Erythropterin, Ekapterin und Lepidopterin. Helv. chim. Acta, 46, 51-56.

Vuilleaume, M. (1968). Pigmentations et variations pigmentaires de trois insectes: Mantis religiosa (L.), Sphodromantis viridis (F.) et Locusta mipatoria (L.). Bull. biol. Fr. Belg. 102, 147-232.

Wachmann, E. (1 969). Multivesikulare und andere Einschlusskorper in den Retinulazel- len der Sumpfgrille Pteronemobius heydeni (Fischer). Z. Zellforsch. 99, 263-276.

Washizu, Y., Burkhardt, D. and Streck, P. (1964). Visual field of single retinula cells and interommatidial inclination in the compound eye of the blowfly Calliphora erythrocephala. Z. vergl. Physiol. 48, 413-418.

Watanabe, M., Watanabe, Y. and Okada, M. (1970a). A new fluorimetric method for the determination of 3-hydroxykynurenine. Clin. chim. Acta, 27, 461466.

Watanabe, M., Tamura, Z. and Okada, M. (1970b). Structure of a product of a fluorescence reaction of 3-hydroxykynurenine. Chem. Phann. Bull. 18, 285-290.

Weber, F. and Wiss, 0. (1966). Am Tryptophan-Stoffwechsel beteiligte Enzyme. In “Handbuch der physiologisch- und pathologisch-chemischen Analyse” (Eds K. Lang and E. Lehnartz), 10th ed., Vol. VI/B, pp. 806-860. Springer-Verlag, Berlin, Heidelberg and New York.

Wehner, R., Cartenmann, C. and Jungi, Th. (1969). Contrast perception in eye colour mutants of Drosophila melanogaster and Drosophila subobscura. J . Insect Physiol.

Weissbach, H. and Katz, E. (1961). Studies on the biosynthesis of actinomycin: enzymic synthesis of the phenoxazone chromophore. /. bioL Chem. 236, PC 17-18.

Wenzl, H. (1969). Saulenchromatographische Abtrennung von Xanthommatin aus biologischen Extrakten. Mikromethode. Hoppe-Seyler’s Z. physioL Chem. 350,

Wessing, A. (1962). Elektronenmikroskopische Studien zur Funktion der Malpighischen Gefasse von Drosophila melanogaster. Protoplasma. 55, 264-293.

Wessing, A. (1963). Elektronenmikroskopische Studien zur Funktion der Malpighischen Gefasse von Drosophila melanogaster. 11. Die Gefasse der Puppe. Protoplasma, 56,

15, 815-823.

446-448.

433-465.

THE TRYPTOPHAN + OMMOCHROME PATHWAY IF1 INSECTS 245

Wessing, A. and Bonse, A. (1962). Untersuchungen iiber die Speicherung und Ausscheidung von freiem Tryptophan durch die Malpighischen Gefasse von Drosophila melanogaster. Z. Naturf. 17b, 620-622.

Wessing, A. and Bonse, A. (1966). Natur und Bildung lies roten Farbstoffes in den Nierentubuli der Mutante “red” von Drosophila mtdanogaster. Z. Naturf. 21b,

Wessing, A. and Danneel, R. (1961). Die Speicherung von Oxykynurenin in den Malpighischen Gefiissen verschiedener Augenfarbenmutanten von Drosophila melanogaster. Z . Naturf. 16b, 388-390.

Wessing, A. and Eichelberg, D. (1968). Die fluoresziereiiden Stoffe aus den Malpi- ghischen Gefassen der Wildform und verschiedener Augenfarbenmutanten von Drosophila melanogaster. Z . Naturf. 23b. 376-386.

Wessing, A. and Eichelberg, D. ( 1 9 72). Elektronenmikrosltopische Untersuchungen an den Nierentubuli (Malpighische Gefasse) von Drosophila melanogaster. 111. Die intrazellulire Speicherung der Aminosaure, 3-Hydroxykynurenin. Z. Zellforsch. 125,

Willig, A. (1969). Die Carotinoide und der Gallenfarbstoff der Stabheuschrecke, Carausius morosus, und ihre Beteiligung an der Entstehung der Farbmodifikationen. J. Insect PhysioL 15, 1907-1927.

Wilson, L. P. (1945). Tolerance of larvae of Drosophila for amino acids: tryptophane. Growth, 9, 145-154.

Wolfram, R. (1949). a e r die Histogenese der Exkretpigmerte bei verschiedenen Rassen von Ptychopoda serMta Schrk. und Ephestia kiihniella Z. Z . VererbLehre, 83,

Wolsky, A. (1960). Oxidative metabolism and ommochrome synthesis in insects. XIth Int. Congr. Entomol. Vienna, 3, 189-193.

Worz, 0. and Scheibe, G. (1969). b e r die Ursacheri der Alggregation von Farbstoffen, das Spektrum der photographischen Sensibilisierung und die biologische Aktivitat. Z . Naturf. 24b, 381-390.

Wyatt, G. R. (1968). Biochemistry of insect metamorphosis. In “Metamorphosis, a Problem in Developmental Biology” (Eds W. Etkin and L. I. Gilbert), pp. 143-184. North Holland, Amsterdam, and Appleton-Century-Crofts. New York.

Yamafuji, K. (1937). Biologie der Seide. In “Tabulae Biologicae” (Eds W. Junk, C. Oppenheimer and W. Weisbach), Vol. 14, pp. 35-50. Uitgeverij Dr W. Junk, Den Haag.

Yamashita, 0. and Hasegawa, K. (1966). Further studies on the mode of action of the diapause hormone in the silkworm, Bombyx mori L. J. InsectPhysiol. 12, 957-962.

Yasuzumi, G. and Deguchi, N. (1958). Submicroscopic structure of the compound eye as revealed by electron microscopy. J . Ultrastruct. Res. 1, :?59-270.

Yoshida, M., Ohtsuki, H. and Suguri, S. (1967). Ommochrome from anthomedusan ocelli and its photoreduction. Photochem. PhotobioL 6, 875-884.

Zeutzschel, B. (1958). Entwicklung und Lage der Augenpigmente bei verschiedenen Drosophilamutanten. Z. VererbLehre. 89, 508-520.

Ziegler, I. (1960). Zur Feinstruktur der Augengranula bei Dt-osophifa melanogaster. Z . VererbLehre, 91,206-209.

1219-1223.

132-142.

254-298.

246 BERNT LINZEN

Ziegler, I. (1961). Genetic aspects of ommochrome and pterine pigments. Adv. Genet.

Ziegler, I. and Feron, M. (1965). Quantitative Bestimmung der hydrierten Pterine und des Xanthommatins in den Augen von Cerutitis cupituta Wild. (Dipt., Trypetidae). 2. Nuturf. 20b, 318-322.

Ziegler, I. and Harmsen, R. (1969). The biology of pteridines in insects. Adv. Insect Ph_ysiol. 6 , 139-203.

Ziegler, I. and Jaenicke, L. (1959). Zur Wirkungsweise des white-Allels bei Drosophilu rnelunogaster. Z. VererbLehre, 90, 53-61.

10, 349-403.

[advances in insect physiology] volume 10 || the tryptophan → omrnochrome pathway in insects

Documents