Involvement of Pteridines in the Body Coloration of the Isopod Armadillidium vulgare

The pteridine content was measured as a function of age in Armadillidium vulgare, and the fine structure of the pteridine-containing granules in the integument was examined in relation to pteridine content. Yellow chromatophores are an essential component of the cream-markings, which are a defining feature of the female A. vulgare. Four kinds of pteridines in the integument including a yellow pigment (sepiapterin) were determined by HPLC. The body color of the red phenotype of A. vulgare varies from dark red to yellowish red and was formerly thought to be due to the quality and quantity of ommochrome pigment. Our analysis of the pteridine content in the integument of this phenotype revealed a significant change in sepiapterin content per body weight with age. Sepiapterin content per body weight decreased gradually with age, while that of biopterin tended to increase with age. Ultrastructural observations of the pigment granules in the yellow chromatophores revealed a corresponding change in the fine structure of pigment granules. In the older adults, some of the electron-dense fibrous materials in the pteridine-containing granules was concentrically arranged, and in the younger adults, most of pteridine-containing granules were electron-lucent. The role of pteridine quality in determining the structure of pteridine-containing granules is discussed.     

Morphological and Biochemical Characterization of the Cream Markings in the Integument of the Female Isopod,Armadillidium vulgare

Cream markings aligned along the dorsal region of the female isopod, A. vulgare, were investigated with light and a fluorescence microscope and an electron microscope. Biochemical studies were also carried out. The cream markings were observed in the dorsal integument as a group of cream-colored chromatophores that emit a yellow fluorescence. These chromatophores, which are distinguishable from ommochrome chromatophores, contained numerous granules in the cytoplasm, and these granules (0.6–3.0 μm in length by 0.4–1.5 μm in width) were electron-lucent and spheroidal in shape with a concentric arrangement of membranes. Based on various biochemical analyses, the principal component of the yellow pigment isolated from the cream markings was identified as sepiapterin. These facts revealed that the cream markings are the chromatophores that contain pteridine granules. The males have no cream markings like those of the females, since the cream-colored chromatophores are externally hidden by the ommochrome chromatophore layer. The content of sepiapterin in the males was about two times greater than that in the females. This quantitative difference in sepiapterin content between males and females suggests that the pteridine formation in this pigment cell may be regulated by hormones associated with sex determination.     

Die rolle der Carotinoide und des Gallenfarbstoffs bei der Farbmodifikation der Puppen von Pieris brassicae

The carotenoids and the bile pigment in larvae and pupae of Pieris brassicae were analysed. Their rôle in the morphological colour adaptation of the pupae was studied by quantitative measurements.The carotenoids are β-carotene, lutein mono-ester, free lutein, and zeaxanthin. Metabolized carotenoids were not found. There are no differences between pupae showing different grades of melanization in the quality of the carotenoids, or in the total amounts, or in the relative portions of each carotenoid fraction. However, the carotenoid content of the integument alone is twofold in the light pupae as compared to dark ones. The integumental carotenoids are deposited mainly in the epidermis. β-Carotene, lutein, and zeaxanthin are selectively absorbed by the larvae from the diet. β-Carotene and lutein ester are localized mainly in the fat body, whereas lutein is predominant in the haemolymph and in the integument.The pupal bile pigment is protobiliverdin-IXγ (pterobilin), which is also known to be the larval pigment. The bile pigment is synthesized mainly during the last larval instar up to the pharate pupal stage. In the pupae the bile pigment content is related to the melanization: pupae exposed to the same light conditions contain less bile pigment the more melanized they are (negative correlation). On the whole there is a strong enhancement by blue light of the bile pigment content besides the known stimulation of melanization (positive correlation). But within such a sample the negative correlation between the amounts of bile pigment and melanin is maintained.     

Fluorescence spectrophotometric analysis of melanins in the house mouse

We extracted the yellow melanin (phaeomelanin), black melanin (eumelanin), and mixed type of melanin from dorsal hair of dominant yellow (A ^y /a), non-agouti (a/a), and agouti (A/A) mice, respectively. Spectrophotometric and fluorescence spectrophotometric analysis demonstrated that the yellow melanin was qualitatively distinct from the black melanin and that the agouti hair contained both types of pigment.This work was supported by Grant 244004 from the Ministry of Education. Part of this work was presented at the X International Pigment Cell Conference.     

A stable,genetically determined colour dimorphism in the dung beetle Aphodius depressus: patterns and mechanisms

1. Melanism – the occurrence of dark morphs – in insects has been attributed to differences in, among other things, thermoregulation and immune defence. Dark individuals are hypothesised to perform better in colder areas, and to exhibit stronger melanin‐based immune defence. 2. In the present study, the geographical distribution of two colour morphs in Aphodius depressus (Kugelann), its climatic correlates, and temporal stability was described. Underlying mechanisms were then targeted through experiments: the inheritance of colour through controlled crosses, heating rates by thermal imaging, physiological tolerance by critical thermal limits, and immune efficiency by melanisation of implants. 3. In A. depressus, colour appears inherited by simple Mendelian principles, with red dominating over black. The frequency of two colour morphs forms a large‐scale cline. In the South West of Finland, all individuals are black, whereas, in the North East, most are red. This pattern has remained constant over 13 years (1996–2008). 4. The geographical pattern was not attributable to thermoregulation: black morphs were more abundant in warmer rather than colder parts of the country. In experiments, we found no differences in the heating rate of the two morphs, or in their upper temperature maxima. Neither did the morphs differ in their response to artificial objects inserted in their haemolymph. 5. Overall, colour variation in A. depressus occurs as a stable, genetically determined dimorphism, governed by Mendelian inheritance. Yet, no support for prevailing theory of factors sustaining melanism was found. The reasons for colour polymorphism in insects may thus be complex, and should be sought on a case‐by‐case basis.     

Blue biliprotein as an effective factor for cryptic colouration in Rhodinia fugax larvae

The fifth instar larva of the saturniid silkworm, Rhodinia fugax, is light yellowish-green on its dorsal surface and dark green on the ventral surface with a lateral demarcation between the two colours. The larva of R. fugax closely resembles the leaves of the host plant, Quercus serrata, in colour and shape. The spectral reflectance of the larval integument of R. fugax corresponds to that of the Q. serrata leaf. In the larval integument, there is more blue biliproteins (BPs) on the ventral surface than on the dorsal surface. Light intensity influences larval colouration. The larval integuments are green under light conditions (1000 lux), whereas larvae kept in dark conditions (10 lux) turn yellow. The BP-I content of the haemolymph is also affected by light intensity. The quantities of BP-I and its blue chromophore are higher under light conditions than under dark conditions. In contrast, there is little difference in the yellow chromophore content between the two light intensities. When larvae are kept in the light, the BP-I content in the cuticle is higher than under dark conditions in both the ventral and dorsal surfaces, and its chromophore content parallels the BP content. However, the amounts of BP-II and its chromophore in the epidermis show no change with the light intensity. Moreover, the quantity of yellow chromophore in the integument is also not affected by light intensity. Therefore, light stimulates the accumulation of BP-I and its chromophore in the haemolymph and cuticle, whereas the accumulation of BP-II and its chromophore in the epidermis are not influenced by light intensity. These results suggest that BPs and their chromophores determine the larval colouration and may play an important role in the cryptic colouration of R. fugax larvae.     

Melanin and novel melanin precursors from Aeromonas media

Many bacteria produce reddish brown to black pigments and some of these have been characterised. This report describes the isolation and characterisation of a diffusible brown melanin-like pigment from the bacterium Aeromonas media. Physico-chemical testing suggested that the pigment is a true melanin. New butanol-soluble yellow, red and brown pigments were isolated from the A. media strain under reducing conditions during melanogenesis and these pigments were shown to be unstable precursors of the polymeric brown melanin product.     

DOPA and tyrosine decarboxylase activity in tissues of Periplanet a americana in relation to cuticle formation and ecdysis

DOPA decarboxylase activity in haemolymph and integument was low in last instar and early pharate adult Periplaneta americana, but began to increase shortly before ecdysis. Decarboxylation rates of l-DOPA, about 10 times the larval level by the start of ecdysis, reached a peak about 6 hr afterward, coinciding with the main period of cuticular sclerotization. Activity decreased rapidly during the next 18 hr, then decreased gradually for several days. Haemolymph DOPA decarboxylase activity was about four times greater than the integument, based on tissue dry weights. The fat body and gut tissues had low DOPA decarboxylase activity in all ages tested, and this did not increase at ecdysis. Tyrosine decarboxylase activity was significant only in the haemolymph and at consistently low levels.DOPA decarboxylase, therefore, apparently plays a major rôle in production of catecholamine derivatives for cuticular sclerotization in P. americana, while tyrosine decarboxylation is minor. Both haemolymph and integument appear to be important sites of dopamine biosynthesis.     

Die Redoxpigmente von Carausius morosus und ihre Bedeutung für den morphologischen Farbwechsel

Summary From the epidermis of Carausius morosus two ommochromes were isolated, and identified by various means as Xanthommatine and Ommine. Their amount was determined by photometry for animals of different colour.Morphological colour change results mainly from changes in ommochrome content.If the lower part of their compound eyes is blackened, green specimens become brown by an increase of ommochrome production.This colour change can be evoked within a single larval instar.It can also be evoked in adult specimens, although to a small degree only.Dark specimens with unblinded eyes become paler under normal illumination in consequence of an increase of bright pigments and of the increase in bodysize, while the amount of ommochrome increases only very slightly.A decrease of ommochrome content or a loss of ommochrome by faeces or offshed cuticles was never observed.At high temperature (28° C) both, ommochrome production in the epidermis and melanin formation in the cuticle are increased.Implantation of supernumerous Corpora allata causes the ommochrome content to increase. After extirpation of the Corpora allata no decrease of ommochrome content is found but the green pigment, insectoverdin vanishes. Apparently in Carausius ommochrome may deposited but is never removed from the integument, which may explain the similar coloration after both experiments.     

Alkaline phosphatase in the integument of Ceratitis capitata: Developmental profile and functional properties

Electrophoretic analysis of alkaline phosphatase from the integument during development, reveals two bands of enzyme activity. One corresponding to phosphatase activity during pupation and just prior to eclosion and the other during the middle of the pupal stages. On the contrary in the haemolymph there is one band on enzyme activity through all the developmental stages. The haemolymph alkaline phosphatase band does not comigrate with any integumental enzyme band. The developmental profile of the integumental alkaline phosphatase activity has also been compared to that of the haemolymph. It was found that the pattern of activity is completely different. In the integument, two peaks of enzyme activity were found: one just prior to pupation and the other during eclosion. These two peaks do not coincide to that of haemolymph alkaline phosphatase activity. The pH optimum for both enzyme forms of third instar larvae, although broad especially for haemolymph form, was clearly in the alkaline range, with a peak at pH 8.5–9.0. The two isozymes have different affinities for the substrate tyrosine-O-phosphate. Tyrosine-O-phosphate is the preferred substrate for the integumental enzyme form with a K_m of 0.4 mM. We suggest that alkaline phosphates from the integument is specific for the hydrolysis of tyrosine-O-phosphate.     

Melanin pigmentation gives rise to black spots on the wings of the silkworm Bombyx mori

Several mutants of the silkworm Bombyx mori show body color variation at the larval and adult stages. The Wild wing spot (Ws) mutant exhibits a phenotype in which the moth has a spot on the apex of the forewing. In this study, we investigated this trait to elucidate the molecular mechanism underlying the color pattern. Microscopy of the black spot of Ws mutants showed that the pigment emerges in the scales of the wing, and accumulation of the pigment becomes strong just before eclosion. We next examined the relationship between the black spot of the Ws mutant and melanin. The spectrophotometry using alkaline extracts from the black spot in the wing showed the highest absorption intensity at 405 nm, which is the absorbance wavelength of melanin. Moreover, inhibition assays for enzymes implicated in melanin synthesis using 3-iodo-l-tyrosine (a tyrosine hydroxylase inhibitor) and L-α-methyl-DOPA (a dopa decarboxylase inhibitor) revealed that treatment with each inhibitor disrupted the pigmentation of the wing of the Ws mutant. On the basis of these results, we analyzed the expression pattern of five genes involved in melanin formation, and found that the expression levels of yellow and laccase2 were increased just before pigmentation, whereas those of DDC, tan, and TH were increased when the apex of the wing turned black. These results showed that melanin pigmentation gives rise to the black spot on the wing.     

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

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

Changes in Myxococcus xanthus pigments induced by phosphate and temperature

Pigment levels have been measured in a number of wild strains and colour mutants of Myxococcus xanthus . The non-carotenoid yellow pigment and carotenoid pigment contents changed according to growth temperature and phosphate concentrations in liquid media. The highest content of both types of pigment was observed at 28°C with all strains. A high phosphate concentration depressed pigment content in both wild and mutant strains at all temperatures, except with two colour mutants, where the pigments levels remained unchanged at 28°C and 33°C.     

Yellow Marking and Pteridine Contents in the Integument of Albino Armadillidium vulgare

The albino mutant strain in the woodlice, Armadillidium vulgare, was investigated with respect to the yellow patterns on the dorsal integument. Pigment cells were observed with electron microscope in order to determine the cell types of yellow markings. Quantitative analyses of pteridines in the albino were carried out by HPLC. The result indicated that the albino integument contain sepiapterin, biopterin, pterin, isoxanthopterin as in the wild type and the red mutant strain. The total amount of the four pteridines in the albino was about half as much as that in the red phenotype for both males and females, respectively. Males and females showed almost the same totals and ratios of the four pteridines in the albino and red phenotypes. Therefore, pteridine contents in both phenotypes of A. vulgare may not be related to the activity of androgenic gland hormone. Yellow chromatophores of the albino and red phenotypes were morphologically identical, emitting a yellow fluorescence. These cells contained numerous electron-lucent pigment organelles which were similar to pteridine granules of the wild type.     

Comparative life history and parasitism of a new colour morph of the walnut aphid in California

1 The walnut aphid Chromaphis juglandicola is a yellow aphid. In 2003, however, a white colour morph was discovered in the Sacramento Valley of California. The colour dimorphism occurs between clone lines and, when white morphs are present, they occur in mixed colour morph colonies on the underside of walnut leaves. 2 Laboratory experiments were undertaken to evaluate the thermal requirements for development, adult longevity and progeny production of the two colour morphs. Host instar preference of Trioxys pallidus, a parasitoid responsible for the successful biological control of the walnut aphid in California, was examined separately for each colour morph, and host colour preference was investigated for the preferred instar. 3 No differences in thermal requirements for development, adult size or mean longevity were detected between yellow and white colour morphs. A small difference in early reproduction was detected: white colour morphs produced more progeny on each of the two first days of adult reproduction than yellow colour morphs. 4 Trioxys pallidus showed a slight preference for the fourth instar of the yellow morph over the second‐ and third‐, but equal preference for second, third and fourth instars of the white morph. When offered equal numbers of fourth instars of the two colour morphs, T. pallidus did not show any colour preference. 5 The differences in early aphid reproduction and host instar preference by T. pallidus were combined in a stage‐structured matrix model. Model analysis showed a greater potential for population growth of the white morph over the yellow morph, with early reproduction having a greater influence than host instar preference.     

Chemical analysis of melanin pigments in feather germs of Japanese quail Bh (black at hatch) mutants.

Bh (black at hatch) is a mutation of Japanese quails which causes darkening or lightening of the plumage in heterozygotes or homozygotes, respectively. We chemically analyzed melanin pigments in feather germs of Bh mutant embryos and in feathers of adult animals. Dark brown dorsal feathers of wild-type adult animals had white barrings, but heterozygous ones lacked clear barrings. The feathers of wild-type and heterozygote animals contained both eumelanins and pheomelanins, the latter being more pheomelanic. On the dorsal skin of 10-day old wild-type embryos, longitudinal stripes from black and yellow rows of feather germs developed; two or three longitudinal rows of black feather germs and then two or three rows of yellow feather germs next to the short central feather germs. Heterozygous embryos appeared black in plumage pigmentation, due to the presence of 'gray' feather germs in rows of dorsal feather germs that corresponded to yellow rows in wild-type embryos. Homozygous dorsal feather germs did not develop the black and yellow longitudinal stripes, but were brown. Chemical analysis showed that embryos of each genotype contained both eumelanins and pheomelanins in the feather germs; however, the eumelanin content in homozygous feather germs was very low. These results suggest that the Bh mutation causes pheomelanic changes in feathers of quails.     

Melanin coloration in New World orioles I: carotenoid masking and pigment dichromatism in the orchard oriole complex

Carotenoids produce the brilliant red, orange, and yellow colors of many animals. However, melanin pigments can also confer some of these same hues. Because carotenoid and melanin colors are produced in different ways and may serve different signaling functions, either within or between species, it is important to establish whether one or both types of pigment are responsible for coloration. We have discovered what appears to be an evolutionary switch from carotenoid- to melanin-based color in two sexually dichromatic New World orioles. Using a combination of reflectance spectrometry and chromatographic analyses of plumage pigments, we found that the chestnut plumage of adult male orchard orioles Icterus spurius is produced predominantly by phaeomelanins. Orchard oriole feathers also contain carotenoids, which appear to be masked by the high concentration of phaeomelanins. In contrast, both carotenoids and phaeomelanins appear to contribute to color in adult male Fuertes's orioles I. fuertesi . Moreover, yellow yearling male and female plumage in both species is produced by carotenoids alone. The masking of carotenoids with phaeomelanins in orchard orioles is interesting in light of the signaling roles that carotenoids are thought to play. In addition, these plumage differences produce a unique case of age and sexual pigment dimorphism in orchard and Fuertes's orioles.