An ultrastructural and carotenoid analysis of the red ventrum of the Japanese newt,Cynops pyrrhogaster

The ventral skin of the wild Japanese newt Cynops pyrrhogaster is creamy at metamorphosis, but turns red when mature. The color of the ventral skin of laboratory (lab)-reared newts stays yellow throughout their life. However, the mechanism for the red coloration of this animal still remains unknown. In this study, we have performed ultrastructural and carotenoid analyses of the red ventrum of wild and lab-reared Japanese newts. Using electron microscopy, we observed a number of xanthophores having ring carotenoid vesicles (rcv) and homogenous carotenoid granules (hcg) in the ventral red skin of the wild newt. In the skin, beta-carotene and five other kinds of carotenoids were detected by thin-layer chromatography (TLC). In the ventral yellow skin of lab-reared newts, however, only beta-carotene and three other kinds of carotenoids were found. The total amount of carotenoids in the red skin of the wild adult newt was six times more than that of the yellow skin of the lab-reared newt. Moreover, rcv were more abundant in xanthophores in red skin, but hcg were more abundant in yellow skin. These results, taken together, suggest that the presence of carotenoids in rcv in xanthophores is one of the critical factors for producing the red ventral coloration of the Japanese newt C. pyrrhogaster.     

Morphological and biochemical changes in carotenoid granules in the ventral skin during growth of the Japanese newt Cynops pyrrhogaster

The color of the ventral skin of the Japanese adult newt Cynops pyrrhogaster is red, whereas that of the small juvenile newts at metamorphosis is creamy. Xanthophores in the red skin have many ring carotenoid vesicles (rcv) and a few homogenous carotenoid granules (hcg), as reported earlier. To understand the reason for this change in coloration of the ventral skin of the newt, we carried out histological and biochemical studies to see whether the size and the number of carotenoid granules (hcg and rcv) in the xanthophores and also carotenoid content in the ventral skin change during the growth of this animal. By electron microscopic observation, only hcg were observed in the creamy skin of larvae at stage 59. The diameter of the hcg in the skin of the larvae was approximately 0.85 microm, but significantly decreased to 0.35 microm in the skin of the small juvenile newt. However, the number of the hcg/100 microm (2) of a xanthophore in the ventral skin was very low in the larva at stage 59, but increased in the small juvenile. The carotenoid content was very low in the creamy skin of small juveniles, but dramatically high in the red skin of the adult newts. In the red skin of the adult newt, many rcv (85%) and a few hcg (15%) were observed. However, the number of carotenoid granules (rcv and hcg)/100 microm(2) of a xanthophore in the red skin of adult newts was not different from that of hcg/100 microm (2) of a xanthophore in the creamy skin of small juveniles. The results, taken together, suggest that the increase in the size and the number of carotenoid granules and also carotenoid content in the ventral skin is very important for red body coloration during the growth of the Japanese newt Cynops pyrrhogaster.     

Pigment cell differentiation in the fire-bellied toad,Bombina orientalis. I. Structural,chemical, and physical aspects of the adult pigment pattern

Wild-collected adults of Bombina orientalis are bright green dorsally and red to red-orange ventrally. As a prelude to an analysis of the differentiation of pigment cells in developing B. orientalis, we describe structural and chemical aspects of the fully differentiated pigment pattern of the “normal” adult. Structurally, differences between dorsal green and ventral red skin are summarized as follows: (1) Dorsal green skin contains a “typical” dermal chromatophore unit comprised of melanophores, iridophores, and xanthophores. Red skin contains predominantly carotenoid-containing xanthophores (erythrophores), and skin from black spot areas contains only melanophores. (2) In ventral red skin, there is also a thin layer of deep-lying iridophores that presumably are not involved in the observed color pattern. (3) Xanthophores of red and green skin are morphologically distinguishable from each other. Dorsal skin xanthophores contain both pterinosomes and carotenoid vesicles; ventral skin xanthophores contain only carotenoid vesicles. Carotenoid vesicles in dorsal xanthophores are much larger but less electron dense than comparable structures in ventral xanthophores. The presence of carotenes in ventral skin accounts for the bright red-orange color of the belly of this frog. Similar pigments are also present in green skin, but in smaller quantities and in conjunction with both colored (yellow) and colorless pteridines. From spectral data obtained for xanthophore pigments and structural data obtained from the size and arrangement of reflecting platelets in the iridophore layer, we attempt to explain the phenomenon of observed green color in B. orientalis.     

Dietary carotenoids change the colour of Southern corroboree frogs

Animal coloration can be the result of many interconnected elements, including the production of colour‐producing molecules de novo, as well as the acquisition of pigments from the diet. When acquired through the diet, carotenoids (a common class of pigments) can influence yellow, orange, and red coloration and enhanced levels of carotenoids can result in brighter coloration and/or changes in hue or saturation. We tested the hypothesis that dietary carotenoid supplementation changes the striking black and yellow coloration of the southern corroboree frog (Pseudophryne corroboree, Amphibia: Anura). Our dietary treatment showed no measurable difference in colour or brightness for black patches in frogs. However, the reflectance of yellow patches of frogs raised on a diet rich in carotenoids was more saturated (higher chroma) and long‐wave shifted in hue (more orange) compared to that of frogs raised without carotenoids. Interestingly, frogs with carotenoid‐poor diets still developed their characteristic yellow and black coloration, suggesting that their yellow colour patches are a product of pteridines manufactured de novo.     

Dissecting the mechanism of Solanum lycopersicum and Solanum chilense flower colour formation

Flowers are the defining feature of angiosperms, and function as indispensable organs for sexual reproduction. Flower colour typically plays an important role in attracting pollinators, and can show considerable variation, even between closely related species. For example, domesticated tomato (S. lycopersicum) has orange/yellow flowers, while the wild relative S. chilense (accession LA2405) has bright yellow flowers. In this study, the mechanism of flower colour formation in these two species was compared by evaluating the accumulation of carotenoids, assessing the expression genes related to carotenoid biosynthetic pathways and observing chromoplast ultrastructure. In S. chilense petals, genes associated with the lutein branch of the carotenoid biosynthetic pathway, phytoene desaturase (PDS), ζ‐carotene desaturase (ZDS), lycopene β‐cyclase (LCY‐B), β‐ring hydroxylase (CRTR‐B) and ε‐ring hydroxylase (CRTR‐E), were highly expressed, and this was correlated with high levels of lutein accumulation. In contrast, PDS, ZDS and CYC‐B from the neoxanthin biosynthetic branch were highly expressed in S. lycopersicum anthers, leading to increased β‐carotene accumulation and hence an orange/yellow colour. Changes in the size, amount and electron density of plastoglobules in chromoplasts provided further evidence of carotenoid accumulation and flower colour formation. Taken together, these results reveal the biochemical basis of differences in carotenoid pigment accumulation and colour between petals and anthers in tomato.     

Food habit of the juvenile of the Japanese newt Cynops pyrrhogaster

The previous study showed that the red coloration of the ventral skin of the Japanese newt Cynops pyrrhogaster was associated with the number of carotenoid vesicles and the content of carotenoid in the pigment cell of the skin. To elucidate the mechanism for the red coloration of the skin of the newt, we studied the food habit of the juvenile from the Japanese newt Cynops pyrrhogaster. Sixty-two juveniles were collected in Fukue Island in Nagasaki Prefecture from November 2000 to May 2002 and divided into 2 groups according to the snout-vent length (SVL). Over 400 prey animals were obtained from the juveniles by stomach flushing. In the larger group (SVL>30.0mm), Collembola (45.4%) and Acari (12.6%), which are very common species of soil animals, were the prey animals dominant in number. In the group with the smaller SVL (<29.9mm), Collembola (30.4%) and Acari (25.4%) were in number as well. We also studied the food habit of the Japanese clouded salamander, Hynobius nebulosus. In the salamander, Doratodesmidae (56.5%) and Amphipoda (13%) were the prey animals dominant in number. Our results, taken together, suggest that the Japanese juvenile C. pyrrhogaster does not change its food habit as it grows, and that it eats soil animals common in its habitat. Moreover, the food habit of juvenile C. pyrrhogaster differs from that of H. nebulosus, although the juveniles of both species live in the same area.     

Carotenogenesis diversification in phylogenetic lineages of Rhodophyta

Carotenoid composition is very diverse in Rhodophyta. In this study, we investigated whether this variation is related to the phylogeny of this group. Rhodophyta consists of seven classes, and they can be divided into two groups on the basis of their morphology. The unicellular group (Cyanidiophyceae, Porphyridiophyceae, Rhodellophyceae, and Stylonematophyceae) contained only β‐carotene and zeaxanthin, “ZEA‐type carotenoids.” In contrast, within the macrophytic group (Bangiophyceae, Compsopogonophyceae, and Florideophyceae), Compsopogonophyceae contained antheraxanthin in addition to ZEA‐type carotenoids, “ANT‐type carotenoids,” whereas Bangiophyceae contained α‐carotene and lutein along with ZEA‐type carotenoids, “LUT‐type carotenoids.” Florideophyceae is divided into five subclasses. Ahnfeltiophycidae, Hildenbrandiophycidae, and Nemaliophycidae contained LUT‐type carotenoids. In Corallinophycidae, Hapalidiales and Lithophylloideae in Corallinales contained LUT‐type carotenoids, whereas Corallinoideae in Corallinales contained ANT‐type carotenoids. In Rhodymeniophycidae, most orders contained LUT‐type carotenoids; however, only Gracilariales contained ANT‐type carotenoids. There is a clear relationship between carotenoid composition and phylogenetics in Rhodophyta. Furthermore, we searched open genome databases of several red algae for references to the synthetic enzymes of the carotenoid types detected in this study. β‐Carotene and zeaxanthin might be synthesized from lycopene, as in land plants. Antheraxanthin might require zeaxanthin epoxydase, whereas α‐carotene and lutein might require two additional enzymes, as in land plants. Furthermore, Glaucophyta contained ZEA‐type carotenoids, and Cryptophyta contained β‐carotene, α‐carotene, and alloxanthin, whose acetylenic group might be synthesized from zeaxanthin by an unknown enzyme. Therefore, we conclude that the presence or absence of the four enzymes is related to diversification of carotenoid composition in these three phyla.     

EVOLUTION OF CAROTENOID PIGMENTATION IN CACIQUES AND MEADOWLARKS (ICTERIDAE): REPEATED GAINS OF RED PLUMAGE COLORATION BY CAROTENOID C4‐OXYGENATION

Many animals use carotenoid pigments to produce yellow, orange, and red coloration. In birds, at least 10 carotenoid compounds have been documented in red feathers; most of these are produced through metabolic modification of dietary precursor compounds. However, it is poorly understood how lineages have evolved the biochemical mechanisms for producing red coloration. We used high‐performance liquid chromatography to identify the carotenoid compounds present in feathers from 15 species across two clades of blackbirds (the meadowlarks and allies, and the caciques and oropendolas; Icteridae), and mapped their presence or absence on a phylogeny. We found that the red plumage found in meadowlarks includes different carotenoid compounds than the red plumage found in caciques, indicating that these gains of red color are convergent. In contrast, we found that red coloration in two closely related lineages of caciques evolved twice by what appear to be similar biochemical mechanisms. The C4‐oxygenation of dietary carotenoids was responsible for each observed transition from yellow to red plumage coloration, and has been commonly reported by other researchers. This suggests that the C4‐oxygenation pathway may be a readily evolvable means to gain red coloration using carotenoids.     

Proximate mechanisms of colour variation in the frillneck lizard: geographical differences in pigment contents of an ornament

Animal coloration has evolved in contexts such as communication, camouflage, and thermoregulation. Most studies of animal coloration focus on its adaptive benefits, whereas its underlying mechanisms have received less attention despite their potential influence on adaptive benefits. In fish and reptiles, for example, colour variation from yellow to red can be produced by carotenoid and/or pteridine pigments, which differ dramatically in the way they are obtained (carotenoids through diet and pteridines synthesized de novo). Hence, potential adaptive benefits could differ greatly depending on the relative contribution to coloration of different pigments. In the present study, we investigate the mechanisms underlying colour variation in the frill of the Australian frillneck lizard (Sauropsida: Chlamydosaurus kingii). Frill colour varies between populations across the species' range (red, orange, yellow or white). We argue that this geographical variation results from different concentrations of carotenoids and pteridines in the frill. Frill carotenoid concentrations were lower in eastern populations (yellow and white forms), and pteridines were present only in the red and orange forms, thereby explaining their redder hues. The observed geographical variation in frill carotenoids suggests variation in carotenoid availability across the species' range, which is backed up by the finding that plasma carotenoid concentrations were higher in the red (western) compared to the yellow (eastern) form. Although no correlations were found between individual colour measurements, frill pigments and plasma carotenoids, our results suggest that selective pressures vary across the species' range and we speculate that predation pressures and/or intrasexual signalling context differ between forms.     

Interspecific variation in the use of carotenoid-based coloration in birds: diet, life history and phylogeny

Birds show striking interspecific variation in their use of carotenoid-based coloration. Theory predicts that the use of carotenoids for coloration is closely associated with the availability of carotenoids in the diet but, although this prediction has been supported in single-species studies and those using small numbers of closely related species, there have been no broad-scale quantitative tests of the link between carotenoid coloration and diet. Here we test for such a link using modern comparative methods, a database on 140 families of birds and two alternative avian phylogenies. We show that carotenoid pigmentation is more common in the bare parts (legs, bill and skin) than in plumage, and that yellow coloration is more common than red. We also show that there is no simple, general association between the availability of carotenoids in the diet and the overall use of carotenoid-based coloration. However, when we look at plumage coloration separately from bare part coloration, we find there is a robust and significant association between diet and plumage coloration, but not between diet and bare part coloration. Similarly, when we look at yellow and red plumage colours separately, we find that the association between diet and coloration is typically stronger for red coloration than it is for yellow coloration. Finally, when we build multivariate models to explain variation in each type of carotenoid-based coloration we find that a variety of life history and ecological factors are associated with different aspects of coloration, with dietary carotenoids only being a consistent significant factor in the case of variation in plumage. All of these results remain qualitatively unchanged irrespective of the phylogeny used in the analyses, although in some cases the precise life history and ecological variables included in the multivariate models do vary. Taken together, these results indicate that the predicted link between carotenoid coloration and diet is idiosyncratic rather than general, being strongest with respect to plumage colours and weakest for bare part coloration. We therefore suggest that, although the carotenoid-based bird plumage may a good model for diet-mediated signalling, the use of carotenoids in bare part pigmentation may have a very different functional basis and may be more strongly influenced by genetic and physiological mechanisms, which currently remain relatively understudied.     

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.     

Synergistic metabolism in hybrid corn indicates bottlenecks in the carotenoid pathway and leads to the accumulation of extraordinary levels of the nutritionally important carotenoid zeaxanthin

Lutein and zeaxanthin cannot be synthesized de novo in humans, and although lutein is abundant in fruit and vegetables, good dietary sources of zeaxanthin are scarce. Certain corn varieties provide adequate amounts because the ratio of endosperm β : ε lycopene cyclase activity favours the β‐carotene/zeaxanthin branch of the carotenoid pathway. We previously described a transgenic corn line expressing the early enzymes in the pathway (including lycopene β‐cyclase) and therefore accumulating extraordinary levels of β‐carotene. Here, we demonstrate that introgressing the transgenic mini‐pathway into wild‐type yellow endosperm varieties gives rise to hybrids in which the β : ε ratio is altered additively. Where the β : ε ratio in the genetic background is high, introgression of the mini‐pathway allows zeaxanthin production at an unprecedented 56 μg/g dry weight. This result shows that metabolic synergy between endogenous and heterologous pathways can be used to enhance the levels of nutritionally important metabolites.     

Ultrastructure and distribution of chromatophores in the skin of the leopard gecko (Eublepharis macularius)

The aim of this study was to describe the ultrastructure and arrangement of pigment cells in the leopard gecko (Eublepharis macularius) skin to explain how wild‐type coloration is formed. The study also attempted to explain, on a morphological level, how skin colour changes occur. Samples of leopard gecko skin were collected from wild‐type coloration adult specimens. The morphology of pigmented cells was determined using light microscopy on haematoxylin and eosin (H&E) stained sections and in transmission electron microscopy. These studies indicate that skin of E. macularis contains xanthophores and melanophores but lacks iridophores and that this is probably related to nocturnal activity. The number and distribution of xanthophores and melanophores determines the skin colour and pigmentation pattern. The colour changes depend on the arrangement of characteristic protrusions of melanophores and the degree of filling them with melanosomes.     

Carotenoid limitation and allocation priorities in asynchronous raptor nestlings

The evolution and maintenance of conspicuous animal traits and communication signals have long fascinated biologists. Many yellow–red conspicuous traits are coloured by carotenoid pigments, and in some species they are displayed at a very young age. In nestling birds, the functions and proximate mechanisms of carotenoid‐pigmented traits are probably different and not as well known as those of adults. Here we investigated how Montagu's harrier (Circus pygargus) nestlings within structured families used a limited resource, carotenoid pigments, and whether they used these for increasing coloration (deposition in integuments) or for mounting a response to a phytohaemagglutinin (PHA) challenge, which measures pro‐inflammatory potential and aspects of cellular immune responsiveness. We manipulated carotenoid availability, using dietary carotenoid supplementations, and show that when supplemented, nestlings primarily allocated supplemental carotenoids to increase their coloration, irrespective of their sex, but depending of their position within the brood. Responses to PHA challenge were condition‐dependent, but depending on carotenoid availability. Moreover, how nestlings allocated carotenoids depended on their rank within the brood, which in turn influenced their level of carotenoid limitation (first‐hatched nestlings being less constrained than later‐hatched nestlings). We discuss why nestlings would use supplemental carotenoids for increasing bare parts coloration rather than for responding to a PHA challenge, and the potential benefits for doing so in a parent–offspring communication context. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 13–24.     

Carotenoid-based colour polyphenism in a moth species: search for fitness correlates

Carotenoid‐based integumental coloration is often associated with individual performance in various animals. This is because the limited amount of the pigment has to be allocated to different vital functions. However, most of the evidence for the carotenoid‐based trade‐off comes from vertebrate studies, and it is unclear if this principle can be applied to insects. This possibility was investigated in Orgyia antiqua L. (Lepidoptera: Lymantriidae). The larvae of this species are polyphenic in their coloration, varying from a highly conspicuous combination of yellow hair tufts on black background to cryptic appearance with brown hair tufts. The conspicuous larvae are aposematic, advertising their aversive hairiness. The maintenance of different colour morphs in O. antiqua requires explanation, as an aposematic signal is expected to evolve towards monomorphism. Chromatographic analysis showed that the yellow coloration of the hair is based on the carotenoid pigment lutein (α‐carotene‐3,3’‐diol). The colour of hair tufts was dependent on their carotenoid content. This justifies an expectation of carotenoid‐based physiological trade‐offs between aposematic coloration and individual performance. To test this hypothesis, we monitored life histories of differently coloured larvae reared on various host plants, recording their body sizes, growth rates, and mortalities in each instar. There was a significant but relatively low heritability of tuft coloration, which allowed us to expect environmental effects. We found no phenotypic associations between hair tuft colour and performance indices in O. antiqua larvae, neither did the quality of host plant affect the frequency of colour morphs. However, the frequency of colour morphs differed between larval instars. Our results suggest that carotenoid‐mediated physiological trade‐offs are not involved in the maintenance of colour morphs in O. antiqua larvae, and factors other than individual condition should be responsible for the observed variability.     

The leaves of the common box,Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid,anhydroeschscholtzxanthin, increases

Carotenoids from the leaves of the common box,Buxus sempervirens (Buxaceae), which turn red in late autumn to winter, were analyzed by reversed-phase HPLC. A novel carotenoid, monoanhydroeschscholtzxanthin (3), was isolated from the red-colored leaves. UV-VIS, MS,¹H-NMR and CD spectral data showed that the structure of 3 was (3S)-2′, 3′, 4′, 5′-tetradehydro-4, 5′-retro-β, β-caroten-3-ol. As well as anhydroeschscholtzxanthin (2), the major red carotenoid in the leaves, eschscholtzxanthin (4) was identified. Very small amounts of yellow carotenoids (neoxanthin, violaxanthin, lutein and β-carotene), which are major components of green leaves, were present in the red-colored leaves. The amounts of chlorophylla andb in the leaves decreased markedly during coloration, even at the early stages, whereas those of the yellow carotenoids decreased gradually. In contrast, the content of 2, a red carotenoid, increased steadily during coloration. The biosynthetic pathway of 2 inB. sempervirens was deduced tentatively on the basis of the individual carotenoid contents during autumnal coloration.     

An appraisal of how the vitamin A‐redox hypothesis can maintain honesty of carotenoid‐dependent signals

The vitamin A‐redox hypothesis provides an explanation for honest signaling of phenotypic quality by carotenoid‐dependent traits. A key aspect of the vitamin A‐redox hypothesis, applicable to both yellow and red coloration, is the hypothesized negative feedback of tightly regulated Vitamin A plasma levels on the enzyme responsible for sequestering both Vitamin A and carotenoids from the gut. We performed a meta‐analysis and find that vitamin A levels are positively related to carotenoid plasma levels (r = 0.50, P = 0.0002). On the basis of this finding and further theoretical considerations, we propose that the vitamin A‐redox hypothesis is unlikely to explain carotenoid‐dependent honest signaling.