Quantitative analysis of eumelanin and pheomelanin in humans,mice, and other animals: a comparative review

The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole-2,3,5-tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.     

Quantitative Analysis of Eumelanin and Pheomelanin in Humans,Mice, and Other Animals: a Comparative Review

The color of hair, skin, and eyes in animals mainly depends on the quantity, quality, and distribution of the pigment melanin, which occurs in two types: black to brown eumelanin and yellow to reddish pheomelanin. Microanalytical methods to quantify the amounts of eumelanin and pheomelanin in biological materials were developed in 1985. The methods are based on the chemical degradation of eumelanin to pyrrole‐2,3,5‐tricarboxylic acid and of pheomelanin to aminohydroxyphenylalanine isomers, which can be analyzed and quantitated by high performance liquid chromatography. This review summarizes and compares eumelanin and pheomelanin contents in various pigmented tissues obtained from humans, mice, and other animals. These methods have become valuable tools to study the functions of melanin, the control of melanogenesis, and the actions and interactions of pigmentation genes. The methods have also found applications in many clinical studies. High levels of pheomelanin are found only in yellow to red hairs of mammals and in red feathers of birds. It remains an intriguing question why lower vertebrates such as fishes do not synthesize pheomelanin. Detectable levels of pheomelanin are detected in human skin regardless of race, color, and skin type. However, eumelanin is always the major constituent of epidermal melanin, and the skin color appears to be determined by the quantity of melanin produced but not by the quality.     

Characterization of Melanogenesis in Normal Human Epidermal Melanocytes by Chemical and Ultrastructural Analysis

Chemical and ultrastructural studies were conducted to define the relationship between type of melanogenesis and fine structures of melanosomes in normal human epidermal melanocytes. Chemical analysis of epidermal melanin demonstrated that the ratio of eumelanin/pheomelanin varied individually, ranging from 1.31 to exclusively eumelanic. Ultrastructural analysis of fine structures of melanosomes revealed that spheroid melanosomes were frequently observed in melanocytes of the epidermis whose eumelanin/pheomelanin ratio was less than 5. Conversely, ellipsoid melanosomes predominated in melanocytes of the epidermis whose ratio was more than 10. On the basis of these findings, it seems reasonable to conclude that 1) normal human epidermal melanocytes synthesize both eumelanin and pheomelanin and 2) pheomelanin synthesis may be characterized by the presence of spheroid melanosomes whereas eumelanin synthesis is ascribed to ellipsoid melanosomes.     

Brain size and the expression of pheomelanin-based colour in birds

Eumelanin and pheomelanin are the most common vertebrate pigments. They generate different colours and are synthesized under different physiological conditions. While pheomelanogenesis requires high levels of a key intracellular antioxidant (glutathione, GSH), eumelanogenesis is inhibited by GSH. This implies that species that present the molecular basis to produce large amounts of pheomelanin might be more limited to perform other costly processes that generate oxidative stress than species that produce eumelanin. Brain development requires large amounts of energy and antioxidants during ontogeny, so that large-brained species may be constrained in their simultaneous synthesis of large amounts of pheomelanin, but not in their synthesis of eumelanin. Here, we tested this hypothesis in a large dataset of 323 bird species. After controlling for the effects of phylogeny, latitude and sexual dichromatism, the proportion of pheomelanic plumage colour was strongly negatively related to the relative brain mass of species, whereas no relationship was found for the proportion of eumelanic colour. This indicates that the production of pheomelanin is a costly process that cannot evolve together with complex neural structures and thus with large cognitive capacity. This is the first time that the expression of melanic traits is found to correlate with another phenotypic character across species.     

Eumelanin levels in rufous feathers explain plasma testosterone levels and survival in swallows

Pigment‐based plumage coloration and its physiological properties have attracted many researchers to explain the evolution of such ornamental traits. These studies, however, assume the functional importance of the predominant pigment while ignoring that of other minor pigments, and few studies have focused on the composition of these pigments. Using the pheomelanin‐based plumage in two swallow species, we studied the allocation of two pigments (the predominant pigment, pheomelanin, and the minor pigment, eumelanin) in relation to physiological properties and viability in populations under a natural and sexual selection. This is indispensable for studying the evolution of pheomelanin‐based plumage coloration. Pheomelanin and eumelanin share the same pathway only during their initial stages of development, which can be a key to unravel the functional importance of pigment allocation and thus of plumage coloration. Using the barn swallow, Hirundo rustica, a migratory species, we found that plasma testosterone levels increased with increasing the proportion of eumelanin pigments compared with pheomelanin pigments, but not with the amount of pheomelanin pigments, during the mating period. In the Pacific swallow Hirundo tahitica, a nonmigratory congener, we found that, during severe winter weathers, survivors had a proportionally smaller amount of eumelanin pigments compared with pheomelanin pigments than that in nonsurvivors, but no detectable difference was found in the pheomelanin pigmentation itself. These results indicated that a minor pigment, eumelanin, matters at least in some physiological measures and viability. Because the major pigment, pheomelanin, has its own physiological properties, a combination of major and minor pigments provides multiple information to the signal receivers, potentially enhancing the signaling function of pheomelanic coloration and its diversification across habitats.     

Morphological and geochemical evidence of eumelanin preservation in the feathers of the Early Cretaceous bird, Gansus yumenensis

Recent studies have shown evidence for the preservation of colour in fossilized soft tissues by imaging melanosomes, melanin pigment containing organelles. This study combines geochemical analyses with morphological observations to investigate the preservation of melanosomes and melanin within feathers of the Early Cretaceous bird, Gansus yumenensis. Scanning electron microscopy reveals structures concordant with those previously identified as eumelanosomes within visually dark areas of the feathers but not in lighter areas or sedimentary matrices. Fourier transform infrared analyses show different spectra for the feathers and their matrices; melanic functional groups appear in the feather including carboxylic acid and ketone groups that are not seen in the matrix. When mapped, the carboxylic acid group absorption faithfully replicates the visually dark areas of the feathers. Electron Paramagnetic Resonance spectroscopy of one specimen demonstrates the presence of organic signals but proved too insensitive to resolve melanin. Pyrolysis gas chromatography mass spectrometry shows a similar distribution of aliphatic material within both feathers that are different from those of their respective matrices. In combination, these techniques strongly suggest that not only do the feathers contain endogenous organic material, but that both geochemical and morphological evidence supports the preservation of original eumelanic pigment residue.     

Environmental constraints for plumage melanization in the northern goshawk Accipiter gentilis

Although it is recognized that certain environmental factors are important determinants of the expression of melanin‐based traits, their influence in wild populations of animals is poorly known. One of these factors is the availability of amino acids that serve as precursors of melanins. Here we measured eumelanin and pheomelanin content in feathers of northern goshawk Accipiter gentilis nestlings, hypothesizing that, if the availability of melanin precursors is related to food abundance and habitat quality, plumage melanization should be affected by those variables. Although the eumelanin content increased with food abundance as predicted, the levels of this variable were higher in low‐quality habitats (homogeneous coniferous forests) and in nestlings in poor condition, and the pheomelanin content and eumelanin:pheomelanin ratio were lower and higher, respectively, in subpopulations where nestlings were in poorer condition. Therefore, environmental availability of melanin precursors seems to determine plumage melanization in goshawks, but our findings may also be explained by the differential effects of environmental oxidative stress on both forms of melanin, as eumelanin and pheomelanin production are favoured under high and low levels, respectively, of oxidative stress.     

Acid hydrolysis reveals a low but constant level of pheomelanin in human black to brown hair

We previously reported a constant ratio of the benzothiazole pheomelanin marker thiazole‐2,4,5‐tricarboxylic acid (TTCA) to the eumelanin marker pyrrole‐2,3,5‐tricarboxylic acid (PTCA) in eumelanic, black human hair. A constant level (20%–25%) of benzothiazole‐type pheomelanin was recently demonstrated in human skin with varying concentrations of melanin. Therefore, in this study, we aimed to investigate the origin of pheomelanin markers in black to brown human hair by developing a method to remove protein components from hair by heating with 6 M HCl at 110°C for 16 hr. For comparison, synthetic melanins were prepared by oxidizing mixtures of varying ratios of dopa and cysteine with tyrosinase. Hair melanins and synthetic melanins were subjected to acid hydrolysis followed by alkaline H₂O₂ oxidation. The results show that the hydrolysis leads to decarboxylation of the 5,6‐di‐hydroxyindole‐2‐carboxylic acid moiety in eumelanin and the benzothiazole moiety in pheomelanin and that eumelanic human hair contains 11%–17% benzothiazole‐type pheomelanin.     

Oxidative physiology is weakly associated with pigmentation in birds

The mechanistic link between avian oxidative physiology and plumage coloration has attracted considerable attention in past decades. Hence, multiple proximal hypotheses were proposed to explain how oxidative state might covary with the production of melanin and carotenoid pigments. Some hypotheses underscore that these pigments (or their precursors, e.g., glutathione) have antioxidant capacities or function as molecules storing the toxic excess of intracellular compounds, while others highlight that these pigments can act as pro‐oxidants under specific conditions. Most studies addressing these associations are at the intraspecific level, while phylogenetic comparative studies are still scarce, though needed to assess the generality of these associations. Here, we tested whether plumage and bare part coloration were related to oxidative physiology at an interspecific level by measuring five oxidative physiology markers (three nonenzymatic antioxidants and two markers of lipid peroxidative damage) in 1387 individuals of 104 European bird species sampled during the breeding season, and by scoring plumage eumelanin, pheomelanin, and carotenoid content for each sex and species. Only the plasma level of reactive oxygen metabolites was related to melanin coloration, being positively associated with eumelanin score and negatively with pheomelanin score. Thus, our results do not support the role of antioxidant glutathione in driving variation in melanin synthesis across species. Furthermore, the carotenoid scores of feathers and bare parts were unrelated to the measured oxidative physiology parameters, further suggesting that the marked differences in pigmentation across birds does not influence their oxidative state.     

Variation in melanin pigmentation of a sexually selected plumage trait and its adaptive value in the Common Snipe Gallinago gallinago

There is increasing evidence that melanin‐based plumage coloration correlates with different components of fitness and that it may act as a social or sexual signal of individual quality. We analysed variation in melanin pigmentation in the outermost tail feathers of the Common Snipe Gallinago gallinago. During courtship flights, male Snipe use their outermost tail feathers to generate a drumming sound, which plays a role in territory establishment and mate choice. As the outermost tail feathers are displayed to females during these flights, we predicted that conspicuous variation in their rusty‐brown (pheomelanin‐based) coloration may act as an honest signal of individual quality. To test this prediction, we spectrophotometrically measured brightness (an indicator of total melanin content) and red chroma (an indicator of pheomelanin content) of the outermost tail feathers in 180 juvenile and adult Common Snipe. An age‐related decline in feather brightness was found exclusively in females, suggesting that melanization could have evolved by natural selection to camouflage incubating birds. In both sexes, brightness of the tail feathers was inversely correlated with their structural quality (as measured with mass–length residuals), suggesting that melanization could increase mechanical properties of feathers and, in males, enhance the quality of courtship sonation. Red chroma positively correlated with total plasma protein concentration, supporting our prediction that pheomelanin pigmentation of tail feathers may act as an honest signal of condition. Our study indicated that variation in the melanin‐based coloration of the outermost tail feathers in the Common Snipe could have evolved as a result of several different selection pressures and it emphasizes the complexity of the processes that underlie the evolution of melanin‐based plumage coloration in birds.     

Cyclic oscillations in melanin composition within hairs of baboons.

The wild-type agouti-banding pattern for hair is well characterized in lower mammals such as mice. The switch between eumelanin and pheomelanin in bands in the hair results from the interaction of alpha-melanocyte stimulating hormone and agouti signal protein through the melanocortin 1 receptor on melanocytes. However, such banding patterns have not been described to date in higher mammals. We now report such 'agouti'-banding patterns that occur in several subspecies of baboons, and characterize those hairs using chemical and immunohistochemical methods. Hair and skin samples were obtained from the dorsa of adult male baboons of different subspecies (Papio cynocephalus hamadryas (PCH) and Papio cynocephalus anubis (PCA)). The hairs were excised with scissors into the gray and the white bands of the PCH subspecies and into the black and the yellow bands of the PCA subspecies, and were analyzed for total melanin, eumelanin, and pheomelanin by spectrophotometric and chemical methods. Hairs in the PCA subspecies oscillate between a eumelanic band (with high melanin content) and a pheomelanic band, while hairs in the PCH subspecies oscillate between a eumelanic band (with low melanin content) and a non-pigmented band. Those chemical data are consistent with the histological appearance of the hair bulbs stained by the Fontana-Masson technique. The difference in the melanin content between PCH and PCA subspecies is most likely related to tyrosinase levels, as suggested by the presence of unpigmented muzzle in the PCH subspecies compared with the black muzzle in the PCA subspecies.     

Spiny Mice Modulate Eumelanin to Pheomelanin Ratio to Achieve Cryptic Coloration in “Evolution Canyon,” Israel

Background

Coat coloration in mammals is an explicit adaptation through natural selection. Camouflaging with the environment is the foremost evolutionary drive in explaining overall coloration. Decades of enquiries on this topic have been limited to repetitive coat color measurements to correlate the morphs with background/habitat blending. This led to an overwhelming endorsement of concealing coloration as a local phenotypic adaptation in animals, primarily rodents to evade predators. However, most such studies overlooked how rodents actually achieve such cryptic coloration. Cryptic coloration could be attained only through optimization between the yellow- to brown-colored “pheomelanin” and gray to black-colored “eumelanin” in the hairs. However, no study has explored this conjecture yet. “Evolution Canyon” (EC) in Israel is a natural microscale laboratory where the relationship between organism and environment can be explored. EC is comprised of an “African” slope (AS), which exhibits a yellow-brownish background habitat, and a “European” slope (ES), exhibiting a dark grayish habitat; both slopes harbor spiny mice (Acomys cahirinus). Here, we examine how hair melanin content of spiny mice living in the opposing slopes of EC evolves toward blending with their respective background habitat.

Methodology/Principal Findings

We measured hair-melanin (both eumelanin and pheomelanin) contents of 30 spiny mice from the EC using high-performance liquid chromatography (HPLC) that detects specific degradation products of eumelanin and pheomelanin. The melanin pattern of A. cahirinus approximates the background color of the slope on which they dwell. Pheomelanin is slightly (insignificantly) higher in individuals found on the AS to match the brownish background, whereas individuals of the ES had significantly greater eumelanin content to mimic the dark grayish background. This is further substantiated by a significantly higher eumelanin and pheomelanin ratio on the ES than on the AS.

Conclusion/Significance

It appears that rodents adaptively modulate eumelanin and pheomelanin contents to achieve cryptic coloration in contrasting habitats even at a microscale.     

Cyclic Oscillations in Melanin Composition within Hairs of Baboons

The wild‐type agouti‐banding pattern for hair is well characterized in lower mammals such as mice. The switch between eumelanin and pheomelanin in bands in the hair results from the interaction of α‐melanocyte stimulating hormone and agouti signal protein through the melanocortin 1 receptor on melanocytes. However, such banding patterns have not been described to date in higher mammals. We now report such ‘agouti’‐banding patterns that occur in several subspecies of baboons, and characterize those hairs using chemical and immunohistochemical methods. Hair and skin samples were obtained from the dorsa of adult male baboons of different subspecies (Papio cynocephalus hamadryas (PCH) and Papio cynocephalus anubis (PCA)). The hairs were excised with scissors into the gray and the white bands of the PCH subspecies and into the black and the yellow bands of the PCA subspecies, and were analyzed for total melanin, eumelanin, and pheomelanin by spectrophotometric and chemical methods. Hairs in the PCA subspecies oscillate between a eumelanic band (with high melanin content) and a pheomelanic band, while hairs in the PCH subspecies oscillate between a eumelanic band (with low melanin content) and a non‐pigmented band. Those chemical data are consistent with the histological appearance of the hair bulbs stained by the Fontana‐Masson technique. The difference in the melanin content between PCH and PCA subspecies is most likely related to tyrosinase levels, as suggested by the presence of unpigmented muzzle in the PCH subspecies compared with the black muzzle in the PCA subspecies.     

Sexual Dimorphism in Melanin Pigmentation,Feather Coloration and Its Heritability in the Barn Swallow (Hirundo rustica)

Melanin is the main pigment in animal coloration and considerable variation in the concentrations of the two melanin forms (pheo- and eumlanin) in pigmented tissues exists among populations and individuals. Melanin-based coloration is receiving increasing attention particularly in socio-sexual communication contexts because the melanocortin system has been hypothesized to provide a mechanistic basis for covariation between coloration and fitness traits. However, with few notable exceptions, little detailed information is available on inter-individual and inter-population variation in melanin pigmentation and on its environmental, genetic and ontogenetic components. Here, we investigate melanin-based coloration in an Italian population of a passerine bird, the barn swallow (Hirundo rustica rustica), its sex- and age-related variation, and heritability. The concentrations of eu- and pheomelanin in the throat (brown) and belly (white-to-brownish) feathers differed between sexes but not according to age. The relative concentration of either melanin (Pheo:Eu) differed between sexes in throat but not in belly feathers, and the concentrations in males compared to females were larger in belly than in throat feathers. There were weak correlations between the concentrations of melanins within as well as among plumage regions. Coloration of belly feathers was predicted by the concentration of both melanins whereas coloration of throat feathers was only predicted by pheomelanin in females. In addition, Pheo:Eu predicted coloration of throat feathers in females and that of belly feathers in males. Finally, we found high heritability of color of throat feathers. Melanization was found to differ from that recorded in Hirundo rustica rustica from Scotland or from H. r. erythrogaster from North America. Hence, present results show that pigmentation strategies vary in a complex manner according to sex and plumage region, and also among geographical populations, potentially reflecting adaptation to different natural and sexual selection regimes, and that some coloration components seem to be highly heritable.     

Excess tyrosine stimulates eumelanin and pheomelanin synthesis in cultured slaty melanocytes from neonatal mouse epidermis

The mouse slaty (Dct(slt)) mutation is known to reduce the activity of dopachrome tautomerase (DCT). The reduced DCT activity inhibits melanosome maturation and reduces the melanin content in the skin, hair and eyes. It is not known whether eumelanin and pheomelanin synthesis in slaty melanocytes is modulated by melanogenic factors. In this study, to address this point, epidermal melanocytes derived from 0.5-, 3.5- and 7.5-day-old wild-type mice (Dct(+)/Dct(+) at the slaty locus) and from congenic mice mutant (Dct(slt) /Dct(slt) at that locus) were cultured in serum-free primary culture with or without additional L-tyrosine (Tyr). The content of melanin was measured by high-performance liquid chromatography in the cultured melanocytes as well as culture supernatants in serum-free primary culture. L-Tyr was found to increase the content of pheomelanin in addition to eumelanin in cultured slaty melanocytes and cuture supernatants at all ages tested. The eumelanin and pheomelanin contents in culture supernatants were greater than in cultured melanocytes. The eumelanin and pheomelanin contents in culture supernatants from 7.5-day-old slaty melanocytes in the presence of L-Tyr were greater than those from wild-type melanocytes. These results suggest that the inhibition of eumelanin synthesis by the slaty mutation can be partly restored by the addition of excess L-Tyr. Eumelanin and pheomelanin may accumulate with difficulty in slaty melanocytes and be easily released from them during skin development. L-Tyr may stimulate this release.     

The adaptive function of melanin-based plumage coloration to trace metals

Trace metals produced by anthropogenic activities are of major importance in urban areas and might constitute a new evolutionary force selecting for the ability to cope with their deleterious effects. Interestingly, melanin pigments are known to bind metal ions, thereby potentially sequestering them in inert body parts such as coat and feathers, and facilitating body detoxification. Thus, a more melanic plumage or coat coloration could bring a selective advantage for animals living in polluted areas. We tested this hypothesis by investigating the link between melanin-based coloration and zinc and lead concentrations in feathers of urban feral pigeons, both at capture time and after one year of captivity in standardized conditions. Results show that differently coloured pigeons had similar metal concentrations at capture time. Metal concentrations strongly decreased after one year in standardized conditions, and more melanic pigeons had higher concentrations of zinc (but not lead) in their feathers. This suggests that more melanic pigeons have a higher ability to store some metals in their feathers compared with their paler counterparts, which could explain their higher success in urbanized areas. Overall, this work suggests that trace metal pollution may exert new selective forces favouring more melanic phenotypes in polluted environments.     

Red-winged blackbirds Agelaius phoeniceus use carotenoid and melanin pigments to color their epaulets

Over the past three decades, the red‐winged blackbird Agelaius phoeniceus has served as a model species for studies of sexual selection and the evolution of ornamental traits. Particular attention has been paid to the role of the colorful red‐and‐yellow epaulets that are striking in males but reduced in females and juveniles. It has been assumed that carotenoid pigments bestow the brilliant red and yellow colors on epaulet feathers, but this has never been tested biochemically. Here, we use high‐performance liquid chromatography (HPLC) to describe the pigments present in these colorful feathers. Two red ketocarotenoids (astaxanthin and canthaxanthin) are responsible for the bright red hue of epaulets. Two yellow dietary precursors pigments (lutein and zeaxanthin) are also present in moderately high concentrations in red feathers. After extracting carotenoids, however, red feathers remained deep brown in color. HPLC tests show that melanin pigments (primarily eumelanin) are also found in the red‐pigmented barbules of epaulet feathers, at an approximately equal concentration to carotenoids. This appears to be an uncommon feature of carotenoid‐based ornamental plumage in birds, as was shown by comparable analyses of melanin in the yellow feathers of male American goldfinches Carduelis tristis and the red feathers of northern cardinals Cardinalis cardinalis, in which we detected virtually no melanins. Furthermore, the yellow bordering feathers of male epaulets are devoid of carotenoids (except when tinged with a carotenoid‐derived pink coloration on occasion) and instead are comprised of a high concentration of primarily phaeomelanin pigments. The dual pigment composition of red epaulet feathers and the melanin‐only basis for yellow coloration may have important implications for the honesty‐reinforcing mechanisms underlying ornamental epaulets in red‐winged blackbirds, and shed light on the difficulties researchers have had to date in characterizing the signaling function of this trait. As in several other birds, the melanic nature of feathers may explain why epaulets are used largely to settle aggressive contests rather than to attract mates.     

Morphology of hair pigmentation in wild red foxes,silver foxes,and their hybrids

The effects of dominant allele Ar of locus Agouti on the morphology of hair pigmentation were described in foxes. The Ar allele was shown to determine the type of melanin and its content in hair with no effect on the morphology of pigment granules and their distribution throughout a hair. Using the method of electron spin resonance (ESR), the types of melanin (eumelanin and pheomelanin) and their content in the hair of red (ArArEE) and silver (aaEE) foxes and their hybrids (AraEE) were determined. In silver foxes, only one type of melanin (eumelanin) was found. In red foxes and their hybrids (which are phenotypically similar but darker than red foxes), both types of melanin (eu- and pheomelanin) were found. The highest melanin content was detected in the coat of silver foxes. In the hybrids, the total melanin content was lower than in silver foxes, but significantly higher than in red foxes. In red foxes, the contribution of pheomelanin to the total hair melanin content was twice as large as in the hybrids.     

Selection on a eumelanic ornament is stronger in the tropics than in temperate zones in the worldwide‐distributed barn owl

Spatial variation in the pattern of natural selection can promote local adaptation and genetic differentiation between populations. Because heritable melanin‐based ornaments can signal resistance to environmentally mediated elevation in glucocorticoids, to oxidative stress and parasites, populations may vary in the mean degree of melanic coloration if selection on these phenotypic aspects varies geographically. Within a population of Swiss barn owls (Tyto alba), the size of eumelanic spots is positively associated with survival, immunity and resistance to stress, but it is yet unknown whether Tyto species that face stressful environments evolved towards a darker eumelanic plumage. Because selection regimes vary along environmental gradients, we examined whether melanin‐based traits vary clinally and are expressed to a larger extent in the tropics where parasites are more abundant than in temperate zones. To this end, we considered 39 barn owl species distributed worldwide. Barn owl species living in the tropics displayed larger eumelanic spots than those found in temperate zones. This was, however, verified in the northern hemisphere only. Parasites being particularly abundant in the tropics, they may promote the evolution of darker eumelanic ornaments.     

Insect cuticular melanins are distinctly different from those of mammalian epidermal melanins

Melanin from several insect samples was isolated and subjected to chemical degradation and HPLC analysis for melanin markers. Quantification of different melanin markers reveals that insect melanins are significantly different from that of the mammalian epidermal melanins. The eumelanin produced in mammals is derived from the oxidative polymerization of both 5,6‐dihydroxyindole and 5,6‐dihydroxyindole‐2‐carboxylic acids. The pheomelanin is formed by the oxidative polymerization of cysteinyldopa. Thus, dopa is the major precursor for both eumelanin and pheomelanin in mammals. But insect eumelanin appears to be mostly made from 5,6‐dihydroxyindole and originates from dopamine. More importantly, our study points out the wide spread occurrence of pheomelanin in many insect species. In addition, cysteinyldopamine and not cysteinyldopa is the major precursor for insect pheomelanin. Thus, both eumelanin and pheomelanin in insects differ from higher animals using dopamine and not dopa as the major precursor.